U.S. patent application number 16/090551 was filed with the patent office on 2019-04-25 for stimuli-responsive nanoparticles for biomedical applications.
The applicant listed for this patent is The Brigham and Women's Hospital, Inc.. Invention is credited to Omid C. Farokhzad, Jinjun Shi, Xiaoding Xu.
Application Number | 20190117799 16/090551 |
Document ID | / |
Family ID | 58672659 |
Filed Date | 2019-04-25 |
View All Diagrams
United States Patent
Application |
20190117799 |
Kind Code |
A1 |
Xu; Xiaoding ; et
al. |
April 25, 2019 |
STIMULI-RESPONSIVE NANOPARTICLES FOR BIOMEDICAL APPLICATIONS
Abstract
Stimuli-responsive NPs with excellent stability, high loading
efficiency, encapsulation of multiple agents, targeting to certain
cells, tissues or organs of the body, can be used as delivery
tools. These NPs contain a hydrophobic inner core and hydrophilic
outer shell, which endows them with high stability and the ability
to load therapeutic agents with high encapsulation efficiency. The
NPs are preferably formed from amphiphilic stimulus-responsive
polymers or a mixture of amphiphilic and hydrophobic polymers or
compounds, at least one type of which is stimuli-responsive. These
NPs can be made so that their cargo is released primarily within
target certain cells, tissues or organs of the body, upon exposure
to endogenous or exogenous stimuli. The rate of release can be
controlled so that it may be a burst, sustained, delayed, or a
combination thereof. The NPs have utility as research tools or for
clinical applications including diagnostics, therapeutics, or
combination of both.
Inventors: |
Xu; Xiaoding; (Malden,
MA) ; Shi; Jinjun; (Newton, MA) ; Farokhzad;
Omid C.; (Waban, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Brigham and Women's Hospital, Inc. |
Boston |
MA |
US |
|
|
Family ID: |
58672659 |
Appl. No.: |
16/090551 |
Filed: |
April 3, 2017 |
PCT Filed: |
April 3, 2017 |
PCT NO: |
PCT/US2017/025772 |
371 Date: |
October 1, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62317033 |
Apr 1, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/65 20170801;
A61K 9/1273 20130101; C08F 220/28 20130101; C08F 2438/01 20130101;
A61K 9/5138 20130101; A61K 49/0093 20130101; C08F 293/005 20130101;
A61K 49/0054 20130101; C08F 220/286 20200201; A61K 49/0021
20130101; A61K 49/0032 20130101; C08F 220/325 20200201; C08F 220/34
20130101; C08F 220/34 20130101; C08F 220/325 20200201; A61K 9/5153
20130101; C08F 220/286 20200201; A61K 31/711 20130101; A61K 9/5146
20130101 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61K 9/51 20060101 A61K009/51; A61K 47/65 20060101
A61K047/65 |
Claims
1. Stimuli responsive amphiphilic polymers which self-assemble to
form nanoparticles, wherein the stimuli are selected from the group
consisting of pH, temperature, light, redox change, over-expressed
enzymes, hypoxia, sound, magnetic force, electrical energy, and
combinations thereof.
2. The polymers of claim 1 wherein the hydrophobic portion of the
amphiphilic polymers changes shape and/or degrades upon exposure to
the stimuli.
3. The polymers of claim 1 wherein the polymers responsive to pH
are selected from the group consisting of poly
(2-(diisopropylamino) ethylmethacrylate (PDPA),
poly(2-(hexamethyleneimino) ethyl methacrylate (PHMEMA), conjugates
and derives thereof thereof.
4. The polymers of claim 3 wherein the polymer derivatives are
selected from the group consisting of methoxyl-polyethylene
glycol-b-poly (2-(diisopropylamino) ethylmethacrylate)
(Meo-PEG-b-PDPA), methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl methacrylate)
(Meo-PEG-b-P(DPA-co-GMA)), methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl
methacrylate-tetraethylenepentamine)
(Meo-PEG-b-P(DPA-co-GMA-TEPA)), methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl
methacrylate-tetraethylenepentamine-C14)
(Meo-PEG-b-P(DPA-co-GMA-TEPA-C14)), methoxyl-polyethylene
glycol-b-poly (2-(diisopropylamino) ethylmethacrylate-co-glycidyl
methacrylate-oligoarginine) (Meo-PEG-b-P(DPA-co-GMA-Rn)),
methoxyl-polyethylene glycol-b-poly(2-(hexamethyleneimino) ethyl
methacrylate) (Meo-PEG-b-PHMEMA), and poly (2-(hexamethyleneimino)
ethyl methacrylate-co-2-aminoethyl methacrylate)
Meo-PEG-b-P(HMEMA-co-AMA).
5. The polymers of claim 1 which are responsive to light selected
from the group consisting of methoxyl-polyethylene glycol-b-poly
(2-(2-oxo-2-phenylacetoxy) ethyl methacrylate) (Meo-PEG-b-POPEMA)
and mixtures thereof.
6. The polymers of claim 1 which are redox responsive selected from
the group consisting of L-cystine-based poly(disulfide) (PDSA)
polymers.
7. Nanoparticles formed by emulsion with a non-aqueous solvent,
solvent extraction, or nanoprecipitation from polymers in
combination with stimuli responsive polymers, wherein the stimuli
are selected from the group consisting of pH, temperature, light,
redox change, over-expressed enzymes, hypoxia, sound, magnetic
force, electrical energy, and combinations thereof.
8. The nanoparticles of claim 7 wherein the polymers comprise a
first amphiphilic polymer containing a polymer represented by
Formula I: (X).sub.m--(Y).sub.n Formula I wherein m and n are
independently integers between one and 1000, inclusive, X is a
hydrophobic polymer and Y is a hydrophilic polymer, and at least
one of X, Y, or both, is stimuli-responsive.
9. The nanoparticles of claim 8 comprising a mixture of polymers
represented by Formula I and a second polymer containing a polymer
represented by Formula II: (Q).sub.c-(R).sub.d Formula II Wherein c
and d are independently integers between zero and 1000, inclusive,
with the proviso that the sum (c+d) is greater than one, and Q and
R are independently hydrophilic or hydrophobic polymers.
10. The nanoparticles of claim 7 wherein the polymer represented by
Formula I, Formula II, or both, contains a ligand.
11. The nanoparticles of claim 10, wherein the ligand is a
targeting ligand, an adhesion ligand, a cell-penetrating ligand, or
an endosomal-penetrating ligand.
12. The nanoparticles of claim 10, wherein the ligand is selected
from the group consisting of a disulfide-based cyclic
arginine-glycine-aspartic acid (RGD) peptide (iRGD), a tumor
targeting moiety
S,S-2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid
(ACUPA), and oligoarginine, and combinations thereof.
13. The nanoparticles of claim 7 further comprising a
stimuli-responsive hydrophobic polymer
14. The nanoparticles of claim 7 wherein the hydrophilic portion of
the amphiphilic polymers is a polyalkylene oxide, or derivative
thereof.
15. The nanoparticles of claim 7 wherein the polymers comprising
polymer responsive to pH selected from the group consisting of poly
(2-(diisopropylamino) ethylmethacrylate (PDPA),
poly(2-(hexamethyleneimino) ethyl methacrylate (PHMEMA), conjugates
and derives thereof thereof, conjugates and derives thereof.
16. The nanoparticles of claim 5 wherein the polymers comprising
polymer responsive to pH selected from the group consisting of
methoxyl-polyethylene glycol-b-poly (2-(diisopropylamino)
ethylmethacrylate) (Meo-PEG-b-PDPA), methoxyl-polyethylene
glycol-b-poly (2-(diisopropylamino) ethylmethacrylate-co-glycidyl
methacrylate) (Meo-PEG-b-P(DPA-co-GMA)), methoxyl-polyethylene
glycol-b-poly (2-(diisopropylamino) ethylmethacrylate-co-glycidyl
methacrylate-tetraethylenepentamine)
(Meo-PEG-b-P(DPA-co-GMA-TEPA)), methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl
methacrylate-tetraethylenepentamine-C14)
(Meo-PEG-b-P(DPA-co-GMA-TEPA-C14)), methoxyl-polyethylene
glycol-b-poly (2-(diisopropylamino) ethylmethacrylate-co-glycidyl
methacrylate-oligoarginine) (Meo-PEG-b-P(DPA-co-GMA-Rn)),
methoxyl-polyethylene glycol-b-poly(2-(hexamethyleneimino) ethyl
methacrylate) (Meo-PEG-b-PHMEMA), and poly (2-(hexamethyleneimino)
ethyl methacrylate-co-2-aminoethyl methacrylate)
Meo-PEG-b-P(HMEMA-co-AMA), conjugates and derives thereof.
17. The nanoparticles of claim 7 comprising therapeutic,
prophylactic, or diagnostic agents selected from the group
consisting of proteins or peptides, nucleic acids, lipids, sugars
or polysaccharides, small molecules, or combinations thereof.
18. The nanoparticles of claim 17 comprising between about 1% and
about 70% weight/weight, between about 5% and about 50%
weight/weight, or between about 10% and about 30% weight/weight of
a therapeutic agent, a prophylactic agent, a diagnostic agent, or
combinations thereof.
19. The nanoparticles of claim 17 release agent primarily within
target certain cells, tissues or organs of the body, upon exposure
to endogenous or exogenous stimuli.
20. The nanoparticles of claim 17 where the agent is released as a
burst, sustained, delayed, or a combination thereof.
21. The nanoparticles of claim 17 wherein the agent is small
interference RNA (siRNA), RNA interference (RNAi), miRNA, or other
regulatory nucleic acid molecules.
22. The nanoparticles of claim 17 wherein the agent is a
chemotherapeutic, or antiinfective for treatment of a disorder
characterized by a stimuli effecting release or which can be
exposed to a stimuli.
23. The nanoparticles of claim 22 releasing a chemotherapeutic, or
antiinfective at a site of low pH caused by cancer or an
infection.
24. The nanoparticles of claim 7 wherein the polymers comprise
polymer selected from the group consisting of methoxyl-polyethylene
glycol-b-poly (2-(diisopropylamino) ethylmethacrylate-co-glycidyl
methacrylate) (Meo-PEG-b-P(DPA-co-GMA-TEPA-C14),
Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5), iRGD-PEG-b-PDPA, and mixtures
thereof.
25. The nanoparticles of claim 7 wherein membrane-penetrating
oligoarginine grafts, and/or an
S,S-2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid
(ACUPA) terminus are bound to the polymer.
26. The nanoparticles of claim 5 comprising a mixture of
Meo-PEG-b-P(DPA-co-GMA-Rn), and ACUPA-PEG-b-PDPA.
27. The nanoparticles of claim 26 comprising a mixture of
Meo-PEG-b-P(DPA-co-GMA-Rn) (90 mol %) and ACUPA-PEG-b-PDPA (10 mol
%).
28. The nanoparticles of claim 7 wherein the stimulus is pH.
29. The nanoparticles of claim 7 wherein the stimulus is
temperature.
30. The nanoparticles of claim 7 wherein the stimuli is a change in
redox, light, sound, oxygen concentration, or electrical
energy.
31. A method of making the nanoparticles of claim 7 comprising
adding polymer and optionally agent to an emulsion of an aqueous
and a non-aqueous solvent to form stimuli responsive
nanoparticles.
32. A method of delivering therapeutic, prophylactic, and/or
diagnostic agents comprising administering the nanoparticles of
claim 17 and exposing the nanoparticles thereafter to the stimulus
causing release of agent to be delivered.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of and priority to U.S.
Provisional Application No. 62/317,033, filed Apr. 1, 2016, which
is hereby incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was supported by the National Institutes of
Health grants EB015419 (O.C.F), CA151884 (O.C.F.), HL127464
(O.C.F.), R00CA160350 (J.S.) and CA200900 (J.S.).
FIELD OF THE INVENTION
[0003] This invention is generally in the field of developing
stimuli-responsive solid polymeric nanoparticles (NPs) which can be
used to deliver therapeutic and diagnostic agents including nucleic
acids, proteins, chemotherapeutic drugs, or other small
molecules.
BACKGROUND OF THE INVENTION
[0004] Nanoparticles have become an important tool in many
industries including healthcare. Biomedical application of NPs has
introduced exciting opportunities for the improvement of disease
diagnosis and treatment. In particular, stimuli-responsive NPs,
which can undergo shape, structure and property change upon to
endogenous or exogenous stimuli, play an increasingly important
role in a diverse range of biomedical applications, such as
controlled release of drugs, gene delivery and diagnostics. The
stimuli-responsive characteristic may offer spatiotemporal control
over the macroscopic properties of NPs, and thus the release of the
encapsulated cargo can be performed directly at the desired site,
minimizing toxic and side effects in surrounding, healthy
tissue.
[0005] For example, the microenvironment in tumor tissue is
different from the normal tissues. Compared to normal tissues, the
pH in tumor tissue is more acidic, the tissue temperature is
relatively higher, and some specific enzymes or chemicals are
over-expressed. Therefore, developing stimuli-responsive NPs that
can specifically respond to tumor microenvironment will accomplish
the targeted delivery of cargos to tumor sites and thus and impair
the toxic and side effects to healthy tissues.
[0006] The problem with the stimuli responsive NPs is that they
often to not make it to area where release is desired, being
phagocytozed, undergoing enzymatic attack, or becoming physically
entrapped on their way to the desired target.
[0007] It is therefore an object of the present invention to
provide stimuli-responsive nanoparticles (NPs) which can be used to
deliver therapeutic and diagnostic agents including nucleic acids,
proteins, chemotherapeutic drugs, or other small molecules, which
have an increased efficacy in getting to the targeted tissue where
release is to occur.
SUMMARY OF THE INVENTION
[0008] Stimuli-responsive NPs with excellent stability, high
loading efficiency, encapsulation of multiple agents, targeting to
specific cells, tissues or organs of the body, can be used as
delivery tools. These NPs contain a hydrophobic inner core and
hydrophilic outer shell, which endows them with high stability in
water, aqueous buffers, serum and other biological fluids, or the
circulatory system in vivo, and the ability to load therapeutic
agents with high encapsulation efficiency. The diameters of the
nanoparticles are between about 50 nm and about 500 nm, preferably
between about 50 nm and about 350 nm. In some embodiments, the
diameters of the nanoparticles are about 100 nm. The zeta potential
of the nanoparticles are between about -50 mV and about +50 mV,
preferably between about -25 mV and +25 mV, most preferably between
about -10 mV and about +10 my.
[0009] Nanoparticles formed from polymers in combination with
stimuli responsive polymers, wherein the stimuli are selected from
the group consisting of pH, temperature, light, redox change,
over-expressed enzymes, hypoxia, sound, magnetic force, electrical
energy, and combinations thereof, are described. Typically, the
nanoparticles are formed by emulsion with a non-aqueous solvent,
solvent extraction, nanoprecipitation, or a combination
thereof.
[0010] Preferably, the nanoparticles are formed by self-assembly in
an emulsion of a non-aqueous solution with an aqueous solution of a
first amphiphilic polymer containing a polymer represented by
Formula I:
(X).sub.m--(Y).sub.n Formula I
[0011] wherein m and n are independently integers between one and
1000, inclusive, X is a hydrophobic polymer and Y is a hydrophilic
polymer, and at least one of X, Y, or both, is
stimuli-responsive.
[0012] In some embodiments, the nanoparticles are formed by
self-assembly of a mixture of polymers represented by Formula I and
a second polymer containing a polymer represented by Formula
II:
(Q).sub.c-(R).sub.d Formula II
[0013] wherein c and d are independently integers between zero and
1000, inclusive, with the proviso that the sum (c+d) is greater
than one. Q and R are independently hydrophilic or hydrophobic
polymers. Optionally, the nanoparticles are formed by self-assembly
of a mixture of polymers represented by Formula I and Formula II,
wherein the polymer represented by Formula I, Formula II, or both,
contains a ligand, wherein the ligand is a targeting ligand, an
adhesion ligand, a cell-penetrating ligand, and/or an
endosomal-penetrating ligand. Preferably, the ligand is conjugated
to the hydrophilic polymer.
[0014] In some embodiments, the nanoparticles are formed by
self-assembly of a mixture of a stimuli-responsive hydrophobic
polymer and, optionally a further polymer containing a polymer
represented by Formula III:
(S).sub.e-(T).sub.f Formula III
[0015] wherein e and f are independently integers between one and
1000, inclusive, S is a hydrophilic polymer and T is a hydrophobic
polymer. In some embodiments, the stimuli-responsive hydrophobic
polymer, and/or the polymer represented by Formula III, contains a
ligand, wherein the ligand is a targeting ligand, an adhesion
ligand, a cell-penetrating ligand, or an endosomal-penetrating
ligand.
[0016] The molecular weights of the polymers are between about 1
kDa and about 100 kDa, preferably between about 2 kDa and about 50
kDa. In some embodiments, the molecular weights of the polymers are
about 2 kDa, 3 kDa, 10 kDa, 20 kDa, 30 kDa, or 00 kDa. In
embodiments in which the polymer is amphiphilic, the amphiphilic
polymer contains between about 5% and about 90% weight/weight of
the hydrophobic polymer, preferably between about 10% and about 80%
weight/weight of the hydrophobic polymer.
[0017] Optionally, the polymers that form the nanoparticles contain
linkers between the blocks of hydrophilic and hydrophobic polymers,
between the hydrophilic polymer and ligand, or both.
[0018] These stimuli-responsive NPs have two main components: 1) a
hydrophobic core that is made with stimuli-responsive hydrophobic
polymers or the hydrophobic end of amphiphilic polymers to
encapsulate therapeutic and diagnostic agents including proteins or
peptides, nucleic acids, lipids, sugars or polysaccharides, small
molecules, or combinations thereof; and 2) a hydrophilic outer
shell that allows the NPs to evade recognition by immune system
components and increase blood circulation half-life. Hydrophobic
polymers making up the hydrophobic core can be modified to
accommodate the active agent to be encapsulated. In some
embodiments, hydrophobic polymers, or hydrophobic segments of
amphiphilic polymers, are modified with charged groups to allow
loading of charged active agents in the hydrophobic core. For
example, conjugating a hydrophobic component of a polymer with
tetraethylenepentamine or 2-aminoethyl methacrylate will impart a
positive charge to the hydrophobic core to help encapsulate
negatively charged molecules such as nucleic acids.
[0019] The stimuli-responsive polymers are hydrophobic or
amphiphilic, and can be, but are not limited to, pH-, hypoxia-,
redox-, light-, temperature-, enzyme-, or ultrasound-responsive
polymers. The NPs may also include a one or more additional
components: 3) a targeting ligand that can specifically bind to its
receptor on certain cells, tissues, or organs of the body;
endosomal or cell penetrating molecule; or adhesion ligand.
[0020] The stimuli-responsive NPs are made by self-assembly in
emulsions of an aqueous solution with a non-aqueous solution,
resulting in a polymeric nanoparticle that may contain non-aqueous
solvent residue. The amphiphilic copolymers are preferably
polyethylene glycol (PEG) based copolymers. In a preferred
embodiment, the NPs are prepared using a mixture of hydrophobic
polymer and amphiphilic compound. The amphiphilic compound can
include naturally derived lipids, lipid-like materials,
surfactants, or synthesized amphiphilic compounds.
[0021] The NPs are useful for delivery of therapeutic,
prophylactic, and/or diagnostic agents. In some embodiments, the
NPs contain between about 1% and about 70% weight/weight of a
therapeutic agent, a prophylactic agent, a diagnostic agent, or
combinations thereof. Preferably, the NPs contain between about 5%
and about 50% weight/weight, most preferably between about 10% and
about 30% weight/weight of a therapeutic agent, a prophylactic
agent, a diagnostic agent, or combinations thereof. These NPs can
be made so that their cargo is released primarily within target
certain cells, tissues or organs of the body, upon exposure to
endogenous or exogenous stimuli (pH, temperature, redox, light,
etc.). The rate of release can be controlled so that it may be a
burst, sustained, delayed, or a combination thereof. The NPs have
utility as research tools or for clinical applications including
diagnostics, therapeutics, or combination of both.
[0022] One specific use of these stimuli-responsive NPs is in the
field of small interference RNA (siRNA) delivery. RNA interference
(RNAi) technology has gained broad interest among academic and
industry investigators for its potential to treat a myriad of
diseases. One major hurdle in clinical translation of RNAi
therapeutics (e.g., siRNA) may be attributed to the lack of
effective and non-toxic delivery vehicles to transport siRNA into
diseased tissues and cells. Due to its polyanionic and
macromolecular characteristics, naked siRNA cannot freely cross
cellular membrane, and thus requires delivery vehicles to
facilitate its intracellular uptake and endosomal escape, as well
as to protect it from degradation during circulation. Specifically
for cancer therapy, the barriers to effective in vivo siRNA
delivery mainly include targeting to tumor, penetrating tumor
tissue and cell membrane, escaping the endosome and releasing
siRNAs in the cytoplasm. The stimuli-responsive NPs can respond to
tumor or intracellular microenvironment, and thus improve the siRNA
ability to target tumor tissue, escape from endosomes/lysosomes or
efficiently release in cytoplasm for high-performance gene
silencing.
[0023] Besides the delivery of siRNA, the stimuli-responsive NPs
can be applied to the delivery of chemotherapeutic drugs or
proteins for cancer therapy. The key principle of cancer therapy is
to improve the therapeutic efficacy and impair the toxic and side
effects. Owing to their scale and distinct physicochemical
properties as well as the specific pathophysiological
characteristics of tumors, NPs offer the potential to significantly
improve cancer therapy. However, lack of active targeting to cancer
cells and the undesired drug release in healthy tissue are the main
barriers to clinical translation. The disclosed NPs that are
selectively responsive to endogenous or exogenous stimuli offer
spatiotemporal control of the delivery of anticancer therapeutics.
Moreover, through surface-modification of these NPs by targeting
ligand, it can accomplish the objective that targeted delivery of
chemotherapeutic drugs or proteins to tumor tissues and then rapid
drug release induced by the tumor microenvironment, which thus can
minimize toxic and side effects to surrounding healthy tissue.
[0024] The delivery of imaging and/or therapeutic agents is an
alternative use of the stimuli-responsive NPs for disease
diagnostics or theranostic. The microenvironment of diseases is
different from the normal tissues. For example, compared to normal
tissues, the pH in tumor tissue is more acidic, the tissue
temperature is relatively higher, and some specific enzymes or
chemicals are over-expressed. Therefore, developing
stimuli-responsive NPs that can specifically respond to tumor
microenvironment will accomplish the site-specific and rapid
release of imaging and/or therapeutic agents at tumor tissue for
cancer diagnostics or theranostic.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIGS. 1A-1C are schematic illustrations of (1A) molecular
structures of the ultra pH-responsive polymer,
Meo-PEG-b-P(DPA-co-GMA-TEPA-C14), and the tumor-penetrating
peptide-conjugated polymer, iRGD-PEG-b-PDPA; (1B) ultra
pH-responsive and tumor-penetrating nanoplatform for siRNA loading
and release; and (1C) the nanoplatform for targeted in vivo siRNA
delivery and cancer therapy.
[0026] FIGS. 2A-2B are graphs showing pH-dependent release from NPs
of PDPA80. (2A) Normalized fluorescence intensity as a function of
pH for the Cy.5.5-labelled NPs of PDPA80. (2B) In vitro siRNA
release from the NPs of PDPA80 at 37.degree. C. from pH 7.4 and pH
6.0.
[0027] FIGS. 3A-3D are (3A) Luciferase expression in Luc-HeLa cells
transfected with siRNA-loaded NPs at a 10 nM siRNA dose. (3B) Flow
cytometry profile of Luc-HeLa cells incubated with the siRNA-loaded
NPs80 and iRGD-NPs80 for 4 h. (3C) A histogram showing relative
survivin expression determined by Western blot analysis in PC3
cells treated by survivin siRNA-loaded NPs80 or survivin
siRNA-loaded iRGD-NPs80. (3D) A graph showing proliferation of PC3
cells incubated with survivin siRNA-loaded NPs80 and iRGD-NPs80 at
a 10 nM siRNA dose. GL3 siRNA-loaded NPs80 were used as a
control.
[0028] FIG. 4 is a bar graph showing cell viability of Luc-HeLa
cells in the presence of 10 nM siRNA dose of the GL3 siRNA-loaded
NPs and Lipo2K-GL3 siRNA complex. Blank: cells incubated with free
medium.
[0029] FIGS. 5A-5D are graphs showing relative fluorescence
intensity of integrins .alpha.v.beta.3 and .alpha.v.beta.5 on
Luc-HeLa (5A, 5C) and PC3 (5B, 5D) cells determined by flow
cytometry analysis. Blank: cells incubated with free medium;
MFI-mean fluorescence intensity.
[0030] FIGS. 6A-6C are graphs showing relative fluorescence
intensity of DY745-siRNA-loaded NPs80 and iRGD-NPs80. (6A) Flow
cytometry profile of PC3 cells incubated with DY745-siRNA-loaded
NPs80 and iRGD-NPs80 for 4 h at a 10 nM siRNA dose. Mean
fluorescence intensity (MFI) of Luc-HeLa 6(B) and PC3 (6C) cells
incubated with DY547-siRNA-loaded NPs80 and iRGD-NPs80 for 4 h at a
10 nM siRNA dose. *p<0.05.
[0031] FIGS. 7A-7D are histograms showing firefly luciferase
expression in Luc-HeLa cells transfected with GL3 siRNA-loaded NPs
of (7A) Meo-PEG113-b-P(DPA80-co-GMA5-TEPA), and (7B)
Meo-PEG113-b-P(MMA80-co-GMA5-TEPA-C14) at a siRNA dose from 0-50
nM; and cytotoxicity of GL3 siRNA-loaded NPs of (7C)
Meo-PEG113-b-P(DPA80-co-GMA5-TEPA) and (7D)
Meo-PEG113-b-P(MMA80-co-GMA5-TEPA-C14) against Luc-HeLa cells at a
siRNA dose 0-50 nM.
[0032] FIGS. 8A-8B are graphs showing (8A) pharmacokinetics of
naked siRNA, and siRNA-loaded NPs; (8B) biodistribution of the NPs
in the PC3 xenograft tumor-bearing mice sacrificed at 24 h
post-injection of naked siRNA, and siRNA-loaded NPs.
[0033] FIG. 9 is a bar graph showing survivin expression in PC3
xenograft tumor of the mice treated by GL3 siRNA-loaded NPs80
(Control NPs), and survivin siRNA-loaded NPs80 and iRGD-NPs80.
[0034] FIG. 10 is a graph showing relative tumor size over time
(days) of the PC3 xenograft tumor-bearing mice after treatment by
PBS, control NPs, and survivin siRNA-loaded NPs. The intravenous
injections are indicated by the arrows. * P<0.05; **
P<0.01.
[0035] FIG. 11 is graph showing body weight over time (days) of the
PC3 xenograft tumor-bearing nude mice treated with PBS, GL3
siRNA-loaded NPs80 (Control NPs), and survivin siRNA-loaded NPs80
and iRGD-NPs80.
[0036] FIGS. 12A-12C are schematic illustrations of (12A) molecular
structures of the oligoarginine-functionalized ultra pH-responsive
polymer, Meo-PEG-b-P(DPA-co-GMA-Rn), and PCa-specific polymer,
ACUPA-PEG-b-PDPA; (12B) endosomal membrane-penetrating and ultra
pH-responsive nanoplatform for siRNA loading and release; and (12C)
the nanoplatform for in vivo PCa-specific siRNA delivery and cancer
therapy.
[0037] FIGS. 13A-13D are graphs showing (13A) size and
polydispersity (PDI) of the GL3 siRNA-loaded NPs of
Meo-PEG-b-P(DPA-co-GMA-Rn) as a function of number of arginine
residues; (13B) Zeta potential (.zeta.) and encapsulation
efficiency (EE %) of the GL3 siRNA-loaded NPs of
Meo-PEG-b-P(DPA-co-GMA-Rn) as a function of number of arginine
residues; (13C) acid-base titration profile of
Meo-PEG-b-P(DPA-co-GMA-R10) at increasing NaOH concentrations.
(13D) In vitro release of DY745-siRNA over time (hours) from the
NPs of Meo-PEG-b-P(DPA-co-GMA-R10) at a pH of 6.0 and 7.4.
[0038] FIG. 14 is a graph showing normalized fluorescence intensity
as a function of pH for the Cy.5.5 labelled NPs of
Meo-PEG-b-P(DPA-co-GMA-R10).
[0039] FIGS. 15A-15C are graphs showing (15A) firefly luciferase
expression in Luc-HeLa cells transfected with GL3 siRNA-loaded NPs
of Meo-PEG-b-P(DPA-co-GMA-Rn) and Lipo2K-siRNA complex at a 10 nM
siRNA dose; (15B) flow cytometry profile of Luc-HeLa cells
incubated with the DY547-siRNA-loaded NPsR10 and ACUPA-NPsR10 for 4
h; and (15C) a graph showing proliferation over time (days) of
LNCaP cells treated with PHB1 siRNA-loaded NPsR10 and ACUPA-NPsR10
at a 10 nM siRNA dose. GL3 siRNA-loaded NPsR10 were used as a
control.
[0040] FIGS. 16A-16F are graphs showing flow cytometry profiles of
PSMA on Luc-HeLa (16A) and PCa cells including PC3 (16B), DU145
(16C), 22RV1 (16D), and LNCaP (16E) determined by flow cytometry
analysis. (16F) is a summary bar graph showing the fluorescence
intensity of PSMA in Luc-HeLa, PC3, DU145, 22RV1, and LNCaP cells.
Blank: cells incubated with free medium; MFI-mean fluorescence
intensity.
[0041] FIGS. 17A-17F are flow cytometry profiles and mean
fluorescence intensity (MFI) of Luc-HeLa (17A, 17D), PC3 (17B, 17E)
and DU145 (17C, 17F) cells incubated with DY546-siRNA loaded NPsR10
and ACUPA-NPsR10 for 4 h at a 10 nM siRNA dose. Blank: cells
incubated with free medium.
[0042] FIG. 17 is a bar graph showing cytotoxicity of the GL2 siRNA
loaded NPs and Lipo1K-GL3 siRNA complex against Luc-HeLa cells at a
10 nM siRNA dose. Control: cells incubated with free medium.
[0043] FIGS. 19A-19B are bar graphs showing (19A) firefly
luciferase expression in Luc-HeLa cells transfected with GL3
siRNA-loaded NPs and ACUPA-NPs of Meo-PEG-b-P(DPA-co-GMA-TEPA) at a
siRNA dose from 0-50 nM; (19B) cytotoxicity of GL3 siRNA-loaded NPs
and ACUPA-NPs of Meo-PEG-b-P(DPA-co-GMA-TEPA) against Luc-HeLa
cells at a siRNA dose of 0-50 nM.
[0044] FIG. 20 is a bar graph showing Mean fluorescence intensity
(MFI) determined by the flow cytometry profiles of LNCaP cells
incubated with DY547-siRNA-loaded NPsR10 and ACUPA-NPsR10 for 4 h,
and anti-PSMA for 30 min followed by ACUPA-NPsR10 for another 4 h
at a 10 nM siRNA dose. Blank: cells incubated with free medium. *
P<0.5
[0045] FIG. 21 is a bar graph showing relative expression of PHB1
determined by Western blot analysis in LNCaP cells treated with
PHB1 siRNA-loaded NPsR10 and ACUPA-NPsR10. GL3 siRNA-loaded NPsR10
were used as a control.
[0046] FIGS. 22A-22B are graphs showing (22A) pharmacokinetics over
time (hours) of naked DY647-siRNA, and DY647-siRNA-loaded NPsR10
and ACUPA-NPsR10; (22B) biodistribution of the NPs in the tumors
and main organs of the LNCaP xenograft tumor-bearing nude mice
sacrificed 24 h post-injection of naked Cy5.5-siRNA,
Cy5.5-siRNA-loaded NPsR10 and ACUPA-NPsR10, and PSMA antibody
followed by Cy5.5-siRNA-loaded ACUPA-NPsR10.
[0047] FIG. 23 is a graph showing relative tumor size over time
(days) of the LNCaP xenograft tumor-bearing nude mice after
treatment by PBS, control NPs, and PHB1 siRNA-loaded NPsR10 and
ACUPA-NPsR10. The intravenous injections are indicated by the
arrows. GL3 siRNA-loaded NPsR10 were used as a control. *
P<0.05; ** P<0.01 FIG. 24 is a bar graph showing PHB1
expression in LNCaP xenograft tumor of the mice treated with GL3
siRNA-loaded NPsR10 (Control NPs), and PHB1 siRNA-loaded NPsR10 and
ACUPA-NPsR10.
[0048] FIG. 25 is a graph showing body weight of the LNCaP
xenograft tumor-bearing nude mice treated with PBS, GL3 siRNA
loaded NPsR10 (Control NPs), and PHB1 siRNA-loaded NPsR10 and
ACUPA-NPsR10.
[0049] FIGS. 26A-26B are schematics of (26A) molecular structure of
Cy5.5 conjugated ultra pH-responsive copolymer,
Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5; (26B) the self-assembly of
Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5) into nanoparticles with the
aggregation of Cy5.5 inside the hydrophobic cores.
[0050] FIGS. 27A and 27B are (27A) a molecular structure of ultra
pH-responsive polymer, Meo-PEG-b-PDPA and (27B) a graph showing
cumulative release profile of PTX from the PTX loaded NPs of
Meo-PEG-b-PDPA in PBS buffer at a pH of 7.4 and 5.0.
[0051] FIGS. 28A-28B are (28A) molecular structure of
light-responsive polymer, Meo-PEG-b-POPEMA; and (28B) GPC profiles
of the light-responsive Meo-PEG-b-POPEMA before and after 365 nm UV
light irradiation.
[0052] FIGS. 29A-29B are (29A) cumulative release profile over time
(hours) of DTX from the DTX loaded NPs of Meo-PEG-b-POPEMA in PBS
buffer (7.4); and (29B) cytotoxicity of free DTX and DTX loaded NPs
of Meo-PEG-b-POPEMA against PC3 cells at increasing concentrations
of DTX. After incubation with the free DTX or DTX loaded NPs for 8
h, the culture medium was replaced and UV irradiation was applied
for 30 min and then the cells were further incubated for another 40
h.
[0053] FIG. 30 is a schematic illustration of the self-assembly of
redox-responsive polymer into spherical NPs for siRNA delivery and
cancer therapy.
[0054] FIGS. 31A-31B are graphs showing (31A) size change over time
(min) of the NPs of PDSA8-1 incubated in PBS buffer containing 10
mM GSH for 4 h; and (31B) firefly luciferase expression in Luc-HeLa
cells transfected with GL3 siRNA loaded NPs of PDSA polymers at a 1
nM siRNA dose.
[0055] FIG. 32 is a graph showing proliferation over time (days) of
PC3 cells treated with KIF11 siRNA loaded NPs of PDSA8-1. GL3 siRNA
loaded NPs were used as a control.
[0056] FIG. 33 is a graph showing pharmacokinetics over time
(hours) of naked DY647-siRNA, and DY647-siRNA loaded NPs of
PDSA8-1.
[0057] FIG. 34 is a bar graph showing relative levels of KIF11
expression by Western blot analysis in the PC3 tumor tissue after
systemic treatment by KIF11 siRNA loaded NPs of PDSA8-1. GL3 siRNA
loaded NPs were used as a control.
[0058] FIG. 35 is a graph showing relative tumor size over time
(days) of the PC3 xenograft tumor-bearing nude mice after treatment
by PBS, control NPs, naked KIF11 siRNA and KIF11 siRNA loaded NPs
of PDSA8-1. The intravenous injections are indicated by the arrows.
GL3 siRNA loaded NPs were used as a control.
[0059] FIG. 36 is molecular structure of ultra pH-responsive
polymer, PDPA; a graph showing cumulative release profile of PTX
from the PTX loaded NPs of PDPA in PBS buffer at a pH of 7.4 and
5.0.
[0060] FIG. 37 a schematic illustration of illustration of the TME
pH-responsive multistaged nanoplatform for systemic siRNA delivery
and effective cancer therapy. After intravenous injection (i), the
siRNA loaded NPs can first extravasate from leaky tumor vasculature
and accumulate in the tumor tissue (ii). Subsequently, the NPs
respond to TME pH to fast release siRNA/TCPA complexes (iii), which
then target and penetrate tumor cells (iv) to eventually achieve
efficient cytosolic siRNA delivery and gene silencing (v).
[0061] FIGS. 38A-38B are (38A) a graph showing emission
fluorescence spectrum of Cy5.5-labelled TME pH-responsive NPs at
different pHs. Ex=675 nm; (38B) a graph showing cumulative siRNA
release from the DY-677 siRNA loaded TCPA2-NPs at pH 7.4 and pH
6.8.
[0062] FIG. 39 is a graph showing fluorescent emission spectra of
naked Luc siRNA, and Luc siRNA loaded TCPA2-NPs incubated with
RNase for 5 min, 10 min, 15 min, and 6 hr. Fluorescein was labelled
at 5'-end of the sense strand and its quencher Dabcyl was labeled
at the 3'-end of the antisense strand.
[0063] FIGS. 40A-40C are (40A) a plot showing count rate of the
siRNA loaded TCPA2-NPs incubated in PBS buffer (pH 6.8) over a
period of 10 min.; (40B) a bar graph showing size distribution of
TCPA2-NPs incubated in PBS buffer at pH 6.8; (40C) a plot showing
cumulative siRNA release from the DY-677 siRNA loaded TCPA2-NPs at
pH 7.4, and pH 6.8.
[0064] FIGS. 41A-41D are (41A) a graph showing flow cytometry
profile, and (41B) a bar graph showing MFI of Luc-HeLa cells
incubated with DY677-siRNA loaded TCPA2-NPs at pH 7.4, and pH 6.8
for 2 hr. (41C) Luc expression in Luc-HeLa cells treated with Luc
siRNA loaded TCPA2-NPs at pH 7.4, and pH 6.8; (41D) a bar graph
showing viability of Luc-HeLa cells treated with Luc siRNA loaded
TCPA2-NPs at different siRNA doses.
[0065] FIGS. 42A-42C are (FIG. 42A) a bar graph quantifying Western
blot analysis of BRD4 expression in LNCaP cells treated with BRD4
siRNA loaded TCPA2-NPs at pH 7.4, and pH 6.8; (FIG. 42B) a bar
graph summarizing flow cytometry analysis of apoptosis of LNCaP
cells treated with BRD4 siRNA loaded TCPA2-NPs at a 20 nM siRNA
dose at pH 7.4, and pH 6.8; (FIG. 42C) a plot showing proliferation
profile of LNCaP cells treated with BRD4 siRNA loaded TCPA2-NPs at
a 20 nM siRNA dose at pH 7.4, and pH 6.8. Luc siRNA loaded
TCPA2-NPs were used as control.
[0066] FIGS. 43A-43B are a plot (FIG. 43A) showing pharmacokinetics
of naked DY677-siRNA and siRNA loaded TCPA2-NPs; and a bar graph
(FIG. 43B) showing biodistribution of the NPs quantified from
fluorescent images of the tumors and main organs of LNCaP xenograft
tumor-bearing nude mice sacrificed 24 h post injection of naked
DY677-siRNA and siRNA loaded TCPA2-NPs.
[0067] FIGS. 44A-44B are graphs showing relative tumor size (44A)
and tumor weight (44B) of the LNCaP xenograft tumor-bearing nude
mice (n=5) after systemic treatment by PBS, naked BRD4 siRNA,
control NPs, and BRD4 siRNA loaded TCPA2-NPs, where intravenous
injections are indicated by the arrows. Luc siRNA loaded TCPA2-NPs
were used as control NPs.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0068] "Binding," as used herein, refers to the interaction between
a corresponding pair of molecules or portions thereof that exhibit
mutual affinity or binding capacity, typically due to specific or
non-specific binding or interaction, including, but not limited to,
biochemical, physiological, and/or chemical interactions.
[0069] "Binding partner" as used herein refers to a molecule that
can undergo binding with a particular molecule.
[0070] "Biological binding" defines a type of interaction that
occurs between pairs of molecules including proteins, peptides,
nucleic acids, glycoproteins, carbohydrates, or endogenous small
molecules.
[0071] "Specific binding" as used herein refers to molecules, such
as polynucleotides, that are able to bind to or recognize a binding
partner (or a limited number of binding partners) to a
substantially higher degree than to other, similar biological
entities.
[0072] A "biocompatible polymer" is used here to refer to a polymer
that does not typically induce an adverse response when inserted or
injected into a living subject, for example, without significant
inflammation and/or acute rejection of the polymer by the immune
system, for instance, via a T-cell response.
[0073] A "copolymer" herein refers to more than one type of repeat
unit present within the polymer defined below.
[0074] "Encapsulation efficiency" (EE) as used herein is the
fraction of initial drug that is encapsulated by the nanoparticles
(NPs).
[0075] "loading" as used herein refers to the mass fraction of
encapsulated agent in the NPs.
[0076] A "polymer," as used herein, is given its ordinary meaning
as used in the art, i.e., a molecular structure including one or
more repeat units (monomers), connected by covalent bonds. The
polymer may be a copolymer. The repeat units forming the copolymer
may be arranged in any fashion. For example, the repeat units may
be arranged in a random order, in an alternating order, or as a
"block" copolymer, i.e., including one or more regions each
including a first repeat unit (e.g., a first block), and one or
more regions each including a second repeat unit (e.g., a second
block), etc. Block copolymers may have two (a diblock copolymer),
three (a triblock copolymer), or more numbers of distinct
blocks.
[0077] A "polymeric conjugate" as used herein refers to two or more
polymers (such as those described herein) that have been associated
with each other, usually by covalent bonding of the two or more
polymers together.
[0078] As used herein, a nanoparticle refers to a polymeric
particle that can be formed using a solvent emulsion, spray drying,
or precipitation in bulk or microfluids, wherein the solvent is
removed to no more than an insignificant residue, leaving a solid
(which may, or may not, be hollow or have a liquid filled interior)
polymeric particle, unlike a micelle whose form is dependent upon
being present in an aqueous solution.
[0079] As used herein, the term "carrier" or "excipient" refers to
an organic or inorganic ingredient, natural or synthetic inactive
ingredient in a formulation, with which one or more active
ingredients are combined.
[0080] As used herein, the term "pharmaceutically acceptable" means
a non-toxic material that does not interfere with the effectiveness
of the biological activity of the active ingredients.
[0081] As used herein, the terms "effective amount" or
"therapeutically effective amount" means a dosage sufficient to
alleviate one or more symptoms of a disorder, disease, or condition
being treated, or to otherwise provide a desired pharmacologic
and/or physiologic effect. The precise dosage will vary according
to a variety of factors such as subject-dependent variables (e.g.,
age, immune system health, etc.), the disease or disorder being
treated, as well as the route of administration and the
pharmacokinetics of the agent being administered.
[0082] As used herein, the term "prevention" or "preventing" means
to administer a composition to a subject or a system at risk for or
having a predisposition for one or more symptom caused by a disease
or disorder to cause cessation of a particular symptom of the
disease or disorder, a reduction or prevention of one or more
symptoms of the disease or disorder, a reduction in the severity of
the disease or disorder, the complete ablation of the disease or
disorder, stabilization or delay of the development or progression
of the disease or disorder.
[0083] The terms "bioactive agent" and "active agent", as used
interchangeably herein, include, without limitation,
physiologically or pharmacologically active substances that act
locally or systemically in the body. A bioactive agent is a
substance used for the treatment (e.g., therapeutic agent),
prevention (e.g., prophylactic agent), diagnosis (e.g., diagnostic
agent), cure or mitigation of disease or illness, a substance which
affects the structure or function of the body, or pro-drugs, which
become biologically active or more active after they have been
placed in a predetermined physiological environment.
[0084] The terms "sufficient" and "effective", as used
interchangeably herein, refer to an amount (e.g. mass, volume,
dosage, concentration, and/or time period) needed to achieve one or
more desired result(s).
[0085] The term "protein" "polypeptide" or "peptide" refers to a
natural or synthetic molecule comprising two or more amino acids
linked by the carboxyl group of one amino acid to the alpha amino
group of another.
[0086] The term "polynucleotide" or "nucleic acid sequence" refers
to a natural or synthetic molecule comprising two or more
nucleotides linked by a phosphate group at the 3' position of one
nucleotide to the 5' end of another nucleotide. The polynucleotide
is not limited by length, and thus the polynucleotide can include
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
II. Stimuli-responsive Nanoparticles
[0087] A long-circulating, optionally cell-penetrating, and
stimuli-responsive NP platform for effective in vivo delivery of
therapeutic, prophylactic and/or diagnostic agents is made of an
amphiphilic polymer, most preferably a PEGylated polymer, which
shows a response to a stimulus such as pH, temperature, or light,
such as an ultra pH-responsive characteristic with a pKa close to
the endosomal pH (6.0-6.5) (Wang Y et al, Nat Mater, 13, 204-212
(2014)). The polymer may include a targeting, cell penetrating,
and/or adhesion molecule such as a tumor-targeting peptide iRGD
(FIGS. 1A-1B). In some embodiments, the targeting, cell
penetrating, and/or adhesion molecule are convalently conjugated to
one or more of the polymer. In other embodiments, the targeting,
cell penetrating, and/or adhesion molecule are associated with the
nanoparticles formed by one or more polymers via non-covalent
association.
[0088] Generally, the disclosed nanoparticles have prolonged
circulation i.e., increased half-life in the blood compared to
controls without stimuli-responsive element, PEGylation, targeting
moiety, or combinations thereof. In some embodiments, the disclosed
nanoparticles have a half-life of about, or more than, 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11
hours, 12 hours, 24 hours, at least a day, or more than a day.
[0089] Typically, the disclosed nanoparticles have increased
accumulation at target site such as tumor sites compared to
controls without stimuli-responsive element, PEGylation, targeting
moiety, or combinations thereof. Preferably, the disclosed
nanoparticles have deeper penetration into the tumor tissues
compared to controls without stimuli-responsive element,
[0090] PEGylation, targeting moiety, or combinations thereof. In
some embodiments, the disclosed nanoparticles have increased
accumulation at target site by about, or more than, 50%, 100%,
200%, 300%, 400%, or 500%.
[0091] In preferred embodiments, the disclosed nanoparticles have
enhanced uptake by tumor cells compared to controls without
stimuli-responsive element, PEGylation, targeting moiety, or
combinations thereof. In some embodiments, the disclosed
nanoparticles have increased uptake by target cells by about, or
more than, 50%, 100%, 200%, 300%, 400%, or 500%. In further
preferred embodiments, the disclosed nanoparticles have greater
intracellular cargo release without stimuli-responsive element,
PEGylation, targeting moiety, or combinations thereof. In some
embodiments, the disclosed nanoparticles have increased
intracellular cargo release in target cells by about, or more than,
50%, 100%, 200%, 300%, 400%, or 500%.
[0092] When tumor cells are targeted, the disclosed nanoparticles
carrying active agents targeting tumor cells can suppress tumor
growth. In some embodiments, the disclosed nanoparticles can reduce
tumor growth by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
When the active agent is siRNA or shRNA to knowndown a therapeutic
target of the tumor cells, the disclosed nanoparticles can
knockdown the particular target by 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, or 90%.
[0093] Generally, stimuli-responsive nanoparticles are prepared
using one or more amphiphilic copolymers through selection of a
hydrophilic or hydrophobic polymer component of the copolymer, or
by modification of the hydrophilic or hydrophobic polymers.
A. Polymers
[0094] Typically, the nanoparticles can be formed by self-assembly
in an emulsion of a non-aqueous solvent with an aqueous solvent of
a first amphiphilic polymer containing a polymer represented by
Formula I:
(X).sub.m--(Y).sub.n Formula I
wherein, m and n are independently integers between one and 1000,
inclusive. X is a hydrophobic polymer and Y is a hydrophilic
polymer, and at least one of X, Y, or both, is
stimuli-responsive.
[0095] In some embodiments, Y is methoxyl-polyethylene glycol
(Meo-PEG). In one embodiment, X is selected from the group
consisting of poly (2-(diisopropylamino) ethyl methacrylate (PDPA),
poly(2-(hexamethyleneimino) ethyl methacrylate (PHMEMA),
L-cystine-based poly(disulfide) (PDSA), and poly
(2-(2-oxo-2-phenylacetoxy) ethyl methacrylate) (POPEMA). In some
embodiments, X is a hydrophobic copolymer and/or Y is a hydrophilic
copolymer. In one embodiment, X is poly (2-(diisopropylamino)
ethylmethacrylate-co-glycidyl methacrylate (P(DPA-co-GMA)).
Generally, the hydrophobic polymer X forms the hydrophobic core of
the nanoparticle, suitable for encapsulating hydrophobic active
agents within the nanoparticle.
[0096] In some embodiments, one or more parts of the hydrophobic
polymer X is further modified with hydrophilic groups to impart
charges such that the hydrophobic core of the nanoparticle contains
an inner hydrophilic core for encapsulating hydrophilic active
agents such as nucleic acids. Examplary modifications include
including 2-aminoethyl methacrylate (AMA), tetraethylenepentamine
(TEPA), TEPA-C14. In one embodiment, X is P(DPA-co-GMA-TEPA),
P(DPA-co-GMA-TEPA-C14), or poly (2-(hexamethyleneimino) ethyl
methacrylate-co-2-aminoethyl methacrylate) (P(HMEMA-co-AMA)).
[0097] In preferred embodiments, the amphiphilic polymer
represented by Formula I is selected from the group consisting of
Meo-PEG-b-P(DPA-co-GMA), Meo-PEG-b-P(DPA-co-GMA-TEPA-C14),
Meo-PEG-b-P(DPA-co-GMA-Rn), Meo-PEG-b-P(DPA-co-GMA-TEPA),
Meo-PEG113-b-PDPA, Meo-PEG-b-PHMEMA, Meo-PEG-b-P(HMEMA-co-AMA),
Meo-PEG-b-POPEMA, and combinations thereof.
[0098] In some embodiments, the first amphiphilic polymer
represented by Formula I contains a ligand, wherein the ligand is a
targeting ligand, an adhesion ligand, a cell-penetrating ligand, or
an endosomal-penetrating ligand, conjugated to X, Y, or both. In
some embodiments, the ligand is oligoarginine
(NH.sub.2--R.sub.n--CONH.sub.2, where n is any integer between
about 6 to about 100, for example n=6, 8, 10, 20, or 30) attached
to the hydrophobic polyer of Formula I, for example,
Meo-PEG-b-P(DPA-co-GMA-R.sub.n). In one embodiment, the ligand is
NH.sub.2--R.sub.8--CONH.sub.2 (SEQ ID NO:15).
[0099] Optionally, the nanoparticles are formed by self-assembly of
a mixture of polymers represented by Formula I, and a second
polymer containing a polymer represented by Formula II:
(Q).sub.c-(R).sub.d Formula II
wherein, c and d are independently integers between zero and 1000,
inclusive, with the proviso that the sum (c+d) is greater than one.
Q and R are independently hydrophilic or hydrophobic polymers.
[0100] In some embodiments, the nanoparticles are formed by
self-assembly of a mixture of polymers represented by Formula I and
Formula II, wherein the polymer represented by Formula I, Formula
II, or both, contains a ligand, wherein the ligand is a targeting
ligand, an adhesion ligand, a cell-penetrating ligand, or an
endosomal-penetrating ligand, with the proviso that the ligand is
conjugated to the hydrophilic polymer. Examplary ligands include a
disulfide-based cyclic arginine-glycine-aspartic acid (RGD) peptide
(iRGD), a tumor targeting moiety
S,S-2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid
(ACUPA), and a fluorescent Cyanine 5.5 (CY5.5.RTM.) dye.
[0101] In one embodiment, the nanoparticles are formed by
self-assembly of a mixture of methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl
methacrylate-tetraethylenepentamine-C14)
(Meo-PEG-b-P(DPA-co-GMA-TEPA-C14)), and iRGD-PEG-b-PDPA. In another
embodiment, the nanoparticles are formed by self-assembly of a
mixture of methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl
methacrylate-oligoarginine) (Meo-PEG-b-P(DPA-co-GMA-Rn)), and
ACUPA-PEG-b-PDPA.
[0102] Besides amphiphilic copolymers, hydrophobic polymers can be
also used to develop stimuli-responsive NPs for various biomedical
applications. In one embodiment, the hydrophobic polymer is poly
(2-(diisopropylamino) ethyl methacrylate (PDPA), or derivatives
thereof. In another embodiment, the hydrophobic polymer is poly
(2-(2-oxo-2-phenylacetoxy) ethyl methacrylate) (POPEMA), or
derivatives thereof. In a further embodiment, the hydrophobic
polymer is poly (2-(2-oxo-2-phenylacetoxy) ethyl methacrylate)
(POPEMA). Any hydrophobic polymers can be used to prepare
amphiphilic polymers by conjugating to one or more hydrophobic
polymers such as polyethylene glycol, or derivatives thereof.
[0103] In some embodiments, the nanoparticles are formed by
self-assembly of a mixture of a stimuli-responsive hydrophobic
polymer, and optionally, a further polymer containing a polymer
represented by Formula III:
(S).sub.e-(T).sub.f Formula III
wherein, e and f are independently integers between zero and 1000,
inclusive, with the proviso that the sum (e+f) is greater than one.
S and T are independently a hydrophilic polymer or a hydrophobic
polymer. In some embodiments, the stimuli-response hydrophobic
polymer, the polymer represented by Formula III, or both contains a
ligand, wherein the ligand is a targeting ligand, an adhesion
ligand, a cell-penetrating ligand, and/or an endosomal-penetrating
ligand, with the proviso that the ligand is conjugated to the
hydrophilic polymer.
[0104] For hydrophobic polymers, their nanoparticles are generally
prepared by using the mixture of the hydrophobic polymer and
amphiphilic polymer or amphiphilic compound. The amphiphilic
compound can include, but is not limited to, one or a plurality of
naturally derived lipids, PEG-modified lipid, lipid-like materials,
surfactants, or synthesized amphiphilic compounds. In one
embodiment, the amphiphilic compound is a lipid-PEG such as 1,
2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene
glycol)-3000 (DSPE-PEG 3000).
[0105] In some embodiments, the nanoparticles are formed by
self-assembly of a mixture of a stimuli-responsive hydrophilic
polymer, and optionally, a further polymer containing a polymer
represented by Formula III:
(S).sub.e-(T).sub.f Formula III
wherein, e and f are independently integers between zero and 1000,
inclusive, with the proviso that the sum (e+f) is greater than one.
S and T are independently a hydrophilic polymer or a hydrophobic
polymer. Optionally, the first stimuli-response hydrophilic
polymer, the polymer represented by Formula III, or both contains a
ligand, wherein the ligand is a targeting ligand, an adhesion
ligand, a cell-penetrating ligand, or an endosomal-penetrating
ligand, with the proviso that the ligand is conjugated to the
hydrophilic polymer.
[0106] Optionally, the polymers that form the nanoparticles contain
linkers between the blocks of hydrophilic and hydrophobic polymers,
between the hydrophilic polymer and ligand, or both.
[0107] Amphiphilic copolymers can spontaneously self-assemble in
aqueous solution to form NPs with hydrophobic inner core and
hydrophilic outer shells. The hydrophobic inner core can be used to
deliver therapeutic, and/or diagnostic agents including nucleic
acids, proteins, chemotherapeutic drugs, or small molecules. The
incorporation of stimuli-responsive moieties to the hydrophobic
core can easily accomplish the spatiotemporal control over the
macroscopic properties of NPs, and thereby the release of the
encapsulated cargo at the desired site.
[0108] The amphiphilic polymers are responsive to a stimulus. This
may be a pH change, redox change, temperature change, exposure to
light or other stimuli, including binding to a target, and sensing
reduction in oxygen concentrations (hypoxia). The responsiveness
may be imparted solely by the hydrophilic polymer, the hydrophobic
polymer, or the conjugate per se. The nanoparticles are formed of a
mixture or blend of polymers. Some may be the amphiphilic polymers,
preferably copolymers of modified polyethylene glycol (PEG) and
polyesters, such as various forms of PLGA-PEG or PLA-PEG
copolymers, collectively referred to herein as "PEGylated
polymers", some hydrophobic polymer such as PLGA, PLA or PGA,
and/or some may be hydrophilic polymer such as a PEG, or PEG
derivative. Some will be modified by conjugation to a targeting
agent, a cell adhesion or a cell penetrating peptide.
[0109] The length of hydrophilic and/or hydrophobic polymers can be
optimized to optimize encapsulation of agent to be delivered, i.e.,
encapsulation efficiency (EE %). As demonstrated in the examples,
as the PDPA length increases, the EE % and size of the resulting
NPs increase (Table 3), possibly because the increased PDPA length
leads to an increase in the size of the hydrophobic core.
Specifically, the EE % reaches almost 100% for the polymer with 80
(PDPA80) or 100 (PDPA100) DPA repeat units. Notably, using a
mixture of Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) (90 mol %) and
tumor-penetrating polymer (iRGD-PEG-b-PDPA, 10 mol %, FIG. 1A) to
prepare NPs does not cause obvious change in the EE % or particle
size.
[0110] The amphiphilic polymers include a hydrophilic polymer. This
is preferably at an end which can orient to the exterior of the
nanoparticles when formed by emulsion techniques such as
self-assembly.
[0111] Polymers and copolymers that can be used to make the
nanoparticles disclosed herein include, but are not limited to,
polymers including glycolic acid units, referred to herein as
"PGA", and lactic acid units, such as poly-L-lactic acid,
poly-D-Iactic acid, poly-D,L-Iactic acid, poly-L-Iactide,
poly-D-Iactide, and poly-D,L-Iactide, collectively referred to
herein as "PLA", and caprolactone units, such as
poly(8-caprolactone), collectively referred to herein as "PCL"; and
copolymers including lactic acid and glycolic acid units, such as
various forms of poly(lactic acid-co-glycolic acid) and
poly(lactide-co-glycolide) characterized by the ratio of lactic
acid:glycolic acid, collectively referred to herein as "PLGA";
polyacrylates, polyanhydrides, poly (ester anhydrides),
poly-4-hydroxybutyrate (P4HB) combinations and derivatives
thereof.
[0112] The polymer is preferably a biocompatible polymer. One
simple test to determine biocompatibility is to expose a polymer to
cells in vitro; biocompatible polymers are polymers that typically
will not result in significant cell death at moderate
concentrations, e.g., at concentrations of 50 micrograms/10.sup.6
cells. For instance, a biocompatible polymer may cause less than
about 20% cell death when exposed to cells such as fibroblasts or
epithelial cells, even if phagocytosed or otherwise uptaken by such
cells.
[0113] The biocompatible polymer is preferably biodegradable, i.e.,
the polymer is able to degrade, chemically and/or biologically,
within a physiological environment, such as within the body.
[0114] Stimuli that the Polymers can be Responsive to
[0115] The polymers can be responsive to changes in pH-, redox-,
light-, temperature-, enzyme-, ultrasound, or other stimuli such as
a conformation change resulting from binding.
[0116] Almeida, et al. J. Applied Pharm.l Sci. 02 (06)01-10 (2012)
is an excellent review of stimuli responsive polymers. The signs or
stimuli that trigger the structural changes on smart polymers can
be classified in three main groups: physical stimuli (temperature,
ultrasound, light, mechanical stress), chemical stimuli (pH and
ionic strength) and biological stimuli (enzymes and bio
molecules).
[0117] Stimuli can be artificially controlled (with a magnetic or
electric field, light, ultrasounds, etc.) or naturally promoted by
internal physiological environment through a feedback mechanism,
leading to changes in the polymer net that allow the drug delivery
without any external intervention (for example: pH changes in
certain vital organs or related to a disease; temperature change or
presence of enzymes or other antigens) or by the physiological
condition. In the presence of a sign or stimuli, changes can happen
on the surface and solubility of the polymer as well as on sol-gel
transition.
[0118] Smart polymers can be classified according to the stimuli
they respond to or to their physical features. Regarding the
physical shape, they can be classified as free linear polymer chain
solutions, reversible gels covalently cross-linked and polymer
chain grafted to the surface.
[0119] Stimuli responsive polymers are also reviewed by James, et
al., Acta Pharma. Sinica B 4(2):120-127 (2014). The following is a
list of exemplary polymers categorized by responsive to various
stimuli:
[0120] Temperature: POLOXAMERS, poly(N-alkylacrylamide)s,
poly(N-vinylcaprolactam)s, cellulose, xyoglucan, and chitosan
[0121] pH: poly(methacrylic acid)s, poly(vinylpyridine)s, and
poly(vinylimmidazole)s
[0122] light: modified poly(acrylamide)s
[0123] electric field: sulfonated polystyrenes, poly(thiophene)s,
and poly(ethyloxazoline)s
[0124] ultrasound: ethylenevinylacetate
[0125] These transitions are reversible and include changes in
physical state, shape and solubility, solvent interactions,
hydrophilic and lipophilic balances and conductivity. The driving
forces behind these transitions include neutralisation of charged
groups by the addition of oppositely charged polymers or by pH
shift, and change in the hydrophilic/lipophilic balance or changes
in hydrogen bonding due to increase or decrease in temperature.
Responses of a stimulus-responsive polymer can be of various types.
Responsiveness of a polymeric solution initiated by physical or
chemical stimuli is limited to the destruction and formation of
various secondary forces including hydrogen bonding, hydrophobic
forces, van der Waals forces and electrostatic interaction.
Chemical events include simple reactions such as oxidation,
acid-base reaction, reduction and hydrolysis of moieties attached
to the polymer chain. In some cases, dramatic conformational change
in the polymeric structure occurs, e.g., degradation of the
polymeric structure due to irreversible bond breakage in response
to an external stimulus. Upon exposure to appropriate stimuli, some
exemplary physicochemical properties include size, zeta potential
and hydrophilic-hydrophobic balance of these nanoparticles.
[0126] pH Dependent Polymers
[0127] Exemplary pH dependent polymers include dendrimers formed of
poly(lysine), poly(hydroxyproline), PEG-PLA, Poly(propyl acrylic
acid), Poly(ethacrylic acid), CARBOPOLL.RTM., Polysilamine,
EUDRAGIT.RTM. S-100 EUDRAGIT.RTM. L-100, Chitosan, PMAA-PEG
copolymer, sodium alginate (Ca2+). The ionic pH sensitive polymers
are able to accept or release protons in response to pH changes.
These polymers contain acid groups (carboxylic or sulfonic) or
basic groups (ammonium salts) so that the pH sensitive polymers are
polyelectrolytes that have in their structure acid or basic groups
that can accept or release protons in response to pH changes in the
surrounding environment. pH values from several tissues and cell
compartments can be used to trigger release in these tissues. For
example, the pH of blood is 7.4-7.5; stomach is 1.0-3.0; duodenum
is 4.8-8.2; colon is 7.0-7.5; lysosome is 4.5-5.0; Golgi complex is
6.4; tumor--extracellular medium is 6.2-7.2.
[0128] Examples of these polymers include polyacrylamide (PAAm),
poly(acrylic acid) (PAA) (CARBOPOL1.RTM.) and derivatives,
poly(methacrylic acid) (PMAA), poly(2-diethylaminoethyl
methacrylate) (PDEAEMA), poly(ethylene imine), poly(L-lysine) and
poly(N,N-dimethylaminoethylmetha crylate) (PDMAEMA). Polymers with
functional acid groups pH sensitive polymers include poly(acrylic
acid) (PAA) or poly(methacrylic) acid (PMAA) are polyanions that
have in their structure a great number of ionizable acid groups,
like carboxylic acid or sulfonic acid. The pH in which acids become
ionized depends on the polymer's pKa (depends on the polymer's
composition and molecular weight). Polymers with functional basic
groups include polycations such as poly(4-vinylpyridine),
poly(2-vinylpyridine) (PVP) and poly(vinylamine) (PVAm), are
protonated at high pH values and positively ionized at neutral or
low pH values, i.e., they go through a phase transition at pH 5 due
to the deprotonation of the pyridine groups. Other polybases are
poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) and
poly(2-diethylaminoethyl methacrylate) (PDEAEMA), with amino groups
in their structure which in acid environments gain protons, and in
basic environments release the protons. Examples of polycationic
polyelectrolyte polymers are poly(N,N-diakyl aminoethyl
methacrylate), poly(lysine) (PL), poly(ethylenimine) (PEI) and
chitosan. Commercially available polymers include EUDRAGIT L.RTM.
and EUDRAGIT S.RTM. from Rohm Pharma GmBH (with methacrylic acid
and methylmethacrylate in their composition), CMEC (a cellulose
derivative) from Freund Sangyo Co., CAP by Wako Pure Chemicals
Ltd., HP-50 and ASM by Shin-Etsu Chemical Co., Ltd.
[0129] There are several natural polymers (for example, albumin,
gelatin and chitosan) that present pH sensibility. Chitosan is a
cationic amino polysaccharide, derivative from chitin, that is
biocompatible and resorbable. Additional examples include the
anionic polymer PEAA (polyethacrylic acid) or by PPAA (polypropyl
acrylic acid), Polypropylacrylic acid (PPAA) and polyethacrylic
acid (PEAA), and poly(ethylene glycol)-poly(aspartame hydrazine
doxorubicin) [(PEG-p(Asp-Hid-dox), and polycationic polymers, such
as poly(2-diethylaminoethyl methacrylate) (PDEAEMA).
[0130] In one embodiment, the pH-sensitive polymer is poly
(2-(diisopropylamino) ethylmethacrylate (PDPA),
poly(2-(hexamethyleneimino) ethyl methacrylate (PHMEMA), or
PEGylated derivatives, and/or copolymers thereof. Examplary
PEGylated derivatives and copolymers include Meo-PEG-b-PDPA,
methoxyl-polyethylene glycol-b-poly (2-(diisopropylamino)
ethylmethacrylate-co-glycidyl methacrylate)
(Meo-PEG-b-P(DPA-co-GMA)), Meo-PEG-b-P(DPA-co-GMA-TEPA-C14),
Meo-PEG-b-P(DPA-co-GMA-R.sub.n), Meo-PEG-b-P(DPA-co-GMA-TEPA),
methoxyl-polyethylene glycol-b-poly(2-(hexamethyleneimino) ethyl
methacrylate) (Meo-PEG-b-PHMEMA), and
Meo-PEG-b-P(HMEMA-co-AMA).
[0131] Temperature Dependent Polymers
[0132] Temperature dependent polymers are sensitive to the
temperature and change their microstructural features in response
to change in temperature. Thermo-responsive polymers present in
their structure a very sensitive balance between the hydrophobic
and the hydrophilic groups and a small change in the temperature
can create new adjustments. If the polymeric solution has a phase
below the critical solution temperature, it will become insoluble
after heating. Above the critical solution temperature (LCST), the
interaction strengths (hydrogen linkages) between the water
molecules and the polymer become unfavorable, it dehydrates and a
predominance of the hydrophobic interaction occurs, causing the
polymer to swell. The LSCT is the critical temperature in which the
polymeric solution shows a phase separation, going from one phase
(isotropic state) to two phases (anisotropic state). The
accumulation of temperature sensitive polymeric systems in solid
tumors is due to the increased impermeability effect to the tumor
vascular net retention and to the use of an external impulse (heat
source) on the tumor area. This temperature increase promotes the
changing of the microstructure of the polymeric system, turning it
into gel and releasing the drug, thus increasing the drug in the
intra-tumoral area and the therapeutic efficiency, and reducing the
side effects (MacEwan et al., 2010).
[0133] Examples of thermosensitive polymers include the
poly(N-substituted acrylamide) polymers such as
poly(N-isopoprylacrilamide) (PNIPAAm), poly (N,N'-diethyl
acrylamide), poly (dimethylamino ethyl methacrylate and poly
(N-(L)-(1-hydroxymethyl) propyl methacrylamide). Other examples of
thermo-responsive polymers are: copolymers blocks of poly(ethylene
glycol)/poly(lactide-coglicolide) (PEG/PLGA, REGEL.RTM.),
polyoxyethylenepolyoxypropylene (PEO/PPO), triple blocks of
copolymers polyoxyethylene-polyoxypropylene-polyoxyethylene
(PEO-PPOPEO) and poly(ethylene glycol)-poly(lactic
acid)-poly(ethylene glycol) (PEG-PLA-PEG). Exemplary polymers and
their LCST: PNIPAAm, LCST 32.degree. C.; PDEAAm, LCST 26-35.degree.
C.; PDMAEMA, LCST 50.degree. C.;
poly(N-(L)-(hydroxymethyl)propylmethacrylamide), LCST 30.degree.
C.
[0134] An increase of the hydrophobic monomers (as, for example,
the butyl methacrylate) or on the molecular weight, results in a
LCST decrease (Jeong, Gutowska, 2002). The incorporation of
hydrophilic monomers such as acrylic acid or hydroxyethyl
methacrylate) fosters the creation of increases LCST. The
co-polymers NIPAAm conjugated with hydrophilic unities such as
acrylic acid promotes the increase of LCST to temperatures around
37.degree. C., i.e., the body temperature. Polymers with
2-hydroxyethyl (methacrylate) (HEMA) promote the increase of LCST
above the body temperature
[0135] POLOXAMERs and derivatives are well known temperature
sensitive polymers. The copolymer blocks based on PEO-PPO sequences
constitutes one family of triple blocks of commercialized
copolymers with the following names: PLURONICS.RTM.,
POLOXAMERS.RTM. AND TETRONICS.RTM.. POLOXAMERS.RTM. are non-ionic
polymers polyoxyethylenepolyoxypropylene-polyoxyethylene
(PEOn-PPOn-PEOn), with many pharmaceutical uses (Ricci et al.,
2005). The triple block of copolymers PEO--PPO-PEO (PLURONICS.RTM.
or POLOXAMERS.RTM.) get into gel at body temperature in
concentrations above 15% (m/m). The POLOXAMERs.RTM. normally used
are: 188 (F-68), 237 (F-87), 338 (F-108) and 407 (F-127). "F"
refers to the polymer in the form of flakes. PLURONICS.RTM. and
TETRONICS.RTM. are polymers approved by FDA to be used as food
additives, pharmaceutical ingredients, drug carriers in parenteral
systems, tissue engineering and agricultural products. PLURONIC
F-127 (Polaxamer 407, PF-127) can also be used as carrier in
several routes of administration, including oral, cutaneous,
intranasal, vaginal, rectal, ocular and parenteral. PLURONIC.RTM.
F127 (PF-127) or POLOXAMER 407 (P407) (copolymer polyoxyethylene
106-polyoxypropylene 70-polyoxyethylene106) contains about 70% of
ethylene oxide which contributes to its hydrophilicity.
[0136] Polymers with Dual Stimuli-Responsiveness
[0137] To obtain a temperature and pH sensitive polymer it is only
necessary to combine temperature sensitive monomers (as, for
example, poly(N-isopropylacrylamide-co-methacrylic acid and PNIPAm)
with pH sensitive monomers (as, for example, AA and MAA).
[0138] Polymers with Binding or Biological Responsiveness
Biologically responsive polymer systems are increasingly important
in various biomedical applications. The major advantage of
bioresponsive polymers is that they can respond to the stimuli that
are inherently present in the natural system. Bioresponsive
polymeric systems mainly arise from common functional groups that
are known to interact with biologically relevant species, and in
other instances the synthetic polymer is conjugated to a biological
component. Bioresponsive polymers are classified into
antigen-responsive polymers, glucose-sensitive polymers, and
enzyme-responsive polymers.
[0139] Glucose-responsive polymeric-based systems have been
developed based on the following approaches: enzymatic oxidation of
glucose by glucose oxidase, and binding of glucose with lectin or
reversible covalent bond formation with phenylboronic acid
moieties. Glucose sensitivity occurs by the response of the polymer
toward the byproducts that result from the enzymatic oxidation of
glucose. Glucose oxidase oxidises glucose resulting in the
formation of gluconic acid and H.sub.2O.sub.2. For example, in the
case of poly (acrylicacid) conjugated with the GOx system, as the
blood glucose level is increased glucose is converted into gluconic
acid which causes the reduction of pH and protonation of PAA
carboxylate moieties, facilitating the release of insulin. Another
system utilizes the unique carbohydrate binding properties of
lectin for the fabrication of a glucose-sensitive system.
Concanavalin A (Con A) is a lectin possessing four binding sites
and has been used frequently in insulin-modulated drug delivery. In
this type of system the insulin moiety is chemically modified by
introducing a functional group (or glucose molecule) and then
attached to a carrier or support through specific interactions
which can only be interrupted by the glucose itself. The
glycosylated insulin-Con A complex exploits the competitive binding
behaviour of Con A with glucose and glycosylated insulin. The free
glucose molecule causes the displacement of glycosylated Con
A-insulin conjugates.
[0140] Another approach includes polymers with phenylboronic groups
and polyol polymers that form a gel through complex formation
between the pendant phenylborate and hydroxyl groups. Instead of
polyol polymers, short molecules such as diglucosylhexadiamine have
been used. As the glucose concentration increases, the crosslinking
density of the gel decreases and as a result insulin is released
from the eroded gel. The glucose exchange reaction is reversible
and reformation of the gel occurs as a result of borate-polyol
crosslinking.
Field-responsive polymers respond to the application of electric,
magnetic, sonic or electromagnetic fields. The additional benefit
over traditional stimuli-sensitive polymers is their fast response
time, anisotropic deformation due to directional stimuli, and also
a controlled drug release rate simply by modulating the point of
signal control.
[0141] Light-Sensitive Polymers
[0142] A light-sensitive polymer undergoes a phase transition in
response to exposure to light. These polymers can be classified
into UV-sensitive and visible-sensitive systems on the basis of the
wavelength of light that triggers the phase transition.
[0143] A variety of materials are known, such as a leuco-derivative
molecule, bis(4-dimethylamino)phenylmethyl leucocyanide, which
undergoes phase transition behaviour in response to UV light.
Triphenylmethane-leuco derivatives dissociate into ion-pairs such
as triphenylmethyl cations upon UV irradiation. At a fixed
temperature these hydrogels swell discontinuously due to increased
osmotic pressure in response to UV irradiation but shrink when the
stimulus is removed. Another example is a thermosensitive
diarylated pluronic F-127.
[0144] Visible light-sensitive polymeric materials can be prepared
by incorporating photosensitive molecules such as chromophores
(e.g., trisodium salt of copper chlorophyllin). When light of
appropriate wavelength is applied, the chromophore absorbs light
which is then dissipated locally as heat by radiationless
transition, increasing the local temperature of the polymeric
material, leading to alteration of the swelling behavior. The
temperature increase directly depends on the chromophore
concentration and light intensity.
[0145] Electric Field-Sensitive Polymers
[0146] Electric field-sensitive polymers change their physical
properties in response to a small change in electric current. These
polymers contain a relatively large concentration of ionisable
groups along the back bone chain that are also pH-responsive.
Electro-responsive polymers transform electric energy into
mechanical energy. The electric current causes a change in pH which
leads to disruption of hydrogen bonding between polymer chains,
causing degradation or bending of the polymer chain. Major
mechanisms involved in drug release from electro-responsive polymer
are diffusion, electrophoresis of charged drug, forced convection
of drug out of the polymer or degradation of the polymer.
[0147] Naturally occurring polymers such as chitosan, alginate and
hyalouronic acid are commonly employed to prepare
electro-responsive materials. Major synthetic polymers that have
been used include allyl amine, vinyl alcohol, acrylonitrile,
methacrylic acid and vinylacrylic acid. In some cases, combinations
of natural and synthetic polymers have been used. Most polymers
that exhibit electro-sensitive behavior are polyelectrolytes and
undergo deformation under an electric field due to anisotropic
swelling or deswelling as the charged ions move towards the cathode
or anode. Neutral polymers that exhibit electro-sensitive behavior
require the presence of a polarisable component with the ability to
respond to the electric field. Another example of a material which
can be used is poly(2-acrylamido-2-methylpropane sulphonic
acid-co-n-butylmethacrylate).
B. Active Agents
[0148] In some embodiments, the NPs contain between about 1% and
about 70% weight/weight of a therapeutic agent, a prophylactic
agent, a diagnostic agent, or combinations thereof. Preferably, the
NPs contain between about 5% and about 50% weight/weight, most
preferably between about 10% and about 30% weight/weight of a
therapeutic agent, a prophylactic agent, a diagnostic agent, or
combinations thereof.
[0149] Active agent cargos to be delivered include therapeutic,
nutritional, diagnostic, and prophylactic agents. The active agents
can be small molecule active agents or biomacromolecules, such as
proteins, polypeptides, sugars or carbohydrates, lipids, nucleic
acids or small molecule compounds (typically 1 kD or less, but may
be larger). Suitable small molecule active agents include organic
and organometallic compounds. The small molecule active agents can
be a hydrophilic, hydrophobic, or amphiphilic compound.
[0150] Active agents include synthetic and natural proteins
(including enzymes, peptide-hormones, receptors, growth factors,
antibodies, signaling molecules), and synthetic and natural nucleic
acids (including RNA, DNA, anti-sense RNA, triplex DNA, inhibitory
RNA (RNAi), and oligonucleotides), and biologically active portions
thereof. Suitable active agents have a size greater than about
1,000 Da for small peptides and polypeptides, more typically at
least about 5,000 Da and often 10,000 Da or more for proteins.
Nucleic acids are more typically listed in terms of base pairs or
bases (collectively "bp"). Nucleic acids with lengths above about
10 bp are typically used. More typically, useful lengths of nucleic
acids for probing or therapeutic use will be in the range from
about 20 bp (probes; inhibitory RNAs, etc.) to tens of thousands of
bp for genes and vectors. The active agents may also be hydrophilic
molecules, preferably having a low molecular weight.
[0151] Exemplary therapeutic agents that can be incorporated into
particles include tumor antigens, CD4+ T-cell epitopes, cytokines,
chemotherapeutic agents, radionuclides, small molecule signal
transduction inhibitors, photothermal antennas, monoclonal
antibodies, immunologic danger signaling molecules, other
immunotherapeutics, enzymes, antibiotics, antivirals (especially
protease inhibitors alone or in combination with nucleosides for
treatment of HIV or Hepatitis B or C), anti-parasites (helminths,
protozoans), growth factors, growth inhibitors, hormones, hormone
antagonists, antibodies and bioactive fragments thereof (including
humanized, single chain, and chimeric antibodies), antigen and
vaccine formulations (including adjuvants), peptide drugs,
anti-inflammatories, immunomodulators (including ligands that bind
to Toll-Like Receptors (including, but not limited to, CpG
oligonucleotides) to activate the innate immune system, molecules
that mobilize and optimize the adaptive immune system, molecules
that activate or up-regulate the action of cytotoxic T lymphocytes,
natural killer cells and helper T-cells, and molecules that
deactivate or down-regulate suppressor or regulatory T-cells),
agents that promote uptake of particles into cells, nutraceuticals
such as vitamins, and oligonucleotide drugs (including DNA, RNAs,
antisense, aptamers, small interfering RNAs, ribozymes, external
guide sequences for ribonuclease P, and triplex forming
agents).
[0152] Exemplary diagnostic agents include paramagnetic molecules,
fluorescent compounds, magnetic molecules, and radionuclides, x-ray
imaging agents, and contrast agents.
[0153] As discussed in more detail below, in some embodiments, the
particles include one or more anti-cancer agents.
[0154] In certain embodiments, the particle includes one or more
immunomodulatory agents. Exemplary immunomodulatory agents include
cytokines, xanthines, interleukins, interferons,
oligodeoxynucleotides, glucans, growth factors (e.g., TNF, CSF,
GM-CSF and G-CSF), hormones such as estrogens (diethylstilbestrol,
estradiol), androgens (testosterone, HALOTESTIN.RTM.
(fluoxymesterone)), progestins (MEGACE.RTM. (megestrol acetate),
PROVERA.RTM. (medroxyprogesterone acetate)), and corticosteroids
(prednisone, dexamethasone, hydrocortisone).
[0155] Examples of immunological adjuvants that can be associated
with the particles include, but are not limited to, TLR ligands,
C-Type Lectin Receptor ligands, NOD-Like Receptor ligands, RLR
ligands, and RAGE ligands. TLR ligands can include
lipopolysaccharide (LPS) and derivatives thereof, as well as lipid
A and derivatives there of including, but not limited to,
monophosphoryl lipid A (MPL), glycopyranosyl lipid A, PET-lipid A,
and 3-O-desacyl-4'-monophosphoryl lipid A. In a specific
embodiment, the immunological adjuvant is MPL. In another
embodiment, the immunological adjuvant is LPS. TLR ligands can also
include, but are not limited to, TLR3 ligands (e.g.,
polyinosinic-polycytidylic acid (poly(I:C)), TLR7 ligands (e.g.,
imiquimod and resiquimod), and TLR9 ligands.
[0156] The particles may also include antigens and/or adjuvants
(i.e., molecules enhancing an immune response). Peptide, protein,
and DNA based vaccines may be used to induce immunity to various
diseases or conditions. Cell-mediated immunity is needed to detect
and destroy virus-infected cells. Most traditional vaccines (e.g.
protein-based vaccines) can only induce humoral immunity. DNA based
vaccine can induce both humoral and cell-mediated immunity. DNA
vaccines are relatively more stable and more cost-effective for
manufacturing and storage. DNA vaccines consist of two major
components, DNA carriers (or delivery vehicles) and DNAs encoding
antigens. DNA carriers protect DNA from degradation, and can
facilitate DNA entry to specific tissues or cells and expression at
an efficient level.
[0157] Under the Biopharmaceutical Classification System (BCS),
drugs can belong to four classes: class I (high permeability, high
solubility), class II (high permeability, low solubility), class
III (low permeability, high solubility) or class IV (low
permeability, low solubility). Suitable active agents also include
poorly soluble compounds; such as drugs that are classified as
class II or class IV compounds using the BCS. Examples of class II
compounds include: acyclovir, nifedipine, danazol, ketoconazole,
mefenamic acid, nisoldipine, nicardipine, felodipine, atovaquone,
griseofulvin, troglitazone glibenclamide and carbamazepine.
Examples of class IV compounds include: chlorothiazide, furosemide,
tobramycin, cefuroxmine, and paclitaxel.
[0158] An imaging, detectable or sensing moiety, i.e., a moiety
that can be determined in some fashion, either directly or
indirectly, may be bound to the NPs or to the polymers forming the
NPs, or encapsulated therein. Representative imaging entities
include, but are not limited to, fluorescent, radioactive,
electron-dense, magnetic, or labeled members of a binding pair or a
substrate for an enzymatic reaction, which can be detected. In some
cases, the imaging entity itself is not directly determined, but
instead interacts with a second entity in order to effect
determination; for example, coupling of the second entity to the
imaging entity may result in a determinable signal. Non-limiting
examples of imaging moieties include, but are not limited to,
fluorescent compounds such as FITC or a FITC derivative,
fluorescein, green fluorescent protein ("GFP"), radioactive atoms
such as .sup.3H, .sup.14C, .sup.33P, .sup.32P, .sup.125I,
.sup.131I, .sup.35S, or a heavy metal species, for example, gold or
osmium. As a specific example, an imaging moiety may be a gold
nanoparticle. A diagnostic or imaging tag such as a fluorescent tag
is chemically conjugated to a polymer to yield a fluorescently
labeled polymer.
[0159] For imaging, radioactive materials such as Technetium99
(.sup.99mTc) or magnetic materials such as Fe.sub.2O.sub.3 could be
used. Examples of other materials include gases or gas emitting
compounds, which are radioopaque.
1. Nucleic Acid-Based Active Agents
[0160] The cargo can be a nucleic acid. An isolated nucleic acid
can be, for example, a DNA, an RNA, or a nucleic acid analog.
Nucleic acid analogs can be modified at the base moiety, sugar
moiety, or phosphate backbone. Such modification can improve, for
example, stability, hybridization, or solubility of the nucleic
acid. Exemplary modifications include, 2'O-methyl, 2'
methoxy-ethyl, phosphoramidate, methylphosphonate, and/or
phosphorothioate backbone chemistry. Other mon-limiting
modifications are discussed in more detail below. The nucleic acid
molecule can exist as a separate molecule independent of other
sequences (e.g., a chemically synthesized nucleic acid, or a cDNA
or genomic DNA fragment produced by PCR or restriction endonuclease
treatment), as well as recombinant DNA that is incorporated into a
vector, an autonomously replicating plasmid, a virus (e.g., a
retrovirus, lentivirus, adenovirus, or herpes virus), etc. The
nucleic acid can be an engineered nucleic acid such as a
recombinant DNA molecule that is part of a hybrid or fusion nucleic
acid.
[0161] The genetic material to be loaded into the particles is
chosen on the basis of the desired effect of that genetic material
on the cell into which it is intended to be delivered and the
mechanism by which that effect is to be carried out. For example,
the nucleic acid may be useful in gene therapy, for example in
order to express a desired gene in a cell or group of cells.
Nucleic acid can also be used in gene silencing. Such gene
silencing may be useful in therapy to switch off aberrant gene
expression. Nucleic acid can also be used for example to express
one or more antigens against which it is desired to produce an
immune response. Thus, the nucleic acid to be loaded into the
particle can encode one or more antigens against which is desired
to produce an immune response, including but not limited to tumour
antigens, antigens from pathogens such as viral, bacterial or
fungal pathogens, such as those discussed in more detail below.
Therapeutic strategies for treating cancer, inflammation, injury,
autoimmunity, and infections are discussed in more detail
below.
a. Functional Nucleic Acids
[0162] In some embodiments, the active agent cargo is a functional
nucleic acid. Functional nucleic acids are nucleic acid molecules
that have a specific function, such as binding a target molecule or
catalyzing a specific reaction. As discussed in more detail below,
functional nucleic acid molecules can be divided into the following
non-limiting categories: antisense molecules, RNAi including siRNA,
miRNA, and piRNA, aptamers, ribozymes, triplex forming molecules,
external guide sequences, and gene editing compositions. The
functional nucleic acid molecules can act as effectors, inhibitors,
modulators, and stimulators of a specific activity possessed by a
target molecule, or the functional nucleic acid molecules can
possess a de novo activity independent of any other molecules.
[0163] Functional nucleic acid molecules can interact with any
macromolecule, such as DNA, RNA, polypeptides, or carbohydrate
chains. Thus, functional nucleic acids can interact with the mRNA
or the genomic DNA of a target polypeptide or they can interact
with the polypeptide itself. Often functional nucleic acids are
designed to interact with other nucleic acids based on sequence
homology between the target molecule and the functional nucleic
acid molecule. In other situations, the specific recognition
between the functional nucleic acid molecule and the target
molecule is not based on sequence homology between the functional
nucleic acid molecule and the target molecule, but rather is based
on the formation of tertiary structure that allows specific
recognition to take place.
i. Antisense
[0164] The functional nucleic acids can be antisense molecules.
Antisense molecules are designed to interact with a target nucleic
acid molecule through either canonical or non-canonical base
pairing. The interaction of the antisense molecule and the target
molecule is designed to promote the destruction of the target
molecule through, for example, RNAse H mediated RNA-DNA hybrid
degradation. Alternatively the antisense molecule is designed to
interrupt a processing function that normally would take place on
the target molecule, such as transcription or replication.
Antisense molecules can be designed based on the sequence of the
target molecule. There are numerous methods for optimization of
antisense efficiency by finding the most accessible regions of the
target molecule. Exemplary methods include in vitro selection
experiments and DNA modification studies using DMS and DEPC. It is
preferred that antisense molecules bind the target molecule with a
dissociation constant (K.sub.d) less than or equal to 10.sup.-6,
10.sup.-8, 10.sup.-10, or 10.sup.-12.
ii. Aptamers
[0165] The functional nucleic acids can be aptamers. Aptamers are
molecules that interact with a target molecule, preferably in a
specific way. Typically aptamers are small nucleic acids ranging
from 15-50 bases in length that fold into defined secondary and
tertiary structures, such as stem-loops or G-quartets. Aptamers can
bind small molecules, such as ATP and theophiline, as well as large
molecules, such as reverse transcriptase and thrombin. Aptamers can
bind very tightly with K.sub.d's from the target molecule of less
than 10.sup.-12 M. It is preferred that the aptamers bind the
target molecule with a K.sub.d less than 10.sup.-6, 10.sup.-8,
10.sup.-10, or 10.sup.-12. Aptamers can bind the target molecule
with a very high degree of specificity. For example, aptamers have
been isolated that have greater than a 10,000 fold difference in
binding affinities between the target molecule and another molecule
that differ at only a single position on the molecule. It is
preferred that the aptamer have a K.sub.d with the target molecule
at least 10, 100, 1000, 10,000, or 100,000 fold lower than the
K.sub.d with a background binding molecule. It is preferred when
doing the comparison for a molecule such as a polypeptide, that the
background molecule be a different polypeptide.
iii. Ribozymes
[0166] The functional nucleic acids can be ribozymes. Ribozymes are
nucleic acid molecules that are capable of catalyzing a chemical
reaction, either intramolecularly or intermolecularly. It is
preferred that the ribozymes catalyze intermolecular reactions.
There are a number of different types of ribozymes that catalyze
nuclease or nucleic acid polymerase type reactions which are based
on ribozymes found in natural systems, such as hammerhead
ribozymes. There are also a number of ribozymes that are not found
in natural systems, but which have been engineered to catalyze
specific reactions de novo. Preferred ribozymes cleave RNA or DNA
substrates, and more preferably cleave RNA substrates. Ribozymes
typically cleave nucleic acid substrates through recognition and
binding of the target substrate with subsequent cleavage. This
recognition is often based mostly on canonical or non-canonical
base pair interactions. This property makes ribozymes particularly
good candidates for target specific cleavage of nucleic acids
because recognition of the target substrate is based on the target
substrates sequence.
iv. Triplex Forming Oligonucleotides
[0167] The functional nucleic acids can be triplex forming
molecules. Triplex forming functional nucleic acid molecules are
molecules that can interact with either double-stranded or
single-stranded nucleic acid. When triplex molecules interact with
a target region, a structure called a triplex is formed in which
there are three strands of DNA forming a complex dependent on both
Watson-Crick and Hoogsteen base-pairing. Triplex molecules are
preferred because they can bind target regions with high affinity
and specificity. It is preferred that the triplex forming molecules
bind the target molecule with a K.sub.d less than 10.sup.-6,
10.sup.-8, 10.sup.-10, or 10.sup.-12.
v. External Guide Sequences
[0168] The functional nucleic acids can be external guide
sequences. External guide sequences (EGSs) are molecules that bind
a target nucleic acid molecule forming a complex, which is
recognized by RNase P, which then cleaves the target molecule. EGSs
can be designed to specifically target an RNA molecule of choice.
RNAse P aids in processing transfer RNA (tRNA) within a cell.
Bacterial RNAse P can be recruited to cleave virtually any RNA
sequence by using an EGS that causes the target RNA:EGS complex to
mimic the natural tRNA substrate. Similarly, eukaryotic EGS/RNAse
P-directed cleavage of RNA can be utilized to cleave desired
targets within eukarotic cells. Representative examples of how to
make and use EGS molecules to facilitate cleavage of a variety of
different target molecules are known in the art.
vi. RNA Interference
[0169] In some embodiments, the functional nucleic acids induce
gene silencing through RNA interference. Gene expression can also
be effectively silenced in a highly specific manner through RNA
interference (RNAi), which can generally be divided into three
major classes based on their processing mechanisms and partner
Argonaute proteins: micro RNAs (miRNAs), small interfering RNAs
(siRNAs), and PIWI-interacting RNA (piRNAs) (Czech and Hannon,
Trends Biochem Sci., 2016 Jan. 19. pii: S0968-0004(15)00258-3. doi:
10.1016/j.tibs.2015.12.008. [Epub ahead of print].
[0170] RNAi silencing was originally observed with the addition of
double stranded RNA (dsRNA) (Fire, et al. (1998) Nature,
391:806-11; Napoli, et al. (1990) Plant Cell 2:279-89; Hannon,
(2002) Nature, 418:244-51). Once dsRNA enters a cell, it is cleaved
by an RNase III--like enzyme, Dicer, into double stranded small
interfering RNAs (siRNA) 21-23 nucleotides in length that contains
2 nucleotide overhangs on the 3' ends (Elbashir, et al. (2001)
Genes Dev., 15:188-200; Bernstein, et al. (2001) Nature, 409:363-6;
Hammond, et al. (2000) Nature, 404:293-6). In an ATP dependent
step, the siRNAs become integrated into a multi-subunit protein
complex, commonly known as the RNAi induced silencing complex
(RISC), which guides the siRNAs to the target RNA sequence
(Nykanen, et al. (2001) Cell, 107:309-21). At some point the siRNA
duplex unwinds, and it appears that the antisense strand remains
bound to RISC and directs degradation of the complementary mRNA
sequence by a combination of endo and exonucleases (Martinez, et
al. (2002) Cell, 110:563-74). However, the effect of iRNA or siRNA
or their use is not limited to any type of mechanism.
[0171] Short Interfering RNA (siRNA) is a double-stranded RNA that
can induce sequence-specific post-transcriptional gene silencing,
thereby decreasing or even inhibiting gene expression. In one
example, a siRNA triggers the specific degradation of homologous
RNA molecules, such as mRNAs, within the region of sequence
identity between both the siRNA and the target RNA. For example, WO
02/44321 discloses siRNAs capable of sequence-specific degradation
of target mRNAs when base-paired with 3' overhanging ends, herein
incorporated by reference for the method of making these
siRNAs.
[0172] Sequence specific gene silencing can be achieved in
mammalian cells using synthetic, short double-stranded RNAs that
mimic the siRNAs produced by the enzyme dicer (Elbashir, et al.
(2001) Nature, 411:494 498) (Ui-Tei, et al. (2000) FEBS Lett
479:79-82). siRNA can be chemically or in vitro-synthesized or can
be the result of short double-stranded hairpin-like RNAs (shRNAs)
that are processed into siRNAs inside the cell. Synthetic siRNAs
are generally designed using algorithms and a conventional DNA/RNA
synthesizer. Suppliers include Ambion (Austin, Tex.), ChemGenes
(Ashland, Mass.), Dharmacon (Lafayette, Colo.), Glen Research
(Sterling, Va.), MWB Biotech (Esbersberg, Germany), Proligo
(Boulder, Colo.), and Qiagen (Vento, The Netherlands). siRNA can
also be synthesized in vitro using kits such as Ambion's
SILENCER.RTM. siRNA Construction Kit.
[0173] The production of siRNA from a vector is more commonly done
through the transcription of a short hairpin RNAse (shRNAs). Kits
for the production of vectors comprising shRNA are available, such
as, for example, Imgenex's GENESUPPRESSOR.TM. Construction Kits and
Invitrogen's BLOCK-IT.TM. inducible RNAi plasmid and lentivirus
vectors.
[0174] Micro RNAs (abbreviated miRNA) are small non-coding RNA
molecules (containing about 22 nucleotides) that functions in RNA
silencing and post-transcriptional regulation of gene expression.
miRNAs resemble siRNAs of the RNA interference (RNAi) pathway,
except miRNAs derive from regions of RNA transcripts that fold back
on themselves to form short hairpins, whereas siRNAs derive from
longer regions of double-stranded RNA (Bartel, et al., Cell,
116:281-297 (2004)).
[0175] The biogenesis of miRNAs and siRNAs typically depends on
RNase III type enzymes that convert their double-stranded RNA
precursors into functional small RNAs. By contrast, piRNAs derive
from single-stranded RNAs and, consequently, require alternative
processing machinery.
[0176] Synthetic piRNAs can be used to block the synthesis of
target proteins by binding to mRNAs, as has been attempted with
miRNAs, might have the advantage of not requiring processing by
enzymes such as Dicer, which is required by miRNAs. Additional
speculative advantages of piRNAs over miRNAs include the
possibility of targets with better specificity because each miRNA
regulates several mRNAs and there is the potential to access
undesirable long non-coding RNAs with possible implications in
disease processes (Assumpcao, et al., Epigenomics, 7(6):975-984
(2015)). miRNA and piRNA can be the therapeutic agent or can be
target sequences for post-transcriptional silencing. For example,
synthetic piRNAs designed to couple to PIWI proteins and exert
genomic silencing on PIWI genes at a transcriptional level is a
possible strategy.
[0177] In some embodiment, the functional nucleic acid is siRNA,
shRNA, miRNA, or piRNA. In some embodiments, the composition
includes a vector expressing the functional nucleic acid. Methods
of making and using vectors for in vivo expression of functional
nucleic acids such as antisense oligonucleotides, siRNA, shRNA,
miRNA, piRNA, EGSs, ribozymes, and aptamers are known in the
art.
vii. Other Gene Editing Compositions
[0178] In some embodiments the functional nucleic acids are gene
editing compositions. Gene editing compositions can include nucleic
acids that encode an element or elements that induce a single or a
double strand break in the target cell's genome, and optionally a
polynucleotide. The compositions can be used, for example, to
reduce or otherwise modify expression of a gene target.
1. Strand Break Inducing Elements
[0179] It will be appreciated that some of the embodiments
discussed below include protein active agents. In some embodiments,
the agents are packaged into particles as nucleic acids encoding
the proteins (e.g., mRNA, expression vectors, etc.).
[0180] CRISPR/Cas
[0181] In some embodiments, the element that induces a single or a
double strand break in the target cell's genome is a CRISPR/Cas
system. CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats) is an acronym for DNA loci that contain multiple, short,
direct repetitions of base sequences. The prokaryotic CRISPR/Cas
system has been adapted for use as gene editing (silencing,
enhancing or changing specific genes) for use in eukaryotes (see,
for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek,
et al., Science, 337(6096):816-21 (2012)). By transfecting a cell
with the required elements including a cas gene and specifically
designed CRISPRs, the organism's genome can be cut and modified at
any desired location. Methods of preparing compositions for use in
genome editing using the CRISPR/Cas systems are described in detail
in WO 2013/176772 and WO 2014/018423, which are specifically
incorporated by reference herein in their entireties.
[0182] In general, "CRISPR system" refers collectively to
transcripts and other elements involved in the expression of or
directing the activity of CRISPR-associated ("Cas") genes,
including sequences encoding a Cas gene, a tracr (trans-activating
CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a
tracr-mate sequence (encompassing a "direct repeat" and a
tracrRNA-processed partial direct repeat in the context of an
endogenous CRISPR system), a guide sequence (also referred to as a
"spacer" in the context of an endogenous CRISPR system), or other
sequences and transcripts from a CRISPR locus. One or more tracr
mate sequences operably linked to a guide sequence (e.g., direct
repeat-spacer-direct repeat) can also be referred to as pre-crRNA
(pre-CRISPR RNA) before processing or crRNA after processing by a
nuclease.
[0183] In some embodiments, a tracrRNA and crRNA are linked and
form a chimeric crRNA-tracrRNA hybrid where a mature crRNA is fused
to a partial tracrRNA via a synthetic stem loop to mimic the
natural crRNA:tracrRNA duplex as described in Cong, Science,
15:339(6121):819-823 (2013) and Jinek, et al., Science,
337(6096):816-21 (2012)). A single fused crRNA-tracrRNA construct
can also be referred to as a guide RNA or gRNA (or single-guide RNA
(sgRNA)). Within an sgRNA, the crRNA portion can be identified as
the `target sequence` and the tracrRNA is often referred to as the
`scaffold`.
[0184] There are many resources available for helping practitioners
determine suitable target sites once a desired DNA target sequence
is identified. For example, numerous public resources, including a
bioinformatically generated list of about 190,000 potential sgRNAs,
targeting more than 40% of human exons, are available to aid
practitioners in selecting target sites and designing the associate
sgRNA to affect a nick or double strand break at the site. See
also, crispr.u-psud.fr/, a tool designed to help scientists find
CRISPR targeting sites in a wide range of species and generate the
appropriate crRNA sequences.
[0185] In some embodiments, one or more vectors driving expression
of one or more elements of a CRISPR system are introduced into a
target cell such that expression of the elements of the CRISPR
system direct formation of a CRISPR complex at one or more target
sites. While the specifics can be varied in different engineered
CRISPR systems, the overall methodology is similar. A practitioner
interested in using CRISPR technology to target a DNA sequence can
insert a short DNA fragment containing the target sequence into a
guide RNA expression plasmid. The sgRNA expression plasmid contains
the target sequence (about 20 nucleotides), a form of the tracrRNA
sequence (the scaffold) as well as a suitable promoter and
necessary elements for proper processing in eukaryotic cells. Such
vectors are commercially available (see, for example, Addgene).
Many of the systems rely on custom, complementary oligos that are
annealed to form a double stranded DNA and then cloned into the
sgRNA expression plasmid. Co-expression of the sgRNA and the
appropriate Cas enzyme from the same or separate plasmids in
transfected cells results in a single or double strand break
(depending of the activity of the Cas enzyme) at the desired target
site.
Zinc Finger Nucleases
[0186] In some embodiments, the element that induces a single or a
double strand break in the target cell's genome is a nucleic acid
construct or constructs encoding a zinc finger nucleases (ZFNs).
ZFNs are typically fusion proteins that include a DNA-binding
domain derived from a zinc-finger protein linked to a cleavage
domain.
[0187] The most common cleavage domain is the Type IIS enzyme Fokl.
Fokl catalyzes double-stranded cleavage of DNA, at 9 nucleotides
from its recognition site on one strand and 13 nucleotides from its
recognition site on the other. See, for example, U.S. Pat. Nos.
5,356,802; 5,436,150 and 5,487,994; as well as Li et al. Proc.,
Natl. Acad. Sci. USA 89 (1992):4275-4279; Li et al. Proc. Natl.
Acad. Sci. USA, 90:2764-2768 (1993); Kim et al. Proc. Natl. Acad.
Sci. USA. 91:883-887 (1994a); Kim et al. J. Biol. Chem. 269:31,
978-31,982 (1994b). One or more of these enzymes (or enzymatically
functional fragments thereof) can be used as a source of cleavage
domains.
[0188] The DNA-binding domain, which can, in principle, be designed
to target any genomic location of interest, can be a tandem array
of Cys.sub.2His.sub.2 zinc fingers, each of which generally
recognizes three to four nucleotides in the target DNA sequence.
The Cys.sub.2His.sub.2 domain has a general structure: Phe
(sometimes Tyr)-Cys-(2 to 4 amino acids)-Cys-(3 amino
acids)-Phe(sometimes Tyr)-(5 amino acids)-Leu-(2 amino
acids)-His-(3 amino acids)-His. By linking together multiple
fingers (the number varies: three to six fingers have been used per
monomer in published studies), ZFN pairs can be designed to bind to
genomic sequences 18-36 nucleotides long.
[0189] Engineering methods include, but are not limited to,
rational design and various types of empirical selection methods.
Rational design includes, for example, using databases including
triplet (or quadruplet) nucleotide sequences and individual zinc
finger amino acid sequences, in which each triplet or quadruplet
nucleotide sequence is associated with one or more amino acid
sequences of zinc fingers which bind the particular triplet or
quadruplet sequence. See, for example, U.S. Pat. Nos. 6,140,081;
6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617;
U.S. Published Application Nos. 2002/0165356; 2004/0197892;
2007/0154989; 2007/0213269; and International Patent Application
Publication Nos. WO 98/53059 and WO 2003/016496.
Transcription Activator-Like Effector Nucleases
[0190] In some embodiments, the element that induces a single or a
double strand break in the target cell's genome is a nucleic acid
construct or constructs encoding a transcription activator-like
effector nuclease (TALEN). TALENs have an overall architecture
similar to that of ZFNs, with the main difference that the
DNA-binding domain comes from TAL effector proteins, transcription
factors from plant pathogenic bacteria. The DNA-binding domain of a
TALEN is a tandem array of amino acid repeats, each about 34
residues long. The repeats are very similar to each other;
typically they differ principally at two positions (amino acids 12
and 13, called the repeat variable diresidue, or RVD). Each RVD
specifies preferential binding to one of the four possible
nucleotides, meaning that each TALEN repeat binds to a single base
pair, though the NN RVD is known to bind adenines in addition to
guanine. TAL effector DNA binding is mechanistically less well
understood than that of zinc-finger proteins, but their seemingly
simpler code could prove very beneficial for engineered-nuclease
design. TALENs also cleave as dimers, have relatively long target
sequences (the shortest reported so far binds 13 nucleotides per
monomer) and appear to have less stringent requirements than ZFNs
for the length of the spacer between binding sites. Monomeric and
dimeric TALENs can include more than 10, more than 14, more than
20, or more than 24 repeats.
[0191] Methods of engineering TAL to bind to specific nucleic acids
are described in Cermak, et al, Nucl. Acids Res. 1-11 (2011). US
Published Application No. 2011/0145940, which discloses TAL
effectors and methods of using them to modify DNA. Miller et al.
Nature Biotechnol 29: 143 (2011) reported making TALENs for
site-specific nuclease architecture by linking TAL truncation
variants to the catalytic domain of Fokl nuclease. The resulting
TALENs were shown to induce gene modification in immortalized human
cells. General design principles for TALE binding domains can be
found in, for example, WO 2011/072246.
2. Gene Altering Polynucleotides
[0192] The nuclease activity of the genome editing systems
described herein cleave target DNA to produce single or double
strand breaks in the target DNA. Double strand breaks can be
repaired by the cell in one of two ways: non-homologous end
joining, and homology-directed repair. In non-homologous end
joining (NHEJ), the double-strand breaks are repaired by direct
ligation of the break ends to one another. As such, no new nucleic
acid material is inserted into the site, although some nucleic acid
material may be lost, resulting in a deletion. In homology-directed
repair, a donor polynucleotide with homology to the cleaved target
DNA sequence is used as a template for repair of the cleaved target
DNA sequence, resulting in the transfer of genetic information from
a donor polynucleotide to the target DNA. As such, new nucleic acid
material can be inserted/copied into the site.
[0193] Therefore, in some embodiments, the genome editing
composition optionally includes a donor polynucleotide. The
modifications of the target DNA due to NHEJ and/or
homology-directed repair can be used to induce gene correction,
gene replacement, gene tagging, transgene insertion, nucleotide
deletion, gene disruption, gene mutation, etc.
[0194] Accordingly, cleavage of DNA by the genome editing
composition can be used to delete nucleic acid material from a
target DNA sequence by cleaving the target DNA sequence and
allowing the cell to repair the sequence in the absence of an
exogenously provided donor polynucleotide.
[0195] Alternatively, if the genome editing composition includes a
donor polynucleotide sequence that includes at least a segment with
homology to the target DNA sequence, the methods can be used to
add, i.e., insert or replace, nucleic acid material to a target DNA
sequence (e.g., to "knock in" a nucleic acid that encodes for a
protein, an siRNA, an miRNA, etc.), to add a tag (e.g., 6xHis, a
fluorescent protein (e.g., a green fluorescent protein; a yellow
fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), to add
a regulatory sequence to a gene (e.g., promoter, polyadenylation
signal, internal ribosome entry sequence (IRES), 2A peptide, start
codon, stop codon, splice signal, localization signal, etc.), to
modify a nucleic acid sequence (e.g., introduce a mutation), and
the like. As such, the compositions can be used to modify DNA in a
site-specific, i.e., "targeted", way, for example gene knock-out,
gene knock-in, gene editing, gene tagging, etc. as used in, for
example, gene therapy.
[0196] In applications in which it is desirable to insert a
polynucleotide sequence into a target DNA sequence, a
polynucleotide including a donor sequence to be inserted is also
provided to the cell. By a "donor sequence" or "donor
polynucleotide" or "donor oligonucleotide" it is meant a nucleic
acid sequence to be inserted at the cleavage site. The donor
polynucleotide typically contains sufficient homology to a genomic
sequence at the cleavage site, e.g., 70%, 80%, 85%, 90%, 95%, or
100% homology with the nucleotide sequences flanking the cleavage
site, e.g., within about 50 bases or less of the cleavage site,
e.g., within about 30 bases, within about 15 bases, within about 10
bases, within about 5 bases, or immediately flanking the cleavage
site, to support homology-directed repair between it and the
genomic sequence to which it bears homology. The donor sequence is
typically not identical to the genomic sequence that it replaces.
Rather, the donor sequence may contain at least one or more single
base changes, insertions, deletions, inversions or rearrangements
with respect to the genomic sequence, so long as sufficient
homology is present to support homology-directed repair. In some
embodiments, the donor sequence includes a non-homologous sequence
flanked by two regions of homology, such that homology-directed
repair between the target DNA region and the two flanking sequences
results in insertion of the non-homologous sequence at the target
region.
b. Peptide and Protein Expression Constructs
[0197] In some embodiments, the active agent is a nucleic acid
encoding a protein or a polypeptide. Although discussed here in the
context of mRNA, it will be appreciated that the nucleic acid
active agent can itself be an mRNA, or can be a DNA or other
oligonucleotide encoding the mRNA (or a functional nucleic acid as
discussed above). As discussed in more detail below, the nucleic
acid active agents, including mRNA and functional nucleic acids,
can be encoded by a nucleic acid that encodes the RNA. The nucleic
acid can be operably linked to an expression control sequence. In
some embodiments, the nucleic acid is a vector, integration
construct, etc., that enables expression of the RNA in a cell.
[0198] The mRNA can be a mature mRNA or pre-mRNA. Thus in some
embodiments, the mRNA includes introns. The mRNA can be a naturally
occurring gene transcript, for example, a human gene transcript.
The mRNA can be an artificial sequence that is not normally
expressed in a naturally occurring organism. An exemplary
artificial sequence is one that contains portions of gene sequences
that are ligated together to form an open reading frame that
encodes a fusion protein. The portions of that are ligated together
can be from a single organism or from more than one organism.
[0199] The mRNA can encode a polypeptide that provides a
therapeutic or prophylactic effect to an organism or that can be
used to diagnose a disease or disorder in an organism. For example,
for treatment of cancer, autoimmune disorders, parasitic, viral,
bacterial, fungal or other infections, the polypeptide can be a
ligand or receptor for cells of the immune system, or can function
to stimulate or inhibit the immune system of an organism.
Typically, it is not desirable to have prolonged ongoing
stimulation of the immune system, nor necessary to produce changes
which last after successful treatment, since this may then elicit a
new problem. For treatment of an autoimmune disorder, it may be
desirable to inhibit or suppress the immune system during a
flare-up, but not long term, which could result in the patient
becoming overly sensitive to an infection. Thus in some
embodiments, delivery of mRNA for transient expression of the
protein (or functional nucleic acid) is preferred to sustained
expression by a vector or gene integration.
[0200] The mRNA can include a 5' cap. A 5' cap (also termed an RNA
cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified
guanine nucleotide that has been added to the "front" or 5' end of
a eukaryotic messenger RNA shortly after the start of
transcription. The 5' cap consists of a terminal group which is
linked to the first transcribed nucleotide. Its presence is
critical for recognition by the ribosome and protection from
RNases. Cap addition is coupled to transcription, and occurs
co-transcriptionally, such that each influences the other. Shortly
after the start of transcription, the 5' end of the mRNA being
synthesized is bound by a cap-synthesizing complex associated with
RNA polymerase. This enzymatic complex catalyzes the chemical
reactions that are required for mRNA capping. Synthesis proceeds as
a multi-step biochemical reaction. The capping moiety can be
modified to modulate functionality of mRNA such as its stability or
efficiency of translation. The 5' cap may, for example, be
m7G(5')ppp(5')G, m7G(5')ppp(5')A, G(5')ppp(5')G or G(5')ppp(5')A
cap analogs, which are all commercially available. The 5' cap can
also be an anti-reverse-cap-analog (ARCA) (see, e.g., Stepinski, et
al., RNA, 7:1468-95 (2001)) or any other suitable analog. The 5'
cap is provided using techniques known in the art and described
herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001);
Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim.
Biophys. Res. Commun., 330:958-966 (2005)).
[0201] The mRNA can contain an internal ribosome entry site (IRES)
sequence. The IRES sequence may be any viral, chromosomal or
artificially designed sequence which initiates cap-independent
ribosome binding to mRNA and facilitates the initiation of
translation.
[0202] The mRNA can include a 5' untranslated region. The 5' UTR is
upsteam from the coding sequence. Within the 5' UTR is a sequence
that is recognized by the ribosome which allows the ribosome to
bind and initiate translation. The mechanism of translation
initiation differs in Prokaryotes and Eukaryotes.
[0203] The mRNA includes an "open reading frame" or "ORF," which is
a series of nucleotides that contains a sequence of bases that
could potentially encode a polypeptide or protein. An open reading
frame is located between the start-code sequence (initiation codon
or start codon) and the stop-codon sequence (termination codon).
The ORF can be from a naturally occurring sequence from the genome
of an organism.
[0204] The mRNA can include a 3' untranslated region. The 3' UTR is
found immediately following the translation stop codon. The 3' UTR
plays an important role in translation termination as well as post
transcriptional gene expression.
[0205] In some embodiments, the mRNA is polyadenylated.
"Polyadenylation" refers to the covalent linkage of a polyadenylyl
moiety, or its modified variant, to a messenger RNA molecule. In
eukaryotic organisms, most messenger RNA (mRNA) molecules are
polyadenylated at the 3' end. The 3' poly(A) tail is a long
sequence of adenine nucleotides (often several hundred) added to
the pre-mRNA through the action of an enzyme, polyadenylate
polymerase. In higher eukaryotes, the poly(A) tail is added onto
transcripts that contain a specific sequence, the polyadenylation
signal. The poly(A) tail and the protein bound to it aid in
protecting mRNA from degradation by exonucleases. Polyadenylation
is also important for transcription termination, export of the mRNA
from the nucleus, and translation. Polyadenylation occurs in the
nucleus immediately after transcription of DNA into RNA, but
additionally can also occur later in the cytoplasm. After
transcription has been terminated, the mRNA chain is cleaved
through the action of an endonuclease complex associated with RNA
polymerase. The cleavage site is usually characterized by the
presence of the base sequence AAUAAA (SEQ ID NO:12) near the
cleavage site. After the mRNA has been cleaved, adenosine residues
are added to the free 3' end at the cleavage site.
[0206] RNA, including mRNA and RNA-based functional nucleic acids,
can be prepared by in vitro transcription using, for example, a
purified linear DNA template containing a promoter, ribonucleotide
triphosphates, a buffer system that includes DTT and magnesium
ions, and an appropriate phage RNA polymerase. The template can be
a vector, PCR product, synthetic oligonucleotide, or cDNA.
3. Vectors
[0207] Nucleic acids, including constructs encoding mRNAs and
functional nucleic acids such as those described above, can be
inserted into vectors for expression in cells. As used herein, a
"vector" is a replicon, such as a plasmid, phage, virus or cosmid,
into which another DNA segment may be inserted so as to bring about
the replication of the inserted segment. Vectors can be expression
vectors. An "expression vector" is a vector that includes one or
more expression control sequences, and an "expression control
sequence" is a DNA sequence that controls and regulates the
transcription and/or translation of another DNA sequence.
[0208] Nucleic acids in vectors can be operably linked to one or
more expression control sequences. As used herein, "operably
linked" means incorporated into a genetic construct so that
expression control sequences effectively control expression of a
coding sequence of interest. Examples of expression control
sequences include promoters, enhancers, and transcription
terminating regions. A promoter is an expression control sequence
composed of a region of a DNA molecule, typically within 100
nucleotides upstream of the point at which transcription starts
(generally near the initiation site for RNA polymerase II). To
bring a coding sequence under the control of a promoter, it is
necessary to position the translation initiation site of the
translational reading frame of the polypeptide between one and
about fifty nucleotides downstream of the promoter. Enhancers
provide expression specificity in terms of time, location, and
level. Unlike promoters, enhancers can function when located at
various distances from the transcription site. An enhancer also can
be located downstream from the transcription initiation site. A
coding sequence is "operably linked" and "under the control" of
expression control sequences in a cell when RNA polymerase is able
to transcribe the coding sequence into mRNA, which then can be
translated into the protein encoded by the coding sequence, or into
a functional nucleic acids.
[0209] The vector can be a viral vector. Nucleic acid molecules
encoding proteins or functional nucleic acids can be packaged into
retrovirus vectors using packaging cell lines that produce
replication-defective retroviruses, as is well-known in the art.
Other virus vectors may also be used, including recombinant
adenoviruses and vaccinia virus, which can be rendered
non-replicating.
[0210] In some embodiments the nucleic acid is designed for
integration into the host cell's genome. Nucleic acids that are
delivered to cells which are to be integrated into the host cell
genome, typically contain integration sequences. These sequences
are often viral related sequences, particularly when viral based
systems are used. Techniques for integration of genetic material
into a host genome are also known and include, for example, systems
designed to promote homologous recombination with the host genome.
These systems typically rely on sequence flanking the nucleic acid
to be expressed that has enough homology with a target sequence
within the host cell genome that recombination between the vector
nucleic acid and the target nucleic acid takes place, causing the
delivered nucleic acid to be integrated into the host genome. These
systems and the methods necessary to promote homologous
recombination are known to those of skill in the art.
4. Nucleic Acid Composition
[0211] The nucleic acid cargos can be DNA or RNA nucleotides which
typically include a heterocyclic base (nucleic acid base), a sugar
moiety attached to the heterocyclic base, and a phosphate moiety
which esterifies a hydroxyl function of the sugar moiety. The
principal naturally-occurring nucleotides comprise uracil, thymine,
cytosine, adenine and guanine as the heterocyclic bases, and ribose
or deoxyribose sugar linked by phosphodiester bonds.
[0212] In some embodiments, the oligonucleotides are composed of
nucleotide analogs that have been chemically modified to improve
stability, half-life, or specificity or affinity for a target
receptor, relative to a DNA or RNA counterpart. The chemical
modifications include chemical modification of nucleobases, sugar
moieties, nucleotide linkages, or combinations thereof. As used
herein `modified nucleotide" or "chemically modified nucleotide"
defines a nucleotide that has a chemical modification of one or
more of the heterocyclic base, sugar moiety or phosphate moiety
constituents. In some embodiments, the charge of the modified
nucleotide is reduced compared to DNA or RNA oligonucleotides of
the same nucleobase sequence. For example, the oligonucleotide can
have low negative charge, no charge, or positive charge.
[0213] Typically, nucleoside analogs support bases capable of
hydrogen bonding by Watson-Crick base pairing to standard
polynucleotide bases, where the analog backbone presents the bases
in a manner to permit such hydrogen bonding in a sequence-specific
fashion between the oligonucleotide analog molecule and bases in a
standard polynucleotide (e.g., single-stranded RNA or
single-stranded DNA). In some embodiments, the analogs have a
substantially uncharged, phosphorus containing backbone.
a. Heterocyclic Bases
[0214] The principal naturally-occurring nucleotides include
uracil, thymine, cytosine, adenine and guanine as the heterocyclic
bases. The oligonucleotides can include chemical modifications to
their nucleobase constituents. Chemical modifications of
heterocyclic bases or heterocyclic base analogs may be effective to
increase the binding affinity or stability in binding a target
sequence. Chemically-modified heterocyclic bases include, but are
not limited to, inosine, 5-(1-propynyl) uracil (pU), 5-(1-propynyl)
cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine,
pseudoisocytosine, 5 and
2-amino-5-(2'-deoxy-.beta.-D-ribofuranosyl)pyridine
(2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine
derivatives.
b. Sugar Modifications
[0215] Oligonucleotides can also contain nucleotides with modified
sugar moieties or sugar moiety analogs. Sugar moiety modifications
include, but are not limited to, 2'-O-aminoetoxy, 2'-O-amonioethyl
(2'-OAE), 2'-O-methoxy, 2'-O-methyl, 2-guanidoethyl (2'-OGE),
2'-O,4'-C-methylene (LNA), 2'-O-(methoxyethyl) (2'-OME) and
2'-O--(N-(methyl)acetamido) (2'-OMA). In some embodiments, the
functional nucleic acid is a morpholino oligonucleotide. Morpholino
oligonucleotides are typically composed of two more morpholino
monomers containing purine or pyrimidine base-pairing moieties
effective to bind, by base-specific hydrogen bonding, to a base in
a polynucleotide, which are linked together by
phosphorus-containing linkages, one to three atoms long, joining
the morpholino nitrogen of one monomer to the 5' exocyclic carbon
of an adjacent monomer. The purine or pyrimidine base-pairing
moiety is typically adenine, cytosine, guanine, uracil or thymine.
The synthesis, structures, and binding characteristics of
morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685,
5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and
5,506,337.
[0216] Important properties of the morpholino-based subunits
typically include: the ability to be linked in a oligomeric form by
stable, uncharged backbone linkages; the ability to support a
nucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil
or inosine) such that the polymer formed can hybridize with a
complementary-base target nucleic acid, including target RNA, with
high T, even with oligomers as short as 10-14 bases; the ability of
the oligomer to be actively transported into mammalian cells; and
the ability of an oligomer:RNA heteroduplex to resist RNAse
degradation.
[0217] In some embodiments, oligonucleotides employ
morpholino-based subunits bearing base-pairing moieties, joined by
uncharged linkages, as described above.
c. Internucleotide Linkages
[0218] Oligonucleotides connected by an internucleotide bond that
refers to a chemical linkage between two nucleoside moieties.
Modifications to the phosphate backbone of DNA or RNA
oligonucleotides may increase the binding affinity or stability
oligonucleotides, or reduce the susceptibility of oligonucleotides
nuclease digestion. Cationic modifications, including, but not
limited to, diethyl-ethylenediamide (DEED) or
dimethyl-aminopropylamine (DMAP) may be especially useful due to
decrease electrostatic repulsion between the oligonucleotide and a
target. Modifications of the phosphate backbone may also include
the substitution of a sulfur atom for one of the non-bridging
oxygens in the phosphodiester linkage. This substitution creates a
phosphorothioate internucleoside linkage in place of the
phosphodiester linkage. Oligonucleotides containing
phosphorothioate internucleoside linkages have been shown to be
more stable in vivo.
[0219] Examples of modified nucleotides with reduced charge include
modified internucleotide linkages such as phosphate analogs having
achiral and uncharged intersubunit linkages (e.g., Sterchak, E. P.
et al., Organic. Chem., 52:4202, (1987)), and uncharged
morpholino-based polymers having achiral intersubunit linkages
(see, e.g., U.S. Pat. No. 5,034,506), as discussed above. Some
internucleotide linkage analogs include morpholidate, acetal, and
polyamide-linked heterocycles.
[0220] The oligonucleotides can be locked nucleic acids. Locked
nucleic acids (LNA) are modified RNA nucleotides (see, for example,
Braasch, et al., Chem. Biol., 8(1):1-7 (2001)). LNAs form hybrids
with DNA which are more stable than DNA/DNA hybrids, a property
similar to that of peptide nucleic acid (PNA)/DNA hybrids.
Therefore, LNA can be used just as PNA molecules would be. LNA
binding efficiency can be increased in some embodiments by adding
positive charges to it. Commercial nucleic acid synthesizers and
standard phosphoramidite chemistry are used to make LNAs.
[0221] In some embodiments, the oligonucleotides are composed of
peptide nucleic acids. Peptide nucleic acids (PNAs) are synthetic
DNA mimics in which the phosphate backbone of the oligonucleotide
is replaced in its entirety by repeating N-(2-aminoethyl)-glycine
units and phosphodiester bonds are typically replaced by peptide
bonds. The various heterocyclic bases are linked to the backbone by
methylene carbonyl bonds. PNAs maintain spacing of heterocyclic
bases that is similar to conventional DNA oligonucleotides, but are
achiral and neutrally charged molecules. Peptide nucleic acids are
comprised of peptide nucleic acid monomers.
[0222] Other backbone modifications include peptide and amino acid
variations and modifications. Thus, the backbone constituents of
oligonucleotides such as PNA may be peptide linkages, or
alternatively, they may be non-peptide peptide linkages. Examples
include acetyl caps, amino spacers such as
8-amino-3,6-dioxaoctanoic acid (referred to herein as 0-linkers),
amino acids such as lysine are particularly useful if positive
charges are desired in the PNA, and the like. Methods for the
chemical assembly of PNAs are well known. See, for example, U.S.
Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336,
5,773,571 and 5,786,571.
[0223] Oligonucleotides optionally include one or more terminal
residues or modifications at either or both termini to increase
stability, and/or affinity of the oligonucleotide for its target.
Commonly used positively charged moieties include the amino acids
lysine and arginine, although other positively charged moieties may
also be useful. Oligonucleotides may further be modified to be end
capped to prevent degradation using a propylamine group. Procedures
for 3' or 5' capping oligonucleotides are well known in the
art.
[0224] The functional nucleic acid can be single stranded or double
stranded.
C. Tissue Targeting Ligands, Cell Adhesion Ligands, and Endosomal
Uptake Ligands
1. Targeting Moieties
[0225] The nanoparticles, cargo they contain, or a combination
thereof can optionally include a targeting moiety, i.e., a moiety
able to bind to or otherwise associate with a biological entity,
for example, a membrane component, a cell surface receptor, or a
molecule. In one embodiment, the targeting moiety has a specificity
(as measured via a disassociation constant) of less than about 1
micromolar, at least about 10 micromolar, or at least about 100
micromolar. Numerous examples of targeting moieties are known, some
of which are more selective than others. The ligand can be selected
based on the disease to be treated, the target cells, tissue or
organ, and the desired delivery strategy (e.g., into a cells or
into the extracellular space). The particles or cargo can include
two, three, or more targeting moieties. In some embodiments, some
polymers of the particle have a targeting moiety attached thereto
and others do not. In this way, the density of the targeting moiety
on the surface of the particle can be manipulated.
[0226] The targeting signal can include a sequence of monomers that
facilitates in vivo localization of the molecule. The monomers can
be amino acids, nucleotide or nucleoside bases, or sugar groups
such as glucose, galactose, and the like which form carbohydrate
targeting signals. Exemplary targeting molecules include small
molecules, peptides, aptamers, polynucleotides, and antibodies and
antigen binding fragments thereof. In certain embodiments, the
antibody is polyclonal, monoclonal, linear, humanized, chimeric or
a fragment thereof. Representative antibody fragments are those
fragments that bind the antibody binding portion such as Fab, Fab',
F(ab'), Fv diabodies, linear antibodies, single chain antibodies
and bispecific antibodies.
[0227] Targeting signals or sequences can be specific for a host,
tissue, organ, cell, organelle, non-nuclear organelle, or cellular
compartment. For example, in some embodiments the particle or a
cargo thereof includes a cell-specific targeting domain, an
organelle specific targeting domain to enhance delivery to a
subcellular organelle, or a combination thereof. For example, the
particle can include targeting moiety that directs the particle to
a microenvironment where the cargo is released. A second targeting
moiety on the cargo can then enhance delivery to cargo into a
target cell or cell(s) in the microenvironment. In some embodiment,
the particle includes a moiety that targets it to a tissue, cell or
organ, and the cargo includes a moiety that enhances delivery of
the cargo to a subcellular location such as an organelle.
[0228] General classes and methods of targeting are discussed here,
and specific exemplary cell, tissue, organ, and microenvironment
specific targets are discussed in more detail and the sections
below devoted to therapeutic strategies and in the working
Examples.
a. Cell Targeting
[0229] The particles, there cargo, or a combination thereof can be
modified to target a specific cell type or population of cells.
[0230] For example, the particles and cargo can be modified with
galactosyl-terminating macromolecules to target the polypeptide of
interest to the liver or to liver cells. The modified particles and
cargo selectively enters hepatocytes after interaction of the
carrier galactose residues with the asialoglycoprotein receptor
present in large amounts and high affinity only on these cells.
[0231] In some embodiments, the targeting signal binds to its
ligand or receptor which is located on the surface of a target cell
such as to bring the composition and cell membranes sufficiently
close to each other to allow penetration of the composition into
the cell.
[0232] The targeting molecule can be, for example, an antibody or
antigen binding fragment thereof, an antibody domain, an antigen, a
T-cell receptor, a cell surface receptor, a cell surface adhesion
molecule, a major histocompatibility locus protein, a viral
envelope protein and a peptide selected by phage display that binds
specifically to a defined cell.
[0233] Targeting the particles or cargo to specific cells can be
accomplished by modifying the particle or cargo to express specific
cell and tissue targeting signals. These sequences target specific
cells and tissues. In some embodiments the interaction of the
targeting signal with the cell does not occur through a traditional
receptor:ligand interaction. The eukaryotic cell comprises a number
of distinct cell surface molecules. The structure and function of
each molecule can be specific to the origin, expression, character
and structure of the cell. Determining the unique cell surface
complement of molecules of a specific cell type can be determined
using techniques well known in the art.
[0234] One skilled in the art will appreciate that the tropism of
the particles and cargo can be altered by changing the targeting
signal. For example, the compositions can be modified to include
cell surface antigen specific antibodies. Exemplary cell surface
antigens are disclosed in Wagner et al., Adv Gen, 53:333-354
(2005). Tumor antigens discussed in more detail below.
[0235] It is known in the art that nearly every cell type in a
tissue in a mammalian organism possesses some unique cell surface
receptor or antigen. Thus, it is possible to incorporate nearly any
ligand for the cell surface receptor or antigen as a targeting
signal. For example, peptidyl hormones can be used a targeting
moieties to target delivery to those cells which possess receptors
for such hormones. Chemokines and cytokines can similarly be
employed as targeting signals to target delivery of the complex to
their target cells. A variety of technologies have been developed
to identify genes that are preferentially expressed in certain
cells or cell states and one of skill in the art can employ such
technology to identify targeting signals which are preferentially
or uniquely expressed on the target tissue of interest
i. Brain Targeting
[0236] The targeting signal can be directed to cells of the nervous
system, including the brain and peripheral nervous system. Cells in
the brain include several types and states and possess unique cell
surface molecules specific for the type. Furthermore, cell types
and states can be further characterized and grouped by the
presentation of common cell surface molecules.
[0237] The targeting signal can be directed to specific
neurotransmitter receptors expressed on the surface of cells of the
nervous system. The distribution of neurotransmitter receptors is
well known in the art and one so skilled can direct the
compositions described by using neurotransmitter receptor specific
antibodies as targeting signals. Furthermore, given the tropism of
neurotransmitters for their receptors, in one embodiment the
targeting signal consists of a neurotransmitter or ligand capable
of specifically binding to a neurotransmitter receptor.
[0238] The targeting signal can be specific to cells of the nervous
system which may include astrocytes, microglia, neurons,
oligodendrites and Schwann cells. These cells can be further
divided by their function, location, shape, neurotransmitter class
and pathological state. Cells of the nervous system can also be
identified by their state of differentiation, for example stem
cells. Exemplary markers specific for these cell types and states
are well known in the art and include, but are not limited to CD133
and Neurosphere.
ii. Muscle Targeting
[0239] The targeting signal can be directed to cells of the
musculoskeletal system. Muscle cells include several types and
possess unique cell surface molecules specific for the type and
state. Furthermore, cell types and states can be further
characterized and grouped by the presentation of common cell
surface molecules.
[0240] For example, the targeting signal can be directed to
specific neurotransmitter receptors expressed on the surface of
muscle cells. The distribution of neurotransmitter receptors is
well known in the art and one so skilled can direct the
compositions described by using neurotransmitter receptor specific
antibodies as targeting signals. Furthermore, given the tropism of
neurotransmitters for their receptors, in some embodiments the
targeting signal consists of a neurotransmitter. Exemplary
neurotransmitters expressed on muscle cells that can be targeted
include but are not limited to acetycholine and norepinephrine.
[0241] The targeting signal can be specific to muscle cells which
consist of two major groupings, Type I and Type II. These cells can
be further divided by their function, location, shape, myoglobin
content and pathological state. Muscle cells can also be identified
by their state of differentiation, for example muscle stem cells.
Exemplary markers specific for these cell types and states are well
known in the art include, but are not limited to MyoD, Pax7 and
MR4.
iii. Antibodies
[0242] Another embodiment provides an antibody or antigen binding
fragment thereof bound to the disclosed proteins of interest acting
as the targeting signal. The antibodies or antigen binding fragment
thereof are useful for directing the vector to a cell type or cell
state. In one embodiment, the polypeptide of interest possesses an
antibody binding domain, for example from proteins known to bind
antibodies such as Protein A and Protein G from Staphylococcus
aureus.
[0243] In some embodiments, the targeting domain includes all or
part of an antibody that directs the vector to the desired target
cell type or cell state. Antibodies can be monoclonal or
polyclonal, but are preferably monoclonal. For human gene therapy
purposes, antibodies are derived from human genes and are specific
for cell surface markers, and are produced to reduce potential
immunogenicity to a human host as is known in the art. For example,
transgenic mice which contain the entire human immunoglobulin gene
cluster are capable of producing "human" antibodies can be
utilized. In one embodiment, fragments of such human antibodies are
employed as targeting signals. In a preferred embodiment, single
chain antibodies modeled on human antibodies are prepared in
prokaryotic culture.
[0244] In preferred embodiments the polypeptide of interest is
itself a fusion protein. The fusion protein can include, for
example, a polynucleotide-binding polypeptide, a protein
transduction domain, and optionally one or more targeting signals.
Other exemplary fusion proteins containing a mitochondrial
transcription factor polypeptide that are suitable for use as a
polypeptide of interest are disclosed in U.S. Pat. Nos. 8,039,587,
8,062,891, 8,133,733.
b. Organelle Targeting
[0245] In some embodiments, the particle, cargo, or a combination
thereof is modified to target a subcellular organelle. Targeting of
the disclosed composition to organelles can be accomplished by
modifying the composition to contain specific organelle targeting
signals. These sequences can target organelles, either specifically
or non-specifically. In some embodiments the interaction of the
targeting signal with the organelle does not occur through a
traditional receptor:ligand interaction.
[0246] The eukaryotic cell comprises a number of discrete membrane
bound compartments, or organelles. The structure and function of
each organelle is largely determined by its unique complement of
constituent polypeptides. However, the vast majority of these
polypeptides begin their synthesis in the cytoplasm. Thus organelle
biogenesis and upkeep require that newly synthesized proteins can
be accurately targeted to their appropriate compartment. This is
often accomplished by amino-terminal signaling sequences, as well
as post-translational modifications and secondary structure.
[0247] Organelles can have single or multiple membranes and exist
in both plant and animal cells. Depending on the function of the
organelle, the organelle can consist of specific components such as
proteins and cofactors. The composition delivered to the organelle
can enhance or inhibit to the functioning of the organelle.
Exemplary organelles include the nucleus, mitochondrion,
chloroplast, lysosome, peroxisome, Golgi, endoplasmic reticulum,
and nucleolus. Some organelles, such as mitochondria and
chloroplasts, contain their own genome. Nucleic acids are
replicated, transcribed, and translated within these organelles.
Proteins are imported and metabolites are exported.
[0248] There can be an exchange of material across the membranes of
organelles. Synthetic organelles can be formed from lipids and can
contain specific proteins within the lipid membranes. Additionally,
the content of synthetic organelles can be manipulated to contain
components for the translation of nucleic acids.
[0249] In certain embodiments the particle, the cargo, or a
combination thereof specifically target mitochondria. Mitochondria
contain the molecular machinery for the conversion of energy from
the breakdown of glucose into adenosine triphosphate (ATP). The
energy stored in the high energy phosphate bonds of ATP is then
available to power cellular functions. Cells with high metabolic
activity, such as heart muscle, have many well developed
mitochondria.
[0250] Mitochondrial targeting agents can include a sequence of
highly positively charged amino acids. This allows the protein to
be targeted to the highly negatively charged mitochondria. Unlike
receptor:ligand approaches that rely upon stochastic Brownian
motion for the ligand to approach the receptor, such targeting
signals are drawn to mitochondria because of charge. Therefore, in
some embodiments, the mitochondrial targeting agent is a protein
transduction domain including but not limited to the protein
transduction domains discussed in more detail below.
[0251] Mitochondrial targeting agents also include short peptide
sequences (Yousif, et al., Chembiochem., 10(13):2131 (2009)), for
example, mitochondrial transporters-synthetic cell-permeable
peptides, also known as mitochondria-penetrating peptides (MPPs),
that are able to enter mitochondria. MPPs are typically cationic,
but also lipophilic; this combination of characteristics
facilitates permeation of the hydrophobic mitochondrial membrane.
For example, MPPs can include alternating cationic and hydrophobic
residues (Horton, et al., Chem Biol., 15(4):375-82 (2008)). Some
MPPs include delocalized lipophilic cations (DLCs) in the peptide
sequence instead of, or in addition to natural cationic amino acids
(Kelley, et al., Pharm. Res., 2011 Aug. 11 [Epub ahead of print]).
Other variants can be based on an oligomeric carbohydrate scaffold,
for example attaching guanidinium moieties due to their delocalized
cationic form (Yousif, et al., Chembiochem., 10(13):2131
(2009).
[0252] Mitochondrial targeting agents also include mitochondrial
localization signals or mitochondrial targeting signals. Many
mitochondrial proteins are synthesized as cytosolic precursor
proteins containing a leader sequence, also known as a presequence,
or peptide signal sequence. Many sequences are known in the art,
see for example, U.S. Pat. No. 8,039,587. The identification of the
specific sequences necessary for translocation of a linked compound
into a mitochondrion can be determined using predictive software
known to those skilled in the art.
[0253] In some embodiments the target moiety directs the
composition to the nucleus. Nuclear localization signals (NLS) or
domains are known in the art and include for example, SV 40 T
antigen or a fragment thereof. The NLS can be simple cationic
sequences of about 4 to about 8 amino acids, or can be bipartite
having two interdependent positively charged clusters separated by
a mutation resistant linker region of about 10-12 amino acids.
2. Endosomal Escape and Membrane Penetration
[0254] In some embodiments, the particles, cargo, or a combination
thereof additionally or alternatively include a moiety that
enhances escape from endosomes or macropinosomes. In some
embodiments, particles enter cells through endocytosis and are
entrapped in endosomes. These early endosomes subsequently fuse
with sorting endosomes, which in turn transfer their contents to
the late endosomes. Late endosomal vesicles are acidified (pH 5-6)
by membrane-bound proton-pump ATPases. If the particles are not
released from the endosome, for example, by pH-induced degradation
and the associated "sponge" effect as discussed in more detail
below, the endosomal content can be relocated to the lysosomes,
which are further acidified (pH .about.4.5) and contain various
nucleases that promote the degradation of nucleic acids. To avoid
lysosomal degradation of cargo, particularly nucleic acid cargo,
the particle including the cargo, or the cargo itself (following
release from the particle) escapes from the endosome into the
cytosol. This is particularly preferred for mRNA and functional
nucleic acid cargos which may rely on cytosolic cellular machinery
for their activity.
[0255] Strategies to promote endosomal release are known in the
art, and include, for example, the use of fusogenic lipids,
polymers with high buffering capacity and membrane-interacting
peptides (exemplary strategies are reviewed in Dominska and
Dykxhoorn, J Cell Sci, 123: 1183-1189 (2010)). In particularly
preferred embodiments, the endosomal escape sequence is a membrane
interacting peptide. In some embodiments, the endosomal escape
sequence is a protein transduction domain. Thus in some embodiments
the endosomal escape sequence is part of, or consecutive with, the
protein transduction domain. In some embodiments, the endosomal
escape sequence is non-consecutive with the protein transduction
domain or provided in the absence of a protein transduction domain.
In some embodiments the endosomal escape sequence includes a
portion of the hemagglutinin peptide from influenza (HA).
[0256] Examples of endosomal escape sequences are known in the art.
See, for example, WO 2013/103972. Hatakeyama, et al., have
described a fusogenic PEG-peptide-DOPE (PPD) construct and a
pH-sensitive fusogenic GALA peptide (Hatakeyama, et al., J Control.
Release 139, 127-132 (2009)) and that PPD constructs can be cleaved
by matrix metalloproteinases that are specifically secreted by
cancer cells, enhancing the delivery of siRNA complexed with this
carrier to tumor cells (Hatakeyama, et al., Gene Ther., 14, 68-77
(2007)).
[0257] Another membrane-destabilization mechanism takes advantage
of the pore-forming ability of viroporins, highly hydrophobic
proteins that create channels and facilitate ion flow across
biological membranes (Gonzalez and Carrasco, FEBS Lett. 552, 28-34
(2003)). For example, peptides derived from the endodomain of the
HIV gp41 envelope glycoprotein (sequence corresponding to residues
783-806 of gp160) form pores in the cell membrane by adopting an
amphipathic .alpha.-helical structure (Costin et al., Virol. J.,
4:123 (2007)) and (Kwon et al., Bioconjugate Chem., 19, 920-927
(2008)).
[0258] The influenza-derived fusogenic peptide diINF-7 has also
been shown to enhance endosomal release (Oliveira et al., Int. J.
Pharm. 331, 211-214 (2007)).
3. Protein Transduction Domains
[0259] The particles, any of the active agents, but particularly
protein and nucleic acid agents, or a combination thereof can
include a protein transduction domain to improve delivery of the
active agent across extracellular membranes, intracellular
membranes, or the combination thereof. As used herein, a "protein
transduction domain" or PTD refers to a polypeptide,
polynucleotide, carbohydrate, organic or inorganic compound that
facilitates traversing a lipid bilayer, micelle, cell membrane,
organelle membrane, or vesicle membrane. A PTD attached to another
molecule facilitates the molecule traversing membranes, for example
going from extracellular space to intracellular space, or cytosol
to within an organelle.
[0260] The protein transduction domain can be a polypeptide. A
protein transduction domain can be a polypeptide including
positively charged amino acids. Thus, some embodiments include PTDs
that are cationic or amphipathic. Protein transduction domains
(PTD), also known as a cell penetrating peptides (CPP), are
typically polypeptides including positively charged amino acids.
PTDs are known in the art, and include but are not limited to small
regions of proteins that are able to cross a cell membrane in a
receptor-independent mechanism (Kabouridis, P., Trends in
Biotechnology (11):498-503 (2003)). Although several PTDs have been
documented, the two most commonly employed PTDs are derived from
TAT (Frankel and Pabo, Cell, 55(6):1189-93(1988)) protein of HIV
and Antennapedia transcription factor from Drosophila, whose PTD is
known as Penetratin (Derossi et al., J Biol Chem., 269(14):10444-50
(1994)). Exemplary protein transduction domains include
polypeptides with 11 Arginine residues, or positively charged
polypeptides or polynucleotides having 8-15 residues, preferably
9-11 residues. The Antennapedia homeodomain is 68 amino acid
residues long and contains four alpha helices. See Derossi, JBC,
1994, 269, 10444) which provides Antp peptide. Oligoarginine is
another preferred PTA (8 arginines) (Goun et al Bioconjugate Chem.
2006, 17, 787)). Penetratin is an active domain of this protein
which consists of a 16 amino acid sequence derived from the third
helix of Antennapedia. TAT protein consists of 86 amino acids and
is involved in the replication of HIV-1 (Vives, et al., JBC, 1997,
272, 16010)) of the parent protein that appears to be critical for
uptake. TAT has been favored for fusion to proteins of interest for
cellular import. Several modifications to TAT, including
substitutions of Glutamine to Alanine, i.e., Q>A, have
demonstrated an increase in cellular uptake anywhere from 90%
(Wender et al., Proc Natl Acad Sci USA., 97(24):13003-8 (2000)) to
up to 33 fold in mammalian cells. (Ho et al., Cancer Res.,
61(2):474-7 (2001)).
4. Linkers
[0261] In different embodiments, the hydrophilic portion of the
polymer can be connected to the hydrophobic portion by a cleavable
linker, the diagnostic, therapeutic or prophylactic agent may be
connected to the amphiphilic polymer by a cleavable linker, and/or
the targeting moiety may be connected to the amphiphilic polymer by
a cleavable linker. The linker may be hydrolyzed by a chemical or
enzymatic process. Preferably, the linker is cleaved by hydrogen
peroxide, which is produced at sites of inflammation or areas of
high neutrophil concentration, thereby increasing the selectivity
of the nanoparticles. For example, the linker may be hydrolyzed by
a chemical or enzymatic process.
5. Exemplary Design Strategy
[0262] It will be appreciated that the stimuli-response particles
and cargo each optionally including targeting moiety, protein
transduction moieties, linkers, and other elements described herein
are modular in nature and can be utilized in various combinations
as selected by the user based on the intended use. Preferred uses
and therapeutic strategies include, but are not limited to, those
described in more detail below. Exemplary particles loaded with
exemplary cargo and optionally including exemplary targeting and
membrane escape elements are provided in, but not limited by the
working Examples below. For example, in one non-limiting design
strategy exemplified in Example 1, after encapsulating the agent(s)
to be delivered, the resulting delivery system shows four unique
features (FIG. 1C):
[0263] i) the surface-encoded iRGD peptide endows the NPs with
tumor-targeting and tumor-penetrating abilities;
[0264] ii) the hydrophilic PEG shells prolong the blood
circulation;
[0265] iii) a small population of cationic lipid-like grafts
randomly dispersed in the hydrophobic poly(2-(diisopropylamino)
ethylmethacrylate) (PDPA) segment can entrap siRNA in the
hydrophobic cores of the NPs; and
[0266] iv) the rapid protonation of the ultra pH-responsive PDPA
segment induces the endosomal swelling via the "proton sponge"
effect, which synergizes with the insertion of the cationic
lipid-like grafts into endosomal membrane to induce membrane
destabilization (Zhu X et al., Proceedings of the National Academy
of Sciences, 112, 7779-7784 (2015)) and efficient endosomal
escape.
III. Nanoparticle Formation
[0267] The nanoparticles are typically formed using an emulsion
process, single or double, using an aqueous and a non-aqueous
solvent. Typically, the nanoparticles contain a minimal amount of
the non-aqueous solvent after solvent removal. Preferred methods of
preparing these nanoparticles are described in the examples.
[0268] In one embodiment, nanoparticles are prepared using emulsion
solvent evaporation method. A polymeric material is dissolved in a
water immiscible organic solvent and mixed with a drug solution or
a combination of drug solutions. The water immiscible organic
solvent is preferably a GRAS ingredient such as chloroform,
dichloromethane, and acyl acetate. The drug can be dissolved in,
but is not limited to, one or a plurality of the following:
acetone, ethanol, methanol, isopropyl alcohol, acetonitrile and
Dimethyl sulfoxide (DMSO). An aqueous solution is then added into
the resulting mixture solution to yield emulsion solution by
emulsification. The emulsification technique can be, but not
limited to, probe sonication or homogenization through a
homogenizer.
[0269] In another embodiment, nanoparticles are prepared using
nanoprecipitation methods or microfluidic devices. A polymeric
material is mixed with a drug or drug combinations in a water
miscible organic solvent. The water miscible organic solvent can be
one or more of the following: acetone, ethanol, methanol, isopropyl
alcohol, acetonitrile and Dimethyl sulfoxide (DMSO). The resulting
mixture solution is then added to an aqueous solution to yield
nanoparticle solution. The agents may be associated with the
surface of, encapsulated within, surrounded by, and/or distributed
throughout the polymeric matrix of the particles.
[0270] In another embodiment, nanoparticles are prepared by the
self-assembly of the amphiphilic polymers, optionally including
hydrophilic and/or hydrophobic polymers, using emulsion solvent
evaporation, a single-step nanoprecipitation method, or
microfluidic devices.
[0271] Two methods to incorporate targeting moieties into the
nanoparticles include: i) conjugation of targeting ligands to the
hydrophilic region (e.g. PEG) of polymers prior to nanoparticle
preparation; and ii) incorporation of targeting molecules into
nanoparticles where the PEG layer on the nanoparticle surface can
be cleaved in the presence of a chemical or enzyme at tissues of
interest to expose the targeting molecules.
[0272] The diameters of the nanoparticles range between about 50 nm
and about 500 nm, preferably between about 50 nm and about 350 nm.
In some embodiments, the diameters of the nanoparticles are about
100 nm. The zeta potential of the nanoparticles ranges between
about -50 mV and about +50 mV, preferably between about -25 mV and
+25 mV, most preferably between about -10 mV and about +10 my.
IV. Formulations and Methods of Administration
A. Formulations
[0273] Formulations are prepared using a pharmaceutically
acceptable "carrier" composed of materials that are considered safe
and effective and may be administered to an individual without
causing undesirable biological side effects or unwanted
interactions. The "carrier" is all components present in the
pharmaceutical formulation other than the active ingredient or
ingredients. The term "carrier" includes but is not limited to
diluents, binders, lubricants, desintegrators, fillers, and coating
compositions.
[0274] Pharmaceutical compositions can be for administration by
parenteral (intramuscular, intraperitoneal, intravenous (IV) or
subcutaneous injection), routes of administration and can be
formulated in dosage forms appropriate for each route of
administration. The compositions are most typically administered
systemically.
[0275] Compounds and pharmaceutical compositions thereof can be
administered in an aqueous solution, by parenteral injection. The
formulation may also be in the form of a suspension or emulsion. In
general, pharmaceutical compositions are provided including
effective amounts of the active agent(s) and optionally include
pharmaceutically acceptable diluents, preservatives, solubilizers,
emulsifiers, adjuvants and/or carriers. Such compositions include
diluents sterile water, buffered saline of various buffer content
(e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and
optionally, additives such as detergents and solubilizing agents
(e.g., TWEEN.RTM. 20, TWEEN.RTM. 80 also referred to as polysorbate
20 or 80), anti-oxidants (e.g., ascorbic acid, sodium
metabisulfite), and preservatives. Examples of non-aqueous solvents
or vehicles are propylene glycol, polyethylene glycol, vegetable
oils, such as olive oil and corn oil, gelatin, and injectable
organic esters such as ethyl oleate. The formulations may be
lyophilized and redissolved/resuspended immediately before use. The
formulation may be sterilized by, for example, filtration through a
bacteria retaining filter, by incorporating sterilizing agents into
the compositions, by irradiating the compositions, or by heating
the compositions.
[0276] Preferably, the aqueous solution is water, physiologically
acceptable aqueous solutions containing salts and/or buffers, such
as phosphate buffered saline (PBS), or any other aqueous solution
acceptable for administration to an animal or human. Such solutions
are well known to a person skilled in the art and include, but are
not limited to, distilled water, de-ionized water, pure or
ultrapure water, saline, phosphate-buffered saline (PBS). Other
suitable aqueous vehicles include, but are not limited to, Ringer's
solution and isotonic sodium chloride. Aqueous suspensions may
include suspending agents such as cellulose derivatives, sodium
alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting
agent such as lecithin. Suitable preservatives for aqueous
suspensions include ethyl and n-propyl p-hydroxybenzoate.
[0277] An emulsion is a preparation of one liquid distributed in
small globules throughout the body of a second liquid. The
dispersed liquid is the discontinuous phase, and the dispersion
medium is the continuous phase. When oil is the dispersed liquid
and an aqueous solution is the continuous phase, it is known as an
oil-in-water emulsion, whereas when water or aqueous solution is
the dispersed phase and oil or oleaginous substance is the
continuous phase, it is known as a water-in-oil emulsion. The oil
phase may consist at least in part of a propellant, such as an HFA
propellant. Either or both of the oil phase and the aqueous phase
may contain one or more surfactants, emulsifiers, emulsion
stabilizers, buffers, and other excipients. Preferred excipients
include surfactants, especially non-ionic surfactants; emulsifying
agents, especially emulsifying waxes; and liquid non-volatile
non-aqueous materials, particularly glycols such as propylene
glycol. The oil phase may contain other oily pharmaceutically
approved excipients. For example, materials such as hydroxylated
castor oil or sesame oil may be used in the oil phase as
surfactants or emulsifiers.
[0278] Buffers are used to control pH of a composition. Preferably,
the buffers buffer the composition from a pH of about 4 to a pH of
about 7.5, more preferably from a pH of about 4 to a pH of about 7,
and most preferably from a pH of about 5 to a pH of about 7.
[0279] Rapid escape and protection from the endosomal degradation
can been achieved by the inclusion of fusogenic lipids such as
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in
pH-sensitive and cationic liposome delivery systems. DOPE is a
helper lipid capable of disrupting the endosomal membrane upon
endosomal acidification by the formation of lipid hexagonal phases.
Endosomal membrane disruption can release the DNA-based therapeutic
and its delivery system into the cytoplasm. Lysosomatropic agents
such as monensin and chloroquine, which raise the endosomal pH,
block acidification, and thus inhibit lysozyme activity, have also
been used to facilitate endosomal release of DNA. Endosomal
degradation of DNA-based therapeutics can also be circumvented by
the incorporation of viral peptides such as hemagglutinin HA2 and
those derived from adenoviruses in their delivery systems.
Hemagglutinin HA2 undergoes conformational transition and leads to
the destruction of the endosome, thereby facilitating the release
of the DNA-based therapeutic. Enhanced rapid endosomal escape and
enhanced transfection have also been achieved using fusogenic
peptides such as poly(L-lysine) (PLL) and cationic polymers such as
polyethylenimine (PEI) and dendrimers.
[0280] Active agent(s) and compositions thereof can be formulated
for pulmonary or mucosal administration. The administration can
include delivery of the composition to the lungs, nasal, oral
(sublingual, buccal), vaginal, or rectal mucosa. In a particular
embodiment, the composition is formulated for and delivered to the
subject sublingually.
[0281] In one embodiment, the compounds are formulated for
pulmonary delivery, such as intranasal administration or oral
inhalation. The respiratory tract is the structure involved in the
exchange of gases between the atmosphere and the blood stream. The
lungs are branching structures ultimately ending with the alveoli
where the exchange of gases occurs. The alveolar surface area is
the largest in the respiratory system and is where drug absorption
occurs. The alveoli are covered by a thin epithelium without cilia
or a mucus blanket and secrete surfactant phospholipids. The
respiratory tract encompasses the upper airways, including the
oropharynx and larynx, followed by the lower airways, which include
the trachea followed by bifurcations into the bronchi and
bronchioli. The upper and lower airways are called the conducting
airways. The terminal bronchioli then divide into respiratory
bronchiole, which then lead to the ultimate respiratory zone, the
alveoli, or deep lung. The deep lung, or alveoli, is the primary
target of inhaled therapeutic aerosols for systemic drug
delivery.
[0282] Pulmonary administration of therapeutic compositions
comprised of low molecular weight drugs has been observed, for
example, beta-androgenic antagonists to treat asthma. Other
therapeutic agents that are active in the lungs have been
administered systemically and targeted via pulmonary absorption.
Nasal delivery is useful for administration of therapeutics since
the nose has a large surface area available for drug absorption due
to the coverage of the epithelial surface by numerous microvilli,
the subepithelial layer is highly vascularized, the venous blood
from the nose passes directly into the systemic circulation and
therefore avoids the loss of drug by first-pass metabolism in the
liver, it offers lower doses, more rapid attainment of therapeutic
blood levels, quicker onset of pharmacological activity, fewer side
effects, high total blood flow per cm.sup.3, porous endothelial
basement membrane, and it is easily accessible.
[0283] The term aerosol as used herein refers to any preparation of
a fine mist of particles, which can be in solution or a suspension,
whether or not it is produced using a propellant. Aerosols can be
produced using standard techniques, such as ultrasonication or
high-pressure treatment.
[0284] Carriers for pulmonary formulations can be divided into
those for dry powder formulations and for administration as
solutions. Aerosols for the delivery of therapeutic agents to the
respiratory tract are known in the art. For administration via the
upper respiratory tract, the formulation can be formulated into a
solution, e.g., water or isotonic saline, buffered or un-buffered,
or as a suspension, for intranasal administration as drops or as a
spray. Preferably, such solutions or suspensions are isotonic
relative to nasal secretions and of about the same pH, ranging
e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0.
Buffers should be physiologically compatible and include, simply by
way of example, phosphate buffers. For example, a representative
nasal decongestant is described as being buffered to a pH of about
6.2. One skilled in the art can readily determine a suitable saline
content and pH for an innocuous aqueous solution for nasal and/or
upper respiratory administration.
B. Methods of Administration
[0285] Suitable parenteral administration routes include
intravascular administration (e.g., intravenous bolus injection,
intravenous infusion, intra-arterial bolus injection,
intra-arterial infusion and catheter instillation into the
vasculature); peri- and intra-tissue injection (e.g., intraocular
injection, intra-retinal injection, or sub-retinal injection);
subcutaneous injection or deposition including subcutaneous
infusion (such as by osmotic pumps); direct application by a
catheter or other placement device (e.g., an implant comprising a
porous, non-porous, or gelatinous material).
[0286] The formulation can be administered in a single dose or in
multiple doses. Certain factors may influence the dosage required
to effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. It will also be appreciated that the effective dosage of
the oligonucleotide used for treatment may increase or decrease
over the course of a particular treatment. Changes in dosage may
result and become apparent from the results of diagnostic
assays.
[0287] Dosing is dependent on severity and responsiveness of the
disease condition to be treated, with the course of treatment
lasting from several days to several months, or until a cure is
effected or a diminution of disease state is achieved. Optimal
dosing schedules can be calculated from measurements of drug
accumulation in the body of the patient. Persons of ordinary skill
can easily determine optimum dosages, dosing methodologies and
repetition rates. Optimum dosages may vary depending on the
relative potency of individual polynucleotides, and can generally
be estimated based on EC50s found to be effective in vitro and in
vivo animal models.
[0288] Dosage levels on the order of about 1 mg/kg to 100 mg/kg of
body weight per administration are useful in the treatment of a
disease. One skilled in the art can also readily determine an
appropriate dosage regimen for administering the disclosed
polynucleotides to a given subject. For example, the formulation
can be administered to the subject once, e.g., as a single
injection, infusion or bolus. Alternatively, the formulation can be
administered once or twice daily to a subject for a period of from
about three to about twenty-eight days, or from about seven to
about ten days.
V. Biomedical Applications and Therapeutic Strategies
[0289] Biomedical application of nanoparticles has introduced
exciting opportunities for the improvement of disease diagnosis and
treatment. Stimuli-responsive nanoparticles, which can undergo
shape, structure and property change upon encountering endogenous
or exogenous stimuli, can be used in diverse range of biomedical
applications, such as drug controlled release, nucleic acid
delivery, imaging, and diagnostics. The stimuli-responsive
characteristic provides spatiotemporal control over the macroscopic
properties of the nanoparticles, and thus the release of the
encapsulated cargo can occur directly at the desired site,
minimizing toxic and side effects in surrounding, healthy tissue.
Dissociation of the particle and release of its cargo, can be
driven by, for example, pH-, redox-, light-, temperature-, enzyme-,
or ultrasound-responsive polymers composing the particles.
[0290] The stimuli that drive a response by the particle can be
present within a cell (e.g., intracellularly) or outside cells in
the extracellular microenvironment, or can be an external stimuli
for example, light, heat, ultrasound, etc., which can be applied by
the user to the target site. The particles can optionally include a
targeting moiety or ligand. For embodiments in which intracellular
release is desired, the targeting moiety or ligand is typically one
that preferentially binds to the surface of a target cell and
induces or allows the particle to be absorbed or internalized by,
for example, endocytosis or micropinocytosis (Vranic et al.,
Particle and Fibre Toxicology, 10(2):(12 page) (2013)). For
embodiments in which extracellular release is desired, the
targeting moiety or ligand can be one that preferentially binds to
an extracellular target in the desired microenvironment.
A. Exemplary Environments for Selective Delivery
1. Acidic Environment
[0291] pH responsive nanoparticles can be used to target tissues
with acidic extracellular pH. Although the nanoparticles can
optionally include a cell, tissue, organ, or extracellular
matrix-specific targeting moiety or ligand, a targeting moiety or
ligand is not requirement. The pH responsive nanoparticles can be
designed to have spherical morphology at a pH above pKa to protect
cargo during systemic circulation and infiltration into tissues
with extracellular pH at or around neutral or physiological pH. The
particles can dissociate at a pH below pKa, releasing its cargo
into the microenvironment. In this way, the particles selectively
release their cargo at the target site.
[0292] pH responsive nanoparticles can also be used to deliver
cargo into cells. Particles, preferable with a targeting moiety or
ligand, can bind to a target cell and be absorbed or internalized.
Upon encountering an acidic intracellular environment such as that
of endosomes, the pH responsive particles can dissociate and
release their cargo. The particles can also optionally include a
moiety that enhances endosomal escape, such as oligoarginine. As
illustrated in the working Examples below, particle dissociation
within the endosome is believe to induce swelling of the endosome
via "sponge" effect, thus achieving fast and high efficacy delivery
of their cargo into the cytosol. Using an intracellular
endosomal-release strategy, virtually any cell with endosomes (or
another equivalently acidic intracellular environment, compartment,
or organelle) can be the target cell. The addition of a targeting
moiety can be used to accomplish selective delivery of the particle
into target cells over non-target cells. pH responsive
intracellular release can be most effective when the extracellular
pH does not induce nanoparticle dissociation thus allowing the
particles to absorbed or internalized by cells.
[0293] In some embodiments, cargo is released below physiological
pH (e.g., 7.4, or 7.2), or below neutral pH (e.g., 7.0), or in a pH
range of about 5.8 to about 7.3, or about 5.8 to about 6.9, or
about 6.0 to about 6.5, or about 6.5 to about 6.9.
2. Temperature
[0294] In embodiments, cargo release is driven by a change in
temperature. In the biomedical setting, a change in temperature
will can be an increase or decrease from the physiological
temperature of the subject being treated. Normal human body
temperature, also referred to as normothermia or euthermia, depends
upon the place in the body at which the measurement is made, the
time of day, as well as the activity level of the person. Typically
values for oral measurement (under the tongue) are
36.8.+-.0.4.degree. C. (98.2.+-.0.72.degree. F.) and internal
(rectal, vaginal) measurement are 37.0.degree. C. (98.6.degree. F.)
(Harrison's Principles of Internal Medicine, 18e, Longo, Editor,
Fauci, et al., Editor, Kasper). Human temperature classifications
can be, for example, Hypothermia <35.0.degree. C. (95.0.degree.
F.); Normal 36.5-37.5.degree. C. (97.7-99.5.degree. F.), Fever
>37.5 or 38.3.degree. C. (99.5 or 100.9.degree. F.),
Hyperthermia >37.5 or 38.3.degree. C. (99.5 or 100.9.degree.
F.), Hyperpyrexia >40.0 or 41.5.degree. C. (104.0 or
106.7.degree. F.). The particles can be designed for release within
one or more of these temperature classifications, or a sub-range
thereof. It will be appreciated that a subject's normal body
temperature can fluctuate, for example, with the time of day, sleep
vs. wake, eating vs. fasting, exercise, the amount of clothing
being worn, the ambient temperature, the anxiety or excitement
level of the subject, etc., as is known in the art. The particles
can be tuned for release when body temperature drops below or
exceeds a predetermined threshold, and therefore selectively
release cargo during certain times of the day or night, caloric
intake (or lack thereof), during exercise, anxiety, etc. The
release can be local so systemic.
[0295] In addition of more global changes in overall body
temperature, such as those introduced above, the particles can be
tuned for release at sites of local temperature changes. For
example, local, tissue-specific increase in tissue temperature can
occur at sites of inflammation, injury, infection, and cancer
(e.g., tumor) (Chapter Nine, Inflammation, Tissue Repair, and
Fever, pages 150-167). The change in temperature can be relative to
unaffected tissue and may occur in the presence or absence of a
global change in body temperature.
3. Reduction-Oxidation (Redox)
[0296] The release of nanoparticle cargo can be induced by a
reduction-oxidation ("redox") reaction. In some embodiments, the
polymers composing the particles include one or more disulfide
bonds. The particles can release their cargo when disulfide bond is
reduced upon exposure to a reducing agent. In some embodiments, the
reducing agent is a glutathione. L-Glutathione (GSH) is a
tripeptide molecule that can also act as an antioxidant. In cells,
GSH reduces the disulfide bonds formed within cytoplasmic proteins
to cysteines and reacts to other oxidized GSH to an oxidized form
of glutathione disulfide (GSSG), also called L(-)-glutathione
(Traverso, et al., Oxidative Medicine and Cellular Longevity,
Volume 2013 (2013), Article ID 972913, 10 pages). As discussed in
more detail below, intracellular levels of glutathione (GSH) are
100-1000 fold higher in cancer cells than in normal tissue, and
thus redox-sensitive particles can be used to selective release
cargo in cells with higher-than-normal GSH, such as cancer cells.
For example, one study showed that intracellular GHS levels in
normal lung cells were about 11.20.+-.0.58 (SEM) nmol GSH/mg
protein (24 patients) with a range from 6.1 to 17.5 nmol GSH/mg
protein, while GHS level in adenocarcinomas was 8.83.+-.0.96
nmol/mg protein (8 patients); large cell carcinomas was
8.25.+-.2.51 nmol/mg protein (3 patients); and squamous cell
carcinomas 23.25.+-.5.99 nmol/mg protein (8 patients) (Cook, et
al., Cancer Research, 51:4287-4294 (1991).
[0297] The Examples below show that cargo can be released
redox-sensitive particles in matter of minutes in the presences of
10 nM GSH.
[0298] In some embodiments, the reducing agent is not endogenous to
the cell, tissue, organ, or other microenvironment. For example, in
some embodiments, the reducing agent is administered locally or
systemically to trigger release of the cargo from the particles in
a local or systemic fashion.
[0299] In addition to GSH, other reducing agents can also induce
release of the cargo, however, it will be appreciated that in some
embodiments, the use, or the amount that can be used, of certain
reducing agents is limited in biological and therapeutic
applications by their toxicity.
4. External Stimuli
[0300] As introduced above, release of nanoparticle cargo can be
induced by external stimuli, such as light, temperature, or
ultrasound. The stimuli can be applied globally, for example to the
entire subject, or preferably to a more limited or local aspect
thereof. For example, light, heat (or cold), or ultrasound can be
administered to a specific tissue(s), location(s), or combination
thereof to modulate selective release of cargo from particles
accumulating or passing through the targeted tissue or location.
For example, heat (or cold) can be applied to the target tissue or
location to cause a local temperature shift that induces
dissociation of the particle and release of its cargo. Radiation at
different frequencies along the electromagnetic spectrum can also
be used to release cargo. For example, particles can be formed that
are sensitive to ionizing radiation, visible light, microwaves, or
radiowaves. In particular embodiments, the particles are sensitive
to visible light (e.g., near ultraviolet, near infrared, mid
infrared, far infrared). Particles can also be formed that are
sensitive to sound waves. For example, in particular embodiments,
the particles release cargo in response to ultrasound.
[0301] In particular embodiments, the particles are sensitive to
ultraviolet light. Ultraviolet (UV) light is an electromagnetic
radiation with a wavelength shorter than that of visible light but
longer than X-rays. The wavelength of UV light is typically from
about 400 nm (750 THz) to about 10 nm (30 PHz). UV radiation can be
divided into five categories: UV-A is about 320-400 nm, UV-B is
290-320 nm, UV-C is 220-209 nm, Far UV is 190-220 nm, and vacuum UV
40-190 nm. In some embodiments, the particles are sensitive to
UV-A, UV-B, UV-C, or a combination thereof. The Examples below
illustrate that particles can be formed that the release their
cargo after exposure to UV light, for example 365 nm UV light (16
W), for different time periods. In some embodiments, the source
provides a specific desired wavelength. In some embodiments, the
source provides a range of wavelength.
[0302] The external stimuli can be provided by the practitioner
using, for example, a piece of equipment that provides the stimuli.
The stimuli can also be provided by the environment and may or may
not be under the control of practitioner or user. For example, the
sun generates visible light, heat, and UV radiation. Thus, in some
embodiments, the particles are designed to release their cargo in
response to the sun.
[0303] Exposure to external stimuli can be carried out over
minutes, hours, days or weeks. In some embodiments, the exposure is
between about 1 and about 120 minutes, for example, 10, 15, 30, 45,
60, 90, or 120 minutes. In some embodiments, the exposure is
between about 1 and 48 hours, for example, 1, 2, 3, 4, 5, 10, 12.5,
15, 20, 24, 36, or 48 hours. In some embodiments, the exposure is
over two or more days.
B. Preferred Tissues to Target and Therapeutic Strategies
[0304] As discussed above, the particles can be used to selectively
target cells, tissues, organs, or microenvironments thereof. The
selective release of cargo at a target site can be used in
strategies to treat a variety of diseases and disorders. Suitable
methods can include administering a subject an effective amount of
nanoparticles containing a therapeutic cargo to reduce or alleviate
one or more symptoms of the disease or disorder to be treated. The
disclosed strategies can include targeting certain intracellular
and/or extracellular environments for selective release based on
response-inducing stimuli alone, or in combination with one or more
targeting moieties that enhance delivery to a desired cell type,
tissue, organ, microenvironment, subcellular organelle, or a
combination thereof 1. Tumor targeting Methods of treating cancer
are provided. The nanoparticles can be designed, for example, for
release in the tumor microenvironment or within a tumor cells, or
in an immune response microenvironment or within an immune cell.
Suitable methods can include administering a subject an effective
amount of nanoparticles containing a therapeutic cargo to reduce or
alleviate one or more symptoms of the cancer. The effect of the
particles on the cancer can be direct or indirect. The compositions
and methods described herein are useful for treating subjects
having benign or malignant tumors by delaying or inhibiting the
growth of a tumor in a subject, reducing the growth or size of the
tumor, inhibiting or reducing metastasis of the tumor, and/or
inhibiting or reducing symptoms associated with tumor development
or growth.
[0305] The tumor microenvironment is the cellular environment in
which the tumor exists, and can include surrounding blood vessels,
immune cells, fibroblasts, bone marrow-derived inflammatory cells,
lymphocytes, signaling molecules, and the extracellular matrix
(ECM). The tumor and the surrounding microenvironment are closely
related and interact constantly. Tumors can modulate the
microenvironment by releasing extracellular signals, promoting
tumor angiogenesis and inducing peripheral immune tolerance, while
the immune cells in the microenvironment can affect the growth and
evolution of cancerous cells. The microenvironment in tumor tissue
is different from the normal tissues. Thus, in some embodiments,
the stimuli-responsive polymers are design to trigger the
structural changes in reponse to stimuli that is unique to the
tumor microenvironment including but not limited to temperature,
pH, ionic strength, composition/organization of the extracellular
matrix (ECM), over-expressed molecules or enzymes, and hypoxia.
[0306] Compared to normal tissues, the pH in tumor tissue is more
acidic, the tissue temperature is relatively higher, oxygen
concentrations are reduced (hypoxia), and some specific enzymes or
chemicals are over-expressed. Hypoxia is an important
characteristic of the tumor microenvironment commonly found in
cancers and a selection force for the glycolytic phenotype. Thus,
in some embodiments, hypoxia-responsive stimula are used to
selectively delivery cargo to an acidic tumor microenvironment. For
example, a hydrophobically modified 2-nitroimidazole derivative
conjugated to the backbone of the carboxymethyl dextran described
to target hypoxia (Thambi T et al., Biomaterials. 2014 February;
35(5):1735-43) can be used with the nanoparticles.
[0307] The interstitial fluid of tumors and abscesses also has
shown pH values of less than 6.0, averaging 0.2-0.6 units lower
than mean extracellular pH of normal tissues (Kraus and Wolf,
Tumour Biol, 17,133-154 (1996)). Tumors commonly have an
extracellular environment with a pH in the range of, for example,
6.5-6.9. See, for example, Balkwill, et al., Journal of Cell
Science, 125(23):5591-6 (2012) and Kato, et al., Cancer Cell
International, 13(89) (8 pages) (2013). Thus, in some embodiments,
pH-sensitive nanoparticles are used to selectively deliver cargo to
an acidic tumor microenvironment.
[0308] Tumors can also have elevated temperatures relative to the
surround or otherwise normal or non-malignant tissue (see, e.g.,
Stefanadis, JCO, 19(3):676-681 (2001)). Therefore,
temperature-responsive particles can also be utilized to
selectively target tumors.
[0309] The intracellular levels of glutathione (GSH) are 100-1000
fold higher in cancer cells than in normal tissue. Redox-sensitive
approach is particularly promising to enhance the exposure of
cancer cells to therapeutic molecules. Thus, in some embodiments,
redox-responsive particles can also be utilized for delivering
cargo to tumor cells.
a. Tumor Targeting Moieties
[0310] In addition or alternative to selectively targeting cancer
cells by targeting an acidic microenvironment, or one with an
elevated temperature, cancer cells or their microenvironment can be
specifically targeted relative to healthy or normal cells by
including a targeting moiety. Tumor or tumor-associated
neovasculature targeting domains can be ligands that bind to cell
surface antigens or receptors that are specifically expressed on
tumor cells or tumor-associated neovasculature or microenvironment,
or are overexpressed on tumor cells or tumor-associated
neovasculature or microenvironment as compared to normal tissue.
Tumors also secrete a large number of ligands into the tumor
microenvironment that affect tumor growth and development.
Receptors that bind to ligands secreted by tumors, including, but
not limited to growth factors, cytokines and chemokines, including
the chemokines provided below, can also be used. Ligands secreted
by tumors can be targeted using soluble fragments of receptors that
bind to the secreted ligands. Soluble receptor fragments are
fragments polypeptides that may be shed, secreted or otherwise
extracted from the producing cells and include the entire
extracellular domain, or fragments thereof. In some embodiments,
the targeting moiety is an antibody, for example a single chain
antibody, the binds to the target.
i. Cancer Antigens
[0311] Cancer antigens that can be targeted are well known in the
art. The antigen expressed by the tumor may be specific to the
tumor, or may be expressed at a higher level on the tumor cells as
compared to non-tumor cells. Antigenic markers such as
serologically defined markers known as tumor associated antigens,
which are either uniquely expressed by cancer cells or are present
at markedly higher levels (e.g., elevated in a statistically
significant manner) in subjects having a malignant condition
relative to appropriate controls, are contemplated for use in
certain embodiments.
[0312] Tumor-associated antigens may include, for example, cellular
oncogene-encoded products or aberrantly expressed
proto-oncogene-encoded products (e.g., products encoded by the neu,
ras, trk, and kit genes), or mutated forms of growth factor
receptor or receptor-like cell surface molecules (e.g., surface
receptor encoded by the c-erb B gene). Other tumor-associated
antigens include molecules that may be directly involved in
transformation events, or molecules that may not be directly
involved in oncogenic transformation events but are expressed by
tumor cells (e.g., carcinoembryonic antigen, CA-125, melonoma
associated antigens, etc.) (see, e.g., U.S. Pat. No. 6,699,475;
Jager, et al., Int. J. Cancer, 106:817-20 (2003); Kennedy, et al.,
Int. Rev. Immunol., 22:141-72 (2003); Scanlan, et al. Cancer
Immun., 4:1 (2004)).
[0313] Genes that encode cellular tumor associated antigens include
cellular oncogenes and proto-oncogenes that are aberrantly
expressed. In general, cellular oncogenes encode products that are
directly relevant to the transformation of the cell, and because of
this, these antigens are particularly preferred targets for
anticancer therapy. An example is the tumorigenic neu gene that
encodes a cell surface molecule involved in oncogenic
transformation. Other examples include the ras, kit, and trk genes.
The products of proto-oncogenes (the normal genes which are mutated
to form oncogenes) may be aberrantly expressed (e.g.,
overexpressed), and this aberrant expression can be related to
cellular transformation. Thus, the product encoded by
proto-oncogenes can be targeted. Some oncogenes encode growth
factor receptor molecules or growth factor receptor-like molecules
that are expressed on the tumor cell surface. An example is the
cell surface receptor encoded by the c-erbB gene. Other
tumor-associated antigens may or may not be directly involved in
malignant transformation. These antigens, however, are expressed by
certain tumor cells and may therefore provide effective targets.
Some examples are carcinoembryonic antigen (CEA), CA 125
(associated with ovarian carcinoma), and melanoma specific
antigens.
[0314] In ovarian and other carcinomas, for example, tumor
associated antigens are detectable in samples of readily obtained
biological fluids such as serum or mucosal secretions. One such
marker is CA125, a carcinoma associated antigen that is also shed
into the bloodstream, where it is detectable in serum (e.g., Bast,
et al., N. Eng. J. Med., 309:883 (1983); Lloyd, et al., Int. J.
Canc., 71:842 (1997). CA125 levels in serum and other biological
fluids have been measured along with levels of other markers, for
example, carcinoembryonic antigen (CEA), squamous cell carcinoma
antigen (SCC), tissue polypeptide specific antigen (TPS), sialyl TN
mucin (STN), and placental alkaline phosphatase (PLAP), in efforts
to provide diagnostic and/or prognostic profiles of ovarian and
other carcinomas (e.g., Sarandakou, et al., Acta Oncol., 36:755
(1997); Sarandakou, et al., Eur. J. Gynaecol. Oncol., 19:73 (1998);
Meier, et al., Anticancer Res., 17(4B):2945 (1997); Kudoh, et al.,
Gynecol. Obstet. Invest., 47:52 (1999)). Elevated serum CA125 may
also accompany neuroblastoma (e.g., Hirokawa, et al., Surg. Today,
28:349 (1998), while elevated CEA and SCC, among others, may
accompany colorectal cancer (Gebauer, et al., Anticancer Res.,
17(4B):2939 (1997)).
[0315] The tumor associated antigen, mesothelin, defined by
reactivity with monoclonal antibody K-1, is present on a majority
of squamous cell carcinomas including epithelial ovarian, cervical,
and esophageal tumors, and on mesotheliomas (Chang, et al., Cancer
Res., 52:181 (1992); Chang, et al., Int. J. Cancer, 50:373 (1992);
Chang, et al., Int. J. Cancer, 51:548 (1992); Chang, et al., Proc.
Natl. Acad. Sci. USA, 93:136 (1996); Chowdhury, et al., Proc. Natl.
Acad. Sci. USA, 95:669 (1998)). Using MAb K-1, mesothelin is
detectable only as a cell-associated tumor marker and has not been
found in soluble form in serum from ovarian cancer patients, or in
medium conditioned by OVCAR-3 cells (Chang, et al., Int. J. Cancer,
50:373 (1992)). Structurally related human mesothelin polypeptides,
however, also include tumor-associated antigen polypeptides such as
the distinct mesothelin related antigen (MRA) polypeptide, which is
detectable as a naturally occurring soluble antigen in biological
fluids from patients having malignancies (see WO 00/50900).
[0316] A tumor antigen may include a cell surface molecule. Tumor
antigens of known structure and having a known or described
function, include the following cell surface receptors: HER1
(GenBank Accession No. U48722), HER2 (Yoshino, et al., J. Immunol.,
152:2393 (1994); Disis, et al., Canc. Res., 54:16 (1994); GenBank
Acc. Nos. X03363 and M17730), HER3 (GenBank Acc. Nos. U29339 and
M34309), HER4 (Plowman, et al., Nature, 366:473 (1993); GenBank
Acc. Nos. L07868 and T64105), epidermal growth factor receptor
(EGFR) (GenBank Acc. Nos. U48722, and KO3193), vascular endothelial
cell growth factor (GenBank No. M32977), vascular endothelial cell
growth factor receptor (GenBank Acc. Nos. AF022375, 1680143, U48801
and X62568), insulin-like growth factor-I (GenBank Acc. Nos.
X00173, X56774, X56773, X06043, European Patent No. GB 2241703),
insulin-like growth factor-II (GenBank Acc. Nos. X03562, X00910,
M17863 and M17862), transferrin receptor (Trowbridge and Omary,
Proc. Nat. Acad. USA, 78:3039 (1981); GenBank Acc. Nos. X01060 and
M11507), estrogen receptor (GenBank Acc. Nos. M38651, X03635,
X99101, U47678 and M12674), progesterone receptor (GenBank Acc.
Nos. X51730, X69068 and M15716), follicle stimulating hormone
receptor (FSH--R) (GenBank Acc. Nos. Z34260 and M65085), retinoic
acid receptor (GenBank Acc. Nos. L12060, M60909, X77664, X57280,
X07282 and X06538), MUC-1 (Barnes, et al., Proc. Nat. Acad. Sci.
USA, 86:7159 (1989); GenBank Acc. Nos. M65132 and M64928) NY-ESO-1
(GenBank Acc. Nos. AJ003149 and U87459), NA 17-A (PCT Publication
No. WO 96/40039), Melan-A/MART-1 (Kawakami, et al., Proc. Nat.
Acad. Sci. USA, 91:3515 (1994); GenBank Acc. Nos. U06654 and
U06452), tyrosinase (Topalian, et al., Proc. Nat. Acad. Sci. USA,
91:9461 (1994); GenBank Acc. No. M26729; Weber, et al., J. Clin.
Invest, 102:1258 (1998)), Gp-100 (Kawakami, et al., Proc. Nat.
Acad. Sci. USA, 91:3515 (1994); GenBank Acc. No. 573003, Adema, et
al., J. Biol. Chem., 269:20126 (1994)), MAGE (van den Bruggen, et
al., Science, 254:1643 (1991)); GenBank Acc. Nos. U93163, AF064589,
U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690,
U10689, U10688, U10687, U10686, U10685, L18877, U10340, U10339,
L18920, U03735 and M77481), BAGE (GenBank Acc. No. U19180; U.S.
Pat. Nos. 5,683,886 and 5,571,711), GAGE (GenBank Acc. Nos.
AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144,
U19143 and U19142), any of the CTA class of receptors including in
particular HOM-MEL-40 antigen encoded by the SSX2 gene (GenBank
Acc. Nos. X86175, U90842, U90841 and X86174), carcinoembryonic
antigen (CEA, Gold and Freedman, J. Exp. Med., 121:439 (1985);
GenBank Acc. Nos. M59710, M59255 and M29540), and PyLT (GenBank
Acc. Nos. J02289 and J02038); p97 (melanotransferrin) (Brown, et
al., J. Immunol., 127:539-46 (1981); Rose, et al., Proc. Natl.
Acad. Sci. USA, 83:1261-61 (1986)).
[0317] Additional tumor associated antigens include prostate
surface antigen (PSA) (U.S. Pat. Nos. 6,677,157; 6,673,545);
.beta.-human chorionic gonadotropin .beta.-HCG) (McManus, et al.,
Cancer Res., 36:3476-81 (1976); Yoshimura, et al., Cancer,
73:2745-52 (1994); Yamaguchi, et al., Br. J. Cancer, 60:382-84
(1989): Alfthan, et al., Cancer Res., 52:4628-33 (1992));
glycosyltransferase .beta.-1,4-N-acetylgalactosaminyltransferases
(GalNAc) (Hoon, et al., Int. J. Cancer, 43:857-62 (1989); Ando, et
al., Int. J. Cancer, 40:12-17 (1987); Tsuchida, et al., J. Natl.
Cancer, 78:45-54 (1987); Tsuchida, et al., J. Natl. Cancer,
78:55-60 (1987)); NUC18 (Lehmann, et al., Proc. Natl. Acad. Sci.
USA, 86:9891-95 (1989); Lehmann, et al., Cancer Res., 47:841-45
(1987)); melanoma antigen gp75 (Vijayasardahi, et al., J. Exp.
Med., 171:1375-80 (1990); GenBank Accession No. X51455); human
cytokeratin 8; high molecular weight melanoma antigen (Natali, et
al., Cancer, 59:55-63 (1987); keratin 19 (Datta, et al., J. Clin.
Oncol., 12:475-82 (1994)).
[0318] Tumor antigens of interest include antigens regarded in the
art as "cancer/testis" (CT) antigens that are immunogenic in
subjects having a malignant condition (Scanlan, et al., Cancer
Immun., 4:1 (2004)). CT antigens include at least 19 different
families of antigens that contain one or more members and that are
capable of inducing an immune response, including but not limited
to MAGEA (CT1); BAGE (CT2); MAGEB (CT3); GAGE (CT4); SSX (CT5);
NY-ESO-1 (CT6); MAGEC (CT7); SYCP1 (C8); SPANXB1 (CT11.2); NA88
(CT18); CTAGE (CT21); SPA17 (CT22); OY-TES-1 (CT23); CAGE (CT26);
HOM-TES-85 (CT28); HCA661 (CT30); NY-SAR-35 (CT38); FATE (CT43);
and TPTE (CT44).
[0319] Additional tumor antigens that can be targeted, including a
tumor-associated or tumor-specific antigen, include, but not
limited to, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8,
beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein,
EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion
protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and
3, neo-PAP, myosin class I, OS-9, pml-RAR.alpha. fusion protein,
PTPRK, K-ras, N-ras, Triosephosphate isomeras, Bage-1, Gage
3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, Mage-A1,2,3,4,6,10,12,
Mage-C2, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA
(MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1,
MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE),
SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL,
H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human
papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,
MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA
19-9, CA 72-4, CAM 17.1, NuMa, K-ras, .beta.-Catenin, CDK4, Mum-1,
p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72,
.alpha.-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA
27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,
G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K,
NY--CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin
C-associated protein), TAAL6, TAG72, TLP, and TPS. Other
tumor-associated and tumor-specific antigens are known to those of
skill in the art and are suitable for targeting the disclosed
nanoparticles.
[0320] In some embodiments, the tumor antigen to be targeted is
prostate-specific membrane antigen (PSMA). Thus, tumor targeting
moieties include any agonist, or antagonists of PSMA, or any
derivatives thereof. In some embodiments, the tumor targeting
moiety is S,S-2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic
acid (ACUPA), or derivatives thereof.
ii. Antigens Associated with Tumor Neovasculature
[0321] The antigen may be specific to tumor neovasculature or may
be expressed at a higher level in tumor neovasculature when
compared to normal vasculature. Exemplary antigens that are
over-expressed by tumor-associated neovasculature as compared to
normal vasculature include, but are not limited to, integrins
including .alpha.v.beta.3, .alpha.v.beta.5, .alpha.v.beta.6,
.alpha.2.beta.1, .alpha.5.beta.1, .alpha.6.beta.1, and
.alpha.6.beta.4, VEGF/KDR, Tie2, vascular cell adhesion molecule
(VCAM), endoglin, and .alpha..sub.5.beta..sub.3
integrin/vitronectin. Other antigens that are over-expressed by
tumor-associated neovasculature as compared to normal vasculature
are known to those of skill in the art and are suitable for
targeting by the nanoparticles.
[0322] In some embodiment, the antigen associated with tumor
neovasculature to be targated is integrin .alpha.v.beta.3. Thus,
tumor targeting moieties include any agonist, or antagonists of
integrin .alpha.v.beta.3, or any derivatives thereof. In some
embodiments, the tumor targeting moiety is a disulfide-based cyclic
arginine-glycine-aspartic acid (RGD) peptide called iRGD, that is,
CRGDRGPDC (SEQ ID NO:11), or derivatives thereof. In some
embodiments, iRGD is conjugated to one or more of the amphiphilic
ploymers, for example, in the form of iRGD-PEG-b-PDPA, as shown
below in the Examples. In further embodiments, one or more of the
amphiphilic ploymers conjugated with RGD include a
membrane-penetrating motif such as oligoarginine. Examples include
C.sub.17H.sub.35CONH-GR8GRGDS-OH (TCPA1);
C.sub.17H.sub.35CONH--(C.sub.17H.sub.35CONH)KR8GRGDS-OH (TCPA2)
shown in Exmaple 8.
iii. Chemokines/Chemokine Receptors
[0323] In another embodiment, the particles contain a domain that
specifically binds to a chemokine or a chemokine receptor.
Chemokines are soluble, small molecular weight (8-14 kDa) proteins
that bind to their cognate G-protein coupled receptors (GPCRs) to
elicit a cellular response, usually directional migration or
chemotaxis. Tumor cells secrete and respond to chemokines, which
facilitate growth that is achieved by increased endothelial cell
recruitment and angiogenesis, subversion of immunological
surveillance and maneuvering of the tumoral leukocyte profile to
skew it such that the chemokine release enables the tumor growth
and metastasis to distant sites. Thus, chemokines are vital for
tumor progression.
[0324] Based on the positioning of the conserved two N-terminal
cysteine residues of the chemokines, they are classified into four
groups namely CXC, CC, CX3C and C chemokines. The CXC chemokines
can be further classified into ELR+ and ELR- chemokines based on
the presence or absence of the motif `glu-leu-arg (ELR motif)`
preceding the CXC sequence. The CXC chemokines bind to and activate
their cognate chemokine receptors on neutrophils, lymphocytes,
endothelial and epithelial cells. The CC chemokines act on several
subsets of dendritic cells, lymphocytes, macrophages, eosinophils,
natural killer cells but do not stimulate neutrophils as they lack
CC chemokine receptors except murine neutrophils. There are
approximately 50 chemokines and only 20 chemokine receptors, thus
there is considerable redundancy in this system of ligand/receptor
interaction.
[0325] Chemokines elaborated from the tumor and the stromal cells
bind to the chemokine receptors present on the tumor and the
stromal cells. The autocrine loop of the tumor cells and the
paracrine stimulatory loop between the tumor and the stromal cells
facilitate the progression of the tumor. Notably, CXCR2, CXCR4,
CCR2 and CCR7 play major roles in tumorigenesis and metastasis.
CXCR2 plays a vital role in angiogenesis and CCR2 plays a role in
the recruitment of macrophages into the tumor microenvironment.
CCR7 is involved in metastasis of the tumor cells into the sentinel
lymph nodes as the lymph nodes have the ligand for CCR7, CCL21.
CXCR4 is mainly involved in the metastatic spread of a wide variety
of tumors.
[0326] Any one or more of the above listed tumor antigens suitable
for targeting the nanoparticles to the site of tumor cells are also
considered suitable to be used for therapeutic, and/or diagnostic
purposes such as knockdown targets by shRNA, and/or siRNA.
[0327] Other suitable oncogenic molecules as therapeutic, and/or
diagnostic targets include molecules involved in tumor-associated
pathways such as those involved in cancer metabolism including
glycolysis, glutaminolysis, autophagy (Galluzzi L et al., Nat Rev
Drug Discov. 12,829-846 (2013); Rubinsztein D C et al., Nat Rev
Drug Discov. 2012 September; 11(9): 709-730.). Other exemplary
pathways associated with tumor cells include PI3/AKT pathway,
Kelch-like ECH-associated protein 1 (KEAP1)/NRF2 (nuclear factor,
erythroid 2-like 2, NFE2L2) pathway, hypoxia-associated pathways,
DNA repair pathways, and other pathways involved in cell division,
apoptosis, cell cycle control.
[0328] Some exemplary metabolic targets for therapeutic, and/or
diagnostic purposes include glucose transporter (GLUTs),
hexokinase, phosphofructokinase inhibitor,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate
mutase (PGM), enolase (ENO), lactate dehydrogenase, pyruvate
dehydrogenase kinase (PDK), glucose-6-phosphate dehydrogenase
(G6PD), tricarboxylic acid (TCA) cycle, monocarboxylate transporter
(MCTs), HSP90 inhibitor, CPT1, oxidative phosphorylation, glutamate
dehydrogenase, mitochondrial citrate transporter SLC25A1 (CIC),
dihydroorotate dehydrogenase, neutral amino acid transporter
SLC1A5, glutamate dehydrogenase 1 (GDH1); glutaminase (GLS);
glutamate oxaloacetate transaminase 2(GOT2);
.gamma.-1-glutamyl-p-nitroanilide (GPNA); glutamate pyruvate
transaminase 2(GPT2); L-type amino acid transporter 1 (LAT1).
[0329] Additional targets for therapeutic, and/or diagnostic
purposes are BET (bromodomain and extra-terminal) proteins
including BRD2, BRD3, BRD4 and BRDT; kinesins including KIF11 (also
known as EG5) and centromere-associated protein E (CENPE);
surviving (an inhibitor of apoptosis protein), and prohibitin
including PHB1 and PHB2.
b. Cancers to be Treated
[0330] The types of cancer that can be treated with the provided
compositions and methods include, but are not limited to, the
following: bladder, brain, breast, cervical, colo-rectal,
esophageal, kidney, liver, lung, nasopharangeal, pancreatic,
prostate, skin, stomach, uterine, ovarian, testicular and the like.
Administration is not limited to the treatment of an existing
tumors but can also be used to prevent or lower the risk of
developing such diseases in an individual, i.e., for prophylactic
use. Potential candidates for prophylactic vaccination include
individuals with a high risk of developing cancer, i.e., with a
personal or familial history of certain types of cancer.
[0331] Malignant tumors which may be treated are classified herein
according to the embryonic origin of the tissue from which the
tumor is derived. Carcinomas are tumors arising from endodermal or
ectodermal tissues such as skin or the epithelial lining of
internal organs and glands. Sarcomas, which arise less frequently,
are derived from mesodermal connective tissues such as bone, fat,
and cartilage. The leukemias and lymphomas are malignant tumors of
hematopoietic cells of the bone marrow. Leukemias proliferate as
single cells, whereas lymphomas tend to grow as tumor masses.
Malignant tumors may show up at numerous organs or tissues of the
body to establish a cancer.
[0332] The cargo can be an anticancer agent for example
anti-proliferative agent, a pro-apoptotic agent, or other cytotoxic
agent, including, but not limited to, chemotherapeutic drugs and
functional nucleic acids.
c. Preferred Cargos
[0333] The preferred cargos for treating cancers are known in the
art and include, for example, anti-cancer agents and
immunotherapeutic agents.
[0334] Representative anti-cancer agents include, but are not
limited to, alkylating agents (such as cisplatin, carboplatin,
oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil,
dacarbazine, lomustine, carmustine, procarbazine, chlorambucil and
ifosfamide), antimetabolites (such as fluorouracil (5-FU),
gemcitabine, methotrexate, cytosine arabinoside, fludarabine, and
floxuridine), antimitotics (including taxanes such as paclitaxel
and decetaxel and vinca alkaloids such as vincristine, vinblastine,
vinorelbine, and vindesine), anthracyclines (including doxorubicin,
daunorubicin, valrubicin, idarubicin, and epirubicin, as well as
actinomycins such as actinomycin D), cytotoxic antibiotics
(including mitomycin, plicamycin, and bleomycin), topoisomerase
inhibitors (including camptothecins such as camptothecin,
irinotecan, and topotecan as well as derivatives of
epipodophyllotoxins such as amsacrine, etoposide, etoposide
phosphate, and teniposide), antibodies to vascular endothelial
growth factor (VEGF) such as bevacizumab (AVASTIN.RTM.), other
anti-VEGF compounds; thalidomide (THALOMID.RTM.) and derivatives
thereof such as lenalidomide (REVLIMID.RTM.); endostatin;
angiostatin; receptor tyrosine kinase (RTK) inhibitors such as
sunitinib (SUTENT.RTM.); tyrosine kinase inhibitors such as
sorafenib (Nexavar.RTM.), erlotinib (Tarceva.RTM.), pazopanib,
axitinib, and lapatinib; transforming growth factor-.alpha. or
transforming growth factor-.beta. inhibitors, and antibodies to the
epidermal growth factor receptor such as panitumumab
(VECTIBIX.RTM.) and cetuximab (ERBITUX.RTM.).
[0335] In some embodiments, the particles include nucleic acid
cargo, including, but not limited to functional nucleic acids,
expression constructs or mRNA, or a combination thereof. For
example, in some embodiments, a functional nucleic acid is designed
to reduce expression of an oncogene, for example a growth factor
(e.g., c-Sis), mitogen, receptor tyrosine kinase (e.g., EGFR,
PDGFR, VEGFR, HER2/neu), cytoplasmic tyrosine kinase (e.g., Src,
Syk-ZAP-70, BTK families) cytoplasmic serine/threonine kinases (or
a regulator subunit thereof) (e.g., Raf, cyclin-dependent kinases),
regulatory GTPases (e.g., Ras), transcription factors (e.g., myc),
angiogenesis (e.g., VEGF).
[0336] In some embodiments, the cargo is a functional nucleic acid
that targets a factor that contributes to chemotherapy resistance,
for example, drug efflux pumps, anti-apoptotic defense mechanisms,
etc. Specific targets include, but are not limited to, glycoprotein
(P-gp), Multidrug resistant protein 1(MRP-1), and B-cell lymphoma
(BCL-2). RNAi-chemotherapeutic drug combinations have also been
found to be effective against different molecular targets as well
and can increase the sensitization of cancer cells to therapy
several folds (Gandhi, et al., J Control Release. 2014 November 28;
0: 238-256).
[0337] Additionally or alternatively, mRNA can be introduced to
enhance the fight against the tumor. For example, in some
embodiments, the mRNA is delivered into the cancer cells. Such mRNA
can enhance apoptosis or sensitivity to drugs or other treatments
such as radiation.
[0338] Functional nucleic acids, mRNA, or a combination thereof can
be introduced into cells that induce, program, or activate
non-cancer cells to attack the cancer cells. For example, in some
embodiments, the cargo is a nucleic acid that primes T cells or
other immune cells for immunotherapy against the cancer.
Immunotherapeutic methods, including CAR T cell therapy and other
strategies for activation of immune cells against target antigens,
and inhibition of immune check points leading to T cell exhaustion,
anergy, or deactivation were well known in the art. The disclosed
particles can be used in vitro or in vivo to introduce nucleic
acids into targets including immune cells, to, for example,
increase antigen-specific proliferation of T cells, enhance
cytokine production by T cells, stimulate differentiation,
stimulate effector functions of T cells, promote T cell survival,
overcome T cell exhaustion, overcome T cell anergy or a combination
thereof. Immune cells, including but not limited to, neutrophils,
lymphocytes, dendritic cells, macrophages, eosinophils, natural
killer cells, can be the target of therapy.
2. Inflammation and Infection
[0339] Methods of treating inflammation and infection are provided.
The nanoparticles can be designed, for example, for release in the
microenvironment of inflammation, injury, and infection, or immune
or pro-inflammatory cells, or within immune or inflammatory cells
themselves. Suitable methods can include administering a subject an
effective amount of nanoparticles containing a therapeutic cargo to
reduce or alleviate one or more symptoms of the inflammation,
injury, or infection. The effect on the inflammation, injury, or
infection can be direct or indirect. Administration is not limited
to the treatment of an existing inflammation, injury, and
infection, but can also be used to prevent or lower the risk of
developing such diseases in an individual, i.e., for prophylactic
use. A characteristic feature of the inflammation is local
acidosis, which is attributed to the local increase of lactic-acid
production by the anaerobic, glycolytic activity of infiltrated
neutrophils and to the presence of short-chain, fatty acid
by-products of bacterial metabolism (Grinstein, et al., Clin.
Biochem. 24,241-247 (1991) and Ehrich, W. E. (1961) Inflammation
Allgower, M. eds. Progress in Surgery vol. 1,1-70 S. Karger Basel,
Switzerland). An acidic extracellular pH is also found in the
epidermis and plays an important protective role against bacterial
infection (Lardner, et al., Journal of Leukocyte Biology,
69(4):522-530 (2001)). As discussed above, local, tissue-specific
increase in tissue temperature can occur at site of inflammation,
injury, and infection. Similar to selectively targeting the tumor
microenvironment, the pH and temperature sensitive particles can be
utilized to delivery and selectively release cargo at sites of
inflammation, injury, and infection.
[0340] As with cancer, in addition or alternative to selectively
targeting cancer cells by targeting an acidic microenvironment, or
one with an elevated temperature, cancer cells or their
microenvironment can be specifically targeted relative to healthy
or normal cells by including a targeting moiety. Preferred
targeting domains target the molecule to areas of inflammation,
injury, or infection. Exemplary targeting domains are antibodies,
or antigen binding fragments thereof that are specific for inflamed
tissue or to a proinflammatory cytokine including but not limited
to IL17, IL-4, IL-6, IL-12, IL-21, IL-22, and IL-23. In the case of
neurological disorders such as Multiple Sclerosis, the targeting
domain may target the molecule to the CNS or may bind to VCAM-1 on
the vascular epithelium. Additional targeting domains can be
peptide aptamers specific for a proinflammatory molecule. In other
embodiments, the particles can include a binding partner specific
for a polypeptide displayed on the surface of an immune cell, for
example a T cell. In still other embodiments, the targeting domain
specifically targets activated immune cells. Preferred immune cells
that are targeted include Th0, Th1, Th17 and Th22 T cells, other
cells that secrete, or cause other cells to secrete inflammatory
molecules including, but not limited to, IL-1.beta., TNF-.alpha.,
TGF-beta, IFN-.gamma., IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs,
and Tregs. For example, a targeting domain for Tregs may bind
specifically to CD25.
[0341] In some embodiments, the target site is neutrophils, which
may phagocytize the particles to release a therapeutic and/or
diagnostic agent at the site of inflammation. Proteins
constitutively expressed on the surface of neutrophils that are
important for recognition of the endothelial inflammatory signals
include the glycoprotein P-selectin glycoprotein ligand-1 (PSGL-1)
and L-selectin.
[0342] Other agents to be targeted include those associated with
the disease. For example, a plaque targeted peptide can be one or
more of the following: Collagen IV, CREKA (SEQ ID NO:13), LyP-I,
CRKRLDRNC (SEQ ID NO:14), or their combinations at various molar
ratios.
[0343] In another embodiment, particles can contain a targeting
domain to target the molecule to an organ or tissue that is being
transplanted. For example, the targeting domain can be an antibody,
antigen binding fragment thereof, or another binding partner
specific for a polypeptide displayed on the surface of cells
specific to the type of organ or tissue being transplanted.
a. Inflammation
[0344] Inflammation is typically a localized physical condition in
which part of the body becomes reddened, swollen, hot, and often
painful, especially as a reaction to injury or infection.
Inflammation is a protective response that involves immune cells,
blood vessels, and molecular mediators, the purpose of which is to
eliminate the cause of cell injury, remove necrotic cells and
tissues damaged from the injury and the inflammatory process, and
to initiate tissue repair. The compositions can be used to treat
acute and chronic inflammation.
[0345] The inflammation can be caused by an infection such as those
described below or can be caused by a non-infectious mechanism. For
example, inflammation is associated with atherosclerosis, type III
hypersensitivity, trauma, and ischaemia. Inflammation can be
associated with autoimmune diseases, transplantation, graft verse
host disease, and conditions driven by immune responses. In some
embodiments, the particles are used to deliver a cargo for
treatment of an inflammatory or autoimmune disease or disorder such
as rheumatoid arthritis, systemic lupus erythematosus, alopecia
areata, anklosing spondylitis, antiphospholipid syndrome,
autoimmune Addison's disease, autoimmune hemolytic anemia,
autoimmune hepatitis, autoimmune inner ear disease, autoimmune
lymphoproliferative syndrome (alps), autoimmune thrombocytopenic
purpura (ATP), Behcet's disease, bullous pemphigoid,
cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome
immune deficiency, syndrome (CFIDS), chronic inflammatory
demyelinating polyneuropathy, cicatricial pemphigoid, cold
agglutinin disease, Crest syndrome, Crohn's disease, Dego's
disease, dermatomyositis, dermatomyositis--juvenile, discoid lupus,
essential mixed cryoglobulinemia, fibromyalgia--fibromyositis,
grave's disease, guillain-barre, hashimoto's thyroiditis,
idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura
(ITP), Iga nephropathy, insulin dependent diabetes (Type I),
juvenile arthritis, Meniere's disease, mixed connective tissue
disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris,
pernicious anemia, polyarteritis nodosa, polychondritis,
polyglancular syndromes, polymyalgia rheumatica, polymyositis and
dermatomyositis, primary agammaglobulinemia, primary biliary
cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome,
rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome,
stiff-man syndrome, Takayasu arteritis, temporal arteritis/giant
cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo,
and Wegener's granulomatosis.
[0346] Preferred cargos for treating inflammation and autoimmune
diseases include, but are not limited to, anti-inflammatory agents
and immunosuppressive agents.
[0347] In some embodiments, the cargo is an immunosuppressive
agents (e.g., antibodies against other lymphocyte surface markers
(e.g., CD40, alpha-4 integrin) or against cytokines), other fusion
proteins (e.g., CTLA-4-Ig (ORENCIA.RTM.), TNFR-Ig (Enbrel.RTM.)),
TNF-.alpha. blockers such as Enbrel, Remicade, Cimzia and Humira,
cyclophosphamide (CTX) (i.e. ENDOXAN.RTM., CYTOXAN.RTM.,
NEOSAR.RTM., PROCYTOX.RTM., REVIMMUNE.TM.), methotrexate (MTX)
(i.e. RHEUMATREX.RTM., TREXALL.RTM.), belimumab (i.e.
BENLYSTA.RTM.), or other immunosuppressive drugs (e.g., cyclosporin
A, FK506-like compounds, rapamycin compounds, or steroids),
anti-proliferatives, cytotoxic agents, or other compounds that may
assist in immunosuppression.
[0348] The cargo can function to inhibit or reduce T cell
activation and cytokine production. In one such embodiment, the
additional therapeutic agent is a CTLA-4 fusion protein, such as
CTLA-4 Ig (ABATACEPT.RTM.). CTLA-4 Ig fusion proteins compete with
the co-stimulatory receptor, CD28, on T cells for binding to
CD80/CD86 (B7-1/B7-2) on antigen presenting cells, and thus
function to inhibit T cell activation. In a preferred embodiment,
the additional therapeutic agent is a CTLA-4-Ig fusion protein
known as BELATACEPT.RTM.. BELATACEPT.RTM. contains two amino acid
substuitutions (L104E and A29Y) that markedly increase its avidity
to CD86 in vivo. In another embodiment, the additional therapeutic
agent is Maxy-4.
[0349] The cargo can treat chronic transplant rejection or GvHD,
whereby the treatment regimen effectively targets both acute and
chronic transplant rejection or GvHD. In a preferred embodiment the
second therapeutic is a TNF-.alpha. blocker.
[0350] The cargo can increase the amount of adenosine in the serum,
see, for example, WO 08/147482. In a preferred embodiment, the
second therapeutic is CD73-Ig, recombinant CD73, or another agent
(e.g. a cytokine or monoclonal antibody or small molecule) that
increases the expression of CD73, see for example WO 04/084933. In
another embodiment the second therapeutic agent is
Interferon-beta.
[0351] The cargo can increase Treg activity or production.
Exemplary Treg enhancing agents include but are not limited to
glucocorticoid fluticasone, salmeteroal, antibodies to IL-12,
IFN-.gamma., and IL-4; vitamin D3, and dexamethasone, and
combinations thereof. Antibodies to other proinflammatory molecules
can also be used. Preferred antibodies bind to IL-6, IL-23, IL-22
or IL-21.
[0352] The cargo can be a rapamycin compound. As used herein the
term "rapamycin compound" includes the neutral tricyclic compound
rapamycin, rapamycin derivatives, rapamycin analogs, and other
macrolide compounds which are thought to have the same mechanism of
action as rapamycin (e.g., inhibition of cytokine function). The
language "rapamycin compounds" includes compounds with structural
similarity to rapamycin, e.g., compounds with a similar macrocyclic
structure, which have been modified to enhance their therapeutic
effectiveness. Exemplary Rapamycin compounds are known in the art
(See, e.g. WO95122972, WO 95116691, WO 95104738, U.S. Pat. Nos.
6,015,809; 5,989,591; 5,567,709; 5,559,112; 5,530,006; 5,484,790;
5,385,908; 5,202,332; 5,162,333; 5,780,462; 5,120,727).
[0353] The language "FK506-like compounds" includes FK506, and
FK506 derivatives and analogs, e.g., compounds with structural
similarity to FK506, e.g., compounds with a similar macrocyclic
structure which have been modified to enhance their therapeutic
effectiveness. Examples of FK506-like compounds include, for
example, those described in WO 00101385. In some embodiments, the
language "rapamycin compound" does not include "FK506-like
compounds."
[0354] Other suitable therapeutics include, but are not limited to,
anti-inflammatory agents. The anti-inflammatory agent can be
non-steroidal, steroidal, or a combination thereof. Representative
examples of non-steroidal anti-inflammatory agents include, without
limitation, oxicams, such as piroxicam, isoxicam, tenoxicam,
sudoxicam; salicylates, such as aspirin, disalcid, benorylate,
trilisate, safapryn, solprin, diflunisal, and fendosal; acetic acid
derivatives, such as diclofenac, fenclofenac, indomethacin,
sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin,
acematacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac,
and ketorolac; fenamates, such as mefenamic, meclofenamic,
flufenamic, niflumic, and tolfenamic acids; propionic acid
derivatives, such as ibuprofen, naproxen, benoxaprofen,
flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen,
pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen,
tioxaprofen, suprofen, alminoprofen, and tiaprofenic; pyrazoles,
such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone,
and trimethazone. Mixtures of these non-steroidal anti-inflammatory
agents may also be employed.
[0355] Representative examples of steroidal anti-inflammatory drugs
include, without limitation, corticosteroids such as
hydrocortisone, hydroxyl-triamcinolone, alpha-methyl dexamethasone,
dexamethasone-phosphate, beclomethasone dipropionates, clobetasol
valerate, desonide, desoxymethasone, desoxycorticosterone acetate,
dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone
valerate, fluadrenolone, fluclorolone acetonide, fludrocortisone,
flumethasone pivalate, fluosinolone acetonide, fluocinonide,
flucortine butylesters, fluocortolone, fluprednidene
(fluprednylidene) acetate, flurandrenolone, halcinonide,
hydrocortisone acetate, hydrocortisone butyrate,
methylprednisolone, triamcinolone acetonide, cortisone,
cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate,
fluradrenolone, fludrocortisone, diflurosone diacetate,
fluradrenolone acetonide, medrysone, amcinafel, amcinafide,
betamethasone and the balance of its esters, chloroprednisone,
chlorprednisone acetate, clocortelone, clescinolone, dichlorisone,
diflurprednate, flucloronide, flunisolide, fluoromethalone,
fluperolone, fluprednisolone, hydrocortisone valerate,
hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone,
paramethasone, prednisolone, prednisone, beclomethasone
dipropionate, triamcinolone, and mixtures thereof.
[0356] In some embodiments, the cargo is a functional nucleic acid
that targets a factor that contributes to inflammation, the
activation or persistence of pro-inflammatory cells, a
pro-inflammatory response, active immune response, an autoimmune
response, etc. Specific targets include, for example,
pro-inflammatory molecules such as IL-1.beta., TNF-.alpha.,
TGF-beta, IFN-.gamma., IL-17, IL-6, IL-23, IL-22, IL-21, and
MMPs.
[0357] Additionally or alternatively, mRNA can be introduced to
reduce the inflammation or autoimmune response.
[0358] Functional nucleic acids, mRNA, or a combination thereof can
be introduced into cells that inhibit the development of naive T
cells into Th1, Th17, Th22 or other cells that secrete, or cause
other cells to secrete, inflammatory molecules. The cargo can
increase the number or activity of Tregs. The cargo can promote or
enhance production of IL-10 or another anti-inflammatory cytokine.
In some embodiments, the cargo enhances the differentiation,
recruitment and/or expansion of Treg cells in the region of
inflammation, autoimmune activity, or tissue engraftment. Exemplary
functional nucleic acid targets for treating autoimmune disease are
reviewed in Pauley and Cha, Pharmaceuticals 2013, 6(3), 287-294;
and discussed in, for example, Kim, et al., Molecular Therapy,
(2010) 18 5, 993-1001, Laroui, et al., Molecular Therapy (2014);
221, 69-80, Ponnappa, et al., Curr Opin Investig Drugs. 2009 May;
10(5):418-24; Abrams, et al., Molecular Therapy, (2010) 18 1,
171-180, Leuschner, et al., Nature biotechnology 29.11 (2011):
1005-1010. PMC. Web. 29 Mar. 2016.
[0359] In some embodiments, the cargo is a nucleic acid that
encodes an anti-inflammatory cytokine, for example, (IL)-1 receptor
antagonist, IL-4, IL-6, IL-10, IL-11, or IL-13 (Opal and DePalo, et
al., Chest. (2000) 117(4):1162-72).
b. Infections
[0360] Similarly, in some embodiments, the disclosed particles are
used to deliver a cargo for treatment of an infectious disease.
Infectious diseases that can be treated, prevented, and/or managed
using the disclosed nanoparticles can be caused by infectious
agents including but not limited to bacteria, fungi, protozae, and
viruses. Viral diseases include, for example, those caused by
hepatitis type A, hepatitis type B, hepatitis type C, influenza
(e.g., influenza A or influenza B), varicella, adenovirus, herpes
simplex type I (HSV-I), herpes simplex type II (HSV-II),
rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial
virus, papilloma virus, papova virus, cytomegalovirus, echinovirus,
arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,
rubella virus, polio virus, small pox, Epstein Barr virus, human
immunodeficiency virus type I (HIV-I), human immunodeficiency virus
type II (HIV-II), and agents of viral diseases such as viral
meningitis, encephalitis, dengue or small pox.
[0361] Bacterial diseases can be caused by bacteria (e.g.,
Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus,
Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans,
and Pseudomonas aeruginosa) include, for example, mycobacteria
rickettsia, mycoplasma, neisseria, S. pneumonia, Borrelia
burgdorferi (Lyme disease), Bacillus antracis (anthrax), tetanus,
streptococcus, staphylococcus, mycobacterium, pertissus, cholera,
plague, diptheria, chlamydia, S. aureus and legionella.
[0362] Protozoal diseases caused by protozoa include, for example,
leishmania, kokzidioa, trypanosome schistosoma or malaria.
Parasitic diseases caused by parasites include chlamydia and
rickettsia.
[0363] Fungal infections include, but are not limited to, Candida
infections, zygomycosis, Candida mastitis, progressive disseminated
trichosporonosis with latent trichosporonemia, disseminated
candidiasis, pulmonary paracoccidioidomycosis, pulmonary
aspergillosis, Pneumocystis carinii pneumonia, cryptococcal
meningitis, coccidioidal meningoencephalitis and cerebrospinal
vasculitis, Aspergillus niger infection, Fusarium keratitis,
paranasal sinus mycoses, Aspergillus fumigatus endocarditis, tibial
dyschondroplasia, Candida glabrata vaginitis, oropharyngeal
candidiasis, X-linked chronic granulomatous disease, tinea pedis,
cutaneous candidiasis, mycotic placentitis, disseminated
trichosporonosis, allergic bronchopulmonary aspergillosis, mycotic
keratitis, Cryptococcus neoformans infection, fungal peritonitis,
Curvularia geniculata infection, staphylococcal endophthalmitis,
sporotrichosis, and dermatophytosis.
[0364] Prepared cargo for treating infections can including
anti-infectives such as Difloxacin Hydrochloride; Lauryl
Isoquinolinium Bromide; Moxalactam Disodium; Ornidazole;
Pentisomicin; Sarafloxacin Hydrochloride; Protease inhibitors of
HIV and other retroviruses; Integrase Inhibitors of HIV and other
retroviruses; Cefaclor (Ceclor); Acyclovir (Zovirax); Norfloxacin
(Noroxin); Cefoxitin (Mefoxin); Cefuroxime axetil (Ceftin); or
Ciprofloxacin (Cipro).
[0365] In some embodiments, the cargo is antibiotic such as a
beta-lactam (e.g., penicillins, cephalosporins, monobactams, and
carbapenems), a cephalosporins, a monobactam, a carbapenem, a
macrolide, a lincosamide, streptogramin, an aminoglycoside, a
quinolone, a sulfonamide, a tetracycline, a glyopeptide,
lipoglycopeptide, rifamycin, a polypeptide, or a
tuberactinomycin.
[0366] In some embodiments, the cargo is a functional nucleic acid
that targets a factor that contributes to anti-infective drug
resistance, for example, drug efflux pumps, anti-apoptotic defense
mechanisms, etc., or infected cells or the pathogens themselves. In
some embodiments, the functional nucleic acid specifically targets
a gene expressed by the pathogen. See, for example, Fischer, et
al., Cell Research, (2004) 14, 460-466, which describes RNAi
strategies for targeting viral infection. Additionally or
alternatively, mRNA can be introduced to enhance the fight against
the infection.
[0367] As in described above in the context of cancer, functional
nucleic acids, mRNA, or a combination thereof can be introduced
into cells that induce, program, or activate cells to resolve an
infection. For example, in some embodiments, the cargo is a nucleic
acid that primes T cells or other immune cells for immunotherapy
against the infection. Immunotherapeutic methods, including CAR T
cell therapy and other strategies for activation of immune cells
against target antigens, and inhibition of immune check points
leading to T cell exhaustion, anergy, or deactivation were well
known in the art. The disclosed particles can be used in vitro or
in vivo to introduce nucleic acids into targets including immune
cells, to, for example, increase antigen-specific proliferation of
T cells, enhance cytokine production by T cells, stimulate
differentiation, stimulate effector functions of T cells, promote T
cell survival, overcome T cell exhaustion, overcome T cell anergy
or a combination thereof. Immune cells, including but not limited
to, neutrophils, lymphocytes, dendritic cells, macrophages,
eosinophils, natural killer cells, can be the target of
therapy.
[0368] The present invention will be further understood by
reference to the following non-limiting examples.
Example 1: Ultra pH-Responsive and Tumor-Penetrating Nanoplatform
for Targeted siRNA Delivery with Robust Anti-Cancer Efficacy
Methods and Materials
Materials
[0369] Methoxyl-polyethylene glycol (Meo-PEG.sub.113-OH) and
hydroxyl polyethylene glycol carboxylic acid (HO-PEG.sub.113-COOH)
were purchased from JenKem Technology and used as received.
Internalizing RGD (iRGD) with the sequence CRGDRGPDC (SEQ ID NO:11)
was obtained from GL Biochem Ltd. 2-(Diisopropyl amino) ethyl
methacrylate (DPA-MA), glycidyl methacrylate (GMA), and methyl
methacrylate (MMA) were provided by Sigma-Aldrich and passed over
an alumina column before use in order to remove the hydroquinone
inhibitors. .alpha.-Bromoisobutyryl bromide, triethylamine (TEA),
N,N,N',N',N'-pentamethyldiethylenetriamine (PMDETA), copper (I)
bromide (CuBr), N,N'-dimethylformamide (DMF),
tetraethylenepentamine (TEPA), 1,2-epoxyhexadecane, isopropyl
alcohol, and dichloromethane (DCM) were acquired from Sigma-Aldrich
and used directly. Lipofectamine 2000 (Lipo2K) was purchased from
Invitrogen. Steady-Glo luciferase assay system was provided by
Promega. GL3, fluorescent dye (DY547, DY647 and DY677) labeled GL3
and survivin siRNAs were acquired from Dharmacon. The siRNA
sequences are as follows: GL3 siRNA, 5'-CUU ACG CUG AGU ACU UCG
AdTdT-3' (sense) (SEQ ID NO:1) and 5'-UCG AAG UAC UCA GCG UAA
GdTdT-3' (antisense)) (SEQ ID NO:2); survivin siRNA, 5'-GGA CCA CCG
CAU CUC UAC AdTdT-3' (sense) (SEQ ID NO:3) and 5'-UGU AGA GAU GCG
GUG GCU CdTdT-3' (antisense) (SEQ ID NO:4). PHB1 siRNA, 5'-GCG ACG
ACC UUA CAG AGC GUU-3' (sense) (SEQ ID NO:5) and 5'-CGC UCU GUA AGG
UCG UCG CUU-3' (antisense) (SEQ ID NO:6); KIF11 siRNA, 5'-GAA UAG
GGU UAC AGA GUU GUU-3' (sense) (SEQ ID NO:7) and 5'-CAA CUC UGU AAC
CCU AUU CUU-3' (antisense) (SEQ ID NO:8). The fluorescent dyes
DY547 and DY647 were labeled at the 5'-end of the sense strand of
GL3 siRNA. DY677 was labeled at the 5'-end of both the sense and
antisense strands of GL3 siRNA. HeLa cells stably expressing
firefly and Renilla luciferase (Luc-HeLa) were obtained from
Alnylam Pharmaceuticals, Inc. The cells were incubated in RPMI-1640
medium (Invitrogen) with 10% fetal bovine serum (FBS,
Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich). All
other reagents and solvents are of analytical grade and used
without further purification.
Synthesis of Meo-PEG-Br and Br-PEG-COOH
[0370] Meo-PEG.sub.113-OH (8 g, 1.6 mmol) and TEA (1.3 mL, 9.6
mmol) were dissolved in 250 mL of DCM. In an ice-salt bath,
.alpha.-bromoisobutyryl bromide (1 mL, 8 mmol) dissolved in 10 mL
of DCM was added dropwise. After stirring for 24 h, the mixture was
washed with 1 M NaOH (3.times.50 mL), 1 M HCl (3.times.50 mL), and
deionized water (3.times.50 mL), respectively. After drying over
anhydrous MgSO.sub.4, the solution was concentrated, and cold ether
was added to precipitate the product. After re-precipitation
thrice, the product was collected as white powder after drying
under vacuum. The synthesis of Br-PEG-COOH was carried out
according to a method similar to that described above, by changing
Meo-PEG.sub.113-OH with HO-PEG.sub.113-COOH. The synthesis scheme
of Br-PEG-COOH is shown below.
Synthesis of methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl methacrylate)
(Meo-PEG-b-P(DPA-co-GMA))
[0371] Meo-PEG-b-P(DPA-co-GMA) copolymers with different
compositions were synthesized by atom transfer radical
polymerization (ATRP). Meo-PEG.sub.113-b-P(DPA.sub.80-co-GMA.sub.5)
is used as an example to illustrate the procedure. DPA-MA (2.6 g,
12 mmol), GMA (0.11 g, 0.75 mmol), Meo-PEG-Br (0.75 g, 0.15 mmol),
and PMDETA (31.5 .mu.L, 0.15 mmol) were added to a polymerization
tube. DMF (3 mL) and 2-propanol (3 mL) were then added to dissolve
the monomer and initiator. After three cycles of freeze-pump-thaw
to remove oxygen, CuBr (21.6 mg, 0.15 mmol) was added under
nitrogen atmosphere and the polymerization tube was sealed under
vacuum. After polymerization at 40.degree. C. for 24 h,
tetrahydrofuran (THF) was added to dilute the product, which was
then passed through a neutral Al.sub.2O.sub.3 column to remove the
catalyst. The resulting THF solution was concentrated and the
residue was dialyzed against THF, followed by deionized water. The
expected copolymer was collected as a white powder after
freeze-drying under vacuum. The synthesis scheme is shown below.
The feed compositions of the copolymers are summarized in Table
1.
TABLE-US-00001 TABLE 1 Feed compositions and characterizations of
Meo-PEG-b-P(DPA-co-GMA) Repeat unit Repeat unit M.sub.n, GPC
M.sub.n, NMR No. (DPA) a (GMA) a (.times.10.sup.-4 Da) b PDI b
(.times.10.sup.-4 Da) a pKa c PDPA40-GMA5 39 5 1.44 1.19 1.42 6.34
PDPA50-GMA5 50 5 1.68 1.12 1.66 6.31 PDPA60-GMA5 58 5 1.69 1.18
1.83 6.29 PDPA70-GMA5 69 5 1.94 1.24 2.06 6.26 PDPA80-GMA5 80 5
2.19 1.29 2.29 6.24 PDPA100-GMA5 99 5 2.87 1.14 2.71 6.21 a
Determined by .sup.1HNMR using CDCl.sub.3 as solvent. b
Number-averaged (Mn) and polydispersity index (PDI) were determined
by GPC using THF as the eluent.
Synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA)
[0372] Meo-PEG-b-P(DPA-co-GMA-TEPA) was synthesized via the ring
opening reaction between TEPA and the epoxy group of GMA repeating
unit. In brief, Meo-PEG-b-P(DPA-co-GMA) (1.5 g) dissolved in DMF
(20 mL) was added dropwise to the DMF solution (5 mL) of TEPA
(30-fold molar excess relative to the GMA repeating unit). After
reaction at 60.degree. C. for 7 h, the mixture was transferred to a
dialysis tube and then dialyzed against deionized water. The
Meo-PEG-b-P(DPA-co-GMA-TEPA) was finally collected as a white
powder after freeze-drying under vacuum. The synthesis route of
Meo-PEG-b-P(DPA-co-GMA-TEPA) is shown below.
Synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA-C14)
[0373] Meo-PEG-b-P(DPA-co-GMA-TEPA) (1 g) and 1,2-epoxyhexadecane
(equal molar amount relative to TEPA repeating unit) were dissolved
in DMF (20 mL) and the solution was stirred at 70.degree. C. for 5
h. Subsequently, the solution was transferred to a dialysis tube
and then dialyzed against DMF, followed by deionized water. The
Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) was obtained as a white powder
after freeze-drying under vacuum. The detailed synthesis of
Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) is shown below. Synthesis of
Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5)
[0374] Meo-PEG-b-P(DPA-co-GMA-TEPA) (0.2 g) and Cy5.5 NHS ester
(1.5-fold molar excess relative to the TEPA repeating unit) were
well dissolved in 5 mL of THF. After constantly stirring in dark
for 48 h, the solution was dialyzed against deionized water and the
product was collected after freeze-drying. The synthesis of
Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5) is shown below.
Synthesis Scheme of Meo-PEG-b-P(DPA-Co-GMA-TEPA-Cy5.5)
##STR00001##
[0375] Synthesis of HOOC-PEG-b-PDPA
[0376] HOOC-PEG-b-PDPA copolymers were also synthesized by the ATRP
method. DPA-MA (1.73 g, 8 mmol), Br-PEG-COOH (0.5 g, 0.1 mmol), and
PMDETA (21 .mu.L, 0.1 mmol) were added to a polymerization tube.
Subsequently, DMF (2 mL) and 2-propanol (2 mL) were added to
dissolve the monomer and initiator. After three cycles of
freeze-pump-thaw to remove oxygen, CuBr (14.4 mg, 0.1 mmol) was
added under nitrogen atmosphere and the polymerization tube was
sealed under vacuum. After polymerization at 40.degree. C. for 24
h, tetrahydrofuran (THF) was added to dilute the product, which was
then passed through a neutral Al.sub.2O.sub.3 column to remove the
catalyst. The obtained THF solution was concentrated and the
residue was dialyzed against deionized water. The HOOC-PEG-b-PDPA
was obtained as a white powder after freeze-drying under vacuum.
The synthesis scheme is shown below. The feed compositions are
summarized in Table 2.
TABLE-US-00002 TABLE 2 Feed compositions and characterizations of
HOOC-PEG-b- PDPA Repeat unit M.sub.n,GPC M.sub.n,NMR No. (DPA) a
(.times.10.sup.-4 ) b PDI b (.times.10.sup.-4 Da) a
HOOC-PEG-.sub.b-PDPA.sub.40 36 1.31 1.34 1.27
HOOC-PEG-.sub.b-PDPA.sub.50 45 1.49 1.28 1.48
HOOC-PEG-.sub.b-PDPA.sub.60 55 1.76 1.29 1.69
HOOC-PEG-.sub.b-PDPA.sub.70 64 1.92 1.27 1.89
HOOC-PEG-.sub.b-PDPA.sub.80 76 2.04 1.24 2.14
HOOC-PEG-.sub.b-PDPA.sub.100 92 2.57 1.19 2.48 a Determined by
.sup.1HNMR using CDCl.sub.3 as solvent. b Number-averaged (Mn) and
polydispersity index (PDI) were determined by GPC using THF as the
eluent.
Synthesis of iRGD-PEG-b-PDPA
[0377] HOOC-PEG-b-PDPA copolymer (0.2 g), iRGD peptide (1.5-fold
molar excess relative to the terminal carboxylic acid group),
EDC.HCl (3-fold molar excess relative to the terminal carboxylic
acid group), and NHS (3-fold molar excess relative to the terminal
carboxylic acid group) were well dissolved in pH 5.0 water. The
mixture was stirred at room temperature for 48 h. The solution was
subsequently dialyzed against deionized water and the expected
iRGD-PEG-PDPA was collected after freeze-drying.
Synthesis Scheme of iRGD-PEG-b-PDPA
##STR00002##
[0378] Synthesis of Control Copolymers
[0379] The control copolymers, methoxyl-polyethylene glycol-b-poly
(methyl methacrylate-co-glycidyl methacrylate)
(Meo-PEG.sub.113-b-P(MMA.sub.80-co-GMA.sub.5))
Meo-PEG.sub.113-b-P(MMA.sub.80-co-GMA.sub.5-TEPA.sub.5),
HOOC-PEG.sub.113-b-PMMA.sub.80, iRGD-PEG.sub.113-b-PMMA.sub.80, and
Meo-PEG.sub.113-b-P(MMA.sub.80-co-GMA.sub.5-TEPA.sub.5-C14) were
synthesized according to the method described above, by changing
the monomer DPA-MA with MMA. The chemical structure of
iRGD-PEG.sub.113-b-PMMA.sub.80 and
Meo-PEG.sub.113-b-P(MMA.sub.80-co-GMA.sub.5-TEPA.sub.5-C14) is
shown below.
##STR00003##
[0380] Gel Permeation Chromatography (GPC)
[0381] Number- and weight-average molecular weights (M.sub.n and
M.sub.w, respectively) of the polymers were determined by a gel
permeation chromatographic system equipped with a Waters 2690D
separations module and a Waters 2410 refractive index detector. THF
was used as the eluent at a flow rate of 0.3 mL/min. Waters
millennium module software was used to calculate molecular weight
on the basis of a universal calibration curve generated by
polystyrene standard of narrow molecular weight distribution.
[0382] .sup.1H Nuclear magnetic resonance (.sup.1HNMR)
[0383] The .sup.1HNMR spectra of the polymers were recorded on a
Mercury VX-300 spectrometer at 400 MHz (Varian, USA), using
CDCl.sub.3 as a solvent and TMS as an internal standard.
[0384] Acid-Base Titration
[0385] Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) was dispersed in deionized
water, and a concentrated HCl aqueous solution was added until
complete dissolution of the copolymer (1 mg/mL). Subsequently, 1 M
NaOH aqueous solution was added in 1-5 .mu.L increments. After each
addition, the solution was constantly stirred for 3 min, and the
solution pH was measured using a pH meter. The pK.sub.a of the
copolymer was determined as the pH at which 50% copolymer turns
ionized.
Preparation and Characterization of Nanoparticles (NPs)
[0386] Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) was dissolved in THF to
form a homogenous solution with a concentration of 4 mg/mL.
Subsequently, a certain volume of this THF solution was taken and
mixed with 1 nmol siRNA (0.1 nmol/4 aqueous solution) in a N/P
molar ratio of 40:1. Under vigorous stirring (1000 rpm), the
mixture was added dropwise to 2.5 mL of deionized water. The NP
dispersion formed was transferred to an ultrafiltration device (EMD
Millipore, MWCO 100 K) and centrifuged to remove the organic
solvent and free compounds. After washing with PBS (pH 7.4)
solution (3.times.5 mL), the siRNA loaded NPs were dispersed in 1
mL of phosphate buffered saline (PBS, pH 7.4) solution. Size and
zeta potential were determined by dynamic light scattering (DLS,
Brookhaven Instruments Corporation). The morphology of NPs was
visualized on a Tecnai G2 Spirit BioTWIN transmission electron
microscope (TEM). Before observation, the sample was stained with
1% uranyl acetate and dried under air. To determine siRNA
encapsulation efficiency, DY547-labelled GL3 siRNA loaded NPs were
prepared according to the method described above. A small volume
(50 .mu.L) of the NP solution was withdrawn and mixed with 20-fold
DMSO. The fluorescence intensity of DY547-labelled GL3 siRNA was
measured using a Synergy HT multi-mode microplate reader (BioTek
Instruments) and compared to the free DY547-labelled GL3 siRNA
solution (1 nmol/mL PBS solution).
[0387] To prepare the iRGD-NPs, Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) (4
mg/mL in THF) was mixed with 1 nmol siRNA (0.1 nmol/4 aqueous
solution) in a N/P molar ratio of 40:1. Then iRGD-PEG-b-PDPA (4
mg/mL in THF, 10 mol % compared to
Meo-PEG-b-P(DPA-co-GMA-TEPA-C14)) was added, and the mixture was
added dropwise to 2.5 mL of deionized water. The iRGD-NPs were
purified by an ultrafiltration device (EMD Millipore, MWCO 100 K)
and finally dispersed in 1 mL of PBS. The siRNA encapsulation
efficiency was examined by replacing the siRNA with DY547-labelled
GL3 siRNA.
[0388] Evaluation of pH Responsiveness
[0389] The THF solution of Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) (4
mg/mL) and Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5) (4 mg/mL) was mixed
in a volume ratio of 8:2. Under vigorously stirring (1000 rpm), 0.5
mL of the mixture was added dropwise to 5 mL of deionized water.
After collection and purification by an ultrafiltration device (EMD
Millipore, MWCO 100 kDa), the NPs formed were dispersed in 1 mL of
deionized water. Subsequently, 1 M NaOH or HCl was added in 1-5
.mu.L increments, and the fluorescence intensity of the NPs was
measured on a Synergy HT multi-mode microplate reader. The
normalized fluorescence intensity (NFI) vs. pH profile was used to
quantitatively assess the pH responsiveness. NFI is calculated as
follows:
NFI=(F-/(F.sub.max-F.sub.mm)
[0390] where F is the fluorescence intensity of the NPs at any
given pH value and F.sub.max and F.sub.min are the maximal and
minimal fluorescence intensity of the NPs, respectively.
[0391] In Vitro siRNA Release
[0392] DY547-labelled GL3 siRNA-loaded NPs were prepared as
described above. Subsequently, the NPs were dispersed in 1 mL of
PBS (pH 7.4) and then transferred to a Float-a-lyzer G2 dialysis
device (MWCO 100 kDa, Spectrum) that was immersed in PBS (pH 7.4)
at 37.degree. C. At a predetermined interval, 5 .mu.L, of the NP
solution was withdrawn and mixed with 20-fold DMSO. The
fluorescence intensity of DY547-labelled siRNA was determined by
Synergy HT multi-mode microplate reader.
[0393] Cell Culture
[0394] Human cervical cancer cell line with the expression of
luciferase (Luc-HeLa) and prostate cancer cell line (PC3) were
incubated in RPMI1640 medium with 10% FBS at 37.degree. C. in a
humidified atmosphere containing 5% CO.sub.2.
[0395] Luciferase Silencing.
[0396] Luc-HeLa cells were seeded in 96-well plates (5,000 cells
per well) and incubated in 0.1 mL of RPMI1640 medium with 10% FBS
for 24 h. Thereafter, the GL3 siRNA-loaded NPs were added. After 24
h incubation, the cells were washed with fresh medium and allowed
to incubate for another 48 h. The expression of firefly luciferase
in HeLa cells was determined using Steady-Glo luciferase assay
kits. Cytotoxicity was measured using alamarBlue assay according to
the manufacturer's protocol. The luminescence or fluorescence
intensity was measured using a microplate reader, and the average
value of three independent experiments was collected. As a control,
the silencing effect of Lipo2K/GL3 siRNA complexes was also
evaluated according to the procedure described above and compared
to that of GL3 siRNA-loaded NPs.
[0397] Determination of the Expression of Integrins
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5
[0398] Luc-HeLa and PC3 cells were seeded in 6-well plates (50,000
cells per well) and incubated in 1 mL of RPMI1640 medium containing
10% FBS for 24 h. Thereafter, 10 .mu.L of FITC-conjugated
anti-human CD51/61 antibody (BioLegend) or FITC-conjugated
anti-human integrin .alpha..sub.v.beta..sub.5 antibody (EMD
Millipore) were added, and the cells were allowed to incubate for
another 4 h. After removing the medium and washing with PBS (pH
7.4) solution thrice, the cells were collected for flow cytometry
quantitative analysis (BD FACSAria.TM. III, USA).
[0399] Confocal Laser Scanning Microscope (CLSM)
[0400] Luc-HeLa and PC3 cells (20,000 cells) were seeded in discs
and incubated in 1 mL of RPMI1640 medium containing 10% FBS for 24
h. Subsequently, the DY547-labelled GL3 siRNA-loaded NPs or
iRGD-NPs were added, and the cells were allowed to incubate for 1
or 4 h. After removing the medium and subsequently washing with PBS
(pH 7.4) solution thrice, the endosomes and nuclei were stained by
lysotracker green and Hoechst 33342, respectively. The cells were
then viewed under a FV1000 CLSM (Olympus).
[0401] Flow Cytometry
[0402] Luc-HeLa and PC3 cells were seeded in 6-well plates (50,000
cells per well) and incubated in 1 mL of RPMI1640 medium containing
10% FBS for 24 h. Subsequently, the DY547-labelled GL3 siRNA-loaded
NPs or iRGD-NPs were added, and the cells were allowed to incubate
for another 4 h. After removing the medium and subsequently washing
with PBS (pH 7.4) solution thrice, the cells were collected for
flow cytometry quantitative analysis.
[0403] In Vitro Survivin Silencing
[0404] PC3 cells were seeded in 6-well plates (50,000 cells per
well) and incubated in 1 mL of RPMI1640 medium containing 10% FBS
for 24 h. Subsequently, the cells were transfected with the
survivin siRNA-loaded NPs or iRGD-NPs for 24 h. After washing the
cells with PBS thrice, the cells were further incubated in fresh
medium for another 48 h. Thereafter, the cells were digested by
trypsin and the proteins were extracted using modified
radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl pH 7.4,
150 mM NaCl, 1% NP-40 substitute, 0.25% sodium deoxycholate, 1 mM
sodium fluoride, 1 mM Na.sub.3VO.sub.4, 1 mM EDTA), supplemented
with protease inhibitor cocktail and 1 mM phenylmethanesulfonyl
fluoride (PMSF). The expression of survivin was examined using the
western blot analysis described below.
[0405] Western Blot Analysis
[0406] Equal amounts of protein, as determined with a bicinchoninic
acid (BCA) protein assay kit (Pierce/Thermo Scientific) according
to the manufacturer's instructions, were added to SDS-PAGE gels and
separated by gel electrophoresis. After transferring the proteins
from gel to polyvinylidene difluoride membrane, the blots were
blocked with 3% BSA in TBST (50 mM Tris-HCl pH 7.4, 150 mM NaCl,
and 0.1% Tween 20) and then incubated with a mixture of survivin
rabbit antibody (Cell Signaling) and beta-actin rabbit antibody
(Cell Signaling). The expression of survivin was detected with
horseradish peroxidase (HRP)-conjugated secondary antibody
(anti-rabbit IgG HRP-linked antibody, Cell Signaling) and an
enhanced chemiluminescence (ECL) detection system (Pierce).
[0407] In Vitro Cell Proliferation
[0408] PC3 cells were seeded in 6-well plates (20,000 cells per
well) and incubated in 1 mL of RPMI1640 medium containing 10% FBS
for 24 h. Thereafter, the cells were transfected with the survivin
siRNA-loaded NPs or iRGD-NPs for 24 h and then washed with fresh
medium for further incubation. At predetermined intervals, the
cytotoxicity was measured by alamarBlue assay according to the
manufacturer's protocol. After each measurement, the alamarBlue
agent was removed and the cells were incubated in fresh medium for
further proliferation.
[0409] Animals
[0410] Healthy male BALB/c mice (4-5 weeks old) were purchased from
Charles River Laboratories. All in vivo studies were performed in
accordance with National Institutes of Health animal care
guidelines and in strict pathogen-free conditions in the animal
facility of Brigham and Women's Hospital. Animal protocol was
approved by the Institutional Animal Care and Use Committees on
animal care (Harvard Medical School).
[0411] PC3 Xenograft Tumor Model
[0412] The tumor model was constructed by subcutaneous injection
with 200 .mu.L of PC3 cell suspension (a mixture of RPMI 1640
medium and Matrigel in 1:1 volume ratio) with a density
1.times.10.sup.7 cells/mL into the back region of healthy male
BALB/c nude mice. When the volume of the PC3 tumor xenograft
reached .about.100 mm.sup.3, the mice were used for the following
in vivo experiments.
[0413] Pharmacokinetics Study
[0414] Healthy male BALB/c mice were randomly divided into three
groups (n=3) and given an intravenous injection of either (i) free
DY647-labelled GL3 siRNA, (ii) DY647-labelled GL3 siRNA-loaded NPs,
or (iii) DY647-labelled GL3 siRNA-loaded iRGD-NP at 650 .mu.g siRNA
dose per kg mouse weight. At predetermined time intervals, orbital
vein blood (20 .mu.L) was withdrawn using a tube containing
heparin, and the wound was pressed for several seconds to stop the
bleeding. The fluorescence intensity of DY647-labelled siRNA in the
blood was determined by microplate reader. The blood circulation
half-life (t1/2) was calculated by first-order decay fit.
[0415] Biodistribution
[0416] PC3 tumor-bearing male BALB/c nude mice were randomly
divided into three groups (n=3) and given an intravenous injection
of either (i) free DY677-labelled GL3 siRNA, (ii) DY677-labelled
GL3 siRNA-loaded NPs or (iii) DY677-labelled GL3 siRNA-loaded
iRGD-NPs at 650 .mu.g siRNA dose per kg mouse weight. Twenty-four
hours after the injection, the mice were imaged using the Maestro 2
In-Vivo Imaging System (Cri Inc). Organs and tumors were then
harvested and imaged. To quantify the accumulation of NPs in tumors
and organs, the fluorescence intensity of each tissue was
quantified by Image-J.
[0417] Immunofluorescence Staining
[0418] PC3 tumor-bearing male BALB/c nude mice were randomly
divided into three groups (n=3) and intravenously injected with
either (i) free DY677-labelled GL3 siRNA, (ii) DY677-labelled GL3
siRNA-loaded NPs or (iii) DY677-labelled GL3 siRNA-loaded iRGD-NPs
at 650 .mu.g siRNA dose per kg mouse weight. Four hours after
injection, the mice were sacrificed and the tumors were harvested,
followed by fixing with 4% paraformaldehyde, embedding in paraffin,
and cutting into sections. To image the tumor vasculature, the
slices were heated at 60.degree. C. for 1 h and washed with xylene,
ethanol, and PBS thrice. After blocking with 10% FBS for 1.5 h, the
slices were incubated with rat anti-mouse CD31 antibody (Abcam) at
4.degree. C. for 1 h. After washing with PBS/0.2% triton X-100
thrice, Alexa Flour 488-conjugated secondary antibody (Goat
anti-rat IgG, Abcam) was added for 1 h to stain the slices.
Thereafter, the slices were washed with PBS thrice and then stained
with Hoechst 33342. The images of the tumor vasculature were viewed
on a FLV1000 CLSM.
[0419] In Vivo Survivin Silencing
[0420] PC3 tumor-bearing male BALB/c nude mice were randomly
divided into two groups (n=3) and intravenously injected with (i)
survivin siRNA-loaded NPs or (ii) survivin siRNA-loaded iRGD-NPs
for three consecutive days. Twenty-four hours after the final
injection, mice were sacrificed and tumors were harvested. The
proteins in the tumor were extracted using modified
radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl pH 7.4,
150 mM NaCl, 1% NP-40 substitute, 0.25% sodium deoxycholate, 1 mM
sodium fluoride, 1 mM Na.sub.3VO.sub.4, 1 mM EDTA), supplemented
with protease inhibitor cocktail and 1 mM phenylmethanesulfonyl
fluoride (PMSF). The expression of survivin was examined using the
aforementioned western blot analysis.
[0421] Inhibition of Tumor Growth
[0422] PC3 tumor-bearing male BALB/c nude mice were randomly
divided into four groups (n=5) and intravenously injected with (i)
PBS, (ii) GL3 siRNA-loaded NPs, (iii) survivin siRNA-loaded NPs or
(iv) survivin siRNA-loaded iRGD-NPs at 650 .mu.g siRNA dose per kg
mouse weight once every two days. All the mice were administrated
by administered five consecutive injections and the tumor growth
was monitored every two days by measuring perpendicular diameters
using a caliper and tumor volume was calculated as follows:
V=W.sup.2.times.L/2
[0423] where W and L are the shortest and longest diameters,
respectively.
[0424] Histology
[0425] Healthy male BALB/c mice were randomly divided into three
groups (n=3) and administered daily intravenous injections of
either (i) PBS, (ii) survivin siRNA-loaded NPs or (iii) survivin
siRNA-loaded iRGD-NPs at 650 siRNA dose per kg mouse weight. After
three consecutive injections, the main organs were collected 2 days
post the final injection, fixed with 4% paraformaldehyde, and
embedded in paraffin. Tissue sections were stained with H&E and
viewed under optical microscope.
Results
[0426] A long-circulating, optionally cell-penetrating, and
stimuli-responsive NP platform for effective in vivo delivery of
therapeutic, prophylactic and/or diagnostic agents is made of an
amphiphilic polymer, most preferably a PEGylated polymer, which
shows a response to a stimulus such as pH, temperature, or light,
such as an ultra pH-responsive characteristic with a pKa close to
the endosomal pH (6.0-6.5) (Wang Y et al, Nat Mater, 13, 204-212
(2014)). The polymer may include a targeting or cell penetrating or
adhesion molecule such as a tumor-penetrating peptide iRGD (FIGS.
1A-1B).
[0427] As demonstrated by example 1, after encapsulating the
agent(s) to be delivered, the resulting delivery system shows four
unique features (FIG. 1C):
[0428] i) the surface-encoded iRGD peptide endows the NPs with
tumor-targeting and tumor-penetrating abilities;
[0429] ii) the hydrophilic PEG shells prolong the blood
circulation;
[0430] iii) a small population of cationic lipid-like grafts
randomly dispersed in the hydrophobic poly(2-(diisopropylamino)
ethylmethacrylate) (PDPA) segment can entrap siRNA in the
hydrophobic cores of the NPs; and iv) the rapid protonation of the
ultra pH-responsive PDPA segment induces the endosomal swelling via
the "proton sponge" effect, which synergizes with the insertion of
the cationic lipid-like grafts into endosomal membrane to induce
membrane destabilization (Zhu X et al., Proceedings of the National
Academy of Sciences, 112, 7779-7784 (2015)) and efficient endosomal
escape.
[0431] The amphiphilic polymer, methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl methacrylate)
(Meo-PEG-b-P(DPA-co-GMA)) was first synthesized (Table 1), which
was further grafted by tetraethylenepentamine (TEPA) and
1,2-epoxyhexadecane to obtain Meo-PEG-b-P(DPA-co-GMA-TEPA-C14).
Synthesis Scheme of Meo-PEG-b-P(DPA-co-GMA-TEPA-C14)
##STR00004##
[0433] The length of PDPA segment was varied to adjust siRNA
encapsulation efficiency (EE %). As the PDPA length increases, the
EE % and size of the resulting NPs increase (Table 3), possibly
because the increased PDPA length leads to an increase in the size
of the hydrophobic core. Specifically, the EE % reaches almost 100%
for the polymer with 80 (PDPA80) or 100 (PDPA100) DPA repeat units.
Notably, using a mixture of Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) (90
mol %) and tumor-penetrating polymer (iRGD-PEG-b-PDPA, 10 mol %,
FIG. 1A) to prepare NPs does not cause obvious change in the EE %
or particle size (Table 4).
TABLE-US-00003 TABLE 3 Size, zeta potential, siRNA encapsulation
efficiency (EE %), and pH responsiveness of the NPs prepared from
Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) Polymer DPA repeating pKa of Size
Zeta potential .DELTA.pH No. abbreviation units a polymer b (nm) c
(mv) EE % d 10%-90% NPs40 PDPA40 39 6.34 62.5 4.79 54.6 0.45 NPs50
PDPA50 50 6.31 69.6 5.26 59.6 0.40 NPs60 PDPA60 58 6.29 75.9 3.13
65.6 0.37 NPs70 PDPA70 69 6.26 66.0 6.44 69.7 0.35 NPs80 PDPA80 80
6.24 69.7 3.81 99.7 0.34 NPs100 PDPA100 99 6.21 82.3 9.26 100 0.33
a Determined by .sup.1HNMR shown in Table 1. b Determined by
acid-base titration c Determined by dynamic light scattering (DLS).
d DY547-labelled GL3 siRNA was used to examine the EE %.
[0434] The polymer, PDPA80 (pKa 6.24, Table 3), was chosen for pH
response evaluation by incorporating a near-infrared dye, Cy5.5,
into its PDPA segment. Due to the quenching of the aggregated
fluorophores inside the hydrophobic cores of the NPs (Wang Y et al,
Nat Mater, 13, 204-212 (2014)), there is no fluorescence signal at
a pH above pKa of PDPA80. In contrast, at a pH below pKa, the
protonated PDPA segment induces the disassembly of the NPs and a
dramatic increase in the fluorescence signal. Measurement of the
fluorescence intensity as a function of pH for the Cy.5.5-labelled
NPs of PDPA80 reveals that the pH difference from 10 to 90%
fluorescence activation (.DELTA.pH10-90%) is 0.34 (FIG. 2A and
Table 3) (Wang Y et al, Nat Mater, 13, 204-212 (2014)), which is
much smaller than that of small molecule dyes (about 2 pH units)
(Urano Y et al., Nat Med, 15, 104-109 (2009)), indicating the
ultra-fast pH response of PDPA80. This characteristic is confirmed
by transmission electron microscope (TEM). The spherical
siRNA-loaded NPs could be visualized at a pH of 6.5, with an
average size of 69.7 nm determined by dynamic light scattering
(DLS, Table 3). If altering pH to 6.0, there are no observable NPs
after 20 min incubation. With this morphological change, the NPs
offer super-fast release of DY547-labelled GL3 siRNA (DY547-siRNA)
(FIG. 2B). Around 90% loaded siRNA has been released within 4 h at
a pH of 6.0. Within the same time frame, less than 30% of the
loaded siRNA is released at a pH of 7.4.
TABLE-US-00004 TABLE 4 Size, zeta potential and siRNA encapsulation
efficiency (EE %) of the iRGD-NPs of prepared from the mixture of
Meo-PEG-b-P(DPA-co- GMA-TEPA-C14) and iRGD-PEG-b-PDPA a No. Size
(nm) b Zeta potential (mv) EE % c iRGD-NPs.sub.40 64.2 3.26 55.1
iRGD-NPs.sub.50 68.3 3.98 59.7 iRGD-NPs.sub.60 82.1 5.69 66.4
iRGD-NPs.sub.70 76.5 7.18 69.6 iRGD-NPs.sub.80 70.7 5.26 99.8
iRGD-NPs.sub.100 86.3 8.93 100 a The molar ratio of
Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) and iRGD-PEG-b-PDPA is 9:1. b
Determined by dynamic light scattering (DLS). c DY547-labelled GL3
siRNA was used to examine the EE%.
[0435] Luciferase-expressing HeLa (Luc-HeLa) cells were used to
evaluate the gene silencing efficacy. GL3 siRNA was employed to
suppress luciferase expression. All the siRNA-loaded NPs show a
reduction in luciferase expression at a 10 nM siRNA dose (FIG. 3A),
with the differential silencing efficacy depending upon the polymer
structure. In comparison, the NPs with iRGD peptide (denoted
iRGD-NPs) offer much better gene silencing efficacy. In particular,
the iRGD-NPs.sub.80 prepared from PDPA80 show the best gene
silencing efficacy, i.e., >90% knockdown in luciferase
expression without obvious cytotoxicity (FIG. 4). Cell viability of
Luc-HeLa cells in the presence of 10 nM siRNA dose of the GL3
siRNA-loaded NPs formed with PDPA40, PDPA50, PDPA60, PDPA70,
PDPA80, or PDPA100; and Lipo2K-GL3 siRNA complex was compared to
cells incubated with free medium. No obvious cytotoxicity was
observed with these NPs (FIG. 4).
[0436] After acquiring the nanoplatform with optimal silencing
efficacy (iRGD-NPs.sub.80), flow cytometry was employed to evaluate
its in vitro tumor-targeting ability. With the specific recognition
between integrins (.alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5, FIGS. 5A-5D) on Luc-HeLa cells and iRGD,
the uptake of DY547-siRNA-loaded iRGD-NPs.sub.80 is more than
3-fold higher than that of iRGD-absent NPs.sub.80 (FIGS. 3B and
6A-6C), demonstrating the excellent tumor-targeting ability of
iRGD-NPs.sub.80. Endosomal escape ability was assessed by staining
the endosomes with lysotracker green. Fluorescent image of Luc-HeLa
cells incubated with the siRNA-loaded iRGD-NPs.sub.80 showed that a
majority of the internalized siRNA-loaded NPs entered the cytoplasm
after 4 h incubation, indicating the effective endosomal escape of
the iRGD-NPs.sub.80. In comparison, for the iRGD-NPs prepared from
polymer without lipid-like grafts or pH response (FIGS. 7A-7D), the
endosome escape ability is relatively weaker, thus leading to a
much lower silencing efficacy (FIGS. 7A-7B).
[0437] The iRGD-NPs80 was further tested on whether it can
downregulate survivin expression, an inhibitor of apoptosis protein
that is over-expressed in most cancers (Altieri D C et al., Nat Rev
Cancer, 3, 46-54 (2003)). PC3 cells, a prostate cancer cell line
showing targeted uptake of iRGD-NPs (FIGS. 5A-5D and 6A-6C) where
the uptake of DY547-siRNA-loaded iRGD-NPs.sub.80 is also about
3-fold higher than that of iRGD-absent NPs.sub.80, were used as a
model cell line. Western blot analysis was carried out for
determining survivin expression in PC3 cells treated by survivin
siRNA-loaded NPs.sub.80 or survivin siRNA-loaded iRGD-NPs.sub.80.
The western blot analysis indicates that the survivin siRNA-loaded
iRGD-NPs.sub.80 significantly suppress survivin expression (>80%
knockdown) at a 10 nM siRNA dose. At a 50 nM siRNA dose, survivin
expression is nearly absent (<3%, FIG. 7C). The similar result
can be also found in the immunofluorescence staining analysis of
PC3 cells treated by survivin siRNA-loaded NPs.sub.80 or survivin
siRNA-loaded iRGD-NPs.sub.80 at a 10 nM siRNA dose. Very weak red
fluorescence corresponding to the residual survivin can be observed
in the cells treated with iRGD-NPs.sub.80 at a 10 nM siRNA dose.
With such suppressed survivin expression, the proliferation rate of
PC3 cells is very slow. There is only 2.5-fold increase in cell
number after 8 days incubation (FIG. 3D).
[0438] After validating the efficient gene silencing of
iRGD-NPs.sub.80, their in vivo tumor-targeting ability was
assessed. Pharmacokinetics was first examined by intravenous
injection of DY647-siRNA-loaded NPs. As shown in FIG. 15A, the
blood half-life (t.sub.1/2) of iRGD-NPs.sub.80 is around 3.56 h,
which is far longer than that of naked siRNA (t.sub.1/2<10 min).
This prolonged blood circulation is mainly due to the protection of
PEG outer layer and small particle size (Knop K et al., Angewandte
Chemie International Edition, 49, 6288-6308(2010)). The in vivo
tumor-targeting ability was evaluated by intravenously injecting
DY677-siRNA-loaded NPs into PC3 xenograft tumor-bearing mice.
Overlaid fluorescent image of PC3 xenograft tumor-bearing mice at
24 h post-injection of naked siRNA and siRNA-loaded NPs showed
that, with the iRGD-mediated tumor-targeting, the iRGD-NPs.sub.80
show a much higher tumor accumulation than that of NPs.sub.80 at 24
h post-injection. The tumors and main organs were harvested and the
biodistribution is shown in FIG. 8B. Naked siRNA has a
characteristic biodistribution, i.e., high accumulation in kidney
but extremely low accumulation in tumor. With the specific
recognition between iRGD and integrins .alpha..sub.v.beta..sub.3
and .alpha..sub.v.beta..sub.5 over-expressed on tumor cells and
angiogenic tumor vasculature (Wang Y et al., Nat Mater, 13, 204-212
(2014); Sugahara K N et al., Cancer Cell, 16, 510-520(2009)), the
tumor accumulation of the iRGD-NPs.sub.80 is around 3-fold higher
that of NPs.sub.80.
[0439] To evaluate the tumor-penetrating ability of the
iRGD-NPs.sub.80, the tumors were collected at 4 h post-injection of
the DY677-siRNA-loaded NPs and then sectioned for
immunofluorescence staining. There is nearly no naked siRNA in the
tumor section. For the NPs.sub.80, the number of NPs in tumor
section is very low. Additionally, most of these NPs are positioned
in the tumor vessels, and only a small number reach the
extravascular tumor parenchyma. In contrast, highly concentrated
iRGD-NPs.sub.80 with bright red fluorescence could be visualized in
the tumor section. Remarkably, a majority of these NPs can cross
tumor vessels and reach the extravascular tumor parenchyma,
strongly demonstrating the deep tumor-penetrating characteristic of
iRGD-NPs.sub.80.
[0440] Finally, the in vivo inhibition of survivin expression and
anti-cancer efficacy was evaluated. The survivin siRNA-loaded NPs
were intravenously injected into the PC3 xenograft tumor-bearing
mice (650 .mu.g/kg siRNA dose, n=3) for three consecutive days.
Western blot analysis of survivin expression in the PC3 tumor
tissue after systemic treatment by control NPs (GL3 siRNA-loaded
NPs.sub.80), survivin siRNA-loaded NPs and survivin siRNA-loaded
iRGD-NPs.sub.80 showed that the siRNA-loaded NPs indeed suppressed
survivin expression in tumor. In particular, the administration of
survivin siRNA-loaded iRGD-NP.sub.80 induces more than 60%
knockdown in survivin expression, whereas survivin siRNA-loaded NPs
induced about 25% knockdown in survivin expression (FIG. 9). Thus,
survivin siRNA-loaded iRGD-NPs.sub.80 showed around 3-fold greater
knockdown in survivin expression than that of NPs.sub.80. Notably,
the administration of NPs shows negligible in vivo side effects. To
confirm whether the NP-mediated survivin silencing has an
anti-cancer effect, the survivin siRNA-loaded NPs were
intravenously injected to the mice once every two days at a 650
.mu.g/kg siRNA dose (n=5). After five consecutive injections (FIG.
10), the tumor growth is inhibited compared to the mice treated
with PBS or GL3 siRNA-loaded NPs (Control NPs). Harvested PC3 tumor
from each group at day 16 was compared with GL3 siRNA-loaded
NPs.sub.80 as a control. Particularly, with the excellent
tumor-targeting and penetrating abilities, the iRGD-NPs.sub.80 can
significantly suppress tumor growth, and there is only around
2-fold increase in tumor size at day 24. The PC3 xenograft
tumor-bearing nude mice treated with PBS, GL3 siRNA-loaded
NPs.sub.80 (Control NPs), or survivin siRNA-loaded NPs.sub.80 and
iRGD-NPs.sub.80 were monitored for body weight but no significant
difference was noticed (FIG. 11).
[0441] In summary, an ultra pH-responsive and tumor-penetrating
nanoplatform for targeted systemic siRNA delivery has been
developed. The in vitro and in vivo results demonstrate that this
polymeric NP has a long blood circulation, and can efficiently
target tumor and penetrate tumor parenchyma, leading to efficient
gene silencing and tumor growth inhibition. The polymeric
nanoplatform reported herein may represent a robust siRNA delivery
vehicle for the treatment of a myriad of important diseases
including cancer.
Example 2: Ultra pH-Responsive and Tumor-Penetrating Nanoplatform
for Targeted siRNA Delivery with Robust Anti-Cancer Efficacy
Methods and Materials
[0442] Materials
[0443] Methoxyl-polyethylene glycol (Meo-PEG.sub.113-OH) and
hydroxyl polyethylene glycol carboxylic acid (HO-PEG.sub.113-COOH)
were purchased from JenKem Technology and used as received.
Oligoarginine (NH2-Rn--CONH2, n=6, 8, 10, 20, 30) was provided by
MIT Biopolymer facility. Allyl protected
S,S-2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid
(ACUPA) was kindly provided by BIND Therapeutics as a gift.
2-(Diisopropyl amino) ethyl methacrylate (DPA-MA) and glycidyl
methacrylate (GMA) were provided by Sigma-Aldrich and passed over
an alumina column before use in order to remove the hydroquinone
inhibitors. .alpha.-Bromoisobutyryl bromide, N,N'-dimethylformamide
(DMF), triethylamine (TEA),
N,N,N',N',N'-pentamethyldiethylenetriamine (PMDETA), copper (I)
bromide (CuBr), tetraethylenepentamine (TEPA), isopropyl alcohol,
p-toluenesulfinate tetrahydrate (PTSF),
tetrakis(triphenylphosphine) palladium (Pd(PPh.sub.3).sub.4) and
dichloromethane (DCM) were acquired from Sigma-Aldrich and used
directly. Lipofectamine 2000 (Lipo2K) was purchased from
Invitrogen. Steady-Glo luciferase assay system was provided by
Promega. GL3, fluorescent dye (DY547, DY647 and Cy5.5) labeled GL3
and PHB1 siRNAs were acquired from Dharmacon. The siRNA sequences
are as follows: GL3 siRNA, 5'-CUU ACG CUG AGU ACU UCG AdTdT-3'
(sense) (SEQ ID NO:1) and 5'-UCG AAG UAC UCA GCG UAA GdTdT-3'
(antisense) (SEQ ID NO:2); PHB1 siRNA, 5'-GCG ACG ACC UUA CAG AGC
GUU-3' (sense) (SEQ ID NO:5) and 5'-CGC UCU GUA AGG UCG UCG CUU-3'
(antisense) (SEQ ID NO:6). The fluorescent dyes DY-547 and DY-647
were labeled at the 5'-end of the sense strand of GL3 siRNA. Cy5.5
was labeled at the 5'-end of both the sense and antisense strands
of GL3 siRNA. HeLa cells stably expressing firefly and Renilla
luciferase (Luc-HeLa) were obtained from Alnylam Pharmaceuticals,
Inc. The cells were incubated in RPMI 1640 medium (Invitrogen) with
10% fetal bovine serum (FBS, Sigma-Aldrich). All other reagents and
solvents are of analytical grade and used without further
purification.
Synthesis of Meo-PEG-Br and Br-PEG-COOH
[0444] Meo-PEG.sub.113-OH (8 g, 1.6 mmol) and TEA (1.3 mL, 9.6
mmol) were dissolved in 250 mL of DCM. In an ice-salt bath,
.alpha.-bromoisobutyryl bromide (1 mL, 8 mmol) dissolved in 10 mL
of DCM was added dropwise. After stirring for 24 h, the mixture was
washed with 1 M NaOH (3.times.50 mL), 1 M HCl (3.times.50 mL), and
deionized water (3.times.50 mL). After drying over anhydrous
MgSO.sub.4, the solution was concentrated, and cold ether was added
to precipitate the product. After re-precipitating thrice, the
product was collected as white powder after drying under vacuum.
The synthesis of Br-PEG-COOH was carried out according to a method
similar to that described above, by changing Meo-PEG.sub.113-OH
with HO-PEG.sub.113-COOH. The synthesis scheme of Meo-PEG-Br is
shown below.
Synthesis of methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl methacrylate)
(Meo-PEG-b-P(DPA-co-GMA))
[0445] Meo-PEG-b-P(DPA-co-GMA) copolymer was synthesized by atom
transfer radical polymerization (ATRP). DPA-MA (2.6 g, 12 mmol),
GMA (0.07 g, 0.45 mmol), Meo-PEG-Br (0.75 g, 0.15 mmol), and PMDETA
(31.5 .mu.L, 0.15 mmol) were added to a polymerization tube. DMF (3
mL) and 2-propanol (3 mL) were then added to dissolve the monomer
and initiator. After three cycles of freeze-pump-thaw to remove
oxygen, CuBr (21.6 mg, 0.15 mmol) was added under nitrogen
atmosphere and the polymerization tube was sealed under vacuum.
After polymerization at 40.degree. C. for 24 h, tetrahydrofuran
(THF) was added to dilute the product, which was then passed
through a neutral Al.sub.2O.sub.3 column to remove the catalyst.
The resulting THF solution was concentrated and the residue was
dialyzed against THF, followed by deionized water. The expected
copolymer was collected as a white powder after freeze-drying under
vacuum. The synthesis scheme is shown below.
Synthesis of Meo-PEG-b-P(DPA-co-GMA-Rn)
[0446] Meo-PEG-b-P(DPA-co-GMA-Rn) was synthesized via the ring
opening reaction between the amino group of
NH.sub.2--Rn--CONH.sub.2 and the epoxy group of the GMA repeating
unit. In brief, Meo-PEG-b-P(DPA-co-GMA) (1 g) dissolved in DMF (15
mL) was added dropwise to the DMF solution (10 mL) of
NH.sub.2--Rn--CONH.sub.2 (10-fold molar excess relative to the GMA
repeating unit). After reaction at 60.degree. C. for 7 h, the
mixture was transferred to a dialysis tube and then dialyzed
against deionized water. The Meo-PEG-b-P(DPA-co-GMA-Rn) was finally
collected as a white powder after freeze-drying under vacuum.
[0447] The synthesis route of Meo-PEG-b-P(DPA-co-GMA-Rn) is shown
below.
##STR00005##
Synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA)
[0448] Meo-PEG-b-P(DPA-co-GMA-TEPA) was synthesized via the ring
opening reaction between TEPA and the epoxy group of the GMA
repeating unit. In brief, Meo-PEG-b-P(DPA-co-GMA) (1 g) dissolved
in DMF (15 mL) was added dropwise to the DMF solution (5 mL) of
TEPA (30-fold molar excess relative to the GMA repeating unit).
After reacting at 60.degree. C. for 7 h, the mixture was
transferred to a dialysis tube and then dialyzed against deionized
water. The Meo-PEG-b-P(DPA-co-GMA-TEPA) was finally collected as a
white powder after freeze-drying under vacuum. The synthesis route
of Meo-PEG-b-P(DPA-co-GMA-TEPA) is shown below.
##STR00006##
Synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5)
[0449] Meo-PEG-b-P(DPA-co-GMA-TEPA) (0.2 g) and Cy5.5 NHS ester
(1.5-fold molar excess relative to the TEPA repeating unit) were
well dissolved in 5 mL of THF. After constantly stirring in dark
for 48 h, the solution was dialyzed against deionized water and the
product was collected after freeze-drying.
The synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5) is shown
below.
##STR00007##
Synthesis of HOOC-PEG-b-PDPA
[0450] HOOC-PEG-b-PDPA copolymers were also synthesized by the ATRP
method. For example, DPA-MA (1.73 g, 8 mmol), Br-PEG-COOH (0.5 g,
0.1 mmol), and PMDETA (21 .mu.L, 0.1 mmol) were added to a
polymerization tube. Subsequently, DMF (2 mL) and 2-propanol (2 mL)
were added to dissolve the monomer and initiator. After three
cycles of freeze-pump-thaw to remove oxygen, CuBr (14.4 mg, 0.1
mmol) was added under nitrogen atmosphere and the polymerization
tube was sealed under vacuum. After polymerization at 40.degree. C.
for 24 h, tetrahydrofuran (THF) was added to dilute the product,
which was then passed through a neutral Al.sub.2O.sub.3 column to
remove the catalyst. The obtained THF solution was concentrated and
the residue was dialyzed against deionized water. The
HOOC-PEG-b-PDPA was obtained as a white powder after freeze-drying
under vacuum. The synthesis route of HOOC-PEG-b-PDPA is shown
below.
Synthesis of Allyl-protected ACUPA-PEG-b-PDPA
[0451] HOOC-PEG-b-PDPA copolymer (1 g), allyl protected ACUPA
(5-fold molar excess relative to the terminal carboxylic acid
group), EDC.HCl (3-fold molar excess relative to the terminal
carboxylic acid group), and NHS (3-fold molar excess relative to
the terminal carboxylic acid group) were well dissolved in 15 mL of
THF. The mixture was stirred at room temperature for 48 h. The
solution was subsequently dialyzed against DMF for 48 h followed by
deionized water. The expected allyl-protected ACUPA-PEG-PDPA was
collected after freeze-drying. The synthesis route of
Allyl-protected ACUPA-PEG-b-PDPA is shown below.
Synthesis of ACUPA-PEG-b-PDPA
[0452] Allyl-protected ACUPA-PEG-PDPA (1 g) was well dissolved in
15 mL of THF and Pd(PPh.sub.3).sub.4 (42 mg) was added. Under
stirring, PTSF (155 mg) dissolved in 2.5 mL of methanol was added
to the suspension of Allyl protected ACUPA-PEG-PDPA and
Pd(PPh.sub.3).sub.4. After reacting in dark for 2 h, the suspension
was transferred to a dialysis tube (MWCO 3500) and dialyzed against
toluene for 48 h. Thereafter, the solution was removed by rotary
evaporation and the residue was dissolved in 15 mL of THF. After
dialyzing against deionized water for 48 h, the ACUPA-PEG-PDPA was
collected through freeze-drying.
The synthesis route of ACUPA-PEG-b-PDPA is shown below.
##STR00008##
[0453] Gel Permeation Chromatography (GPC)
[0454] Number- and weight-average molecular weights (Mn and Mw,
respectively) of the polymers were determined by a gel permeation
chromatographic system equipped with a Waters 2690D separations
module and a Waters 2410 refractive index detector. THF was used as
the eluent at a flow rate of 0.3 mL/min. Waters millennium module
software was used to calculate molecular weight on the basis of a
universal calibration curve generated by polystyrene standard of
narrow molecular weight distribution.
[0455] .sup.1H Nuclear Magnetic Resonance (.sup.1HNMR)
[0456] The .sup.1HNMR spectra of the polymers were recorded on a
Mercury VX-300 spectrometer at 400 MHz (Varian, USA), using
CDCl.sub.3 as a solvent and TMS as an internal standard.
[0457] Acid-Base Titration
[0458] Meo-PEG-b-P(DPA-co-GMA-Rn) was dispersed in deionized water,
and a concentrated HCl aqueous solution was added until the
copolymer was completely dissolved (1 mg/mL). Subsequently, 1 M
NaOH aqueous solution was added in 1-5 .mu.L increments. After each
addition, the solution was constantly stirred for 3 min, and the
solution pH was measured using a pH meter. The pKa of the copolymer
was determined as the pH at which 50% of the copolymer turns
ionizes.
Preparation and Characterization of Nanoparticles (NPs)
[0459] Meo-PEG-b-P(DPA-co-GMA-Rn) was dissolved in THF to form a
homogenous solution with a concentration of 4 mg/mL. Subsequently,
a certain volume of this THF solution was taken and mixed with 1
nmol siRNA (0.1 nmol/4 aqueous solution) in an N/P molar ratio of
80:1. Under vigorously stirring (1000 rpm), the mixture was added
dropwise to 4 mL of deionized water. The NP dispersion formed was
transferred to an ultrafiltration device (EMD Millipore, MWCO 100
K) and centrifuged to remove the organic solvent and free
compounds. After washing with PBS (pH 7.4) solution (3.times.5 mL),
the siRNA loaded NPs were dispersed in 1 mL of phosphate buffered
saline (PBS, pH 7.4) solution. Size and zeta potential were
determined by dynamic light scattering (DLS, Brookhaven Instruments
Corporation). The morphology of NPs was visualized on a Tecnai G2
Spirit BioTWIN transmission electron microscope (TEM). Before
observation, the sample was stained with 1% uranyl acetate and
dried under air. To determine the siRNA encapsulation efficiency,
DY547-labelled GL3 siRNA (DY547-siRNA) loaded NPs were prepared
according to the method described above. A small volume (50 .mu.L)
of the NP solution was withdrawn and mixed with 20-fold DMSO. The
fluorescence intensity of DY547-siRNA was measured using a Synergy
HT multi-mode microplate reader (BioTek Instruments) and compared
to the free DY-547 labelled GL3 siRNA solution (1 nmol/mL PBS
solution.)
[0460] To prepare the ACUPA-NPs, Meo-PEG-b-P(DPA-co-GMA-Rn) (4
mg/mL in THF) was mixed with 1 nmol siRNA (0.1 nmol/4 aqueous
solution) in a N/P molar ratio of 80:1. Then ACUPA-PEG-b-PDPA (4
mg/mL in THF, 10 mol % compared to Meo-PEG-b-P(DPA-co-GMA-Rn)) was
added, and the mixture was added dropwise to 4 mL of deionized
water. The ACUPA-NPs were purified by an ultrafiltration device
(EMD Millipore, MWCO 100 K) and finally dispersed in 1 mL of PBS.
The siRNA encapsulation efficiency was examined by replacing the
siRNA with DY-547 labelled GL3 siRNA.
[0461] Evaluation of pH Sensitivity
[0462] The THF solution of Meo-PEG-b-P(DPA-co-GMA-Rn) (4 mg/mL) and
Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5) (4 mg/mL) was mixed in a volume
ratio of 8:2. Under vigorously stirring (1000 rpm), 0.2 mL of the
mixture was added dropwise to 2 mL of deionized water. After
collection and purification by an ultrafiltration device (EMD
Millipore, MWCO 100 kDa), the NPs formed were dispersed in 1 mL of
deionized water. Subsequently, 1 M NaOH or HCl was added in 1-5
.mu.L increments, and the fluorescence intensity of the NPs was
measured on a Synergy HT multi-mode microplate reader. The
normalized fluorescence intensity (NFI) vs. pH profile was used to
quantitatively assess the pH responsiveness. NFI is calculated as
follows:
NFI=(F-Fmin)/(Fmax-Fmin)
[0463] where F is the fluorescence intensity of the NPs at any
given pH value and Fmax and Fmin are the maximal and minimal
fluorescence intensity of the NPs, respectively.
[0464] In Vitro siRNA Release
[0465] DY547-siRNA-loaded NPs were prepared as described above.
Subsequently, the NPs were dispersed in 1 mL of PBS (pH 7.4) and
then transferred to a Float-a-lyzer G2 dialysis device (MWCO 100
kDa, Spectrum) that was immersed in PBS (pH 7.4) at 37.degree. C.
At a predetermined interval, 5 .mu.L of the NP solution was
withdrawn and mixed with 20-fold DMSO. The fluorescence intensity
of DY547-siRNA was determined by Synergy HT multi-mode microplate
reader.
[0466] Cell Culture
[0467] Human cervical cancer cell line with the expression of
luciferase (Luc-HeLa) and prostate cancer (PCa) cell lines (LNCaP,
PC3, DU145, 22RV1) were incubated in RPMI 1640 medium with 10% FBS
at 37.degree. C. in a humidified atmosphere containing 5%
CO.sub.2.
[0468] Luciferase Silencing
[0469] Luc-HeLa cells were seeded in 96-well plates (5,000 cells
per well) and incubated in 0.1 mL of RPMI 1640 medium with 10% FBS
for 24 h. Thereafter, the GL3 siRNA-loaded NPs were added. After
incubating for 24 h, the cells were washed with fresh medium and
allowed to incubate for another 48 h. The expression of firefly
luciferase in HeLa cells was determined using Steady-Glo luciferase
assay kits. Cytotoxicity was measured using the alamarBlue assay
according to the manufacturer's protocol. The luminescence or
fluorescence intensity was measured using a microplate reader, and
the average value of five independent experiments was collected. As
a control, the silencing effect of Lipo2K/GL3 siRNA complexes was
also evaluated according to the procedure described above and
compared to that of GL3 siRNA-loaded NPs.
[0470] Determination of the Expression of Prostate Specific
Membrane Antigen (PSMA)
[0471] The PCa cell lines were seeded in 6-well plates (50,000
cells per well) and incubated in 1 mL of RPMI 1640 medium
containing 10% FBS for 24 h. Thereafter, 10 .mu.L of PE-conjugated
anti-human PSMA antibody (BioLegend) was added, and the cells were
allowed to incubate for another 4 h. After removing the medium and
washing with PBS (pH 7.4) solution thrice, the cells were collected
for flow cytometry quantitative analysis (DXP11 Analyzer).
[0472] Evaluation of Endosomal Escape
[0473] Luc-HeLa cells (20,000 cells) were seeded in discs and
incubated in 1 mL of RPMI 1640 medium containing 10% FBS for 24 h.
Subsequently, the DY547-siRNA-loaded NPs were added, and the cells
were allowed to incubate for 1 or 2 h. After removing the medium
and subsequently washing with PBS (pH 7.4) solution thrice, the
endosomes and nuclei were stained with lysotracker green and
Hoechst 33342, respectively. The cells were then viewed under a
FV1000 confocal laser scanning microscope (CLSM, Olympus).
[0474] Flow Cytometry
[0475] Luc-HeLa and PCa cell lines (LNCaP, PC3, DU145) were seeded
in 6-well plates (50,000 cells per well) and incubated in 1 mL of
RPMI 1640 medium containing 10% FBS for 24 h. Subsequently, the
DY547-siRNA-loaded NPs or ACUPA-NPs were added, and the cells were
allowed to incubate for another 4 h. After removing the medium and
subsequently washing with PBS (pH 7.4) solution thrice, the cells
were collected for flow cytometry quantitative analysis.
[0476] In Vitro PHB1 Silencing
[0477] LNCaP cells were seeded in 6-well plates (50,000 cells per
well) and incubated in 1 mL of RPMI 1640 medium containing 10% FBS
for 24 h. Subsequently, the cells were transfected with the PHB1
siRNA-loaded NPs or ACUPA-NPs for 24 h. After washing the cells
with PBS thrice, the cells were further incubated in fresh medium
for another 48 h. Thereafter, the cells were digested by trypsin
and the proteins were extracted using modified
radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl pH 7.4,
150 mM NaCl, 1% NP-40 substitute, 0.25% sodium deoxycholate, 1 mM
sodium fluoride, 1 mM Na3VO4, 1 mM EDTA), supplemented with
protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride
(PMSF). The expression of PHB1 was examined using the following
western blot analysis.
[0478] Western Blot Analysis
[0479] Equal amounts of protein, as determined with a bicinchoninic
acid (BCA) protein assay kit (Pierce/Thermo Scientific) according
to the manufacturer's instructions, were added to SDS-PAGE gels and
separated by gel electrophoresis. After transferring the proteins
from gel to polyvinylidene difluoride membrane, the blots were
blocked with 3% BSA in TBST (50 mM Tris-HCl pH 7.4, 150 mM NaCl,
and 0.1% Tween 20) and then incubated with a mixture of PHB1 rabbit
antibody (Cell Signaling) and .beta.-actin rabbit antibody (Cell
Signaling). The expression of PHB1 was detected with horseradish
peroxidase (HRP)-conjugated secondary antibody (anti-rabbit IgG
HRP-linked antibody, Cell Signaling) and an enhanced
chemiluminescence (ECL) detection system (Pierce).
[0480] In Vitro Cell Proliferation
[0481] LNCaP cells were seeded in 6-well plates (20,000 cells per
well) and incubated in 1 mL of RPMI 1640 medium containing 10% FBS
for 24 h. Thereafter, the cells were transfected with the PHB1
siRNA-loaded NPs or ACUPA-NPs for 24 h and then washed with fresh
medium for further incubation. At predetermined intervals, the
cytotoxicity was measured using the alamarBlue assay according to
the manufacturer's protocol. After each measurement, the alamarBlue
agent was removed and the cells were incubated in fresh medium for
further proliferation.
[0482] LNCaP Xenograft Tumor Model
[0483] The tumor model was constructed by subcutaneous injection
with 200 .mu.l of LNCaP cell suspension (a mixture of RPMI 1640
medium and Matrigel in 1:1 volume ratio) with a density of
3.times.10.sup.7 cells/mL into the back region of healthy male
BALB/c nude mice. When the volume of the PC3 tumor xenograft
reached .about.50 mm.sup.3, the mice were used for the following in
vivo experiments.
[0484] Pharmacokinetics Study
[0485] Healthy male BALB/c mice were randomly divided into three
groups (n=3) and given an intravenous injection of either (i) free
DY647-labelled GL3 siRNA (DY647-siRNA), (ii) DY647-siRNA-loaded
NPs, or (iii) DY647-siRNA-loaded ACUPA-NPs at a 650 .mu.g/kg siRNA
dose. At predetermined time intervals, orbital vein blood (20
.mu.L) was withdrawn using a tube containing heparin, and the wound
was pressed for several seconds to stop the bleeding. The
fluorescence intensity of DY-647 labelled siRNA in the blood was
determined using a microplate reader. The blood circulation
half-life (t1/2) was calculated by first-order decay fit.
[0486] Biodistribution
[0487] LNCaP tumor-bearing male BALB/c nude mice were randomly
divided into four groups (n=3) and given an intravenous injection
of either (i) free Cy5.5-labelled GL3 siRNA (Cy5.5-siRNA), (ii)
Cy5.5-siRNA-loaded NPs, (iii) Cy5.5-siRNA-loaded ACUPA-NPs or (iv)
PSMA antibody (5 mg/kg dose) 15 min followed by Cy5.5-siRNA loaded
ACUPA-NPs at a 650 .mu.g/kg siRNA dose. Twenty-four hours after the
injection, the mice were imaged using the Maestro 2 In-Vivo Imaging
System (Cri Inc). Main organs and tumors were then harvested and
imaged. To quantify the accumulation of NPs in tumors and organs,
the fluorescence intensity of each tissue was quantified by
Image-J.
[0488] In Vivo PHB1 Silencing
[0489] LNCaP tumor-bearing male BALB/c nude mice were randomly
divided into two groups (n=3) and intravenously injected with (i)
PHB1 siRNA-loaded NPs or (ii) PHB1 siRNA-loaded ACUPA-NPs for three
consecutive days. Twenty-four hours post the final injection, mice
were sacrificed and tumors were harvested. The proteins in the
tumor were extracted using modified radioimmunoprecipitation assay
lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40
substitute, 0.25% sodium deoxycholate, 1 mM sodium fluoride, 1 mM
Na3VO4, 1 mM EDTA), supplemented with protease inhibitor cocktail
and 1 mM phenylmethanesulfonyl fluoride (PMSF). The expression of
PHB1 was examined using the aforementioned western blot
analysis.
[0490] Inhibition of Tumor Growth
[0491] LNCaP tumor-bearing male BALB/c nude mice were randomly
divided into four groups (n=5) and intravenously injected with (i)
PBS, (ii) GL3 siRNA-loaded NPs, (iii) PHB1 siRNA-loaded NPs or (iv)
PHB1 siRNA-loaded ACUPA-NPs at a 650 .mu.g/kg siRNA dose once every
two days. All the mice were administrated five consecutive
injections and the tumor growth was monitored every two days by
measuring perpendicular diameters using a caliper and tumor volume
was calculated as follows:
V=W.sup.2.times.L/2
[0492] where W and L are the shortest and longest diameters,
respectively.
[0493] Histology
[0494] Healthy male BALB/c mice were randomly divided into three
groups (n=3) and administered daily intravenous injections of
either (i) PBS, (ii) PHB1 siRNA-loaded NPs or (iii) PHB1
siRNA-loaded ACUPA-NPs at a 650 .mu.g/kg siRNA dose. After five
consecutive injections (once every two days), the main organs were
collected 2 days post the final injection, fixed with 4%
paraformaldehyde, and embedded in paraffin. Tissue sections were
stained with hematoxylin-eosin (H&E) and viewed under an
optical microscope.
[0495] Results
[0496] A high loading, biosafe and long-circulating siRNA delivery
nanoplatform that shows high prostate specificity and excellent
endosomal escape capability for PCa therapy is developed. To
construct this robust nanoplatform, a library of ultra
pH-responsive PEGylated polymers were developed, containing
membrane-penetrating oligoarginine grafts and an
S,S-2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid
(ACUPA) terminus. ACUPA is a small molecule target ligand that can
specifically bind to prostate specific membrane antigen (PSMA),
which is abundantly expressed in PCa, in both its metastatic form
and the hormone-refractory form (Israeli, R et al., Cancer
Research, 53, (2), 227-230 (1993); Murphy, G P et al., Cancer, 83,
(11), 2259-2269 (1998); Dhar, S et al., Proceedings of the National
Academy of Sciences, 105, (45), 17356-17361 (2008)). The resulting
polymer/siRNA nanoassembly is expected to have the following unique
features (FIG. 12): i) the surface-encoded ACUPA moieties endow the
NPs with high PCa specificity and selectivity; ii) the hydrophilic
PEG shells allow the NPs to escape immunological recognition, thus
improving blood circulation (Knop, K et al., Angewandte Chemie
International Edition, 49, (36), 6288-6308 (2010); Guo, X et al.,
Accounts of Chemical Research, 45, 971-979 (2012); Bertrand, N et
al., Advanced Drug Delivery Reviews, 66, 2-25(2014); iii) a small
population of cationic membrane-penetrating oligoarginine grafts
randomly dispersed in the hydrophobic poly(2-(diisopropylamino)
ethylmethacrylate) (PDPA) segment can strongly entrap a high amount
of siRNA into the hydrophobic cores of the NPs; iv) the rapid
protonation of the ultra pH-responsive PDPA segment with a pKa
close to endosomal pH (6.0-6.5) causes the swelling of endosomes
via the "proton sponge" effect (Yu, H et al., ACS Nano, 5,
9246-9255 (2011); Zhou, K et al., Angewandte Chemie International
Edition, 50, 6109-6114 (2011)), which works alongside the
membrane-penetrating oligoarginine grafts to induce efficient and
fast release of siRNA in cytoplasm to inhibit tumor growth (Chen, J
X et al., ACS Applied Materials & Interfaces, 6, (1), 593-598
(2014); Chen, J X et al., Biomaterials, 32, (6), 1678-1684 (2011);
Lim, Y B et al., M. Angewandte Chemie International Edition, 46,
9011-9014(2007).
[0497] Atom-transfer radical polymerization (ATRP) was employed to
synthesize the PEGlyated polymer, methoxyl-polyethylene
glycol-b-poly (2-(diisopropylamino) ethylmethacrylate-co-glycidyl
methacrylate) (Meo-PEG-b-P(DPA-co-GMA)). The epoxy group was
subsequently subjected to attack by oligoarginine (R.sub.n, n=6, 8,
10, 20, 30) to endow the resulting polymer
(Meo-PEG-b-P(DPA-co-GMA-R.sub.n) with siRNA loading and endosomal
membrane-penetrating abilities. The PCa-specific PEGylated polymer,
ACUPA-PEG-b-PDPA was also prepared by ATRP, followed by conjugation
with ACUPA.
[0498] The length of the oligoarginine grafts was varied to adjust
the siRNA loading ability and physiochemical properties of the NPs.
The siRNA-loaded NPs were prepared by mixing siRNA aqueous solution
with the tetrahydrofuran (THF) solution of
Meo-PEG-b-P(DPA-co-GMA-Rn) at a N/P molar ratio of 80:1. The
amphiphilic nature of the polymers induces self-assembly into NPs
with siRNA entrapped in the hydrophobic cores. As the number of
arginine residues increases from 6 to 30, the size of the resulting
NPs increases from 56.6 to 189.9 nm (FIG. 13A, Table 5), but siRNA
encapsulation efficiency (EE %) decreases from 90.6% to 49.7% (FIG.
13B). One possible reason is that enhancing overall hydrophilicity
of the amphiphilic polymers by increasing the length of the
oligoarginine grafts leads to the formation of looser NPs with
weaker siRNA loading ability. This also results in an increased
zeta potential (FIG. 13B). Notably, there is no obvious change in
the EE % or size of the NPs made with the mixture of
Meo-PEG-b-P(DPA-co-GMA-Rn) (90 mol %) and ACUPA-PEG-b-PDPA (10 mol
%) (Table 6).
TABLE-US-00005 TABLE 5 Size, zeta potential, siRNA encapsulation
efficiency (EE %), and pH responsiveness of the NPs prepared from
Meo-PEG-b-P(DPA-co-GMA-Rn) Size Zeta (nm) potential .DELTA.pH10-
No. a (mv) EE % b pKa b 90% NPsR6 56.6 7.09 90.6 6.24 0.32 NPsR8
83.4 8.26 84.4 6.27 0.36 NPsR10 90.8 9.13 72.7 6.31 0.39 NPsR20
117.8 13.74 54 6.42 0.46 NPsR30 179.9 14.01 49.7 6.49 0.51 a
Determined by dynamic light scattering (DLS). b DY-547-labelled GL3
siRNA was used to examine the EE %. c Corresponding to the pKa of
the polymer determined by acid-base titration.
TABLE-US-00006 TABLE 6 Size, zeta potential and siRNA encapsulation
efficiency (EE %) of the iRGD-NPs of prepared from the mixture of
Meo-PEG-b-P(DPA-co- GMA-Rn) and ACUPA-PEG-b-PDPA a No. Size (nm) b
Zeta potential (mv) EE % c ACUPA-NPsR6 58.7 6.97 92.1 ACUPA-NPsR8
85.9 7.92 86.9 ACUPA-NPsR10 93.6 8.87 76.1 ACUPA-NPsR20 119.4 13.46
58.2 ACUPA-NPsR30 184.1 13.78 51.8 a The molar ratio of
Meo-PEG-b-P(DPA-co-GMA-Rn) and ACUPA-PEG-b-PDPA is 9:1. b
Determined by dynamic light scattering (DLS). c DY-547-labelled GL3
siRNA was used to examine the EE %.
[0499] The amphiphilic polymer, Meo-PEG-b-P(DPA-co-GMA-R10) (pKa
6.31, FIG. 13C) was used to investigate its pH sensitivity. The
transmission electron microscope (TEM) image of the GL3
siRNA-loaded NPs of Meo-PEG-b-P(DPA-co-GMA-R10) incubated in PBS
buffer at a pH of 6.5 indicated that this amphiphilic copolymer was
able to assemble with siRNA to form spherical NPs at a pH of 6.5,
with an average size of 90.8 nm determined by dynamic light
scattering (DLS, FIG. 13A). When the solution pH decreases to 6.0,
there are no observable NPs after 20 min incubation using TEM
imaging, indicating a very fast pH sensitivity. To further evaluate
the pH sensitivity, a near-infrared dye, Cy5.5-conjugated PEGylated
polymer, was mixed with Meo-PEG-b-P(DPA-co-GMA-R10) to prepare the
NPs with the aggregation of fluorophores inside the hydrophobic
cores. Fluorescent images of the Cy.5.5 labelled NPs of
Meo-PEG-b-P(DPA-co-GMA-R10) at different pH indicated that, with
the quenching of the fluorophores, fluorescence signal is absent at
a pH above pKa. However, protonation of the PDPA segment at pH
below pKa causes the NPs to disassemble, leading to a dramatic
increase in the fluorescence signal. Measuring the fluorescence
intensity upon the pH change reveals that the pH difference from 10
to 90% fluorescence activation (ApH10-90%) is 0.39 (FIG. 14) (Wang,
Y et al., Nat Mater, 13, (2), 204-212 (2014)). This value is much
smaller than that of small molecule dyes (about 2 pH units) with
the same degree of fluorescence intensity change (Urano, Y et al.,
Nat Med, 15, (1), 104-109 (2009)), demonstrating the ultra-fast pH
response rate of Meo-PEG-b-P(DPA-co-GMA-R10). This characteristic
allows the NPs of this polymer to show a super-fast release of
DY547-labelled GL3 siRNA (DY547-siRNA) at a pH below pKa. As shown
in FIG. 13D, around 80% of the loaded siRNA has been released
within 3 h at a pH of 6.0. Within the same time frame, less than
30% of the loaded siRNA is released at a pH of 7.4.
[0500] Luciferase-expressing HeLa (Luc-HeLa) cells, which are
genetically modified to stably express both firefly and Renilla
luciferase, were employed to evaluate the gene silencing efficacy
of the siRNA-loaded NPs. The GL3 siRNA was used to selectively
suppress firefly luciferase expression. Renilla luciferase
expression was used as an internal cell viability control. As shown
in FIG. 15A, all the siRNA-loaded NPs can suppress the firefly
luciferase expression at a 10 nM siRNA dose, with the differential
silencing efficacy depending on the length of the oligoarginine
grafts. However, there is no obvious difference between the NPs
with and without the ACUPA ligand. The main reason is the extremely
low PSMA expression in HeLa cells (FIGS. 16A-16F), which leads to a
lack of any significant difference in cellular uptake between these
two types of NPs (FIGS. 17A-17F). Among these nanoplatforms, the
NPs self-assembled from Meo-PEG-b-P(DPA-co-GMA-R8) or
Meo-PEG-b-P(DPA-co-GMA-R10) show a better gene silencing efficacy.
In Particular, the NPs made with Meo-PEG-b-P(DPA-co-GMA-R10) can
reduce the firefly luciferase expression by about 90%, which is
significantly more than the commercial lipofectamine 2000 (Lipo2K)
treatment, which is capable of around 70% knockdown in luciferase
expression. Notably, there is no obvious cytotoxicity of NPs used
for these in vitro transfection experiments (FIG. 18). Cytotoxicity
of the GL3 siRNA loaded NPs with varying length of the
oligoarginine grafts, R6, R8, R10, R20, and R30; and Lipo2K-GL3
siRNA complex, against Luc-HeLa cells at a 10 nM siRNA dose was
compared with free medium. No obvious cytotoxicity of these NPs was
observed.
[0501] To validate the contention that the optimal silencing
efficacy of the NPs prepared from Meo-PEG-b-P(DPA-co-GMA-R10)
(NPsR10 and ACUPA-NPsR10) is attributable to their excellent
endosomal escape capability, lysotracker green was used to label
the endosomes and examined the intracellular distribution of the
DY547-siRNA-loaded NPsR10. The confocal laser scanning microscope
(CLSM) images of Luc-HeLa cells incubated with the
DY547-siRN-loaded NPsR10 for 2 h showed that a majority of the
internalized siRNA-loaded NPs enter the cytoplasm after 2 h
incubation, dramatically demonstrating the excellent endosomal
escape ability of the NPs. If the R10 grafts are replaced by
tetraethylenepentamine (Meo-PEG-b-P(DPA-co-GMA-TEPA), the endosomal
escape ability of the resulting NPs is comparatively weaker,
leading to a much lower silencing efficacy (FIGS. 19A-19B). This
suggests that the "proton sponge" effect alone is insufficient for
endosomal escape (Yu, H et al., ACS Nano, 5, 9246-9255 (2011); Won,
Y Y et al., Journal of Controlled Release, 139, (2), 88-93 (2009)).
Additionally, the better silencing efficacy of NPsR8 and NPsR10
also agrees with the contention that the length of oligoarginine
for the most efficient membrane penetration is between 8 and 10
arginine residues (Mitchell, D J et al., The Journal of Peptide
Research, 56, (5), 318-325 (2000); Suzuki, T et al., Journal of
Biological Chemistry, 277, 2437-2443 (2002); Fuchs, S M et al.,
Cell. Mol. Life Sci., 63, 1819-1822 (2006))
[0502] After screening the nanoplatform with optimal silencing
efficacy, its PCa specificity was evaluated. LNCaP cells, a PCa
cell line with over-expressed PSMA (FIG. 16E) (Farokhzad, O C et
al., Proceedings of the National Academy of Sciences, 103, (16),
6315-6320 (2006)), were chosen for incubation with the NPs. From
the flow cytometry profile in FIG. 15B, unlike the Luc-HeLa cells,
LNCaP cells showed around 5-fold stronger uptake of the
DY547-siRNA-loaded ACUPA-NPsR10 than that of NPsR10 (FIG. 20). If
the cells are pre-treated with the anti-PSMA antibody for 30 min
followed by ACUPA-NPsR10 for another 4 h at a 10 nM siRNA dose,
there is no obvious difference in cellular uptake between
ACUPA-NPsR10 and NPsR10, indicating that the high cellular uptake
of ACUPA-NPsR10 is built on the specific recognition between the
ACUPA ligand and the over-expressed PSMA on LNCaP cells. To further
validate this ACUPA-mediated PCa specificity, two other PCa cell
lines with extremely low PSMA expression, PC3 and DU145 cells,
similar to that of HeLa cells (FIGS. 16A-16C), were also incubated
with the DY547-siRNA-loaded NPs. With the absence of specific
interaction between the ACUPA ligand and PSMA, HeLa, PC3, and DU145
cell lines show similar ability to internalize the ACUPA-NPsR10 and
NPsR10 (FIGS. 17A-17F). A summary bar graph showing the
fluorescence intensity of PSMA in Luc-HeLa, PC3, DU145, 22RV1, and
LNCaP cells (FIG. 16F). DU145 cells express moderate amount of PSMA
but at less 30% of that of LNCaP cells.
[0503] Based on the high PCa specificity of ACUPA-NPsR10, it was
further examined whether this siRNA delivery nanoplatform can be
used to silence a potential therapeutic target in LNCaP cells.
Prohibitin1 (PHB1) is a highly conserved and multifunctional 32 kDa
protein that regulates various cell behaviors such as
proliferation, apoptosis, and transcription (Thuaud, F et al.,
Chemistry & Biology 20, (3), 316-331; Theiss, A L et al.,
Biochimica et Biophysica Acta (BBA)--Molecular Cell Research, 1813,
(6), 1137-1143 (2011)). Upregulation of PHB1 has been found in most
cancers including PCa and is associated with drug resistance
(Kapoor, S. Human Pathology 44, (4), 678-679; Gregory-Bass, R C et
al., International Journal of Cancer, 122, (9), 1923-1930 (2008)).
Western blot was employed to investigate the knockdown efficacy of
PHB1 siRNA-loaded ACUPA-NPsR10. Western blot analysis of PHB1
expression in LNCaP cells treated with PHB1 siRNA-loaded NPsR10 and
ACUPA-NPsR10 indicated that this siRNA delivery nanoplatform can
knock down PHB1 by around 90% at a 10 nM siRNA dose. Additionally,
the PHB1 expression is nearly absent (<2%) at a 50 nM siRNA
dose. However, more than 30% of PHB is still expressed in the cells
incubated with the siRNA-loaded NPsR10 at a 10 nM siRNA dose (FIG.
21). A similar tendency can be also found in the immunofluorescence
staining analysis. Immunofluorescence analysis of the LNCaP cells
treated by PHB1 siRNA-loaded NPsR10 at a 10 nM siRNA dose showed
that red fluorescence corresponding to residual PHB1 expression can
be observed in the LNCaP cells treated by siRNA-loaded NPsR10 at a
10 nM siRNA dose. In contrast, there is nearly no red fluorescence
in the cells treated by siRNA-loaded ACUPA-NPsR10. With this
suppressed PHB1 expression, LNCaP cells show a very slow
proliferation rate (FIG. 15C). After 8 days incubation, there is
only roughly a 3-fold increase in the cell number at a 10 nM siRNA
dose. In contrast, there is around a 7-fold or 11-fold increase in
the number of cells treated with PHB1 or GL3 siRNA-loaded
NPsR10.
[0504] After proving the in vitro PCa-specificity of the
ACUPA-NPsR10, their pharmacokinetics and in vivo PCa-specificity
was evaluated. The pharmacokinetics of the ACUPA-NPsR10 was
examined by intravenous injection of DY647 labelled GL3 siRNA
(DY647-siRNA) loaded NPs to healthy mice (650 .mu.g/kg siRNA dose,
n=3). As shown in FIG. 22, the blood half-life (t1/2) of
ACUPA-NPsR10 is around 4.56 h, far longer than naked siRNA
(t1/2<30 min). This better stability is mainly attributed to
protection by the PEG outer layer and small particle size (Knop, K
et al., Angewandte Chemie International Edition, 49, 6288-6308
(2010); Guo, X et al., Accounts of Chemical Research, 45, 971-979
(2012); Bertrand, N et al., Advanced Drug Delivery Reviews, 66,
2-25(2014). Moreover, due to the negative nature of the
surface-encoded ACUPA ligand with three carboxylic acid groups, the
ACUPA-NPsR10 show a much longer blood circulation than NPsR10
(t1/2=4.18 h). The in vivo PCa-specificity of ACUPA-NPsR10 was
assessed by intravenously injecting Cy5.5 labelled GL3 siRNA
(Cy5.5-siRNA) loaded NPs to LNCaP xenograft tumor-bearing mice (650
.mu.g/kg siRNA dose, n=3). Overlaid fluorescent image of the LNCaP
xenograft tumor-bearing nude mice 24 h post-injection of naked
Cy5.5-siRNA, Cy5.5-siRNA-loaded NPsR10 and ACUPA-NPsR10, and PSMA
antibody followed by Cy5.5-siRNA-loaded ACUPA-NPsR10 showed the
fluorescent image of the mice at 24 h post-injection. There is
almost no accumulation of naked siRNA in the tumor. However, the
ACUPA-NPsR10 shows high accumulation in the tumor corresponding to
the bright fluorescence. In the absence of the PSMA-specific ACUPA
ligand, the accumulation of NPsR10 in the tumor is much lower
compared to ACUPA-NPsR10. If first injecting the PSMA antibody (5
mg/kg dose) followed by ACUPA-NPsR10, the blocked PSMA leads to a
decrease in the accumulation of ACUPA-NPsR10 in tumor, highlighting
the important effect of specific interaction between PSMA and the
ACUPA ligand on the PCa-specificity of ACUPA-NPsR10. To analyze the
accumulation of NPs in tumor and other organs, the tumor and main
organs of mice 24 h post-injection were harvested and the
biodistribution of the NPs determined. The naked siRNA presents a
characteristic biodistribution, i.e., high accumulation in kidney
but extremely low accumulation in tumor (Zhu X et al., Proceedings
of the National Academy of Sciences, 112, (25), 7779-7784 (2015)).
With the specific recognition between the ACUPA ligand and PSMA
over-expressed on LNCaP xenograft tumor, the accumulation of
ACUPA-NPsR10 in tumor is around 3-fold higher than that of NPsR10
or that found in mice pre-treated with PSMA antibody.
[0505] Finally, the in vivo inhibition of PHB1 expression and
anti-tumor efficacy was evaluated. To examine the inhibition of
PHB1 expression in tumor tissue, PHB1 siRNA-loaded NPs were
intravenously injected to LNCaP xenograft tumor-bearing mice (650
.mu.g/kg siRNA dose, n=3) on three consecutive days and in vivo
PHB1 expression was examined by western blot. Western blot analysis
of PHB1 expression in the LNCaP tumor tissue after systemic
treatment by control NPs, PHB1 siRNA-loaded NPsR10 and PHB1
siRNA-loaded ACUPA-NPsR10 indicated that the siRNA loaded NPs
inhibited PHB1 expression. With the ACUPA ligand targeting tumor
tissues, injection of siRNA-loaded ACUPA-NPsR10 leads to more than
70% knockdown of PHB1 expression. In contrast, there is only around
33% knockdown for mice treated with siRNA-loaded NPsR10 (FIG. 24).
In addition, the administration of NPs shows neglectable in vivo
side effects. After five consecutive injections of the NPs to
healthy mice (once every two days at a 650 .mu.g/kg siRNA dose,
n=3), there are no noticeable histological changes in the tissues
from heart, liver, spleen, lung or kidney. To determine whether
this NP-mediated PHB1 silencing has an anti-tumor effect, the PHB1
siRNA-loaded NPs were intravenously injected to the LNCaP xenograft
tumor-bearing mice (once every two days at a 650 .mu.g/kg siRNA
dose, n=5). As shown in FIG. 23, the siRNA loaded NPs do indeed
inhibit tumor growth. In particular, due to their excellent PCa
specificity, the siRNA-loaded ACUPA-NPsR10 significantly suppress
tumor growth after five consecutive injections and there is less
than a 3-fold increase in the tumor size at 30 days after the first
injection However, for the mice treated with GL3 siRNA-loaded
NPsR10 (Control NPs) or PBS, more than 6-fold or 8-fold increase in
the tumor size can be found at 18 days after the first injection.
Moreover, the administration of the siRNA-loaded ACUPA-NPsR10 shows
no obvious influence on body weight (FIG. 25), demonstrating the
good biocompatibility of this nanoplatform.
[0506] In conclusion, an oligoarginine-functionalized and ultra
pH-responsive nanoplatform for PCa-specific siRNA delivery has been
developed. This nanoplatform can specifically deliver siRNA to PCa
through the recognition between the ACUPA ligand and over-expressed
PSMA on PCa cells. With the endosome swelling induced by ultra
pH-responsive characteristic along with the oligoarginine-mediated
endosomal membrane penetration, this nanoplatform can efficiently
escape from endosomes and rapidly release therapeutic siRNA in the
cytoplasm, leading to a significant inhibition of cancer-associated
PHB1 expression and tumor growth. The targeted membrane-penetrating
and ultra pH-responsive nanoplatform is effective as a robust siRNA
delivery vehicle for PCa-specific therapy.
Example 3: Ultra pH-Responsive Nanoparticles (NPs) as Nanoprobe for
Cancer Diagnostics
Methods and Materials
Synthesis of Meo-PEG-Br and Br-PEG-COOH
[0507] The detailed synthesis is same as the description in
Examples 1 and 2.
Synthesis of methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate-co-glycidyl methacrylate)
(Meo-PEG-b-P(DPA-co-GMA))
[0508] Meo-PEG113-b-P(DPA80-co-GMA5) copolymer was synthesized
according to the same method described in Example 1.
Synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA)
[0509] Meo-PEG-b-P(DPA-co-GMA-TEPA) was synthesized according to
the same method described in Example 1.
Synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5)
[0510] Meo-PEG-b-P(DPA-co-GMA-TEPA) (0.2 g) and Cy5.5 NHS ester
(1.5-fold molar excess relative to the TEPA repeating unit) were
well dissolved in 5 mL of THF. After constantly stirring in dark
for 48 h, the solution was dialyzed against deionized water and the
product was collected after freeze-drying. The synthesis scheme is
shown above.
Synthesis of Meo-PEG-b-P(DPA-co-GMA-TEPA-C14)
[0511] Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) was synthesized according
to the same method described in Example 1.
Preparation of Cy5.5-labelled NPs
[0512] The THF solution of Meo-PEG-b-P(DPA-co-GMA-TEPA-C14) (4
mg/mL) and Meo-PEG-b-P(DPA-co-GMA-TEPA-Cy5.5) (4 mg/mL) was mixed
in a volume ratio of 8:2. Under vigorously stirring (1000 rpm), 0.5
mL of the mixture was added dropwise to 5 mL of deionized water.
After collection and purification by an ultrafiltration device (EMD
Millipore, MWCO 100 kDa), the NPs formed were dispersed in 1 mL of
PBS buffers (pH 7.4). The size and zeta potential of the
Cy5.5-labelled NPs were determined by DLS. The morphology of NPs
was visualized on TEM. Before observation, the sample was stained
with 1% uranyl acetate and dried under air.
[0513] Evaluation of pH Responsiveness
[0514] The Cy5.5-labelled NPs were prepared as described above and
then dispersed in 1 mL of deionized water. Subsequently, 1 M NaOH
or HCl was added in 1-5 .mu.L increments, and the fluorescence
intensity of the NPs was measured on a Synergy HT multi-mode
microplate reader. The normalized fluorescence intensity (NFI) vs.
pH profile was used to quantitatively assess the pH responsiveness.
NFI is calculated as follows:
NFI=(F-F.sub.min)/(F.sub.max-Fmin)
[0515] where F is the fluorescence intensity of the NPs at any
given pH value and F.sub.max and F.sub.min are the maximal and
minimal fluorescence intensity of the NPs, respectively.
Results
[0516] Imaging agents such as fluorescent dye can also be
conjugated to the ultra pH-responsive copolymer to prepare
dye-labelled NPs for disease diagnostics. The fluorescent dye can
be, but is not limited to, Cy5.5. Cy5.5 was conjugated to the
structure of the ultra pH-responsive copolymer (FIG. 26A). This
dye-labelled copolymer can self-assemble into NPs with the
aggregation of fluorophores inside the hydrophobic cores (FIG.
26B). Due to the quenching of the fluorophores, fluorescence signal
is absent at a pH above pKa. However, protonation of the PDPA
segment at pH below pKa causes the NPs to disassemble, leading to a
dramatic increase in the fluorescence signal. Measuring the
fluorescence intensity upon the pH change reveals that the pH
difference from 10 to 90% fluorescence activation (ApH10-90%) is
0.39 (FIG. 2A). This value is much smaller than that of small
molecule dyes (about 2 pH units) with the same degree of
fluorescence intensity change, demonstrating the ultra-fast pH
response characteristic of the NPs. Compared to normal tissue, the
microenvironment of tumor tissue is weakly acidic. Therefore, the
dye-labelled ultra pH-responsive NPs can be applied for targeted
cancer diagnostics.
Example 4: Ultra pH-Responsive Nanoplatform for Anticancer Drug
Delivery and Cancer Therapy
Methods and Materials
Synthesis of Meo-PEG-Br and Br-PEG-COOH
[0517] The detailed synthesis is same as the description in
Examples 1 and 2.
Synthesis of methoxyl-polyethylene glycol-b-poly
(2-(diisopropylamino) ethylmethacrylate) (Meo-PEG-b-PDPA)
[0518] Meo-PEG113-b-PDPA80 copolymer was synthesized by using ATRP
method. In brief, DPA-MA (2.6 g, 12 mmol), Meo-PEG-Br (0.75 g, 0.15
mmol), and PMDETA (31.5 4, 0.15 mmol) were added to a
polymerization tube. DMF (3 mL) and 2-propanol (3 mL) were then
added to dissolve the monomer and initiator. After three cycles of
freeze-pump-thaw to remove oxygen, CuBr (21.6 mg, 0.15 mmol) was
added under nitrogen atmosphere and the polymerization tube was
sealed under vacuum. After polymerization at 40.degree. C. for 24
h, tetrahydrofuran (THF) was added to dilute the product, which was
then passed through a neutral Al.sub.2O.sub.3 column to remove the
catalyst. The resulting THF solution was concentrated and the
residue was dialyzed against THF, followed by deionized water. The
expected copolymer was collected as a white powder after
freeze-drying under vacuum.
[0519] Acid-Base Titration
[0520] Meo-PEG-b-PDPA was dispersed in deionized water, and a
concentrated HCl aqueous solution was added until the copolymer was
completely dissolved (1 mg/mL). Subsequently, 1 M NaOH aqueous
solution was added in 1-5 .mu.L increments. After each addition,
the solution was constantly stirred for 3 min, and the solution pH
was measured using a pH meter. The pKa of the copolymer was
determined as the pH at which 50% of the copolymer turns
ionizes.
Preparation and Characterization of Nanoparticles (NPs)
[0521] Meo-PEG-b-PDPA was dissolved in THF to form a homogenous
solution with a concentration of 5 mg/mL. Subsequently, a certain
volume of this THF solution was taken and added dropwise to 4 mL of
deionized water under vigorously stirring (1000 rpm). The NP
dispersion formed was transferred to an ultrafiltration device (EMD
Millipore, MWCO 100 K) and centifuged to remove the organic solvent
and free compounds. After washing with PBS (pH 7.4) solution
(3.times.5 mL), the NPs were dispersed in 1 mL of phosphate
buffered saline (PBS, pH 7.4) solution. Size and zeta potential
were determined by DLS. The morphology of NPs was visualized on
TEM. Before observation, the sample was stained with 1% uranyl
acetate and dried under air.
[0522] To prepare the PTX loaded NPs, a certain volume of polymer
solution (5 mg/mL in THF) was taken and mixed with PTX (20 .mu.L,
20 mg/mL THF solution). Under vigorously stirring (1000 rpm), the
mixture was added dropwise to 4 mL of deionized water. The NPs were
collected and purified according to the same method describe above.
To determine the PTX encapsulation efficiency, a small volume (50
.mu.L) of the NP solution was withdrawn and mixed with 20-fold
DMSO. The UV absorption was examined on a UV-Vis spectrometer and
compared to the free PTX solution (5 .mu.L stock solution mixed
with 20-fold DMSO).
[0523] In Vitro Drug Release
[0524] The PTX loaded NPs were prepared as described above.
Subsequently, the NPs were dispersed in 1 mL of PBS (pH 7.4) and
then transferred to a Float-a-lyzer G2 dialysis device (MWCO 100
kDa, Spectrum) that was immersed in PBS (pH 7.4) at 37.degree. C.
At a predetermined interval, 5 .mu.L of the NP solution was
withdrawn and mixed with 20-fold DMSO. The UV absorption was
examined on a UV-Vis spectrometer and compared to the standard PTX
work curve. The average value of three independent experiments was
collected and the cumulative PTX release was calculated as
follows:
Cumulative PTX release (%)=(M.sub.t/M.sub..infin.).times.100
[0525] where M.sub.t is the amount of PTX released from the
micelles and is the amount of PTX loaded in the micelles.
[0526] In Vitro Cytotoxicity
[0527] Prostate cancer cells (PC3, DU145 and LNCaP) were seeded in
a 96-well plate with a density of 5000 cells/well. After the
incubation in 100 .mu.L of RPMI-1640 containing 10% FBS for 24 h, a
fixed amount of PTX loaded NPs dispersed in 100 .mu.L of RPMI-1640
was added and the cells were allowed to incubate for another 48 h.
After replacing the medium with 100 pt of fresh RPMI-1640, 10 .mu.L
of alamarBlue agent was added to each well and the cells were
further incubated for 1 h. The cytotoxicity was measured using the
alamarBlue assay according to the manufacturer's protocol. The
average value of six independent experiments was collected and the
cell viability was calculated as follows:
Viability (%)=(OD.sub.treated/OD.sub.control).times.100
[0528] where OD.sub.control is obtained in the absence of the PTX
loaded NPs and OD.sub.treated is obtained in the presence of the
PTX loaded NPs.
Results
[0529] Chemotherapeutic drugs can be also encapsulated into the
ultra pH-responsive NPs for disease treatment. The chemotherapeutic
drugs can be, but are not limited to, docetaxel (DTX), paclitaxel
(PTX), doxorubicin (DOX), mitoxantrone (MTX), etc. Ultra
pH-responsive PEGylated copolymer was synthesized (FIG. 27A), which
can co-assemble with anticancer drug PTX to form spherical NPs with
PTX loaded in their hydrophobic core as observed in TEM images
taken of the NPs of Meo-PEG-b-PDPA in PBS buffer at a pH of 7.4.
The PTX loading efficacy is more than 10% and the size of the PTX
loaded NPs is around 100 nm. TEM images of the NPs of
Meo-PEG-b-PDPA in PBS buffer at a pH of 5.0 showed that with the
rapid protonation of the ultra pH-responsive copolymer, there are
no observable NPs at a pH below pKa, thus leading to a super-fast
PTX release (FIG. 27B).
Example 5: Light-Responsive Nanoplatform for Anticancer Drug
Delivery and Cancer Therapy
Methods and Materials
Synthesis of 2-(2-oxo-2-phenylacetoxy) ethyl methacrylate
(OPEMA)
[0530] Phenylglyoxylic acid (PGA, 13.5 g, 90 mmol), 2-hydroxyethyl
methacrylate (HEMA, 21.06 g, 162 mmol), and 4-dimethylaminopyridine
(DMAP) were well dissolved 200 mL of DCM. In an ice-salt bath,
N,N'-dicyclohexylcarbodiimide (DCC, 22.2 g, 108 mmol) dissolved in
110 mL of DCM was added. After reaction at room temperature
overnight, the mixture was filtered and filtration was washed with
water (3.times.50 mL), 10% HCl (3.times.50 mL), and saturated
Na2CO3 solution (3.times.50 mL). After drying over anhydrous
Na2SO4, the solvent was removed and the final product was collected
as powder. The synthesis scheme is shown below.
Synthesis of Meo-PEG-Br and Br-PEG-COOH
[0531] The detailed synthesis is same as the description in
Examples 1 and 2.
Synthesis of methoxyl-polyethylene glycol-b-poly
(2-(2-oxo-2-phenylacetoxy) ethyl methacrylate)
(Meo-PEG-b-POPEMA)
[0532] Meo-PEG113-b-POPEMA80 copolymer was synthesized using ATRP
method. In brief, OPEMA (3.15 g, 12 mmol), Meo-PEG-Br (0.75 g, 0.15
mmol), and PMDETA (31.5 .mu.L, 0.15 mmol) were added to a
polymerization tube. DMF (3 mL) and 2-propanol (3 mL) were then
added to dissolve the monomer and initiator. After three cycles of
freeze-pump-thaw to remove oxygen, CuBr (21.6 mg, 0.15 mmol) was
added under nitrogen atmosphere and the polymerization tube was
sealed under vacuum. After polymerization at 40.degree. C. for 24
h, tetrahydrofuran (THF) was added to dilute the product, which was
then passed through a neutral Al.sub.2O.sub.3 column to remove the
catalyst. The resulting THF solution was concentrated and the
residue was dialyzed against THF, followed by deionized water. The
expected copolymer was collected as a white powder after
freeze-drying under vacuum.
[0533] The synthesis scheme is shown below.
##STR00009##
Preparation and Characterization of Nanoparticles (NPs)
[0534] Meo-PEG-b-POPEMA was dissolved in THF to form a homogenous
solution with a concentration of 10 mg/mL. Subsequently, a certain
volume of this THF solution was taken and added dropwise to 5 mL of
deionized water under vigorously stirring (1000 rpm). The NP
dispersion formed was transferred to an ultrafiltration device (EMD
Millipore, MWCO 100 K) and centrifuged to remove the organic
solvent and free compounds. After washing with PBS (pH 7.4)
solution (3.times.5 mL), the NPs were dispersed in 1 mL of
phosphate buffered saline (PBS, pH 7.4) solution. Size and zeta
potential were determined by DLS. The morphology of NPs was
visualized on TEM. Before observation, the sample was stained with
1% uranyl acetate and dried under air.
[0535] Determination of Light-Sensitivity
[0536] The NPs of Meo-PEG-b-POPEMA were prepared as described above
and then dispersed in 1 mL of deionized water. The solution of the
NPs was placed under 365 nm UV light (16 W) for different time
periods. The size of the NPs was examined at pre-determined time
points. After 24 h UV irradiation, the solution was freeze-dried
and the sample was dissolved in DMF for GPC analysis.
[0537] Preparation of Drug Loaded NPs
[0538] To prepare the drug loaded NPs, a certain volume of the
polymer solution (10 mg/mL in THF) was taken and mixed with PTX (20
.mu.L, 20 mg/mL THF solution). Under vigorously stirring (1000
rpm), the mixture was added dropwise to 4 mL of deionized water.
The NPs were collected and purified according to the same method
describe above. To determine the PTX encapsulation efficiency, a
small volume (50 .mu.L) of the NP solution was withdrawn and mixed
with 20-fold DMSO. The UV absorption was examined on a UV-Vis
spectrometer and compared to the free PTX solution (5 .mu.L stock
solution mixed with 20-fold DMSO).
[0539] In Vitro Drug Release
[0540] The PTX loaded NPs were prepared as described above.
Subsequently, the NPs were dispersed in 1 mL of PBS (pH 7.4) and
then transferred to a Float-a-lyzer G2 dialysis device (MWCO 100
kDa, Spectrum) that was immersed in PBS (pH 7.4) at 37.degree. C.
At a predetermined interval, 5 .mu.L of the NP solution was
withdrawn and mixed with 20-fold DMSO. The UV absorption was
examined on a UV-Vis spectrometer and compared to the standard PTX
work curve. The average value of three independent experiments was
collected and the cumulative PTX release was calculated as
follows:
Cumulative PTX release (%)=(M.sub.t/M.sub..infin.).times.100
[0541] where M.sub.t is the amount of PTX released from the
micelles and is the amount of PTX loaded in the micelles.
[0542] In Vitro Cytotoxicity
[0543] Prostate cancer cells (PC3, DU145 and LNCaP) were seeded in
a 96-well plate with a density of 5000 cells/well. After the
incubation in 100 .mu.L of RPMI-1640 containing 10% FBS for 24 h, a
fixed amount of PTX loaded NPs dispersed in 100 .mu.L of RPMI-1640
was added and the cells were allowed to incubate for another 48 h.
After replacing the medium with 100 .mu.L of fresh RPMI-1640, 10
.mu.L of alamarBlue agent was added to each well and the cells were
further incubated for 1 h. The cytotoxicity was measured using the
alamarBlue assay according to the manufacturer's protocol. The
average value of six independent experiments was collected and the
cell viability was calculated as follows:
Viability (%)=(OD.sub.treated/OD.sub.control).times.100
[0544] where OD.sub.control is obtained in the absence of the PTX
loaded NPs and OD.sub.treated is obtained in the presence of the
PTX loaded NPs.
Results
[0545] Stimuli-responsive amphiphilic copolymers can be used to
prepare the NPs for delivery of therapeutic and diagnostic agents
including but not limited to genes, chemotherapeutic drugs, or
other small molecules. These amphiphilic polymers can be, but not
limited to, light-, redox-, and temperature-responsive polymers.
The light-sensitive monomer, 2-(2-oxo-2-phenylacetoxy) ethyl
methacrylate (OPEMA) was synthesized, and atom-transfer radical
polymerization (ATRP) was used to synthesize the PEGylated
light-sensitive copolymer: (FIG. 28A). Under 365 nm UV light
irradiation, this copolymer can be degraded and there is
significant decrease in its molecular weight (FIG. 28B). Due to the
amphiphilic nature, this copolymer can self-assemble into spherical
NPs with an average size of 80 nm as seen in TEM images of the NPs
of Meo-PEG-b-POPEMA in PBS buffer (pH 7.4) before 365 nm UV light
irradiation. Under 365 nm UV light irradiation for 30 min, there
are no observed NPs under transmission electron microscope observed
in the TEM images of the NPs of Meo-PEG-b-POPEMA in PBS buffer (pH
7.4). This morphological change leads to a rapid release of loaded
anticancer drug DTX (FIG. 29A) and efficient anticancer activity
(FIG. 29B).
Example 6: Fast Redox-Responsive Nanoplatform for siRNA Delivery
with Robust Anti-Cancer Efficacy
Methods and Materials
Synthesis of the L-cystine-based poly(disulfide) (PDSA)
polymers
[0546] PDSA polymers were prepared by one-step polycondensation of
L-cystine dimethyl ester dihydrochloride ((H-Cys-OMe)2.2HCl) and
dichlorides or Bis-nitrophenol esters of different fatty diacids. A
standard synthesis procedure was carried out as follows:
(H-Cys-OMe)2.2HCl (10.0 mmol) and triethylamine (15 mmol) were
dissolved in 20.0 mL DMSO, then the dichloride of fatty acid (10.0
mmol) DMSO solution (10.0 mL) was added into the cystine mixture
solution dropwise. The solution was stirred for 15 mins to obtain a
uniform mixture, precipitated twice in 250 mL of cold ethyl ether,
and dried under reduced atmosphere. The final product was a yellow
or brown yellow powder. The synthesis scheme is shown below.
##STR00010##
[0547] Redox-Responsive Behavior of the PDSA Polymers
[0548] GPC analysis was used to study the redox-responsive behavior
of the PDSA polymers. The polymer (1 mg) was dissolved in 2 mL of
DMF/H2O (9:1, V/V) and then GSH (6.2 mg, 0.02 mmol) was added to
obtain a solution with GSH concentration of 10 mM. At predetermined
intervals, 100 .mu.L of the solution was taken for GPC
analysis.
Preparation and Characterization of Nanoparticles (NPs)
[0549] The PDSA polymers were dissolved in DMF or DMSO to form a
homogenous solution with a concentration of 20 mg/mL. Subsequently,
200 .mu.L of this solution was taken and mixed with 140 .mu.L of
DSPE-PEG3000 (20 mg/mL in DMF), 50 .mu.L of G0-C14 (5 mg/mL in DMF)
and 1 nmol siRNA (0.1 nmol/.mu.L aqueous solution). Under
vigorously stirring (1000 rpm), the mixture was added dropwise to 5
mL of deionized water. The NP dispersion formed was transferred to
an ultrafiltration device (EMD Millipore, MWCO 100 K) and
centrifuged to remove the organic solvent and free compounds. After
washing with PBS (pH 7.4) solution (3.times.5 mL), the siRNA loaded
NPs were dispersed in 1 mL of phosphate buffered saline (PBS, pH
7.4) solution. Size and zeta potential were determined by DLS. The
morphology of NPs was visualized on TEM. To determine the siRNA
encapsulation efficiency, DY547-labelled GL3 siRNA (DY547-siRNA)
loaded NPs were prepared according to the method described above. A
small volume (50 .mu.L) of the NP solution was withdrawn and mixed
with 20-fold DMSO. The fluorescence intensity of DY547-siRNA was
measured using a Synergy HT multi-mode microplate reader (BioTek
Instruments) and compared to the free DY-547 labelled GL3 siRNA
solution (1 nmol/mL PBS solution).
[0550] Redox-Responsive Behavior of the NPs
[0551] The siRNA loaded NPs were prepared as described above and
dispersed in PBS containing 10 mM GSH. At pre-determined time
point, the particle size was examined by DLS and the particle
morphology was observed on TEM. To evaluate the intracellular
redox-responsive behavior, the NPs with Nile red and coumarin 6
encapsulated in their hydrophobic cores were prepared and then
incubated with HeLa cells for different time. The fluorescence of
Nile red and coumarin 6 was observed a FV1000 confocal laser
scanning microscope (CLSM, Olympus). If the NPs respond to redox
stimulus, the Nile red and coumarin 6 will release and only green
fluorescence of coumarin 6 can be observed under CLSM. If the NPs
are intact, the fluorescence of coumarin 6 will be quenched by Nile
red and only red fluorescence can be observed under CLSM.
[0552] Evaluation of Endosomal Escape
[0553] Luc-HeLa cells (20,000 cells) were seeded in discs and
incubated in 1 mL of RPMI 1640 medium containing 10% FBS for 24 h.
Subsequently, the DY547-siRNA-loaded NPs were added, and the cells
were allowed to incubate for 1 or 2 h. After removing the medium
and subsequently washing with PBS (pH 7.4) solution thrice, the
endosomes and nuclei were stained with lysotracker green and
Hoechst 33342, respectively. The cells were then viewed under
CLSM.
[0554] In Vitro siRNA Release
[0555] DY547-siRNA-loaded NPs were prepared as described above.
[0556] Subsequently, the NPs were dispersed in 1 mL of PBS (pH 7.4)
and then transferred to a Float-a-lyzer G2 dialysis device (MWCO
100 kDa, Spectrum) that was immersed in PBS (pH 7.4) at 37.degree.
C. At a predetermined interval, 5 .mu.L of the NP solution was
withdrawn and mixed with 20-fold DMSO. The fluorescence intensity
of DY547-siRNA was determined by Synergy HT multi-mode microplate
reader.
[0557] Luciferase Silencing
[0558] Luc-HeLa cells were seeded in 96-well plates (5,000 cells
per well) and incubated in 0.1 mL of RPMI 1640 medium with 10% FBS
for 24 h. Thereafter, the GL3 siRNA-loaded NPs were added. After
incubating for 24 h, the cells were washed with fresh medium and
allowed to incubate for another 48 h. The expression of firefly
luciferase in HeLa cells was determined using Steady-Glo luciferase
assay kits. Cytotoxicity was measured using the alamarBlue assay
according to the manufacturer's protocol. The luminescence or
fluorescence intensity was measured using a microplate reader, and
the average value of five independent experiments was collected. As
a control, the silencing effect of Lipo2K/GL3 siRNA complexes was
also evaluated according to the procedure described above and
compared to that of GL3 siRNA-loaded NPs.
[0559] In Vitro KIF11 Silencing
[0560] Prostate cancer cells (PC3, LNCaP, DU145 and 22Rv1) were
seeded in 6-well plates (50,000 cells per well) and incubated in 1
mL of RPMI 1640 medium containing 10% FBS for 24 h. Subsequently,
the cells were transfected with the KIF11 siRNA-loaded NPs for 24
h. After washing the cells with PBS thrice, the cells were further
incubated in fresh medium for another 48 h. Thereafter, the cells
were digested by trypsin and the proteins were extracted using
modified radioimmunoprecipitation assay lysis buffer (50 mM
Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40 substitute, 0.25% sodium
deoxycholate, 1 mM sodium fluoride, 1 mM Na3VO4, 1 mM EDTA),
supplemented with protease inhibitor cocktail and 1 mM
phenylmethanesulfonyl fluoride (PMSF). The expression of KIF11 was
examined using the western blot analysis.
[0561] In Vitro Cell Proliferation
[0562] PC3 cells were seeded in 6-well plates (20,000 cells per
well) and incubated in 1 mL of RPMI 1640 medium containing 10% FBS
for 24 h. Thereafter, the cells were transfected with the KIF11
siRNA-loaded NPs for 24 h and then washed with fresh medium for
further incubation. At predetermined intervals, the cytotoxicity
was measured using the alamarBlue assay according to the
manufacturer's protocol. After each measurement, the alamarBlue
agent was removed and the cells were incubated in fresh medium for
further proliferation.
[0563] PC3 Xenograft Tumor Model
[0564] The tumor model was constructed by subcutaneous injection
with 200 .mu.L of LNCaP cell suspension (a mixture of RPMI 1640
medium and Matrigel in 1:1 volume ratio) with a density of
2.times.10.sup.6 cells/mL into the back region of healthy male
BALB/c nude mice. When the volume of the PC3 tumor xenograft
reached .about.50 mm.sup.3, the mice were used for the following in
vivo experiments.
[0565] Pharmacokinetics Study
[0566] Healthy male BALB/c mice were randomly divided into two
groups (n=3) and given an intravenous injection of either (i) free
DY647-labelled GL3 siRNA (DY647-siRNA) and (ii) DY647-siRNA-loaded
NPs at a 650 .mu.g/kg siRNA dose. At predetermined time intervals,
orbital vein blood (20 .mu.L) was withdrawn using a tube containing
heparin, and the wound was pressed for several seconds to stop the
bleeding. The fluorescence intensity of DY-647 labelled siRNA in
the blood was determined using a microplate reader. The blood
circulation half-life (t1/2) was calculated by first-order decay
fit.
[0567] Biodistribution
[0568] PC3 tumor-bearing male BALB/c nude mice were randomly
divided into two groups (n=3) and given an intravenous injection of
either (i) free DY677-labelled GL3 siRNA (DY677-siRNA) or (ii)
DY677-siRNA-loaded NPs at a 650 .mu.g/kg siRNA dose. Twenty-four
hours after the injection, the mice were imaged using the Maestro 2
In-Vivo Imaging System (Cri Inc). Main organs and tumors were then
harvested and imaged. To quantify the accumulation of NPs in tumors
and organs, the fluorescence intensity of each tissue was
quantified by Image-J.
[0569] In Vivo KIF11 Silencing
[0570] PC3 tumor-bearing male BALB/c nude mice were randomly
divided into two groups (n=3) and intravenously injected with (i)
KIF11 siRNA-loaded NPs or (ii) GL3 siRNA-loaded NPs at a 650
.mu.g/kg siRNA dose for three consecutive days. Twenty-four hours
post the final injection, mice were sacrificed and tumors were
harvested. The proteins in the tumor were extracted using modified
radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl pH 7.4,
150 mM NaCl, 1% NP-40 substitute, 0.25% sodium deoxycholate, 1 mM
sodium fluoride, 1 mM Na.sub.3VO.sub.4, 1 mM EDTA), supplemented
with protease inhibitor cocktail and 1 mM phenylmethanesulfonyl
fluoride (PMSF). The expression of KIF11 was examined using the
aforementioned western blot analysis.
[0571] Inhibition of Tumor Growth
[0572] PC3 tumor-bearing male BALB/c nude mice were randomly
divided into four groups (n=5) and intravenously injected with (i)
PBS, (ii) Naked KIF11 siRNA, (iii) Blank NPs, and (iv) KIF11
siRNA-loaded NPs at a 650 .mu.g/kg siRNA dose. All the mice were
administrated four injections and the tumor growth was monitored
every two days by measuring perpendicular diameters using a caliper
and tumor volume was calculated as follows:
V=W.sup.2.times.L/2
[0573] where W and L are the shortest and longest diameters,
respectively.
[0574] Histology
[0575] Healthy male BALB/c mice were randomly divided into three
groups (n=3) and administered daily intravenous injections of
either (i) PBS or (ii) KIF11 siRNA-loaded NPs at a 650 .mu.g/kg
siRNA dose. After four consecutive injections (once every two
days), the main organs were collected 2 days post the final
injection, fixed with 4% paraformaldehyde, and embedded in
paraffin. Tissue sections were stained with hematoxylin-eosin
(H&E) and viewed under an optical microscope.
[0576] Results
[0577] Besides amphiphilic copolymers, hydrophobic polymers can be
also used to develop stimuli-responsive NPs for various biomedical
applications. For these hydrophobic polymers, their NPs are
prepared by using the mixture of the hydrophobic polymer and
amphiphilic compound. The amphiphilic compound can be, but is not
limited to, one or a plurality of the following: naturally derived
lipids, lipid-like materials, surfactants, synthesized amphiphilic
compounds, or combinations thereof.
[0578] Redox-responsive hydrophilic polymer was synthesized which
could co-assemble with lipid-PEG to form spherical NPs for gene
delivery and cancer therapy (FIG. 30). The intracellular levels of
glutathione (GSH) are 100-1000 fold higher in cancer cells than in
normal tissue. Redox-sensitive approach is particularly promising
to enhance the exposure of cancer cells to therapeutic molecules.
In this example, L-cystine dimethyl ester and fatty diacid were
used to synthesize a library of L-cystine-based poly(disulfide
amide) polymers (PDSA).
[0579] Feed compositions and molecular weight of the PDSA polymers
are summarized in Table 7. Taking PDSA8-1, for example, with the
presence of many disulfide bonds, there is a significant decrease
in the molecule weight of PDSA8-1 incubated in 10 mM glutathione
(GSH) solution. When mixing this redox-responsive polymer with
DSPE-PEG3000, siRNA and cationic lipid (Xiaoyang Xu et al. Proc
Natl Acad Sci USA, 110, 18638-18643(2013)) in water miscible
solvent such as DMF, DMSO, THF, etc., spherical NPs with an average
size of .about.100 nm (as seen in TEM image of the NPs of the
PDSA8-1) can be formed via nanoprecipitation method, in which
hydrophilic PEG chains are on the outer shell and siRNA is
encapsulated in the hydrophobic core. The physiochemical properties
of other PDSA polymers are summarized in Table 8. With the
redox-responsive characteristic to induce the breakage of the NPs
of PDSA8-1 (FIG. 31A and observed by TEM imaging), the siRNA loaded
NPs of PDSA8-1 show efficient endosomal escape ability as seen in
fluorescent images of HeLa cells incubated with the siRNA loaded
NPs of tPDSA8-1 for 1 hour and 4 hour time points; and high
efficacy in down-regulation of luciferase expression in HeLa cells
(>90% knockdown at 1 nM siRNA dose, FIG. 31B). These
redox-responsive NPs can be used as a robust nanoplatform to
deliver therapeutic siRNA for prostate cancer therapy. After
treatment with the NPs loading kinesin family member 11 (KIF11)
siRNA, there is a significant decrease in the expression of KIF11
in four prostate cancer cell lines (PC3, LNCaP, 22Rv1 and DU145) at
a very low siRNA dose by Western blot analysis of KIF11 expression
in prostate cancer cells treated with KIF11 siRNA loaded NPs of
PDSA8-1. Especially for PC3 cells, there is nearly no KIF11
expression at an extremely low siRNA dose (5 nM) as seen by
immunofluorescence analysis of PC3 cells treated by KIF11 siRNA
loaded NPs of PDSA8-1 at a 0 and 5 nM siRNA dose. With this
down-regulated KIF11 expression, the proliferation rate of PC3
cells is significantly inhibited and there is around 80% decrease
in the cell number decreases at a siRNA dose of 10 nM (FIG. 32). In
vivo experiment results demonstrated that these NPs have a long
blood circulation (FIG. 33) and show high accumulation in PC3
xenograft tumor of mice as seen in overlaid fluorescent image of
the PC3 xenograft tumor-bearing nude mice 24 h post systemic
injection of naked DY677-siRNA, and DY677-siRNA loaded NPs of
PDSA8-1. This lead to around 90% knockdown of KIF11 expression in
tumor tissue assessed by Western blot analysis of KIF11 expression
in the PC3 tumor tissue after systemic treatment by KIF11 siRNA
loaded NPs of PDSA8-1 (FIG. 34), and significant inhibition of
tumor growth within 52 days (FIG. 35).
TABLE-US-00007 TABLE 7 Feed compositions and molecular weight of
the PDSA polymers. Poly(disulfide amide) M.sub.n.sup.a
M.sub.w.sup.a Polydispersity .sup.a m = 4 PDSA4 2900 4300 1.48 m =
6 PDSA6 3900 5700 1.48 m = 8 PDSA8-1 5700 7300 1.43 m = 10 PDSA10
9100 13200 1.45 m = 8 PDSA8-2 4700 7800 1.66 m = 8 PDSA8-3 9300
15200 1.63 m = 8 PDSA8-4 11700 16600 1.42 .sup.aDetermined by GPC
using DMF as the client.
TABLE-US-00008 TABLE 8 Size, siRNA encapsulation efficiency (EE %)
and zeta potential of the NPs of PDSA polymers. PDSA4 PDSA6 PDSA8-1
PDSA10 PDSA8-2 PDSA8-3 PDSA8-4 Size (nm) .sup.a 155.7 134.5 102.9
87.6 118.9 99.4 93.4 EE % .sup.b 29.7 35.1 55.9 82.9 46.3 79.4 88.2
.xi. (mV) -6.79 -8.08 -11.21 -15.05 -9.79 -12.05 -20.01 .sup.a N:P
ratio is 20:1; .sup.b siRNA encapsulation efficiency.
Example 7: Ultra pH-Responsive Nanoplatform for Anticancer Drug
Delivery and Cancer Therapy
Methods and Materials
Synthesis of poly (2-(diisopropylamino) ethylmethacrylate
(PDPA)
[0580] DPA was synthesized by radical polymerization using
2-aminoethanethiol hydrochloride (AET.HCl) as a chain transfer
agent. In brief, DPA-MA (4.27 g, 20 mmol), AET.HCl (0.88 mmol, 0.1
g), and AIBN (18 mg, 0.11 mmol) were dissolved in 15 mL of DMF. The
solution was degassed by bubbling with nitrogen for 30 min. The
mixture reacted at 70.degree. C. for 6 h under nitrogen. Then, the
product was precipitated by the addition of chilled methanol. The
final PDPA was collected after drying in vacuum for 24 h.
Preparation and Characterization of Nanoparticles (NPs)
[0581] The PDPA polymer was dissolved in THF to form a homogenous
solution with a concentration of 5 mg/mL. Subsequently, 250 .mu.L
of this solution was taken and mixed with 125 .mu.L of DSPE-PEG3000
solution (5 mg/mL in DMF). Under vigorously stirring (1000 rpm),
the mixture was added dropwise to 5 mL of deionized water. The NP
dispersion formed was transferred to an ultrafiltration device (EMD
Millipore, MWCO 100 K) and centrifuged to remove the organic
solvent and free compounds. After washing with PBS (pH 7.4)
solution (3.times.5 mL), the final NPs were dispersed in 1 mL of
phosphate buffered saline (PBS, pH 7.4) solution. Size and zeta
potential were determined by DLS. The morphology of NPs was
visualized on TEM.
[0582] Ultra pH-Responsive Behavior of the NPs
[0583] The pH-responsive behavior of the NPs was evaluated by
examining the particle size change at a pH below pKa. In brief, the
NPs of PDPA were prepared as described above and then dispersed in
deionized water. After adding concentrated HCl solution to adjust
the solution pH to a value of 5.0, the particle size was examined
by DLS.
Preparation of PTX Loaded Nanoparticles (NPs)
[0584] To prepare the paclitaxel (PTX) loaded NPs, a certain volume
of polymer solution (5 mg/mL in THF) was taken and mixed with 125
.mu.L of DSPE-PEG3000 solution (5 mg/mL in DMF) and PTX (20 .mu.L,
20 mg/mL THF solution). Under vigorously stirring (1000 rpm), the
mixture was added dropwise to 5 mL of deionized water. The NPs were
collected and purified according to the same method describe above.
To determine the PTX encapsulation efficiency, a small volume (50
.mu.L) of the NP solution was withdrawn and mixed with 20-fold
DMSO. The UV absorption was examined on a UV-Vis spectrometer and
compared to the free PTX solution (54 stock solution mixed with
20-fold DMSO).
[0585] In Vitro Drug Release
[0586] The PTX loaded NPs were prepared as described above.
Subsequently, the NPs were dispersed in 1 mL of PBS (pH 7.4) and
then transferred to a Float-a-lyzer G2 dialysis device (MWCO 100
kDa, Spectrum) that was immersed in PBS (pH 7.4) at 37.degree. C.
At a predetermined interval, 54 of the NP solution was withdrawn
and mixed with 20-fold DMSO. The UV absorption was examined on a
UV-Vis spectrometer and compared to the standard PTX work curve.
The average value of three independent experiments was collected
and the cumulative PTX release was calculated as follows:
Cumulative PTX release (%)=(Mt/M.infin.).times.100
[0587] where Mt is the amount of PTX released from the micelles and
Moo is the amount of PTX loaded in the micelles.
[0588] In Vitro Cytotoxicity
[0589] Prostate cancer cells (PC3, DU145 and LNCaP) were seeded in
a 96-well plate with a density of 5000 cells/well. After the
incubation in 100 .mu.L of RPMI-1640 containing 10% FBS for 24 h, a
fixed amount of PTX loaded NPs dispersed in 100 .mu.L of RPMI-1640
was added and the cells were allowed to incubate for another 48 h.
After replacing the medium with 100 .mu.L of fresh RPMI-1640, 10
.mu.L of alamarBlue agent was added to each well and the cells were
further incubated for 1 h. The cytotoxicity was measured using the
alamarBlue assay according to the manufacturer's protocol. The
average value of six independent experiments was collected and the
cell viability was calculated as follows:
Viability (%)=(ODtreated/ODcontrol).times.100
[0590] where ODcontrol is obtained in the absence of the PTX loaded
NPs and ODtreated is obtained in the presence of the PTX loaded
NPs
Results
[0591] Other than the previously discussed redox-responsive
polymer, other hydrophobic polymers can be also used to mix with
one or more amphiphilic polymers to prepare stimuli-responsive NPs
for delivery of therapeutic and diagnostic agents including genes,
chemotherapeutic drugs, or other small molecules. These hydrophobic
polymers can be, but not limited to pH-, light-, and
temperature-responsive polymers. Ultra pH-responsive polymer,
poly(2-(diisopropylamino) ethylmethacrylate) (PDPA) was
synthesized.
##STR00011##
[0592] This polymer is hydrophobic at a pH above pKa but becomes
hydrophilic at a pH below pKa. Mixing this polymer with
DSPE-PEG3000 and anticancer drug PTX in water miscible solvent,
spherical NPs with an average size of 90 nm can be formed with PTX
encapsulated into their hydrophobic core as seen by TEM imaging of
the NPs of PDPA in PBS buffer at a pH of 7.4. Due to the ultra
pH-responsive characteristic, these NPs show a super-fast PTX
release at a pH below pKa seen by TEM imaging of the NPs of PDPA in
PBS buffer at a pH of 5.0 (FIG. 22).
Example 8: Tumor Microenvironment (TME) pH-Responsive Multistaged
Nanoparticle Platform for siRNA Delivery and Cancer Therapy
Methods and Materials
[0593] Materials 2-(Hexamethyleneimino) ethanol, methacryloyl
chloride, and hydroquinone were purchased from Alfa Aesar Company
and used directly. .alpha.-Bromoisobutyryl bromide,
N,N'-dimethylformamide (DMF), triethylamine (TEA),
N,N,N',N',N'-pentamethyldiethylenetriamine (PMDETA), copper (I)
bromide (CuBr), isopropyl alcohol, dichloromethane (DCM),
tetrahydrofuran (THF), and diethyl ether were provided by
Sigma-Aldrich and used as received. Methoxyl-polyethylene glycol
(Meo-PEG.sub.113-OH) was purchased from JenKem Technology.
Tumor-targeting and cell-penetrating peptide-amphiphiles (TCPA1:
C.sub.17H.sub.35CONH-GR.sub.8GRGDS-OH; TCPA2:
C.sub.17H.sub.35CONH--(C.sub.17H.sub.35CONH)--KR.sub.8GRGDS-OH)
were obtained from GL Biochem Ltd. 2-Aminoethyl methacrylate (AMA)
were purchased from Polyscience Company. Cyanine5.5 NHS ester was
purchased from Lumiprobe. Lipofectamine 2000 (Lipo2K) was purchased
from Invitrogen. Steady-Glo luciferase assay system was provided by
Promega. Fluorescent dye DY677-labelled Luc and BRD4 siRNAs were
acquired from GE Dharmacon. The siRNA sequences are as follows: Luc
siRNA, 5'-CUU ACG CUG AGU ACU UCG AdTdT-3' (sense) (SEQ ID NO:1)
and 5'-UCG AAG UAC UCA GCG UAA GdTdT-3' (antisense) (SEQ ID NO:2);
BRD4 siRNA, 5'-AAA CAC AAC UCA AGC AUC GUU-3' (sense) (SEQ ID NO:9)
and 5'-CGA UGC UUG AGU UGU GUU UUU-3' (antisense) (SEQ ID NO:10).
DY677 was labelled at the 5'-end of both the sense and antisense
strands of Luc siRNA. Fluorescein and its quencher
(Dabcyl)-labelled Luc siRNA was also provided by GE Dharmacon.
Fluorescein was labelled at the 5'-end of the sense strand and
Dabcyl was labelled at 3'-end of the antisense strand. HeLa cells
stably expressing firefly luciferase (Luc-HeLa) were obtained from
Alnylam Pharmaceuticals, Inc. The cells were incubated in RPMI 1640
medium (Invitrogen) with 10% fetal bovine serum (FBS,
Sigma-Aldrich). All other reagents and solvents are of analytical
grade and used without further purification.
Synthesis of 2-(hexamethyleneimino) ethyl methacrylate (HMEMA)
[0594] 2-(Hexamethyleneimino) ethanol (0.1 mol, 14.3 g), TEA (0.12
mol, 12.1 g), and inhibitor hydroquinone (0.001 mol, 0.11 g) were
dissolved in 100 mL of THF and then methacryloyl chloride (0.1 mol,
10.5 g) was added dropwise. After refluxing for 2 h, the
precipitation was removed and the THF solvent was removed by rotary
evaporator. The resulting residue was distilled under vacuum as a
colorless liquid. The synthesis of HMEMA is shown below.
Synthesis Scheme of HMEMA
##STR00012##
[0595] Synthesis of Meo-PEG-Br
[0596] Meo-PEG-Br was synthesized according to the same method
described in Example 1.
Synthesis of methoxyl-polyethylene
glycol-b-poly(2-(hexamethyleneimino) ethyl methacrylate)
(Meo-PEG-b-PHMEMA)
[0597] Meo-PEG-b-PHMEMA block copolymer was synthesized by atom
transfer radical polymerization (ATRP). HMEMA (12 mmol), Meo-PEG-Br
(0.15 mmol), and PMDETA (0.15 mmol) were added to a polymerization
tube. DMF (3 mL) and 2-propanol (3 mL) were then added to dissolve
the monomer and initiator. After three cycles of freeze-pump-thaw
to remove oxygen, CuBr (0.15 mmol) was added under nitrogen
atmosphere and the polymerization tube was sealed under vacuum.
After polymerization at 40.degree. C. for 24 h, tetrahydrofuran
(THF) was added to dilute the product, which was then passed
through a neutral Al.sub.2O.sub.3 column to remove the catalyst.
The resulting THF solution was concentrated and the residue was
dialyzed against THF, followed by deionized water. The expected
polymer was collected as a white powder after freeze-drying under
vacuum. The synthesis of Meo-PEG-b-PHMEMA is shown below. The
molecular weight was determined by gel permeation chromatography
(GPC) using THF as eluent. M.sub.n,GPC=2.34.times.10.sup.4
(PDI=1.25); M.sub.n,NMR=2.15.times.10.sup.4.
Synthesis Scheme of Meo-PEG-b-PHMEMA
##STR00013##
[0598] Synthesis of methoxyl-polyethylene glycol-b-poly
(2-(hexamethyleneimino) ethyl methacrylate-co-2-aminoethyl
methacrylate) (Meo-PEG-b-P(HMEMA-co-AMA))
[0599] Meo-PEG-b-P(HMEMA-co-AMA) copolymer was synthesized by ATRP.
HMEMA (6 mmol), Meo-PEG-Br (0.075 mmol), and PMDETA (0.075 mmol)
were added to a polymerization tube. DMF (1.5 mL) and 2-propanol
(1.5 mL) were then added to dissolve the monomer and initiator.
After three cycles of freeze-pump-thaw to remove oxygen, CuBr
(0.075 mmol) was added under nitrogen atmosphere and the
polymerization tube was sealed under vacuum. After polymerization
at 40.degree. C. for 24 h, tetrahydrofuran (THF) was added to
dilute the product, which was then passed through a neutral
Al.sub.2O.sub.3 column to remove the catalyst. The resulting THF
solution was concentrated and the residue was dialyzed against THF,
followed by deionized water. The expected polymer was collected as
a white powder after freeze-drying under vacuum. The synthesis of
Meo-PEG-b-P(HMEMA-co-AMA) is shown below. The molecular weight was
determined by gel permeation chromatography (GPC) using THF as
eluent. M.sub.n,GPC=2.42.times.10.sup.4 (PDI=1.33);
M.sub.n,NMR=2.23.times.10.sup.4.
Synthesis of Meo-PEG-b-P(HMEMA-co-AMA-Cy5.5)
[0600] Meo-PEG-b-P(HMEMA-co-AMA) (0.5 g) and Cy5.5 NHS ester
(1.5-fold molar excess relative to the AMA repeating unit) were
well dissolved in 15 mL of DMF. After constantly stirring in dark
for 48 h, the solution was dialyzed against DMF for 48 h followed
deionized water for 72 h. The product was collected after
freeze-drying. The synthesis of Meo-PEG-b-P(HMEMA-co-AMA-Cy5.5) is
shown below.
Synthesis Scheme of Meo-PEG-b-P(HMEMA-co-AMA) and
Meo-PEG-b-P(HMEMA-co-AMA-Cy5.5)
##STR00014##
[0602] Gel Permeation Chromatography (GPC)
[0603] Number- and weight-average molecular weights (M.sub.n and
M.sub.w, respectively) of the polymers were determined by a gel
permeation chromatographic system according to the same method
described in Example 1.
[0604] .sup.1H Nuclear Magnetic Resonance (.sup.1HNMR)
[0605] The .sup.1HNMR spectra of the polymers were recorded
according to the same method described in Example 1.
[0606] Acid-Base Titration
[0607] Meo-PEG-b-PHMEMA was dispersed in deionized water, and a
concentrated HCl aqueous solution was added until the copolymer was
completely dissolved (1 mg/mL). Subsequently, 1 M NaOH aqueous
solution was added in 1-5 .mu.L increments. After each addition,
the solution was constantly stirred for 3 min, and the solution pH
was measured using a pH meter. The pKa of the copolymer was
determined as the pH at which 50% of the copolymer turns
ionized.
[0608] Evaluation of pH Sensitivity
[0609] A DMF solution of Meo-PEG-b-PHMEMA (5 mg/mL) and
Meo-PEG-b-P(HMEMA-co-AMA-Cy5.5) (5 mg/mL) was mixed in a volume
ratio of 1:1. Under vigorously stirring (1000 rpm200 .mu.L of the
mixture was added dropwise to 5 mL of deionized water. After
collection and purification using ultrafiltration device (EMD
Millipore, MWCO 100 kDa), the NPs formed were dispersed in 1 mL of
phosphate buffered saline (PBS, pH 7.4). Subsequently, 1 M NaOH or
HCl aqueous solution was added in 1-5 .mu.L increments, and
fluorescence intensity with an excitation of 675 nm was measured on
a Synergy HT multi-mode microplate reader (BioTek Instruments). The
normalized fluorescence intensity (NFI) vs. pH profile was used to
quantitatively assess the pH responsiveness. NFI is calculated as
follows:
NFI=(F-F.sub.min)/(F.sub.max-F.sub.min)
[0610] where F is the fluorescence intensity of the NPs at any
given pH value and Fmax and Fmin are the maximal and minimal
fluorescence intensity of the NPs, respectively.
Preparation of the siRNA Loaded Nanoparticles (NPs)
[0611] Meo-PEG-b-PHMEMA was dissolved in DMF to form a homogenous
solution with a concentration of 10 mg/mL. Subsequently, a mixture
of 1 nmol siRNA (0.1 nmol/4 aqueous solution) and TCPA (5 mg/mL in
DMF) in an N/P molar ratio of 1:20 was prepared and mixed with 200
.mu.L of Meo-PEG-b-PHMEMA solution. Under vigorously stirring (1000
rpm), the mixture was added dropwise to 5 mL of deionized water.
The NP dispersion formed was transferred to an ultrafiltration
device (EMD Millipore, MWCO 100 K) and centrifuged to remove the
organic solvent and free compounds. After washing with PBS buffer
(pH 7.4) (3.times.5 mL), the siRNA loaded NPs were dispersed in 1
mL of PBS buffer (pH 7.4).
[0612] Characterizations of NPs
[0613] Size and zeta potential were determined by dynamic light
scattering (DLS, Brookhaven Instruments Corporation). The
morphology of NPs was visualized on a Tecnai G2 Spirit BioTWIN
transmission electron microscope (TEM). Before observation, the
sample was stained with 1% uranyl acetate and dried under air. To
determine siRNA encapsulation efficiency (EE %), DY677-labelled Luc
siRNA (DY677-siRNA) loaded NPs were prepared according to the
method aforementioned. A small volume (5 .mu.L) of the NP solution
was withdrawn and mixed with 20-fold DMSO. The standard was
prepared by mixing 54 of naked DY677-siRNA solution (1 nmol/mL in
pH 7.4 PBS buffer) with 20-fold DMSO. The fluorescence intensity of
DY677-siRNA was measured using a microplate reader and the siRNA EE
% is calculated as: EE
%=(FI.sub.NPs/FI.sub.Standard).times.100.
[0614] Digestion Assay
[0615] NPs loaded with fluorescein- and Dabcyl-labelled Luc siRNA
were prepared according to the method aforementioned, and then
dispersed in 1 mL of PBS buffer. Subsequently, 20 U RNase was added
and the sample was incubated in 37.degree. C. At predetermined time
intervals, the fluorescent emission spectra were examined using a
microplate reader with excitation at 480 nm and emission data range
between 490 and 650 nm.
[0616] In Vitro siRNA Release
[0617] DY677-labelled Luc siRNA loaded NPs were prepared as
described above. Subsequently, the NPs were dispersed in 1 mL of
PBS (pH 7.4) and then transferred to a Float-a-lyzer G2 dialysis
device (MWCO 100 kDa, Spectrum) that was immersed in PBS buffer (pH
7.4 or 6.8) at 37.degree. C. At a predetermined interval, 5 .mu.L
of the NP solution was withdrawn and mixed with 20-fold DMSO. The
fluorescence intensity of DY677-labelled siRNA was determined using
a microplate reader.
[0618] Flow Cytometry
[0619] Luc-HeLa (50,000 cells) were seeded in 6-well plate and
incubated in 2 mL of RPMI1640 medium (pH 7.4) containing 10% FBS
for 24 h. After replacing the medium with 2 mL of fresh medium at
pH 7.4 or 6.8, DY677-labelled Luc siRNA loaded NPs were added, and
the cells were allowed to incubate for 2 h. After removing the
medium and subsequently washing with PBS buffer (pH 7.4) thrice,
the cells were digested by trypsin and collected for flow cytometry
quantitative analysis (DXP11 Analyzer).
[0620] Confocal Laser Scanning Microscope (CLSM)
[0621] Luc-HeLa (50,000 cells) were seeded in round discs and
incubated in 2 mL of RPMI1640 medium (pH7.4) containing 10% FBS for
24 h. After replacing the medium with 2 mL of fresh medium at pH
7.4 or 6.8, DY677-labelled Luc siRNA loaded NPs were added, and the
cells were allowed to incubate for 2 h. After removing the medium
and subsequently washing with PBS buffer (pH 7.4) thrice,
lysotracker green was added to stain the endosomes and the nuclei
were stained by Hoechst 33342. The uptake of siRNA loaded NPs were
viewed under a FV1000 CLSM (Olympus).
[0622] Luc Silencing
[0623] Luc-HeLa cells were seeded in 96-well plates (5,000 cells
per well) and incubated in 0.1 mL of RPMI1640 medium (pH 7.4) with
10% FBS for 24 h. Thereafter, the medium was replaced by fresh
medium at Luc siRNA-loaded NPs were added. After 24 h incubation,
the cells were washed with PBS buffer (pH 7.4) and allowed to
incubate in fresh medium (pH 7.4) for another 48 h. The Luc
expression in HeLa cells was determined using Steady-Glo luciferase
assay kits. Cytotoxicity was measured using AlamarBlue assay
according to the manufacturer's protocol. The luminescence or
fluorescence intensity was measured using a microplate reader, and
the average value of five independent experiments was
collected.
[0624] In Vitro BRD4 Silencing
[0625] LNCaP cells were seeded in 6-well plates (50,000 cells per
well) and incubated in 2 mL of RPMI1640 medium (pH 7.4) containing
10% FBS for 24 h. Subsequently, the medium was replaced by fresh
medium at pH 7.4 or 6.8, and then BRD4 siRNA loaded NPs were added.
After incubation for 24 h, the cells were washed with PBS buffer
(pH 7.4) and further incubated in fresh medium (pH 7.4) for another
48 h. Thereafter, the cells were digested by trypsin and the
proteins were extracted using modified radioimmunoprecipitation
assay lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40
substitute, 0.25% sodium deoxycholate, 1 mM sodium fluoride, 1 mM
Na3VO4, 1 mM EDTA), supplemented with protease inhibitor cocktail
and 1 mM phenylmethanesulfonyl fluoride (PMSF). The BRD4 expression
was examined using the western blot analysis.
[0626] Western Blot Analysis
[0627] Western blot analysis was carried according to the same
method described in Example 1. BRD4 rabbit antibody (Abcam) and
beta-actin rabbit antibody (Cell Signaling) were used. The BRD4
expression was detected with horseradish peroxidase
(HRP)-conjugated secondary antibody (anti-rabbit IgG HRP-linked
antibody, Cell Signaling) and an enhanced chemiluminescence (ECL)
detection system (Pierce).
[0628] Apoptosis Analysis
[0629] LNCaP cells were seeded in 6-well plates (50,000 cells per
well) and incubated in 2 mL of RPMI1640 medium (pH 7.4) containing
10% FBS for 24 h. Subsequently, the medium was replaced by fresh
medium at pH 7.4 or 6.8, and then BRD4 siRNA loaded NPs were added.
After incubation for 24 h, the cells were washed with PBS buffer
(pH 7.4) and further incubated in fresh medium (pH 7.4) for another
48 h. Thereafter, the cells were digested by trypsin and the cells
were collected for 7-amino-actinomycin (7-AAD) and PE Annexin V
staining using PE Annexin V Apoptosis Detection Kit I (BD
Pharmingen.TM.). The apoptosis analysis was performed using a DXP11
Flow Cytometry Analyzer.
[0630] Immunofluorescence Staining
[0631] LNCaP cells (50,000 cells) were seeded in round disc and
incubated in 2 mL of RPMI1640 medium (pH 7.4) containing 10% FBS
for 24 h. After replacing the medium with fresh medium (pH 7.4 or
6.8), BRD4 siRNA loaded NPs were added and the cells were allowed
to incubated for 24 h. Subsequently, the cells were washed with PBS
buffer (pH 7.4) and fresh medium (pH 7.4) was added. After 48 h
incubation, the cells were fixed with 4% paraformaldehyde. The
cells were then permeabilized by incubation in 0.2% Triton X-100 in
PBS buffer (pH 7.4) for 5 minutes, followed by washing with pH 7.4
PBS buffer (3.times.5 min). Thereafter, the cells were blocked with
blocking buffer (2% normal goat serum, 2% BSA, and 0.2% gelatin in
pH 7.4 PBS buffer) at room temperature for 1 h. After washing the
cells with pH 7.4 PBS buffer (3.times.5 min), BRD4 rabbit antibody
(Abcam) diluted in 1% BSA solution was added and the cells were
incubated for 1 h. Subsequently, the cells were with pH 7.4 PBS
buffer (3.times.5 min), and then further incubated with Alex Fluro
647-linked secondary antibody and Alex Fluro 488-conjugated
phalloidin for another 1 h. After washing with pH 7.4 PBS buffer
(3.times.5 min), the cells were viewed under a FV1000 CLSM.
[0632] In Vitro Cell Proliferation
[0633] LNCaP cells were seeded in 6-well plates (20,000 cells per
well) and incubated in 2 mL of RPMI1640 medium (pH 7.4) containing
10% FBS for 24 h. Thereafter, the cells were treated with the BRD4
siRNA loaded NPs at pH 7.4 or 6.8 for 24 h and then washed with PBS
buffer (pH 7.4) for further incubation. At predetermined intervals,
the cytotoxicity was measured by AlamarBlue assay according to the
manufacturer's protocol. After each measurement, the AlamarBlue
agent was removed and 2 mL of fresh medium (pH 7.4) was added for
further incubation.
[0634] Pharmacokinetics Study
[0635] Healthy male BALB/c mice were randomly divided into two
groups (n=3) and given an intravenous injection of either (i)
DY677-labelled naked Luc siRNA or (ii) DY677-labelled Luc siRNA
loaded NPs at a 1 nmol siRNA dose per mouse. At predetermined time
intervals, orbital vein blood (20 .mu.L) was withdrawn using a tube
containing heparin, and the wound was pressed for several seconds
to stop the bleeding. The fluorescence intensity of DY677-labelled
siRNA in the blood was determined by microplate reader. The blood
circulation half-life (t.sub.1/2) was calculated according to
previous report (Winter H et al., Antimicrob. Agents Chemother. 57,
5516-5520 (2013)).
[0636] LNCaP Xenograft Tumor Model
[0637] LNCaP xenograft tumor model was constructed by subcutaneous
injection with 200 .mu.L of LNCaP cell suspension (a mixture of
RPMI 1640 medium and Matrigel in 1:1 volume ratio) with a density
1.times.10.sup.7 cells/mL into the back region of healthy male
Athymic nude mice. When the volume of the LNCaP tumor xenograft
reached .about.70 mm.sup.3, the mice were used for the following in
vivo experiments.
[0638] Biodistribution
[0639] LNCaP tumor-bearing male Athymic nude mice were randomly
divided into three groups (n=3) and given an intravenous injection
of either (i) DY677-labelled naked Luc siRNA or (ii) DY677-labelled
Luc siRNA loaded NPs at a 1 nmol siRNA dose per mouse. Twenty-four
hours after the injection, the mice were imaged using the Maestro 2
In-Vivo Imaging System (Cri Inc). Organs and tumors were then
harvested and imaged. To quantify the accumulation of NPs in tumors
and organs, the fluorescence intensity of each tissue was
quantified by Image-J.
[0640] In Vivo BRD4 Silencing
[0641] LNCaP tumor-bearing male Athymic nude mice were randomly
divided into two groups (n=2) and intravenously injected with (i)
Luc siRNA loaded NPs or (ii) BRD4 siRNA loaded NPs for three
consecutive days. Twenty-four hours after the final injection, mice
were sacrificed and tumors were harvested for western blot
analysis, and immunohistochemistry and TUNEL staining. For the
western blot analysis, the proteins in the tumor were extracted
using modified radioimmunoprecipitation assay lysis buffer (50 mM
Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40 substitute, 0.25% sodium
deoxycholate, 1 mM sodium fluoride, 1 mM Na.sub.3VO.sub.4, 1 mM
EDTA), supplemented with protease inhibitor cocktail and 1 mM
phenylmethanesulfonyl fluoride (PMSF). Western blot was performed
according to the method described above.
[0642] Immunohistochemistry (IHC) Staining
[0643] IHC staining was performed on formalin-fixed
paraffin-embedded tumor sections. Briefly, tumor slides were first
heated to 60.degree. C. for 1 h, desparaffinized with xylene
(3.times.5 min), and washed with different concentrations of
alcohol. After retrieval of antigen using DAKO target retrieval
solution at 95-99.degree. C. for 40 min, followed by washing, the
slides were blocked with peroxidase blocking buffer (DAKO Company)
for 5 min. After washing buffer (DAKO Company), the slides were
incubated with BRD4 rabbit antibody (Abcam) diluted in DAKO
antibody solution for 1 h. The slides were then washed and
incubated with peroxidase-labeled polymer for 30 min. After washing
and staining with DAB+ substrate-chromogen solution and
hematoxylin, the slides we remounted and viewed under a MVX10
MacroView Dissecting scope equipped with OlympusDP80 camera.
[0644] Immune Response
[0645] Healthy male BALB/c mice were randomly divided into three
groups (n=3) and given an intravenous injection of either (i) PBS,
(ii) naked BRD4 siRNA or (iii) BRD4 siRNA loaded NPs at a 1 nmol
siRNA dose per mouse. Twenty-four hours after injection, blood was
collected and serum isolated for measurements of representative
cytokines (TNF-.alpha., IL-6, IL-12, and IFN-.gamma.) by
enzyme-linked immunosorbent assay or ELISA (PBL Biomedical
Laboratories and BD Biosciences) according to the manufacturer's
instructions.
[0646] Histology
[0647] Healthy male BALB/c mice were randomly divided into three
groups (n=3) and administered daily intravenous injections of
either (i) PBS, (ii) naked BRD4 siRNA or (iii) BRD4 siRNA loaded
NPs at a 1 nmol siRNA dose per mouse. After three consecutive
injections, the main organs were collected 24 h post the final
injection, fixed with 4% paraformaldehyde, and embedded in
paraffin. Tissue sections were stained with hematoxylin-eosin
(H&E) and then viewed under an optical microscope.
[0648] Inhibition of Tumor Growth
[0649] LNCaP tumor-bearing male Athymic nude mice were randomly
divided into four groups (n=4) and intravenously injected with (i)
PBS, (ii) naked BRD4 siRNA, (iii) Luc siRNA loaded NPs, or (iv)
BRD4 siRNA loaded NPs at a 1 nmol siRNA dose per mouse once every
three days. All the mice were administrated by administered four
consecutive injections and the tumor growth was monitored every two
days by measuring perpendicular diameters using a caliper and tumor
volume was calculated as follows:
V=W.sup.2.times.L/2
[0650] where W and L are the shortest and longest diameters,
respectively.
[0651] Statistical Analysis
[0652] Statistical significance was determined by a two-tailed
Student's t test assuming equal variance. A p value <0.05 is
considered statistically significant.
Results
[0653] Most of current delivery systems can overcome one or a few
barriers, but unfortunately encounter various dilemmas (e.g., long
circulation vs. weak uptake and active targeting vs. unfavorable
circulation) to induce suboptimal therapeutic effect. RNAi
technology has demonstrated the potential to make a huge impact on
cancer treatment by silencing the expression of target gene(s),
especially those that encode "undruggable" proteins (Kay, M A, Nat
Rev Genet 2011, 12, (5), 316-328; Yin H et al., Nat Rev Genet 2014,
15, (8), 541-555; Grimm, D. Advanced Drug Delivery Reviews 2009,
61, (9), 672-703; Zhu X et al., Proceedings of the National Academy
of Sciences 2015, 112, (25), 7779-7784; Xu X et al., Angewandte
Chemie International Edition 2016, 55, (25), 7091-7094).24-29
Nevertheless, systemic delivery of RNAi agents (e.g., siRNA) to
solid tumor followed by sufficient cytosolic siRNA release has
remained a barrier to the clinical translation of RNAi therapy
(Pack D W et al., Nat Rev Drug Discov 2005, 4, (7), 581-593;
Whitehead K A et al., Nat Rev Drug Discov 2009, 8, (2), 129-138;
Tseng Y C et al., Advanced Drug Delivery Reviews 2009, 61, (9),
721-731; Pan D W et al., Bioconjugate Chemistry 2015, 26, (8),
1791-1803). Currently, the de-PEGylation technique is a commonly
used strategy to promote siRNA delivery efficacy, in which PEG
chains can be cleaved by the acidic pH34 or over-expressed
metalloprotease (MMP) in tumor tissues to simultaneously achieve
high tumor accumulation and enhanced cellular uptake (Hatakeyama H
et al., Biomaterials 2011, 32, (18), 4306-4316; Wang H X et al.,
Biomaterials 2014, 35, (26), 7622-7634;). However, the complicated
TME stimuli-responsive chemistry involved in this strategy may
introduce additional complexities in the synthesis and scale-up of
therapeutic formulations.
[0654] Here, a tumor microenvironment (TME) pH-responsive
multistaged NP platform for systemic siRNA delivery and effective
cancer therapy has been developed. This NP platform is composed of
a polyethylene glycol (PEG) outer shell and a super-fast TME
pH-responsive core that can entrap the complex formed between siRNA
and a tumor cell-targeting and -penetrating peptide-amphiphile
(TCPA). After encapsulating siRNA, the resulting NP platform shows
the following features for multistaged siRNA delivery (FIG. 37): i)
polyethylene glycol (PEG) outer shell prolongs blood circulation
and thus enhances tumor accumulation; ii) super-fast TME pH
response of the hydrophobic poly(2-(hexamethyleneimino) ethyl
methacrylate) (PHMEMA) induces the rapid exposure of siRNA/TCPA
complexes at tumor site; iii) tumor cell-targeting ability of TCPA
improves the uptake of the exposed siRNA/TCPA complexes by tumor
cells; iv) cell-penetrating ability of TCPA enhances the cytosolic
siRNA delivery to achieve efficient gene silencing; and v) ease of
polymer synthesis and commercial available TCPA facilitate the
scale-up of this multistaged NP platform using standard unit
operations.
[0655] First, classic acid-base titration was used to examine the
pKa of the TME pH-responsive polymer, methoxyl-polyethylene
glycol-b-poly(2-(hexamethyleneimino) ethyl methacrylate)
(Meo-PEG-b-PHMEMA), and the pKa value is determined as .about.6.9,
which is close to the pH of tumor extracellular fluid
(6.5.about.6.8) (Wang Y et al., Nat Mater 2014, 13, (2), 204-212.).
This result suggests that a TME pH-responsive cargo release can be
achieved when using a carrier formulated with the Meo-PEG-b-PHMEMA
polymer. To further support this, a near-infrared dye, Cy5.5, was
incorporated into the hydrophobic PHMEMA moiety
(Meo-PEG-b-P(HMEMA-AMA-Cy5.5)). When mixing this Cy5.5-labelled
polymer with Meo-PEG-b-PHMEMA (1:1 in molar ratio), they can
self-assemble into well-dispersed NPs visualized by transmission
electron microscopy (TEM), with an average size of .about.40 nm
determined by dynamic light scattering (DLS). Due to the quenching
of the aggregated fluorophores inside the hydrophobic cores of
these NPs, there is no fluorescence signal at a pH above pKa of
Meo-PEG-b-PHMEMA (FIG. 38A). In contrast, at a pH below pKa, the
protonated PHMEMA moiety leads to the disassembly of the NPs,
visualized by TEM, and a dramatic increase in the fluorescence
signal (FIG. 38A). Measurement of the fluorescence intensity upon
pH change shows that the pH difference from 10 to 90% fluorescence
activation (.DELTA.pH10-90%) is 0.24 (FIG. 38B), which is close to
the previous report and much smaller than that of small molecule
dyes (about 2 pH units), demonstrating the super-fast TME pH
response of the Meo-PEG-b-PHMEMA polymer (Zhou K et al., Angewandte
Chemie International Edition 2011, 50, (27), 6109-6114; Urano Y et
al., Nat Med 2009, 15, (1), 104-109).
[0656] The siRNA loading ability and TME pH-responsive behavior of
the siRNA loaded NPs was investigated. Nanoprecipitation method was
employed to prepare the NPs by using a mixture of siRNA aqueous
solution and dimethylformamide (DMF) solution of Meo-PEG-b-PHMEMA
and TCPA. Two TCPAs (TCPA1: C17H35CONH-GR8GRGDS-OH; TCPA2:
C17H35CONH--(C17H35CONH)-KR8GRGDS-OH, chemical structures shown
below) were used to adjust the siRNA loading ability and
physiochemical properties of the NPs (denoted TCPA1-NPs and
TCPA2-NPs). Under the same conditions, the siRNA encapsulation
efficiency (EE %, Table 9) of the TCPA1-NPs (.about.39%) is lower
than that of TCPA2-NPs (.about.52%). In contrast, the size of the
TCPA1-NPs (.about.90.1 nm, with PDI 0.279) is larger than that of
the TCPA2-NPs (.about.72.8 nm, with PDI 0.194), determined by DLS.
The possible reason is that the two hydrophobic tails of TCPA2
facilitate the formation of more compact TCPA2/siRNA complexes to
improve the siRNA loading ability and decrease the size of the NPs
(Lim Y B et al., Angewandte Chemie International Edition 2007, 46,
(47), 9011-9014). In addition, the TCPA2-NPs show a strong ability
to protect the siRNA stability. When encapsulating fluorescein and
its quencher (Dabcyl)-labelled siRNA into the NPs, there is nearly
no fluorescence change after 6 h incubation with RNase (FIG. 39).
However, naked siRNA can be rapidly degraded by RNase at 5 min, 10
min, and 15 min, which induces the dissociation between fluorescein
and Dabcyl, and thereby significant increase of the fluorescence
intensity.
TABLE-US-00009 TABLE 9 Size, zeta potential, and siRNA
encapsulation efficiency (EE %) of the siRNA loaded NPs made with
of Meo-PEG-b-PHMEMA and TCPA1 or TCPA2. Zeta potential (mv) NPs
siRNA EE (%) Size (nm) pH 7.4 pH 6.8 TCPA1-NPs 39 90.1 9.27 29.2
TCPA2-NPs 52 72.8 5.69 27.6
[0657] The TCPA2-NPs were chosen to evaluate their TME
pH-responsive behavior. The DY-677 siRNA loaded TCPA2-NPs showed a
spherical morphology at pH 7.4 under TEM. After adjusting the
solution pH to 6.8, there is a significant decrease in the NP
number within 1 min (FIG. 40A), indicating the super-fast TME pH
response of the siRNA loaded NPs. Transmission electron microscopy
(TEM) measurements show that there are some large amorphous
aggregates and small size particles in the solution, which possibly
correspond to the ionized polymer and exposed TCPA2/siRNA
complexes. This result is further confirmed by DLS analysis, in
which particles ranging from several nanometers to thousand
nanometers can be detected (FIG. 40B). With this rapid disassembly
upon pH change, the TCPA2-NPs offer a fast release of
DY677-labelled siRNA (DY677-siRNA) (FIG. 40C). More than 80% of
loaded siRNA has been released within 4 hours at pH 6.8. Within the
same time frame, less than 20% of the loaded siRNA is released at
pH 7.4.
Molecular Structures of TCPA1 and TCPA2
##STR00015##
[0659] Next, the ability of this TME pH-triggered NP disassembly to
improve cellular uptake of loaded siRNA and to enhance gene
silencing was investigated. Luciferase-expressing HeLa (Luc-HeLa)
cells were incubated with the DY677-siRNA loaded TCPA2-NPs at pH
6.8 or 7.4 for 2 h, and the cellular uptake was observed by
confocal laser scanning microscopy (CLSM). Endosomes were stained
by lysotracker green; nuclei were stained by Hoechst 33342.
Compared to the cells incubated at pH 7.4, the brighter red
fluorescence indicates a higher siRNA uptake at pH 6.8. More
importantly, unlike the cells incubated at pH 7.4 with the
internalized siRNA co-localizing with lysosomes and endosomes, lots
of the internalized siRNA molecules at pH 6.8 are distributed in
the cytoplasm where siRNA functions. Flow cytometry was used to
quantitatively examine the uptake at different pHs. As shown in
FIGS. 41A-41B, the siRNA uptake at pH 6.8 is more than 5-fold
stronger than that of the cells incubated at pH 7.4. All these
results strongly demonstrate that the TME pH-triggered disassembly
of the TCPA2-NPs induces the rapid exposure of the TCPA2/siRNA
complexes, which subsequently use their tumor cell-targeting and
-penetrating functions to dramatically increase the cytosolic siRNA
delivery (Sun C Y et al., Journal of the American Chemical Society
2015, 137, (48), 15217-15224; Xu X D et al., Polymer Chemistry
2012, 3, (9), 2479-2486; Ren Y et al., Science translational
medicine 2012, 4, (147), 147ra1 12; Xu X et al., ACS Nano
2017).
[0660] Next, Luc siRNA was encapsulated into the TCPA2-NPs and
their gene silencing efficacy was evaluated using Luc-HeLa cells.
As shown in FIG. 41C, the siRNA loaded NPs show a reduction in Luc
expression at both pH 7.4 and 6.8. In comparison, due to rapid
disassembly of the NPs at pH 6.8 to increase the cytosolic siRNA
delivery (FIGS. 41A-41C), they offer much better gene silencing
efficacy and can silence .about.90% Luc expression without obvious
cytotoxicity at a 10 nM siRNA dose (FIG. 41D). The ability of the
TCPA2-NPs to silence the expression of BRD4 was examined. BRD4 is a
conserved member of the BET family of chromatin readers that
exhibits anti-proliferation effect in metastatic
castration-resistant prostate cancer (mCRPC). LNCaP cells, an
Androgen Receptor (AR) positive PCa cell line with high level of
BRD4 expression compared to other PCa cells including PC3, 22RV1,
and DU145. Thus, LNCaP cells were used as a model cell line. As
shown in FIG. 42A, the BRD4 siRNA loaded NPs showed a higher
efficacy in BRD4 silencing at pH 6.8, determined by Western blot.
Around 60% BRD4 can be knocked down at a 10 nM siRNA dose and this
BRD4 silencing reaches .about.90% at a 20 nM siRNA dose. In
comparison, for the cells treated with the BRD4 siRNA loaded NPs at
pH 7.4, there is still a high level of BRD4 expression (>60%) at
a 20 nM siRNA dose. Similar results can be also found in the
immunofluorescence staining analysis. At a 20 nM siRNA dose, bright
red fluorescence corresponding to the residual BRD4 was observed in
the cells treated with the siRNA loaded NPs at pH 7.4. However,
very weak red fluorescence is observable in the cells treated with
the siRNA loaded NPs at pH 6.8. With this efficient BRD4 silencing
at pH 6.8, the percentage of apoptotic (Annexin-V positive) or
necrotic (Annexin V-negative and 7-ADD-postivie) cells increases
markedly to 39.5% or 36.3% (FIG. 42B), which is around 2.5-flod
higher than that of the cells treated with the siRNA loaded NPs at
pH 7.4. In addition, the BRD4 silencing also induces significant
inhibition of cell proliferation. Only 20% of the LNCaP cells are
alive after 6 days incubation (FIG. 42C). However, there is about
8-fold increase in the number of cells treated with the siRNA
loaded NPs at pH 7.4.
[0661] The pharmacokinetics and biodistribution of the TCPA2-NPs
was subsequent assessed. Pharmacokinetics was examined by
intravenous injection of DY677-siRNA loaded NPs to health mice (1
nmol siRNA dose per mouse, n=3). As shown in FIG. 43A, with the
protection of PEG outer layer, (Knop K et al., Angewandte Chemie
International Edition 2010, 49, (36), 6288-6308) the TCPA2-NPs show
long blood circulation with a half-life (t.sub.112) of around 4.38
h. In contrast, the naked siRNA is rapidly cleared from the blood
and its blood half-life (t.sub.1/2) is less than 10 min. The
biodistribution was evaluated by intravenously injecting
DY677-siRNA loaded NPs into LNCaP xenograft tumor-bearing mice. Due
to the long blood circulation characteristic of the TCPA2-NPs, they
show a much higher tumor accumulation than naked siRNA when
visualized using the fluorescent image of the LNCaP xenograft
tumor-bearing nude mice 24 hours post injection of naked
DY677-siRNA and siRNA loaded TCPA2-NPs. The tumors and main organs
were harvested 24 h post injection and the biodistribution is shown
in FIG. 43B. Naked siRNA has a high accumulation in kidney but very
low accumulation in tumor. However, the TCPA-NPs show an
approximately 10-fold higher tumor accumulation than the naked
siRNA.
[0662] The results of above in vitro and in vivo experiments
demonstrate that the TCPA2-NPs have a high tumor accumulation via
long blood circulation, and can respond to TME pH to target and
penetrate tumor cells to induce efficient gene silencing, which is
a typical multi-staged delivery characteristic (Wang S. et al., ACS
Nano 2016, 10, (3), 2991-2994; Wang S. et al., Advanced materials
2016, 28, (34), 7340-64; Chen B et al., Theranostics 2017, 7, (3),
538-558).
[0663] As a proof of concept, bromodomain 4 (BRD4) was chosen as a
therapeutic target and systematically evaluated the BRD4 siRNA
delivery and its anticancer efficacy. BRD4 is a conserved member of
the bromodomain and extraterminal (BET) family of chromatin
readers, which plays a critical role in tran-scription by RNA
polymerase II (RNA Pol II) by facilitating recruitment of the
positive transcription elongation factor b (P-TEFb) (Jang, M K et
al., Molecular Cell 2005, 19, (4), 523-534; Yang, Z et al.,
Molecular Cell 2005, 19, (4), 535-545.). For mCRPC, BRD4 physically
interacts with the N-terminal domain of androgen receptor (AR), a
key factor that predominantly drives primary prostate cancer (PCa)
to mCRPC after androgen-deprivation therapy (Taylor B S et al.,
Cancer Cell 2010, 18, (1), 11-22; Chen C D et al., Nat Med, 2004,
10, (1), 33-39; Visakorpi T et al., Nat Genet, 1995, 9, (4),
401-406). Recent studies demonstrated that BRD4 inhibition can
disrupt AR recruitment to target gene loci and exhibits much more
effective mCRPC treat-ment than direct AR antagonism (i.e.,
enzalutamide) (Asangani, I A. et al., Nature 2014, 510, (7504),
278-282).
[0664] Motivated by the important role of BRD4 to regulate AR
signaling pathway and PCa progression, BRD4 siRNA was encapsulated
in the multi-staged NP platform. It was evaluated whether this
multi-staged siRNA delivery platform can silence the BRD4
expression in vivo and show anticancer effect. To assess the in
vivo BRD4 silencing, the BRD4 siRNA loaded NPs were intravenously
injected into the LNCaP xenograft tumor-bearing mice (1 nmol siRNA
dose per mouse, n=3) for three consecutive days. Western blot
analysis of the tumor tissue showed that the administration of BRD4
siRNA loaded NPs leads to around 85% knockdown in BRD4 expression
compared to the control NPs loaded with Luc siRNA. A similar
tendency was also observed in the immunohistochemistry (IHC)
staining analysis. With this suppressed BRD4 expression, there is a
significant increase in tumor cell apoptosis confirmed by TUNEL
staining. Additionally, the administration of the TCPA2-NPs shows
negligible in vivo side effects. After three consecutive injections
of the NPs to healthy mice (once every two days at a 1 nmol siRNA
dose per mouse, n=3), there were no noticeable histological changes
in the tissues from heart, liver, spleen, lung or kidney. Blood
serum analysis shows that TNF-.alpha., IFN-.gamma., IL-6, and IL-12
levels are in the normal range 24 hour post injection. To confirm
whether the NP-mediated BRD4 silencing has an anti-cancer effect,
the BRD4 siRNA loaded NPs were intravenously injected into the
LNCaP xenograft tumor-bearing mice once every three days at a 1
nmol siRNA dose per mouse (n=5). After four consecutive injections,
the tumor growth is significantly inhibited compared to the mice
treated with PBS, naked BRD4 siRNA or Luc siRNA loaded NPs (FIGS.
44A-45B). There is less than 1.5-fold increase (from .about.63 to
.about.81 mm3) in tumor size of the mice treated with the BRD4
siRNA loaded NPs at day 16 (FIG. 44A). However, for the mice
treated with other formulations, their tumor size (FIG. 44A) and
weight (FIG. 44B) are more than 4-fold larger than that of mice
treated with the BRD4 siRNA loaded NPs. In addition, similar as the
histological analysis results, the BRD4 siRNA loaded NPs shows no
obvious influence on mouse body weight, implying good
biocompatibility of this NP platform. The results show that the
systemic delivery of BRD4 siRNA can efficiently silence BRD4
expression in the tumor tissue and significantly inhibit PCa tumor
growth with negligible toxicities.
[0665] Thus, a TME pH-responsive multi-staged NP platform for
systemic siRNA delivery and effective cancer therapy has
successfully developed. In vitro and in vivo results show that this
multi-staged NP platform can first highly accumulate at tumor site
via long blood circulation and then respond to TME pH to fast
expose siRNA/TCPA complex, which subsequently target and penetrate
to induce strong cytosolic siRNA delivery and efficient in vivo
gene silencing.
Sequence CWU 1
1
15119RNAArtificial Sequencesynthetic
polynucleotidesiRNAmisc_feature(19)..(19)dTdT - Double nucleotide
overhang TT 1cuuacgcuga guacuucga 19219RNAArtificial
Sequencesynthetic polynucleotide siRNAmisc_feature(19)..(19)dTdT -
Double nucleotide overhang TT 2ucgaaguacu cagcguaag
19319RNAArtificial Sequencesynthetic polynucleotide
siRNAmisc_feature(19)..(19)dTdT - Double nucleotide overhang TT
3ggaccaccgc aucucuaca 19419RNAArtificial Sequencesynthetic
polynucleotide siRNAmisc_feature(19)..(19)dTdT - Double nucleotide
overhang TT 4uguagagaug cgguggcuc 19521RNAArtificial
Sequencesynthetic polynucleotide siRNA 5gcgacgaccu uacagagcgu u
21621RNAArtificial Sequencesynthetic polynucleotide siRNA
6cgcucuguaa ggucgucgcu u 21721RNAArtificial Sequencesynthetic
polynucleotide siRNA 7gaauaggguu acagaguugu u 21821RNAArtificial
Sequencesynthetic polynucleotide siRNA 8caacucugua acccuauucu u
21921RNAArtificial Sequencesynthetic polynucleotide siRNA
9aaacacaacu caagcaucgu u 211021RNAArtificial Sequencesynthetic
polynucleotide 10cgaugcuuga guuguguuuu u 21119PRTArtificial
Sequencesynthetic polypeptide 11Cys Arg Gly Asp Arg Gly Pro Asp
Cys1 5126RNAArtificial Sequencesynthetic polynucleotide - cleavage
site 12aauaaa 6135PRTArtificial Sequencesynthetic polypeptide 13Cys
Arg Glu Lys Ala1 5149PRTArtificial Sequencesynthetic polypeptide
14Cys Arg Lys Arg Leu Asp Arg Asn Cys1 5158PRTArtificial
Sequencesynthetic polypeptideMISC_FEATURE(8)..(8)-NH2 modification
at C-terminus 15Arg Arg Arg Arg Arg Arg Arg Arg1 5
* * * * *