U.S. patent application number 12/941938 was filed with the patent office on 2011-04-28 for particles coated with zwitterionic polymers.
This patent application is currently assigned to University of Washington. Invention is credited to Guangwei Jia, Shaoyi Jiang, Hong Xue, Wei Yang, Lei Zhang.
Application Number | 20110097277 12/941938 |
Document ID | / |
Family ID | 51351673 |
Filed Date | 2011-04-28 |
United States Patent
Application |
20110097277 |
Kind Code |
A1 |
Jiang; Shaoyi ; et
al. |
April 28, 2011 |
PARTICLES COATED WITH ZWITTERIONIC POLYMERS
Abstract
Nanoparticles zwitterionic polymers grafted thereto or grafted
therefrom, and methods for making and using the nanoparticles.
Zwitterionic nanogels, and methods for making and using the
nanogels.
Inventors: |
Jiang; Shaoyi; (Redmond,
WA) ; Jia; Guangwei; (Tianjin, CN) ; Xue;
Hong; (Seattle, WA) ; Yang; Wei; (Seattle,
WA) ; Zhang; Lei; (Seattle, WA) |
Assignee: |
University of Washington
Seattle
WA
|
Family ID: |
51351673 |
Appl. No.: |
12/941938 |
Filed: |
November 8, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12020998 |
Jan 28, 2008 |
7879444 |
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12941938 |
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PCT/US2006/298988 |
Jul 25, 2006 |
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12020998 |
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61259081 |
Nov 6, 2009 |
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60711613 |
Aug 25, 2005 |
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Current U.S.
Class: |
424/9.322 ;
424/497; 525/123; 525/301; 525/330.2; 977/742; 977/762;
977/774 |
Current CPC
Class: |
C09D 5/1637 20130101;
C08J 3/075 20130101; C08F 2438/01 20130101; C07C 317/50 20130101;
C09D 7/62 20180101; C08K 3/16 20130101; C07C 317/08 20130101; C08F
220/365 20200201; C08F 120/36 20130101; C07C 317/28 20130101; C08F
292/00 20130101; A61K 49/18 20130101; C09D 7/68 20180101; B82Y
30/00 20130101; C07C 317/44 20130101; C08K 3/34 20130101; C08K 9/08
20130101; C09D 7/67 20180101 |
Class at
Publication: |
424/9.322 ;
424/497; 525/330.2; 525/123; 525/301; 977/762; 977/774;
977/742 |
International
Class: |
A61K 49/18 20060101
A61K049/18; A61K 9/50 20060101 A61K009/50; C08F 8/00 20060101
C08F008/00 |
Goverment Interests
STATEMENT OF GOVERNMENT LICENSE RIGHTS
[0002] This invention was made with Government support under
Contract No. N000140910137 awarded by the Office of Naval Research
and Contract No. DMR-0705907 awarded by the National Science
Foundation. The Government has certain rights in the invention.
Claims
1. A particle, comprising: (a) a core, and (b) a surface having a
plurality of zwitterionic polymers grafted thereto or grafted
therefrom.
2. The particle of claim 1, wherein the particle has nanoscale
dimensions.
3. The particle of claim 1, wherein the core comprises a metal, a
metal oxide, a ceramic, a synthetic polymer, a natural polymer,
silicon dioxide, a crystal, a semiconductor material, a hydrogel, a
liposome, a micelle, or a carbon-based material.
4. The particle of claim 1, wherein the core comprises gold,
silver, iron, or platinum, cadmium sulfide, cadmium selenide, or
combinations thereof.
5. The particle of claim 1, wherein the core comprises titanium
oxide, iron oxide, zinc oxide, aluminum oxide, copper oxide, or
tantalum oxide, or combinations thereof.
6. The particle of claim 1, wherein the core comprises a carbon
fiber, a carbon nanotube, a carbon nanosheet, a carbon nanobelt, a
carbon nanorod, a carbon nanowire, and a carbon nanodish.
7. The particle of claim 1, wherein the core comprises
polyurethane, polyethylene, polystyrene, poly(methyl methacrylate),
or silicone.
8. The particle of claim 1, wherein the core comprises calcium
fluoride or quartz.
9. The particle of claim 1, wherein the core is a quantum dot.
10. The particle of claim 1, wherein the zwitterionic polymer has
the formula:
PB-(L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd.O)--OM).su-
b.n (X).sub.n wherein PB is a polymer backbone having n pendant
groups L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd.O)--OM);
N.sup.+ is a cationic center; R.sub.a and R.sub.b are independently
optional as necessary to provide a cationic center and
independently selected from alkyl and aryl; A(.dbd.O)--OM is the
anionic center, wherein A is C, S, SO, P, or PO, and wherein M is a
counterion; L.sub.1 is a linker that covalently couples the
cationic center to the polymer backbone; L.sub.2 is a linker that
covalently couples the cationic center to the anionic center;
X.sup.- is the counter ion associated with the cationic center; and
n is an integer from 1 to about 10,000.
11. The particle of claim 1, wherein the zwitterionic polymer has
the formula:
PB-[L.sub.1-N.sup.+(R.sub.a)(R.sub.b)(R.sub.c)].sub.n[L.sub.2-A-
(.dbd.O)--OM].sub.p(X.sup.-).sub.n wherein PB is a polymer backbone
having n pendant groups L.sub.1-N.sup.+(R.sub.a)(R.sub.b)(R.sub.c)
and p pendant groups L.sub.2-A(.dbd.O)--OM; N.sup.+ is a cationic
center; R.sub.a, R.sub.b, and R.sub.c are independently optional as
necessary to provide a cationic center and independently selected
from alkyl and aryl; A(.dbd.O)--OM is the anionic center, wherein A
is C, S, SO, P, or PO, and wherein M is a counterion; L.sub.1 is a
linker that covalently couples the cationic center to the polymer
backbone; L.sub.2 is a linker that covalently couples the anionic
center to the polymer backbone; X.sup.- is the counter ion
associated with the cationic center; n is an integer from 1 to
about 10,000; and p is an integer from 1 to about 10,000.
12. The particle of claim 1 further comprising one or more
targeting agents.
13. A nanogel, comprising a zwitterionic polymer having the
formula:
PB-(L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd.O)--OM).sub.n(X.sup.-
-).sub.n wherein PB is a polymer backbone having n pendant groups
L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd.O)--OM) comprises
crosslinks for those nanogels that are crosslinked; N.sup.+ is a
cationic center; R.sub.a and R.sub.b are independently optional as
necessary to provide a cationic center and independently selected
from alkyl and aryl; A(.dbd.O)--OM is the anionic center, wherein A
is C, S, SO, P, or PO, and wherein M is a counterion; L.sub.1 is a
linker that covalently couples the cationic center to the polymer
backbone; L.sub.2 is a linker that covalently couples the cationic
center to the anionic center; X.sup.- is the counter ion associated
with the cationic center; and n is an integer from 1 to about
10,000.
14. The nanogel of claim 13 further comprising one or more
therapeutic agents.
15. The nanogel of claim 13 further comprising one or more
diagnostic agents.
16. The nanogel of claim 14 further comprising one or more
targeting agents.
17. The nanogel of claim 15 further comprising one or more
targeting agents.
18. The nanogel of claim 16 further comprising one or more
diagnostic agents.
19. A nanogel, comprising a zwitterionic polymer having the
formula:
PB-[L.sub.1-N.sup.+(R.sub.a)(R.sub.b)(R.sub.c)].sub.n[L.sub.2-A(.dbd.O)---
OM].sub.p(X.sup.-).sub.n wherein PB is a polymer backbone having n
pendant groups L.sub.1-N.sup.+(R.sub.a)(R.sub.b)(R.sub.c) and p
pendant groups L.sub.2-A(.dbd.O)--OM; N.sup.+ is a cationic center;
R.sub.a, R.sub.b, and R.sub.c are independently optional as
necessary to provide a cationic center and independently selected
from alkyl and aryl; A(.dbd.O)--OM is the anionic center, wherein A
is C, S, SO, P, or PO, and wherein M is a counterion; L.sub.1 is a
linker that covalently couples the cationic center to the polymer
backbone; L.sub.2 is a linker that covalently couples the anionic
center to the polymer backbone; X.sup.- is the counter ion
associated with the cationic center; n is an integer from 1 to
about 10,000; and p is an integer from 1 to about 10,000.
20. The nanogel of claim 19 further comprising one or more
therapeutic agents.
21. The nanogel of claim 19 further comprising one or more
diagnostic agents.
22. The nanogel of claim 20 further comprising one or more
targeting agents.
23. The nanogel of claim 21 further comprising one or more
targeting agents.
24. The nanogel of claim 22 further comprising one or more
diagnostic agents.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/259,081, filed Nov. 6, 2009, and is a
continuation-in-part of U.S. patent application Ser. No.
12/020,998, filed Jan. 28, 2008, which is a continuation of
PCT/US2006/0298988, filed Jul. 25, 2006, which claims the benefit
of U.S. Provisional Application No. 60/711,613, filed Aug. 25,
2005. Each application is expressly incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] Nanoparticle-based biotechnology is quickly heading to the
forefront of drug delivery, diagnosis and other areas. One of the
largest obstacles to these applications is nonspecific protein
adsorption, which can result in cellular uptake, nanoparticle
aggregation, immune system response and other disastrous problems
for in vivo applications. This lack of a versatile effective
nonfouling material is thus a crucial issue for many
nanoparticle-based biomedical applications. Poly(ethylene glycol)
(PEG) and oligo(ethylene glycol) (OEG) are the most commonly
studied nonfouling materials. However, PEG or OEG can auto-oxidize
rapidly in the presence of oxygen and transition metal ions.
Another class of nonfouling materials is based on phosphorylcholine
(PC), but these are harder to synthesize. In addition to fouling
resistance, many biomedical applications require a functionalizable
surface. This is necessary to immobilize a bio-recognition element
for targeting specific disease areas or selectively interacting
with cells or biomolecules. There are few reports about directly
functionalizing PEG surfaces. However, these involve complex
reactions.
[0004] Although performance of low fouling materials and coatings
has been demonstrated for relative expansive macroscopic surfaces,
surface chemistries are still challenging for nanoparticles used in
diagnostics and therapeutics, particularly in complex media such as
blood.
[0005] Therefore, a need exists for low fouling materials and
coatings for application to nanoparticles, particularly in complex
media. The present invention seeks to fulfill this need and
provides further related advantages.
SUMMARY OF THE INVENTION
[0006] In one aspect, the invention provides zwitterionic particles
in which the particle surface has zwitterionic polymers grafted
from the surface or zwitterionic polymers grafted to the surface.
Methods for making and using the zwitterionic particles are also
provided.
[0007] In one embodiment, the invention provides a particle
comprising a core and a surface having a plurality of zwitterionic
polymers grafted thereto or grafted therefrom. In one embodiment,
the particle has nanoscale dimensions. In one embodiment, the core
comprises a metal, a metal oxide, a ceramic, a synthetic polymer, a
natural polymer, silicon dioxide, a crystal, a semiconductor
material, a hydrogel, a liposome, a micelle, or a carbon-based
material. In one embodiment, the core comprises gold, silver, iron,
or platinum, cadmium sulfide, cadmium selenide, or combinations
thereof. In one embodiment, the core comprises titanium oxide, iron
oxide, zinc oxide, aluminum oxide, copper oxide, or tantalum oxide,
or combinations thereof. In one embodiment, the core comprises a
carbon fiber, a carbon nanotube, a carbon nanosheet, a carbon
nanobelt, a carbon nanorod, a carbon nanowire, and a carbon
nanodish. In one embodiment, the core comprises polyurethane,
polyethylene, polystyrene, poly(methyl methacrylate), or silicone.
In one embodiment, the core comprises calcium fluoride or quartz.
In one embodiment, the core is a quantum dot.
[0008] In one embodiment, the zwitterionic polymer has the
formula:
PB-(L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd.O)OM).sub.n(X.sup.--
).sub.n
[0009] wherein PB is a polymer backbone having n pendant groups
L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd.O)OM);
[0010] N.sup.+ is a cationic center;
[0011] R.sub.a and R.sub.b are independently optional as necessary
to provide a cationic center and independently selected from alkyl
and aryl;
[0012] A(.dbd.O)OM is the anionic center, wherein A is C, S, SO, P,
or PO, and wherein M is a counterion;
[0013] L.sub.1 is a linker that covalently couples the cationic
center to the polymer backbone;
[0014] L.sub.2 is a linker that covalently couples the cationic
center to the anionic center;
[0015] X.sup.- is the counter ion associated with the cationic
center; and
[0016] n is an integer from 1 to about 10,000.
[0017] In one embodiment, the zwitterionic polymer has the
formula:
PB-[L.sub.1-N.sup.+(R.sub.a)(R.sub.b)(R.sub.c)].sub.n[L.sub.2-A(.dbd.O)O-
M].sub.p(X.sup.-).sub.n
[0018] wherein PB is a polymer backbone having n pendant groups
L.sub.1-N.sup.+(R.sub.a)(R.sub.b)(R.sub.c) and p pendant groups
L.sub.2-A(.dbd.O)OM;
[0019] N.sup.+ is a cationic center;
[0020] R.sub.a, R.sub.b, and R.sub.c are independently optional as
necessary to provide a cationic center and independently selected
from alkyl and aryl;
[0021] A(.dbd.O)--OM is the anionic center, wherein A is C, S, SO,
P, or PO, and wherein M is a counterion;
[0022] L.sub.1 is a linker that covalently couples the cationic
center to the polymer backbone;
[0023] L.sub.2 is a linker that covalently couples the anionic
center to the polymer backbone;
[0024] X.sup.- is the counter ion associated with the cationic
center;
[0025] n is an integer from 1 to about 10,000; and
[0026] p is an integer from 1 to about 10,000.
[0027] In certain embodiments, the particle further comprises one
or more targeting agents.
[0028] In another aspect, the invention provides zwitterionic
nanogels. Methods for making and using the zwitterionic nanogels
are also provided.
[0029] In one embodiment, the invention provides a nanogel,
comprising a zwitterionic polymer having the formula:
PB-(L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd.O)OM).sub.n(X.sup.--
).sub.n
[0030] wherein PB is a polymer backbone having n pendant groups
L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd.O)OM) and
comprises crosslinks for those nanogels that are crosslinked;
[0031] N.sup.+ is a cationic center;
[0032] R.sub.a and R.sub.b are independently optional as necessary
to provide a cationic center and independently selected from alkyl
and aryl;
[0033] A(.dbd.O)OM is the anionic center, wherein A is C, S, SO, P,
or PO, and wherein M is a counterion;
[0034] L.sub.1 is a linker that covalently couples the cationic
center to the polymer backbone;
[0035] L.sub.2 is a linker that covalently couples the cationic
center to the anionic center;
[0036] X.sup.- is the counter ion associated with the cationic
center; and
[0037] n is an integer from 1 to about 10,000.
[0038] In another embodiment, the invention provides a nanogel,
comprising a zwitterionic polymer having the formula:
PB-[L.sub.1-N.sup.+(R.sub.a)(R.sub.b)(R.sub.c)].sub.n[L.sub.2-A(.dbd.O)O-
M].sub.p(X.sup.-).sub.n
[0039] wherein PB is a polymer backbone having n pendant groups
L.sub.1-N.sup.+(R.sub.a)(R.sub.b)(R.sub.c) and p pendant groups
L.sub.2-A(.dbd.O)--OM and comprises crosslinks for those nanogels
that are crosslinked;
[0040] N.sup.+ is a cationic center;
[0041] R.sub.a, R.sub.b, and R.sub.c are independently optional as
necessary to provide a cationic center and independently selected
from alkyl and aryl;
[0042] A(.dbd.O)OM is the anionic center, wherein A is C, S, SO, P,
or PO, and wherein M is a counterion;
[0043] L.sub.1 is a linker that covalently couples the cationic
center to the polymer backbone;
[0044] L.sub.2 is a linker that covalently couples the anionic
center to the polymer backbone;
[0045] X.sup.- is the counter ion associated with the cationic
center;
[0046] n is an integer from 1 to about 10,000; and
[0047] p is an integer from 1 to about 10,000.
[0048] In certain embodiments, the nanogels further comprises one
or more therapeutic agents and/or one or more diagnostic
agents.
[0049] In another aspect, the invention provides methods for
delivering a diagnostic agent and/or a therapeutic agent to a
subject. In the method, a nanogel of the invention comprising one
or more therapeutic agents and/or one or more diagnostic agents is
administered to the subject.
DESCRIPTION OF THE DRAWINGS
[0050] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0051] The foregoing aspects and many of the attendant advantages
of this invention will become more readily appreciated as the same
become better understood by reference to the following detailed
description, when taken in conjunction with the accompanying
drawings.
[0052] FIGS. 1A and 1B are tunneling electron microscopy (TEM)
images of silica nanoparticles before coating (1A) and
representative zwitterionic polymer coated silica particles of the
invention, polyCBAA-SiP1 (1B).
[0053] FIG. 2 compares hydrodynamic diameter change of uncoated
(bare) silica nanoparticles to representative zwitterionic polymer
coated silica particles of the invention, polyCBAA-SiP (135.5 and
221.3 nm) in BSA/PBS solution (10 mg/mL).
[0054] FIG. 3 compares the polydispersity indexes (PDI) of
representative zwitterionic polymer coated silica particles of the
invention, polyCBAA-SiP (135.5 and 221.3 nm) before and after
incubation in BSA/PBS solution (10 mg/mL).
[0055] FIG. 4 compares hydrodynamic diameter change of uncoated
(bare) silica nanoparticles to representative zwitterionic polymer
coated silica particles of the invention, polyCBAA-SiP (135.5 and
221.3 nm) in Lyz/PBS solution (10 mg/mL).
[0056] FIG. 5 compares the polydispersity indexes (PDI) of
representative zwitterionic polymer coated silica particles of the
invention, polyCBAA-SiP (135.5 and 221.3 nm) before and after
incubation in Lyz/PBS solution (10 mg/mL).
[0057] FIG. 6 is a schematic illustration of grafting a
representative zwitterionic polymer of the invention, polyCBAA,
onto a GNP surface covered with initiators via surface-initiated
ATRP,
[0058] FIGS. 7A-7C are illustrations of representative zwitterionic
polymer particles of the invention, polyCBAA-coated GNPs,
presenting an abundance of functional groups for ligand
immobilization in an ultra-low fouling background. PolyCBAA-coated
GNPs (pCBAA-GNPs) (7A); PCBAA-GNPs immobilized with antibodies
(7B); and PolyCBAA surfaces are highly resistant to nonspecific
protein adsorption after antibody immobilization (7C).
[0059] FIG. 8 is a TEM image of representative zwitterionic polymer
particles of the invention, polyCBAA coated GNPs (pCBAA-GNPs).
[0060] FIGS. 9A and 9B compare hydrodynamic size of GNPs coated
with different polymers along with bare GNPs in complex media: bare
GNPs and polymer-coated GNPs in 10% blood serum (PBS) (9A); and
bare GNPs and polymer-coated GNPs in 100% blood serum (9B). These
GNPs were separated from serum and re-suspended in buffer before
detection.
[0061] FIG. 10 compares the stability of gold nanoparticles in 10%
and 100% serum from UV-vis spectroscopy. Serum
induced-agglomeration was determined by measuring the red shift in
the absorbance of nanoparticles after 72 h incubation. The
absorbance was integrated from 600-750 nm.
[0062] FIG. 11 compares UV-vis spectra of GNPs coated with
different amounts of anti-ALCAM and their binding with different
levels of ALCAM measured from 400 to 800 nm. Black and red lines
represent pCBAA-GNPs functionalized with 2 and 25 .mu.gml.sup.-1
anti-ALCAM, respectively; solid, dash and dot lines represent
pCBAA-GNPs functionalized with anti-ALCAM in the presence of 0, 10
and 25 .mu.gml.sup.-1 ALCAM, respectively.
[0063] FIG. 12 illustrates the size distribution of representative
zwitterionic polymer particles of the invention, polyCBAA coated
GNPs (pCBAA-GNPs).
[0064] FIG. 13 is a schematic illustration of the preparation of a
useful initiator (DOPA.sub.2(TBDMS).sub.4-Br) and the preparation
of a useful zwitterionic material (DOPA.sub.2-pCBMA) for coating
particles to provide zwitterionic polymer coated particles of the
invention.
[0065] FIG. 14 is a schematic illustration of preparation of
representative zwitterionic polymer coated magnetic nanoparticles
of the invention, pCBMA-DOPA.sub.2-MNPs, and their magnetization in
the presence of a permanent magnet.
[0066] FIG. 15 is a TEM image of representative zwitterionic
polymer coated magnetic nanoparticles of the invention,
pCBMA-DOPA.sub.2-MNPs, scale bar=50 nm
[0067] FIG. 16 illustrates magnetic properties of representative
zwitterionic polymer coated magnetic nanoparticles of the
invention, pCBMA-DOPA.sub.2-MNPs: hysteresis loop of the MNPs
measured by a SQUID magnetometer (most MNPs were collected by the
magnetic about 1 min under a permanent magnet).
[0068] FIG. 17 illustrates R2 relaxivity of representative
zwitterionic polymer coated magnetic nanoparticles of the
invention, pCBMA-DOPA.sub.2-MNPs, as a function of Fe
concentration.
[0069] FIG. 18 compares stability of representative zwitterionic
polymer coated magnetic nanoparticles of the invention,
pCBMA-DOPA.sub.2-MNPs, in 10% NaCl and PBS solution by DLS
(n=3).
[0070] FIG. 19 compares stability of uncoated MNPs, dextran-coated
MNPs and representative zwitterionic polymer coated magnetic
nanoparticles of the invention, pCBMA-DOPA.sub.2-MNPs, in 100%
human blood serum, continuously measured by DLS at 37.degree. C.
(n=3).
[0071] FIG. 20 compares cytotoxicity of representative zwitterionic
polymer coated magnetic nanoparticles of the invention,
pCBMA-DOPA.sub.2-MNPs, to HeLa, macrophage, and HUVEC cells by MTT
assay (n=3).
[0072] FIG. 21 compares macrophage cell uptake of uncoated MNPs,
dextran-coated MNPs, and representative zwitterionic polymer coated
magnetic nanoparticles of the invention, pCBMA-DOPA.sub.2-MNPs, at
the Fe concentration of 10 .mu.g Fe/mL (n=3).
[0073] FIG. 22 compares HUVEC cell uptake of representative
zwitterionic polymer coated magnetic nanoparticles of the
invention, pCBMA-DOPA.sub.2-MNPs, with or without RGD peptide at
two different Fe concentrations (10 .mu.g Fe/mL and 20 .mu.g Fe/mL,
n=3). The insert figure shows T2-weighted MR images of cell samples
treated with pCBMA-DOPA.sub.2-MNPs with or without RGD peptide at
20 .mu.g Fe/mL.
[0074] FIG. 23 compares the stability of representative
zwitterionic nanogels of the invention, pCBMA nanogels with 3% MBAA
(dot line) and 1.5% MBAA (black solid line), in 100% fetal bovine
serum at 37.degree. C., as a function of time.
[0075] FIG. 24 illustrates in vitro FITC-dextran release from
representative zwitterionic nanogels of the invention, pCBMA
nanogels with 1.5% MBAA. FITC-dextran release was measured by a
fluorescence spectrophotometer. The results are averaged from three
replicates.
[0076] FIG. 25 compares cytotoxicity of representative zwitterionic
nanogels of the invention, pCBMA nanogels with 1.5, 3, and 5% MBAA,
as a function of concentration on HUVECs determined by MTT
assay.
[0077] FIGS. 26A and 26B compare flow cytometric analyses of the
uptake of representative zwitterionic nanogels of the invention,
pCBMA nanogels: (5% MBAA) red (no pCBMA nanogels) green (pCBMA
nanogels), and blue (pCBMA nanogels conjugated with RGD), at the
concentration of 0.2 mg/mL (26A) and 1 mg/mL (26B).
[0078] FIG. 27 is an illustration of the formation and degradation
of a representative zwitterionic nanogel of the invention, a
degradable pCBMA nanogel.
[0079] FIG. 28 is an SEM image of representative zwitterionic
nanogels of the invention degradable, pCBMA nanogels (scale bar=1
.mu.m).
[0080] FIG. 29 illustrates the stability of representative
zwitterionic nanogels of the invention in PBS.
[0081] FIG. 30 illustrates the results of a cytotoxicity test of
representative zwitterionic nanogels of the invention as a function
of Fe concentration on macrophage cells and HUVEC cells.
[0082] FIG. 31 illustrates the results of a macrophage uptake test
of representative zwitterionic nanogels of the invention as a
function of Fe concentration.
[0083] FIG. 32 illustrates the results of a degradation test (DLS)
of representative zwitterionic nanogels of the invention.
[0084] FIG. 33 illustrates the results of a degradation test (MRI)
of representative zwitterionic nanogels of the invention.
[0085] FIG. 34 compares the release of FITC-dextran from
representative zwitterionic nanogels of the invention with and
without 10 mM DTT at 37.degree. C.
[0086] FIG. 35 compares the HUVEC cell uptake of representative
zwitterionic nanogels of the invention, with or without RGD peptide
at two different Fe concentrations (5 .mu.g Fe/mL and 10 .mu.g
Fe/mL, n=3).
[0087] FIG. 36 compares hydrodynamic size as a function of time for
polymer coated gold nanoparticles of the invention in 100% human
blood serum.
[0088] FIG. 37 compares hydrodynamic size as a function of time for
polymer coated gold nanoparticles of the invention in 100% human
blood serum.
DETAILED DESCRIPTION OF THE INVENTION
[0089] The invention provides particles having low fouling
properties, methods for making the particles, and methods for using
the particles.
[0090] In one aspect, the invention provides zwitterionic particles
in which the particle surface has zwitterionic polymers grafted
from the surface or zwitterionic polymers grafted to the surface.
Methods for making and using the zwitterionic particles are also
provided.
[0091] In another aspect, the invention provides zwitterionic
nanogels. Methods for making and using the zwitterionic nanogels
are also provided.
Zwitterionic Particles
[0092] In one aspect, the present invention provides particles
having zwitterionic polymers on their surfaces that impart low
fouling properties to the particles. In one embodiment, the
zwitterionic polymers on the surface of the particles are grafted
from the particle surface by polymerization processes. In another
embodiment, the zwitterionic polymers on the surface of the
particles are grafted to the particle surface by associating a
suitably functionalized zwitterionic polymer with the particle
surface. In another embodiment, the zwitterionic polymers or
hydrogels are coated onto particle surfaces.
[0093] Particles
[0094] The nature of the particle, which is advantageously treated
by the methods of the invention to provide the particle having a
surface to which the zwitterionic polymers are associated, can be
widely varied in size as well as composition. In one embodiment,
the particle is a nanoparticle, which is a particle having
nanometer scale dimensions.
[0095] Representative particle surfaces that can be advantageously
treated with the polymers include metal and metal oxide surfaces,
ceramic surfaces, synthetic and natural polymeric surfaces, glass
surfaces, fiber glass surface, silicon/silica surfaces, and
carbon-based material surfaces. Suitable particle surfaces include
semiconductor particle surfaces (e.g., quantium dots, cadmium
sulfide and cadmium selenide), hydrogel surfaces, liposome
surfaces, and micelle surfaces. Representative natural polymeric
surfaces include collagen, fibrins, and other carbohydrate
surfaces. Representative particle carbon-based surfaces include
carbon fiber surfaces, carbon nanotube surfaces, bulky ball
surfaces, carbon nanosheet surfaces, carbon nanotube surfaces,
carbon nanowire surfaces, carbon nanorod surfaces, and carbon
nanodish surfaces.
[0096] Representative metals particle surfaces to which the
polymers can be attached include gold, silver, iron, and platinum
surfaces. Representative metal oxide particle surfaces to which the
polymers can be attached include titanium oxide, iron oxide, zinc
oxide, aluminum oxide, copper oxide, and tantalum oxide surfaces.
Representative silicon oxide particle surfaces to which the
polymers can be attached include glass surfaces, and silica wafers.
Representative organic particle surfaces to which the polymers can
be attached include organic surfaces such as organic polymer
surfaces including polyurethane, polyethylene, polystyrene,
poly(methyl methacrylate) and silicone. Particles having surfaces
comprising mixtures of the above can also be advantageously treated
with the zwitterionic polymers of the invention.
[0097] The polymers of the invention can be advantageously adhered
to fiber particle surfaces. Representative fibers and fibrous
materials to which the polymers can be adhered include nylon,
polyvinyl nitrile, and polyester.
[0098] The polymers of the invention can also be advantageously
adhered to crystalline particle surfaces. Representative
crystalline surfaces include calcium fluoride and quartz
surfaces.
[0099] Particles Having Zwitterionic Polymers Grafted Therefrom
[0100] In one aspect, the invention provides particle surfaces
having zwitterionic polymers grafted therefrom and methods for
grafting the polymers from surfaces (i.e., polymers grafted from a
surface). As used herein, the term, "grafted therefrom" or "grafted
from" refers to polymers that are prepared by polymerizing monomers
from polymerization initiators associated with the particle
surface. In certain embodiments, the polymers are grafted from
surfaces to which a polymerization initiator has been adhered
through an adhesive group.
[0101] In one embodiment, representative zwitterionic polymers
grafted from a particle surface have formula (I):
PB-(L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd.O)--OM).sub.n(X).su-
b.n (I)
wherein PB is the polymer backbone having n pendant groups
L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd.O)--OM); N.sup.+
is the cationic center; R.sub.a and R.sub.b are independently
optional as necessary to provide a cationic center and
independently selected from alkyl and aryl; A(.dbd.O)--OM is the
anionic center, where M is a counterion and A is C, S, SO, P, or
PO; L.sub.1 is a linker that covalently couples the cationic center
to the polymer backbone; L.sub.2 is a linker that covalently
couples the cationic center to the anionic center; X.sup.- is the
counter ion associated with the cationic center; and n is an
integer from 1 to about 10,000.
[0102] In another embodiment, representative zwitterionic polymers
grafted from a particle surface have formula (II):
PB-[L.sub.1-N.sup.+(R.sub.a)(R.sub.b)(R.sub.c)].sub.n[L.sub.2-A(.dbd.O)--
-O.sup.-M.sup.+)].sub.p(X.sup.-).sub.n (II)
[0103] wherein PB, L.sub.1, R.sub.a, R.sub.b, L.sub.2,
A(.dbd.O)O.sup.-, M.sup.+, X.sup.-, and n are as described above,
and R.sub.c,
[0104] is as for R.sub.a and R.sub.b, and p is an integer from 1 to
about 10,000.
[0105] In the above formulas, PB is the polymer backbone.
Representative polymer backbones include vinyl backbones (i.e.,
--C(R')(R'')--C(R''')(R'''')--, where R', R'', R''', and R''' are
independently selected from hydrogen, alkyl, and aryl) derived from
vinyl monomers (e.g., acrylate, methacrylate, acrylamide,
methacrylamide, styrene). In one embodiment, the polymer backbone
comprises --[CH.sub.2--C(R.sub.d)].sub.n--, wherein R.sub.d is
selected from the group consisting of hydrogen, fluorine,
trifluoromethyl, and C1-C6 alkyl, and n is from 1 to about
10,000.
[0106] For the polymers, the degree of polymerization (DP or n),
number average molecular weight (M.sub.n), and the ratio of weight
average and number average molecular weights (M.sub.w/M.sub.n.),
also known as polydispersity index, can vary. In one embodiment,
the polymers have a degree of polymerization (n) from 1 to about
10,000. In one embodiment, n is from about 10 to about 5,000. In
another embodiment, n is from about 100 to about 3,500. In one
embodiment, the polymers have a number average molecular weight
(M.sub.n) of from about 200 to about 200,000. In one embodiment,
M.sub.n is from about 2,000 to about 100,000. In another
embodiment, M.sub.n is from about 20,000 to about 80,000. In one
embodiment, the polymers of have a ratio of weight average and
number average molecular weight (M.sub.w/M.sub.n.) of from about
1.0 to about 2.0. In one embodiment, M.sub.w/M.sub.n. is from about
1.1 to about 1.5. In another embodiment, M.sub.w/M.sub.n. is from
about 1.2 to about 2.0.
[0107] In the above formulas, N.sup.+ is the cationic center. In
certain embodiments, the cationic center is a quaternary ammonium
(e.g., N bonded to L.sub.1; R.sub.a, R.sub.b, and L.sub.2). In
addition to ammonium, other useful cationic centers include
imidazolium, triazaolium, pyridinium, morpholinium, oxazolidinium,
pyrazinium, pyridazinium, pyrimidinium, piperazinium, and
pyrrolidinium. In these embodiments, R.sub.a and R.sub.b are absent
because the four valencies of the positively-charged nitrogen are
taken up by the ring structure of the cationic center and bonds to
L.sub.1 and L.sub.2. In another embodiment, the cationic center is
a phosphonium center.
[0108] When present, R.sub.a, R.sub.b, and/or R.sub.c are
independently selected from hydrogen, alkyl, and aryl groups.
Representative alkyl groups include C1-C5 straight chain and
branched alkyl groups. In certain embodiments, the alkyl group is
further substituted with one of more substituents including, for
example, an aryl group (e.g., --CH.sub.2C.sub.6H.sub.5, benzyl). In
one embodiment, R.sub.a and R.sub.b are methyl. Representative aryl
groups include C6-C12 aryl groups including, for example,
phenyl.
[0109] In the above formulas, L.sub.1 is a linker that covalently
couples the cationic center to the polymer backbone. In certain
embodiments, L.sub.1 includes a functional group (e.g., ester or
amide) that couples the remainder of L.sub.1 to the polymer
backbone. In addition to the functional group, L.sub.1 can include
an C1-C10 alkylene chain. Representative L.sub.1 groups include
--C(.dbd.O)O--(CH.sub.2).sub.n-- and
--C(.dbd.O)NH--(CH.sub.2).sub.n--, where n is 1-10 (e.g., 2 or 3).
In one embodiment, n is 2. In one embodiment, n is 3.
[0110] In the above formulas, L.sub.2 is a linker that covalently
couples the cationic center to the anionic center. L.sub.2 can be a
C1-C25 alkylene chain. Representative L.sub.2 groups include
--(CH.sub.2).sub.n--, where n is 1-5. In one embodiment, n is 2. In
one embodiment, n is 3.
[0111] In the above formulas, A(.dbd.O)--OM is the anionic center,
where A is C, S, SO, P, or PO. The anionic center is an acid. M is
counterion ion. Representative counterions include metals ions
(e.g., lithium sodium, potassium, calcium, magnesium),
nitrogen-containing ions (e.g., ammonium, imidazolium, triazolium,
pyridinium), and organic ions.
[0112] In the above formulas, X.sup.- is the counter ion associated
with the cationic center. The counter ion can be the counter ion
that results from the synthesis of the polymers (e.g., Cl.sup.-,
Br.sup.-, I.sup.-). The counter ion that is initially produced from
the synthesis of the cationic center can also be exchanged with
other suitable counter ions to provide polymers having controllable
hydrolysis properties and other biological properties.
Representative counter ions include halides; carboxylates, such as
benzoic acid and fatty acid anions (e.g.,
CH.sub.3(CH.sub.2).sub.nCO.sub.2.sup.- where n=1-19); alkyl
sulfonates (e.g., CH.sub.3 (CH.sub.2).sub.nSO.sub.3.sup.- where
n=1-19); salicylate; lactate; bis(trifluoromethylsulfonyl)amide
anion (N(SO.sub.2CF.sub.3).sub.2); and derivatives thereof. Other
counter ions also can be chosen from sulfate, nitrate, perchlorate
(ClO.sub.4), tetrafluoroborate (BF.sub.4), hexafluorophosphate
(PF.sub.6), trifluoromethylsulfonate (SO.sub.3 CF.sub.3),
bis(trifluoromethylsulfonyl)amide, lactate, salicylate, and
derivatives thereof.
[0113] In another aspect, the invention provides a method for
making a particle surface having zwitterionic polymers grafted
therefrom. In the method, a radical initiator terminated monolayer
is formed on a particle surface. The radical initiator comprises
one or more groups effective to adhere the initiator to the
surface. A zwitterionic monomer is then polymerized on the radical
initiator terminated monolayer to provide a surface having
zwitterionic polymers grafted therefrom.
[0114] In one embodiment, zwitterionic polymers are grafted from
self-assembly monolayers (SAMs) terminated with initiators through
atom transfer radical polymerization (ATRP) by polymerization of
suitable zwitterionic monomers. In the process, the particle
surface is coated with the SAMs terminated with radical initiator
followed by zwitterionic monomer polymerization onto the SAMs to
form a zwitterionic polymer coating on the particle surface. The
atom transfer radical polymerization is initiated by the radical
initiator at the terminus of the SAMs.
[0115] The radical terminated SAMs can be formed by a one-step or a
two-step method. In a one-step method, an initiator SAM is formed
by attaching radical initiator-terminated molecules to the particle
surface through interaction with the radical initiator's attaching
group. In a two-step method, a functional group-terminated SAM is
formed by attaching functional group-terminated molecule to the
surface through covalent or noncovalent bonding. The functional
group-terminated SAM is subsequently converted to the
initiator-terminated SAM by chemical reaction.
[0116] Suitable polymerization methods include atom transfer
radical polymerization (ATRP), reversible addition fragmentation
chain transfer (RAFT) polymerization, and free radical
polymerization. Any conventional radical initiators for
polymerization may be used to practice the invention. The
representative initiators for normal thermal or photochemical free
radical polymerization include benzoyl peroxide,
2,2'-azo-bis(2-methylproionitrile) and benzoin methyl ether.
Representative initiators for ATRP include alkyl halides, such as
bromoisobutyryl bromide (BIBB). Representative initiators for RAFT
polymerization (i.e., free radical initiators with chain reversible
agency (CTA)) include thiocarbonylthio compounds.
[0117] As noted above, in the grafted from method, the radical
initiator terminated monolayer formed on a particle surface
comprises a radical initiator that includes one or more groups
effective to adhere the initiator to the surface.
[0118] In one embodiment, the radical initiator comprises one or
more dihydroxyphenyl groups effective to adhere the initiator to
the particle surface. A zwitterionic monomer is then polymerized on
the radical initiator terminated monolayer to provide a surface
having zwitterionic polymers grafted therefrom.
[0119] As noted above, in the grafted from method, the radical
initiator terminated monolayer formed on a particle surface
comprises a radical initiator that includes one or more
dihydroxyphenyl groups effective to adhere the initiator to the
surface. In one embodiment, representative radical initiators of
the invention have formula (III):
(DHP).sub.m-L.sub.4-NH--C(.dbd.O)--C(CH.sub.3).sub.2--Br (III)
wherein L.sub.4 is a linker moiety that covalently couples the m
dihydroxyphenyl (DHP) groups to the amide nitrogen (m is an integer
from 1 to 20, for example, 1, 2, 3, or 4). Linker moiety L.sub.4
can include up to about 20 atoms.
[0120] In one embodiment, zwitterionic monomers useful in the
invention have formula (IV):
CH.sub.2.dbd.C(R.sub.d)-L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd-
.O)--OMX.sup.- (IV)
wherein L.sub.1, N.sup.+, R.sub.a, R.sub.b, A(.dbd.O)OM, and
L.sub.2, and X.sup.- are as described above for the zwitterionic
polymers, and R.sub.d is selected from the group consisting of
hydrogen, fluorine, trifluoromethyl, and C1-C6 alkyl.
[0121] Methods for making carboxybetaine and sulfobetaine polymers
and their uses described in WO 2007/024393, expressly incorporated
herein by reference in its entirety.
[0122] The preparation and characterization of representative
zwitterionic polymer coated particles, silica nanoparticles, having
the polymer grafted from the particle surface is described in
Example 1.
[0123] The preparation and characterization of representative
zwitterionic polymer coated particles, gold nanoparticles, are
described in Example 2.
[0124] Particle Surfaces Having Zwitterionic Polymers Grafted
Thereto
[0125] In another aspect, the invention provides particle surfaces
having zwitterionic polymers grafted thereto and methods for
grafting the polymers to particle surfaces (i.e., polymers grafted
to the surface). As used herein, the term "grafted thereto" or
"grafted to" refers to polymers that are first prepared and then
associated with a particle surface, which is in contrast to
polymers grafted from a particle surface.
[0126] Zwitterionic polymers suitable for grafting to particle
surfaces include one or more adhesive groups. In one embodiment,
the adhesive group is a dihydroxyphenyl group.
[0127] Representative zwitterionic polymers useful for grafting to
particle surfaces have formula (V):
(DHP).sub.m-L.sub.3-PB-(L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd-
.O)--OM).sub.n(X.sup.-).sub.n (V)
wherein DHP, L.sub.3, PB, L.sub.1, N.sup.+, R.sub.a, R.sub.b,
A(.dbd.O)OM, m, n, and X.sup.- are as described above.
[0128] In one embodiment, the dihydroxyphenyl group is a
3,4-dihydroxyphenyl group (i.e., a catechol group). In certain
embodiments, the polymer includes a 3,4-dihydroxyphenyl group
derived from 3,4-dihydroxyphenyl alanine (i.e., DOPA). In one
embodiment, m is 1 and, in another embodiment, m is 2. In formula
(I), L.sub.3 is a linker moiety that covalently couples the m
dihydroxyphenyl groups to the polymer backbone. The linker moiety
is a group of atoms that is effective to covalently couple the m
dihydroxyphenyl groups to the polymer backbone.
[0129] In another aspect, the invention provides methods for
treating particle surfaces with the polymers (i.e., polymers
grafted to a surface). In the methods, a particle surface is
treated with a polymer by applying the polymer to the surface or
contacting the surface with the polymer. In one embodiment,
applying the polymer to the surface comprises contacting a surface
with a solution comprising the polymer. In one embodiment, applying
the polymer to the surface comprises flowing a solution comprising
the polymer over the surface.
[0130] Conditions for effectively adhering the polymers depend on
the nature of the polymer and the particle surface to which the
polymer is to be adhered. In certain embodiments, effective
adhesion of the polymer to the surface involves presenting the
polymer's adhesive group to the surface. Presenting the adhesive
group to the surface can involve using a polymer solution or
composition that allows the polymer to assume a conformation that
reveals or exposes the adhesive group for binding to the surface
(e.g., extends the polymer away from the adhesive group).
[0131] Compositions for adhering a polymer to a surface include the
polymer and a solvent. Suitable solvents include aqueous solvents,
organic solvents, and combinations thereof. Representative aqueous
solvents include aqueous buffers such as MOPS, Tris, and PBS
buffers. Representative organic solvents include acetone,
acetonitrile, methanol, ethanol, isopropanol, n-propanol,
dimethylformamide, dimethylsulfoxide, tetrahydrofuran, and
trifluoroethanol. Representative combinations of aqueous and
organic solvents include organic solvents that are miscible in
water. Suitable water-miscible organic solvents include acetone,
acetonitrile, methanol, ethanol, isopropanol, n-propanol,
dimethylformamide, dimethylsulfoxide, tetrahydrofuran, and
trifluoroethanol. Suitable compositions include water and
water-miscible solvents combined in a ratio of from about 1:20 v/v
to about 20:1 v/v. In one embodiment, the surface is contacted with
a polymer in a MOPS buffer. In one embodiment, the surface is
contacted with a polymer in a Tris buffer. In one embodiment, the
surface is contacted with a polymer in aqueous tetrahydrofuran
(e.g., THF:water, 1:2).
[0132] The pH of the polymer composition can affect the
effectiveness of polymer adhesion to a surface. For carboxybetaine
polymers, the pH is from about 2 to about 10. For sulfobetaine
polymers, the pH is from about 1 to about 12.
[0133] The preparation and characterization of representative
zwitterionic polymer coated particles, magnetic iron oxide
nanoparticles, are described in Example 3.
[0134] Targeting Agents
[0135] Particles useful for therapeutic and diagnostic purposes can
be advantageously treated with the polymers of the invention. In
certain embodiments, the surface further comprises a plurality of
target binding partners covalently coupled to a portion of the
plurality of polymers adhered to the surface. In this embodiment,
the target binding partner has affinity toward a target molecule.
In these embodiments, the surfaces can be used in diagnostic
assays.
[0136] The binding affinity of a target molecule toward to the
surface results from the target binding partners immobilized on the
surface. The target binding partner and the target molecule, each
termed a binding pair member, form a binding pair. Each binding
pair member is a molecule that specifically binds the other member.
In one embodiment, the target binding partner has affinity to a
target molecule with K.sub.d less than about 10.sup.-8.
[0137] A binding pair member can be any suitable molecule
including, without limitation, proteins, peptides, proteins, poly-
or oligo-saccharides, glycoproteins, lipids and lipoproteins, and
nucleic acids, as well as synthetic organic or inorganic molecules
having a defined bioactivity, such as an antibiotic,
anti-inflammatory agent, or a cell adhesion mediator.
[0138] Examples of proteins that can be immobilized on the surfaces
of the present invention include ligand-binding proteins, lectins,
hormones, receptors, and enzymes. Representative proteins include
antibodies (monoclonal, polyclonal, chimeric, single-chain or other
recombinant forms), their protein/peptide antigens, protein-peptide
hormones, streptavidin, avidin, protein A, proteins G, growth
factors and their respective receptors, DNA-binding proteins, cell
membrane receptors, endosomal membrane receptors, nuclear membrane
receptors, neuron receptors, visual receptors, and muscle cell
receptors. Representative oligonucleotides that can be immobilized
on the surfaces of the present invention include DNA (genomic or
cDNA), RNA, antisense, ribozymes, and external guide sequences for
RNase P, and can range in size from short oligonucleotide primers
up to entire genes.
[0139] Other target binding partners that bind specifically to a
target compound include poly- or oligosaccharides on glycoproteins
that bind to receptors, for example, the carbohydrate on the ligand
for the inflammatory mediators P-selectin and E-selectin, and
nucleic acid sequences that bind to complementary sequences, such
as ribozymes, antisense, external guide sequences for RNase P, and
aptamers.
[0140] In one embodiment, the target binding partner is an
antibody, and the target molecule is an antigen against the
antibody. In this embodiment, the surface of the invention
specifically binds to the antigen and resists non-specific protein
adsorption. In one embodiment, the target binding partner is a
protein capable of promoting cell adhesion, and the target molecule
is a cell. In this embodiment, the surface of the invention
specifically binds to the cell and resists non-specific protein
adsorption and non-specific cell adhesion.
[0141] The use of carboxybetaine polymer surfaces for immobilizing
target binding partners is described in WO 2008/083390, expressly
incorporated herein by reference in its entirety.
[0142] The following is a description of representative
zwitterionic coated nanoparticles of the invention, their
preparation, characterization, and advantageous uses.
[0143] Zwitterionic Polymer Coated Silica Particles
[0144] Surface Modification of Silica Nanoparticles. There is a
difference expected in the size before and after surface
modification. The results determined by dynamic light scattering
(DLS), including the particle size and polydispersity index (PDI),
are listed in Table 1.
TABLE-US-00001 TABLE 1 Results of dynamic light scattering
measurements of silica nanoparticles. Sample d.sup.c (nm)
Polydispersity index.sup.d Bare SiP.sup.a 66 0.047 Initiator-coated
SiP.sup.a 74.1 0.053 PolyCBAA-SiP1.sup.b 135.5 0.118
PolyCBAA-SiP2.sup.b 221.3 0.172 .sup.aMeasured in ethanol.
.sup.bMeasured in water. .sup.cAverage hydrodynamic diameter.
.sup.dBased on the cumulant method.
[0145] As can be seen from Table 1, there is an increase in size by
8 nm (from 66 nm to 74.1 nm) after silane modification. This
indicates that the initiator was successfully anchored onto the
surfaces of the nano silica particles. The sizes of the
representative particles increase to around 135.5 (polyCBAA-SiP1)
and 221.3 nm (polyCBAA-SiP2) after polyCBAA polymerization for 12 h
and 24 h, respectively. This significant increase in the average
hydrodynamic diameter of the nanoparticles from DLS measurements
confirmed the presence of the polyCBAA shell on the nanoparticles.
Furthermore, the size distribution is quite narrow (all PDI is
lower than 0.2), indicating the high stability of the nanoparticles
during the experimental process.
[0146] FIG. 1 displays the TEM images of polyCBAA-SiP1 (Table 1)
and particles before polyCBAA coating. Compared with the pre-coated
nanoparticles (a), the boundaries among the coated particles become
unclear. This is due to the fused polymer shell under the
measurement condition of TEM (voltage 200 KV). This further
confirmed that polyCBAA polymers were incorporated on the surface
of the nanoparticles. In addition, due to the hydration layer in
solution, the sizes determined from TEM in FIG. 1 are slightly
smaller than those determined from DLS in Table 1.
[0147] Stability in protein solution. To evaluate the stability of
nanoparticles in protein solutions, DLS is used to track the size
change of the nanoparticles during their incubation in protein
solutions. Lysozyme and bovine serum albumin, representative of
positively and negatively charged proteins at neutral pH, were
chosen for protein binding tests. Stability of the polyCBAA coated
silica nanoparticles was tested in 10 mg/mL protein/PBS solution
and incubated at room temperature. After nanoparticles are added,
transparent protein solutions changed to light blue because of the
light scattering of the nanoparticles.
[0148] FIG. 2 shows the hydrodynamic diameters of the bare
nanoparticles and nanoparticles coated with polyCBAA in 10 mg/ml
BSA/PBS solution. All the particles show excellent stability
without obvious size increase during a 72 hours incubation period.
Moreover, the size distribution of the nanoparticles after
incubation is similar to that before incubation (see FIG. 3). This
indicates that polyCBAA indeed offers a robust coating around the
silica nanoparticles, protecting them against aggregation in
complex physiological conditions. It is well known that the bare
silica nanoparticles carry negative charge on their surface at
neutral pH. Thus, there are repulsive interactions between the
silica surface and the negative BSA. This is why the bare silica
nanoparticles are also stable for a long time in the BSA
solution.
[0149] The size change of the bare and coated nanoparticles in
Lyz/PBS solution was shown in FIG. 4. As can be seen from FIG. 4,
two representative polyCBAA coated nanoparticles show excellent
stability during the 72 hour incubation. The similar size
distribution before incubation and after incubation in FIG. 5
provides further evidence for the stability of the coated
nanoparticles in the Lyz/PBS solution. Due to the negative surface
and its attraction to positively charged Lyz, bare silica
nanoparticles formed white precipitate when exposed to the Lyz/PBS
solution. The excellent stability of polyCBAA coated nanoparticles
in both negative and positive protein solution shows that polyCBAA
layer is highly effective to protect silica nanoparticles from
nonspecific protein binding.
[0150] Functionalization. To test the functionalization of these
particles, preliminary experiments were carried out. After
activated in a fresh prepared solution of NHS (0.05M) and EDC (0.2
M) in MilliQ water (pH of the final NHS/EDC solution was about
5.5), the polyCBAA-SiP1 nanoparticles were re-dispersed in the
solution of anti-ALCAM with a concentration at 50 .mu.g/mL in 10 mM
sodium borate buffer (pH .about.8.5). DLS results showed that the
size of functionalized particles is 166 nm, as compared with bare
polyCBAA-SiP1 of 135.5 nm. A 30 nm increase in diameter was
observed after functionalization. This is equivalent to the size of
two antibodies in diameter, indicating successful antibody
immobilization. Therefore, the presence of the multifunctional
polyCBAA shell makes these particles to be easily
functionalized.
[0151] In summary, zwitterionic polyCBAA was used to prepare
biocompatible and functionalizable silica nanoparticles via silane
chemistry. The modified silica nanoparticles with two different
thicknesses of polyCBAA are stable at least 72 hours in both
negative and positive protein solutions, demonstrating the high
efficiency of the polyCBAA layers to improve the biointerfacial
properties of silica nanoparticles. Moreover, abundant functional
groups in polyCBAA make these coated particles to be easily
functionalized for future applications in targeted drug delivery
vehicle and diagnostics.
[0152] Zwitterionic Polymer Coated Gold Particles
[0153] Structure of CBAA monomer and pCBAA-GNPs. The structure of
CBAA monomer is shown in FIG. 6. PCBAA-GNPs, as illustrated in FIG.
7 were synthesized via ATRP method, transmission electron
microscope (TEM) image of small pCBAA-GNPs (as shown in FIG. 8)
showed the monodisperse nanoparticles without any aggregated
structures. The average diameter of GNPs cores was 18.5 nm. The
hydrodynamic diameter of pCBAA-GNPs conjugates measured by dynamic
light scattering (DLS) showed an average diameter of 58.4 nm,
indicating that the pCBAA coating thickness was around 20 nm.
[0154] Stability of bare polymer-coated GNPs in salt and common
protein solutions. The stability of bare GNPs, PEG-GNPs, OEGMA-GNPs
and pCBAA-GNPs with two different sizes were first evaluated in 1
mgml.sup.-1 lysozyme (14 kD, pI=12) solution and 20% NaCl solution.
UV-vis spectroscopy was applied to examine nonspecific protein
adsorption onto the surface of these nanoparticles or salt effect
on their stability. Aggregation of colloid particles and/or protein
adsorption on their surface will result in a shift in the surface
plasmon absorption. Results showed (see Table 2), after mixing with
lysozyme or NaCl solution, the plasmon resonance peak of bare GNPs
had a dramatically red shift. Only a slightly shift was observed
for PEG-GNPs and OEGMA-GNPs, whereas the peak was the same for
pCBAA-GNPs of two different sizes. This indicates that pCBAA-GNPs
were intact. Dynamic light scattering (DLS) was also applied to
test their stability. The hydrodynamic size of PEG-GNPs and
OEGMA-GNPs in water were 53.4 nm and 74.3 nm, respectively. But,
after the addition of lysozyme or NaCl solution, nanoparticles
showed an increase of about 20 nm in size compared to those in
water. In the case of pCBAA-GNPs, their diameters did not change,
indicating the high in vitro stability of pCBAA-GNPs in high ionic
strength or in the presence of proteins under physiological
conditions.
TABLE-US-00002 TABLE 2 Plasmon resonance peak and hydrodynamic size
of bare and polymer-coated GNPs after they are mixed with water,
20% NaCl and 1 mg ml.sup.-1 lysozyme solutions. Plasmon resonance
peak (nm) Hydrodynamic size (nm) 1 mg ml.sup.-1 1 mg ml.sup.-1
H.sub.2O lysozyme 20% NaCl H.sub.2O lysozyme 20% NaCl Bare GNPs
523.2 .+-. 0.3 580.8 .+-. 0.2 697.4 .+-. 0.1 18.5 .+-. 1.4 160.2
.+-. 1.1 387.7 .+-. 0.5 PEG-GNPs 527.7 .+-. 0.1 530.0 .+-. 0.1
530.2 .+-. 0.2 53.4 .+-. 1.3 85.0 .+-. 0.4 77.3 .+-. 3.0 OEGMA-GNPs
534.1 .+-. 0.1 534.9 .+-. 0.4 538.3 .+-. 0.2 74.3 .+-. 1.4 93.1
.+-. 1.1 91.2 .+-. 3.6 Small pCBAA-GNPs 532.2 .+-. 0.1 532.2 .+-.
0.1 532.2 .+-. 0.1 58.4 .+-. 1.7 58.9 .+-. 1.4 57.2 .+-. 0.4 Large
pCBAA-GNPs 568.0 .+-. 0.1 568.0 .+-. 0.1 568.0 .+-. 0.1 105.9 .+-.
3.0 105.5 .+-. 0.4 105.8 .+-. 3.4
[0155] Stability of and bare polymer-coated GNPs in 10% and 100%
serum solution. The stability of bare GNPs and polymer-coated GNPs
was further evaluated in PBS plus 10% human blood serum. FIG. 9A
shows the hydrodynamic diameters of bare GNPs and GNPs coated with
different polymers in PBS plus 10% human blood serum. Previous
studies have shown that the addition of serum increases the
stability of the unmodified particles due to nonspecific protein
adsorption. Referring to FIG. 9A, after 1 h, the bare GNPs showed
an increase of about 60 nm in size. This value increased to 80 nm
after 72 h, which was attributed to the interactions of
nanoparticles with proteins in the incubation serum medium.
However, with polymers coatings, there is no agglomeration and all
four samples showed good stability without obvious size increase
during the test period of 72 h.
[0156] Protein adsorption onto GNPs coated with different polymers
in undiluted (100%) human blood serum was further studied. 100%
serum is far more challenging than 10% serum. Due to high protein
concentrations, these nanoparticles were separated from human blood
serum proteins by centrifugation and re-dispersed in PBS buffer.
The average diameter of the nanoparticles was then evaluated by
DLS. As shown in FIG. 9B, bare GNPs showed a size increase of about
80 nm in a very short period of time. At the end of 72 h, the
diameter increased to about 380 nm, indicating significant protein
adsorption and particulate aggregation. PEG-GNPs and OEGMA-GNPs
were not stable in such extreme situation either. Their diameter
increments were 300 nm and 210 nm, respectively, after an
incubation period of 72 h. Precipitates could be observed in the
above solutions. However, with the protection of polyCBAA coating,
the interactions between proteins and nanoparticles did not cause
any agglomeration and the particle sizes after their separation
from human blood serum proteins was almost the same as those
without serum (58.4 and 105.9 nm), again indicating their excellent
stability.
[0157] The stability in 10% and 100% blood serum was also evaluated
by UV-vis spectroscopy. Aggregation of gold nanoparticles results
in a red-shifted absorbance profile. Therefore, aggregation was
quantified by integrating particle absorbance from 600 nm to 750 nm
(FIG. 10). After their incubation in 100% serum for 72 h, the
integrated absorbance values of bare GNPs, PEG-GNPs and OEGMA-GNPs
presented notable increases. The values in 100% serum are higher
than that in 10% serum, which is consistent with the DLS data.
Similar to DLS results in FIG. 9, pCBAA-GNPs were stable in 10%
serum and 100% serum with integrated absorbance<2. Similar
phenomena were observed previously on flat sensor surfaces. For
example, despite the excellent nonfouling capabilities of short OEG
self-assembled monolayers (SAMs) in single protein solutions and
10% human blood serum, they failed when exposed to complex media
such as 100% human blood serum. Thus, 10% serum commonly used to
evaluate the stability of nanoparticles is not sufficient and
undiluted blood serum is recommended to screen nanoparticles before
their in vivo experiments.
[0158] Functionalization of pCBAA-GNPs. Besides the enhanced
stability of GNPs in different environments, the polyCBAA coating
also provided abundant functional groups for ligand immobilization.
To demonstrate that antibody is immobilized onto GNPs and its
immobilized density can be adjusted, antibody-coated pCBAA-GNPs
with two immobilized antibody densities were probed by the targeted
antigen. A candidate cancer biomarker (activated leukocyte cell
adhesion molecule, ALCAM or CD 166) was applied as a model
antibody. Polyclonal anti-ALCAM and ALCAM were employed because
each polyclonal anti-ALCAM contains multiple ALCAM binding sites,
by adding ALCAM, it is expected that anti-ALCAM-modified pCBAA-GNPs
can aggregate, causing a red shift in the absorbance spectrum of
the GNPs. As shown in FIG. 11, the degree of antigen-induced
aggregation of nanoparticles increased with the concentration of
antigen at a given antibody concentration, demonstrating that the
ligand density on the surface of GNPs can be controlled. With the
same antigen concentration, the degree of aggregation of
nanoparticles increased with the antibody concentration. Therefore,
antibody/antigen ligand density on GNPs can be easily controlled by
the antibody/antigen concentration. Unreacted activated sites of
polyCBAA can be converted back to nonfouling carboxylate anions
groups via hydrolysis, ensuring the ultra-low fouling properties of
post-functionalized surfaces in undiluted blood plasma and serum
(as shown in FIG. 6C).
[0159] In summary, a functionalizable and stable surface platform
for nanoparticles has been demonstrated. Results show that polyCBAA
coated GNPs have superior performance in undiluted blood serum over
GNPs with other conventional coatings including PEG, although their
performance in 10% blood serum is comparable. This indicates that
10% serum commonly used to evaluate the stability of nanoparticles
is not sufficient. Undiluted blood serum is recommended to screen
nanoparticles before in vivo experiments. This new criterion will
allow one to screen NPs effectively before in vivo experiments and
save unnecessary in vivo experiments. Furthermore, bio-recognition
elements such as anti-ALCAM can be easily conjugated to polyCBAA
via NHS/EDC method. There are many more functional groups available
for ligand immobilization onto polyCBAA. Ligand immobilization
density can be varied by adjusting antibody/antigen concentrations.
The uniqueness of polyCBAA (i.e., ultra low fouling and multiple
functionalities) makes this zwitterionic biopolymer useful for
nanoparticle coatings for in vivo targeting drug delivery and
diagnostics.
[0160] Zwitterionic Polymer Coated Iron Oxide (Magnetic)
Particles
[0161] Magnetic nanoparticles (MNPs) have many attractive
properties, often combining low toxicity with excellent magnetic
properties. Recently, "theranostics", which incorporate both
therapy and diagnosis, are attracting significant attention and may
revolutionize current medical treatments. To achieve this goal,
MNPs can work as multifunctional carriers to selectively accumulate
at the target site, cure disease by certain mechanisms (either
hyperthermia or drug release) and be detected using non-invasive
diagnosis modality such as magnetic resonance imaging (MRI).
Multifunctional MNPs can typically be formed from magnetic cores
and surface coating. Magnetic cores are iron oxide nanoparticles
which are detectable by MRI and can be manipulated by a magnetic
field, while an ideal surface coating can carry a therapeutic
reagent, prevent MNPs from being cleared from the blood
circulation, and provide functional groups for conjugation of
targeting ligands. Thus, the surface coating plays a key role in
achieving multifunctional MNPs.
[0162] As described above, surfaces coated with zwitterionic
polymers can be prepared via atom transfer radical polymerization
(ATRP) to achieved surface coatings with excellent ultra low
fouling properties. This process provides surfaces in which the
polymer is grafted from the surface (referred to as the
"graft-from-surface" method). However, ATRP reactions require
surface-grafted initiators, catalysts, and oxygen-free conditions
which limit its practical application. The present invention
provides an alternative method, in which the polymer is grafted to
the surface (referred to as the "graft-to-surface" method). In this
method, polymers carrying adhesive moieties with strong surface
affinity are synthesized and then grafted onto the surface through
their adhesive moieties.
[0163] The invention provides a convenient method to coat particles
(e.g., MNPs) with the dual-functional pCBMA polymer via two DOPA
groups. Results show that representative zwitterionic polymer
coated particles of the invention, pCBMA-MNPs, presented high
saturation magnetization and long-term stability in bio-relevant
media such as 100% human blood serum. Moreover, pCBMA-MNPs can be
easily conjugated to a RGD peptide for their enhanced ability to
enter targeted cells.
[0164] Preparation and Physical Properties of
pCBMA-DOPA.sub.2-MNPs. Co-precipitation and thermal decomposition
are the two major categories of methods to prepare MNPs. In
previous studies, DOPA-conjugated molecules were normally coated
onto MNPs prepared by thermal decomposition. In the present
invention, pCBMA-DOPA.sub.2 was attached onto MNPs prepared by
co-precipitation. The composition of the magnetic core and the
formation of pCBMA-DOPA.sub.2-MNPs are illustrated in FIG. 14. The
TEM image (FIG. 15) confirms the structure of
pCBMA-DOPA.sub.2-MNPs. Each magnetic core is formed by a number of
Fe.sub.3O.sub.4 nanocrystals with a single crystal size of about 15
nm. The hydrodynamic size of the magnetic cores is about 70 nm, as
measured by DLS. With a DOPA.sub.2-pCBMA coating, the hydrodynamic
size of the nanoparticles increased to about 130 nm. Multi-crystal
cores are preferred for magnetic targeting, since it has been
reported that without inter-particle aggregation, the small
Fe.sub.3O.sub.4 nanocrystals have very poor mobility under a normal
magnetic gradient. Negligible hysteresis in the magnetization curve
in FIG. 16 reveals that at room temperature pCBMA-DOPA.sub.2-MNPs
possess superparamagnetic property, indicating that the
nanoparticles present no coercivity (Hc) or remnant magnetization
(Mr) in the absence of an external magnetic field. The SQUID
magnetometer test also proves that our product has a saturation
magnetization (Ms) of 110.2 emu/g Fe, which is 1.6 times higher
than that of the commercial product, Feridex.RTM.. Ms is mainly
determined by the Fe.sub.3O.sub.4 nanocrystal size, and the strong
Ms of the particle of the invention is due to the size of
nanocrystals (about 15 nm) is much larger than that of Feridex.RTM.
(about 4.8 nm), but still in the range of superparamagnetic size
(<25 nm). The multi-crystal cores with larger crystal sizes
render pCBMA-DOPA.sub.2-MNPs highly responsive to a magnetic field.
Most nanoparticles were attracted to the permanent magnet side
about 1 min.
[0165] To ascertain the ability of pCBMA-DOPA.sub.2-MNPs to enhance
magnetic resonance imaging, the R.sub.2 transverse relaxivity was
measured by a clinical 3T MRI instrument. The quantitative results
in FIG. 17 show that the R.sub.2 relaxivity is 428 mM.sup.-1
s.sup.-1, which is about two times higher than that of Feridex.RTM.
at a 3T magnetic field. The high relaxivity is also due to the
strong Ms of pCBMA-DOPA.sub.2-MNPs. These results reveal the
ability of pCBMA-DOPA.sub.2-MNPs to be used as a T.sub.2-weighted
MR contrast agent.
[0166] Stability Studies. Uncoated MNPs can achieve long-term
stability in DI water due to their surface charge. However, when
mixed in solutions of higher ionic strengths such as PBS or 10%
NaCl solution, they aggregate immediately and their hydrodynamic
size increases to several thousand nanometers because their surface
electronic double layer was significantly compressed by the ionic
environment. With the DOPA.sub.2-pCBMA coating, MNPs are stable in
PBS or high ionic strength solutions such as 10% NaCl for at least
6 months without any size change monitored by DLS, as shown in FIG.
18. This result also verifies the stable formation of the
pCBMA-coated MNPs.
[0167] To evaluate the stability of pCBMA-DOPA.sub.2-MNPs in blood,
the particles were suspended in 100% human blood serum at
37.degree. C. Dextran-coated MNPs and uncoated MNPs were used as
controls. Results are shown in FIG. 19. The size of uncoated MNPs
increased to about 250 nm as soon as they entered the simulated
blood environment. However, their size did not continue increasing
to several thousand nanometers as they did in PBS and 10% NaCl
solutions. This phenomenon is likely due to the formation of
relatively stable particles coated with serum proteins with a size
of about 250 nm. Dextran-coated MNPs showed notable size increase
soon after they entered the 100% human blood serum. The size
increase could be due to proteins adsorption from blood serum
because the anti-fouling ability of dextran is limited. In
contrast, no obvious size change could be observed for the
pCBMA-DOPA.sub.2-MNPs sample over the entire time-course of the
test, indicating the excellent stability and ultra-lowfouling
ability of the nanoparticles.
[0168] In Vitro Studies. Resistance to macrophage cell uptake is
also important to evaluate nanoparticles in vitro, because it can
indicate the in vivo response of the innate immune system to the
nanoparticles. Before cell uptake studies, the cytotoxicity of
pCBMA-DOPA.sub.2-MNPs was evaluated by an MTT assay and results are
as shown in FIG. 20. No significant cell viability decrease can be
observed at the tested concentration range. Mouse macrophage cell
line, RAW 264.7 cell, was used in this work. As shown in FIG. 21,
uptake of pCBMA-DOPA.sub.2-MNPs by macrophage cell is much lower
than that of dextran-coated MNPs and uncoated MNPs. This test
further shows the advantage of the pCBMA coating.
pCBMA-DOPA.sub.2-MNPs achieve a longer circulation half-life time
than dextran coated MNPs.
[0169] Another important issue in the development of
multifunctional MNPs is that the nanoparticles should be
functionalizable. The abundant carboxyl groups in pCBMA can be
efficiently and easily conjugated to biomolecules by conventional
EDC/NHS chemistry. Furthermore, activated but unreacted NHS groups
will be hydrolyzed back to carboxyl groups as a part of non-fouling
zwitterionic groups, ensuring that the excellent nonfouling
properties of the coating is maintained in post-functionalized
surfaces. A RGD peptide, Cyclo[Arg-Gly-Asp-.sub.D-Tyr-Lys], was
used as a targeting ligand and conjugated to pCBMA-DOPA.sub.2-MNPs.
HUVEC cells were used to test the targeting efficiency of the MNPs
by means of measuring intracellular iron concentrations. As shown
in FIG. 22, at both Fe concentrations (10 .mu.g Fe/mL and 20 .mu.g
Fe/mL) tested, non-functionalized pCBMA-DOPA.sub.2-MNPs have a very
low uptake level, similar to macrophage cell studies. In contrast,
RGD-pCBMA-DOPA.sub.2-MNPs show much higher uptake levels.
T.sub.2-weighted MR images visually confirmed the uptake of MNPs,
as shown by the insert figure in FIG. 22, the cell sample treated
with functionalized MNPs at 20 .mu.g Fe/mL shows much higher
contrast compared with the nonfunctionlized one. These results
demonstrate the successful conjugation of RGD with the
nanoparticles and the notable active targeting efficacy of
pCBMA-DOPA.sub.2-MNPs after loaded with a targeting ligand.
[0170] In summary, the invention provides a convenient method to
efficiently coat MNPs with the zwitterionic pCBMA with adhesive
3,4-dihydroxyphenyl-L-alanine linkages. The superior stability of
pCBMA-coated MNPs in ionic solutions and undiluted human blood
serum, along with their ultra-low macrophage cell uptake suggest
that pCBMA-DOPA.sub.2-MNPs could achieve long blood circulation
half-life in vivo. In addition, these nanoparticles possess high
mobility in the presence of an external magnetic field due to their
multi-crystal cores. Importantly, the pCBMA coating can be easily
functionalized by target ligands via simple NHS/EDC chemistry.
These features enable them to take advantages of both passive
targeting (by a magnetic field) and active targeting (by targeting
ligands). Thus, multifunctional pCBMA-DOPA.sub.2-MNPs hold great
promise as a MRI detectable, high efficient targeting delivery
carrier.
Zwitterionic Nanogels
[0171] Hydrogels have been broadly used as implantable tissue
scaffolds, surface coatings for implantable sensors, wound
dressings, and drug delivery vectors due to their high water
content, biocompatibility, and low cytotoxicity. Recently, there is
an increased interest in developing hydrogel particles at the
nanometer scale (i.e., nanogels) as drug delivery carriers due to
their high drug-loading capacity, excellent biocompatibility, and
responsiveness to environmental factors such as temperature and
pH.
[0172] One of the major challenges of current nanoparticle drug
delivery carriers is limited blood circulation time after
intravenous (IV) systemic administration and quick uptake by the
liver and spleen due to nonspecific protein adsorption onto the
particles from blood. The stability of nanoparticles in blood is
critical to the success of drug delivery or nanoparticle-based
diagnostics. In order to achieve a prolonged blood circulation
time, nanoparticles are modified with neutral and hydrophilic
materials to reduce nonspecific protein adsorption from blood.
Although many materials have been developed to resist non-specific
protein adsorption, very few materials can achieve ultra-low
fouling level, which is defined as less than 5 ng/cm.sup.2 adsorbed
fibrinogen.
[0173] In another aspect, the invention provides zwitterionic
nanogels. The nanogels are useful as delivery vehicles for
therapeutic and diagnostic agents.
[0174] In one embodiment, the zwitterionic nanogels are crosslinked
nanogels prepared by copolymerization of zwitterionic monomers and
a polymerizable crosslinking agent. The crosslinking agent can be a
conventional crosslinking agent, a zwitterionic crosslinking agent,
or a degradable conventional or zwitterionic crosslinking
agent.
[0175] In one embodiment, representative zwitterionic nanogels of
the invention have formula (VI):
PB-(L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(.dbd.O)--OM).sub.n(X.sup-
.-).sub.n (VI)
and, in another embodiment, representative zwitterionic nanogels of
the invention have formula (VII):
PB-[L.sub.1-N.sup.+(R.sub.a)(R.sub.b)(R.sub.c)].sub.n
[L.sub.2-A(.dbd.O)--O.sup.-M.sup.+)].sub.p(X.sup.-).sub.n (VII)
wherein PB, L.sub.1, R.sub.a, R.sub.b, R.sub.c, L.sub.2,
A(.dbd.O)O.sup.-, M.sup.+, X.sup.-, n, and p are as described
above. For the nanogels of the invention, PB is the polymer
backbone and includes crosslinks for those hydrogels that are
crosslinked.
[0176] The nanogels of the invention are effective in delivering
cargo. In certain embodiments, the nanogel includes one or more
therapeutic agents. In certain embodiments, the nanogel includes
one or more diagnostic agents. In certain embodiments, the nanogel
includes one or more therapeutic agents and one or more diagnostic
agents.
[0177] Therapeutic Agents. Representative therapeutic agents that
can be incorporated into and advantageously delivered by the
nanogels of the invention include small molecules, nucleic acids,
proteins (including multimeric proteins, protein complexes,
peptides), lipids, carbohydrates, metals, radioactive elements,
and/or combinations thereof.
[0178] In some embodiments, the therapeutic agent is a small
molecule and/or organic compound with pharmaceutical activity. In
some embodiments, the therapeutic agent is a clinically-used drug.
In some embodiments, the drug is an anti-cancer agent, antibiotic,
anti-viral agent, anti-HIV agent, anti-parasite agent,
anti-protozoal agent, anesthetic, anticoagulant, inhibitor of an
enzyme, steroidal agent, steroidal or non-steroidal
anti-inflammatory agent, antihistamine, immunosuppressant agent,
anti-neoplastic agent, antigen, vaccine, antibody, decongestant,
sedative, opioid, analgesic, anti-pyretic, birth control agent,
hormone, prostaglandin, progestational agent, anti-glaucoma agent,
ophthalmic agent, anti-cholinergic, analgesic, anti-depressant,
anti-psychotic, neurotoxin, hypnotic, tranquilizer,
anti-convulsant, muscle relaxant, anti-Parkinson agent,
anti-spasmodic, muscle contractant, channel blocker, miotic agent,
anti-secretory agent, anti-thrombotic agent, anticoagulant,
anti-cholinergic, .beta.-adrenergic blocking agent, diuretic,
cardiovascular active agent, vasoactive agent, vasodilating agent,
anti-hypertensive agent, angiogenic agent, modulators of
cell-extracellular matrix interactions (e.g. cell growth inhibitors
and anti-adhesion molecules), inhibitor of DNA, RNA, or protein
synthesis.
[0179] In certain embodiments, a small molecule agent can be any
drug. In some embodiments, the drug is one that has already been
deemed safe and effective for use in humans or animals by the
appropriate governmental agency or regulatory body. For example,
drugs approved for human use are listed by the FDA under 21 C.F.R.
.sctn..sctn.330.5, 331 through 361, and 440 through 460,
incorporated herein by reference; drugs for veterinary use are
listed by the FDA under 21 C.F.R. .sctn..sctn.500 through 589,
incorporated herein by reference. All listed drugs are considered
acceptable for use in accordance with the present invention.
[0180] A more complete listing of classes and specific drugs
suitable for use in the present invention may be found in
Pharmaceutical Drugs: Syntheses, Patents, Applications by Axel
Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999 and the
Merck Index: An Encyclopedia of Chemicals, Drugs and Biologicals,
Ed. by Budavari et al, CRC Press, 1996, both of which are
incorporated herein by reference.
[0181] In certain embodiments of the invention, the therapeutic
agent is a nucleic acid (e.g., DNA, RNA, derivatives thereof). In
some embodiments, the nucleic acid agent is a functional RNA. In
general, a "functional RNA" is an RNA that does not code for a
protein but instead belongs to a class of RNA molecules whose
members characteristically possess one or more different functions
or activities within a cell. It will be appreciated that the
relative activities of functional RNA molecules having different
sequences may differ and may depend at least in part on the
particular cell type in which the RNA is present. Thus the term
"functional RNA" is used herein to refer to a class of RNA molecule
and is not intended to imply that all members of the class will in
fact display the activity characteristic of that class under any
particular set of conditions. In some embodiments, functional RNAs
include RNAi-inducing entities (e.g., short interfering RNAs
(siRNAs), short hairpin RNAs (shRNAs), and microRNAs), ribozymes,
tRNAs, rRNAs, RNAs useful for triple helix formation.
[0182] In some embodiments, the nucleic acid agent is a vector. As
used herein, the term "vector" refers to a nucleic acid molecule
(typically, but not necessarily, a DNA molecule) which can
transport another nucleic acid to which it has been linked. A
vector can achieve extra-chromosomal replication and/or expression
of nucleic acids to which they are linked in a host cell. In some
embodiments, a vector can achieve integration into the genome of
the host cell.
[0183] In some embodiments, vectors are used to direct protein
and/or RNA expression. In some embodiments, the protein and/or RNA
to be expressed is not normally expressed by the cell. In some
embodiments, the protein and/or RNA to be expressed is normally
expressed by the cell, but at lower levels than it is expressed
when the vector has not been delivered to the cell. In some
embodiments, a vector directs expression of any of the functional
RNAs described herein, such as RNAi-inducing entities,
ribozymes.
[0184] In some embodiments, the therapeutic agent may be a protein
or peptide. The terms "protein," "polypeptide," and "peptide" can
be used interchangeably. In certain embodiments, peptides range
from about 5 to about 5000, 5 to about 1000, about 5 to about 750,
about 5 to about 500, about 5 to about 250, about 5 to about 100,
about 5 to about 75, about 5 to about 50, about 5 to about 40,
about 5 to about 30, about 5 to about 25, about 5 to about 20,
about 5 to about 15, or about 5 to about 10 amino acids in
size.
[0185] Polypeptides may contain L-amino acids, D-amino acids, or
both and may contain any of a variety of amino acid modifications
or analogs known in the art. Useful modifications include, e.g.,
terminal acetylation, amidation. In some embodiments, polypeptides
may comprise natural amino acids, unnatural amino acids, synthetic
amino acids, and combinations thereof, as described herein.
[0186] In some embodiments, the therapeutic agent may be a hormone,
erythropoietin, insulin, cytokine, antigen for vaccination, growth
factor. In some embodiments, the therapeutic agent may be an
antibody and/or characteristic portion thereof. In some
embodiments, antibodies may include, but are not limited to,
polyclonal, monoclonal, chimeric (i.e., "humanized"), or single
chain (recombinant) antibodies. In some embodiments, antibodies may
have reduced effector functions and/or bispecific molecules. In
some embodiments, antibodies may include Fab fragments and/or
fragments produced by a Fab expression library (e.g. Fab, Fab',
F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments).
[0187] In some embodiments, the therapeutic agent is a
carbohydrate. In certain embodiments, the carbohydrate is a
carbohydrate that is associated with a protein (e.g. glycoprotein,
proteoglycan). A carbohydrate may be natural or synthetic. A
carbohydrate may also be a derivatized natural carbohydrate. In
certain embodiments, a carbohydrate may be a simple or complex
sugar. In certain embodiments, a carbohydrate is a monosaccharide,
including but not limited to glucose, fructose, galactose, and
ribose. In certain embodiments, a carbohydrate is a disaccharide,
including but not limited to lactose, sucrose, maltose, trehalose,
and cellobiose. In certain embodiments, a carbohydrate is a
polysaccharide, including but not limited to cellulose,
microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC),
methylcellulose (MC), dextrose, dextran, glycogen, xanthan gum,
gellan gum, starch, and pullulan. In certain embodiments, a
carbohydrate is a sugar alcohol, including but not limited to
mannitol, sorbitol, xylitol, erythritol, malitol, and lactitol.
[0188] In some embodiments, the therapeutic agent is a lipid. In
certain embodiments, the lipid is a lipid that is associated with a
protein (e.g., lipoprotein). Exemplary lipids that may be used in
accordance with the present invention include, but are not limited
to, oils, fatty acids, saturated fatty acid, unsaturated fatty
acids, essential fatty acids, cis fatty acids, trans fatty acids,
glycerides, monoglycerides, diglycerides, triglycerides, hormones,
steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin
E), phospholipids, sphingolipids, and lipoproteins.
[0189] In some embodiments, the lipid may comprise one or more
fatty acid groups or salts thereof. In some embodiments, the fatty
acid group may comprise digestible, long chain (e.g., C8-C50),
substituted or unsubstituted hydrocarbons. In some embodiments, the
fatty acid group may be one or more of butyric, caproic, caprylic,
capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or
lignoceric acid. In some embodiments, the fatty acid group may be
one or more of palmitoleic, oleic, vaccenic, linoleic,
alpha-linolenic, gamma-linoleic, arachidonic, gadoleic,
arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid.
[0190] Diagnostic Agents. Representative diagnostic agents that can
be incorporated into and advantageously delivered by the nanogels
of the invention include commercially available imaging agents used
in positron emissions tomography (PET), computer assisted
tomography (CAT), single photon emission computerized tomography,
x-ray, fluoroscopy, and magnetic resonance imaging (MRI);
anti-emetics; and contrast agents. Examples of suitable materials
for use as contrast agents in MRI include gadolinium chelates, as
well as iron, magnesium, manganese, copper, and chromium. Examples
of materials useful for CAT and x-ray imaging include iodine-based
materials.
[0191] In some embodiments, a diagnostic and/or therapeutic agent
may be a radionuclide. Among the radionuclides used,
gamma-emitters, positron-emitters, and X-ray emitters are suitable
for diagnostic and/or therapeutic purposes, while beta emitters and
alpha-emitters may also be used for therapy. Suitable radionuclides
for use in the invention include, but are not limited to, 1231,
1251, 1301, 1311, 1331, 1351, 47Sc, 72As, 72Se, 90Y, 88Y, 97Ru,
100Pd, 101mRh, 119Sb, 128Ba, 197Hg, 211At, 212Bi, 212Pb, 109Pd,
111In, 67Ga, 68Ga, 67Cu, 75Br, 77Br, 99 mTc, 14C, 13N, 150, 32P,
.sup.33P, and 18F.
[0192] In some embodiments, a diagnostic agent may be a
fluorescent, luminescent, or magnetic moiety. Fluorescent and
luminescent moieties include a variety of different organic or
inorganic small molecules commonly referred to as "dyes," "labels,"
or "indicators." Examples include fluorescein, rhodamine, acridine
dyes, Alexa dyes, cyanine dyes. Fluorescent and luminescent
moieties may include a variety of naturally occurring proteins and
derivatives thereof, e.g., genetically engineered variants. For
example, fluorescent proteins include green fluorescent protein
(GFP), enhanced GFP, red, blue, yellow, cyan, and sapphire
fluorescent proteins, reef coral fluorescent protein. Luminescent
proteins include luciferase, aequorin and derivatives thereof.
Numerous fluorescent and luminescent dyes and proteins are known in
the art (see, e.g., U.S. Patent Application Publication
2004/0067503; Valeur, B., "Molecular Fluorescence: Principles and
Applications," John Wiley and Sons, 2002; Handbook of Fluorescent
Probes and Research Products, Molecular Probes, 9th edition, 2002;
and The Handbook-A Guide to Fluorescent Probes and Labeling
Technologies, Invitrogen, 10th edition, available at the Invitrogen
web site).
[0193] The preparation and characterization of representative
zwitterionic nanogels are described in Example 4.
[0194] The preparation and characterization of representative
degradable zwitterionic nanogels are described in Example 5.
[0195] pCBMA nanogels were synthesized by inverse mini-emulsion
free radical polymerization. Because the potential application of
pCBMA nanogels as carriers involves temperature-sensitive
biologically active compounds such as such as protein, DNA or RNA,
polymerization was initiated by using a low-temperature free
radical imitator V-70 at 40.degree. C. The size of nanoparticles
can greatly influence their blood circulation time. It is reported
that nanoparticles smaller than 200 nm have less chance to be
cleared by Kupffer cells and splenic filtration. Furthermore, it is
also reported that small particles (<200 nm) can more
effectively extravasate into tumors. In the present invention, the
size of nanogels was maintained below 200 nm by adjusting the ratio
and the concentration of the two surfactants (Tween 80 and Span
80). The hydrodynamic size and polydispersity of pCBMA nanogels
(Table 3) were analyzed by dynamic light scattering in DI water and
PBS (pH 7.4). The sizes of pCBMA nanogels with 1.5%, 3% and 5% MBAA
(molar concentration) were 103.63 nm, 117.47 nm, and 99.30 nm,
respectively. The size distribution of nanogels with 1.5% MBAA is
the narrowest among these nanogels.
TABLE-US-00003 TABLE 3 Sizes of pCBMA nanogels with or without
encapsulated FITC-dextran in DI water. Diameter (nm) in DI water
Sample (polydispersity index) 1.5% MBAA 103.63 (0.12) 1.5% MBAA +
Dextran 100.29 (0.16) 3% MBAA 117.47 (0.25) 3% MBAA + Dextran
109.47 (0.15) 5% MBAA 99.30 (0.20) 5% MBAA + Dextran 93.58
(0.21)
[0196] The stability of zwitterionic nanogels in the complex medium
was investigated (FIG. 23) by measuring the size change of nanogels
as a function of time in 100% fetal bovine serum (FBS). Both
nanogels with 1.5% and 3% MBAA retained their original sizes after
an 18-hour incubation in 100% FBS. The major challenge for
intravenous administration of nanogels is that the adsorption of
blood proteins on the nanogels can destabilize the nanogel and lead
to fast clearance by liver, spleen, and macrophage cells before the
nanogel can reach its intended target. Neutral and hydrophilic
materials have been coated on nanoparticles to reduce nonspecific
protein adsorption.
[0197] FITC-dextran was encapsulated in pCBMA nanogels as a model
drug. FIG. 24 shows a controlled release of FITC-dextran from pCBMA
nanogel as a function of time. 25% of FITC-dextran initially
encapsulated in pCBMA hydrogel was released after 18 days. Due to
their strong hydration and excellent biocompatibility, hydrogels
have been extensively studied as controlled release drug delivery
vectors. Macromolecules can be released from the matrix through
diffusion or environmental stimuli such as the change in pH or
temperature. Environmental stimuli can lead to faster release due
to the decomposition of the matrix or the increased pore size of
the matrix. In order to investigate the intrinsic capacity and
property of novel zwitterionic pCBMA as the drug delivery carrier,
the release rate of the encapsulated FITC-dextran is simply
controlled by diffusion, which is determined by the molecular
weight of the encapsulated drug and the pore size of the nanogels.
FITC-dextran with a molecular weight of 10 kD and 1.5% crosslinker
was used. The release rate can be adjusted by controlling the ratio
between MBAA and CBMA monomers in aqueous stock solutions,
depending on the size and hydrophilicity of a drug used for a
specific application.
[0198] The cytotoxicity of nanogels was assessed by measuring the
viability of primary HUVECs as a function of the concentration of
pCBMA nanogels. The effect of the concentration of the crosslinker,
MBAA, was also investigated. Results in FIG. 25 show that pCBMA
nanogels with 1.5% MBAA as the crosslinker have minimal
cytotoxicity even at high concentrations (2 mg/mL).
[0199] Cyclo-RGD as a targeting ligand was conjugated to
FITC-dextran-containing-pCBMA nanogels using EDC/Sulfo-NHS
chemistry in water. The cellular uptake of FITC-labeled nanogels
was quantified with a flow cytometer. pCBMA nanogels conjugated
with cyclo-RGD showed a higher uptake by HUVECs than pCBMA nanogels
without cRGD (FIGS. 26A and B). pCBMA nanogels at a concentration
of 1 mg/mL lead to a higher uptake of pCBMA nanogels than at a
concentration of 0.2 mg/mL. The ratio of mean fluorescent
intensities between cells containing cyclo-RGD-conjugated nanogels
and cells containing bare pCBMA nanogels is 4.99 and 3.87 for the
nanogels at the concentration of 1 mg/mL and 0.2 mg/mL,
respectively. For conventional coatings on nanoparticles, mixed
functional and nonfouling groups, such as the hydroxyl
terminated-poly(ethylene glycol) (PEG) and carboxylate-terminated
PEG, are commonly used. Due to the limitation of conjugation
efficiency, unreacted functional groups such as carboxylate group
and amine groups will cause nonspecific protein adsorption onto the
nanoparticle surfaces in these traditional systems. However, pCBMA
does not have such a problem since one pCBMA can do both jobs of
nonfouling and functionalization in one material. Any unreacted
functional groups in pCBMA can be hydrolyzed back into nonfouling
zwitterionic groups. Thus, the density of targeting agents on pCBMA
nanogels can be controlled simply by adjusting the concentration of
the targeting agent during ligand conjugation.
[0200] In summary, multifunctional nanogels based on pCBMA were
synthesized by an inverse microemulsion free radical polymerization
method. pCBMA nanogels exhibited excellent biophysical stability in
100% fetal bovine serum and had minimal cytotoxicity. Controlled
release of FITC-dextran encapsulated within pCBMA nanogels was
demonstrated. The release rate depends on the particular hydrogels
and drugs used and can be readily controlled. Furthermore, each
side-chain of the pCBMA nanogels contains a carboxylate group for
ligand immobilization. Results obtained from flow cytometry
indicated that nanogels conjugated with cyclo-RGD-ligands
dramatically increased the uptake of nanogels by human umbilical
vein endothelium cells. These functionalizable zwitterionic
nanogels are of great potentials as targeted drug delivery vectors
due to their ultra stability and multiple functionalities all in
one chemically-uniform particle.
[0201] Multifunctional nanoparticles have been extensively studied
in the field of targeting drug delivery due to their great
potential to work as an intelligent carrier for both therapy and
imaging, however, a successful and sophisticated multifunctional
nanoparticle-based drug delivery system should have the following
properties: first, it should have high therapeutic drug loading and
could release its payload at the target cite; second, it should
have long in vivo circulation half-life and can target to specific
site after administrated to reduce side effects to other healthy
tissue; third, it should load imaging reagent for non-invasive
imaging to monitor the targeting and therapeutic efficiency;
fourth, after these NPs complete their missions, they should be
either digested or downgraded to small fragments that can be
removed from the body (e.g., via renal clearance) to avoid possible
toxicity and side-effects.
[0202] Multifunctional pCBMA nanogels encapsulating monodisperse
Fe.sub.3O.sub.4 magnetic NPs (MNPs) as MRI contrast reagent and
fluorescence labeled dextran as a model drug were synthesized by
using a reducing sensitive crosslinker. Results show that, the
nanogels were degraded after entering reducing environment (similar
to intracellular environment), resulting in the spontaneously
release of monodisperse Fe.sub.3O.sub.4 NPs and dextran. The final
degraded parts could be either digested by the body or removed from
body by renal filtration.
[0203] SEM image shows the morphology of the nanogels, as presented
in FIG. 28. In PBS solution, the nanogels possess a hydrodynamic
size of about 110 nm. FIG. 29 shows the long term stability of the
nanogels in PBS (for at least 6 months), this result is expected
because it has been shown that if the NPs were well coated with
pCBMA polymer, they can stay in saline solutions (PBS or NaCl) for
very long time without any size change.
[0204] FIG. 30 shows the cytotoxicity of the nanogels on macrophage
cells, in all the concentration tested, the nanogels didn't present
obvious cytotoxicity to the cells. Macrophage cell uptake test is
an important method to evaluate the response of the innate immune
system to nanoparticles. FIG. 31 shows that, at two different Fe
concentration levels (5 ppm and 10 ppm), the nanogels present very
low cell uptake amount, indicating the nanogels could possibly have
long circulation half-life after in vivo administration to the
body.
[0205] Degradation of the nanogels was firstly tested by DLS, as
shown in FIG. 32, after mixed with PBS solution containing 10 mM
DDT at 37.degree. C. and incubated for 24 hours, the nanogels
(about 110 nm) were degraded to small pieces with a hydrodynamic
size of about 12 nm, which is almost the same as the water soluble
monodisperse MNPs. This result indicates the degraded solution are
composed of the original water soluble monodisperse MNPs and pCBMA
polymer chains.
[0206] Degradation of the nanogels was further evaluated by MRI
test. FIG. 33 shows the T2 tests and MR images of different
samples. The original monodisperse MNPs have a transverse
relaxivity (R2 relaxivity, reciprocal of the proton spin-spin
relaxation time) of 39.531 mM.sup.-1 s.sup.-1, while the MNPs
loaded nanogels have a R2 relaxivity of 113.12 mM.sup.-1 s.sup.-1,
this phenomenon is due to the encapsulating of several monodisperse
MNPs could enhance the R2 relaxivity. R2 relaxivity of degraded
samples decreased to 41.56 mM.sup.-1 s.sup.-1, indication the
completely disassemble of the encapsulated monodisperse MNPs. MR
images in FIG. 33 visually present the different contrast of the
three samples, showing the different MR contrast ability of the
samples, which is consist with the T2 tests.
[0207] The release of encapsulated FITC-dextran is shown in FIG.
34, result shows the significant difference between the samples
with or without the reducing reagent (DTT). The nanogels incubated
with DTT shows an efficient release of FITC-dextran, about 80% of
the payload with release over a period of 48 hours. In contrast,
nanogels incubate without DTT could only release their payload by
physical diffusion, which is much less efficient, only about 3% was
released during the same 48 hours.
[0208] These results elucidated the nanogels presented in this work
hold great promise to serve as intelligent targeting drug delivery
carriers: the ultra-low fouling pCBMA polymer chains could
efficiently stabilize the particles and resist the clearance of the
particles after systemic administration, meanwhile, the pCBMA
polymer chains could be easily functionalized with targeting
ligands to enhance the accumulation of the nanogels. Before the
nanogels arrive at the target site and internalized into the target
cells, the loaded therapeutic reagents has a very slow release
profile, while after the nanogels enter the intracellular
environment which is much more reducing, the disulfide crosslinkers
will be cleaved, resulting in the fast release of the therapeutic
payload, also the loaded imaging reagent (nanoparticles, such as
MNPs). A further benefit of this design is, not like some other
non-degradable nano-carriers that accumulate in the body after
injection, the nanogels of the invention can be removed from the
body either by degradation of the MNPs or by the renal filtration
of the degraded pCBMA polymer chains.
[0209] The following examples are provided for the purpose of
illustrating, not limiting, the invention.
EXAMPLES
Materials
[0210] Tetraethoxysilane (TEOS), copper (I) bromide (99.999%),
bromoisobutyryl bromide (BIBB, 98%), 3-(trimethoxysilyl)propylamine
(TMOSPA, 97%), .beta.-propiolactone (90%), tetrahydrofuran (THF,
HPLC grade) and 2,2'-bipyridine (BPY 99%) were purchased from
Sigma-Aldrich (Milwaukee, Wis.) and used as received.
N-[3-(dimethylamino)propyl]acrylamide (DMAPA, 98%) was purchased
from TCI (America, OR). Human monoclonal antibodies against
activated leukocyte cell molecule (anti-ALCAM) were purchased from
R&D Systems (Minneapolis, Minn.). Bovine serum albumin (BSA)
and lysozyme (Lyz) were purchased from Sigma-Aldrich (Milwaukee,
Wis.). Phosphate buffered saline (PBS: 0.01M phosphate, 0.138 M
sodium chloride, 0.0027 M potassium chloride, pH 7.4) was purchased
from Sigma Chemical Co. and used at 0.15M. THF for reactions and
washings were dried by sodium before use. Chicken egg white
lysozyme were purchased from Sigma-Aldrich (Milwaukee, Wis.).
Pooled human blood serum was purchased from BioChemed Services
(Winchester, Va.). Human polyclonal antibodies against activated
leukocyte cell molecule (anti-ALCAM) and human recombinant ALCAM/Fc
chimera were purchased from R&D Systems (Minneapolis, Minn.).
PEG.sub.5000-thiol was purchased from Nektar (Huntsville, Ala.).
.beta.-propiolactone (85-90%), copper(I) bromide (99.999%),
methanol (99.9%), bromoisobutyryl bromide (98%),
11-mercapto-1-undecanol (97%), sodium citrate (98%), HAuCl.sub.4
(99.999%), N,N-dimethylformamide (DMF, 99%), oligo(ethylene glycol)
methyl methacrylate (OEGMA) (95%), N-hydroxysuccinimide (NHS) and
N-ethyl-N'-(3-diethylaminopropyl) carbodiimide hydrochloride (EDC)
were purchased from Sigma-Aldrich (Milwaukee, Wis.). Ethanol
(absolute 200 proof) was purchased from AAPER Alcohol and Chemical
Co. Water used in these experiments was purified using a Millipore
water purification system with a minimum resistivity of 18.0
M.OMEGA.cm. The ATRP initiator, .omega.-mercaptoundecyl
bromoisobutyrate, was synthesized through the reaction of
bromoisobutyryl bromide and 11-mercapto-1-undecanol as described in
Jones D M, Brown A A, Huck W T S. Langmuir 2002; 18(4): 1265-1269.
FeCl.sub.2 4H.sub.2O (>99%), FeCl.sub.3 6H.sub.2O (>99%),
NH.sub.3H.sub.2O(NH.sub.3 content: 28-30%), dextran (M.W. 10,000)
from Leuconostoc mesenteroides, 3,4-dihydroxyphenyl-L-alanine,
2-bromoisobutyric acid, and dicyclohexyl carbodiimide (DCC) were
purchased from Sigma-Aldrich. Tetrabutylammonium fluoride (TBAF, 1
M solution in THF containing ca 5% water),
1,3-diamino-2-hydropropane, diisopropylethylamine (DIEA), and
tert-butyl chlorodimethylsilane (TBDMS, 98%) were purchased from
Acros Organics. Hexane was purchased from J. T. Baker.
2-(Dimethylamino)ethyl methacrylate (DMAEMA),
N,N'-methylene-bis-acrylamide (MBAA), sodium salicylate,
acetonitrile, ethyl ether, SPAN 80, TWEEN 80 and fluorescein
isothiocyanate-dextran (FITC-dextran) (MW 10,000 and MW 20,000)
were purchased from Sigma-Aldrich Chemical Co. (MO, USA).
N-hydroxysulfosuccinimide (Sulfo-NHS) was purchased from Acros
Organics (USA). Cyclo[Arg-Gly-Asp-D-Tyr-Lys] (cRGD) was purchased
from Peptides International (KY, USA).
2,2'-Azobis(4-methoxy-2.4-dimethyl valeronitrile) (V-70) was
purchased from Wako Pure Chemical Industries (VA, USA). Oleic acid
(90%), dithiothreitol (DTT, 99%), and cetyltrimethylammonium
bromide (CTAB, 95%) were purchased from Sigma-Aldrich; L-Cystine
(98%), sodium oleate (95%) were purchased from TCI America. The
disulfide crosslinker, L-cystine bisacrylamide (BACy), was
synthesized according to a reported method.
Example 1
Preparation and Characterization of Representative Zwitterionic
Coated Particles: Silica Nanoparticles
[0211] In this example, the preparation and characterization of
representative zwitterionic polymer coated particles, silica
nanoparticles, are described.
[0212] Synthesis of a CBAA monomer. A CBAA monomer,
(3-Acryloylamino-propyl)-(2-carboxyethyl)dimethyl-ammonium was
synthesized as described in Vaisocherova, H. Yang, W. Zhang, Z.
Cao, Z. Cheng, G. Piliarik, M. Homola, J. Jiang, S. Anal. Chem.
2008, 80, 7894-7901. Typically, 1.54 g of DMAPA was reacted with
0.99 g of .beta.-propiolactone in 50 mL of anhydrous acetone at
0.degree. C. for 2 h under nitrogen protection. The product (white
precipitate) was washed with anhydrous ether three times, dried in
vacuum, and stored as 4.degree. C. Yield: 81%. .sup.1H NMR (Bruker
500 MHZ. DMSO-d.sub.6): 8.61 (t, 1H, N--H), 6.28 (t, 1H,
CHH.dbd.CH), 6.13 (t, 1H, CHH.dbd.CH), 5.61 (t, 1H, CHH.dbd.CH),
3.44 (t, 2H, N--CH.sub.2--CH.sub.2--COO), 3.21 (m, 4H,
NH--CH.sub.2--CH.sub.2--CH.sub.2), 2.97 (s, 6H,
N--(CH.sub.3).sub.2), 2.25 (t, 2H, CH.sub.2-000), 1.87 (t, 2H,
NH--CH.sub.2--CH.sub.2--CH.sub.2).
[0213] Synthesis of a surface initiator. The ATRP initiator,
2-bromo-2-methyl-N-3-[(trimethoxysilyl)propyl]propanamide (BrTMOS)
was synthesized as described in Zhang, Z. Chao, T. Chen, S. Jiang,
S. Langmuir 2006, 22, 10072-10077. Typically, TMOSPA (1.76 g, 10
mmol) was mixed with triethylamine (1.01 g, 10 mmol) in 50 mL of
dried THF. BIBB (3.45 g, 15 mmol) was added dropwise into the
solution for 30 min with stirring. The reaction was kept for 12 h
under nitrogen protection with stirring. The precipitate was
filtered off using a frit funnel. The product was redissolved with
CH.sub.2Cl.sub.2 (20 mL) and washed with 0.01 N HCl (2.times.20 mL)
and cold water (2.times.20 mL). The organic phase was dried with
anhydrous CaCl.sub.2. After the removal of the solvent, the final
product was colorless oil with a yield of 90.5%. .sup.1H NMR (300
MHz, CDCl.sub.3): .delta. 6.91 (s, 1H, NH), 3.49 (s, 9H,
SiOCH.sub.3), 3.24 (t, 2H, CH.sub.2N), 1.94 (s, 6H, CH.sub.3), 1.68
(m, 2H, CH.sub.2), 0.67 (t, 2H, SiCH.sub.2). .sup.13C NMR (600 MHZ,
CDCl.sub.3): .delta. 171.98, 62.57, 50.29, 42.62, 32.44, 22.52,
7.64.
[0214] Preparation and modification of silica particles (SiP). The
colloid silica particles of 66 nm in diameter were synthesized
according to the well-known Stober method as described in Stober,
W. Fink, A. J. Colloid. Interf. Sci. 1968, 26, 62-69. A 52 mL
portion of absolute ethanol, 2.3 mL of ammonia, and 0.9 mL of water
were introduced in a 100-mL, three-neck, and round-bottom flask
equipped with a refrigerating system and heated to 50.degree. C.
under stirring. Then, 1.95 mL of TEOS was added into the solution
and stirred at 50.degree. C. for 24 h.
[0215] Colloid silica was coated with silane according to the
Philipse and Vrij method as described in Philipse, A. P. Vrij, A.
J. Colloid. Interf. Sci. 1989, 128, 121-136. A mixture of silica
sol and BrTMOS was stirred for 30 min at room temperature, solvents
were slowly distilled off during a period of 2 h, and the mixture
was diluted with toluene. The dispersion of modified silica was
purified from free silane and water or ammonia by centrifugation
and redispersion in absolute ethanol. The final product was stored
in absolute ethanol for further use.
[0216] Surface-initiated ATRP on SiP. Prior to polymerization, the
initiator-coated SiP in ethanol was solvent-exchanged to
methanol/water (vol/vol=3/1). The predetermined amounts of Cu(I) Br
(30 mg), CBAA (120 mg) and BPY (66 mg) were added into a glass
tube. The mixture was immediately degassed by three
freeze-pump-thaw cycles. The degassed SiP solutions were added to
the mixture to start reaction. The polymerization was carried out
overnight at room temperature.
[0217] The rest of the reaction mixture was diluted by methanol and
centrifuged to collect polymer-grafted SiP. This cycle of
centrifugation and redispersion in methanol was repeated two times.
The samples were then washed with water for three times to obtain
polymer-grafted SiPs free of unbounded polymer. The products were
stored in PBS for further use.
[0218] Nanoparticle characterization. The hydrodynamic diameters of
the nanoparticles were measured by Malvern Zeta Sizer Nano-90
dynamic light scattering (DLS) instrument. Transmission electron
microscope (TEM) measurements were taken on a Tecnai G2 F20 (200
kV). The samples were prepared by placing a drop of colloidal
solutions on a 400-mesh carbon-coated copper grid and air-drying
the grid at 25.degree. C.
[0219] Resistance to Nonspecific Protein Adsorption. Particle
stability in protein solution was assessed by Malvern Zeta Sizer
Nano-90 dynamic light scattering (DLS) instrument. Experiments were
done by re-suspending 0.1 mg modified nanoparticles with 10 mg/mL
protein in phosphate buffer solution (pH 7.4). Then, the size
change of the nanoparticles during the incubation was tracked by
DLS.
Example 2
Preparation and Characterization of Representative Zwitterionic
Coated Particles: Gold Nanoparticles
[0220] In this example, the preparation and characterization of
representative zwitterionic polymer coated particles, gold
nanoparticles, are described.
[0221] Synthesis of CBAA monomer.
(3-Acryloylamino-propyl)-(2-carboxy-ethyl)-dimethyl-ammonium (CBAA)
was prepared as described in Example 1.
[0222] Preparation of Bare Gold Nanoparticles (Gnps). Gnps with an
Average Diameter of 18.5 nm were prepared by reduction of
HAuCl.sub.4 with citric sodium as described in Storhoff J J,
Elghanian R, Mucic R C, Mirkin C A, Letsinger R L. J. Am. Chem.
Soc. 1998; 120(9): 1959-1964. An aqueous solution (1.75 ml) of 1%
(w/v) sodium citrate was added quickly to a boiling aqueous
solution (100 ml) of 0.01% (w/v) HAuCl.sub.4 under stirring,
resulting in a change in solution color from pale yellow to deep
red. After the color change, the solution was refluxed for an
additional 15 min, allowed to cool to room temperature, and
subsequently filtered through a Micron Separations Inc. 0.45 .mu.m
nylon filter. A typical solution of 18.5 nm diameter gold particles
exhibited a characteristic surface plasmon band centered at 523.2
nm.
[0223] Preparation of CBAA coated GNPs (pCBAA-GNPs). As shown in
FIG. 6, prior to the reaction, bare GNPs (8.5 ml) was mixed with
the initiator (10 .mu.l, 100 mM) in DMF (1.5 ml) and stirred for 24
h at room temperature. The initiator-functionalized GNPs were then
purified with methanol three times by centrifugation (8000 r.p.m.,
15 min) to obtain 1.5 ml solution A. 600 mg CBAA monomer, 28.533 mg
copper(I) bromide and were dissolved in 3 ml degassed methanol and
1.5 ml water under nitrogen atmosphere to obtain solution B.
Solution A was deoxygenated by bubbling nitrogen before mixed with
solution B. The final mixture was stirred (100 r.p.m.) at room
temperature for 90 min or 3 h to control the coating thickness.
After the polymerization, pCBAA-GNPs were washed several times by
centrifuging/redispersing in water.
[0224] Preparation of PEG.sub.5000 coated gold nanoparticles
(PEG-GNPs). PEG.sub.5000-thiol was added in excess and reacted with
the gold nanoparticles (GNPs) for 30 min at room temperature. The
modified particles were centrifuged at 5000.times.g for 5 min to
remove unreacted PEG modifiers and resuspended in the appropriate
solvent.
[0225] Preparation of OEGMA coated gold nanoparticles (OEGMA-GNPs).
According to the same procedure as pCBAA-GNPs, the
initiator-functionalized GNPs were purified with Milli-Q water
three times by centrifugation to obtain 1 ml solution A. 47.7 mg
copper(I) bromide and 104 mg 2,2-bipyridine were dissolved in 4 ml
degassed methanol under nitrogen atmosphere to obtain solution B.
Solution A was deoxygenated by bubbling nitrogen before directly
mixed with solution B. 2 g macromonomer OEGMA was added and the
final mixture was stirred at room temperature for 90 min. After the
polymerization, OEGMA-GNPs were washed several times by
centrifuging/redispersing in Milli-Q water.
[0226] PolyCBAA Functionalization Method. The carboxyl groups on
small pCBAA-GNPs were first activated by 0.05 M NHS and 0.2 M EDC
(pH of final NHS/EDC solution was about 5.5). After centrifuging at
8000 r.p.m. for 15 min, the pCBAA-GNPs were dispersed in the
polyclonal anti-ALCAM (R&D Systems, Minneapolis, Minn.)
solution in 10 mM sodium borate buffer (pH 8.5-9.0). After all
noncovalently bound ligands were removed by centrifugation at 8000
r.p.m. for 15 min, the pCBAA-GNPs were re-suspended in PBS
containing ALCAM (R&D Systems, Minneapolis, Minn.) of different
concentrations. The antigen-induced aggregation of GNPs was
observed by spectrometer from 400-800 nm.
Example 3
Preparation and Characterization of Representative Zwitterionic
Coated Particles: Magnetic Iron Oxide Gold Nanoparticles
[0227] In this example, the preparation and characterization of
representative zwitterionic polymer coated particles, magnetic iron
oxide nanoparticles, are described.
[0228] Synthesis of DOPA.sub.2(TBDMS).sub.4--Br initiator. FIG. 13
illustrates the synthesis of DOPA.sub.2(TBDMS).sub.4--Br initiator.
DOPA.sub.2(TBDMS).sub.4--NHS and 2-aminoethyl 2-bromoisobutyrate
were synthesized as described in Dalsin J L, Lin L J, Tosatti S,
Voros J, Textor M, Messersmith P B. Langmuir 2005; 21(2):640-646;
Sever M J, Wilker J J. Tetrahedron 2001 July: 57(29):6139-6146; Lu
C W, Hung Y, Hsiao J K, Yao M, Chung TH, Lin Y S, et al. Nano Lett
2007 January: 7(1):149-154. To prepare the initiator,
DOPA.sub.2(TBDMS).sub.4--NHS (1032 mg, 1.00 mM) was dissolved in mL
dry N,N-dimethylformamide (DMF), and the trifluoroacetic acid salt
of 2-aminoethyl-2-bromoisobutyrate (339 mg, 1.00 mM) was added
under nitrogen. The mixture was stirred on an ice bath before DIEA
(385 .mu.L, 2.2 mM) was added via a syringe. After 1 h, the mixture
was warmed to room temperature and stirred overnight, and then 40
mL 5% aqueous HCl was added. The mixture was extracted with 30 mL
ethyl acetate, and the organic phase was washed with 30 mL DI
water, dried, and concentrated in vacuo. The crude product was
purified on a silica gel column with chloroform and 1% methanol as
an eluent. The product, 2-bromoisobutyric acid
DOPA.sub.2(TBDMS)-4-amino ethyl ester was obtained as a white foam,
(1.03 g, 91%). .sup.1H NMR (CDCl.sub.3) .delta.: 6.60-6.82 (m, 6H),
6.38-6.44 (m, 2H), 4.64-4.67 (m, 2H), 4.12-4.19 (m, 2H), 4.09-4.11
(m, 1H), 3.14-3.60 (m, 3H), 2.66-3.04 (m, 3H), 1.95 (d, 6H), 1.31
(s, 9H), 1.0 (m, 36H) 0.2 (m, 24 H).
[0229] Synthesis of DOPA.sub.2-pCBMA. DOPA.sub.2(TBDMS).sub.4--Br
initiator (52 mg, 0.05 mM), BPY (44 mg, 0.29 mM), CuBr (13.6 mg,
0.094 mM), and CuBr.sub.2 (1.03 mg, 0.005 mM) were placed in a 50
mL flask and degassed three times. 1 mL degassed DMF was then added
to dissolve the reactants. Then, 1.0 g CBMA, dissolved in degassed
H.sub.2O/DMF (2 mL/7 mL), was added into the flask while stirring
to start the reaction. Polymerization was conducted at room
temperature for 10 h. The resultant was purified by dialysis for
three days against pure water. The purified polymer was lyophilized
to white powder.
[0230] Both DOPA groups of the DOPA.sub.2-pCBMA polymer were
protected by TBDMS groups. Before the polymer was coated onto MNPs,
the TBDMS groups were removed with TBAF in THF and reacted for 12
h. The deprotected polymer was purified by THF three times and
dried under vacuum at room temperature. The molecular weight and
molecular weight distribution of the polymer were measured with gel
permeation chromatography (GPC). The number average molecular
weight (Mn) was 80.8 kDa (using PEG standards) and the
polydispersity index (PDI) was 1.22.
[0231] Preparation of Uncoated, Dextran Coated, and
DOPA.sub.2-pCBMA Coated MNPs. Water-soluble uncoated MNPs
(Fe.sub.3O.sub.4) were prepared by a co-precipitation method.
Briefly, FeCl.sub.2 4H.sub.2O and FeCl.sub.3 6H.sub.2O were
precipitated by adding NH.sub.3H.sub.2O under the protection of
nitrogen gas. The resultant was washed 5 times by DI water and
collected with a permanent magnet. During this procedure, any small
particles with poor mobility to the magnet were removed. The
homogenous colloid was filtered by a 0.2 .mu.m membrane and stored
for further use. Similar to the preparation of uncoated NPs,
dextran-coated MNPs were prepared by adding NH.sub.3H.sub.2O to
precipitate FeCl.sub.2 4H.sub.2O and FeCl.sub.3 6H.sub.2O at the
presence of dextran. To prepare DOPA.sub.2-pCBMA coated MNPs, 20 mg
unprotected DOPA.sub.2-pCBMA polymer was dissolved in 5 mL DI water
and stirred for 1 h before 5 mg uncoated MNPs were added. The
mixture was stirred for another 2 h and then washed three times
with DI water.
[0232] Characterization of MNPs. The morphology of
pCBMA-DOPA.sub.2-MNPs was characterized by transmission electron
microscope (TEM, Tecnai G2 F20, FEI). Magnetic properties were
measured by a superconducting quantum interference device (SQUID)
(MPMS-5S, Quantum Design). The hydrodynamic size of the particles
was analyzed with a dynamic light scattering (DLS) particle sizer
(Nano ZS, Zetasizer Nano, Malvern, Pa.). The concentration of all
MNP samples was determined by inductively coupled plasma atomic
emission spectroscopy (ICP-AES, Elan DRC-e, PerkinElmer).
[0233] Stability Studies. To evaluate the stability of various
MNPs, uncoated, dextran-coated and DOPA.sub.2-pCBMA coated MNPs
were mixed in PBS, 10% NaCl, or 100% human blood serum. The
particle size was continuously monitored by DLS. Tests in serum
were conducted at 37.degree. C. to mimics physiological
conditions.
[0234] Cytotoxicity Assay. The cell viability of HeLa, macrophage,
and HUVEC cells was tested by a typical MTT method using a
Vybrant.RTM. MTT Cell Proliferation Assay Kit (Molecular Probes).
Cells were seeded in 96-well cell culture plates in 200 .mu.L
medium with serum under 5% CO.sup.2 at 37.degree. C. to allow
80-90% confluence. On the day of the test, cells were washed with
PBS and incubated with 200 .mu.L fresh medium containing
nanoparticles at various concentrations. After 24 h, cells were
washed with PBS and incubated with 100 .mu.L medium and 50 .mu.l of
12 mM MTT stock solution for another 4 h. Then, the medium was
removed and 150 .mu.L DMSO was added and incubated for 10 min. The
absorbance at 570 nm was read with a 96-well plate reader
(SpectraMax M5, Molecular Devices).
[0235] Macrophage Cell Uptake. RAW 264.7 cells were cultured in
DMEM medium with 10% FBS and 1% antibiotics in a 6-well plate.
Prior to the test, cells were washed with PBS three times, and then
various types of nanoparticles in culture media (concentration 10
mg Fe/mL) were added. After 4 h incubation at 37.degree. C., 5%
CO.sub.2, cells were washed three times with PBS and lysed with 1
mL of 50 mM NaOH solution. Intracellular iron content was
determined by the ICP-MS method.
[0236] Functionalization of pCBMA-DOPA.sub.2-MNPs. 5 mg
pCBMA-DOPA.sub.2-MNPs were dispersed in 2 mL DI water. 3 mg EDC and
0.5 mg NHS were then added successively. The mixture was stirred
for 0.5 h and then washed two times by DI water. After that, the
nanoparticles were re-dispersed in 2 mL DI water, and 0.05 mg of
RGD peptide Cyclo[Arg-Gly-Asp-.sub.D-Tyr-Lys] was added. The
mixture was stirred for another 3 h at room temperature. The final
product was washed three times with DI water.
[0237] Magnetic Resonance Imaging. All MRI studies were conducted
on a 3 T whole body scanner (Philips Achieva R2.6.1, Best,
Netherlands). An eight-channel receive-only head coil was used for
signal acquisition because of its high signal-to-noise ratio (SNR).
The spin-spin (T.sub.2) transverse relaxation time was acquired by
a multi-echo turbo spin echo (TSE) sequence. PCBMA-DOPA.sub.2-MNPs
at various concentrations were scanned using the following
parameters: TR 3000 ms, TE 7-224 ms in steps of 7 ms, field of view
(FOV) 140.times.120 mm.sup.2, matrix size 188.times.160, slice
thickness 10 mm, number of signal average 1, acquisition bandwidth
250 Hz/pixel, and total scan time is 5'21''.
[0238] T.sub.2 maps were generated from the multi-echo TSE images
using a custom-programmed algorithm coded in MATLAB (Mathworks,
Natick, Mass.). The T.sub.2 relaxation time of each sample was
measured using a custom-made image processing software CASCADE.
Images were loaded into the software and then a region of interest
(ROI) of no smaller than 2 cm.sup.2 was carefully delineated within
the boundary of the samples of interest. The average T.sub.2
relaxation time of the sample was then measured automatically by
CASCADE.
[0239] HUVEC Cell Targeting. HUVEC cells were cultured in Medium
200 supplemented with low serum growth supplement in a 6-well
plate. First, cells were washed by PBS for three times. Then,
pCBMA-DOPA.sub.2-MNPs with or without RGD peptide in fresh culture
media (concentration 10 or 20 mg Fe/mL) were added. After 4 h
incubation, cells were washed three times with PBS and lysed with 1
mL of 50 mM NaOH solution. Intracellular iron content was
determined by the ICP-MS method. MRI images of different cell
samples were also taken by the 3T MRI instrument using the similar
T.sub.2-weighted sequence as described above.
Example 4
Preparation and Characterization of Representative Zwitterionic
Nanogels
[0240] In this example, the preparation and characterization of
representative zwitterionic nanogels are described.
[0241] Inverse microemulsion polymerization of pCBMA nanogels. In a
typical reaction, pCBMA nanogels were prepared via inverse
microemulsion polymerization. The continuous phase solution
contains 40 ml hexane, 1.4 g of TWEEN 80, 1.6 g of SPAN 80, and 8
mg of V-70. The solution was kept on ice. Aqueous monomer stock
solutions were prepared by dissolving 229 mg (1 mmole) of CBMA, 4.6
mg (0.03 mmole) of MBAA in 0.5 mL of DI water. Then aqueous stock
solution was added into a 100 mL flask containing 40 mL of
continuous phase solution followed by vigorous shaking and a
2-minute sonication. The flasks were purged with nitrogen at
4.degree. C. for 30 minutes to remove dissolved oxygen. During
polymerization, the reaction was kept at 40.degree. C. with
stirring, and the reaction was protected under nitrogen for 4 h.
For the synthesis of pCBMA nanogels containing FITC-Dextran, the
conditions are the same as those for pCBMA nanogels without Dextran
except that 10 mg of FITC-dextran was added to the aqueous stock
solution.
[0242] Purification of nanogels. 10 mL of the reaction solution was
mixed with 30 mL of THF and stirred for 5 hours to remove
surfactants. The mixture was centrifuged for 40 minutes at 4400
rpm. The supernatant was discarded, and the precipitate was washed
twice with 30 ml of THF. The final precipitate was resuspended in 4
mL of DI water for 4 hours, and the aqueous solution was placed
into a 100 kD molecular weight cutoff Amicon Ultra centrifugal
filter devices (Millipore, MA, USA) to remove the liquid. pCBMA
nanogels were resuspended in 4 mL of DI water. The wash was
repeated 10 times at room temperature. Then, the aqueous solution
containing nanogels was filtered through a sterile 0.45 .mu.m PTFE
syringe filter and stored at 4.degree. C. for further
characterization. The concentration of nanogels was measured by
weighing the material before and after lyophilization. The yield
for nanogels containing no dextran with 1.5%, 3% and 5% crosslinker
(molar concentration) is 36.2, 51.3, and 40.0%, respectively.
[0243] Hydrodynamic diameter and polydispersity of pCBMA nanogels.
The hydrodynamic diameter and polydispersity of pCBMA nanogels were
analyzed by a dynamic light scattering (DLS) Zetasizer Nano ZS,
Malvern, UK) at the wavelength of 633 nm. The scattering angle of
173.degree. was used and the temperature was 25.degree. C. The
values of dispersant refractive index and viscosity of water were
taken as 1.330 and 0.8872 cP, respectively.
[0244] Release of encapsulated dextran from pCBMA nanogels. The
release of encapsulated FITC-dextran from pCBMA nanogels with 1.5%
crosslinker was determined. 40 mg of the purified pCBMA nanogels
with FITC-dextran were resuspended in 20 mL of DI water. At time
zero, 1 mL of solution was taken from pCBMA nanogel solution, and
its total fluorescent density was measured. At different time
points, 1 mL of solution was placed into a 100 kD molecular weight
cutoff Amicon Ultra centrifugal filter devices (Millipore, MA,
USA), and centrifuged at a speed of 4400 rpm for 90 minutes to
collect flow-through for FITC fluorescent detection. The
fluorescent density at 515 nm of the filtrate was measured at
25.degree. C. with a fluorescence spectrophotometer (F-4500
Fluorescence Spectrophotometer, Hitachi, Japan) with an excitation
wavelength of 495 nm and a cut-off wavelength of 500 nm. The
percentage of the released FITC-dextran was defined as the ratio of
the fluorescent density of flow-through at different time points to
the total fluorescent density at time zero.
[0245] Functionalization of pCBMA nanogels. 50 mg pCBMA nanogels
with 5% MBAA were resuspended in 2 mL of DI water. 153 mg of EDC
and 23 mg of Sulfo-NHS were added to pCBMA nanogel solution and the
solution was incubated at 25.degree. C. for 30 minutes to activate
the carboxylate group of pCBMA nanogel. Then, 1.3 mg of cRGD was
added to the activated pCBMA nanogel solution and the pH value of
the solution was adjusted to 8.5-9.0. The reaction was incubated at
25.degree. C. for 3 hours. The reaction solution was placed into a
100 kD molecular weight cutoff Amicon Ultra centrifugal filter
device (Millipore, MA, USA) to remove reactants. The nanogels were
resuspended in 10 ml of DI water and again passed through a 100 kD
molecular weight cutoff Amicon Ultra centrifugal filter device. The
wash was repeated 10 times at room temperature.
[0246] Nanogel cytotoxicity assay. Cell viability was assessed
using a Vybrant MTT Cell Proliferation Assay Proliferation Assay
Kit (Invitrogen, USA). Human umbilical cord vascular endothelial
cells (HUVEC) were seeded in 96-well tissue culture plates at a
density of 7000 cells/well and cultured in 100 .mu.L of Medium 200
supplemented with low serum growth supplement (Invitrogen, USA).
Cells were incubated in 100 .mu.L of Medium 200 with nanogels at
various concentrations for 4 h. Then, the medium was removed, and
50 .mu.L of DMSO was added and incubated for 10 min. The absorbance
at 570 nm was read with a 96-well plate reader (SpectraMax M5,
Molecular Devices, USA). Cell viability was expressed as the
percentage of absorbance of treated cells relative to the
absorbance of cells which were not incubated with pCBMA nanogels.
Each measurement had 5 replicate wells.
[0247] Flow cytometry. HUVEC cells were seeded in 24-well tissue
culture plates at a density of 10,000 cells/well and cultured in
500 .mu.L of Medium 200 supplemented with low serum growth
supplement (Invitrogen, USA). Then, cells were incubated with 500
.mu.L of Medium 200 with 10 mg/mL or 2 mg/ml of solutions of pCBMA
nanogels (5% MBAA) conjugated with or without cRGD for 4 h. After
the medium was replaced with 500 .mu.L of free Medium 200, HUVEC
cells were incubated for 12 hours. Then, the medium was removed and
the cells were washed three times with PBS. After detachment by
trypsin, HUVEC cells were resuspended in PBS with 1% fetal bovine
serum. The cellular uptake of pCBMA nanogels was analyzed by flow
cytometry (FACScan, BD, USA).
Example 5
Preparation and Characterization of Representative Degradable
Zwitterionic Nanogels
[0248] In this example, the preparation and characterization of
representative degradable zwitterionic nanogels are described.
[0249] Synthesis of monodisperse MNPs. Monodisperse MNPs (9 nm)
were synthesized by the thermal decomposition method. Iron-oleate
complex was firstly synthesized by reacting 5.4 g iron chloride and
18.25 g sodium oleate in a mixture solvent composed of 40 ml
ethanol, 30 ml distilled water and 70 ml hexane at 70.degree. C.
for 4 h. The product was washed 3 times with DI water, the hexane
was then evaporated off. After that, 18 g iron-oleate complex and
2.85 g oleic acid were dissolved in 100 g 1-octadecene, the
solution was stirred vigorously and gradually heated to 320.degree.
C., and then kept at this temperature for 20 min. After the mixture
cooled to room temperature, pure ethanol was used to precipitate
the NPs, the final MNPs were dispersed in hexane.
[0250] To prepare water soluble monodisperse MNPs, 1 mL NPs hexane
solution (10 mg/mL) was mixed with 10 mL water and 0.5 g CTAB. The
mixture was sonicated and stirred vigorously for 30 min, the hexane
solvent was then evaporated from the mixture. The resulting water
soluble NPs were washed 3 times by DI water using a 100 kD
molecular-weight-cutoff Amicon Ultra centrifugal filter device
(Millipore) and filtered by a 0.2 .mu.m syringe filter.
[0251] Synthesis of nanogels. PCBMA nanogels loaded with MNPs were
prepared by inverse microemulsion polymerization as illustrated in
FIG. 27. Briefly, 0.7 g Tween 80, 0.8 g Span 80 and 4 mg V-70 were
dissolved in 20 mL of hexane and kept in ice bath. 10 mg
monodisperse MNPs, 10 mg FITC-dextran, 115 mg CBMA and 5 mg
disulfide crosslinker were dissolve in 0.5 mL of DI water. The two
stock solutions were mixed in a 100 mL flask with vigorous
stirring, then strong sonication was applied to form the
microemulsion. The flask was purged with nitrogen at 4.degree. C.
for 30 min to remove dissolved oxygen. During polymerization, the
reaction was kept at 40.degree. C. with stirring and was protected
under nitrogen for 4 h. After the reaction, the product was washed
by tetrahydrofuran (THF) for 3 times to remove the surfactants,
then the product was dispersed in DI water and remaining impurities
were removed by using a 100 kD molecular-weight-cutoff Amicon Ultra
centrifugal filter. The final nanogels loaded with MNPs were
collected by using a permanent magnet.
[0252] Nanogel Characterization. The morphology of nanogels was
characterized by scanning electron microscope (SEM, Sirion, FEI).
The hydrodynamic size of all the particles was analyzed with a
dynamic light scattering (DLS) particle sizer (Nano ZS, Zetasizer
Nano, Malvern). The Fe concentration nanogel samples was determined
by inductively coupled plasma atomic emission spectroscopy
(ICP-AES, Elan DRC-e, PerkinElmer).
[0253] Cytotoxicity Assay. The cell viability of macrophage cells
and HUVEC cells was tested by a typical MTT method using a
Vybrant.RTM. MTT Cell Proliferation Assay Kit (Molecular Probes).
Cells were seeded in 96-well cell culture plates in 200 mL medium
with serum under 5% CO.sub.2 at 37.degree. C. to allow 80-90%
confluence. On the day of the test, cells were washed with PBS and
incubated with 200 mL fresh medium containing nanogels at various
concentrations. After 24 h, cells were washed with PBS and
incubated with 100 mL medium and 50 mL of 12 mM MTT stock solution
for another 4 h. Then, the medium was removed and 150 mL DMSO was
added and incubated for 10 min. The absorbance at 570 nm was read
with a 96-well plate reader (SpectraMax M5, Molecular Devices).
[0254] Macrophage Uptake Test
[0255] RAW264.7 cells were cultured in DMEM medium with 10% FBS and
1% antibiotics in a 6-well plate. Prior to the test, cells were
washed with PBS three times, and MNPs loaded nanogels at Fe
concentration of 5 ppm or 10 ppm in culture media were added. After
4 h incubation at 37.degree. C., 5% CO.sub.2, cells were washed
three times with PBS and lysed with 1 mL of 50 mM NaOH solution.
Intracellular iron content was determined by the ICP-AES
method.
[0256] Functionalization of Nanogels and HUVEC Cells Targeting
[0257] 5 mg nanogels were dispersed in 5 mL DI water. 6 mg EDC and
1 mg NHS were then added successively. The mixture was stirred for
0.5 h and then washed two times by DI water. After that, the
nanoparticles were re-dispersed in 2 mL DI water, and 0.1 mg of RGD
peptide Cyclo[Arg-Gly-Asp-D-Tyr-Lys] was added. The mixture was
stirred for another 3 h at room temperature. The final product was
washed three times with DI water.
[0258] HUVEC cells were cultured in Medium 200 supplemented with
low serum growth supplement in a 6-well plate. First, cells were
washed by PBS for three times. Then, nanogels with or without RGD
peptide in fresh culture media (concentration 5 or 10 mg Fe/mL)
were added. After 4 h incubation, cells were washed three times
with PBS and lysed with 1 mL of 50 mM NaOH solution. Intracellular
iron content was determined by the ICP-MS method.
[0259] Degradation Test by DLS. 0.1 mg of MNPs and FITC-dextran
loaded nanogels was dispersed in 5 mL PBS solution containing 10 mM
DTT, the hydrodynamic size was monitored by DLS at 37.degree. C.
Nanogels dispersed in PBS without DTT served as the control.
[0260] Degradation Test by MRI. All MRI studies were conducted on a
3 T whole body scanner (Philips Achieva R2.6.1, Best, Netherlands).
An eight-channel receive-only head coil was used for signal
acquisition because of its high signal-to-noise ratio (SNR). The
spin-spin (T2) transverse relaxation time was acquired by a
multi-echo turbo spin echo (TSE) sequence.
[0261] Monodisperse MNPs, nanogels and degraded nanogels at various
Fe concentrations were scanned using the following parameters: TR
3000 ms, TE 7-224 ms in steps of 7 ms, field of view (FOV)
140.times.120 mm.sup.2, matrix size 188.times.160, slice thickness
10 mm, number of signal average 1, acquisition band width 250
Hz/pixel, and total scan time is 5'21''.
[0262] T2 maps were generated from the multi-echo TSE images using
a custom-programmed algorithm coded in MATLAB (Mathworks, Natick,
Mass.). The T2 relaxation time of each sample was measured using
image processing software CASCADE. Images were loaded into the
software and then a region of interest (ROI) of no smaller than 2
cm.sup.2 was carefully delineated within the boundary of the
samples of interest. The average T2 relaxation time of the sample
was then measured automatically by CASCADE.
[0263] Release Test. Two PBS stock solutions (10 mL, with or
without 10 mM DTT) both containing 5 mg nanogels loaded with MNPs
and FITC-dextran were incubated at 37.degree. C. At different time
points, 0.5 mL solution from each sample solution was collected and
the released FITC-dextran was obtained by using a 100 kD
molecular-weight-cutoff Amicon Ultra centrifugal filter. The
fluorescence intensity of was determined by a fluorescence
spectrophotometer (F-4500 fluorescence spectrophotometer,
Hitachi).
Example 6
Zwitterionic Poly(Carboxybetaine) Hydrogels for Gold
Nanoparticles
[0264] In this example, the use of a representative zwitterionic
crosslinked hydrogel of the invention, CBMA/CBMAX (CBMAX is
carboxybetaine dimethacrylate), in a glucose biosensor is
described.
[0265] Synthesis of Initiator-Modified Gold Nanoparticles (GNPs). 5
mM aqueous solution of either HAuCl.sub.4 (30 mL) was added to a 4
mM solution of tetraoctylammonium bromide (TOAB) in toluene (80 mL)
under stirring for 10 min. Aqueous solution NaBH.sub.4 (0.4 M, 25
mL) was then added dropwise to this solution while vigorously
stirring. The dark orange solution turned red within a minute, and
the stirring was continued for 3 h to make sure the reaction was
complete. Then the two phases were separated, and the organic phase
was subsequently washed with 0.1 M H.sub.2SO.sub.4, 0.1 M NaOH, and
water (three times each). Then, initiator, 274.2 mg of
11-mercaptoundecyl 2-bromoisobutyrate
(Br(CH.sub.3).sub.2COO(CH.sub.2).sub.11SH) (0.808 mmol, dissolved
in 1 mL toluene) were added to the solution in a dropwise fashion
within 15 min. The reaction was allowed to proceed for overnight.
Methanol (60 mL) was added to the system to precipitate the Au-NPs.
The precipitate was collected and re-dispersed in toluene and
precipitated again into ethanol. This precipitation and
re-dispersion cycle was repeated twice before the pure Au-NPs (i.e.
free of reaction byproducts) were obtained. The NPs were well
dispersed in acetone without aggregation and the average diameter
of the Au-NPs was about 5 nm.
[0266] Preparation of CBMA coated GNPs via ATRP (CA-GNPs). 300 mg
CBMA monomer, 61.707 mg 2,2-bipyridine, and 28.533 mg copper(I)
bromide were dissolved in 3 ml degassed acetone and 0.5 ml methanol
under nitrogen atmosphere. 1 mL initiator-modified GNPs solution
was deoxygenated by bubbling nitrogen before mixed with above
solution. The final mixture was stirred (100 rpm) at room
temperature for 2 h. After the polymerization, CA-GNPs were washed
several times by centrifuging/redispersing in water. The average
diameter of the CA-NPs was 69.8 nm in water.
[0267] Preparation of OEGMA coated GNPs via ATRP (OA-GNPs). 47.7 mg
copper(I) bromide, 7.43 mg copper(II) bromide, and 104 mg
2,2-bipyridine were dissolved in 4 ml degassed acetone under
nitrogen. 1 mL initiator-modified GNPs solution was deoxygenated by
bubbling nitrogen before directly mixed with the above solution. 2
g macromonomer OEGMA was added and the final mixture was stirred at
room temperature for 6 h. After the polymerization, OA-GNPs were
washed several times by centrifuging/redispersing in Milli-Q water.
The average diameter of the OA-NPs was 72.4 nm in water.
[0268] Preparation of OEGMA coated GNPs via ATRP with addition of
EGDMA crosslinker (OC-GNPs). 47.7 mg copper(I) bromide, 7.43 mg
copper(II) bromide, and 104 mg 2,2-bipyridine were dissolved in 4
ml degassed acetone under nitrogen. 1 mL initiator-modified GNPs
solution was deoxygenated by bubbling nitrogen before directly
mixed with the above solution. 2 g macromonomer OEGMA and 126.4
.mu.L EGDMA was added and the final mixture was stirred at
50.degree. C. for 6 h. After the polymerization, OC-GNPs were
washed several times by centrifuging/redispersing in Milli-Q water.
The average diameter of the OC-NPs was 71.9 nm in water.
[0269] Preparation of CBMA coated GNPs via ATRP with addition of
CBMAX crosslinker (CC-GNPs). 300 mg CBMA monomer, 3.0 mg CBMAX,
61.7 mg 2,2-bipyridine, 4.4 mg copper(II) bromide and 28.533 mg
copper(I) bromide were dissolved in 3 ml degassed acetone and 0.5
ml methanol under nitrogen atmosphere. 1 mL initiator-modified GNPs
solution was deoxygenated by bubbling nitrogen before directly
mixed with the above solution. The final mixture was stirred at
50.degree. C. for 6 h. After the polymerization, CCE-GNPs were
washed several times by centrifuging/redispersing in Milli-Q water.
The average diameter of the CC-NPs was 80 nm in water.
[0270] Stability test of polymer-coated GNPs. The stability of
polymer-coated GNPs was further evaluated in 100% human blood serum
at 37.degree. C. Due to high protein concentrations, these
nanoparticles were separated from human blood serum proteins by
centrifugation and re-dispersed in PBS buffer. The average diameter
of the nanoparticles was then evaluated by DLS at 37.degree. C. All
the solutions were mixed with 100% human blood serum at 37.degree.
C. before the next test at different incubating time. As shown in
FIG. 36, OA-GNPs showed a size increase of about 50 nm in a very
short period of time. At the end of 72 h, the diameter increased to
about 140 nm, indicating significant protein adsorption and
particulate aggregation. Although OC-GNPs were not stable in such
extreme situation, the addition of EGDMA helped to enhance the
stability. The diameter increments were 6 nm and 30 nm after an
incubation period of 6 h and 72 h, respectively. Precipitates could
be observed in the above solutions. However, three kinds of GNPs
with the protection of polyCBAA coating (CA-GNPs, CCE-GNPs, and
CCC-GNPs), the interactions between proteins and nanoparticles did
not cause any agglomeration and the particle sizes after their
separation from human blood serum proteins was almost the same as
those without serum (70 nm, 50 nm and 105.9 nm), indicating their
excellent stability.
[0271] Next, polymer-coated nanoparticles were mixed with human
blood serum at a very high concentration and incubated at
37.degree. C. The average diameter of the nanoparticles was then
evaluated by DLS at 37.degree. C. As shown in FIG. 37, the OA-GNPs
showed an increase of about 20 nm in size after 6 h. This value
increased to 200 nm after 72 h, which was attributed to the
interactions of nanoparticles with proteins in the incubation serum
medium. Again, the addition of EGDMA increased the stability. The
diameter increment was 70 nm after an incubation period of 72 h.
However, with polyCBMA coating, there is no agglomeration and all
three samples showed good stability without obvious size increase
during the test period of 72 h.
[0272] While illustrative embodiments have been illustrated and
described, it will be appreciated that various changes can be made
therein without departing from the spirit and scope of the
invention.
* * * * *