U.S. patent application number 15/010784 was filed with the patent office on 2016-08-11 for polymer-grafted nanobins.
The applicant listed for this patent is Northwestern University. Invention is credited to Anthony J. Chipre, SonBinh T. Nguyen.
Application Number | 20160228363 15/010784 |
Document ID | / |
Family ID | 56565725 |
Filed Date | 2016-08-11 |
United States Patent
Application |
20160228363 |
Kind Code |
A1 |
Chipre; Anthony J. ; et
al. |
August 11, 2016 |
POLYMER-GRAFTED NANOBINS
Abstract
Provided herein are small unilaminar vesicles with
surface-displayed polymer moieties, and methods of use and
manufacture thereof. In particular, provided herein are
polymer-grafted nanobins, and methods of drug delivery
therewith.
Inventors: |
Chipre; Anthony J.;
(Rochester, NY) ; Nguyen; SonBinh T.; (Evanston,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Northwestern University |
Evanston |
IL |
US |
|
|
Family ID: |
56565725 |
Appl. No.: |
15/010784 |
Filed: |
January 29, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62110055 |
Jan 30, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/704 20130101;
A61K 45/06 20130101; A61K 9/1271 20130101; A61K 31/704 20130101;
A61K 2300/00 20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/704 20060101 A61K031/704 |
Goverment Interests
STATEMENT REGARDING FEDERAL FUNDING
[0002] This is invention was made with government support under P30
CA060553, U01 CA151461 and U54 CA151880 awarded by the National
Institutes of Health; and FA9550-11-1-0275 awarded by the Air Force
Office of Scientific Research. The government has certain rights in
the invention.
Claims
1. A composition comprising a polymer-grafted nanobin (PGN),
wherein said PGN comprises a small unilaminer vesicle comprising a
phospholipid-based bilayer with surface-exposed polymers extending
therefrom.
2. The composition of claim 1, wherein said small unilaminer
vesicle is between 15 and 50 nm in diameter.
3. (canceled)
4. The composition of claim 1, wherein said small unilaminer
vesicle comprises 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC), Dipalmitoylphosphatidylcholine (DPPC), and/or
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC).
5. The composition of claim 1, wherein said surface-exposed
polymers comprise poly(acrylic acid) extending from
cholesterol-terminated poly(acrylic acid) (Chol-PAA), wherein the
cholesterol portion of the Chol-PAA is inserted into the
bilayer.
6. (canceled)
7. The composition of claim 1, wherein said PGN further comprises a
molecular payload encapsulated within said small unilaminar
vesicle.
8. The composition of claim 7, wherein said molecular payload
comprises a small molecule, peptide, or nucleic acid.
9. The composition of claim 7, wherein, said PGN releases less than
20% of said payload over the course of one month at normal
physiologic conditions, and said PGN releases at least 50% of said
payload over the course of less than one hour at a pH between 4.0
and 6.0.
10. (canceled)
11. The composition of claim 1, wherein said surface-exposed
polymers are not cross-linked.
12. The composition of claim 1, comprising a polymer-grafted
nanobin (PGN), wherein said PGN comprises a small
phospholipid-based unilaminer vesicle (SUV) comprising
cholesterol-terminated poly(acrylic acid) groups (Chol-PAA),
wherein said Chol-PAA is oriented such that said poly(acrylic acid)
is surface exposed and extends from said SUV into the surrounding
environment, wherein said SUV encompasses a molecular payload
comprising one or more peptides, nucleic acids, and/or small
molecules.
13. A method of drug delivery to a low-pH microenvironment within a
subject comprising: (a) administering a polymer-grafted nanobin
(PGN) of claim 12 to said subject; and (b) allowing said PGN to
migrate from physiologic conditions to an acidic
microenvironment.
14. The method of claim 13, wherein the PGN is administered locally
at or near the acidic microenvironment.
15. The method of claim 13, wherein the PGN is administered
systemically.
16. The method of claim 13, wherein said acidic microenvironment is
a tumor.
17. The method of claim 16, wherein said therapeutic agent is a
chemotherapeutic.
18. The method of claim 16, wherein said payload is a nucleic
acid-based therapy.
19. The method of claim 13, wherein said PGN releases less than 20%
of said payload over the course of one month at normal physiologic
conditions and releases at least 50% of said payload over the
course of less than one hour at a pH between 4.0 and 6.0.
20. The method of claim 13, wherein the PGN is co-administered with
an additional therapeutic agent.
21. The method of claim 16, further comprising a step of surgically
removing the tumor.
21. (canceled)
22.-29. (canceled)
30. A method of preparing a polymer-grafted nanobin (PGN)
comprising: (a) preparing a lipid mixture by dissolving selected
lipids in an organic solvent; (b) hydrating the product of step (a)
with an aqueous hydration solvent to form liposomes; (c) sizing the
liposomes to yield small unilaminar vesicles (SUVs); and (d)
incubating the SUVs with lipid-anchored polymer to generate PGNs
with surface exposed polymer.
31.-46. (canceled)
47. The method of claim 21, wherein the administration step is
performed after surgical removal of the tumor.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the priority benefit of U.S.
Provisional Patent Application 62/110,055, filed Jan. 30, 2015,
which is incorporated by reference in its entirety.
FIELD
[0003] Provided herein are unilaminar vesicles with
surface-displayed polymer moieties, and methods of use and
manufacture thereof. In particular, provided herein are
polymer-grafted nanobins, and methods of drug delivery
therewith.
BACKGROUND
[0004] The hydrodynamic diameter (DH) of a nanoparticle strongly
influences its behavior in vivo. A series of recent reports
indicate that particles with DH of 15-50 nm may be ideal for
various biomedical applications including delivery of therapeutics
to hypovascularized cancers (e.g., pancreatic cancer) (Cabral et
al. Nat. Nanotechnol. 2011, 6 (12), 815-823; herein incorporated by
reference in its entirety), mediating therapeutic transport across
the blood-brain barrier (Fresta et al. Pharm. Res. 1995, 12 (11),
1769-1774; herein incorporated by reference in its entirety),
serving as carriers for transdermal medicine (Sonavane et al.
Colloids Surf. B 2008, 65 (1), 1-10.: herein incorporated by
reference in its entirety), and selectively targeting lymph nodes
for vaccination (Reddy et al. Nat. Biotechnol. 2007, 25 (10),
1159-1164.). Particles smaller than 15 nm are rapidly excreted via
kidney filtration (Choi et al. Nat. Biotechnol. 2007, 25 (10),
1165-1170; herein incorporated by reference in its entirety),
whereas particles larger than 50 nm accumulate in organs (De Jong
et al. Biomaterials 2008, 29 (12), 1912-1919;herein incorporated by
reference in its entirety) and poorly penetrate hypovascularized
tissues (Cabral et al. Nat. Nanotechnol. 2011, 6 (12), 815-823;
herein incorporated by reference in its entirety). Thus, a major
direction in current biodelivery research is the development of
stable, biocompatible nanoparticles in the 15-50 nm size range that
can be used in the tissue-specific delivery of a therapeutic
payload.
[0005] The liposome, a self-assembled, spherical lipid bilayer, is
arguably the perfect bio-delivery system due to its inherent
biocompatibility, tunable size, robust and easy preparation,
ability to encapsulate a therapeutic payload, and proven clinical
success as a delivery vehicle for small-molecule drugs (Torchilin.
Nat. Rev. Drug Discovery 2005, 4 (2), 145-160;herein incorporated
by reference in its entirety). Thus far, most liposome-based
systems investigated for biodelivery have diameters greater than
100 nm, even though small unilamellar vesicles (SUVs), which are
liposomes in the 15-50 nm size range (Silva & Cavaco-Paulo.
Biomacromolecules 2011, 12 (10), 3353-3368; herein incorporated by
reference in its entirety), can easily be generated by sonicating
larger liposome vesicles. This dearth of applications for SUVs
stems from their poor colloid stability, high fusogenicity, and
uncontrolled payload leakage. All of these limitations can be
attributed to the highly strained membrane curvature of SUVs and a
large level of molecular disorder in their lipid bilayers, which
can induce inter-particle fusion and lead to flocculation during
storage (Lin et al. Langmuir 2012, 28 (1), 689-700; herein
incorporated by reference in its entirety). The rapid fusion of
SUVs has enabled their use as a model system for biomembrane
research (Franzin & Macdonald. Biochemistry 1997, 36 (9),
2360-2370; herein incorporated by reference in its entirety), but
strongly hindered their application in biodelivery. Indeed, due to
their poor colloid stabilities and inherent leakage of encapsulated
cargo (Mcconnell & Schullery. Biochim. Biophys. Acta 1985, 818
(1), 13-22; herein incorporated by reference in its entirety),
these particles have only been employed as a carrier for in vivo
therapeutic delivery in a handful of cases over the last three
decades (Forssen et al. Cancer Res. 1992, 52 (12), 3255-3261;
Fresta et al. Pharm. Res. 1995, 12 (11), 1769-1774; herein
incorporated by reference in their entireties). While a few
strategies for overcoming the limitations of SUVs have been
investigated, most have limited success. For example, the
incorporation of a negatively charged lipid into SUVs reduced the
leakage of an encapsulated fluorescent dye (Mercadal et al.
Biochim. Biophys. Acta, Biomembr. 1995, 1235 (2), 281-288; herein
incorporated by reference in its entirety), but did not confer
long-term stability. The only reported example of stabilization of
fusogenic SUVs is by Wunder and coworkers, who employed Si0.sub.2
nanoparticles as a surface coating that inhibits vesicle
aggregation (Savarala et al. ACS Nano 2011, 5 (4), 2619-2628;
herein incorporated by reference in its entirety). However, these
SiO.sub.2-stabilized SUVs readily aggregate in the presence of mM
concentrations of sodium chloride, which would limit their
application as delivery agents.
SUMMARY
[0006] Provided herein are unilaminar vesicles with
surface-displayed polymer moieties, and methods of use and
manufacture thereof. In particular, provided herein are
polymer-grafted nanobins, and methods of drug delivery therewith.
In some embodiments, the polymer-grafted nanobins comprise small
phospholipid-bilayer-based vesicles with lipid-terminated polymers
embedded in and extending from the bilayer, and methods of drug
delivery therewith.
[0007] In some embodiments, provided herein are compositions
comprising a polymer-grafted nanobin (PGN), wherein said PGN
comprises a small unilaminer vesicle with surface-exposed polymers
extending thereform. In some embodiments, the small unilaminer
vesicle is between 15 and 50 nm in diameter (e.g., 15 nm, 20 nm, 25
nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm and ranges therein). In some
embodiments, the small unilaminer vesicle comprises a
phospholipid-containing bilayer. In some embodiments, the small
unilaminer vesicle comprises
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
Dipalmitoylphosphatidylcholine (DPPC), and/or
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some
embodiments, the surface-exposed polymers comprise poly(acrylic
acid). In some embodiments, the poly(acrylic acid) extends from
cholesterol-terminated poly(acrylic acid) (Chol-PAA), wherein the
cholesterol portion of the Chol-PAA is inserted into the bilayer.
In some embodiments, the PGN further comprises a molecular payload
encapsulated within said small unilaminar vesicle. In some
embodiments, the molecular payload comprises a small molecule,
peptide, or nucleic acid. In some embodiments, the PGN releases 20%
or less of said payload over the course of one month at normal
physiologic conditions (e.g., 20%, 15%, 10%, 5%, 1%, or less, and
ranges therein). In some embodiments, the PGN releases 0.1 to 20%
of said payload over the course of one month at normal physiologic
conditions (e.g., 0.1% to 5%, 0.5% to 3%, 1% to 5%, 2% to 15%, 3%
to 20%, or other combinations of range endpoints therein). In some
embodiments, the PGN releases at least 50% (e.g., 50%, 60%, 70%,
80%, 85%, 90%, 95%, 99%, and ranges therein) of said payload over
the course of less than one hour at a pH between 4.0 and 6.0. In
some embodiments, the PGN releases between 50% and 99% (e.g., 50%
to 70%, 60% to 95%, 70% to 85%, 80% to 90%, 85% to 99%, 90% to 99%,
75% to 95%, or other combinations of range endpoints therein) of
said payload over the course of less than one hour at a pH between
4.0 and 6.0. In some embodiments, the PGN releases at least 50%
(e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, and ranges therein)
of said payload over the course of less than one hour at a pH below
6.0 (e.g., pH 5.8, pH 5.6, pH 5.4, pH 5.2, pH 5.0, pH 4.8, pH 4.6,
pH 4.4, pH 4.2, pH 4.0, or lower, and ranges therein). In some
embodiments, the surface-exposed polymers are not cross-linked.
[0008] In some embodiments, provided herein are compositions
comprising a polymer-grafted nanobin (PGN), wherein said PGN
comprises a small phospholipid-based unilaminer vesicle (SUV)
comprising cholesterol-terminated poly(acrylic acid) groups
(Chol-PAA), wherein said Chol-PAA is oriented such that said
poly(acrylic acid) is surface exposed and extends from said SUV
into the surrounding environment, wherein said SUV encompasses a
molecular payload comprising one or more peptides, nucleic acids,
and/or small molecules.
[0009] In some embodiments, provided herein are methods of drug
delivery to a low-pH microenvironment within a subject or cell
comprising: (a) administering a polymer-grafted nanobin (PGN) to
said subject or cell, wherein said PGN comprises a small
phospholipid-based unilaminer vesicle (SUV) comprising
cholesterol-terminated poly(acrylic acid) groups (Chol-PAA),
wherein said Chol-PAA is oriented such that said poly(acrylic acid)
is surface exposed and extends from said SUV into the surrounding
environment, wherein said SUV encompasses a molecular payload
comprising one or more therapeutic agents; and (b) allowing said
PGN to migrate from physiologic conditions to an acidic
microenvironment. In some embodiments, the PGN is administered
locally at or near the acidic microenvironment. In some
embodiments, the PGN is administered systemically. In some
embodiments, the acidic microenvironment is a tumor. In some
embodiments, the therapeutic agent is a chemotherapeutic. In some
embodiments, payload is a nucleic acid-based therapy (e.g., siRNA,
antisense RNA, miRNA, ribozyme, vector encoding a gene, etc.). In
some embodiments, the PGN releases less than 20% of said payload
over the course of one month at normal physiologic conditions and
releases at least 50% of said payload over the course of less than
one hour at a pH between 4.0 and 6.0. In some embodiments, the PGN
is co-administered with an additional therapeutic agent. In some
embodiments, methods further comprise a step of surgically removing
the tumor (e.g., the administration step is performed before,
during, or after surgical removal of the tumor). In some
embodiments, methods further comprise a step of radiating or
ablating the tumor (e.g., the administration step is performed
before, during, or after radiating or ablating the tumor).
[0010] In some embodiments, provided herein are methods of
triggering the release of a molecular payload comprising: (a)
encapsulating the molecular payload within a polymer-grafted
nanobin (PGN); (b) placing the PGN under approximately physiologic
conditions; and (c) triggering the release of the molecular payload
by lowering the pH to below 6.0. In some embodiments, the PGN
comprises a small phospholipid-based unilaminer vesicle (SUV)
comprising cholesterol-terminated poly(acrylic acid) groups
(Chol-PAA), wherein said Chol-PAA is oriented such that said
poly(acrylic acid) is surface exposed and extends from said SUV
into the surrounding environment. In some embodiments, release is
triggered by lowering the pH below 5.5. In some embodiments,
lowering the pH comprises allowing the PNG to migrate into a low pH
environment. In some embodiments, lowering the pH comprises adding
a reagent to lower the pH of the PGN's environment.
[0011] In some embodiments, provided herein are polymer-grafted
nanobin (PGN)-encapsulated therapeutics for use as medicaments. In
some embodiments, provided herein is the use of a polymer-grafted
nanobin (PGN)-encapsulated therapeutic for the treatment of a
disease or condition. In some embodiments, provided herein is the
use of a PGN for the manufacture of a medicament for a therapeutic
application.
[0012] In some embodiments, provided herein are methods of
preparing a polymer-grafted nanobin (PGN) comprising: (a) preparing
a lipid mixture by dissolving selected lipids in an organic
solvent; (b) hydrating the product of step (a) with an aqueous
hydration solvent to form liposomes; (c) sizing the liposomes to
yield small unilaminar vesicles (SUVs); and (d) incubating the SUVs
with lipid-anchored polymer to generate PGNs with surface exposed
polymer. In some embodiments, the sizing step comprises one or more
of: (i) high sheer mixing, (ii) extruding through one or more
filters, and (iii) sonicating. In some embodiments, methods further
comprise a step of removing the organic solvent (e.g., prior to
step (b), after step (b), etc.). In some embodiments, the lipid
mixture comprises phospholipids. In some embodiments, lipid mixture
comprises at least 70% phosphiolipids (e.g., 70%, 75%, 80%, 85%,
90%, 95%, 99%, ranges therein). In some embodiments, the lipid
mixture comprises dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
Dipalmitoylphosphatidylcholine (DPPC), and/or
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some
embodiments, sizing results in SUVs between 15 and 50 nm in
diameter (e.g., 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50
nm, ranges therein). In some embodiments, sizing results in SUVs
with a PDI of less than 0.3 (e.g., 0.25, 0.2, 0.15, 0.1, 0.05, or
less, and ranges therein). In some embodiments, the lipid
constituent of the lipid-anchored polymer is a sterol. In some
embodiments, the sterol is cholesterol. In some embodiments, the
polymer constituent of the lipid-anchored polymer is poly(acrylic
acid). In some embodiments, the lipid-anchored polymer is
cholesterol-terminated poly(acrylic acid) (Chol-PAA). In some
embodiments, methods further comprise a step of hydrating with
hydrophilic molecular agent to yield an encapsulated molecular
payload. In some embodiments, the step of hydrating with
hydrophilic molecular agent is performed between steps (b) and (c).
In some embodiments, the step of hydrating with hydrophilic
molecular agent is performed between steps (c) and (d).
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. A schematic illustration of the drop-in synthesis of
PGN: a batch of preformed liposomes are sonicated into SUVs using a
probe sonicator, separated from heavy impurities by
ultracentrifugation, and stabilized by the addition of
Chol-PAA.
[0014] FIG. 2. Comparative stability and cargo-retention properties
of SUVs and PGNs derived from DOPC, DMPC, and DPPC. (A-C)
Hydrodynamic diameters (DH) of three independent batches of SUVs
and PGNs, as monitored by dynamic light scattering (DLS) over
sixteen weeks. The shown DH values are the volume-average
distribution data for the most significant population, which
emphasizes the presence of aggregation compared to data derived
based on a number distribution. The error bars represent the
standard deviations between three different batches. (D-F) The mean
calcein leakage profiles over a six-week period for three batches
of SUVs and PGNs. The error bars represent the average standard
deviation between three different batches.
[0015] FIG. 3. (A) TE micrograph and photographic image (inset) of
DPPC-SUVs three months after synthesis, illustrating the
instability of this formulation. (B) TE micrograph and photographic
image (inset) of DPPC-PGNs three months after synthesis,
illustrating the exceptional stability of this formulation.
[0016] FIG. 4. The release of DXR, as measured by fluorescence,
from DOPC-PGN and SUVs in response to in-situ acidification of pH
7.4 to pH 5.0.
[0017] FIG. 5. A schematic illustration of the proposed mechanism
by which clustering of the grafted Chol-PAA chains on the surface
of SUV-derived PGNs creates large defects on the already highly
curved membranes, leading to spontaneous release of the
payload.
[0018] FIG. 6. The arithmetic mean DH for PEG2000-, PEG3000-, and
PEG600-grafted DPPC-based SUVs over eight weeks of storage, as
quantified by DLS.
[0019] FIG. 7. Photographic image of PEG-SUVs after 3 months of
storage at 4.degree. C.: Chol-PEG.sub.600 modified DPPC (left),
DPPC-PEG.sub.2000 (middle), and DPPC-PEG.sub.3000 (right).
[0020] FIG. 8. Photographic images of SUV and PGN samples after six
months of storage at 4.degree. C. (A) DPPC-PGN (left), DPPC-SUVs
(right). (B) DMPC-PGN (left), DMPC-SUVs (right). (C) DOPC-PGN
(left), DOPC-SUVs (right). (D) DPPC-SUVs with 5 (left), 10 (left
middle), 15 (right middle), and 20 (right) mol % cholesterol
addition.
[0021] FIG. 9. TEM images of a sample of PEG.sub.2000-grafted
DPPC-SUVs stored at 4.degree. C. two days after preparation (A) and
two months after preparation (B).
[0022] FIG. 10. TEM images of a sample of DPPC-SUVs stored at
4.degree. C. two days after preparation (A) and six months after
preparation (B).
[0023] FIG. 11. TEM images of a sample of DPPC-PGN stored at
4.degree. C. two days after preparation (A) and six months after
preparation (B).
DEFINITIONS
[0024] As used herein, the term "lipid" refers to a variety of
compounds that are characterized by their solubility in organic
solvents. Such compounds include, but are not limited to, fats,
waxes, steroids, sterols, glycolipids, glycosphingolipids
(including gangliosides), phospholipids, terpenes, fat-soluble
vitamins, prostaglandins, carotenes, and chlorophylls. As used
herein, the terms "lipid-based materials" and "lipid assemblies"
refers to any material that contains lipids. In some embodiments,
"lipid assemblies" are structures including, but not limited to
vesicles, liposomes, films, micelles, dendrimers, monolayers,
bilayers, tubules, rods, and coils.
[0025] As used herein, the term "vesicle" refers to a small,
membrane-bilayer-enclosed structure. Membranes of vesicles may
comprise lipids, proteins, glycolipids, steroids or other
components associated with membranes. Vesicles can be naturally
generated (e.g., the vesicles present in the cytoplasm of cells
that transport molecules and partition specific cellular functions)
or can be synthetic (e.g., liposomes).
[0026] As used herein, the term "liposome" refers to
artificially-produced spherical lipid complexes that are induced to
segregate out of aqueous media. Liposomes are composed of
amphiphilic lipids arranged in a spherical bilayer. Liposomes are
unilamellar (e.g., contained within a single bilayer of amphiphilic
components (e.g., lipids)) or multilamellar vesicles.
[0027] As used herein, the term "surface exposed" refers to
molecules (e.g., polymers) that are present at the surface of a
structure (e.g., a lipid assembly) and are accessible to the
solvent surrounding the structure as well as being accessible to
other agents contained in the solvent. The terms "payload" and
"molecular payload" refer to any chemical entity, pharmaceutical,
drug (such drug can be, but not limited to, a small molecule, an
inorganic solid, a polymer, or a biopolymer), small molecule,
nucleic acid (e.g., DNA, RNA, siRNA, etc.), protein, peptide and
the like that is encompassed within a liposomal or vesicular
formulation described herein. A "therapeutic payload" refers to a
payload with the intended effect of providing a subject with
treatment or prevention of a disease or condition.
[0028] As used herein, the term "nanobin" refers to a nanoscale
molecular delivery vehicle wherein a molecular payload is
encapsulated with a lipid bilayer and polymeric exterior. Exemplary
nanobins are less than 100 nm in diameter, have an aqueous core
within a lipid bilayer for encapsulation of hydrophilic payloads
(e.g., small molecules, nucleic acids, peptides etc.), and have
surface accessible polymers anchored to the bilayer by a conjugated
lipid components (e.g., cholesterol-terminated poly(acrylic acid),
etc.).
[0029] As used herein, the term "physiologic conditions" refers to
solution or reaction conditions roughly simulating those most
commonly found in mammalian organisms, particularly humans (e.g.,
not relating to specific micorenvironments within organisms (e.g.,
not the acidic conditions (pH 5.0) commonly found in tumor
microenvironments and cellular late endosomes) or other rare
conditions). While variables such as temperature, availability of
cations, and pH ranges may vary, "physiologic conditions" typically
mean a temperature of 35-40.degree. C., with about 37.degree. C.
being particularly preferred, and a pH of 7.0-8.0, with about 7.5
being particularly preferred. The conditions may also include the
availability of cations, preferably divalent and/or monovalent
cations, with a concentration of about 2-15 mM Mg.sup.2+ and 0 1.0
M Na.sup.+ being particularly preferred.
DETAILED DESCRIPTION
[0030] Provided herein are unilaminar vesicles with
surface-displayed polymer moieties, and methods of use and
manufacture thereof. In particular, provided herein are
polymer-grafted nanobins, and methods of drug delivery therewith.
In some embodiments, the polymer-grafted nanobins comprise small
phospholipid-bilayer-based vesicles with lipid-terminated polymers
embedded in and extending from the bilayer, and methods of drug
delivery therewith.
[0031] In some embodiments, provided herein are fusogenic SUVs and
experiments demonstrating their long-term stabilization in
biologically relevant media (pH 7.4, 150 mM NaCl). By utilizing
polymer surface grafts (e.g., short poly(acrylic acid) (PAA)
surface grafts), metastable cargo-loaded SUVs were converted into
highly stable polymer-grafted nanobins (PGNs). Typically, the
potentials of these PGNs are quite negative (-45.+-.3 mV, Table 5),
which is predicted by the Derjaguin, Landau, Verwey and Overbeek
(DLVO) theory to be in the right repulsive range for the observed
stabilization (Laaksonen et al. ChemPhysChem 2006, 7 (10),
2143-2149; herein incorporated by reference in its entirety). Not
only can the polymer (e.g., PAA) surface grafts stabilize the PGNs
for over six months with minimal cargo leakage, these grafts also
cause PGNs to spontaneously release their cargos under the acidic
conditions (pH 5.0) commonly found in tumor microenvironments,
cellular late endosomes, and other acidic environemnts (Lee et al.
ACS Nano 2011, 5 (5), 3961-3969; herein incorporated by reference
in its entirety). This switchable combination of properties makes
PGNs a class of "smart" nanocarriers for biological
applications.
[0032] Experiments conducted during development of embodiments
described herein demonstrate that biocompatible Chol-PAA
polymer-grafts endow metastable SUVs with a high potential that
enables them to remain dispersed in biologically relevant solutions
for long periods. Additionally, the CholPAA-grafted PGNs exhibit a
combination of properties that are highly desirable for smart
nanocarriers, including: remarkable cargo retention under
physiologically relevant conditions along with a facile, abrupt
release of the payload in response to acidification. As Chol-PAA
grafting endows a large number of carboxyl groups
(.about.20,000/particle) on the PGN surface, this simple drop-in
modification extends the utility of SUVs beyond the immediate
benefits of their small sizes. Such functionalization can be used
as a robust conjugation handle for other functional groups such as
cellular-targeting ligands (Lee et al. J. Am. Chem. Soc. 2009, 131
(26), 9311-9320; herein incorporated by reference in its entirety),
therapeutics (Lee et al. J. Am. Chem. Soc. 2010, 132 (48),
17130-17138; herein incorporated by reference in its entirety), and
imaging agents (Lee et al. Angew. Chem., Int. Ed. 2010, 49 (51),
9960-9964; herein incorporated by reference in its entirety).
Together, the desirable combination of high stabilities, triggered
release capability, and functionalization handles enable PGN as a
robust and versatile scaffold for the smart and efficient delivery
of cargos for both targeted biological and non-biological
applications.
[0033] In some embodiments, the liposomes and/or vesicles (e.g.,
SUVs, PGNs, etc.) described herein are composed of any suitable
lipids, phospholipids, steroids (e.g., sterols), and other
components useful or suitable for the formation of such structures.
For example, suitable phospholipds for the formation of liposomes
include: 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
Dipalmitoylphosphatidylcholine (DPPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-Bis(dimethylphosphino)ethane (DMPE),
1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE),
1,2-ditetradecanoyl-sn-glycero-3-phosphate (DMPA),
1,2-Dipalmitoyl-sn-glycero-3-phosphate (DPPA),
1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphate (DOPA),
1,2-Dimyristoyl-sn-Glycero-3-PhosphoGlycerol (DMPG),
1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG),
1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS),
1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS),
1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS),
1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine (DSPE),
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), etc. Similarly,
suitable sterols for use in the formation of liposomes and other
vesicles described herein include, but are not limited to:
cholesterol, ergosterol, hopanoids, phytosterol, stanol, etc.
Further, any of the aforementioned components of liposomes and
vesicles may be appropriately modified (e.g., terminally modified)
with moieties, e.g., for interaction with the solvent surrounding
the structure or componentns therein. For example, one or more
liposome or vesicle components may be terminally modified, with a
suitable moiety such as: poly(ethylene glycol) (PEG), poly(ethylene
oxide)diacrylate (PEODA), polyacrylic acid, poly vinyl alcohol,
collagen, poly(D,L-lactide-co-glycolide (PLGA), polyglactin,
alginate, polyglycolic acid (PGA), other polyesters (e.g.,
poly-(L-lactic acid) (PLLA), polyanhydrides, poly(diol citrate)s,
etc.), etc. Examples of polymer modified lipids include
cholesterol-terminated poly(acrylic acid) (Chol-PAA) and
poly(ethylene glycol) modified DSPE (e.g., PE-PEG600, PE-PEG2000,
PE-PEG3000, etc.), poly(ethylene glycol) modified cholesterol
(Chol-PEG), etc.
[0034] In some embodiments, PGNs and other liposomal and/or
vesicular formulations described herein comprise between 70 mol %
and 100 mol % phospholipid content (e.g., 70 mol %, 75 mol %, 80
mol %, 85 mol %, 90 mol %, 95 mol %, 99 mol %, and ranges therein
(e.g., 85-95 mol %) within the lipid bilayer. In some embodiments,
a single type of phospholipid is present (e.g., DPPC, DMPC, DOPC,
etc.). In some embodiments, multiple types of the phospholipids
described herein make up the bilayer. In some embodiments,
lipid-terminated polymer (e.g., Chol-PAA) comprises 1-30 mol % of
the content of the lipid bilayer (e.g., 1 mol %, 2 mol %, 5 mol %,
10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, and ranges
therein (e.g., 5-20 mol %)).
[0035] In some embodiments, PGN and/or other liposomal/vesicular
formulations further comprise a cryo- and/or lyo-protecting agent.
During storage of liposomes the phospholipids may undergo
hydrolysis. One simple way of preventing decomposition of the
phospholipids in the liposome formulation is by freezing or
freeze-drying. Freezing may however induce leakage of the liposome
formulation and result in release of the encapsulated drug.
Addition of a cryo-protecting agent may prevent or reduce leakage
from a preparation after freezing. Examples of agents that may be
used as cryo-protecting agents may without limitation be
disaccharides such as sucrose, maltose and/or trehalose. Such
agents may be used at various concentrations depending on the
preparation and the selected agent such as to obtain an isotonic
solution. In some embodiments, PGN and/or other liposomal/vesicular
formulations are freeze-dried, stored and the reconstituted such
that a substantial portion of the internal contents are retained.
Dehydration generally requires use of a lyo-protecting agent such
as a disaccharide (sucrose, maltose or trehalose) at both the
inside and outside interfaces of the bilayer. This hydrophilic
compound prevents the rearrangement of the lipids in the
formulation, so that the size and contents are maintained during
the drying procedure and through subsequent reconstitution.
Appropriate qualities for such drying protecting agents are that
they possess stereo chemical features that preserve the
intermolecular spacing of the liposome bilayer components.
[0036] In some embodiments, PGNs comprise one or more functional
surface moieties to confer one or more beneficial functionalities
to the PGNs. Exemplary functional moieties may include, but are not
limited to: a detectable moiety (e.g., fluorophore, chromophore,
contrast agent, radionuclide, etc.), a
targeting/binding/interaction moiety (e.g., antibody, antibody
fragment, binding peptide (e.g., recognized by a cell surface
receptor), etc.), etc. For example, suitabale functional moieties
may include: one or more small molecules (e.g., drugs, drug-like
molecules), biomolecules, a peptide or polypeptide (protein)
including an antibody or a fragment thereof, a His-tag, a FLAG tag,
a Strep-tag, an enzyme, a cofactor, a coenzyme, a substrate for an
enzyme, a suicide substrate, a receptor, double stranded or single
stranded nucleic acid (e.g., RNA or DNA), e.g., capable of binding
a protein, a glycoprotein, a polysaccharide, a peptide-nucleic acid
(PNA), a solid support (e.g., a sedimental particle such as a
magnetic particle, a sepharose or cellulose bead, a membrane, a
glass slide, cellulose, alginate, plastic or other synthetically
prepared polymer (e.g., an eppendorf tube or a well of a multi-well
plate, etc.), etc.), a drug (e.g., chemotherapeutic), pH sensor, a
radionuclide, a contrast agent, a chelating agent, a cross-linking
group (e.g., a succinimidyl ester or aldehyde, maleimide, etc.),
glutathione, biotin, streptavidin, one or more dyes (e.g., a
xanthene dye, a calcium sensitive dye (e.g.,
1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)-phenoxy]-2-(2'-am-
- -ino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (Fluo-3),
etc.), a sodium sensitive dye (e.g., 1,3-benzenedicarboxylic acid,
4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-
-6,2-benzofurandiyl)]bis (PBFI), etc.), a NO sensitive dye (e.g.,
4-amino-5-methylamino-2',7'-difluorescein), or other fluorophore, a
hapten or an immunogenic molecule (e.g., one which is bound by
antibodies specific for that molecule), etc.
[0037] Functional moieties may be attached to the polymer portion
of the lipid-terminated polymer components of PGNs (e.g., the
poly(acrylic acid) portion of Chol-PAA), may be attached to the
head group of phospholipids within the bilayer, may be attached to
lipophilic moieties embedded within the bilayer (e.g., cholesterol
groups), etc. Functional moieties may be directly attached to
components of the PGN or may be connected by a suitable linker
(e.g., carbon-containing chain, peptide, cleavable linker,
etc.).
[0038] In some embodiments, PGN's comprise one or more functional
moieties to direct and/or localize the PGNs to intended locations
of drug delivery (e.g., acidic tumor microenvironments). In some
embodiments, PGNs display a tumor-targeting ligand to direct the
PGNs to tumor microenvironments, such tumor-targeting ligands
areselected from folic acid, retinoic acid, a peptide, an estrogen
analog, transferrin, and granulocyte-macrophage colony stimulating
factor. In some embodiments, the targeting moiety comprises an
antibody. In some embodiments, the antibody is selected from
RITUXAN, HERCEPTIN, CAMPATH-1H, HM1.24, anti-HER2, Anti-CD38,
HuM195, HP67.6, TRAIL mAb, transferin, ATN-291, and prolactin. Any
moiety capable of directing a PGN to a tumor may find use in
emboidments herein.
[0039] In some embodiments, provided herein are methods of
generating or synthesizing liposomes and/or vesicles from component
elements. Suitable techniques for assembling such components into
liposomes are understood in the field. For example, an exemplary
method comprises the steps of: (a) preparing a lipid mixture by
dissolving selected lipids in an organic solvent; (b) hydrating the
product of step (a) with an aqueous hydration solvent so as to form
liposomes; and (c) removing the organic solvent of step (a) either
before addition of the aqueous hydration solvent or after the
addition of the aqueous hydration solvent. Suitable methods may
further comprise steps of, for example, (1) high sheer mixing to
reduce the size of the liposomes, (2) extruding the liposomes
through filter(s) to produce liposomes of a certain mean size,
and/or (3) sonicating the liposomal formulation to produce
liposomes of a certain size.
[0040] PGNs, SUVs, and/or other liposomal/vesicular formulations
may be loaded with at least one molecular payload (e.g.,
therapeutic agent) by suitable methods understood in the field. For
example, by solubilizing the compound in the organic solvent or
hydration solvent used to prepare the liposomes. Alternatively, an
ionizable therapeutic agent can be loaded into vesicles by
establishing an electrochemical potential, e.g., by way of a pH
gradient, across the outermost bilayer, and then adding the
ionizable therapeutic agent to the aqueous medium external to the
liposome.
[0041] In some embodiments, methods further comprise a step of
changing the exterior aqueous phase of the formulation. In some
embodiments, the aqueous phase initially comprises the hydration
solvent. The exterior aqueous phase may be changed by
centrifugation, ultrafiltration, dialysis or similar in order to
prepare a liposomal formulation comprising vesicles (e.g. PNG) in a
solution of defined composition of the ex terior aqueous phase. In
some embodiments, bioactive compounds (therapeutic agents) are only
present inside or attached to the vesicles (e.g. PNG) and not as
free compounds in solution. Preferably, the drug encapsulation in
the vesicles (e.g. PNG) is >70%, more preferably >95% and
most preferably >99%. The degree of drug encapsulation is the
ratio of drug encapsulated to the total amount of drug in the
formulation.
[0042] In some embodiments, the PGN and/or other
liposomal/vesicular formulations described herein exhibit
hydrodynamic diameters of 15-50 nm (e.g., 15 nm, 20 nm, 15 nm, 20
nm, 15 nm, 20 nm, 15 nm, 20 nm, and ranges therein (e.g., 30-50
nm)).
[0043] In some embodiments, the Poly Dispersity Index (PDI) of the
PGN and/or other liposomal/vesicular formulations described herein
do not exceed 0.3 (e.g., 0.3, 0.25, 0.2, 0.15, 0.10, 0.05, 0.01,
and ranges therein (e.g., 0.05-0.15 PDI)). A PDI value in the
ranges described herein represented a relatively narrow particle
size-distribution in the formulations. One object of the
embodiments described herein is to provide liposomal and/or
vesicular formulations with increased stability during storage. In
some embodiments, the PGNs described herein exhibit dispersion
and/or degradation of less than 20% of PGNs in a sample (e.g.,
<20%, <15%, <10%, <5%, <4%, <3%, <2%, <1%,
and ranges therein (e.g., 1-5%)) over a time period of at least one
month (e.g., >1 month, >2 months, >3 months, >6 months,
>1 year, and ranges therein (e.g., 6 months to 1 year)).
[0044] Liposomes and vesicles can be used to carry various
compounds such as, e.g., drugs, encapsulated within the interior
aqueous compartment (e.g., encapsulated within the
liposome/vesicle) and/or embedded within the bilayer (e.g.,
embedded in the liposome/vesicle). Depending on the chemical nature
of the compound to be encapsulated it will be localized to either
of the compartments. Currently, there are several parenteral
liposome-drug formulations available on the market. Water soluble
drugs tend to be encapsulated in the aqueous compartment of
liposomes, and examples of drugs encapsulated in liposome's are,
e.g., doxorubicin (Doxil), doxorubicin (Myocet) and daunorubicin
(DaunoXone). Examples of drugs intercalated in the liposome
membrane are, e.g., amphotericin B (AmBisome), amphotericin
(Albelcet B), benzoporphyrin (Visudyne) and
muramyltripeptide-phosphatidylethanolamine (Junovan). Embodiments
described herein are not limited by the variety of drugs or other
molecular payloads that can be encapsulated within or embedded in
the PGN.
[0045] Unless specifically stated, the embodiments described herein
are not to be limited by the identity of the potential agents that
can be delivered therewith. In some embodiments, agents are
embedded within the bilayer or linked to hydrophobic moieties
within the bilayer but the agent is surface exposed. Due to the
triggered release functionality of the PGNs and/or
liposomal/vesicular formulations described herein, the compositions
are of particular utility for the encapsulation of water soluble
agents (or solubilized agents) within the PGN (e.g., as a molecular
payload) or other formulation and/or delivery/release of those
agents at a desired time/location. Suitable molecular payloads
include small molecules (e.g., drugs or drug-like molecules),
peptides, polypeptides, nucleic acids (e.g., DNA (e.g., genes,
microgenes, DNAzymes, etc.), RNA (e.g., siRNA, miRNA, antisense
RNA, RNA decoys, ribozymes), aptamers (e.g., DNA or RNA), nucleic
acid vectors (e.g., encoding genes), etc.), etc. In some
embodiments, PGNs are useful for the delivery of a nucleic acid
therapeutic (or other agent) to a desired cell type or
microenvironment (e.g., acidic micorenvironemnt) followed by
release of the nucleic acid (or other agent). A description of
nucleic acid therapeutics, for which the delivery systems described
herein may provide utility are described, for example, by
Pushpendra et al. in V. A. Erdmann and J. Barciszewski (eds.), From
Nucleic Acids Sequences to Molecular Medicine, RNA Technologies,
DOI 10.1007/978-3-642-27426-8_2, Springer-Verlag Berlin Heidelberg
2012; herein incorporated by reference in its entirety). In some
embodiments, the molecular payload comprises a peptide therapeutic;
descriptions of useful peptide therapeutics include Boohaker et al.
Curr Med Chem. 2012;19(22):3794-804; Kaspar and Reichert. Drug
Discov Today. 2013 September;18(17-18):807-17; herein incorporated
by reference in their entireties).
[0046] An object of the embodiments described herein is to provide
formulations for drug delivery that deliver their payload (e.g., a
drug) to the target site, and release the payload at the target
site (e.g., >25% release, >30% release, >40% release,
>50% release, >60% release, >70% release, >80% release,
>90% release, and ranges therein (e.g., 40-70%)) with low
uncontrolled/non-specific delivery and/or leakiness (e.g., <1%,
<2%, <5%, <10%, <20%, <25%, and ranges therein
(e.g., 1-10%)) at a non-target site and/or under non-target
conditions (e.g., normal physiologic conditions).
[0047] The PGNs and other liposomal/vesicular formulations provided
herein find use in a variety of applications. As described
throughout, PGNs exhibit a variety of characteristics that make
them particularly well suited for drug delivery. In particular, due
to the pH dependence of their payload release (e.g., little leakage
at physiologic pH and bulk release at acidic pH (e.g., <pH 6.0,
<pH 5.8, <pH5.6, <pH 5.4, <pH 5.2, or lower), etc.),
certain PNGs described herein are particularly well suited for
delivery of a payload (e.g., therapeutic agent) through a
physiologic environment for release of payload (e.g., delivery of
therapeutic) in an acidic environment (e.g., acidic extracellular
microenvirnment. Therefore, in some embodiments, PGNs and other
liposomal/vesicular formulations provided herein find use in the
treatment of cancer (e.g., solid tumor cancer). In some
embodiments, compositions (e.g., comprising PGNs) described herein
find use in drug delivery and the treatment of cancers, for
example, adenomas, carcinomas or sarcomas, and including but not
limited to: melanoma, brain tumors, neuroblastomas, breast cancer,
lung cancer, prostate cancer, cervix cancer, uterine cancer,
ovarian cancer, colon cancer, rectum cancer, cancer of the testis,
cancer of the kidney, cancer of the liver, cancer of the lip,
cancer of the tongue, cancer of the stomach, skin cancer,
mesotheliomas, bladder cancer, bone tumors, malignant pleural
effusions, ascites, meningeal carcinomatosis, head and neck
cancers, cancers of endocrine organs such as: thyroid gland,
pituitary gland and suprarenal gland, etc.
[0048] As addressed herein, PGNs and/or other liposomal/vesicular
formulations may display, for example, various targeting moieties
that direct the drug-delivery vehicle to the appropriate tumor and
allow release of therapeutic (e.g., chemotherapeutic) payload in
the acidic tumor microenvironment. In alternative embodiments, PGNs
and/or other liposomal/vesicular formulations are delivered
directly to a tumor microenvironment.
[0049] Drug-delivery utilizing PGNs and/or other
liposomal/vesicular formulations described herein are not limited
to cancer treatment. For example, the compositions described herein
find use in the delivery to any acidic locations (e.g., vaginal
mucosa, stomach, fallopian tubes, etc.).
[0050] In some embodiments, because of the ability of the
compositions described herein to stably contain a molecular payload
with minimal leakage, such compositions find use in containment of
molecular agents and or bulk release of such agents at a desired
time point. Such functionality may be particularly useful in the
containment of toxic substances and/or the precise timing of the
release of a chemical reactant.
[0051] Applications for PGNs and/or other liposomal/vesicular
formulations are not limited to drug-delivery. For example,
compositions described herein may find use in: the formulation of
nutritional/dietary supplements, cosmetics, and lubricants;
regenerative medicine; industrial applications; agricultural
applications (e.g., pesticide delivery); veterinary applications;
etc.
Experimental
EXAMPLE 1
Materials
[0052] All lipids--1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (ammonium salt) (PE-PEG.sub.2000), and
2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-3000] (ammonium salt) (PE-PEG.sub.3000)--were purchased
from Avanti Polar Lipids, Inc., as either a dry powder or a
chloroform solution. Cholesterol-PEG.sub.600,
N-tert-butyl-O-[1-[4-(chloromethyl)phenyl]ethyl]-N-(2-methyl-1-phenylprop-
yl)hydroxylamine, 2,2,5-trimethyl-4-phenyl-3-azahexane-3-nitroxide,
cholesterol, and tent-butyl acrylate were purchased from
Sigma-Aldrich (Milwaukee, Wis.) and used as received. Doxorubicin
hydrochloride was purchased from PolyMed Therapeutics (Houston,
Tex.). Ultrapure deionized water (18.2 MS) cm resistivity) was
obtained from a Millipore system (Billerica, Mass.).
[0053] HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid)-buffered saline (HBS) solution (20 mM HEPES, 150 mM NaCl, pH
7.4) was prepared using standard protocols. Chol-PAA (M.sub.n=10.7
kDa and PDI=1.12) was prepared following a literature procedure
(Leevet al. J. Am. Chem. Soc. 2007, 129(49), 15096; herein
incorporated by reference in its entirety). A 20 mM solution of
calcein in HBS was prepared by sonicating the appropriate amount of
powdered calcein (Sigma-Aldrich) in HBS for 10 minutes using a
probe sonicator at room temperature.
EXAMPLE 2
Instrumentation
[0054] Phosphorus concentrations of the synthesized materials were
determined using a Varian Vista MPX (Varian, Inc., Palo Alto,
Calif.) simultaneous inductively coupled plasma optical emission
spectrometer (ICP-OES).
[0055] Polymer molecular weights were determined relative to
polystyrene standards on a Varian PL-GPC 50 Plus (Varian, Inc.,
Palo Aalto, Calif.) gel-permeation chromatography (GPC) system
equipped with Cirrus software, a PL-50 RT GPC autosampler, both RI
and UV detectors, Agilent Resipore guard column, and Agilent
Mesopore and Resipore columns (both 300.times.7.5 mm in size) in
series. HPLC-grade chloroform was used as an eluent at a flow rate
of 1.0 mL/min and the instrument was calibrated using polystyrene
standards (Aldrich Chemical Co., 6 standards, 2,330-18,700
Daltons).
[0056] Dynamic light scattering (DLS) and zeta potential
measurements were performed on a Zetasizer Nano ZS (Malvern
Instruments, Malvern, UK) equipped with a He-Ne laser (633 nm).
Non-invasive backscatter method (detection at 173.degree.
scattering angle) was used. Correlation data were fitted, using the
method of cumulants, to the logarithm of the correlation function,
yielding the diffusion coefficient (D). The hydrodynamic diameters
(D.sub.H) of the small unilamellar vesicles (SUVs) and PCNs were
calculated using D and the Stokes-Einstein equation
(D.sub.H=k.sub.BT/3.pi..eta.D, where k.sub.B is the Boltzmann
constant, T is the absolute temperature, and .eta. is the solvent
viscosity (.eta.=0.8872 cP for water)). The polydispersity index
(PDI) of liposomes--represented as 2c/b.sup.2, where b and c are
first- and second-order coefficients, respectively, in a polynomial
of a semi-log correlation function--was calculated by cumulants
analysis. Size distribution of vesicles was obtained by
non-negative least squares (NNLS) analysis..sup.S2 Typically, a
sizing sample was prepared by adding a small aliquot (10 .mu.L) of
the lipid dispersion into a low-volume disposable sizing cuvette
(Malvern Instruments, Malvern, UK) filled with 20 mM HBS (750
.mu.L) and the dispersion was briefly mixed with a pipet before
data was collected.
[0057] Typically, a potential measurement sample was prepared by
adding a small aliquot (20 .mu.L, twice as concentrated as that for
the sizing sample to increase count rate) of the lipid dispersion
into a disposable capillary cell (Malvern Instruments, Malvern, UK)
filled with 20 mM HBS (740 .mu.L) and the dispersion was briefly
mixed with a pipet before data was collected. The reported data
represents the statistical average of the volume particle size
distribution (volume PSD) from five measurements with 25 scans
each, the calculated standard deviation is the error associated
with each measurement. If two or more significant (>1%),
populations occur within one sample, they are reported as relative
percent population (Tables 1-4).
TABLE-US-00001 TABLE 1 Volume particle size distribution (volume
PSD) data collected for three separate batches for each DPPC-,
DMPC-, and DOPC-SUVs. If two or more significant (>1%)
populations occur within one sample, they are reported as relative
percent populations. Errors are represented as average standard
deviations of the volume PSD. DPPC-SUV DMPC-SUV DOPC-SUV Time
Volume % Volume % Volume % (days) PSD (nm) population PDI PSD (nm)
population PDI PSD (nm) population PDI 0 48 .+-. 3 86 0.57 26 .+-.
3 100 0.33 32 .+-. 5 100 0.28 646 .+-. 35 14 15 58 .+-. 1 65 0.68
34 .+-. 2 100 0.45 34 .+-. 3 100 0.25 1450 .+-. 84 35 30 1450 .+-.
204 70 1 615 .+-. 110 88 0.39 35 .+-. 2 100 0.26 52 .+-. 5 30 28
.+-. 3 11 60 920 .+-. 150 100 1 545 .+-. 230 100 0.33 39 .+-. 2 100
0.31 90 1330 .+-. 200 100 0.89 654 .+-. 200 100 0.47 42 .+-. 1 100
0.35 120 1330 .+-. 250 100 1 715 .+-. 250 100 0.55 35 .+-. 2 100
0.31 150 1330 .+-. 200 100 1 740 .+-. 230 100 0.54 46 .+-. 6 100
0.35 180 1330 .+-. 250 100 1 720 .+-. 250 100 0.55 36 .+-. 5 100
0.32
TABLE-US-00002 TABLE 2 Volume particle size distribution (volume
PSD) data collected for three separate batches for each DPPC-,
DMPC-, and DOPC-PGNs. If two or more significant (>1%)
populations occur within one sample, they are reported as a
relative percent populations. Error is represented as .+-. average
standard deviations of the volume PSD. DPPC-PGN DMPC-PGN DOPC-PGN
Volume Volume Volume Time PSD % PSD % PSD % (days) (nm) population
PDI (nm) population PDI (nm) population PDI 0 46 .+-. 1 100 0.33 38
.+-. 4 100 0.35 41 .+-. 2 100 0.28 15 52 .+-. 4 100 0.22 37 .+-. 4
100 0.36 42 .+-. 5 100 0.31 30 51 .+-. 3 100 0.27 36 .+-. 5 100
0.33 35 .+-. 3 100 0.26 60 53 .+-. 2 100 0.26 38 .+-. 4 100 0.40 41
.+-. 2 100 0.28 90 56 .+-. 5 100 0.24 37 .+-. 7 100 0.39 40 .+-. 2
100 0.28 120 55 .+-. 5 100 0.29 38 .+-. 3 100 0.52 38 .+-. 5 100
0.27 150 54 .+-. 2 100 0.32 39 .+-. 6 100 0.46 36 .+-. 3 100 0.25
180 54 .+-. 2 100 0.27 38 .+-. 6 100 0.50 34 .+-. 4 100 0.25
TABLE-US-00003 TABLE 3 Volume particle size distribution (volume
PSD) data collected for three separate batches for each PEG-
incorporated DPPC-based SUV: PEG.sub.600-, PEG.sub.2000-, and
PEG.sub.3000-SUVs. If two or more significant (>1%) populations
occur within one sample, they are reported as relative percent
populations. Errors are represented as average standard deviations
of the volume PSD. PEG.sub.600-SUV PEG.sub.2000-SUV
PEG.sub.3000-SUV Volume Volume Volume Time PSD % PSD % PSD % (days)
(nm) population PDI (nm) population PDI (nm) population PDI 0 86
.+-. 8 85 0.45 40 .+-. 4 60 0.20 60 .+-. 3 82 0.43 612 .+-. 41 15
173 .+-. 7 40 183 .+-. 9 18 15 760 .+-. 67 74 0.56 45 .+-. 5 60
0.25 109 .+-. 14 70 0.41 153 .+-. 3 26 162 .+-. 8 40 543 .+-. 43 30
30 670 .+-. 40 55 0.59 162 .+-. 12 72 0.22 86 .+-. 7 55 0.42 65
.+-. 8 45 55 .+-. 2 28 825 .+-. 57 45 60 527 .+-. 52 82 0.40 190
.+-. 10 67 0.41 92 .+-. 8 57 0.21 98 .+-. 9 18 64 .+-. 2 33 715
.+-. 45 43
TABLE-US-00004 TABLE 4 Volume particle size distribution (volume
PSD) data collected for three separate batches for each
cholesterol-incorporated DPPC-based SUV: Chol.sub.5%-,
Chol.sub.10%-, Chol.sub.15%-, and Chol.sub.20%-SUV. If two or more
significant (>1%) populations occur within one sample, they are
reported as relative percent populations. Errors are represented as
average standard deviations of the volume PSD. Chol.sub.5%-SUV
Chol.sub.10%-SUV Chol.sub.15%-SUV Chol.sub.20%-SUV Volume % Volume
Volume % Time PSD pop- PSD % PSD % Volume pop- (days) (nm) ulation
PDI (nm) population PDI (nm) population PDI PSD (nm) ulation PDI 0
68 .+-. 11 100 0.33 80 .+-. 1 100 0.35 55 .+-. 1 100 0.28 60 .+-. 1
100 0.33 15 1505 .+-. 176 73 0.65 825 .+-. 227 100 1 710 .+-. 120
52 0.76 564 .+-. 192 95 0.88 76 .+-. 81 27 45 .+-. 6 48 50 .+-. 6 5
30 711 .+-. 81 55 0.76 1511 .+-. 170 100 1 752 .+-. 95 70 1 3115
.+-. 307 100 1 54 .+-. 7 45 45 .+-. 5 30
[0058] Fourier-transformed nuclear magnetic resonance (NMR)
spectroscopy of Chol-PAA was performed on a Varian INOVA-500 MHz
spectrometer (Varian, Inc., Palo Alto, Calif.). Chemical shifts of
.sup.1H NMR spectra are reported in ppm against residual solvent
resonance as the internal standard (CHCl.sub.3=7.27 ppm,
CHD.sub.2COCD.sub.3=2.05 ppm, CHD.sub.2OD=3.31 ppm, D.sub.2O=4.8
ppm. Fluorescence emission spectra were obtained on a Jobin Yvon
Fluorolog fluorometer (.lamda..sub.ex=480 nm, slith width=3 nm for
Doxorubicin and .lamda..sub.ex=515 nm for Calcein).
[0059] Ultracentrifugation was carried out on a refrigerated
Beckmann-Coulter Optima.TM. XPN ultracentrifuge (Beckmann-Coulter,
Inc., Indianapolis, Ind.). Lyophilization was carried out on a
Freezone lyophilizer (Labconco, Kansas City, Mo.). High-power
sonication was carried out using a titanium-alloy solid probe
ultrasonicator (500 watt Vibra-Cell.TM. VC 505, Sonics &
Materials, Inc., Newtown, Conn.) set at 20 kHz, 40% intensity
without pulsing. All tangential flow filtration (TFF) was manually
carried out using a 50 kD pore size 8 cm.sup.2 polystyrene (0.5 mm)
MicroKros.TM. Module (Spectrum Labs, Rancho Dominguez, Calif.).
EXAMPLE 3
Preparation of SUV-Based Materials.
[0060] Preparation of DPPC-, DMPC-, and DOPC-SUVs. The appropriate
lipid (40 .mu.mol) was dissolved in HPLC-grade chloroform (1.0 mL)
in a 20 mL vial and then gently evaporated by a stream of nitrogen.
The resulting lipid film was thoroughly dried via lyophilization
for 24 hours and then hydrated with of 20 mM HBS (5.0 mL) followed
by vigorous vortexing for 5 minutes. The liposome suspension was
then submerged in an ice bath and probe-sonicated for 20 minutes
with the tip submerged .about.1 cm into the sample and without
pulsing. The resulting suspension was then diluted with HBS to 10
mL and then ultracentrifuged at 104,986 g and 4.degree. C. for 1
hour. To avoid contamination by the metal filings from the probe
and large liposomal pellet, only the top 9 mL of the supernatant
was collected using a serological pipet; this portion contains the
desired SUV dispersion.
[0061] To monitor particle size distribution as a function of
storage time, three batches of each SUV were stored in 20 mL vials:
the surfaces of the dispersions were blanketed briefly with a
stream of nitrogen, the vial were capped with a plastic screw cap,
and the samples were stored at 4.degree. C. Lipid concentrations
(2.45 mM for DPPC, 2.62 mM for DMPC, and 4.60 mM for DOPC) were
calculated using phosphorous ICP-AES.
Preparation of Polymer-Grafted Nanobins (PGNs).
[0062] In a typical preparation, Chol-PAA (10 mol % of the lipid)
was dissolved in 20 mM HBS (.about.20 .mu.L), corrected to pH 7.4
using aqueous NaOH (1 N), and added dropwise to a stirring solution
of SUVs (lipid concentrations shown above) in a 20 mL vial. The
resulting solution was allowed to stir at room temperature for 24
hours to form the desired PGN dispersion. To monitor particle size
distribution as a function of storage time, three batches of each
PGN were stored in 20 mL vials: the surfaces of the dispersions
were blanketed briefly with a stream of nitrogen, the vials were
capped with a plastic screw cap, and the samples were stored at
4.degree. C. As the addition of Chol-PAA results in a minimal
volume change, the final lipid concentrations of these dispersions
were assumed to be the same as those for the starting SUV
dispersions (2.45 mM for DPPC, 2.62 mM for DMPC, and 4.60 mM for
DOPC).
Preparation of SUV-PEG600, SUV-PEG2000 and SUV-PEG3000
[0063] These SUV-PEG dispersions were prepared using the same
method described above for the lipid-only SUVs except with
different lipid formulations (90 mol % of either DPPC or DMPC and
10 mol % of either PE-PEG2000 or PE-PEG3000). There is a persistent
layer of foam on the surface of these dispersions after
preparation; this layer increases in volume after sonication. The
surface of these dispersions was blanketed briefly with a stream of
nitrogen, capped with a plastic screw cap, and stored under inert
atmosphere at 4.degree. C. The volume size distributions data are
tabulated in Table 3. SUV-PEG600 were prepared using the same
method described above for the PGN polymer insertion except that
Chol-PEG600 (10 mol % of the lipid concentration) was inserted into
the SUVs.
Preparation of Calcein-Loaded SUVs and PGNs
[0064] This procedure is similar to the preparation of SUVs
described above except that the lyophilized lipid film (40 .mu.mol)
was first hydrated with the 20 mM calcein solution in HBS (1 mL).
After vortexing for 5 minutes, this dispersion was then diluted to
5 mL with 20 mM HBS.
[0065] The resulting calcein-loaded SUVs were purified by
tangential flow filtration (TFF) during which time the particles
were washed with extra 20 mMHBS solution (40 mL). The resulting
purified calcein-loaded SUVs (-1 mL) were used immediately for PGN
preparation or stored at 4.degree. C. for leakage evaluation.
Calcein-loaded PGNs were formed using the purified calcein-loaded
SUVs using the same procedure described above for PGNs.
Preparation of Doxorubicin-Loaded SUVs and PGNs
[0066] In a typical experiment, DOPC (40 .mu.mol) was dissolved in
HPLC-grade chloroform in a 20 mL vial and then gently evaporated by
a stream of nitrogen. The resulting lipid film was thoroughly dried
via lyophilization for 24 hours and then hydrated with of 200 mM
ammonium sulfate (5.0 mL) followed by vigorous vortexing for 5
minutes. The liposome suspension was then submerged in an ice bath
and probe-sonicated for 20 minutes with the tip submerged .about.1
cm into the sample and without pulsing. The resulting suspension
was then diluted with 20 mM HBS to 10 mL and then ultracentrifuged
at 104,986 g and 4.degree. C. for 1 hour. To avoid contamination by
the metal filings from the probe and large liposomal pellet, only
the top 9 mL of the supernatant was collected using a serological
pipet; this portion contains the desired SUV dispersion. Using TFF
the ammonium sulfate-loaded SUVs were washed with 20 mM HBS (40
mL). The encapsulation of DXR was evaluated by calculating the
drug-to-lipid ratio which was consistently 0.22 (mol DXR/ mol
lipid).
[0067] Into a 20 ml vial containing a stirring solution of the
ammonium sulfate-loaded DOPC particles (5.0 mL, DOPC
concentration=5.6 mM) was slowly added an HBS solution of
doxorubicin hydrochloride (17.5 mg, 1.8 equiv of DOPC) over 15
minutes. The vial was then wrapped in aluminum foil and allowed to
stir at room temperature for 24 hours. The unincorporated
doxorubicin hydrochloride was separated from the loaded dispersions
via gel-filtration chromatography (1/4.times.5 inch plug of
Sepharose CL-B4 and HBS elution). The purified DXR-loaded SUVs
(DOPC-SUVDXR) were collected as the first ten 1 mL fractions.
DXR-loaded PGN (DOPC-PGNDXR) were prepared from DOPC-SUVDXR using
the same Chol-PAA incorporation procedure described above for PGN
preparation.
EXAMPLE 4
Synthesis and Colloidal Stability of PGN vs SUV
[0068] SUVs with narrow size distributions were prepared from the
appropriate lipid (FIG. 1) using a modification of a previously
reported protocol (Wong et al. Biochemistry 1982, 21 (17),
4126-4132; herein incorporated by reference in its entirety). To
cover a broad range of phase transition temperatures (T.sub.m's),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), or
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were selected
(T.sub.m=41.degree. C., 23.degree. C., and -21.degree. C.,
respectively). The SUVs were then grafted with a
cholesterol-terminated poly(acrylic acid) (Chol-PAA, M.sub.n=4670
Da, PDI=0.12) following a "drop-in" method (FIG. 1) (Lee et al. J.
Am. Chem. Soc. 2007, 129 (49), 15096-+; herein incorporated by
reference in its entirety). The resulting PGNs were then stored at
4.degree. C., below the T.sub.m's of DPPC and DMPC, which enhances
the fusogenicity of the SUVs derived from these lipids (Ellens et
al. Biochemistry 1989, 28 (9), 3692-3703; herein incorporated by
reference in its entirety). Their long-term colloidal stabilities
in 20 mM HEPES buffered saline (HBS, 150 mM NaCl) were regularly
monitored and compared to the unmodified SUVs (FIG. 2).
[0069] For DPPC- and DMPC-based PGN formulations (DPPC-PGN and
DMPC-PGN, respectively), the dynamic light scattering (DLS, FIGS.
2a and 2b) and transmission electron microscopy (TEM, FIG. 3a) data
clearly indicate that these dispersions are stable at 4.degree. C.
for at least six months after preparation. In stark contrast, the
corresponding unmodified SUVs, which are expected to be fusogenic
at temperatures below the lipid T.sub.m's, show poor
dispersability, with aggregation occurring within three weeks of
preparation (FIGS. 2a, 2b, and 3b), and formation of a visible
flocculent after four weeks (FIG. 3b inset). This is consistent
with previous observations that the r; herein incorporated by
reference in its entiretyates of fusion and aggregation of SUVs are
directly correlated with the T.sub.m's of the lipids that make up
these vesicles, and these rates increase at temperatures below
T.sub.m (Bentz & Ellens. Colloids Surf 1988, 30 (1-2),
65-112.). While the low T.sub.m of DOPC may explain the better
dispersability of DOPC-SUVs, in comparison to DPPC- and DMPC-SUVs,
when stored at 4.degree. C., it does not fully explain the
consistently high cargo leakage rates observed for all three
formulations (FIGS. 2d, 2e, and 2f).
EXAMPLE 5
Cargo Leakage
[0070] Sustained colloidal stability and cargo retention are key
criteria for biodelivery carriers. To evaluate the cargo-retention
capability of the three exemplary PGN formulations against the
unmodified SUVs, the carriers were loaded with the fluorescent
small-molecule calcein and compared their leakage profiles when
stored at 4.degree. C., which is the recommended storage
temperature for the liposome-based drug Doxil (Doxil Storage.
doxil.com/hcp/iv-preparation;
[0071] herein incorporated by reference in its entirety).
Remarkably, DMPC- and DPPC-PGNs both retain over 95% of their
calcein payloads over a one-month period while the corresponding
SUV analogs displayed significant leakage (30% and 73%,
respectively) (FIGS. 2d and 2e).
[0072] Even the less fusogenic DOPC-based SUVs still leak 15% of
the encapsulated calcein after one month, during which time the
corresponding DOPC-PGNs show less than 1% leakage (FIG. 2f). This
observation is consistent with a previous result that showed rapid
calcein leakage occurring from egg phosphatidylcholine SUVs when
these were stored above the lipid transition temperature
(T.sub.m=-10.degree. C.), where fusion should be minimized
(Mercadal et al. Biochim. Biophys. Acta, Biomembr. 1995, 1235 (2),
281-288; herein incorporated by reference in its entirety).
Although the mechanism associated with leakage from cargo-bearing
SUVs is currently unknown, it is likely related to the high degree
of membrane disorder, which has recently been computationally
modeled; however, the embodiments described herein are not limited
to any particular mechanism of action and an understanding of the
mechanism of action is not necessary to practice the embodiments.
Therefore, the enhanced payload retention of the negatively charged
PGNs (.zeta. potential=-45.+-.3 mV) was attributed to a combination
of cholesterol-induced membrane ordering and reduction in
interparticle fusion that does not exist in the unmodified,
near-neutral SUVs (.zeta. potential=.+-.2 mV).
[0073] In some embodiments, a biodelivery platform is capable of
and/or configured for releasing its therapeutic payload in response
to external stimuli such as changes in biological milieu. To this
end, experiments were conducted during development of embodiments
of the present invention to investigate the ability of PGNs to a
payload of the anti-cancer drug DXR in response to in situ
acidification. DXR-loaded DOPC-PGNs were incubated at 37.degree. C.
in pH 7.4 HBS for 200 minutes, the solution was quickly acidified
to pH 5.0, and regularly monitored with fluorescence spectroscopy
over a period of 72 hours. Remarkably, and in stark contrast to
DXR-loaded DOPC-SUVs, the drug-release profile for DXR-loaded
DOPC-PGNs clearly indicates a near-immediate bulk release of DXR
upon acidification to pH 5.0 (FIG. 4). It is contemplated that this
rapid release is triggered by an acid-induced phase change of the
surface bound Chol-PAA (Chen & Hoffman. Nature 1995, 373
(6509), 49-52; herein incorporated by reference in its entirety),
which catastrophically destabilizes the high-curvature lipid
membrane of the small-sized PGNs and causes them to rupture. This
property appears to be specific to the small size (D.sub.H<50
nm) of the SUV-derived PGNs, as a larger version (D.sub.H.about.100
nm) releases its payloads very slowly upon acidification and
requires the cross-linking of grafted PAA polymer chains into a
shell around the lipid template before acid-triggered release can
be induced (Laaksonen et al. ChemPhysChem 2006, 7 (10), 2143-2149;
herein incorporated by reference in its entirety). Acidification
causes the grafted Chol-PAA chains to cluster (Ringsdorf et al.
Angew. Chem., Int. Ed. 1991, 30 (3), 315-318; herein incorporated
by reference in its entirety), creating large defects on the
already highly curved membranes and leading to spontaneous release
of the payload (FIG. 5). Together with their small sizes, this
pH-triggered payload release property enables SUVs to serve as
"smart" therapeutic carriers that selectively release cargo at
acidic environments such as the tumor interstitium (Smallbone et
al. J. Theor. Biol. 2008, 255 (1), 106-112; herein incorporated by
reference in its entirety) and the lumen of late endosomes (Huotari
& Helenius. EMBO J. 2011, 30 (17), 3481-3500; herein
incorporated by reference in its entirety).
EXAMPLE 6
Mechanism of Chol-PAA Stabilization
[0074] To test whether charge-induced repulsion and/or the steric
bulk provided by Chol-PAA grafts is the primary cause of enhanced
PGN dispersability and improved cargo retention, a series of
poly(ethylene glycol) (PEG)-grafted DPPC-SUVs were prepared using
both PEGylated lipids (PE-PEG.sub.2000 and PE-PEG.sub.3000) and
Chol-PEG.sub.600. PEG-grafts were implemented as a charge-neutral
polymer that closely resembles the Chol-PAA grafts of the PGN
particles. PEG.sub.2000-grafts were specifically selected because
it has the same degree of polymerization as the Chol-PAA, while
PEG.sub.600 and PEG.sub.3000 were selected to provide a range of
steric environments for comparison.
[0075] Chol-PEG.sub.600-, PE-PEG.sub.2000-, and
PE-PEG.sub.3000-grafted SUVs all exhibited a steady increase in
particle size over a four-week period (FIG. 6; Table 3), after
which rapid aggregation occurred along with visible flocculent
formation. This aggregation has been previously reported for
DOPC-based SUVs (Evans & Lentz. Biochemistry 2002, 41 (4),
1241-1249; herein incorporated by reference in its entirety) and is
not surprising considering the near-neutral .zeta. potential (.+-.2
mV) of these PEG-grafted SUVs, following the prediction of DLVO
theory. These observations suggest that simple PEGylation of SUVs,
as employed in Doxil.TM. to improve stabilization and engender in
vivo stealth capabilities and longer circulation, will not lead to
a robust ultra-small liposome carrier.
[0076] To elucidate the stabilizing effects contributed by
cholesterol, which is known to prevent SUV fusion (Mercadal et al.
Biochim. Biophys. Acta, Biomembr. 1995, 1235 (2), 281-288; herein
incorporated by reference in its entirety), in the
Chol-PAA-stabilized PGN platform, experiments were conducted during
development of embodiments described herein to examine the
colloidal stability of DPPC-SUVs composed of 5, 10, 15, and 20 mol
% of cholesterol (See Tables 4). Faster particle aggregation was
observed for all of these formulations compared to the unmodified
DPPC-SUVs, which is consistent with previous reports (Roy et al.
Langmuir 2010, 26 (24), 18967-18975; Mcconnell & Schullery.
Biochim. Biophys. Acta 1985, 818 (1), 13-22; herein incorporated by
reference in their entireties). Together, these experimental
controls further indicate PGN stabilization by Chol-PAA grafts is
primarily conferred by charge-induced repulsion between particles;
although the present invention is not limited to any particular
mechanism of action and an understanding of the mechanism of action
is not necessary to practice the present invention.
EXAMPLE 7
Methods
Calcein Leakage Evaluation
[0077] To evaluate the calcein leakage as a function of storage
time, three batches of calcein-loaded PGN and SUV dispersions were
stored in sealed 20 mL vials under inert atmosphere at 4.degree. C.
The lipid/calcein ratio was then monitored directly after
synthesis, after two weeks of storage, and after a period of 1
month. At each evaluation, the calcein-loaded samples were
re-purified using TFF (same purification method as described
above), and the calcein/lipid ratio was revaluated as well as size
measurements. The concentration of calcein in solution was measured
directly by fluorescence spectroscopy against a calibration curve,
and the concentration of lipid was calculated from measured
phosphorous content by ICP-OES.
Cholesterol Addition Experiments
[0078] To a stirring batch of DPPC and DMPC SUV particles, 5, 10,
15, or 20 mol % of cholesterol dissolved in 20 mM HBS was added
dropwise over 15 minutes. The SUV mixtures were allowed to stir at
room temperature for 24 hours. The volume-average particle size
distribution was monitored over several months.
Doxorubicin Release Experiments
[0079] A quartz fluorescence cuvette (1 mL) equipped with a
magnetic micro-stir bar was loaded with 20 mM HBS (900 .mu.L) and
of DOPC-PGN.sub.DXR (100 .mu.L of a 3.5 mM lipid solution in 20 mM
HBS) The cuvette was placed in a temperature-controlled flourimeter
mount which enabled warming to 37.degree. C. and constant stirring
during real-time measurements. The fluorescence was measured
regularly at pH 7.4 at 10-minute intervals, within the first 200
minutes. At the 200-minute mark, an aliquot (10 .mu.L) of 1 M HCl,
a pre-determined amount to acidify solution to pH 5.0, was added to
the cuvette and measurements continued at 10-minute intervals for
the next 100 minutes. After 720 minutes of fluorescence
measurements, an aliquot (50 uL) of Triton-X 100 solution (10% in
water) is added and the resulting mixture was allowed to stir for
five minutes. The fluorescence that was measured represents the
value for 100% release of DXR, which was used to normalize release
values. We attribute an increase in fluorescence to an increased
release of doxorubicin because the fluorescence of doxorubicin
encapsulated within liposomes is self-quenched and is only restored
upon release..sup.S4
TABLE-US-00005 TABLE 5 .zeta. potential (mV) of DPPC, DMPC, and
DOPC-based PGN and SUV samples after preparation (0 days) and after
180 days of storage at 4.degree. C. .zeta. potential (mV) Time
DPPC-based DMPC-based DOPC-based (days) PGN SUV PGN SUV PGN SUV 0
-37 .+-. 3 -4 .+-. 1 -43 .+-. 2 +1 .+-. 1 -45 .+-. 3 -2 .+-. 1 180
-41 .+-. 4 -2 .+-. 1 -40 .+-. 4 +3 .+-. 1 -42 .+-. 2 -3 .+-. 1
[0080] All publications and patents listed below and/or provided
herein are hereby incorporated by reference in their entireties.
Various modifications and variations of the described compositions
and methods of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention that are obvious to
those skilled in the relevant fields are intended to be within the
scope of the present invention.
* * * * *