U.S. patent application number 15/025060 was filed with the patent office on 2016-08-25 for surface functionalized, host-guest polymer nano-assemblies and methods thereof.
The applicant listed for this patent is UNIVERSITY OF MASSACHUSETTS. Invention is credited to Sankaran Thayumanavan, Hui Wang, Jiaming Zhuang.
Application Number | 20160243047 15/025060 |
Document ID | / |
Family ID | 53524459 |
Filed Date | 2016-08-25 |
United States Patent
Application |
20160243047 |
Kind Code |
A1 |
Thayumanavan; Sankaran ; et
al. |
August 25, 2016 |
SURFACE FUNCTIONALIZED, HOST-GUEST POLYMER NANO-ASSEMBLIES AND
METHODS THEREOF
Abstract
The invention generally relates to polymer-based
nano-structures. More particularly, the invention relates to novel,
surface-functionalized, guest-host polymer nano-assemblies and
nano-delivery vehicles useful in diverse fields including drug
delivery, diagnostics and specialty materials. The nano-assemblies
and nano-delivery vehicles of the invention are afforded via
simplify and reliable approaches.
Inventors: |
Thayumanavan; Sankaran;
(Amherst, MA) ; Zhuang; Jiaming; (Amherst, MA)
; Wang; Hui; (Sunderland, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF MASSACHUSETTS |
Boston |
MA |
US |
|
|
Family ID: |
53524459 |
Appl. No.: |
15/025060 |
Filed: |
October 2, 2014 |
PCT Filed: |
October 2, 2014 |
PCT NO: |
PCT/US2014/058751 |
371 Date: |
March 25, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61886016 |
Oct 2, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/5138 20130101;
Y10S 977/906 20130101; A61K 9/5146 20130101; A61K 47/6903 20170801;
A61P 35/00 20180101; Y10S 977/773 20130101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; A61K 47/32 20060101 A61K047/32; A61K 9/06 20060101
A61K009/06 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] The United States Government has certain rights to the
invention pursuant to Grant No. CHE-1307118 from National Science
Foundation (NSF), and Grant No. 5RO1GM065255 from National
Institutes of Health (NIH), Grant No. W911NF1010313 from U. S. Army
Research Office, Grant No. CMMI-1025020 from NSF-NSEC and Grant No.
DMR-0820506 from NSF-MRSEC.
Claims
1. A nano-assembly comprising: a host crosslinked polymer network
having a functionalized surface with one or more functional groups;
and a guest molecular cargo non-covalently encapsulated in the host
crosslinked polymer network, wherein the host crosslinked polymer
network is addressable by a biological or chemical intervention
resulting in partial or complete decrosslinking of the host polymer
network and release of the guest molecular cargo from the
nano-assembly.
2. The nano-assembly of claim 1, wherein the host crosslinked
polymer network is a nanogel.
3. (canceled)
4. The nano-assembly of claim 2, wherein the host crosslinked
polymer network is formed from a random copolymer comprising
##STR00019## wherein each of Z.sub.1 and Z.sub.2 is independently
##STR00020## wherein Z' is selected from O, NH, NR
(R.dbd.C.sub.1-C.sub.6 alkyl group or R.sub.F); each of R.sub.1 and
R.sub.2 is independently selected from a hydrogen, C.sub.1-C.sub.12
alkyl group, or halogen; each of R.sub.L1 and R.sub.L2 is
independently a single bond or a spacer group; R.sub.F is --H or a
functional group; R.sub.x is a crosslinking group capable of inter-
or intra-molecular crosslinking; R.sub.x' is an inter- or
intra-molecularly crosslinked group; and i=an, j=xn, and k=bn-xn,
wherein each of a and b is a positive number with a+b =1, each of n
and x is independently an integer from about 0.1% to about 100% of
b.
5. The nano-assembly of claim 4, wherein each of R.sub.1 and
R.sub.2 is methyl.
6. The nano-assembly of claim 4, wherein each of R.sub.il and
R.sub.L2 is selected from --(CH.sub.2).sub.m--, wherein m is an
integer from about 1 to about 15, and
--(CH.sub.2CH.sub.2--O).sub.q--, wherein q is an integer from about
1 to about 50.
7. The nano-assembly of claim 4, wherein R.sub.F is selected from
the group consisting of amines, carboxylates, hydroxyl, halides,
acyl halides, esters, azides, nitriles, amides, epoxides,
aldehydes, furans, alkenes and alkynes.
8. The nano-assembly of claim 7, wherein R.sub.F is an amine.
9. The nano-assembly of claim 7, wherein R.sub.F is an activated
carboxylic ester.
10. The nano-assembly of claim 4, wherein R.sub.x comprises a
reactive group selected from coumarin, alkenes, thiols, reactive
disulfides, alkynes, furans, aldehydes, amines, activated esters,
maleimides, and epoxides.
11. (canceled)
12. The nano-assembly of claim 2, wherein the host crosslinked
polymer network is formed from a homopolymer comprising
##STR00021## wherein Z.sub.3 is ##STR00022## wherein Z' is selected
from O, NH, NR (R.dbd.C.sub.1-C.sub.6 alkyl group or R.sub.F);
R.sub.3 is selected from a hydrogen, C.sub.1-C.sub.12 alkyl group,
or halogen; R.sub.L3 is CR.sub.4, N or a trivalent group, wherein
R.sub.4 is selected from a hydrogen, C.sub.1-C.sub.12 alkyl group,
or halogen; R.sub.F is --H or a functional group; R.sub.x is a
crosslinking group capable of inter- or intra-molecular
crosslinking or an inter- or intra-molecularly crosslinked group;
and r is an integer from about 10 to about 1000.
13. The nano-assembly of claim 12, wherein R.sub.3 is --H or
-methyl.
14. The nano-assembly of claim 12, wherein R.sub.L3 is CH.
15. The nano-assembly of claim 12, wherein R.sub.F is selected from
the group consisting of amines, carboxylates, hydroxyl, halides,
acyl halides, esters, azides, nitriles, amides, epoxides,
aldehydes, furans, alkenes and alkynes.
16. The nano-assembly of claim 15, wherein R.sub.F comprises an
amine group.
17. The nano-assembly of claim 15, wherein R.sub.F comprises a
carboxylate group.
18. The nano-assembly of claim 12, wherein R.sub.x comprises a
reactive group selected from coumarin, alkenes, thiols, reactive
disulfides, alkynes, furans, aldehydes, and epoxides.
19. The nano-assembly of claim 12, wherein the polymer network is
formed from a homopolymer via a controlled crosslinking.
20. The nano-assembly of claim 1, wherein the biological, physical
or chemical intervention is a change in pH, redox reagent, redox
potential, ionic strength, enzymatic activity, protein
concentration, light, heat, or mechanical stress.
21-32. (canceled)
33. A method for controlled delivery of an agent to a target
biological site, comprising: providing a nano-assembly of a host
crosslinked polymer network non-covalently encapsulating therein a
guest molecular cargo, wherein the host crosslinked polymer network
is capable of partial or complete decrosslinking by a biological or
chemical intervention resulting in release of the guest molecular
cargo from the nano-assembly; delivering the nano-assembly to the
target biological site; and causing a biological or chemical
intervention resulting in a partial or complete decrosslinking
resulting in release of the guest molecular cargo from the
nano-assembly.
34-51. (canceled)
52. A nanoparticle comprising: a shell of a crosslinked polymer
network having a surface functionalized with one or more functional
groups; and a core comprising a host polymer network and a guest
agent non-covalently encapsulated therein, wherein the core, the
shell, or one or more intervening layers of the crosslinked polymer
network are independently addressable by a biological or chemical
intervention resulting in partial or complete disassembly of the
shell, the one or more intervening layers, and/or the core thereby
releasing of the guest agent.
53-92. (canceled)
Description
PRIORITY CLAIMS AND RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/886,016, filed Oct. 2, 2013, the entire content
of which is incorporated herein by reference in its entirety.
TECHNICAL FIELDS OF THE INVENTION
[0003] The invention generally relates to polymer-based
nano-structures. More particularly, the invention relates to novel,
surface-functionalized, guest-host polymer nano-assemblies and
nano-delivery vehicles useful in diverse fields including drug
delivery, diagnostics and specialty materials.
BACKGROUND OF THE INVENTION
[0004] Nanoparticles have had a significant impact on a variety of
areas such as microelectronics, multiphase catalysis, sensing and
therapeutics. (Nanoparticles: From Theory to Application; Schmid,
Ed.; Wiley-VCH: Essen, 2004; Zhang, et al. Self-Assembled
Nanostructures; Nanostructure Science and Technology Series;
Springer: 2002; Nanoparticles: Building Blocks for Nanotechnology;
Rotello, Ed.; Springer: 2003; Daniel, et al. 2004 Chem. Rev. 104,
293.) For most applications, facile modulation of the nanoparticle
surface is critical in order to obtain appropriate interfacial
properties. The ability to encapsulate and release guest molecules
within the nanoparticle interior is also required for applications
such as sensing and therapeutics.
[0005] A platform that affords both surface functionalization and
guest encapsulation in a single nanoscopic scaffold is highly
desirable. Nanoscale materials, such as metallic or semiconductor
nanoparticles and dendrimers, are excellent scaffolds for
displaying surface functional groups. (Giljohann, et al. 2010
Angew. Chem. Int. Ed. 49, 3280-3294; Saha, et al. 2012 Chem. Rev.
112, 2739-2779; Khandare, et al. 2012 Chem. Soc. Rev. 41,
2824-2848; Grayson, et al. 2001 Chem. Rev. 101, 3819-3867; Arima,
et al. 2012 Pharmaceutics 4, 130-148.) For example, monolayer
protection of gold nanoparticles is easily achieved with
thiol-bearing molecules due to the high affinity of thiol moiety
toward gold nanoparticles. However, these scaffolds generally lack
features that allow for favorable non-covalent host-guest
interactions. In contrast, amphiphilic molecules readily
self-assemble into nanoassemblies, such as micelles and liposomes,
which can encapsulate guest molecules within their interior spaces.
(Harada, et al. 2006 Progress in Poly. Sci. 31, 949-982; O'Reilly,
et al. 2006 Chem. Soc. Rev. 35, 1068-1083; Zhu, et al. 2012 J. Mat.
Chem. 22, 7667-7671; Owen, et al. 2012 Nano Today 7, 53-65; Sawant,
et al. 2010 Soft Matter 6, 4026-4044; Micheli, et al. 2012 Recent
Patents on CNS Drug Discovery 7, 71-86.)
[0006] Nevertheless, modifying their surface functional groups are
challenging because these modifications often result in changes in
the hydrophilic-lipophilic balance that is necessary for retention
of the fidelity of the assembly. Surface functionalizable polymeric
nanoparticles often require rigorous processing conditions.
Additionally, size variations are limited and difficult to achieve
and control.
[0007] Polymer vesicles, or polymersomes have been generated and
extensively investigated as drug delivery vehicles, diagnostics,
nanoreactors and artificial organelles. (Tanner, et al. 2011 Acc.
Chem. Res. 44, 1039-1049; Vriezema, et al. 2007 Angew. Chem. Int.
Ed. 46, 7378-7382; Wang, et al. 2012 Angew. Chem. Int. Ed. 51,
11122-11125; Lomas, et al. 2007 Adv. Mater. 19, 4238-4243;
Choucair, et al. 2005 Langmuir 21, 9308-9313; Ben-Haim, et al. 2008
Nano Lett. 8, 1368-1373; Ghoroghchian, et al. 2005 PNAS 102,
2922-2927; Choi, et al. 2005 Nano Lett. 5, 2538-2542; Sanson, et
al. 2011 ACS Nano 5, 1122-1140; Ahmed, et al. 2006 1 Control.
Releas. 116, 150-158.)
[0008] The potential advantages of polymeric vesicle compared with
their lipid counterparts arise from the enhanced colloidal
stability, tunable membrane thickness and permeability, and
engineered surface functionalities. However, a remaining challenge
involves the unstable nature of the non-covalently organized
supramolecular assembly of the polymersome, which causes
morphological changes. The morphological changes can be due to the
change in solvent composition during the dialysis to remove organic
solvent in polymersome formation process or due to the
post-functionalization that changes hydrophilic lipophilic balance
(HLB) of the precursor polymer resulting in loss of fidelity of the
assembly. (Du, et al. 2009 Soft Matter 5, 3544-3561; Su, et al.
2014 ACS Macro Lett. 3, 534-539.)
[0009] Additionally, like other self-assembled systems polymersomes
also undergo disassembly upon dilution, solvent exposure and
interaction with their surroundings depending on the specific
applications. (Savariar, et al. 2008 J. Am. Chem. Soc. 130,
5416-5417.) To address these issues, the formation of vesicle by
solvation of polymer in aqueous media followed by the chemical
crosslinking can be a good solution. Unfortunately, chemistries
developed for polymersome post-functionalization, direct
dissolution method and efficient cross-linking chemistry for
aqueous solution are all quite limited. (Egli, et al. 2011 J. Am.
Chem. Soc. 133, 4476-4483; Opsteen, et al. 2007 Chem. Commun. 30,
3136-3138; Li, et al. 2007 Chem. Commu. 30, 5217-5219; Rosselgong,
et al. 2012 ACS Macro Lett. 1, 1041-1045.)
[0010] Another challenge for polymersome preparation is to control
the size that is critical for their applications. (Harashima, et
al. 1996 Adv. Drug Delivery Rev. 19,425-444.) Although polymersomes
with tunable size can be achieved by varying the block length,
concentration of copolymers and ratio of solvents, the preparation
of vesicle-forming copolymers with complex architectures like
block, graft or dendritic copolymer requires demanding synthetic
effort even with the aid of the robust polymerization techniques.
(Xu, et al. 2009 J. Mater. Chem. 19, 4183-4190; Anraku, et al. 2010
J. Am. Chem. Soc. 132, 1631-1636; Blanazs, et al. 2012
Macromolecules 45, 5099-5107; Zhou, et al. 2004 Angew. Chem. Int.
Ed. 43, 4896-4899; Azzam, et al. 2006 Angew. Chem. Int. Ed, 45,
7443-7447; Sun, et al. 2009 ACS Nano 3, 673-681; Wang, et al. 2001
J. Colloid. Interf. Sci. 237, 200-207.)
[0011] Capability of facile formation of polymersome from easily
accessible polymers with simple architectures will open a new
avenue to prepare customized polymersomes. Directed by the
potential applications, the "intelligent" polymersomes that are
capable of adapting to the environmental changing are really
desired. (Bellomo, et al. 2004 Nat. Mater. 3, 244-248; Napoli, et
al. 2004 Nat. Mater. 3. 183-189; Yan, et al. 2013 Angew. Chem. Int.
Ed. 52, 5070-5073; Liu, et al. 2014 J. Am. Chem. Soc. 136,
7492-7497; Liu, et al. 2006 Angew. Chem. Int. Ed. 45,
3846-3850.)
[0012] Thus, there is an urgent need for simplified and reliable
approaches to functionalizable polymer nanoparticles and delivery
vehicles. Additionally, challenges remain for "intelligent"
polymersomes and simple methods to their preparation.
SUMMARY OF THE INVENTION
[0013] The invention relates to novel,
surface-functionalized/functionalizable, guest-host polymer
nano-assemblies and nano-delivery vehicles useful in diverse fields
including drug delivery, diagnostics and specialty materials. The
functionalized/functionalizable polymer nanoparticles exhibit the
advantages of surface functionalization capabilities available in
dendrimers and metallic nanoparticles as well as the host-guest
features presented in micelles and vesicles. Improtantly, the
nano-assemblies and nano-delivery vehicles of the invention are
afforded via simple and reliable approaches.
[0014] The invention also relates to novel novel polymer-based
cross-linked nanoparticle with vesicular structures, or VesiGel,
and methods of their preparation. Herein disclosed is an easy and
generally applicable method in which a simple and easily obtained
amphiphilic homopolymer electrolyte self-assembles into vesicular
structure assisted by a variety of multivalent salts based on a
salt-bridging mechanism. The VesiGel disclosed herein have been
demonstrated to capture the features including: i) organic solvent
free formation; ii) tunable nanoscaled sizes; ii) variable surface
functionalities; iii) functionalizable corona and membrane; iv)
reversible crosslinking stabilization; v) capable of encapsulating
hydrophobic and hydrophilic small molecules as well as proteins; v)
stimuli-responsive controlled release of encapsulants.
[0015] In one aspect, the invention generally relates to a
nano-assembly. The nano-assembly includes: (1) a host crosslinked
polymer network having a functionalized surface with one or more
functional groups; and (2) a guest molecular cargo non-covalently
encapsulated in the host crosslinked polymer network. The host
crosslinked polymer network is addressable by a biological,
physical or chemical intervention resulting in partial or complete
decrosslinking of the host polymer network and release of the guest
molecular cargo from the nano-assembly.
[0016] In another aspect, the invention generally relates to a
composition comprising the nano-assembly disclosed herein.
[0017] In yet another aspect, the invention generally relates to a
method for controlled delivery of an agent to a target biological
site. The method includes: (1) providing a nano-assembly of a host
crosslinked polymer network non-covalently encapsulating therein a
guest molecular cargo, wherein the host crosslinked polymer network
is capable of partial or complete decrosslinking by a biological,
physical or chemical intervention resulting in release of the guest
molecular cargo from the nano-assembly; (2) delivering the
nano-assembly to the target biological site; and (3) causing a
biological, physical or chemical intervention resulting in a
partial or complete decrosslinking resulting in release of the
guest molecular cargo from the nano-assembly.
[0018] In yet another aspect, the invention generally relates to a
nanoparticle. The nanoparticle includes: (1) a shell of a
crosslinked polymer network having a surface functionalized with
one or more functional groups; and (2) a core comprising a host
polymer network and a guest agent non-covalently encapsulated
therein. The core and shell of crosslinked polymer network are
independently addressable by a biological, physical or chemical
intervention resulting in partial or complete disassembly of the
shell and/or the core thereby releasing of the guest agent.
[0019] In yet another aspect, the invention generally relates to a
polymer-based nanoparticle having vesicular structures stabilized
by intraparticular crosslinking, wherein the polymer is an
amphiphilic homopolymer comprising a hydrophilic head group and a
hydrophobic tail group.
[0020] In certain embodiments, the hydrophilic head group is
selected from charged functional groups such as amino, ammonium,
sulfonium, phosphonium, carboxylate, phosphate, phosphonate,
sulfate, and sulfonate, groups and charge-neutral functional groups
such as carboxy betaine, sulfo betaine, phosphoryl choline,
phosphonyl choline, saccharides, and polyethylene glycol groups and
the hydrophobic tail group is selected from linear and branched
alkyl chains, linear and branched fluoro-substituted alkyl chains,
and alkyl chains containing aromatic or heteromatic functional
groups.
[0021] In yet another aspect, the invention generally relates to a
nano-assembly. The nano-assembly includes: a host crosslinked
polymer-based nanoparticle disclosed herein; and a guest molecular
cargo non-covalently encapsulated in the host crosslinked
polymer-based nanoparticle. The host crosslinked polymer network is
addressable by a biological or chemical intervention resulting in
partial or complete decrosslinking of the host polymer network and
release of the guest molecular cargo from the nano-assembly.
[0022] In yet another aspect, the invention generally relates to a
method for forming a polymer-based nanoparticle disclosed herein.
The method includes: providing an amphiphilic homopolymer by a ring
opening reaction; causing self-assembly of the amphiphilic
homopolymer, induced or assisted by one or more salt, to form a
polymersome comprising vesicle structures; and performing an
intra-particular crosslinking on the polymersome resulting in
intra- and inter-molecularly crosslinked polymer-based nanoparticle
having vesicle structures.
[0023] In yet another aspect, the invention generally relates to a
method for controlled delivery of a molecular cargo to a target
biological site. The method includes: providing a nano-assembly
disclosed herein; delivering the nano-assembly to the target
biological site; and causing a biological or chemical intervention
resulting in a partial or complete decrosslinking resulting in
release of the guest molecular cargo from the nano-assembly.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1. Schematic representation of the polymer nanoparticle
with surface functionalization and guest binding abilities.
[0025] FIG. 2. Size distributions of (a) non-crosslinked, (b)
cross-linked, and (c) de-crosslinked random copolymer 1 by DLS. (d)
Absorption spectra of non-crosslinked and cross-linked random
copolymer 1.
[0026] FIG. 3. Absorption spectra of guest molecules in
non-crosslinked and crosslinked random copolymer 1.
[0027] FIG. 4.(a) Surface charges of nanoparticles by zeta
potential (b) Contact angle measurements of unmodified
nanoparticles (top) and nanoparticles modified by lauric acid NHS
ester (bottom left) and dodecyl isocyanate (bottom right). (c) IR
spectra of azidoacetic acid NHS ester (top), unmodified
nanoparticles (middle), and nanoparticles functionalized with
azidoacetic acid NHS ester (bottom). (d) Fluorescence emission
intensity of nanoparticles treated with excess fluorescamine after
reacting with different functional groups.
[0028] FIG. 5. Nanoparticles (.about.180 nm by DLS) functionalized
with capric acid NHS ester (.about.190 nm by TEM).
[0029] FIG. 6.(a) Size distribution of nanoparticles cross-linked
at different pHs in water. The DLS measurements were all done at pH
3. (b) Percentage of amine available for functionalization on
different nanoparticle sizes accessed by fluorescamine assay.
[0030] FIG. 7. Nanoparticles reacted with different concentrations
of capric acid NHS ester monitored by fluorescamine.
[0031] FIG. 8. Synthesis of amphiphilic homopolymer
[0032] FIG. 9. Size of nanoassembly of amphiphilic homopolymer P1
(left) and P2 (right) in a variety of pHs
[0033] FIG. 10. Size of nanoassembly of P1 (left) in phosphate
solution and P2 in CaCl2 solution
[0034] FIG. 11. Size of nanoassembly of P1 (left) in the presence
of divalent anions and P2 in the presence of divalent cations.
[0035] FIG. 12. Size of nanoassembly of P3 in different temperature
(left) and in sodium carbonate solution (right).
[0036] FIG. 13. Size of cross-linked nanoassembly of P1 (top), P2
(middle) and P3 (bottom).
[0037] FIG. 14. Reductant triggered guest release from cross-linked
nano-assembly prepared from P1 (top), P2 (middle) and P3 (bottom).
Left panel: control w/o reductant; Right panel: w/reductant.
[0038] FIG. 15.(a) Schematic presentation of slat-induced formation
of polymersome; (b) Na.sub.2HPO.sub.4 concentration dependent sizes
of polymersomes prepared from P1; (c) TEM images of polymersomes
prepared in Na.sub.2HPO.sub.4 (left: 1.5 mM; middle: 2.0 mM; right:
2.5 mM); (d) AFM image of polymersome prepared from P1 in 2.5 mM
Na.sub.2HPO.sub.4; (e) MgCl.sub.2 concentration dependent sizes of
polymersomes from P2; (f) TEM images of polymersomes prepared in
MgCl.sub.2 (left: 2.4 mM; middle: 3.6 mM; right: 4.8 mM); (g) AFM
image of polymersome prepared from P2 in 4.2 mM MgCl.sub.2.
[0039] FIG. 16.(a) Sizes of P1 in different salts; (b) TEM image of
cationic VesiGel; (c) sizes of P2 in different salts; (d) TEM image
of anionic VesiGel. Scale bars are 100 nm, unless noted.
[0040] FIG. 17(a) Schematic illustration of salt-bridging mechanism
probed by fluorescent dyes; (b) UV-vis of probes mixed with
polymers with opposite charge; (c) UV-vis of probes mixed with
polymers with same charges; (d) spectroscopic evolution on
calcein-P1 complex absorbance titrated by Na.sub.2SO.sub.4; (e)
fluorescence of probes mixed with polymers with opposite charge;
(f) fluorescence of probes mixed with polymers with same charges;
(g) spectroscopic evolution on calcein-P1 complex emission titrated
by Na.sub.2SO.sub.4.
[0041] FIG. 18.(a) Changes on the surface charge of VesiGels upon
peglation; (b) measurement of functionalizable amine by
fluorescamine assay; (c) absorbance evolution of FRET pair
functionalized VesiGel upon DTT treatment; (d) emission evolution
of FRET pair functionalized VesiGel upon DTT treatment.
[0042] FIG. 19.(a) Hydrophobic guest released triggered by DTT; (b)
R6G released from cationic VesiGel; (c) Calcein released from
anionic VesiGel; (d) Encapsulation of Myoglobin in cationic VesGel
followed by Uv-vis; (e) Lysozyme released from anionic VesGel
followed by MALDI after trypsin digestion.
[0043] FIG. 20. DLS of P1 in Na.sub.2HPO.sub.4 solution with
different concentrations before crosslinking.
[0044] FIG. 21. DLS of P2 in MgCl.sub.2 solution with different
concentrations before crosslinking.
[0045] FIG. 22. Salt concentration dependant nanoassembly size of
P1 in a variety of salt after crosslinking.
[0046] FIG. 23. Salt concentration dependant nanoassembly size of
P2 in CaCl.sub.2 and BaCl.sub.2 after crosslinking.
[0047] FIG. 24. Size of P1 in 10 mM slat solution with different
cations (Top) and P2 in 20 mM salt solution with different anions
(bottom).
[0048] FIG. 25. TEM image of cationic VesiGel prepared in 1.5 mM
(left), 2.0 mM (middle), and 2.5 mM (right) Na.sub.2HPO.sub.4. The
scale bar is 100 nm.
[0049] FIG. 26. TEM image of cationic VesiGel prepared in 1.5 Mm
(left), 2.0 mM (right) Na2SO4 solution. The scale bar is 100
nm.
[0050] FIG. 27. TEM image of cationic VesiGel prepared in 1.25 mM
(left, scale bar: 100 nm) and 1.5 mM (right, scale bar: 500 nm)
Na.sub.2SO.sub.3 solution.
[0051] FIG. 28. TEM image of cationic VesiGel prepared in 2.0 mM
(left) and 2.5 mM (right) Na.sub.2SO.sub.3 solution. The sacle bar
is 100 nm.
[0052] FIG. 29. TEM image of anionic VesiGel prepared in 2.4 mM
(top left), 3.0 mM (top middle), 3.6 mM (top right), 4.2 mM (bottom
left) and 4.8 mM (bottom right) MgCl.sub.2 solution. The sacle bar
is 100 nm.
[0053] FIG. 30. TEM image of anionic VesiGel prepared in 2.4 mM
BaCl.sub.2 (left, scale bar: 100 nm) and 2.2 mM CaCl.sub.2 (right,
scale bar: 500 nm) MgCl.sub.2 solution.
DEFINITIONS
[0054] Definitions of specific functional groups and chemical terms
are described in more detail below. General principles of organic
chemistry, as well as specific functional moieties and reactivity,
are described in "Organic Chemistry", Thomas Sorrell, University
Science Books, Sausalito: 2006. It will be appreciated that the
compounds, as described herein, may be substituted with any number
of substituents or functional moieties.
[0055] As used herein, "C.sub.x-C.sub.y" refers in general to
groups that have from x to y (inclusive) carbon atoms. Therefore,
for example, C.sub.1-C.sub.6 refers to groups that have 1, 2, 3, 4,
5, or 6 carbon atoms, which encompass C.sub.1-C.sub.2,
C.sub.1-C.sub.3, C.sub.1-C.sub.4, C.sub.1-C.sub.5, C.sub.2-C.sub.3,
C.sub.2-C.sub.4, C.sub.2-C.sub.5, C.sub.2-C.sub.6, and all like
combinations. "C.sub.1-C.sub.20" and the likes similarly encompass
the various combinations between 1 and 20 (inclusive) carbon atoms,
such as C.sub.1-C.sub.6, C.sub.1-C.sub.12 and C.sub.3-C.sub.12.
[0056] As used herein, the term "alkyl", refers to a hydrocarbyl
group, which is a saturated hydrocarbon radical having the number
of carbon atoms designated and includes straight, branched chain,
cyclic and polycyclic groups. The term "hydrocarbyl" refers to any
moiety comprising only hydrogen and carbon atoms. Hydrocarbyl
groups include saturated (e.g., alkyl groups), unsaturated groups
(e.g., alkenes and alkynes), aromatic groups (e.g., phenyl and
naphthyl) and mixtures thereof.
[0057] As used herein, the term "C.sub.x-C.sub.y" alkyl refers to a
saturated linear or branched free radical consisting essentially of
x to y carbon atoms, wherein x is an integer from 1 to about 10 and
y is an integer from about 2 to about 20. Exemplary C.sub.x-C.sub.y
alkyl groups include "C.sub.1-C.sub.20 alkyl," which refers to a
saturated linear or branched free radical consisting essentially of
1 to 20 carbon atoms and a corresponding number of hydrogen atoms.
Exemplary C.sub.1-C.sub.20 alkyl groups include methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, dodecanyl, etc.
[0058] As used herein, the term "halogen" refers to fluorine (F),
chlorine (Cl), bromine (Br), or iodine (I).
[0059] As used herein, the term "biological, physical or chemical
interventions" includes a change in pH, redox reagent, redox
potential, ionic strength, enzymatic activity, protein
concentration, light (e.g., UVA, UVB or UVC), heat, or mechanical
stress.
[0060] As used herein, "VesiGels" refers to polymer-based
cross-linked nanoparticle with vesicular structures disclosed
herein.
DETAILED DESCRIPTION OF THE INVENTION
[0061] The invention provides
surface-functionalized/functionalizable, guest-host polymer
nano-assemblies and nano-delivery vehicles useful in diverse fields
including drug delivery, diagnostics and specialty materials.
[0062] The nano-assemblies and nano-delivery vehicles of the
invention can be prepared via simple and reliable synthetic
techniques. The nanoparticles of the invention capture the
advantages of surface functionalization capabilities available in
dendrimers and metallic nanoparticles as well as the host-guest
features presented in micelles and vesicles.
[0063] In one aspect, the invention generally relates to a
nano-assembly. The nano-assembly includes: (1) a host crosslinked
polymer network having a functionalized surface with one or more
functional groups; and (2) a guest molecular cargo non-covalently
encapsulated in the host crosslinked polymer network. The host
crosslinked polymer network is addressable by a biological,
physical or chemical intervention resulting in partial or complete
decrosslinking of the host polymer network and release of the guest
molecular cargo from the nano-assembly.
[0064] In certain embodiments, the host crosslinked polymer network
is a nanogel.
[0065] In certain embodiments, the host crosslinked polymer network
is formed from a random copolymer via a controlled
crosslinking.
[0066] In certain embodiments, the polymer network is formed from a
homopolymer via a controlled crosslinking.
[0067] Depending on the nature of the polymer network and
crosslinking, the biological or chemical intervention may be a
change in pH, redox reagent, redox potential, ionic strength,
enzymatic activity, protein concentration, light, heat, or
mechanical stress, which intervention leads to a breaking and/or
forming of a chemical bond.
[0068] The nano-assembly may take any suitable dimensions, for
example, having a diameter from about 3 nm to about 300 nm (e.g.,
about 3 nm to about 200 nm, about 3 nm to about 100 nm, about 3 nm
to about 50 nm, about 3 nm to about 30 nm, about 10 nm to about 300
nm, about 30 nm to about 300 nm, about 50 nm to about 300 nm, about
100 nm to about 300 nm).
[0069] The non-covalently encapsulated guest molecular cargo may be
present in any suitable amounts, for example, accounting for from
about 1 wt % to about 45 wt % (e.g., about 1 wt % to about 35 wt %,
about 1 wt % to about 25 wt %, about 1 wt % to about 20 wt %, about
1 wt % to about 15 wt %, about 1 wt % to about 10 wt %, about 5 wt
% to about 45 wt %, about 10 wt % to about 45 wt %, about 15 wt %
to about 45 wt %, about 20 wt % to about 45 wt %) of the
nano-assembly.
[0070] The guest molecular cargo may be any sutiable material, for
example, selected from therapeutic, diagnostic or imaging agents.
For example, the guest molecular cargo is a small molecule, a
peptide or an oligonucleotide. In certain embodients, the guest
molecular cargo is an antitumor agent.
[0071] In certain preferred embodiments, the guest molecular cargo
is a hydrophobic molecule.
[0072] The functionalized surface of the non-covalently may display
one or more reactive groups at any suitable density, for example,
from very sparingly (e.g., about 0.1%) to full coverage (e.g.,
about 100%). Thus, for example, the functionalized surface of the
non-covalently may display reactive groups at a density of 1, 2, 5,
10, 20, 30, 40, 50, 60, 70, 80, 90, 100%.
[0073] In another aspect, the invention generally relates to a
composition comprising the nano-assembly disclosed herein.
[0074] In yet another aspect, the invention generally relates to a
method for controlled delivery of an agent to a target biological
site. The method includes: (1) providing a nano-assembly of a host
crosslinked polymer network non-covalently encapsulating therein a
guest molecular cargo, wherein the host crosslinked polymer network
is capable of partial or complete decrosslinking by a biological,
physical or chemical intervention resulting in release of the guest
molecular cargo from the nano-assembly; (2) delivering the
nano-assembly to the target biological site; and (3) causing a
biological, physical or chemical intervention resulting in a
partial or complete decrosslinking resulting in release of the
guest molecular cargo from the nano-assembly.
[0075] Depending on the nature of the polymer network and
crosslinking, the biological, physical or chemical intervention is
a change in pH, redox reagent, redox potential, ionic strength,
enzymatic activity, protein concentration, light, heat, or
mechanical stress.
[0076] In certain preferred embodiments, the guest molecular cargo
is selected from a therapeutic, diagnostic or imaging agent. For
example, the guest molecular cargo is a small molecule, a peptide
or an oligonucleotide. In certain embodients, the guest molecular
cargo is an antitumor agent.
[0077] In certain embodiments, the target biological site comprises
a site inside a cell (e.g., a tumor cell).
[0078] In certain embodiments, the target biological site comprises
a site extracellular to a tumor cell. In certain embodiments, the
nano-assembly is preferably taken up by a tumor tissue in a
physiological environment.
[0079] In yet another aspect, the invention generally relates to a
nanoparticle. The nanoparticle includes: (1) a shell of a
crosslinked polymer network having a surface functionalized with
one or more functional groups; and (2) a core comprising a host
polymer network and a guest agent non-covalently encapsulated
therein. The shell of crosslinked polymer network is addressable by
a biological, physical or chemical intervention resulting in
partial or complete dissociation of the shell thereby releasing of
the guest agent.
[0080] In certain embodiments, the host crosslinked polymer network
is formed from a random copolymer via a controlled
crosslinking.
[0081] In certain embodiments, the host crosslinked polymer network
is formed from a homopolymer via a controlled crosslinking.
[0082] In certain embodiments, the one or more functional groups
are selected from the group consisting of amines, carboxylates,
hydroxyl, halides, acyl halides, esters, azides, nitriles, amides,
epoxides, aldehydes, furans, alkenes and alkynes.
[0083] In certain embodiments, the biological, physical or chemical
intervention is a change in pH, redox potential, ionic strength,
enzymatic activity, protein concentration, light, heat, or
mechanical stress.
[0084] For example, certain copolymer-based nanoparticles can be
rapidly formed by ultraviolet or visible irradiation without the
need to use any chemical crosslinkers or agents. The encapsulated
guest molecules can be released by ultraviolet or visible
irradiation or in the presence of a chemical stimulus such as
glutathione if disulfide-bond-forming moieties (or sulfhydryl
groups) are also incorporated into the copolymer structure.
[0085] In certain preferred embodiments, the guest molecular cargo
is selected from a therapeutic, diagnostic or imaging agent. For
example, the guest molecular cargo is a small molecule, a peptide
or an oligonucleotide. In certain embodients, the guest molecular
cargo is an antitumor agent.
I. Surface-Functionalized Polymer Nanoparticle Formed from
Amphiphilic Copolymers
[0086] Unique features of the functionalizable copolymer-based
nanoparticles include: (i) the precursor polymer is based on a
random copolymer; (ii) the polymer self-assembles in a solvent,
which can then be converted to nanoparticle in one step without the
need for any additional processing; (iii) the nanoparticle contains
a surface functional group, which can be further manipulated
easily; (iv) the size of the nanoparticle is tunable; and (iv) the
interior of the nanoparticle is capable of sequestering guest
molecules.
[0087] In certain embodiments, the host crosslinked polymer network
is formed from a random copolymer via a controlled
crosslinking.
[0088] In certain preferred embodiments, the random copolymer has
the formula of
##STR00001##
wherein
[0089] each of Z.sub.1 and Z.sub.2 is independently
##STR00002##
wherein Z' is selected from O, NH, NR (R.dbd.C.sub.1-C.sub.6 alkyl
group or R.sub.F);
[0090] each of R.sub.1 and R.sub.2 is independently selected from a
hydrogen, C.sub.1-C.sub.12 alkyl group, or halogen;
[0091] each of R.sub.L1 and R.sub.L2 is independently a single bond
or a spacer group;
[0092] R.sub.F is --H or a functional group;
[0093] R.sub.x is a crosslinking group capable of inter- or
intra-molecular crosslinking;
[0094] R.sub.x' is an inter- or intra-molecularly crosslinked
group; and
[0095] i=an, j=xn, and k=bn-xn, wherein each of a and b is a
positive number with a+b=1, each of n and x is independently an
integer from about 0.1% to about 100% of b.
[0096] In certain preferred embodiments, each of R.sub.1 and
R.sub.2 is methyl.
[0097] In certain preferred embodiments, each of R.sub.L1 and
R.sub.L2 is selected from --(CH.sub.2).sub.m--, wherein m is an
integer from about 1 to about 15 (e.g., from about 1 to about 10,
from about 1 to about 6, from about 1 to about 3, from about 3 to
about 6, from about 3 to about 15), and
--(CH.sub.2CH.sub.2--O).sub.q--, wherein q is an integer from about
1 to about 50 (e.g., from about 1 to about 30, from about 1 to
about 20, from about 1 to about 10, from about 1 to about 6, from
about 1 to about 3, from about 3 to about 10, from about 6 to about
20).
[0098] R.sub.F can be any suitable functional groups, for example,
selected from the group consisting of amines, carboxylates,
sulfates, sulfonates, phosphates, phosphonates, hydroxyl, halides,
acyl halides, esters, azides, nitriles, amides, epoxides,
aldehydes, furans, alkenes and alkynes.
[0099] In certain preferred embodiments, R.sub.F includes an amine
gorup.
[0100] In certain preferred embodiments, R.sub.F includes an
activated carboxylic ester group.
[0101] R.sub.x can be any crosslinking group group, for examples,
selected from coumarin, alkenes, thiols, reactive disulfides,
esters, reactive esters, maleimides, alkynes, furans, aldehydes and
epoxides.
[0102] A unique aspect of the invention is that the molecular
design involves self-assembly of amphiphilic random copolymers. In
the aqueous phase, the surface functional groups of such an
assembly would be dictated by the hydrophilic moiety of the
polymer. Here, for example, the use of a primary amine based
monomer as the hydrophilic moiety, combined with a reactive
hydrophobic monomer as the crosslinkable moiety, led to a
functionalizable polymer nanoparticle. The amphiphilic nature of
the nano-assembly also allows for incorporation of guest molecules
within the hydrophobic interior of the assembly prior to
crosslinking. (FIG. 1)
[0103] Disclosed herein are the design, synthesis,
characterization, and further functionalization of various
functionalized polymeric nanoparticles. Primary amines are employed
as examples of the surface functional groups. The reactivity of
primary amines complements a wide range of functional groups such
as alkyl halides, Michael acceptors, carboxylic acid, acid
chlorides, activated esters, epoxides, anhydride and aldehydes.
[0104] Referring to Scheme 1, polymer 1, a poly(methacrylamide), is
derived from the co-polymerization of 2-aminoethylmethacylamide and
3-(9-methylcoumarinoxy)propyl methacrylamide (Scheme 1). This
co-polymer self-assembles into an amphiphilic aggregate, where the
hydrophilic amino moieties are exposed to the aqueous phase, while
the coumarin moieties are tucked in the hydrophobic interior. The
propensity of coumarins to undergo photochemically driven [2+2]
cylcoaddition reaction (Muthuramu, et al. 1982 J. Org. Chem. 47,
3976; Gnanaguru, et al. 1985 J. Org. Chem. 50, 2337-2346; He, et
al. 2011 Soft Matter, 7, 2380-2386; Trenor, et al. 2004 Chem. Rev.
104, 3059-3078; Raghupathi, et al. 2011 Chem. Eur. J. 17,
11752-11760) was utilized to achieve the targeted polymer
nanoparticles.
##STR00003##
[0105] To synthesize the targeted polymer 1, the precursor random
copolymer, which contains 30% of N-Boc-aminoethylmethacrylamide and
70% of 3-(9-methylcoumarinoxy)propylmethacrylamide was first
synthesized; this polymer was prepared by reversible
addition-fragmentation chain transfer (RAFT) polymerization. The
Boc group was deprotected using trifluoroacetic acid in
dichloromethane to yield the random copolymer 1. An aqueous
solution of this polymer forms an aggregate of .about.22 nm at 1
mg/mL concentration, as discerned by dynamic light scattering
(DLS). This solution was then irradiated at 365 nm for 10 minutes
to generate the crosslinked nanoparticle. Several features of this
reaction are noteworthy: (i) the intensity of the absorption peak
centered at 320 nm, which corresponds to the coumarin moiety,
reduces within this irradiation time--confirming the photochemical
reaction of the coumarin moiety; (ii) the size of the nanoparticle
is the same as the aggregate, suggesting that the coumarin
dimerization process is exclusively intra-aggregate--note that
inter-aggregate reactions would result higher nanoparticle sizes;
(iii) there is no discernible nanoaggregate of the uncrosslinked
polymer in 10% water in DMSO, while the crosslinked nanoparticle's
size is slightly increased in this solvent mixture (FIG. 2)--this
swelling feature further confirms the crosslinked nature of the
nanoparticle. The degree of swelling should inversely vary with the
degree of crosslinking. It is known that irradiation of coumarin
dimers at 250 nm causes it to revert to the monomer. (Muthuramu, et
al. 1982 J. Org. Chem. 47, 3976; Gnanaguru, et al. 1985 J. Org.
Chem. 50, 2337-2346; He, et al. 2011 Soft Matter, 7, 2380-2386;
Trenor, et al. 2004 Chem. Rev. 104, 3059-3078; Raghupathi, et al.
2011 Chem. Eur. 1 17, 11752-11760). This reaction is often not
complete because of the photo-stationary state between the monomer
and the dimer at this irradiation wavelength. Therefore, this
reaction should cause the crosslink density to lower. Accordingly,
crosslinked nanoparticle in 10% water in DMSO was irradiataed at
250 nm for 30 minutes. DLS study of this solution indeed showed a
further increase in size (FIG. 2).
[0106] Hydrophobic dye molecules, such as
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI) or 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) were
encapsulated in the polymer aggregates successfully and guest
molecules were retained in the interiors of nanoparticles after
photoinduced crosslinking (FIG. 3).
[0107] The versatility of the amine functionality provides a ready
handle for various surface functionalizations of the nanoparticles.
First, amines were reacted with an activated ester and a cyclic
anhydride to provide amides with complementary surface
characteristics. Reaction of the amine-functionalized nanoparticles
with a peg-2000 NHS ester converted the positively charged surface
of the polymer nanoparticle to a charge neutral surface, while the
reaction with succinic anhydride should convert the charge to
negative. Zeta potential measurements of the reactants and the
products confirmed surface charge modification (FIG. 4a).
[0108] Second, the nanoparticles were modified by a NHS ester (of
lauric acid) and an isocyanate (dodecyl). The size of the
nanoparticles stayed similar after modification, which was
confirmed by TEM (FIG. 5). Both modifications would change the
surface nanoparticles from hydrophilic to hydrophobic. Evaluation
of the nanoparticle surface hydrophobicity by contact angle showed
that the unmodified nanoparticles exhibited a contact angle of
33.degree., while the modified nanoparticles have a contact angle
of 103.degree. and 98.degree., respectively (FIG. 4b).
[0109] Next, the surface functionalization was monitored by FTIR.
The nanoparticles were reacted with the NETS-ester of azidoacetic
acid. The FTIR spectrum of modified nanoparticles showed the
appearance of a peak at 2100 cm.sup.-1, characteristic of the azido
group, with the concurrent disappearance of the NETS ester peaks at
1812 cm.sup.-1 and 1783 cm.sup.-1 (FIG. 4c). To directly analyze
the conversion of the amino moiety on the nanoparticle surface, a
well-established fluorescamine assay was used, in which selective
reaction of primary amines with fluorescamine provides a
fluorescent derivative. (Udenfriend, et al. 1972 Science 178,
871-872.)
[0110] The relative fluorescence was used to ascertain the extent
of surface functionalization. Nanoparticles were first reacted with
molecules with different amine-reactive functional groups
(pentafluorophenol (PFP) ester, NHS ester, epoxide, and
isocyanate). The extent of functionalization was analyzed by
comparing the fluorescence from the functionalized amine
nanoparticles and the unreacted amine nanoparticle. The
fluorescence from three of the functionalized nanoparticles (PFP
ester, NHS ester, and isocyanate) was found to be similar to that
found with the negative control, indicating that the reaction was
quantitative in these cases (FIG. 4d).
[0111] Experiments were performed to investigate the tuning of the
nanoparticle sizes. While variations such as polymer MW,
concentration, and monomer ratio could afford different aggregate
sizes, a simpler variation with the same polymer was also studied
as a way to tune the nanoparticle sizes.
[0112] The pH of the solution and the ensuing variation the
hydrophilic-lipophilic balance of the polymer was studied. Aqueous
solutions of polymer 1 at different pH were prepared. The polymer
precipitates out at pH.about.9, consistent with the pK.sub.a of
amine groups. Also observed was that the aggregate sizes were not
significantly different between pH 3.0 and 6.5. Interestingly, the
greatest size differences were observed with subtle pH changes
between 7.0 and 8.5, indicating that subtle changes in the degree
of protonation of the amines lead to significant size differences.
This is presumably due to the difference in hydrophilic lipophilic
balance of the polymer at these pHs. These differences can be
utilized to systematically tune the size of the nanoparticles by
photochemically locking these aggregates, as shown in FIG. 6a.
[0113] Although the fluorescamine assay showed that all the
accessible amines can be utilized for surface decoration, it is
important to investigate the percentage of amines in the polymer
nanoparticle that are inherently accessible. Fluorescamine assay
was carried out in 1:3 water/DMSO mixture, in which no aggregation
was observed for the non-crosslinked polymer. The fluorescence
generated from the uncrosslinked polymer was utilized as a true
indicator of the amine moieties available in the polymer.
Evaluation of nanoparticles of different sizes, using this as the
standard, indicated that nearly all the amine moieties seem to be
available at a particle size of 22 nm. However, only 85% and 65% of
the amine moieties were available for functionalization in 45 nm
and 118 nm particles respectively (FIG. 6b). This supports the
notion that the smaller surface area of larger nanoparticles leads
to decreased availability of surface functionalities.
[0114] Thus, the versatile polymer nanoparticles provide a number
of key advantages as they (i) display versatile functional groups
on its surface, which can be further manipulated with a variety of
complementary reactive moieties; (ii) are capable of non-covalently
binding hydrophobic guest molecules; (iii) afford size tunability
by simply altering the pH at which the nanoparticle is synthesized;
(iv) have a very high percentage of the accessible surface moieties
at smaller sizes. Overall, the simplicity and versatility of the
surface functionalizable soft nanoparticles with host-guest
capabilities have implications in a variety of applications from
materials to biology.
Experimental
Materials and Methods
[0115] All chemicals and reagents were purchased from commercial
sources and were used as received, unless otherwise mentioned.
.sup.1H-NMR spectra were recorded on a 400 MHz Bruker NMR
spectrometer using the residual proton resonance of the solvent as
the internal standard. .sup.13C-NMR spectra were recorded on a 400
MHz Bruker NMR spectrometer using carbon signal of the deuterated
solvent as the internal standard. .sup.19F-NMR spectra were
collected on a 300 MHz Bruker NMR spectrometer. Molecular weights
of the polymers were estimated by gel permeation chromatography
(GPC) using PMMA standard with a refractive index detector. Dynamic
light scattering (DLS) and zeta potential were determined by
Nano-ZS (Malvern Instrument) Zetasizer. The fluorescence spectra
were obtained from a JASCO FP-6500 spectrofluorimeter. UV-visible
absorption spectra were collected using a Cary 100
spectrophotometer. FTIR spectra were recorded on a Perkin Elmer
spectrometer. Contact angles of water were examined on a Rame-Hart
telescopic goniometer. Transmission electron microscopy (TEM)
images were taken from JEOL 100CX at 100 KV.
Synthetic Methodologies
Synthesis of N-2-[(tert-butoxycarbonyl)amino]ethyl methacrylamide
Boc-AEMA
##STR00004##
[0116] Synthesis of 4-Methylcoumarin-7-oxypropyl methacrylamide
(CPMA)
##STR00005##
[0117] Synthetic Scheme for Random copolymer 1
##STR00006## ##STR00007##
[0118] Synthesis of N-Boc-ethylenediamine
##STR00008##
[0120] Di-tert-butyl dicarbonate (8.0 g, 36.7 mmol) was dissolved
in chloroform (50 mL) and added dropwise to a solution of
ethylenediamine (13.2 g, 220 mmol) in chloroform (250 mL) at
0.degree. C. The mixture was allowed to warm to room temperature.
After stirring for 12 hrs, the reaction crude was filtered and
washed with chloroform. The filtrates were collected and the
solvent was evaporated. The crude was re-dissolved in ethyl acetate
and washed with brine (3.times.100 mL) and water (100 mL). The
organic solution was dried over anhydrous Na.sub.2SO.sub.4,
filtered and concentrated under reduced pressure to afford N-Boc
ethylenediamine (2.97 g, 51%) as a colorless oil. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta.: 4.95 (bs, 1H), 3.20 (q, 2H), 2.82 (t,
2H), 1.99 (s, 2H). (Roy, et al. 2012 ACS Macro Lett. 1,
529-532.)
Synthesis of N-2-[(tert-butoxycarbonyl)amino]ethyl methacrylamide
(Boc-AEMA)
##STR00009##
[0122] To a solution of N-Boc-ethylenediame (2.0 g, 12.5 mmol) in
20 mL of dry dichloromethane was added 1.5 g (15.0 mmol) of
triethylamine and the mixture was cooled in an ice-bath. To this
cold mixture, a solution of methacryloyl chloride (1.3 g, 12.5
mmol) in 10 mL dichloromethane was added dropwise with continuous
stirring. After the addition, the reaction mixture was stirred at
room temperature for 6 hr. The stirring was stopped and the
reaction mixture was washed with 3.times.30 mL distilled water and
then with 30 mL of brine. The organic layer was collected, dried
over anhydrous Na.sub.2SO.sub.4 and concentrated to get the crude
product as a white solid. It was purified by column chromatography
using silica gel as stationary phase and mixture of ethyl
acetate/hexane as eluent. Yield: 2.52 g (88%). .sup.1H NMR (400
MHz, CDCl.sub.3) .delta.: 6.70 (bs, 1H), 5.75 (s, 1H), 5.33 (s,
1H), 4.92 (bs, 1H), 3.41 (q, 2H) 3.33 (q, 2H), 1.96 (s, 3H), 1.44
(s, 9H). (Sun, et al. 2007 Chem. Eur. J. 13, 7701-7707.)
Synthesis of Compound 3a
##STR00010##
[0124] To a solution of 3-aminopropanol (2.0 g, 26.6 mmol) in
chloroform (50 mL) was added di-tert-butyl dicarbonate (7.0 g, 31.9
mmol) at 0.degree. C. and stirred for 6 hrs at room temperature.
Chloroform was evaporated and the residue was re-dissolved in ethyl
acetate and washed with saturated NaHCO.sub.3 aqueous solution (100
mL) and brine (2.times.100 mL) The organic solution was dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo to
afford N-boc-3-aminopropanol (4.5 g, 97% yield). .sup.1H NMR (400
MHz, CDCl.sub.3) .delta.: 3.66 (t, 2H), 3.29 (t, 2H), 1.66 (p, 2H),
1.44 (s, 9H). (Mehlich, et al. 2011 Org. Biomol. Chem. 9,
4108-4115.)
Synthesis of Compound 3b
##STR00011##
[0126] N-boc-3-aminopropanol (4.0 g, 22.8 mmol) was dissolved in
100 mL of dry dichloromethane and 2.7 g (27.4 mmol) of
triethylamine was added to it. To this mixture, a solution of
p-toluenesulfonyl chloride (5.2 g, 27.4 mmol) and
4-dimethylaminopyridine (catalytic amount) in 20 mL dry
dicholoromethane was added. The reaction mixture was allowed to
stir at room temperature overnight. Solvent was evaporated to get
the crude product, which was purified by flash column
chromatography using silica gel as stationary phase and mixture of
ethyl acetate/hexane as eluent. Yield: 4.46 g (59%). .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta.: 7.80 (d, 2H), 7.36 (d, 2H), 4.10 (t,
2H), 3.16 (t, 2H), 2.45 (s, 3H), 1.84 (p, 2H), 1.42 (s, 9H).
(Simoni, et al. 2012 J. Med. Chem. 55, 9708-9721.)
Synthesis of Compound 3
##STR00012##
[0128] In a two-neck round bottom flask, compound 3b (3.0 g, 9.1
mmol) was mixed with 4-methylumbelliferone (1.76 g, 10.0 mmol),
K.sub.2CO.sub.3 (1.38 g, 10.0 mmol), and 18-crown-6 (0.48 g, 1.82
mmol) in acetone (300 mL) under argon atmosphere. The reaction
mixture was refluxed for 12 hours. Then, the crude reaction mixture
was filtered and washed with acetone. The filtrates were collected
and the solvent was evaporated. The crude was then poured into
water and extracted with ethyl acetate (3.times.100 mL). The
organic layers were dried over anhydrous Na.sub.2SO.sub.4,
filtered, and concentrated in vacuo. The crude product was purified
by flash column chromatography using silica gel as stationary phase
and mixture of ethyl acetate/hexane as eluent. Yield: 2.36 g (78%
yield). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 7.48 (d, 1H),
6.84 (dd, 1H), 6.80 (d, 1H), 6.13 (s, 1H), 4.73 (bs, 1H), 4.07 (t,
2H), 3.34 (q, 2H), 2.39 (s, 3H), 2.01 (p, 2H), 1.44(s, 9H).
.sup.13C NMR (400 MHz, CDCl.sub.3) .delta.: 162.0, 161.5, 156.1,
155.4, 152.7, 125.7, 113.8, 112.7, 112.2, 101.58, 79.8, 66.3, 38.0,
29.7, 28.5, 18.8.
Synthesis of 4-Methylcoumarin-7-oxypropyl methacrylamide (CPMA)
##STR00013##
[0130] To deprotect the N-boc amine functionality, compound 3 (2.36
g, 7.1 mmol) was dissolved in 10 mL of 1:1 v/v
dichloromethane/trifluoroacetic acid mixture. After stirring at
room temperature for 2 hrs, solvent mixture was removed by
evaporation, and the oil residue was rinsed two times with diethyl
ether (20 mL). The resultant precipitate was collected and dried in
vacuo. To a solution of the dried precipitate in 50 mL of dry
dichloromethane was added 2.15 g (21.3 mmol) of triethylamine and
the mixture was cooled in an ice-bath. To this cold mixture, a
solution of methacryloyl chloride (0.82 g, 7.8 mmol) in 10 mL
dichloromethane was added drop-wise with continuous stirring. After
the addition, the reaction mixture was stirred at room temperature
for 6 hrs. The reaction mixture was then washed with 3.times.30 mL
distilled water and then with 30 mL of brine. The organic layer was
collected, dried over anhydrous Na.sub.2SO.sub.4 and concentrated
to get the crude product as a yellow solid. It was purified by
column chromatography using silica gel as stationary phase and
mixture of ethyl acetate/hexane as eluent. Yield: 1.18 g (55%).
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 7.50 (d, 1H), 6.84 (dd,
1H), 6.80 (d, 1H), 6.19 (bs, 1H), 6.14 (s, 1H), 5.71 (s, 1H), 5.34
(s, 1H), 4.12 (t, 2H), 3.55 (q, 2H), 2.39 (s, 3H), 2.10 (p, 2H),
1.97 (s, 3H); .sup.13C NMR (400 MHz, CDCl.sub.3) .delta.: 168.7,
161.8, 161.4, 155.3, 152.7, 140.0, 125.7, 119.8, 113.8, 112.4,
112.1, 101.6, 67.0, 37.5, 28.9, 18.8.
Synthesis of random copolymer 1:
[0131] A mixture of 4-cyano-4-(phenylcarbonothioylthio)pentanoic
acid (7.8 mg, 0.028 mmol), Boc-AEMA (194 mg, 0.85 mmol), CPMA (600
mg, 1.99 mmol) and AIBN (0.92 mg, 0.0019 mmol) was dissolved in DMF
(10 ml) and degassed by performing three freeze-pump-thaw cycles.
The reaction mixture was sealed and then heated with a pre-heated
oil bath at 75.degree. C. for 12 h. The resultant mixture was
precipitated in ethyl acetate (200 mL) to remove unreacted
monomers. The precipitate was further dissolved in dichloromethane
(5 mL) and re-precipitated in ethyl acetate (200 mL) to yield
purified random copolymer as a yellow solid. Yield: 21%. .sup.1H
NMR (400 MHz, CDCl.sub.3/MeOD) .delta.: 6.8-7.4, 6.5-6.8, 5.8-6.0,
3.8-4.1, 3.0-3.4, 2.1-2.4, 1.5-2.0, 1.2-1.4, 0.7-1.2. GPC
(THF)M.sub.n: 3000 Da. PDI: 1.3. The molar ratio between two blocks
was determined by integrating the Boc group protons in Boc-AEMA and
an aromatic proton in the coumarin and found to be 3:7
(Boc-AEMA:CPMA). To remove the Boc groups, the resulting random
copolymer was dissolved in 10 mL of 1:1 v/v trifluoroacetic
acid/dichloromethane mixture and stirred overnight at room
temperature. Solvent mixture was then removed by evaporation, and
the oil residue was rinsed three times with diethyl ether. The
resultant precipitate was collected and dried overnight in vacuum
to afford random copolymer 1. Yield: 87%. .sup.1H NMR (400 MHz,
DMSO-d6) .delta.: 7.3-8.2, 6.7-7.1, 6.0-6.2, 3.9-4.2, 3.0-3.3,
2.7-2.9, 2.2-2.4, 1.5-2.1, 0.7-1.2.Complete disappearance of the
methyl proton signal of the Boc group at 1.2-1.4 ppm confirmed that
all the Boc groups have been removed.
Synthesis of azidoacetic acid
[0132] To a solution of sodium azide (2.3 g, 36 mmol) in water (50
mL) was added bromoacetic acid (1.0 g, 7.2 mmol) slowly. The
reaction mixture was stirred overnight at room temperature. The
reaction was quenched with 1 M of HCl aqueous solution. The crude
material was mixed with water (50 mL) and extracted with ethyl
acetate (3.times.100 mL). The organic solution was dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced
pressure to afford azidoacetic acid (0.39 g, 54% yield). .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta.: 10.62 (bs, 1H), 3.98 (s, 2H).
(Ghosh, et al. 2012 Chem. Eur. 1 18, 2361-2365.)
Synthesis of [2-(2-methoxyethoxy)ethoxy]acetic acid
pentafluorophenyl ester (Peg178 PFP ester)
[0133] To a solution of [2-(2-methoxyethoxy)ethoxy]acetic acid (1
equivalent) and pentafluorophenol (1.2 equivalent) in dry
dichloromethane was added
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2
equivalent) and catalytic amount of 4-dimethylaminopyridine at
0.degree. C. The reaction mixture was stirred for 6 h at room
temperature. The reaction mixture was washed with saturated
NaHCO.sub.3 aqueous solution and then with brine. The organic
solution was dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated under reduced pressure to afford the crude product. It
was purified by column chromatography using silica gel as the
stationary phase and mixture of ethyl acetate/hexane as eluent.
Yield: 50%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 4.54 (s,
2H), 3.83 (m, 2H), 3.74 (m, 2H), 3.65 (m, 2H), 3.57 (m, 2H), 3.38
(s, 3H). .sup.19F NMR (300 MHz, CDCl3) .delta.: -152.5 (2F), -157.3
(1F), -161.9 (2F).
General Procedure for the Synthesis of N-hydroxysuccinimide (NHS)
ester
[0134] To a solution of carboxylic acid (1 equivalent) and
N-hydroxysuccinimide (1.2 equivalent) in dry dichloromethane was
added 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(1.2 equivalent) at 0.degree. C. and stirred for 12 hours at room
temperature. The stirring was stopped and the reaction mixture was
washed with saturated NaHCO.sub.3 aqueous solution and then with
brine. The organic solution was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure
to afford the N-hydroxysuccinimide ester.
Methoxypolyethylene glycol 2,000 acetic acid NHS ester (PEG2000 NHS
ester)
[0135] Synthesis of PEG2000 NHS ester was done in dry DMF and the
crude reaction mixture was directly taken to next step without any
purification.
Azidoacetic acid NHS ester
[0136] Yield: 61%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 4.24
(s, 2H), 2.88 (s, 4H). (Ghosh, et al. 2012 Chem. Eur. J. 18,
2361-2365)
Lauric acid NHS ester
[0137] Yield: 71%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 2.84
(s, 4H), 2.60 (t, 2H), 1.74 (p, 2H), 1.40 (p, 2H), 1.20-1.35 (m,
14H), 0.88 (t, 3H). (Kapdi, et al. 2013 New J. Chem. 37,
961-964)
Capric acid NHS ester
[0138] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 2.84 (s, 4H),
2.60 (t, 2H), 1.74 (p, 2H), 1.17-1.48 (m, 12H), 0.88 (t, 3H).
(Schulze, et al. 2004 Adv. Synth. Catal. 346, 252-256)
Encapsulation of Guest Molecules
[0139] 50 .mu.L of 1 mg/mL DiO/DiI (in acetone) was added to a
vial, followed by evaporating the acetone with mild blow of air. To
this was added 2 mL of nanoparticle solution (1 mg/mL) and
sonicated at room temperature for 2 hrs. The resultant mixture was
then passed through 0.22 .mu.m filter to remove the
non-encapsulated DIO/DiI, followed by stirring the solution at room
temperature overnight to remove any residual acetone present in the
solution. This stock solution was accordingly diluted with milliQ
water (pH 3) to achieve required concentration of the
nanoparticles.
General Procedures for Surface Charge, Contact Angle and FTIR
Measurements
[0140] To 1 mL of nanoparticle stock solution (1 mg/mL) at basic pH
was added the functionalization agents (10 equivalents) dissolved
in DMF. After stirring overnight at room temperature, the excess
functional group reagents were removed by dialysis. For the
functionalization with succinic anhydride, the reaction was done in
0.1 M NaCl solution to minimize the aggregation of opposite charge
nanoparticles that could prevent the reaction from going to
completion.
Surface Charge Measurements:
[0141] The reaction mixtures were first dialyzed in acetone to
remove excess reagents and then were switched to aqueous medium.
Solutions after dialysis were accordingly diluted with milliQ water
to achieve a final concentration of 0.35 mg/mL. All solutions were
adjusted to pH 7.1 and then filtered through a 0.22 .mu.m filter
before performing surface charge measurements.
Contact Angle Measurements:
[0142] To prepare samples for contact angle measurements, stock
solution of nanoparticles dissolved in water and
dodecyl-functionalized nanoparticles dissolved in dichloromethane
were dropped onto a silicon slides and dried at room temperature
overnight.
Functionalization of Nanoparticles for Emission Spectrum
Measurements
[0143] Emission spectra were recorded on a JASCO (FP-6500)
spectrofluorimeter using quartz cuvettes. To 100 .mu.L of
nanoparticle (1 equivalent) stock solution at basic pH, functional
groups (3 equivalents) dissolved in DMSO (800 .mu.L) were added and
stirred overnight at room temperature. Fluorescamine (10
equivalents) dissolved in DMSO (100 .mu.L) was then added and
stirred for another 2 h at room temperature. All solutions were
directly taken to the spectrofluorimeter for measurement without
further purification. The emission spectra for fluorescamine-amine
adduct were recorded by exciting at 390 nm, with both excitation
and emission bandwidths set at 3 nm.
Quantifying the Amounts of Amine Available for
Functionalization
[0144] Different aliquots of nanoparticle (200 .mu.g/mL) were
pipetted into a 96 well microplate in triplicates. Different
volumes of water and DMSO were added to adjust the final water/DMSO
(1:2, v:v) volume to 150 .mu.L. The microplate was placed on a
microplate shaker and 50 .mu.l of 3.6 mM (1 mg/mL) fluorescamine
dissolved in DMSO was added to each well. Following the addition of
fluorescamine the plate was shaken for one minute and then allowed
to stand at room temperature for 2 h. The fluorescence was then
determined using a SpectraMax M5 plate reader with a 400 nm
excitation filter and a 460 nm emission filter. The sensitivity
setting was at 6 and the data collected from the top.
Different Percentage of Amines on the Nanoparticles can be
Functionalized
[0145] 100 .mu.L aliquots of nanoparticle (200 .mu.g/mL) were
pipetted into a 96 well microplate in triplicates, followed by
addition of different equivalents of capric acid NHS ester
dissolved in DMSO to each well. Different volumes of water and DMSO
were added accordingly to adjust the final water/DMSO (1:2, v:v)
volume to 150 .mu.L. The microplate was allowed to stay at room
temperature for 6 hours during which it was shaken on a microplate
shaker for the frequency of one minute per hour (FIG. 7).
Size Control
[0146] 2 mL polymer solutions (1 mg/mL) in scintillation vials were
adjusted to the required pH using NaOH and HCl aqueous solution.
After sonicating for 2 min, all solutions were irradiated under UV
light (365 nm) for 10 min to crosslink the polymers. The
nanoparticle solutions were dialyzed in milliQ water to remove
residual DMSO. All nanoparticle solutions were adjusted to pH 3 and
then filtered through a 0.22 .mu.m filter before performing dynamic
light scattering measurements.
II. Surface-Functionalized Polymer Nanoparticles Self-Assembled
from Amphiphilic Homopolymers
[0147] Amphiphilic homopolymers have been shown to have predictable
solution self-assembly behavior and applications in peptide
extraction and guest delivery. Although it is relatively easily to
homopolymerize an amphiphilic monomer, the multistep synthesis of
monomer itself is laborious. A new methodology with a simple
polymer preparation is strongly desired that can profoundly enhance
the application of amphiphilic polymers.
[0148] In yet another aspect, the invention generally relates to a
polymer-based nanoparticle having vesicular structures stabilized
by intraparticular crosslinking, wherein the polymer is an
amphiphilic homopolymer comprising a hydrophilic head group and a
hydrophobic tail group.
[0149] In certain embodiments, the hydrophilic head group is
selected from charged functional groups such as amino, ammonium,
sulfonium, phosphonium, carboxylate, phosphate, phosphonate,
sulfate, and sulfonate groups and charge-neutral functional groups
such as carboxy betaine, sulfo betaine, phosphoryl choline,
phosphonyl choline, saccharides, and polyethylene glycol groups and
the hydrophobic tail group is selected from linear and branched
alkyl chains, linear and branched fluoro-substituted alkyl chains,
and alkyl chains containing aromatic or heteromatic functional
groups
[0150] In certain embodiments, the intraparticular crosslinking is
via a group selected from pyrdinyl disulfide, activated esters,
coumarin derivatives, alkynes or alkenes along with thiols,
maleimdes with thiols, amines and epoxides, amines, thiols or
alcohols along with perforophenyl esters, and alkynes along with
azides. In certain embodiments, the amphiphilic homopolymer is a
cationic homopolymer. In certain embodiments, the cationic
homopolymer includes one or more groups selected from amino,
ammonium, sulfonium, phosphonium, pyridinium, imidazolium and other
heteroaromatic cationic functional groups. In certain embodiments,
the amphiphilic homopolymer is an anionic homopolymer. In certain
embodiments, the anionic homopolymer includes one or more groups
selected from carboxylate, phosphate, phosphonate, sulfate, and
sulfonate groups. In certain embodiments, the amphiphilic
homopolymer is a charge-neutral homopolymer. In certain
embodiments, the charge-neutral homopolymer is selected from
carboxy betaine, sulfo betaine, phosphoryl choline, phosphonyl
choline, saccharides, and polyethylene glycol groups.
[0151] In certain embodiments, the nanoparticle is surface
functionalized. In certain embodiments, the nanoparticle comprises
one or more surface functional groups, for example, selected from
the group consisting of amines, carboxylates, hydroxyl, halides,
acyl halides, esters, azides, nitriles, amides, epoxides,
aldehydes, furans, alkenes and alkynes. In certain embodiments, the
surface functionalization results in the surface being
functionalized by one or more groups selected from polyethylene
glycol, amines, carboxylates, epoxides, activated esters, thiols,
dopamines, and zwitterionic functional groups. In certain
embodiments, the surface functionalization results in the surface
being functionalized by peptides (including proteins and
antibodies), nucleic acids (including aptamers), and small or large
molecule ligands (including targeting ligands with desired binding
specificity and selectivity).
[0152] In certain embodiments, the intraparticular crosslinking of
the polymer-based nanoparticle is addressable by a biological or
chemical intervention resulting in partial or complete disassembly
of the polymer-based nanoparticle. In certain embodiments, the
biological or chemical intervention is a change in pH, redox
reagent, redox potential, ionic strength, enzymatic activity,
protein concentration, light, heat, or mechanical stress.
[0153] In yet another aspect, the invention generally relates to a
nano-assembly. The nano-assembly includes: a host crosslinked
polymer-based nanoparticle disclosed herein; and a guest molecular
cargo non-covalently encapsulated in the host crosslinked
polymer-based nanoparticle. The host crosslinked polymer network is
addressable by a biological or chemical intervention resulting in
partial or complete decrosslinking of the host polymer network and
release of the guest molecular cargo from the nano-assembly.
[0154] In certain preferred embodiments, the nano-assembly is a
nanogel. In certain embodiments, the non-covalently encapsulated
guest agent accounts for from 0.1 wt % about to about 45 wt % of
the nanoparticle. In certain embodiments, the guest molecular cargo
is selected from a therapeutic, diagnostic or imaging agent. In
certain embodiments, the guest molecular cargo is a small molecule.
In certain embodiments, the guest molecular cargo is a peptide
(e.g., a polypeptide, a protein, an antibody). In certain
embodiments, the molecular cargo is an oligonucleotide (e.g., DNA
or RNA). In certain embodiments, the molecular cargo is an
antitumor agent.
[0155] In yet another aspect, the invention generally relates to a
method for forming a polymer-based nanoparticle disclosed herein.
The method includes: providing an amphiphilic homopolymer by a ring
opening reaction; causing self-assembly of the amphiphilic
homopolymer, induced or assisted by one or more salt, to form a
polymersome comprising vesicle structures; and performing an
intraparticular crosslinking on the polymersome resulting in
intramolecularly crosslinked polymer-based nanoparticle having
vesicle structures.
[0156] Any suitable salt may be used to induce or assist the
formation of the polymersome.
[0157] In certain embodiments, the salt is one or more of
Na.sub.2SO.sub.4, Na.sub.2S.sub.2O.sub.3, and Na.sub.2SO.sub.3,
Na.sub.2HPO.sub.4. Na.sub.3PO.sub.4, and similar multivalent anions
with various counterions. In certain embodiments, the salt is one
or more of MgCl.sub.2, CaCl.sub.2, BaCl.sub.2, BeCl.sub.2,
ZnCl.sub.2, CuCl.sub.2, BiCl.sub.3, and similar multivalent cations
with various counterions.
[0158] In yet another aspect, the invention generally relates to a
method for controlled delivery of a molecular cargo to a target
biological site. The method includes: providing a nano-assembly
disclosed herein; delivering the nano-assembly to the target
biological site; and causing a biological or chemical intervention
resulting in a partial or complete decrosslinking resulting in
release of the guest molecular cargo from the nano-assembly.
[0159] In certain preferred embodiments, the homopolymer has the
formula of
##STR00014##
wherein Z.sub.3 is
##STR00015##
wherein Z' is selected from O, NH, NR (R.dbd.C.sub.1-C.sub.6 alkyl
group or R.sub.F);
[0160] R.sub.3 is selected from a hydrogen, C.sub.1-C.sub.12 alkyl
group, or halogen;
[0161] R.sub.L3 is CR.sub.4, N or a trivalent group, wherein
R.sub.4 is selected from a hydrogen, C.sub.1-C.sub.12 alkyl group,
or halogen;
[0162] R.sub.F is --H or a functional group;
[0163] R.sub.x is a crosslinking group capable of inter- or
intra-molecular crosslinking or an inter- or intra-molecularly
crosslinked group; and
[0164] r is an integer from about 10 to about 1000.
[0165] In certain preferred embodiments, R.sub.3 is methyl.
[0166] In certain preferred embodiments, R.sub.L3 is CH.
[0167] The trivalent group may be any suitable group. A trivalent
group may include a linear or cyclic portion, serving as a spacer
or linker, leading to the trivalent branching point. The branching
may be at a single atom or may be at different atoms, for example,
three branches from a cyclic or a linear scaffold.
[0168] R.sub.F can be any suitable functional groups, for example,
R.sub.F is selected from the group consisting of amines,
carboxylates, hydroxyl, halides, acyl halides, esters, azides,
nitriles, amides, epoxides, aldehydes, furans, alkenes and
alkynes.
[0169] In certain preferred embodiments, R.sub.F is an amine.
[0170] In certain preferred embodiments, R.sub.F is an activated
carboxylic ester.
[0171] R.sub.x can be any crosslinking group group, for examples,
selected from coumarin, alkenes, thiols, reactive disulfides,
alkynes, furans, aldehydes and epoxides. In certain embodiments,
R.sub.x may be selected to be suitable for and provide chemical
reactivities, for example, to incorporate functional diversity.
[0172] r can be an integer from about 10 to about 1000 (e.g., from
about 10 to about 800, from about 10 to about 500, from about 10 to
about 300, from about 10 to about 100, from about 10 to about 50,
from about 50 to about 800, from about 100 to about 800, from about
200 to about 800).
[0173] Unique features of the functionalizable homopolymer-based
nanoparticles include a rapid synthesis method affording targeted
amphiphilic polymer within 2 to 3 steps from monomer. The
functional polymers can be facilely achieved because the fidelity
of thiolactone chemistry makes it possible to introduce various
functionalities to the polymer by reacting with amino containing
functional molecules. It is also worth noting that the chemistry
also provides an avenue to incorporate a secondary functionality
simultaneously based on thiol chemistry.
Synthesis of Amphiphilic Homopolymer
[0174] The amphiphilic homopolymer was achieved by
post-polymerization functionalization, shown in FIG. 8. Briefly,
N-acryloyl homocysteine-based thialactone polymer (P0), which was
prepared from RAFT polymerization was used as precursor. Amine
bearing a protected functional group was used to react with the
precursor opening lactone ring and generating thiol, which was
captured by the aldithiol in situ affording cross-linkable
disulfide. The targeted polymer (P1-3) was achieved followed by TFA
deprotection if required.
[0175] Examples of amphiphilic homopolymers include:
##STR00016## ##STR00017## ##STR00018##
Self-Assembly of Amphiphilic Homopolymer
1. Tune the Size of Nanoassembly of Ionic Amphiphilic Homopolymer
(P1, P2)
[0176] Tuning the size by changing pH:
[0177] The driving force for the solution assembly of amphiphilic
polymer is hydrophilic lipophilic balance (HLB), which offers an
opportunity for us to manipulate the self-assembly of polymer. The
polymer precursors have carboxylic acid or amine functionalities
which are involved in protonation-deprotonation equilibrium. By
varying the pH of polymer solution, the extent to which the
functionalities are ionized can be tuned to shift the hydrophilic
lipophilic balance. As a result of changing in HLB, the polymer
forms nano-assembly with different sizes in aqueous solution (FIG.
9).
Tuning the Size by the Addition of Multivalent Counter Ion:
[0178] An alternate way to change the hydrophilicity of ammonium or
carboxylate functionalities involves interaction with counter ions.
A complementary divalent or trivalent counter ion was employed to
polymer solution to form intra-particle salt bridges transiently
crosslinking the polymer assembly. Various concentrations of
counter ions were added to tune the degree of crosslinking density
to tune the size of nanoparticles (FIG. 10).
Tuning the Size by the Addition of Different Type of Counter
Ions:
[0179] The binding affinity of counter ion pairs is quite different
from each other, which suggests that the type of counter ions can
regulate the self-assembly of polymer to different extent. A series
of counter ions are shown that can tune the size of polymer
nano-assembly in FIG. 11.
2. Tune the Size of Nanoassembly of Neutral Amphiphilic Homopolymer
(P3):
Tuning the Size by Changing Temperature:
[0180] Oligoethylene glycol containing polymers are well known for
their LCST behaviors. It is no surprise that oligoethylene glycol
containing neurtal amphiphilic homopolymers also have LCST
behavior. However, the LCST behavior can be utilized to control the
size of the nanoassembly (FIG. 12).
Tuning the Size by the Addition of Salt:
[0181] A series of salt have Hofmeister effect on oligoethylene
glycol containing polymer have been demonstrated. Here, salt was
used to manipulate the assembly of the amphiphilic homopolymer. The
size of the nanoassembly was found to increase when salt
concentration is increased (FIG. 12).
Cross-Linked Nanoassembly of Amphiphilic Homopolymer
Nanoassembly:
[0182] Regarding the application of nano-assembly, structural and
morphological stability of the assembly are highly desired. In
order to stabilize the nano-assembly, chemical cross-linking was
used to covalently stabilize the nano-assembly via the addition of
DTT. The addition of DTT results in the formation of disulfide bond
inside the nano-assembly locking the nano-assembly. The approach
applied to regulate the self-assembly of amphiphilic homopolymer
can be translated to prepare the cross-linked particles. By varying
pH, salt concentration, salt species and polymer solution
temperature, the size of cross-linked nano-assembly can be
controlled in a wide range (FIG. 13).
Post-Functionalization:
[0183] The performance of nanoparticles for biomedical application
is highly dependent on their surface properties. The ability to
functionalize the hydrophilic corona of polymersome allows one to
optimize its performance and also expand the polymersome inventory
for other potential applications. Pegylation of both cationic and
anionic VesiGels were used to demonstrate the surface
functionalization of hydrphoilic corona. NHS ester and amine
terminated polyethylene glycol monomethyl ether were used to treat
amino and carboxylate-containing VesiGel respectively. The
successful peglation of the two types of VesiGels was confirmed by
following their surface charges before and after the
functionalization.
[0184] As shown in FIG. 18a, a neutral zeta potential was observed
after the pegylation from both original cationic or anionic
VesiGel. The degree of functionalization of cationic VesiGel was
measured by fluorescamine assay as shown in FIG. 18b. 14.7 nmol out
of 36.6 nmol of amine groups on cationic VesiGels can be accessed
for fluorescamine functionalization indicating 40% of
functionalizable amine groups. 86% of this 40% (2.0 nmol out of
14.7 nmol can not be accessed) of functionalizable amines on
VesiGel can be pegylated. The lack of full pegylation when compared
to fluorescamine is likely because of the limited accessibility of
PEG (Mn: 2000 Da) to amines.
[0185] It is desirable to control over the permeability of
polymersomes to tune the mass transportation between media and
polymersome lumen for the applications like nanoreactor or
artificial organelles where the disruption of the vesicular
structure is not desired. A potential solution to manipulate the
transportation process can be tuning the hydrophobicity of vesicle
wall by post-functionalization. This can also be an effective way
to covalently incorporate the active components into the
nanoassembly for therapeutic delivery applications. Independent
functionalization of the hydrophilic corona and hydrophobic wall of
the VesiGel would help demonstrate the versatility of the VesiGel
platform for post-functionalization.
[0186] Taking the advantage of amino group and the left over
pyridinyl disulfide, the VesiGel can be orthogonally functionalized
with a FRET pair by reacting with the complementary isocynate
containing rhodamine and thiol bearing fluorescein. The
functionalization permanently immobilized rhodamine onto the corona
with a thiourea linkage, but fluorescein onto hydrophobic wall with
a cleavable disulfide bond. Concurrent observation of the
absorbance of both dyes and FRET after extreme dialysis suggests
the success of the functionalization (FIGS. 18c and 18d).
[0187] To further test the difference on the reversibility of the
two chemistries applied for functionalization, the VesiGel was
treated with DTT which selectively cleave fluorescein from the
nanoparticle. It was envisioned that the cleavage of fluorescein
will result in precipitation out from solution, therefore, the
decrease of absorbance while the absorbance of rhodamine remains
the same because it still attached. This was indeed observed. The
increase in the emission of fluorescein after the addition of DTT
also indicated the cleavage of fluorescein. The increasing
fluorescence can be attributed to the disappearance of FRET due to
the lack of proximity between rhodamine and fluorescein molecules.
Another possible reason for the fluorescence recovery of
fluorescein can be due to the decrease in self-quenching of
fluorescein after cleavage from VesiGel. All these observations
suggest orthogonal functionalization of hydrophilic corona and
hydrophobic wall can be done on the disclosed VesiGel.
Guest Loading and Stimuli-Triggered Release
[0188] The amphiphilicity of polymer allows the nano-assembly to
encapsulate guest molecules. Due to the disulfide functionality in
the nano-assembly, it should have responsiveness toward applied
trigger, reductant, which reduces the disulfide bond and
destabilizes the nano-assembly causing the release of loaded guest
molecules. The ross-linked nano-assemblies are indeed found to
release the loaded guest molecule when a redundant was added into
nano-assembly solution (FIG. 14).
[0189] The unique structural features having hydrophobic wall and
hydrophilic lumen simultaneously, gives the polymersome the
capability of serving as a reservoir for both hydrophobic and
hydrophilic cargos. Bearing this structural characteristic along
with the disulfide crosslinks, the disclosed VesiGels are expected
to be able to encapsulate hydrophilic and hydrophobic cargos and
have features of redox-triggered cargo release. Initially, DiI, a
hydrophobic dye was encapsulated in VesiGels. The release of DiI
was observed in the presence of DTT, which disrupts the VesiGel by
breaking the disulfide crosslinks (FIG. 19a). In contrast, no
observation of encapsulated DiI was found without external
stimuli.
[0190] To test the ability to load the hydrophilic cargos, cationic
and anionic VesiGels was formed in rodamine 6G and calcein solution
respectively, where the hydrophilic cargos were expected to be
engulfed into the cavity of polymersome in situ. So, even after the
extensive dialysis, the local concentration of dye in VesiGel lumen
is the same as that of the original dye solution which is high
enough for self-quenching. Fluorescence recovery from both cationic
and anionic VesiGels was observed upon the treatment of DTT (FIGS.
19b and 19c). The reason for the observation of fluorescence
recovery is that disruption of vesicular structure by DTT reduction
liberates dye molecules from VesiGel lumen into bulk solution. As a
result of release from VesiGel, the dye concentration is diluted to
be lower than the concentration that is required for
self-quenching. Therefore, an increase in fluorescence can be
observed in response to the treatment of DTT.
[0191] Formulating the biomacromolecules into nanostructure for
therapeutic or catalysis application is of much more interest.
Here, myoglobin (Mw: 17 kDa, pI: 7.2) was chosen as a model protein
to load into cationic VesiGels following the same procedure of
rhodamine 6G encapsulation. As shown in FIG. 19d, the loading of
myoglobin after removal of free myoglobin was followed by measuring
the absorbance of myoglobin at 408 nm attributed to the porphyrin
absorption. By fitting in the calibration curve generating by pure
myoglobin solution, 3 wt % of myoglobin can be encapsulated in the
VesiGel. Pepsin (Mw: 35 kDa, pI 4.2) was also encapsulated in to
anionic VesiGels using the same protocol. While not exclusive,
positively charged proteins are encapsulated more efficiently in
positively charged vesigels and negatively charged proteins with
negatively charged vesigels. This is an unusual combination.
[0192] The success of encapsulation was evident by the observation
of tyrosine absorption and emission. The release of pepsin
triggered by the DTT was demonstrated by trypsin digestion assay
detected by mass spectrometry. The basic design on this assay is
that pepsin is prevented from being digested when it is trapped
inside an intact VesiGel, while the disruption of VesiGel by DTT
makes the pepsin accessible for digestion.
[0193] In FIG. 19e, signal with an m/z of 34709.605 that originates
from pepsin was observed from pepsin encapsulated in VesiGel
without DTT exposure after trypsin digestion. However, the
corresponding signal was not observed from pepsin itself or pepsin
encapsulated in VesiGel followed by DTT treatment after trypsin
digestion indicating the consumption of pepsin by trypsin
digestion. All of these evidences together suggest the release of
pepsin from VesiGel can be obtained by applying DTT to
VesiGels.
[0194] Thus, the invention discloses a general methodology for the
preparation of size-tunable VesiGel platform directly from
synthetically easily accessible homopolymers aqueous solution in
the assistance of multivalent counter ions via salt-bridging
mechanism. This method provides a variety of physical and chemical
properties including: i) variable surface functionalities: ii)
capability of simultaneous encapsulation of hydrophilics and
hydrophobics; iii) engineerable corona and membrane; iv)
redox-modulated programmable host-guest property into a single
VesiGel system. The simplicity and versatility of this method to
prepare VesiGel with engineerable properties will profoundly
facilitate and extend the applications of the vesicular
nanostructures.
Functional VesiGels
[0195] As disclosed herein, instead of using copolymers with
complex architectures, typically block copolymers, simple
homopolymers are used as polymersome precursor synthetically saving
tremendous efforts on polymer preparation. The targeted polymers
are amphiphilic homopolymers comprised of a hydrophilic head group
(amino or carboxylate) and a hydrophobic and cross-linkable group
(pyrdinyl disulfide), as shown in FIG. 15a.
[0196] Thus, the approach takes advantage of the fidelity of the
thiolatone ring opening reaction to prepare amphiphilic
homopolymer. (Reinicke, et al. 2013 ACS Macro Lett. 2, 539-543;
Espeel, et al. 2012 Polym. Chem. 3, 1007-1015.) The ring opening
reaction of poly (thiolactone acrylamide) (PT1a) homopolymer was
initiated by N-Boc ethylenediamine or .beta.-alanine t-butyl ester
generating an amide and thiol which was captured by excessive
amount of aldrithiol in situ. Removal of protecting group by TFA
gives the targeted amphiphilic homopolymers bearing ionic head
groups and crosslinkable hydrophobic disulfide tails.
[0197] Single tailed small molecule surfactants with opposite
charges that normally form micelles have been mixed at appropriate
ratio to construct so-called "catanionic" vesicles. (Segota, et al.
2006 Adv. Colloid Interface Sci. 121, 51-75; Iampietro, et al. 1998
J. Phys. Chem. B. 102, 3105-3113.) Besides, multivalent counter
ions have also been employed to induce the formation of vesicular
structures. (Lee, et al. 2013 Soft Mater 9, 200-207; Li, et al.
2014 Adv. Funct. Mater. DOI: 10.1002/ADFM.201400569.) The formation
of vesicular structure is attributed to the reduction in the area
of hydrophilic head group due to the counter ion pairing changing
the molecular packing parameters.
[0198] The herein disclosed approach takes advantage of the
interaction between polyions and their counter ions to modulate the
self-assemble process. The addition of divalent counterions adjusts
the hydrophilic lipophilic balance of the homopolymer, along with
the molecular packing parameters, to adopt the vesicular type
assembly. The cationic homopolymer, P1 was found to form
polymersomes in Na.sub.2HPO.sub.4 solution with a low millimolar
salt concentration. The size of polymersomes readily increases from
40 nm to 90 nm when the concentration of Na.sub.2HPO.sub.4
increases from 1.5 mM to 2.5 mM (shown in SP).
[0199] The assembled polymersome can be further covalently
stabilized by intraparticle disulfide crosslinking to lock the
vesicular morphology. The size of VesiGel, shown in FIG. 15b,
retains almost the same after the crosslinking suggesting the
intra-particular crosslinking occurs. The TEM images indicate the
nanoassemblies of P1 adopt vesicular structures in all investigated
Na.sub.2HPO.sub.4 concentrations. Size of the VesiGel measured by
DLS is coincident with that determined by TEM and AFM.
[0200] Similarly, when anionic polymer, P2 was used as a precursor,
MgCl.sub.2 can be used to induce the formation of anionic
polymersome, which was confirmed by TEM. The size of obtained
anionic polymersome was also found to be MgCl.sub.2 concentration
dependant. Tunable size ranging from 45 nm to 120 nm was obtained
when concentration of MgCl.sub.2 was increased from 2.4 mM to 4.8
mM. The crosslinking of anionic polymersome was also
intra-particular indicated by the observation of similar particle
size after and before crosslinking.
[0201] To further confirm VesiGels do have vesicular structure,
radius of gyration (R.sub.g) of the VesiGels prepared in 2.4 mM and
4.8 mM MgCl.sub.2 solution were by SLS to compare with their
hydrodynamic radius (R.sub.h) obtained from DLS. It turns out that
R.sub.g/R.sub.h of VesiGels prepared in 2.4 mM and 4.8 mM is
respectively measured to 1.19 and 0.99. The measured
R.sub.g/R.sub.h values are closed to 1.0, which is the theoretical
value for a vesicle. (Benoit, H., Froehlich, Light scattering from
polymer solutions, edited by Huglin, M. B. (Academic Press, London,
1972).) A larger R.sub.g/R.sub.h value for small VesiGels prepared
in 2.4 mM MgCl.sub.2 solution indicates a loose vesicular structure
that was also confirmed by the blurred structure from TEM
image.
[0202] To test if salt-induced formation of the polymersome can be
generally applied, a variety of salts were employed to polymer
solution followed by the measurement of the size of the
nanoassembly. The size of nanoassembly from P1 in the presence of
Na.sub.2SO.sub.4, Na.sub.2S.sub.2O.sub.3, and Na.sub.2SO.sub.3 was
found to be significantly affected by the salt species (FIG. 16a).
The morphology of nanoassemblies obtained from these salts was also
found to be vesicular from TEM (FIG. 16b). Similar to MgCl.sub.2,
the influence of CaCl.sub.2 and BaCl.sub.2 on self-assembly of P2
was also observed and vesicular structure was adopted in both cases
evident by the TEM. Importantly, the size of nanoassemblies of P1
and P2 in these salts solutions all increase with increasing salt
concentration.
[0203] In addition to these divalent salts, the effect of
monovalent salts on self-assembly of both polymers was also
investigated. Nanostructures around 6 nm were obtained in the
presence of monovalent salts. These nanostructures with much
smaller size are likely single chain nanoparticles due to the
polymer chain collapse after cross-linking. It is worth noting that
the concentration of monovalent salts used in both case are much
higher than that of divalent salts (4-5 times). It is clear that
the salts having divalent counter ions to corresponding polymers
remarkably affect their self-assembly. Along with the lack of
influence of monovalent salts on polymer assembly, the observation
of divalent counterions mediated self-assembly suggests that
operating mechanism of polymersome formation assisted by these
salts can potentially be through salt-bridging.
Mechanistic Study on VesiGels Formation
[0204] It was proposed that the divalent counter ion interacts with
two hydrophilic head groups of the polymer reducing the
hydrophilicity of the polymer, consequently adjusting the packing
parameters to meet the requirement for polymersome formation. On
the other hand, salt-bridging also stabilize the lamellar packing
of polymer chains on the vesicle wall. This hypothesis is also
supported by the observation of salt-bridging induced morphological
transformation of block copolymer assemblies from micelle to
vesicle. (Zhang, et al. 1996 Science, 272, 1777-1779; Zhang, et al.
1996 Macromolecules 29, 8805-8815.) However, the proposed
salt-bridging mechanism was not yet systematically
investigated.
[0205] To test this theory, the polyions were mixed with a
water-soluble dye, as a substitute of salt, which has multivalent
counter ions to interact with the polymer. As shown in FIG. 17a, if
the interaction causes the aggregation, the probe molecules will be
brought closer by the polymer leading to a red-shift on absorbance
as well as fluorescence quenching. (Grohn, et al. 2008 Chem. Eur. 1
14, 6866-6869; Yildiz, et al. 2009 Macromol. Chem. Phys. 210,
1678-1690.) Probes used were calcein and lysine modified pyrene
respectively for P1 and P2. As shown in FIG. 17b, observed was a
clear red-shift of probes on absorbance spectrum after the addition
of polymers with opposite charge. Concurrently, an obvious
fluorescence quenching was also observed in both probes.
[0206] Interestingly, excimer emission was observed from dimerized
pyrene due to the proximity of the probe in polymer nanoaggregate.
In addition, the lack of observation of the absorbance red shifting
and fluorescence quenching of probes when polyions are mixed with
probes of the same charge further supported the
electrostatics-based hypothesis (FIGS. 17d and 17e). Furthermore,
increases of the size of polymer assembly in the presence of the
probes were also found as previously observed, when divalent or
trivalent salts were to induce the assembly.
[0207] To further test this theory, a salt competition experiment
was also performed, in which change on absorbance and fluorescence
of a pre-mixed P1/calcein solution was followed, when the solution
was titrated by Na.sub.2SO.sub.4. Here, it was envisioned that
sulfate anions act as a competetiver binder with the cationic
polymer and liberating the calcein probe from polymer/calcein
complex, therefore, changing the photophysical properties of the
probe. As released from the complex, the maximum absorption of
calcein will be gradually blue shifted to its original one when it
is completely freed from the complex. This was indeed observed and
the data are shown in FIG. 17. On the other hand, the
self-quenching of calcein will be also eliminated due to the lack
of proximity of probes in the presence of competing sulfate ions.
Fluorescence recovery of calein was also clearly observed in the
course of sulfate titration.
Experimental
Materials and Methods
[0208] Polymer stock solution preparation. 15 mg of P1 or P2 was
directly dissolved in 2 mL of milliQ water. To make P2 dissolved,
1.5 eq. of NaOH was added to the solution. The obtained solution
was then dialyzed against DI water using membrane with a MWCO of
3500 Da. The final concentration of solution was fixed to 5
mg/mL.
[0209] Polymersome and VesiGel formation. Calculated amount of salt
stock solution (100 mM) was added to 300 .mu.L of milliQ water. To
the above slat solution, 200 .mu.L of P1 stock solution was added
to make final polymer solution with a concentration of 2 mg/mL. For
P2, 400 .mu.L of salt solution was prepared and added with 100
.mu.L of P2 stock solution giving the polymer solution with a
concentration of 1 mg/mL. The final polymer solutions were left for
3 hours to form polymersomes. The VesiGels were obtained by
cross-linking the polymersome solution through the addition of
calculated amount of DTT. The cross-linking reaction was allowed to
undergo for 4 hours. The VesiGel was then purified by dialysis
against water.
[0210] Guest molecules encapsulation. For hydrophobic guest
encapsulation, 1 wt % of guest solution (1 mg/mL) in acetone was
added to the polymersome solutions followed by the addition of DTT.
The unloaded guest molecule was removed by filtration using syringe
filter with a pore size of 0.4 .mu.m. The hydrophilic guests were
dissolved in the salt solution with a desired salt and guest
concentration. To the solution, polymer stock solutions were added
to form polymersomes which encapsulate the hydrophilic guest in
situ. The free guest was removed by extensive dialysis against
water after DTT crosslinking.
Polymer Stock Solution Preparation.
[0211] 15 mg of P1 or P2 was directly dissolved in 2 mL of milliQ
water. To make P2 dissolved, 1.5 eq. of NaOH was added to the
solution. The obtained solution was then dialyzed against DI water
using membrane with a MWCO of 3500 Da. The final concentration of
solution was fixed to 5 mg/mL by adding milli Q water.
Polymersome and VesiGel Formation.
[0212] Calculated amount of salt stock solution (100 mM) was added
to 300 .mu.L of milliQ water. To the above salt solution, 200 .mu.L
of P1 stock solution was added to make final polymer solution with
a concentration of 2 mg/mL. For P2, 400 uL of salt solution was
prepared and added with 100 .mu.L of P2 stock solution giving the
polymer solution with a concentration of 1 mg/mL. The final polymer
solutions were left for 3 hours to form polymersomes. The VesiGels
were obtained by cross-linking the polymersome solution through the
addition of calculated amount of DTT. The cross-linking reaction
was allowed to take place for 4 hrs. The VesiGel was then purified
by dialysis against water.
Small Guest Molecules Encapsulation.
[0213] For hydrophobic guest encapsulation, 1 wt % of guest
solution (1 mg/mL) in acetone was added to the polymersome
solutions followed by the addition of DTT. The unloaded guest
molecule was removed by filtration using syringe filter with a pore
size of 0.4 um. The hydrophilic guests were dissolved in the salt
solution with a desired salt and guest concentration. To the
solution, polymer stock solutions were added to form polymersomes
which encapsulate the hydrophilic guest in situ. The free guest was
removed by extensive dialysis against water after DTT
crosslinking.
Myoglobin Encapsulation.
[0214] 2 mg/mL of myoglobin was dissolved in 1 mL of milliQ water.
11 .mu.L of Na.sub.2HPO.sub.4 solution (100 mM) was added to 289
.mu.L of myoglobin solution. 100 .mu.L of P1 solution was added to
myoglobin-Na.sub.2HPO.sub.4 solution followed by the addition of
0.1 equivalent of DTT after 2 hrs. The free myoblobin was removed 4
hrs later after the addition of DTT by dialysis against water using
membrane with MWCO of 100 kDa.
Lysozyme Encapsulation.
[0215] 10 mg of lysozyme was dissolved in 1 mL of milliQ water. 21
.mu.L of MgCl.sub.2 solution (100 mM) was added to 379 .mu.L of
lysozyme solution. Then, 100 .mu.L of P2 solution was added into
lysozyme-MgCl.sub.2 solution followed by the addition of 0.1 eq of
DTT after 2 hrs. The cross-linking reaction was allowed to undergo
for 4 hrs. The unloaded lysozyme was removed by extensively
dialysis against water using membrane with MWCO of 100 kDa.
[0216] VesiGel functionalization. Cationic VesiGel solution was
added to 1 mL of DMSO solution. Then, the solution was adjusted to
pH 9 by the addition of NaHCO.sub.3 solution. Then 0.2 equivalent
of TRITC (to the amine) in DMSO was added to the nanoparticle
solution. After 24 hours, 0.2 eq. thiol functionalized fluorescein
solution in DMSO was also added to the reaction mixture. The
reaction mixture was dialysis against MeOH using membrane with MWCO
of 11000 Da.
[0217] PEGylation of cationic VesiGel. To 500 uL of 2 mg/mL
cationic VesiGel solution (0.0022 mmol repeat unit), 500 .mu.L of
DMSO was added with 0.95 .mu.L of triethylamine (0.0066 mmol, 3
eq.). 8.8 mg (0.0044 mmol, 2 eq.) of PEG-NHS ester (Mn: 2000 Da)
was dissolved in 500 .mu.L of DMSO and then added to the VesiGel
solution. The reaction was allowed to go overnight. The free PEG
was removed by dialysis against water.
[0218] PEGylation of anionic VesiGel. To 2 mL of 1 mg/mL of anionic
VesiGel solution (0.0054 mmol repeat unit), 2.07 mg EDC (0.0108
mmol, 2 eq.) was added. Then, 6 mg (0.0108 mmol, 2 eq.) of
PEG-NH.sub.2 (Mn: 550 Da) in 500 .mu.L of water solution was added
to VesiGel solution. The reaction was kept for 24 hrs. The free PEG
was removed by dialysis against water.
[0219] In this specification and the appended claims, the singular
forms "a," "an," and "the" include plural reference, unless the
context clearly dictates otherwise. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood too one of ordinary skill in the art. Unless
defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art. Methods recited herein may be carried out in any
order that is logically possible, in addition to a particular order
disclosed.
Incorporation by Reference
[0220] References and citations to other documents, such as
patents, patent applications, patent publications, journals, books,
papers, web contents, have been made in this disclosure. All such
documents are hereby incorporated herein by reference in their
entirety for all purposes. Any material, or portion thereof, that
is said to be incorporated by reference herein, but which conflicts
with existing definitions, statements, or other disclosure material
explicitly set forth herein is only incorporated to the extent that
no conflict arises between that incorporated material and the
present disclosure material. In the event of a conflict, the
conflict is to be resolved in favor of the present disclosure as
the preferred disclosure.
Equivalents
[0221] The representative examples are intended to help illustrate
the invention, and are not intended to, nor should they be
construed to, limit the scope of the invention. Indeed, various
modifications of the invention and many further embodiments
thereof, in addition to those shown and described herein, will
become apparent to those skilled in the art from the full contents
of this document, including the examples and the references to the
scientific and patent literature included herein. The examples
contain important additional information, exemplification and
guidance that can be adapted to the practice of this invention in
its various embodiments and equivalents thereof.
* * * * *