U.S. patent application number 12/780194 was filed with the patent office on 2010-09-30 for hemostatic wound dressing.
This patent application is currently assigned to WASHINGTON, UNIVERSITY OF. Invention is credited to Gang Cheng, Shaoyi Jiang, Hong Xue.
Application Number | 20100247614 12/780194 |
Document ID | / |
Family ID | 40254461 |
Filed Date | 2010-09-30 |
United States Patent
Application |
20100247614 |
Kind Code |
A1 |
Jiang; Shaoyi ; et
al. |
September 30, 2010 |
HEMOSTATIC WOUND DRESSING
Abstract
Hemostatic wound dressings that include cationic polymers and
related hydrogels, methods for making and using the wound
dressings.
Inventors: |
Jiang; Shaoyi; (Redmond,
WA) ; Xue; Hong; (Seattle, WA) ; Cheng;
Gang; (Akron, OH) |
Correspondence
Address: |
Christensen O'Connor Johnson Kindness PLLC
1420 Fifth Avenue, Suite 2800
Seattle
WA
98101-2347
US
|
Assignee: |
WASHINGTON, UNIVERSITY OF
Seattle
WA
|
Family ID: |
40254461 |
Appl. No.: |
12/780194 |
Filed: |
May 14, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2008/084099 |
Nov 19, 2008 |
|
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12780194 |
|
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60989073 |
Nov 19, 2007 |
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Current U.S.
Class: |
424/445 ;
424/78.06; 525/54.1 |
Current CPC
Class: |
C08F 220/38 20130101;
C08F 120/38 20130101; C08F 20/36 20130101; C08F 120/36 20130101;
A61K 2800/5426 20130101; C08F 20/38 20130101; A61K 8/368 20130101;
A61P 17/02 20180101; A61P 31/10 20180101; A61Q 19/00 20130101; A61P
31/00 20180101; C08F 20/54 20130101; A61K 8/8158 20130101; C08F
220/36 20130101; A61P 31/04 20180101 |
Class at
Publication: |
424/445 ;
424/78.06; 525/54.1 |
International
Class: |
A61K 31/785 20060101
A61K031/785; A61P 17/02 20060101 A61P017/02; A61K 47/48 20060101
A61K047/48 |
Goverment Interests
STATEMENT OF GOVERNMENT LICENSE RIGHTS
[0002] This invention was made with Government support under Grant
Nos. N00014-04-1-0409 and N00014-07-1-1036 awarded by the Office of
Naval Research, Grant No. AB06BAS759 awarded by the Defense Threat
Reduction Agency, and Grant No. DMR0705907 awarded by the National
Science Foundation. The Government has certain rights in the
invention.
Claims
1. A wound dressing, comprising a cationic polymer comprising: (a)
polymer backbone; (b) a plurality of cationic centers, each
cationic center covalently coupled to the polymer backbone by a
first linker; (c) a counter ion associated with each cationic
center; and (d) a hydrolyzable group covalently coupled to each
cationic center through a second linker, wherein the hydrolyzable
group is hydrolyzable to an anionic center to provide a
zwitterionic polymer having the anionic center covalently coupled
to the cationic center through the second linker.
2. The wound dressing of claim 1, wherein the polymer has the
formula:
PB-(L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(=O)--OR.sub.c).sub.n(X.su-
p.-).sub.n 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(=O)--OR.sub.c);
N.sup.+ is the cationic center; R.sub.a and R.sub.b are
independently selected from hydrogen, alkyl, and aryl;
A(=O)--OR.sub.c is the hydrolyzable group, wherein A is selected
from the group consisting of C, S, SO, P, or PO, and R.sub.c is an
alkyl, aryl, acyl, or silyl group that may be further substituted
with one or more substituents; 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
hydrolyzable group; X.sup.- is the counter ion associated with the
cationic center; and n is an integer from about 10 to about
10,000.
3. The wound dressing of claim 1, wherein the counter ion is a
hydrophobic organic counter ion.
4. The wound dressing of claim 1, wherein the counter ion is
selected from the group consisting of C1-C20 carboxylates and
C1-C20 alkylsulfonates.
5. The wound dressing of claim 1, wherein the counter ion is a
therapeutic agent.
6. The wound dressing of claim 1, wherein the counter ion is
selected from the group consisting of an antimicrobial, an
antibacterial, and an antifungal agent.
7. The wound dressing of claim 1, wherein the counter ion is
selected from the group consisting of amino acids, proteins, and
peptides.
8. The wound dressing of claim 1, wherein the hydrolyzable group
releases a hydrophobic organic group on hydrolysis.
9. The wound dressing of claim 1, wherein the hydrolyzable group
releases a C1-C20 carboxylate on hydrolysis.
10. The wound dressing of claim 1, wherein the hydrolyzable group
releases a therapeutic agent on hydrolysis.
11. The wound dressing of claim 1, wherein the hydrolyzable group
releases an antimicrobial, an antibacterial, or an antifungal agent
on hydrolysis.
12. The wound dressing of claim 1, wherein the cationic center is
selected from the group consisting of ammonium, imidazolium,
triazaolium, pyridinium, morpholinium, oxazolidinium, pyrazinium,
pyridazinium, pyrimidinium, piperazinium, and pyrrolidinium.
13. The wound dressing of claim 2, wherein R.sub.a and R.sub.b are
independently selected from the group consisting of C1-C10 straight
chain and branched alkyl groups.
14. The wound dressing of claim 2, wherein L.sub.1 is selected from
the group consisting of --C(.dbd.O)O--(CH.sub.2).sub.n-- and
--C(.dbd.O)NH--(CH.sub.2).sub.n--, wherein n is an integer from 1
to 20.
15. The wound dressing of claim 2, wherein L.sub.2 is
--(CH.sub.2).sub.n--, where n is an integer from 1 to 20.
16. The wound dressing of claim 2, wherein A is selected from the
group consisting of C, SO, and PO.
17. The wound dressing of claim 2, wherein R.sub.c is C1-C20
alkyl.
18. The wound dressing of claim 2, wherein R.sub.c is an amino
acid.
19. The wound dressing of claim 2, wherein X.sup.- is selected from
the group consisting of halide, carboxylate, alkylsulfonate,
sulfate; nitrate, perchlorate, tetrafluoroborate,
hexafluorophosphate, trifluoromethylsulfonate,
bis(trifluoromethylsulfonyl)amide, lactate, and salicylate.
20. The wound dressing of claim 1, wherein the cationic polymer is
a hydrogel.
21. The wound dressing of claim 20, wherein the hydrogel is a
chemical hydrogel.
22. The wound dressing of claim 20, wherein the hydrogel is an
interpenetrating network hydrogel.
23. The wound dressing of claim 20, wherein the hydrogel comprises
first and second polymers, wherein the first polymer is a cationic
polymer hydrolyzable to provide a zwitterionic polymer, and wherein
the second polymer is a zwitterionic polymer.
24. The wound dressing of claim 23, wherein the first and second
polymers are crosslinked.
25. The wound dressing of claim 23, wherein the hydrogel is
prepared by copolymerizing a first cationic monomer having a
hydrolyzable group and second zwitterionic monomer.
26. The wound dressing of claim 24, wherein the hydrogel is
prepared by copolymerizing a first cationic monomer having a
hydrolyzable group and second zwitterionic monomer.
27. The wound dressing of claim 25, wherein the hydrogel is
prepared by polymerizing the first monomer to provide a cationic
polymer having hydrolyzable groups, adding the second monomer to
the cationic polymer, and polymerizing the second monomer in the
presence of the cationic polymer.
28. The wound dressing of claim 25, wherein the hydrogel is
prepared by polymerizing the second monomer to provide a
zwitterionic polymer, adding the first monomer having a
hydrolyzable group to the zwitterionic polymer, and polymerizing
the first monomer in the presence of the zwitterionic polymer.
29. The wound dressing of claim 20, wherein the hydrogel is
prepared by polymerizing a first cationic monomer having a
hydrolyzable group to provide a cationic polymer having
hydrolyzable groups, and hydrolyzing at least a portion of the
hydrolyzable groups of the cationic polymer.
30. A method for treating a wound, comprising applying the wound
dressing of claim 1 to a wound.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of International Patent
Application No. PCT/US2008/084099, filed Nov. 19, 2008, which
claims the benefit of U.S. Provisional Application No. 60/989,073,
filed Nov. 19, 2007. Each application is incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] Uncontrolled bleeding remains the leading cause of
preventable death in the battlefield and effective control of
hemorrhage can improve outcome. Early and effective hemorrhage
control is obviously important and can save more lives than any
other measure. The use of effective hemostatic dressings will
benefit most combat injuries. Furthermore, the nature of combat
injuries is such that bacterial contamination is frequently present
in traumatic wounds. Infections that develop in traumatic and
surgical wounds remain a major problem. There has been an increased
effort to develop better hemostatic agents and dressings, some of
which also have antimicrobial capability. Over the last decade,
many hemostatic agents and dressings such as collagen, dry fibrin
thrombin, chitosan, quaternary amine-containing compound, silver,
and antimicrobial-loaded poly(ethylene glycol) (PEG) polymer have
been tested with a variable degree of success. While some of them
do not adequately prevent and limit bleeding, hemorrhaging, or
bacterial infection, others may present side effects or
antimicrobial-resistance. Some dressings can also be very expensive
(e.g., $ 1,000 per fibrin dressing).
[0004] Two hemostatic products designed to control severe
hemorrhage are used by the military and commercially available: (1)
chitosan-based HemCon.RTM. (HC) Bandage (HemCon Inc., Tigard,
Oreg.) and (2) granular zeolite, QuickClot.RTM. (QC) (Z-Medica,
Newington, Conn.). The chitosan dressing is a deacetylated complex
carbohydrate derived from the naturally occurring substance chitin
(N-acetyl D-glucosamine). Because chitosan has a positive charge,
it attracts negatively charged red blood cells and offers an
antibacterial barrier. Both dressings were judged to be effective
based on study findings to date, but the Committee on Tactical
Combat Casualty Care (COTCCC) was not able to identify a clear
winner based on efficacy. According to the committee's
recommendations, all the bleeding wounds should initially be
treated with standard of care (e.g., pressure dressings and
tourniquets). If the bleeding continues, application of HemCon.RTM.
with manual compression should be the next step. Finally, if
bleeding still does not stop, then HemCon.RTM. should be removed
and QuickClot.RTM. applied with manual compression for 5 minutes as
a lifesaving measure. There is no single perfect hemostatic
dressing. Each has its drawbacks and benefits. While QuickClot.RTM.
is effective, granular zeolite becomes markedly exothermic in blood
and may result in thermal injury to tissues. Chitosan-based wound
dressings also have several issues: (a) HC chitosan has limited
solubility; (b) chitosan-based wound dressings are not as efficient
as hydrogel-based counterparts and have limited capability to
absorb wound fluids, wound fluids are absorbed by other means
before HC bandages are applied; (c) HC chitosan is prepared and
compressed on a pad and is a fairly rigid wafer and, as a result,
these HC dressings work well on planar surfaces, but some
investigators have reported difficulties in conforming them to
deep, narrow wounds or wounds of more irregular shapes; (d) the
amines in chitosan are not as effective as quaternary amines
against bacterial infection; (e) the difficulties in production
(e.g., batch to batch variability) need to be resolved.
[0005] Despite the advancement of hemostatic dressings, there
exists a need for new hemostatic wound dressings having improved
properties. The present invention seeks to fulfill this need and
provides further related advantages.
SUMMARY OF THE INVENTION
[0006] The invention provides hemostatic wound dressings that
include cationic polymeric materials hydrolyzable to zwitterionic
polymeric materials. Methods for making and using the cationic
polymeric materials and wound dressings are also provided.
[0007] In one aspect, the invention provides a wound dressing that
includes a cationic polymer comprising:
[0008] (a) polymer backbone;
[0009] (b) a plurality of cationic centers, each cationic center
covalently coupled to the polymer backbone by a first linker;
[0010] (c) a counter ion associated with each cationic center;
and
[0011] (d) a hydrolyzable group covalently coupled to each cationic
center through a second linker, wherein the hydrolyzable group is
hydrolyzable to an anionic center to provide a zwitterionic polymer
having the anionic center covalently coupled to the cationic center
through the second linker.
[0012] In one embodiment, the polymer has the formula:
PB-(L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(=O)--OR.sub.c).sub.n(X.s-
up.-).sub.n
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(=O)--OR.sub.c); N.sup.+
is the cationic center; R.sub.a and R.sub.b are independently
selected from hydrogen, alkyl, and aryl; A(=O)--OR.sub.c is the
hydrolyzable group, wherein A is selected from the group consisting
of C, S, SO, P, or PO, and R.sub.c is an alkyl, aryl, acyl, or
silyl group that may be further substituted with one or more
substituents; 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 hydrolyzable group;
X.sup.- is the counter ion associated with the cationic center; and
n is an integer from about 10 to about 10,000.
[0013] In one embodiment, the counter ion is a hydrophobic organic
counter ion. Representative counter ions include is C1-C20
carboxylates and C1-C20 alkylsulfonates.
[0014] In one embodiment, the counter ion is a therapeutic agent.
Representative therapeutic counter ions include antimicrobial,
antibacterial, and antifungal agents.
[0015] In certain embodiments, the counter ion is an amino acid,
protein, or peptide.
[0016] In one embodiment, the hydrolyzable group releases a
hydrophobic organic group on hydrolysis. Representative hydrophobic
groups include C1-C20 carboxylates.
[0017] In one embodiment, the hydrolyzable group releases a
therapeutic agent on hydrolysis. Representative therapeutic agents
include antimicrobial, antibacterial, and antifungal agents.
[0018] In certain embodiments, the cationic center is selected from
ammonium, imidazolium, triazaolium, pyridinium, morpholinium,
oxazolidinium, pyrazinium, pyridazinium, pyrimidinium,
piperazinium, and pyrrolidinium groups.
[0019] In one embodiment, R.sub.a and R.sub.b are independently
selected from C1-C10 straight chain and branched alkyl groups.
[0020] In one embodiment, L.sub.1 is selected from the group
consisting of --C(.dbd.O)O--(CH.sub.2).sub.n-- and
--C(.dbd.O)NH--(CH.sub.2).sub.n--, wherein n is an integer from 1
to 20.
[0021] In one embodiment, L.sub.2 is --(CH.sub.2).sub.n--, where n
is an integer from 1 to 20.
[0022] In one embodiment, A is selected from the group consisting
of C, SO, and PO.
[0023] In one embodiment, R.sub.c is C1-C20 alkyl. In another
embodiment, R.sub.c is an amino acid.
[0024] In certain embodiments, X.sup.- is selected from halides,
carboxylates, alkylsulfonates, sulfate; nitrate, perchlorate,
tetrafluoroborate, hexafluorophosphate, trifluoromethylsulfonate,
bis(trifluoromethylsulfonyl)amide, lactate, and salicylate.
[0025] In one embodiment, the cationic polymer is a hydrogel.
Representative hydrogels include chemical hydrogels and
interpenetrating network hydrogels.
[0026] In one embodiment, the hydrogel comprises first and second
polymers, wherein the first polymer is a cationic polymer
hydrolyzable to provide a zwitterionic polymer, and wherein the
second polymer is a zwitterionic polymer. In one embodiment, the
first and second polymers are crosslinked.
[0027] In one embodiment, the hydrogel is prepared by
copolymerizing a first cationic monomer having a hydrolyzable group
and second zwitterionic monomer, optionally with a crosslinking
agent.
[0028] In one embodiment, the hydrogel is prepared by polymerizing
the first monomer to provide a cationic polymer having hydrolyzable
groups, optionally with a crosslinking agent; adding the second
monomer to the cationic polymer, and polymerizing the second
monomer in the presence of the cationic polymer, optionally with a
crosslinking agent. In another embodiment, the hydrogel is prepared
by polymerizing the second monomer to provide a zwitterionic
polymer, optionally with a crosslinking agent; adding the first
cationic monomer having a hydrolyzable group to the zwitterionic
polymer, and polymerizing the first monomer in the presence of the
zwitterionic polymer, optionally with a crosslinking agent.
[0029] In one embodiment, the hydrogel is prepared by polymerizing
a first cationic monomer having a hydrolyzable group, optionally
with a crosslinking agent, to provide a cationic polymer having
hydrolyzable groups, and hydrolyzing at least a portion of the
hydrolyzable groups of the cationic polymer.
[0030] In another aspect of the invention, a method for treating a
wound is provided. In one embodiment, the method includes applying
a wound dressing of the invention to a wound.
DESCRIPTION OF THE DRAWINGS
[0031] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0032] 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, wherein:
[0033] FIG. 1 illustrates the structures of three representative
cationic monomers useful for making cationic polymers useful in the
invention: three acrylamide monomers with different carboxybetaine
ester groups; CBAA-1-ester, CBAA-3-ester, and CBAA-5-ester.
[0034] FIG. 2 illustrates the hydrolysis of a representative
cationic polymer of the invention: hydrolysis of a cationic
polycarboxybetaine ester to zwitterionic polycarboxybetaine.
[0035] FIG. 3 compares the 'H NMR spectra of the hydrolysis of a
representative cationic polymer of the invention, polyCBAA-3-ester,
after one-hour treatment in a solution with the sodium hydroxide
concentration of (a) 10 mM (3% hydrolysis), (b) 100 mM (82%
hydrolysis), and (c) 1 M (100% hydrolysis).
[0036] FIG. 4 compares the hydrolysis rates of representative
cationic polymers useful in the invention at 10 mM and 100 mM
aqueous sodium hydroxide.
[0037] FIGS. 5A-5C are SPR sensorgrams for fibrinogen adsorption on
the surfaces grafted with representative polymers useful in the
invention: polycarboxybetaine esters before and after hydrolysis;
(a) polyCBAA-1-ester, (b) polyCBAA-3-ester, and (c)
polyCBAA-5-ester. The surfaces with polymer brushes were hydrolyzed
with a 100 mm NaOH solution for 1-2 h.
[0038] FIG. 6 is a graph comparing antimicrobial activities of
three representative cationic polymers useful in the invention,
polyCBAA-esters, before and after hydrolysis. E. coli (10.sup.8
cells/mL) was incubated with each polymer solution (repeat unit
molar concentration: 2 mM) for 30 min. PBS buffer (pH 7.4 and 150
mM) is used as a negative control.
[0039] FIG. 7 is a schematic illustration of a representative
surface of the invention coated with a cationic polymer. The
surface switches from an antibacterial surface to a non-fouling
surface upon hydrolysis: (a) antimicrobial cationic pCBMA-1 C2
effectively kills bacteria, (b) pCBMA-1 C2 is converted to
non-fouling zwitterionic pCBMA-1 upon hydrolysis, (c) killed
bacteria remaining on the surface is released from non-fouling
zwitterionic pCBMA-1 demonstrating that (d) zwitterionic pCBMA-1
itself is highly resistant to bacterial adhesion.
[0040] FIG. 8 illustrates the chemical structures of a
representative cationic polymer of the invention, switchable
pCBMA-1 C2; antimicrobial cationic pC8NMA; and non-fouling
zwitterionic pCBMA-2.
[0041] FIG. 9 is a graph comparing bactericidal activity of pCBMA-1
C2 and pC8NMA against E. coli K12. The percentage of live E. coli
K12 colonies that grew on the surfaces coated with antimicrobial
polymers is relative to the number of colonies that grew on the
pCBMA-2 control (n=3).
[0042] FIGS. 10A-10F are fluorescence microscopy images of attached
E. coli K12 cells (red color) from a suspension with 10.sup.10
cellsmL.sup.-1 for one-hour exposure to the surfaces covered with
various polymers: (a), (c), and (e) are for pCBMA-1 C2, pC8NMA and
pCBMA-2, respectively, before hydrolysis and (b), (d), and (f) are
for the same polymers, respectively, after hydrolysis. Hydrolysis
was for 8 days with 10 mM CAPS (pH 10.0).
[0043] FIG. 11 is a graph comparing the attachment of E. coli K12
from a suspension with 10.sup.10 cells mL.sup.-1 for one-hour
exposure to pCBMA-1 C2, pC8NMA, and pCBMA-2 before and after
hydrolysis (n=3).
[0044] FIG. 12A compares SPR sensorgrams showing the adsorption of
1 mg mL.sup.-1 fibrinogen in PBS buffer on the surfaces grafted
with pCBMA-1 C2 via ATRP (a) before hydrolysis, and (b), (c) and
(d) after 24 hr hydrolysis with water, 10 mM CEHS at pH 9.0, and 10
mM CAPS at pH 10.0, respectively; FIG. 12B compares SPR sensorgrams
showing the adsorption of 1 mgmL.sup.-1 fibrinogen in PBS buffer on
the surfaces grafted with pC8NMA (a) before and (b) after 24 hr
incubation with 10 mM CAPS at pH 10.0, and on the surfaces grafted
with pCBMA-2 (c) before hydrolysis and (d) after 24 h of hydrolysis
with 10 mM CAPS at pH 10.0.
[0045] FIG. 13 illustrates the structure of a representative
cationic monomers useful for making cationic polymers useful in the
invention: CBMA-1 C2 SA, the ethyl ester of CBMA-1 having a
salicylate counter ion.
[0046] FIG. 14 compares the release rate (mg/h) of salicylic acid
over time (12 h, 39 h, and 63 h) at 25.degree. C. under four
conditions from hydrogels prepared by polymerizing CBMA-1 C2 SA:
(a) water, neutral pH; (b) phosphate buffered saline (PBS); (c)
water, pH 10; and (d) 0.15 M aqueous sodium chloride, pH 10.
[0047] FIG. 15 compares the release rate (mg/h) of salicylic acid
over time (12 h, 39 h, and 63 h) at 37.degree. C. under four
conditions from hydrogels prepared by polymerizing CBMA-1 C2 SA:
(a) water, neutral pH; (b) phosphate buffered saline (PBS); (c)
water, pH 10; and (d) 0.15 M aqueous sodium chloride, pH 10.
[0048] FIG. 16 illustrates schematic representative wound dressing
hydrogels of the invention containing both zwitterionic and
cationic zwitterionic precursor polymers. The cationic zwitterionic
precursor polymers are useful for hemostatic and antimicrobial
actions and the zwitterionic polymers are useful for wound fluid
adsorbents. After action, cationic zwitterionic precursor polymers
are converted to nontoxic, non-sticky, and biocompatible
zwitterionic polymers by hydrolysis. These wound dressing hydrogels
can be prepared from two monomers (e.g., CBMA and CBMA ester) via
polymerization or from just one monomer (CBMA ester) by partially
hydrolyzing the corresponding pCBMA ester hydrogel.
[0049] FIGS. 17A-17B illustrate the SPR response for nonspecific
adsorption of 10% human serum in PBS and 100% human serum (17A) and
non-specific adsorption of 10% human plasma in PBS and 100% human
serum (17B). Error bars represent the standard error of the mean.
An adsorbed protein monomer is equivalent to 2,500 pg/mm.sup.2.
[0050] FIG. 18 illustrates the resistance of a representative
zwitterionic polymer, CBAA-2, to nonspecific protein adsorption
from 100% blood plasma and 100% serum (<0.3 ng/cm.sup.2 adsorbed
proteins).
[0051] FIGS. 19A-19H compares microscopy images of accumulated P.
aeruginosa on surfaces treated with pCBMA (FIGS. 19C-19H, days 1,
3, 5, 7, 10, and 11, respectively) with untreated (FIG. 19A) and
OEG SAM-modified glass substrates (FIG. 19B) (as references) in
growth medium over an 11-day growth period.
[0052] FIG. 20 is a schematic illustration of the preparation of a
zwitterionic CBMA monomer.
[0053] FIG. 21 is a schematic illustration of the preparation of
representative cationic polymers useful in the invention, CBMA
ester monomers (m=1-20 and n=1-5).
[0054] FIG. 22 is a schematic illustration of the preparation of
representative hydrogels of the invention from the zwitterionic
monomer illustrated in FIG. 20 and the cationic monomer illustrated
in FIG. 21: pCBMA ester/pCBMA chemical hydrogels.
[0055] FIG. 23 is a schematic illustration of the preparation of
representative IPN hydrogels of the invention from the zwitterionic
monomer illustrated in FIG. 20 and the cationic monomer illustrated
in FIG. 21: CBMA ester/pCBMA IPN hydrogels.
[0056] FIG. 24 is a schematic illustration of the preparation of
representative hydrogels of the invention from the cationic monomer
illustrated in FIG. 21 by partial hydrolysis: partially hydrolyzed
pCB ester hydrogels.
[0057] FIG. 25 illustrates the chemical structure of a
representative cationic polymer of the invention having a glycine
leaving group: pCBMA with a glycine leaving group.
[0058] FIG. 26 illustrates a representative wound dressing of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The invention provides hemostatic wound dressings that
include cationic polymeric materials hydrolyzable to zwitterionic
polymeric materials. Methods for making and using the cationic
polymeric materials and wound dressings are also provided.
[0060] In one aspect of the invention, hemostatic wound dressings
that include cationic polymeric materials hydrolyzable to
zwitterionic polymeric materials are provided. The cationic
polymers useful in the invention include hydrolyzable groups that
can be hydrolyzed to provide zwitterionic polymers. Zwitterionic
polymers are polymers having a balance of positive and negative
charge. Zwitterionic polymers can be highly resistant to protein
adsorption and bacterial adhesion. Due to their biomimetic nature,
zwitterionic polymers, such as phosphobetaine, sulfobetaine, and
carboxybetaine polymers, exhibit high biocompatibility.
[0061] Controlled Hydrolysis. The variation of the structural
features of the cationic polymers allows for their controlled
hydrolysis and the control of the biological, chemical, and
mechanical properties. The rate of hydrolysis can be significantly
affected by and controlled by the selection of the nature of the
hydrolyzable group (e.g., for esters, --OR).
[0062] As described below, in certain embodiments, the cationic
polymers useful in the invention advantageously release functional
groups on hydrolysis. For example, for cationic esters of the
invention, hydrolysis ester releases an --OR group. In these
embodiments, the released group can be a therapeutic agent (e.g.,
an antimicrobial, antibacterial, an antifungal agent). Similarly,
in certain embodiments, the cationic polymers can release their
counter ions (X.sup.-), which can also be therapeutic agents (e.g.,
nucleic acids, amino acids, peptides, proteins, and
salicylate).
[0063] For applications as antimicrobial agents, antimicrobial
cationic polymers can be converted to zwitterionic polymers,
leaving no toxic residues in the environment or no killed microbes
on a surface.
[0064] It will be appreciated that the hydrolyzable group can be
cleaved not only by hydrolysis, but also by cleavage (e.g.,
degradation or erosion) that occurs by other means. The cationic
polymers can be converted to their corresponding zwitterionic
polymers by environmental changes due to enzymatic catalysis,
redox, heat, light, ionic strength, pH, and hydrolysis, among
others.
[0065] Representative cationic polymers useful in the invention and
their corresponding zwitterionic polymer counterparts are described
below.
[0066] Cationic Polymers
[0067] The cationic polymers useful in the invention include
hydrolyzable groups that, when hydrolyzed, provide anionic groups
that render the polymer zwitterionic. In each polymer, the number
of hydrolyzable groups is substantially equal to the number of
cationic groups such that, when the hydrolyzable groups are
hydrolyzed, in the resulting polymer is zwitterionic. As used
herein, the term "zwitterionic polymer" refers to a polymer having
substantially equal numbers of cationic groups and anionic
groups.
[0068] Representative cationic polymers useful in the invention
have formula (I):
PB-(L.sub.1-N.sup.+(R.sub.a)(R.sub.b-L.sub.2-A(=O)--OR.sub.c).sub.n(X.su-
p.-).sub.n (I)
wherein PB is the polymer backbone having n pendant groups (i.e.,
L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(=O)--OR.sub.c); N.sup.+
is the cationic center; R.sub.a and R.sub.b are independently
selected from hydrogen, alkyl, and aryl groups; A(=O)--OR.sub.c) is
the hydrolyzable group, wherein A is C, S, SO, P, or PO, and
R.sub.c is an alkyl, aryl, acyl, or silyl group that may be further
substituted with one or more substituents; 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 hydrolyzable group; X.sup.- is the counter ion associated with
the cationic center; and n is from about 10 to about 10,000. The
average molecular weight of the polymers of formula (I) is from
about 1 kDa to about 1,000 kDa.
[0069] Hydrolysis of the cationic polymer of formula (I) provides
zwitterionic polymer having formula (II):
PB-(L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(=O)O.sup.-).sub.n
(II)
wherein PB, L.sub.1, N.sup.+, R.sub.a, R.sub.b, L.sub.2, A, and n
are as described above, and A(=O)O.sup.- is the anionic group.
[0070] In this embodiment, the polymer of formula (I) includes n
pendant groups and can be prepared by polymerization of monomers
having formula (III):
CH.sub.2.dbd.C(R.sub.d)-L.sub.1-N.sup.+(R.sub.a)(R.sub.b)-L.sub.2-A(=O)--
-OR.sub.cX.sup.- (III)
wherein L.sub.1, N.sup.+, R.sub.a, R.sub.b, A(=O)OR.sub.c, and
L.sub.2, and X.sup.- are as described above, R.sub.d is selected
from hydrogen, fluorine, trifluoromethyl, C1-C6 alkyl, and C6-C12
aryl groups.
[0071] In formulas (I) and (II), 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). Other suitable backbones include polymer
backbones that provide for pendant cationic groups that include
hydrolyzable groups that can be converted to zwitterionic groups,
and backbones that include cationic groups and that provide for
pendant hydrolyzable groups that can be converted to zwitterionic
groups. Other representative polymer backbones include peptide
(polypeptide), urethane (polyurethane), and epoxy backbones.
[0072] Similarly, in formula (III), CH.sub.2.dbd.C(R.sub.d)-- is
the polymerizable group. It will be appreciated that other
polymerizable groups, including those noted above, can be used to
provide the monomers and polymers of the invention.
[0073] The following is a description of the polymers and monomers
of formulas (I)-(III) described above.
[0074] In formulas (I)-(III), N.sup.+ is the cationic center. In
certain embodiments, the cationic center is a quaternary ammonium
(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.
[0075] R.sub.a and R.sub.b are independently selected from
hydrogen, alkyl, and aryl groups. Representative alkyl groups
include C1-C10 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. For certain embodiments of
formulas (I)-(III), R.sub.2 or R.sub.3 is absent.
[0076] 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 (or polymerizable
moiety for the monomer of formula (III)). In addition to the
functional group, L.sub.1 can include an C1-C20 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 1-20 (e.g., 3).
[0077] L.sub.2 is a linker that covalently couples the cationic
center to the hydrolyzable group (or anionic group for the
zwitterionic polymer of formula (II)). L.sub.2 can be a C1-C20
alkylene chain. Representative L.sub.2 groups include
--(CH.sub.2).sub.n--, where n is 1-20 (e.g., 1, 3, or 5).
[0078] The hydrophobicity and the rate of hydrolysis of the
cationic polymers of formula (I) can be controlled by L.sub.1
and/or L.sub.2. The greater the hydrophobicity of L.sub.1 or
L.sub.2, the slower the hydrolysis of the hydrolyzable group and
the conversion of the cationic polymer to the zwitterionic
polymer.
[0079] A(=O)--OR.sub.c is the hydrolyzable group. The hydrolyzable
group can be an ester, such as a carboxylic acid ester (A is C), a
sulfinic acid ester (A is S), a sulfonic acid ester (A is SO), a
phosphinic acid ester (A is P), or a phosphonic acid ester (A is
PO). The hydrolyzable group can also be an anhydride. R.sub.c is an
alkyl, aryl, acyl, or silyl group that may be further substituted
with one or more substituents.
[0080] Representative alkyl groups include C1-C30 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
certain embodiments, R.sub.c is a C1-C20 straight chain alkyl
group. In one embodiment, R.sub.c is methyl. In another embodiment,
R.sub.c is ethyl. In one embodiment, R.sub.c is a C3-C20 alkyl. In
one embodiment, R.sub.c is a C4-C20 alkyl. In one embodiment,
R.sub.c is a C5-C20 alkyl. In one embodiment, R.sub.c is a C6-C20
alkyl. In one embodiment, R.sub.c is a C8-C20 alkyl. In one
embodiment, R.sub.c is a C10-C20 alkyl. For applications where
relatively slow hydrolysis is desired, R.sub.c is a C4-C20 n-alkyl
group or a C4-C30 n-alkyl group.
[0081] Representative aryl groups include C6-C12 aryl groups
including, for example, phenyl including substituted phenyl groups
(e.g., benzoic acid).
[0082] Representative acyl groups (--C(.dbd.O)R.sub.e) include acyl
groups where R.sub.e is C1-C20 alkyl or C6-C12 aryl.
[0083] Representative silyl groups (--SiR.sub.3) include silyl
groups where R is C1-C20 alkyl or C6-C12 aryl.
[0084] In certain embodiments of the invention, the hydrolysis
product R.sub.cO.sup.- (or R.sub.cOH) is a therapeutic agent (e.g.,
an antimicrobial agent, such as salicylic acid (2-hydroxybenzoic
acid), benzoate, lactate, and the anion form of antibiotic and
antifungal drugs).
[0085] In certain other embodiments, the hydrolysis product
R.sub.cO.sup.- (or R.sub.cOH) is lactate, glycolate, or an amino
acid.
[0086] The rate of hydrolysis of the cationic polymers of formula
(I) can also be controlled by R.sub.c. The slower the hydrolysis of
the hydrolyzable group due to, for example, steric and/or kinetic
effects due to R.sub.c, the slower the conversion of the cationic
polymer to the zwitterionic polymer.
[0087] 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 cationic polymer of formula (I) or the
monomers of formula (III) (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.
[0088] The rate of hydrolysis of the cationic polymers of formula
(I) can be controlled by the counter ion. The more hydrophobic the
counter ion, the slower the hydrolysis of the hydrolyzable group
and the slower the conversion of the cationic polymer to the
zwitterionic polymer. Representative hydrophobic counter ions
include 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.sup.-(SO.sub.2CF.sub.3).sub.2); and derivatives thereof.
Other counter ions also can be chosen from chloride, bromide,
iodide, sulfate; nitrate; perchlorate (ClO.sub.4);
tetrafluoroborate (BF.sub.4); hexafluorophosphate (PF.sub.6);
trifluoromethylsulfonate (SO.sub.3CF.sub.3); and derivatives
thereof.
[0089] Other suitable counter ions include hydrophobic counter ions
and counter ions having therapeutic activity (e.g., an
antimicrobial agent, such as salicylic acid (2-hydroxybenzoic
acid), benzoate, lactate, and the anion form of antibiotic and
antifungal drugs).
[0090] For the monomer of formula (III), R.sub.d is selected from
hydrogen, fluoride, trifluoromethyl, and C1-C6 alkyl (e.g., methyl,
ethyl, propyl, butyl). In one embodiment, R.sub.d is hydrogen. In
one embodiment, R.sub.d is methyl. In another embodiment, R.sub.d
is ethyl.
[0091] The variation of the structural features of the cationic
polymers allows for their controlled hydrolysis and the control of
the biological, chemical, and mechanical properties. The structural
features of the cationic polymers noted above that can be varied to
achieve the desired controlled hydrolysis of the polymer include
L.sub.1, L.sub.2, R.sub.a, R.sub.b, A, R.sub.c, and X.sup.-. In
general, the more hydrophobic the polymer or the noted structural
feature, the slower the hydrolysis of the cationic polymer to the
zwitterionic polymer.
[0092] Homopolymers, Random Copolymers, Block Copolymers. The
cationic polymers useful in the invention include homopolymers,
random copolymers, and block copolymers.
[0093] In one embodiment, the invention provides cationic
homopolymers, such as defined by formula (I), prepared by
polymerizing a cationic monomer, such as defined by formula (III).
It will be appreciated that the advantageous properties associated
with cationic polymers useful in the invention including those
polymers defined by formula (I) can be imparted to other polymeric
materials.
[0094] In one embodiment, the invention provides random copolymers
prepared by copolymerizing two different cationic monomers of
formula (III).
[0095] In another embodiment, the invention provides random
copolymers that include cationic repeating units prepared by
copolymerizing one or more cationic monomers of the invention
defined by formula (III) with one or more other monomers (e.g.,
hydrophobic monomers, anionic monomers, or zwitterionic monomers,
such as phosphorylbetaine, sulfobetaine, or carboxybetaine
monomers).
[0096] In one embodiment, the invention provides block copolymers
having one or more blocks comprising cationic repeating units and
one or more other blocks. In this embodiment, the one or more
blocks that include cationic repeating units include only cationic
repeating units (e.g., homo- or copolymer prepared from cationic
monomers of formula (III)). Alternatively, the one or more blocks
that include cationic repeating units include cationic repeating
units and other repeating units (e.g., hydrophobic, anionic,
zwitterionic repeating units).
[0097] Other Suitable Polymers
[0098] The invention also provides the following polymers.
[0099] In one embodiment, the cationic polymer has the following
structure:
##STR00001##
[0100] R.sub.1=--H, --CH.sub.3, --C.sub.2H.sub.5
[0101] R.sub.2=no atom, --H, --CH.sub.3, --C.sub.2H.sub.5
[0102] R.sub.3=--H, --CH.sub.3, --C.sub.2H.sub.5
[0103] x=1-8.
[0104] R=any alkyl chain, aromatic or lactate or glycolate
##STR00002##
[0105] R.sub.4=--H, --CH.sub.3, --C.sub.2H.sub.5
[0106] Y=1-10
[0107] Z=0-22
[0108] or C(.dbd.O)R'
[0109] R'=any alkyl chain or aromatic group.
[0110] In another embodiment, the cationic polymer has the
following structure:
##STR00003##
[0111] n>5
[0112] x=1-5
[0113] y=1-5
[0114] R.sub.1=H, or alkyl chain
[0115] R.sub.2=no atom, H, or alkyl chain
[0116] R.sub.3=alkyl chain.
[0117] In another embodiment, the invention provides a polymer
having the following structure:
##STR00004##
[0118] R.sub.1 is any alkyl chain
[0119] R.sub.3 is any alkyl chain
[0120] R.sub.2, R.sub.4 is any alkyl chain
[0121] x=1-18
[0122] y=1-18
[0123] n>3.
[0124] In another embodiment, the invention provides a polymer
having the following structure:
##STR00005##
[0125] R is alkyl chain
[0126] x=1-18
[0127] y=1-18
[0128] n>3.
[0129] In another embodiment, the invention provides a polymer
having the following structure:
##STR00006##
[0130] R=any alkyl chain
[0131] x=0-11
[0132] n>3.
[0133] In another embodiment, the invention provides a polymer
having the following structure:
##STR00007##
[0134] n>3
[0135] x=1-10
[0136] R=any alkyl chain, aromatic or lactate or glycolate.
##STR00008##
[0137] R.sub.4=--H, --CH.sub.3, --C.sub.2H.sub.5
[0138] y=1-10
[0139] z=0-22
[0140] or C(.dbd.O)R'
[0141] R'=any alkyl chain, aromatic group.
[0142] In another embodiment, the invention provides polymers
having the following structure:
##STR00009##
[0143] n>3
[0144] x=1-6
[0145] y=0-6
[0146] R=any alkyl chain, aromatic or lactate or glycolate)
##STR00010##
[0147] R.sub.4=--H, --CH.sub.3, --C.sub.2H.sub.5
[0148] y=1-10
[0149] z=0-22
[0150] or C(.dbd.O)R'
[0151] R'=any alkyl chain, aromatic group.
[0152] In another embodiment, the invention provides a polymer
having the following structure:
##STR00011##
[0153] n>5
[0154] x=0-5.
[0155] In another embodiment, the invention provides a polymer
having the following structure:
##STR00012##
[0156] x=0-17
[0157] n>5
[0158] R.dbd.H or alkyl chain.
[0159] In another embodiment, the invention provides a polymer
having the following structure:
##STR00013##
[0160] n>5
[0161] R.sub.2=H or any alkyl chain, e.g., methyl
[0162] x, y=1-6
[0163] R.sub.1=any alkyl chain,
##STR00014##
[0164] R.sub.4=--H, --CH.sub.3, --C.sub.2H.sub.5
[0165] y=1-10
[0166] z=0-22
[0167] In another embodiment, the invention provides a polymer
having the following structure:
##STR00015##
[0168] n>3
[0169] R.sub.1=any alkyl chain.
[0170] Three representative cationic monomers of formula (III)
useful for making cationic polymers of formula (I), and ultimately
the zwitterionic polymers of formula (II) are illustrated in FIG.
1. Referring to FIG. 1, three positively charged polyacrylamides
having pendant groups that bear cationic carboxybetaine ester
groups are illustrated. The three monomers have different spacer
groups (L.sub.2: --CH.sub.2).sub.n--) between the quaternary
ammonium groups (cationic center) and the ester (hydrolyzable)
groups: CBAA-1-ester (n=1); CBAA-3-ester (n=3); and CBAA-5-ester
(n=5). Polymerization of the monomers provides the corresponding
cationic polymers. The three monomers were polymerized using free
radical polymerization to form linear polymers, or using
surface-initiated ATRP to prepare polymer brushes on SPR sensors.
The polymers with different spacer groups (L.sub.2) and ester
groups were expected to have different chemical, physical and
biological properties. The synthesis of the three monomers and
their polymerizations are described in Example 1.
[0171] For the linear polymers polymerized via free radical
polymerization, their molecular weights were measured using gel
permeation chromatography (GPC) in aqueous solutions.
PolyCBAA-1-ester, polyCBAA-3-ester, and polyCBAA-5-ester had
average molecular weights of 14 kDa, 13 kDa, and 9.6 kDa,
respectively
[0172] Hydrolysis of the cationic polymers provides the
zwitterionic polymers (i.e., zwitterionic polycarboxybetaines). The
hydrolysis of representative cationic polymer of the invention is
described in Example 2 and illustrated schematically in FIG. 2. In
FIG. 2, n is 1, 3, or 5 (corresponding to polyCBAA-1-ester,
polyCBAA-3-ester, and polyCBAA-5-ester, respectively). The three
carboxybetaine ester polymers were dissolved under different sodium
hydroxide concentrations and their hydrolysis behavior was studied.
After a period of time, the hydrolysis rate of the polymers was
analyzed by measuring the retaining ester groups on the polymer
using .sup.1H NMR. All the three polymers are stable in water and
no evident hydrolysis was detected after four days. The
concentration of NaOH is crucial for the hydrolysis of the
carboxybetaine ester polymers. FIG. 3 illustrates the NMR spectra
of polyCBAA-3-ester after a one-hour treatment with three different
concentrations of NaOH. For NaOH solution with a concentration of
10 mM, only slightly hydrolysis was detected (ca. 3%). For 100 mM
NaOH solution, about 82% polymer was hydrolyzed. For the NaOH
concentration of 1 M, the polymer was totally hydrolyzed in one
hour. FIG. 4 graphs the hydrolysis rate under 100 mM or 10 mM NaOH
as a function of time. Referring to FIG. 4, under these two NaOH
concentrations, most hydrolysis happens in the first hour. After
that, the hydrolysis rate changes only slightly with the time.
[0173] As noted above, the hydrolysis rate of the cationic polymers
useful in the invention can be controlled by modifying their
structures. To obtain the different hydrolysis behavior, cationic
polymers having varying structure parameters such as ester groups
(hydrolyzable groups), spacer groups (L.sub.1 and L.sub.2), and
counter ions (X.sup.-). Hydrolysis behavior can also be controlled
by varying polymer molecular weight or copolymerizing with other
monomers. Hydrolyzable ester groups (such as t-butyl and alkyl
substituted silyl) or anhydride groups can be easily hydrolyzed
under acidic or basic condition. Changing spacer groups (L.sub.2:
--CH.sub.2).sub.n--) between the quaternary ammonium groups
(cationic center) and the ester (hydrolyzable) groups also can tune
the hydrolysis rate. Short spacers can increase the hydrolysis
rate. In addition, counter ions, such as hydrophilic anions (e.g.,
Cl.sup.-, Br.sup.-, I.sup.-, SO.sub.4) also increase the hydrolysis
rate, and low polymer molecular weight and copolymerization with
other hydrophilic monomers also help to increase the hydrolysis
rate.
[0174] Protein Adsorption
[0175] The hydrolyzable cationic polymers useful in the invention
can advantageously be used as materials effective in reducing
protein adsorption to surfaces treated with the polymers. The
cationic polymers can be used to prepare low-fouling surfaces.
These surfaces can be advantageously employed for devices in
environments where the protein adsorption to device surfaces are
detrimental.
[0176] To demonstrate the utility of representative cationic
polymers useful in the invention in providing surfaces having low
protein adsorption, polymer brushes were prepared from
representative cationic polymers as described in Example 3 and
their protein adsorption measured.
[0177] The three monomers (CBAA-1-ester, CBAA-3-ester, and
CBAA-S-ester) were grafted on the surfaces of a SPR sensor using
surface-initiated ATRP. The polymer brushes had a thickness of 5-20
nm estimated from XPS analysis. Protein adsorption from a 1 mg/mL
fibrinogen solution on the three polymer brushes was measured using
SPR. Fibrinogen is a sticky protein and plays an important role in
platelet aggregation and blood clotting on biomaterials. Fibrinogen
adsorption was 195 ng/cm.sup.2, 255 ng/cm.sup.2, and 600
ng/cm.sup.2 for polyCBAA-1-ester, polyCBAA-3-ester, and
polyCBAA-5-ester, respectively (see FIGS. 5A-5C). All three
polymers have evident protein adsorption due to their positive
charges. PolyCBAA-1-ester had relatively lower fibrinogen
adsorption due to its higher hydrophilicity compared to the other
two esters having more hydrophobic L.sub.2 (i.e., C3 and C5,
respectively). With the increase in L.sub.2 from methylene to
propylene to pentylene, the hydrophobicity of the polymer
increases, leading to higher fibrinogen adsorption.
[0178] After hydrolysis at 100 mM for 1-2 hours, surface properties
were dramatically changed. FIGS. 5A-5C illustrate that the surfaces
grafted with each of the three polymers were converted to surfaces
that were highly resistant to fibrinogen adsorption. On the
surfaces with hydrolyzed polyCBAA-1-ester and hydrolyzed
polyCBAA-3-ester, fibrinogen adsorption is less than 0.3
ng/cm.sup.2, which is the detection limit of the SPR. Fibrinogen
adsorption on hydrolyzed polyCBAA-5-ester was about 1.5
ng/cm.sup.2. By controlling hydrolysis, the polymer-grafted
surfaces can change their properties from high protein adsorption
to strongly resistant to protein adsorption. Moreover, resulting
surfaces with zwitterionic polymers after hydrolysis are
biocompatible and highly resistant to nonspecific protein
adsorption from blood plasma/serum and bacterial adhesion/biofilm
formation.
[0179] Antimicrobial Properties
[0180] The hydrolyzable cationic polymers useful in the invention
exhibit antimicrobial properties. The evaluation of antimicrobial
properties of representative cationic polymers useful in the
invention is described in Example 4.
[0181] To evaluate the antimicrobial properties of the cationic
polycarboxybetaine esters, polymer solutions of polyCBAA-1-ester,
polyCBAA-3-ester, and polyCBAA-5-ester were incubated with E. coli.
It was found that at a concentration of 2 mM (repeat unit molar
concentration), polyCBAA-1-ester, polyCBAA-3-ester, and
polyCBAA-5-ester present a live cell percentage of 95%, 87.3%, and
46.2%, respectively (see FIG. 6). Antimicrobial activities appears
to increase with the increase in the length of L.sub.2. After
hydrolysis, the zwitterionic polymers, polyCBAA-1, polyCBAA-3, and
polyCBAA-5, exhibit a live cell percentage of 93.7%, 96.3% and
95.3%, respectively, indicating that the antimicrobial activity
decreases with the hydrolysis of the cationic polymers (i.e.,
polycarboxybetaine esters) to the zwitterionic polymers (i.e.,
polycarboxybetaines).
[0182] Several amphiphilic polycations have been investigated for
their antibacterial activities. The alkyl pendent chain length of
the polycations was studied to compare the bactericidal efficiency
of different polycations. It is found that the polymers with
quaternary amine groups and longer hydrophobic pendant chains have
better antimicrobial activities due to higher hydrophobicity. Small
molecular quaternary ammonium compounds (QMCs) with carboxybetaine
esters were found to have rapid bactericidal action when they have
longer hydrocarbon groups. These QMCs could bind to the outer
membrane and cytoplasmic membrane of enterobacteria and permeate
into the bacterial membranes. The antimicrobial effect is increased
with increasing the spacer length (L.sub.2) of the cationic
polymers (e.g., polycarboxybetaine esters) of the invention.
[0183] The antimicrobial efficacy of the polyCBAA-5-ester is
comparable to that of other quaternized polymers with similar alkyl
chain length. Higher antimicrobial efficacy can be achieved with
longer alkyl chain lengths (e.g., C1-C20).
[0184] For conventional antimicrobial coatings, the killed microbes
and adsorbed proteins usually accumulate on the surfaces and
dramatically decrease their antimicrobial activities. In contrast,
antimicrobial coatings made from the cationic polymers useful in
the invention are hydrolyzed to zwitterionic polymers to provide
surfaces that are highly resistant to the adsorption of various
biomolecules. These zwitterionic polymers are nontoxic,
biocompatible, and nonfouling, both as bulk materials and surface
coatings.
[0185] Representative crosslinked zwitterionic polymers useful in
the invention, polycarboxybetaines hydrogels, were non-cytotoxic
and contain less than 0.06 units (EU)/mL of endotoxin using a
Limulus Amebocyte Lysate (LAL) endotoxin assay kit (Cambrex
Bioscience. Walkerville, Md.). The polycarboxybetaine hydrogels
were implanted subcutaneously within mice for up to four weeks. The
results showed that the polycarboxybetaines have in vivo
biocompatibility comparable to that of poly(2-hydroxyethyl
methacrylate (polyHEMA) hydrogels, a well-accepted model
biomaterial for implantation. The nontoxic properties of the
zwitterionic polymers convert the toxicity of their cationic
polymer precursors and further provide nonfouling properties that
can prevent dead microbes and adsorbed proteins from accumulating
on the surface.
[0186] Switchable Polymer Coatings and their Use in Wound
Dressings
[0187] The cationic polymers useful in the invention, hydrolyzable
to zwitterionic polymers, can be advantageously incorporated into
hemostatic wound dressings. The cationic polymers useful in the
invention provide switchable biocompatible polymer surfaces having
self-sterilizing and nonfouling capabilities.
[0188] FIG. 7 is a schematic illustration of a switchable
biocompatible polymer surfaces having self-sterilizing and
nonfouling capabilities. Referring to FIG. 7, antimicrobial surface
(a) is a surface coated with a representative cationic polymer of
the invention (i.e., pCBMA-1 C2, see FIG. 8) that effectively kills
bacteria. On hydrolysis (b) the representative cationic polymer is
converted to a nonfouling zwitterionic polymer (i.e., pCBMA-1, the
carboxylate corresponding to pCBMA-1 C2 ester) and dead bacteria
remaining on the surface are released (c) from the nonfouling
zwitterionic polymer (i.e., pCBMA-1) to provide a surface coated
with the zwitterionic polymer, which is highly resistant to
bacterial adhesion (d).
[0189] The materials of the invention (e.g., polymers, hydrogels)
are advantageously used to coat surfaces to provide biocompatible,
antimicrobial, and nonfouling surfaces. Accordingly, in another
aspect, the invention provides wound dressings and related
materials having a surface (i.e., one or more surfaces) to which
have been applied (e.g., coated, covalently coupled, ionically
associated, hydrophobically associated) one or more materials of
the invention. Representative wound dressings and related materials
devices are advantageously treated with a material of the
invention, modified to include a material of the invention, or
incorporate a material of the invention.
[0190] Microbial adhesion onto implanted biomaterials and the
subsequent formation of biofilms is one of the major causes of
biomedical device failure. The use of antimicrobial and nonfouling
coatings are two strategies for the prevention of the attachment
and spreading of microorganisms on the surfaces of implantable
materials. Antimicrobial surfaces containing covalently linked
quaternary ammonium compounds (QACs) have proved to be able to
efficiently kill a variety of microorganisms. A major problem with
QAC surfaces is the attachment of dead microorganisms remaining on
antimicrobial coatings, which can trigger an immune response and
inflammation, and block its antimicrobial functional groups. In
addition, such antimicrobial coatings can not fulfill the
requirements of nonfouling and biocompatibility as implantable
biomaterials. Poly(ethylene glycol) (PEG) derivatives or
zwitterionic polymers have been extensively used as nonfouling
materials to reduce bacterial attachment and biofilm formation.
However, the susceptibility of PEG to oxidation damage has limited
its long-term application in complex media. Zwitterionic materials
such as poly(sulfobetaine methacrylate) (pSBMA) are able to
dramatically reduce bacterial attachment and biofilm formation and
are highly resistant to nonspecific protein adsorption, even from
undiluted blood plasma and serum. Although zwitterionic coatings
can reduce the initial attachment and delay colonization of
microbes on surfaces, there is a possibility of introducing
pathogenic microbes into the patient during implantation operations
and catheter insertions, which results in the failure of implanted
devices; the use of antimicrobial agents will then be necessary to
eliminate these microbes. Surface-responsive materials have been
developed for a broad spectrum of applications, but it is still a
great challenge to develop biocompatible materials that have both
antimicrobial and nonfouling capabilities.
[0191] As noted above, in one embodiment, the present invention
provides a switchable polymer surface coating that combines the
advantages of both nonfouling surface and that can kill greater
than 99.9% of Escherichia coli K12 in one hour, with 98% of the
dead bacterial cells released when the cationic derivatives are
hydrolyzed to nonfouling zwitterionic polymers. pCBMA-1-C2
(cationic polymer of formula (I) where L.sub.1 is
--C(.dbd.O)OCH.sub.2CH.sub.2--, L.sub.2 is --CH.sub.2--, R.sub.c is
CH.sub.2CH.sub.3, and X.sup.- is Br.sup.-) control coatings were
grafted by surface-initiated atom transfer radical polymerization
(ATRP) onto a gold surface covered with initiators. The thicknesses
of the obtained polymer coatings, as measured by atomic force
microscopy (AFM), were 26-32 nm (Table 1).
TABLE-US-00001 TABLE 1 Film thicknesses (av .+-. std dev.) of
pCBMA-1 C2, pC8NMA, and pCBMA-2 grafted onto gold-coated glass
slides by ATRP and fibrinogen adsorption on these surfaces measured
by SPR before and after hydrolysis under different conditions.
pCBMA-1 C1 pC8NMA pCBMA-2 polymer brush thickness (31.2 .+-. 2.4)
(27.8 .+-. 2.8) (26.1 .+-. 2.5) (nm) protein adsorption (ng
cm.sup.-2) 0 h 229.2 243.4 1.5 24 h H.sub.2O 189.9 -- -- 24 h CHES
(pH 9.0) 114.9 -- -- 24 h CAPS (pH 10.0) 0 285.1 0.7
[0192] The bactericidal activity of pCBMA-1 C2 surfaces was
determined using E. coli K12, according to a modified literature
procedure (Tiller et al., Proc. Natl. Acad. Sci. USA 98:5981,
2001). The permanently cationic
poly(methacryloyloxyethyl-dimethyloctylammonium bromide) (pC8NMA,
cationic control, (see FIG. 8) and the zwitterionic
poly(2-carboxy-N,N-dimethyl-N-[2'-(methacryloyloxy)ethyl]ethanaminium)
(pCBMA-2, zwitterionic control, see FIG. 8) were used as the
positive and the negative control surfaces, respectively. The
antimicrobial efficiency was defined as the amount of live cells on
the tested surfaces relative to those on the pCBMA-2 surface. FIG.
9 shows that pCBMA-1 C2 and pC8NMA surfaces kill greater than 99.9%
and 99.6%, respectively, of the E. coli in one hour relative to
pCBMA-2 surfaces. The total number of live bacterial cells on the
gold surface, which was also used as a negative-control surface, is
similar to that on the pCBMA-2 surface.
[0193] The attachment and release of E. coli K12 were tested on the
pCBMA-1 C2 surfaces before and after hydrolysis. Cationic pC8NMA
and zwitterionic pCBMA-2 were used as the negative and the positive
nonfouling control surfaces, respectively, and as the positive and
the negative antimicrobial control surfaces, respectively. FIGS.
10A-10F show that large amounts of bacteria were attached to the
cationic pCBMA-1 C2 and pC8NMA surfaces before hydrolysis, whereas
very few bacterial cells were attached to the zwitterionic pCBMA-2
surface. In contrast to pC8NMA, pCBMA-1 C2 released the majority of
cells after hydrolysis while pCBMA-2 remained nonfouling. FIG. 11
shows quantitative data for the amount of bacterial cells remaining
on all three polymer surfaces before and after hydrolysis. There
were similar amounts of bacterial residues on both cationic pCBMA-1
C2 and pC8NMA surfaces before hydrolysis, while the amount of
attached cells on the pCBMA-2 surface is less than 0.3% of that on
both cationic pCBMA-1 C2 and pC8NMA surfaces. To test the release
of bacterial residues, the three surfaces were incubated in
N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer (10 mM, pH
10.0) at 37.degree. C. for 8 days. The pCBMA-1 C2 surfaces were
hydrolyzed to
poly(N-(carboxymethyl)-N,N-dimethyl-2-[(2-methyl-1-oxo-2-propen-1-yl)-oxy-
]ethanaminium) (pCBMA-1) and 98% of the dead bacterial cells were
released. In contrast, no release of the dead cells was observed on
pC8NMA surfaces (p>0.1) while pCBMA-2 surfaces retained very low
bacterial adhesion.
[0194] The release of the attached bacterial cells is dependent on
the conversion of cationic pCBMA-1 C2 into zwitterionic pCBMA-1.
Hydrolysis rate of betaine esters is influenced by several factors,
such as the length of the spacer (L.sub.2) between the quaternary
amine and the carboxyl groups, the nature of the hydrolyzable
group, temperature,.sup.1 and pH value. The majority of polymer
chains of the ester group used were hydrolyzed. The hydrolysis rate
of the betaine esters is also slower after bacterial cells and
proteins are attached to the surface. pCBMA-1 C2, which has one
methylene spacer (L.sub.2), was chosen and the experimental
temperature was set at 37.degree. C. to achieve a fast hydrolysis
rate and to provide a physiologically relevant temperature. The
protein adsorption results (see Table 2) showed that the clean,
cationic pCBMA-1 C2 surface was hydrolyzed into a nonfouling
zwitterionic surface after only 24 h at 37.degree. C. and pH 10.0,
while it took 48 h to form a nonfouling surface and release
bacterial residues after the attachment of bacteria from an E. coli
K12 suspension of 10.sup.7 cells mL.sup.-1. When bacterial cells
were attached to the pCBMA-1 C2 surface from a suspension of
10.sup.10 cells mL.sup.-1, the release of attached bacteria took
eight days under the same hydrolysis conditions.
[0195] Nonspecific protein adsorption on various surfaces was
measured by a surface plasmon resonance (SPR) sensor to determine
the nonfouling characteristics of the surfaces (see Table 2).
Hydrolysis conditions for pCBMA-1 C2 and control surfaces were
investigated in situ in the SPR sensor. FIGS. 12A and 12B show
representative SPR sensorgrams for fibrinogen adsorption on pCBMA-1
C2 and control surfaces over time. The fibrinogen adsorption on
pCBMA-1 C2 before hydrolysis was 229.2 ng cm.sup.-2. After 24 h of
incubation with CAPS buffer (pH 10.0), there was no measurable
protein adsorption on the pCBMA-1 C2 surface, which indicated that
pCBMA-1 C2 was completely hydrolyzed to nonfouling zwitterionic
pCBMA-1. In contrast, hydrolysis of pCBMA-1 C2 was not complete
after 24 h incubation in either water or
N-cyclohexyl-2-aminoethanesulfonic acid (CEHS) buffer (pH 9.0). As
shown in FIG. 12B, high fibrinogen adsorption was observed on the
pC8NMA surface before and after the surface was incubated with CAPS
buffer (pH 10.0) for 24 h at 37.degree. C. However, under identical
conditions, the pCBMA-2 surface still exhibited excellent
nonfouling properties, with less than 2 ng cm.sup.-2 fibrinogen
absorption. This result indicates that the obtained zwitterionic
surfaces are highly resistant to protein adsorption and are
qualified as ultralow fouling surfaces, which are required for the
surface coatings of implantable medical devices.
[0196] In this embodiment, the invention provides a switchable
polymer surface that integrates antimicrobial and nonfouling
properties and is biocompatible. The representative cationic
polymer (i.e., precursor of pCBMA) is able to kill bacterial cells
effectively and switches to a zwitterionic nonfouling surface and
releases dead bacterial cells upon hydrolysis. Moreover, the
resulting nonfouling zwitterionic surface can further prevent the
attachment of proteins and microorganisms and reduce the formation
of a biofilm on the surface. The switchable process from
antimicrobial to nonfouling surfaces can be tuned through adjusting
the hydrolysis rate of these polymers for specific requirements of
applications.
[0197] As noted above, the cationic polymers useful in the
invention can include a hydrophobic counter ion or a counter ion
having therapeutic activity (e.g., antimicrobial or antibacterial
activity. A representative polymer having a salicylate counter ion
(polyCBMA-1 C2) can be prepared from the monomer illustrated in
FIG. 13: CBMA-1 C2 ("1" indicates one carbon between two charged
groups and "C2" indicates C2 ester). PolyCBMA-1 C2 hydrogel loaded
with salicylic acid (SA) as its counter ion was prepared by
copolymerizing 1 mM CBMA-1 C2 SA monomer (FIG. 13) with 0.05 mM
tetraethylenglycoldimethacrylate in 1 ml of solvent (ethylene
glycol:water:ethanol=1:2:1) at 65.degree. C. for 2 hours. The
resulting hydrogel was soaked in DI water for 12 hours. The
hydrogel was cut into round disks with 1 cm diameter. The hydrogel
disks were then transferred into solutions with different pH and
ionic strength and incubated at 25.degree. C. or 37.degree. C. At
different time points the aqueous phase was completely removed and
new solutions were added. The release of SA into the aqueous phase
was measured by high performance liquid chromatography (HPLC). The
release rate of SA is defined as the amount of released SA divided
by time (mg/h). The release rate of SA from pCBMA-1 C2 SA hydrogel
depends on temperature, ionic strength, and pH. FIG. 14 and FIG. 15
indicated that higher pH promotes the release of SA and that
increased ionic strength can slightly increase the release rate of
SA. By comparing FIG. 14 and FIG. 15, it can be observed that the
elevated temperature results in a faster release of SA in water and
phosphate buffered saline (PBS). The release rate of SA decreases
as a function of time for all the conditions.
[0198] Cationic Polymers and their Use in Wound Dressings
[0199] The cationic polymers useful in the invention, hydrolyzable
to zwitterionic polymers, can be advantageously used as coatings
for the surfaces of hemostatic wound dressings. In this embodiment,
the cationic polymers useful in the invention provide switchable
biocompatible polymer surfaces having self-sterilizing and
nonfouling capabilities. These polymers are useful in hemostasis
devices optionally in combination with one or more conventional
hemostasis components.
[0200] As noted above, the present invention provides switchable
biocompatible polymer surfaces having self-sterilizing and
non-fouling capabilities based on the cationic polymers useful in
the invention (e.g., pCBMA ester). See FIG. 7. Antimicrobial
cationic pCBMA ester can prevent bacterial infection and is
converted to biocompatible and water-absorbent zwitterionic pCBMA
upon hydrolysis.
[0201] In one embodiment, the wound dressing includes a hydrogel.
In this embodiment, the hydrogel includes both a zwitterionic
polymer as well as a cationic polymer (i.e., a cationic
zwitterionic precursor polymer). The cationic zwitterionic
precursor polymers are used for hemostatic and antimicrobial
actions while the zwitterionic polymers are used as wound fluid
adsorbents. After action, cationic zwitterionic precursor polymers
are converted to nontoxic, non-sticky, and biocompatible
zwitterionic polymers upon hydrolysis (controllable hydrolysis
rates). In one embodiment, the wound dressing hydrogels can be
prepared from two monomers (CBMA and CBMA ester) via polymerization
or from just one monomer (CBMA ester) by partially hydrolyzing the
pCBMA ester hydrogel.
[0202] A representative wound dressing hydrogel of the invention is
schematically illustrated in FIG. 16.
[0203] In one embodiment, the invention provides wound dressings
based on an integrated formulation containing both cationic (e.g.,
pCBMA ester) and zwitterionic (e.g., pCBMA) polymers. These wound
dressings containing both cationic and zwitterionic polymers
improve hemorrhage control and survival, promote wound healing, and
remove bacteria. The advantages of the wound dressings include (a)
multiple hemostatic, antimicrobial, wound fluid-absorbent, and
wound healing functions with high efficacy since cationic polymers
(e.g., pCBMA ester) can attract red blood cells and kill bacteria
while the zwitterionic polymer (e.g., pCBMA) can absorb wound
fluids; (b) add-on wound fluid adsorbent capability, non-sticky,
biocompatibility, and nontoxicity even at high concentrations when
cationic polymers are converted to zwitterionic polymers upon
hydrolysis; (c) simplicity, reproducibility, and low-cost since
they can be prepared from only one or two monomers (i.e., CBMA
ester and CBMA). The wound dressings of the invention are based on
cationic polymers hydrolyzable to zwitterionic polymers. The wound
dressings include switchable polymer surfaces described above have
both self-sterilizing and non-fouling/biocompatible capabilities.
This cationic zwitterionic precursor polymer has unique performance
as coatings on a surface or as additives to a solution. On
hydrolysis, the cationic polymers are converted to zwitterionic
polymers that are highly resistant to nonspecific protein
adsorption from undiluted blood plasma and serum and bacterial
adhesion/biofilm formation. The zwitterionic polymer hydrolysis
products are also highly biocompatible with tissues from in vivo
animal studies. The wound dressing includes a switchable polymer
surface integrating antimicrobial and nonfouling/biocompatible
properties. The antimicrobial cationic zwitterionic precursor
surface can effectively kill bacterial cells and then switch to a
nonfouling and biocompatible zwitterionic surface (see FIG. 7).
[0204] Six representative cationic polymers useful in the invention
and SAM modifications were evaluated for their interactions with
human serum and plasma. Human serum and plasma are components of
human blood, comprised of a complex mixtures of hundreds of
proteins. FIGS. 17A and 17B show sensor responses to human serum
and human plasma, respectively. pCBMA had the best non-specific
resistance to the test media. A pCBMA surface had an improved
resistance to nonspecific protein adsorption from human plasma or
serum over the poly[oligo(ethylene glycol) methacrylate] (pOEGMA)
and poly(sulfobetaine methacrylate) (pSBMA) (See FIGS. 17A and
17B). While protein adsorption from 10% serum is generally low, the
difference among several surfaces studied to resist nonspecific
protein adsorption from 100% plasma and serum is enormous as shown
in FIGS. 17A and 17B. With the optimization of film thickness and
density, pCBMA can highly resist 100% blood plasma and serum to the
level that is undetectable by SPR (See FIG. 18).
[0205] Microbial adhesion and the subsequent formation of biofilm
are critical issues for many biomedical applications. Therefore,
the development of surfaces that resist the initial adhesion of
bacteria is the first step towards the effective prevention of
long-term biofilm formation. The accumulation of P. aeruginosa on
pSBMA modified glass chips formed with a silane initiator and
surface-initiated ATRP indicates that pSBMA grafted surfaces show
strong resistance to bacterial adhesion and biofilm formation for
one day. Pseudomonas aeruginosa PAO1 with a GFP expressing plasmid
was used for long-term adhesion and biofilm formation studies.
There is an absence of attached bacteria after exposure to P.
aeruginosa for a long period of time, while P. aeruginosa is
readily attached to the unmodified portion of glass or
oligo(ethylene glycol) (OEG) self-assembled monolayer (SAM)
modified substrates. These studies were performed in a laminar flow
chamber in situ.
[0206] FIGS. 19A-19H are representative microscopy images of the
accumulated P. aeruginosa on pCBMA-treated surfaces in the growth
medium over an 11-day growth period. On the bare and OEG-modified
glass surfaces, very quick bacterial adhesion and subsequent
biofilm formation of P. aeruginosa were observed (see FIGS. 19A and
19B, respectively). A confluent biofilm was formed by the second
day on these two control surfaces. However, the surface
concentration of adherent P. aeruginosa on the pCBMA-coated glass
was very small (<<10.sup.6 cell/mm.sup.2). Over the 11-day
growth mode experiments, there was no observed biofilm formation
(see FIGS. 19C-19H). It is believed that the ability of
zwitterionic pCBMA materials to resist protein adsorption and
inhibit bacterial adhesion is due to its strong hydration via ionic
solvation.
[0207] In one embodiment, the wound dressings of the invention
include hydrogels containing cationic polymers (zwitterionic
precursors) of the invention and zwitterionic polymers.
[0208] Dried hydrogels as wound dressings can be prepared in two
ways: (1) forming a chemical or IPN gel from zwitterionic monomers
(e.g., CBMA) and cationic monomers (e.g., CBMA ester monomers) (a
more controllable way) and (2) forming a chemical gel from the
cationic monomer (pCB ester monomer) first and then partially
hydrolyzing the product hydrogel (e.g., pCBMA ester hydrogel) to
mixed cationic and zwitterionic hydrogel (pCBMA ester/pCBMA
hydrogel) (a more economic way).
[0209] A representative hydrogel can be prepared from
carboxybetaine methacrylate (CBMA) and cationic zwitterionic
precursor monomers. CBMA monomer can be synthesized by the reaction
of 2-(N,N'-dimethylamino)ethyl methacrylate (DMAEMA) and
.beta.-propiolactone with anhydrous acetone as solvent at low
temperature (see FIG. 20). The purity (>99%) can be monitored by
nuclear magnetic resonance (NMR) and elemental analysis.
Alternatively, carboxybetaine acrylamide (CBAA) can be similarly
prepared and used. Similar to CBMA, CBAA monomer can be synthesized
by the reaction of N-(3-dimethylaminopropyl)acrylamide (DMAPAA) and
.beta.-propiolactone with anhydrous acetone as solvent at low
temperature.
[0210] Cationic zwitterionic precursor monomer (i.e., CBMA ester)
can be synthesized by quaternization reaction from DMAEMA and alkyl
chloro (or bromo) carboxylates using anhydrous acetonitrile as
solvent (see FIG. 21).
[0211] CBMA was synthesized by DMAEMA and .beta.-propiolactone with
anhydrous acetone as solvent at low temperature using ice-bath as
cooling method (yield .about.90%).
[0212] Representative hydrogels can be prepared from zwitterionic
(e.g., CBMA) and cationic (e.g., CBMA ester) monomers. The
polymerization of the monomers is illustrated schematically in FIG.
22. CBMA, CBMA ester and 4 mol % N,N'-methylene-bisacrylamide (MBA,
as a cross-linker) are dissolved in deionized water ([M] 10 wt %).
To this solution, 0.2 wt % ammonium peroxodisulfate and 1.0 wt %
N,N,N',N'-tetramethylethylenediamine (TMEDA, as an accelerator) is
added as redox initiators. TMEDA is chosen since it is an initiator
for reaction at room temperature. Polymerization is performed at
room temperature for 24 hr. After the gelation is completed, the
gel is immersed in an excess amount of deionized water for 3 days
to remove the residual unreacted monomers. Swollen polymer gels are
dried at room temperature for 1 day, and these samples are further
dried with freeze drying method or in vacuum oven for 2 days at
60.degree. C.
[0213] Representative interpenetrating network (IPN) hydrogels can
be prepared from zwitterionic (e.g., CBMA) and cationic (e.g., CBMA
ester) monomers. The polymerization of the monomers is illustrated
schematically in FIG. 23. As a mixture of two or more crosslinked
networks that are dispersed or mixed at a molecular segmental
level, IPNs can help improve the mechanical strength and resiliency
of the polymer gels. An example of the preparation of such an IPN
hydrogel is as follows. CBMA and 4 mol % MBA (as a cross-linker)
are dissolved in deionized water ([M] 10 wt %). To this solution,
0.2 wt % ammonium peroxodisulfate and 1.0 wt % TMEDA (as an
accelerator) are added as redox initiators. Polymerization is
performed at room temperature for 24 hr. After the gelation is
completed, the gel is immersed in an excess amount of deionized
water for 3 days to remove the residual unreacted monomer and
initiator. The degree of crosslinking is defined as the molar ratio
of the crosslinking agent (MBA) to CBMA.
[0214] Representative interpenetrating network (IPN) hydrogels can
be prepared from zwitterionic (e.g., pCBMA) and cationic (e.g.,
pCBMA ester) polymers. pCBMA/pCBMA ester IPN gels are prepared
according to the following procedure. A pCBMA gel is immersed in 10
ml of the pCBMA ester monomer solution of the prescribed
concentration containing redox initiators mentioned above, and left
for 5 days at 4.degree. C. to let pCBMA ester penetrate into pCBMA
gel. pCBMA ester inside the pCBMA gel is polymerized at 30.degree.
C. for 24 hr to give the IPN gels comprising pCBMA ester linear
polymer and pCBMA gel. After the polymerization, the gel is
immersed in an excess amount of deionized water for 3 days to
remove the residual unreacted monomer and initiator. These samples
are dried with freeze drying method or in vacuum oven for 2 days at
60.degree. C.
[0215] While the mixed (chemical or IPN) hydrogel from zwitterionic
(e.g., CBMA) and cationic (e.g., CBMA ester) monomers can be
prepared in a controllable way, it is possible to achieve mixed
zwitterionic and cationic hydrogels from a single cationic (e.g.,
CBMA ester) monomer. This is done by preparing cationic (e.g.,
pCBMA ester) hydrogel first and then partially hydrolyzing the
hydrogel to a mixed cationic/zwitterionic hydrogel (e.g., pCBMA
ester/pCBMA).
[0216] Hydrophilic cationic (e.g., pCBMA ester) hydrogels can be
used as antimicrobial wound dressing once they are partially
hydrolyzed. The gels after partial hydrolysis are a mixture of
zwitterionic pCBMA and cationic pCBMA ester compounds, which
integrates both hemostatic, antimicrobial, and water-adsorbent
functions.
[0217] A representative procedure for preparing to a partially
hydrolyzed cationic (e.g., pCBMA ester) hydrogel is described below
and illustrated schematically in FIG. 24. CBMA and 4 mol % MBA as
crosslinker) is dissolved in deionized water ([M] 10 wt %). To this
solution, 0.2 wt % ammonium peroxodisulfate and 1.0 wt % TMEDA (as
an accelerator) are added as redox initiators. Polymerization is
performed at room temperature for 24 hr. After the gelation is
completed, the gel is immersed in an excess amount of deionized
water for 3 days to remove the residual unreacted monomer and
initiator. Then, the gel is immersed in the buffer solution (pH
8.about.12) for 3 to 12 hr, then is washed with deionized water,
and immersed in an excess amount deionized water and left for 5
days at 4.degree. C. to remove the residual salts. These samples
are dried with freeze drying method or in vacuum oven for 2 days at
60.degree. C.
[0218] The hydrogels of the invention prepared as described above
can be optimized and evaluated for their antimicrobial, hemostatic,
water-adsorbent, and other physical/chemical properties in vitro by
adjusting the degree of cross-linking, the ratio of cationic
polymer (e.g., pCBMA ester to pCBMA, and hydrolysable groups.
Hydrogel pastes are coated onto a polymeric pad to form wound
dressing-pad assemblies (or bandages) for in vivo experiments and
practical applications. Standard gauze dressing and commercial HC
dressing will be used as negative and positive controls whenever is
possible.
[0219] In one embodiment, the invention provides a wound
dressing-pad assembly that includes the wound dressing of the
invention. The wound dressing-pad assembly is sized and configured
for easy manipulation by the caregiver's fingers and hand. The
backing isolates a caregiver's fingers and hand from the dressing
gels. The backing permits the dressing matrix to be handled,
manipulated, and applied at the tissue site, without adhering or
sticking to the caregiver's fingers or hand. The backing can
include low-modular meshes and/or films and/or weaves of synthetic
and naturally occurring polymers. For temporary external wound
applications, the backing includes a fluid impermeable polymer
material, e.g., polyethylene (3M 1774T polyethylene foam medical
tape, 0.056 cm thick). Other polymers may be used, including
cellulose polymers, polyethylene, polypropylene, metallocene
polymers, polyurethanes, polyvinylchloride polymers, polyesters,
polyamides or their combinations. The backing can be attached or
bonded by directed adhesion with a top layer of the wound dressing
gel. The dried hydrogel samples can be moistened with 1.about.3 wt
% of sterile physiological saline, which can turn into a sticky
paste and have sufficient adhesive properties for attaching to the
backing materials. If needed, an adhesive such as 3M 9942 Acrylate
Skin Adhesive, or fibrin glue, or cyanoacrylate glue can be
employed to enhance the adhesion between the wound dressing and the
pad. The wound dressing matrix is desirably vacuum packaged before
use with low moisture content, preferably 5% moisture or less, in
an air-tight heat sealed foil-lined pouch. The antimicrobial wound
dressing pad assembly is subsequently terminally sterilized within
the pouch by the use of gamma irradiation. Once removed from the
pouch, the wound dressing pad assembly is immediately ready to be
adhered to the targeted tissue site. These wound dressings are
non-irritating and non-sticky to the wound, absorb wound exudate,
enhance the sterile environment around the wound, stem blood loss,
and promote wound healing. The wound dressing gels can be removed
without leaving any gel residue on the wound due to the nonfouling
properties of these wound dressing hydrogels.
[0220] A representative wound dressing of the invention is
illustrated in FIG. 26. Referring to FIG. 26, representative wound
dressing 100 includes backing 200 and gel 300.
[0221] The hydrolysis rate of the cationic polymers (e.g., esters)
is influenced by several factors, such as the length of the spacer
between the quaternary amine and the carboxyl group, the type of
the hydrolyzable group, temperature, and pH. For a representative
cationic polymer, pCBMA ester with n=1, the ester is an ethyl ester
that produces ethanol on hydrolysis. A limited amount of ethanol
will be generated upon hydrolysis, some of which will be trapped
within the hydrogel, and some ethanol will be released, but will
not affect wound healing. Alternatively, in one embodiment, the
cationic polymer has a glycine leaving group (hydrolyzable bond is
an anhydride) as illustrated in FIG. 25.
[0222] The following examples are provided for the purpose of
illustrating, not limiting, the claimed invention.
EXAMPLES
Example 1
The Synthesis and Characterization of Representative Cationic
Polymers
[0223] Materials. N-(3-dimethylaminopropyl)acrylamide (>98%) was
purchased from TCI America, Portland, Oreg. Methyl bromoacetate
(97%), ethyl 4-bromobutyrate (.gtoreq.97.0%), ethyl
6-bromohexanoate (99%), copper (I) bromide (99.999%),
bromoisobutyryl bromide (BIBB 98%), 11-mercapto-1-undecanol (97%),
and 2,2'-bipyridine(BPY 99%), and
2,2'-azobis(2-methylpropionitrile) (AIBN 98%) were purchased from
Sigma-Aldrich. Fibrinogen (fraction I from bovine plasma) and
phosphate buffer saline (PBS, pH7.4, 0.15 M, 138 mM NaCl, 2.7 mM
KCl) were purchased from Sigma Chemical Co. Ethanol (absolute 200
proof) was purchased from AAPER Alcohol and Chemical Co. Water used
in experiments was purified using a Millipore water purification
system with a minimum resistivity of 18.0 M.OMEGA..cm.
[0224] .omega.-Mercaptoundecyl bromoisobutyrate (1) was synthesized
through reaction of BIBB and 2 using a method described in Ilker,
M. F.; Nuesslein, K.; Tew, G. N.; Coughlin, E. B., "Tuning the
Hemolytic and Antibacterial Activities of Amphiphilic
Polynorbornene Derivatives," Journal of the American Chemical
Society 126:(48):15870-15875, 2004). 1H NMR (300 MHz, CDCl.sub.3):
4.15 (t, J=6.9, 2H, OCH.sub.2), 2.51 (q, J=7.5, 2H, SCH.sub.2),
1.92 (s, 6H, CH.sub.3), 1.57-1.72 (m, 4H, CH.sub.2), and 1.24-1.40
(m, 16H, CH.sub.2).
[0225] Cationic Monomer Syntheses
CBAA-1-ester: (2-carboxymethyl)-3-acrylamidopropyldimethylammonium
bromide, methyl ester.
[0226] N-(3-dimethylaminopropyl)acrylamide (25 mmol), methyl
bromoacetate (37.5 mmol), and acetonitrile (25 mL) were added into
a 100-mL round-bottom flask. The mixture was stirred under a
nitrogen atmosphere for 6 hr at room temperature. The precipitate
was collected, washed with ca 500 mL of anhydrous acetone. The
solvent was removed on a rotary evaporator to get a white powder
(96% yield). 1H NMR (300 MHz, D.sub.2O): 2.02 (m, 2H,
--CH.sub.2--), 3.25 (s, 6H, N.sup.+(CH.sub.3).sub.2), 3.37 (t, 2H,
CH.sub.2--N.sup.+), 3.58 (m, 2H, CH.sub.2--N), 3.79 (s, 3H,
O--CH3), 4.29 (s, 2H, CH.sub.2--C.dbd.O), 5.77 (m, 1H,
CH.dbd.C--CON-trans); 6.19 (m, 1H, CH.dbd.C--CON-- cis), 6.23 (m,
1H, .dbd.CH--CON--).
[0227] CBAA-3-ester:
(4-carboxypropyl)-3-acrylamidopropyldimethylammonium bromide, ethyl
ester.
[0228] N-(3-dimethylaminopropyl)acrylamide (50 mmol), ethyl
4-bromobutyrate (60 mmol), and acetonitrile (25 mL) were added into
a 100-mL round-bottom flask. The mixture was stirred under a
nitrogen atmosphere for three days at room temperature. The solvent
was removed on a rotary evaporator to get a colorless oil (92%
yield). 1H NMR (300 MHz, D.sub.2O): 1.22 (t, 3H CH.sub.3), 2.00 (m,
4H, C--CH.sub.2--C), 2.47 (t, 2H, CH.sub.2--C.dbd.O), 3.06 (s, 6H,
N.sup.+(CH.sub.3).sub.2), 3.28-3.35 (6H, CH.sub.2--N and
CH.sub.2--N.sup.+---(CH.sub.2), 4.14 (q, 2H, O--CH.sub.2), 5.75 (m,
1H, CH.dbd.C--CON-trans); 6.19 (m, 1H, CH.dbd.C--CON-- cis), 6.26
(m, 1H, .dbd.CH--CON--).
[0229] CBAA-5-ester:
(6-carboxypentyl)-3-acrylamidopropyldimethylammonium bromide, ethyl
ester.
[0230] N-(3-dimethylaminopropyl)acrylamide (50 mmol), ethyl
6-bromohexanoate (55 mmol), and acetonitrile (25 mL) were added
into a 100-mL round-bottom flask. The mixture was stirred under a
nitrogen atmosphere for five days at 45.degree. C. The solvent was
removed on a rotary evaporator to get a slightly yellowish oil (87%
yield). 1H NMR (300 MHz, D.sub.2O): 1.20 (t, 3H CH.sub.3), 1.34 (m,
2H, C--C--CH.sub.2--C--C), 1.60-1.72 (4H,
C--CH.sub.2--C--CH.sub.2--C), 2.00 (m, 2H, N--C--CH.sub.2--C--N),
2.34 (t, 2H, CH.sub.2--C.dbd.O), 3.04 (s, 6H,
N.sup.+(CH.sub.3).sub.2), 3.24-3.37 (6H, CH.sub.2--N and
CH.sub.2--N.sup.+--CH.sub.2), 4.12 (q, 2H, O--CH.sub.2), 5.75 (m,
1H, CH.dbd.C--CON-trans); 6.20 (m, 1H, CH.dbd.C--CON-- cis), 6.24
(m, 1H, .dbd.CH--CON--).
[0231] Representative Cationic Polymer Syntheses
[0232] Surface-Initiated ATRP. Three monomers, CBAA-1-ester,
CBAA-3-ester, and CBAA-5-ester, were grafted onto gold-coated SPR
sensor chips or gold-coated silicon chips using surface-initiated
ATRP. The preparation and characterization of the polymer brushes
is described in Zhang, Z.; Chen, S.; Chang, Y.; Jiang, S., Surface
Grafted "Sulfobetaine Polymers Via Atom Transfer Radical
Polymerization as Superlow Fouling Coatings," Journal of Physical
Chemistry B 110(22):10799-10804, 2006, and Zhang, Z.; Chen, S.;
Jiang, S., "Dual-Functional Biomimetic Materials: Nonfouling
Poly(carboxybetaine) With Active Functional Groups for Protein
Immobilization," Biomacromolecules 7(12):3311-3315, 2006. previous
publications. Briefly, CuBr (1 mmol) and a SPR chip or a gold disk
with a Br-thiol SAM was placed in a nitrogen-purged reaction tube.
Degassed solution (pure water and methanol in a 1:1 volume ratio,
10 mL) with CBAA ester (6.5 mmol), and BPY (2 mmol, in 5 mL
degassed methanol) were transferred to the tube using a syringe.
After reaction for more than 1 hour under nitrogen, the SPR chip or
gold disk was removed and rinsed with ethanol, water and PBS
solution. The samples were stored in PBS solutions before
testing.
[0233] Polymer Synthesis and Characterization
[0234] CBAA-1-ester solution of ca. 0.3 M in methanol was purged
with nitrogen for 30 min. The polymerization was then performed at
60.degree. C. for ca 48 hours under nitrogen using 3 mol % AIBN as
an initiator to provide polyCBAA-1-ester. Similar methods were
applied for preparation of polyCBAA-3-ester or polyCBAA-5-ester
using ethanol as a solvent. The polymers were washed with ethyl
ether and the solvent was then removed. The structures of the
polymers were confirmed by NMR. 1H NMR (300 MHz, D.sub.2O):
polyCBAA-1-ester: 1.62 (br, 2H), 2.05 (br, 3H), 3.25-3.32 (br, 8H),
3.62 (br, 2H), 3.83 (s, 3H), 4.38 (s, 2H); polyCBAA-3-ester 1.21
(t, 3H), 1.61 (br, 2H), 2.04 (br, 5H), 2.50 (t, 2H), 3.37 (br, 6H),
3.12 (s, 6H), 4.14 (q, 2H); polyCBAA-5-ester: 1.22 (t, 3H), 1.37
(m, 2H), 1.62-1.80 (br m, 6H), 2.01 (br, 3H), 2.39 (t, 2H), 3.03
(s, 6H), 3.24 (br m, 6H), 4.12 (q, 2H).
[0235] The molecular weight of linear polyCBAA was estimated using
a Waters Alliance 2695 Separations Module equipped with a Waters
Ultrahydrogel 1000 column and detected with a Waters 2414 Reflex
Detector. The mobile phase was an aqueous solution at a flow rate
of 0.5 mL/min. The instrument and column were calibrated with
poly(ethylene oxide) standards from Polymer Laboratories. All
measurements were performed at 35.degree. C. The molecular weight
of the polymer was calculated using Empower Pro from Waters.
Example 2
Representative Cationic Polymer Hydrolysis
[0236] The cationic polymers prepared as described in Example 1
were dissolved in NaOH solutions with different concentration (10
mM, 100 mM, and 1 M) in a concentration of 50 mg/mL. After an
appropriate time interval, the polymer solutions were neutralized
with dilute HCl solution and the water was removed by vacuum. 1H
NMR spectroscopy (D.sub.2O) was performed to measure the
degradation rate by determining the amount of intact ester groups
and comparing with other non-hydrolyzable pendant groups as inner
standards. The results are illustrated in FIG. 3.
Example 3
Representative Cationic Polymer Protein Adsorption and Release
[0237] The cationic polymers prepared as described in Example 1
were evaluated for protein adsorption by surface plasmon resonance
(SPR).
[0238] Protein adsorption was measured with a custom-built SPR
sensor, which is based on wavelength interrogation. A SPR chip was
attached to the base of the prism, and optical contact was
established using refractive index matching fluid (Cargille). A
dual-channel flow cell with two independent parallel flow channels
was used to contain liquid sample during experiments. A peristaltic
pump (Ismatec) was utilized to deliver liquid sample to the two
channels of the flow cell. Fibrinogen solution of 1.0 mg/mL in PBS
(0.15M, pH 7.4) was flowed over the surfaces at a flow rate of 0.05
mL/min. A surface-sensitive SPR detector was used to monitor
protein-surface interactions in real time. Wavelength shift was
used to measure the change in surface concentration (mass per unit
area). The results are illustrated in FIGS. 5A-5C.
Example 4
Representative Cationic Polymer Antimicrobial Properties
[0239] The cationic polymers prepared as described in Example 1
were evaluated for their antimicrobial properties.
[0240] E. coli K12 were first cultured in separate pure cultures
overnight at 37.degree. C. on LB agar plates, which was then
incubated with shaking at 37.degree. C. for 24 h. Cultures on agar
plates can be used for two weeks, if kept at 4.degree. C. Several
colonies were used to inoculate 25 ml of LB (20 g/L). These initial
cultures were incubated at 37.degree. C. with shaking at 100 rpm
for 18 hours and were then used to inoculate a second culture of
each species in 200 ml of appropriate medium. When the second
suspended culture reached an optical density of 1.0 at 600 nm,
bacteria were collected by centrifugation at 8,000.times.g for 10
min at 4.degree. C. Cell pellets were washed three times with
sterile phosphate buffered saline (PBS, pH7.4) and subsequently
suspended in PBS to a final concentration of 10.sup.8 cells/mL.
[0241] Exposure of bacterial cells to representative polymer
solutions was started when the culture containing bacterial cells
was added to above polymer suspension which was pre-equilibrated
and shaken at 30.degree. C., and the mixture were incubated at room
temperature for 30 min. The final solution contains ca. 10.sup.8
cells/mL E. coli and 2 mM repeat unit concentration, which is the
molar concentration of the repeat unit of the polymers (ca.
0.6-0.76 mg/mL based on molecular weight of CBAAs and CBAA-esters).
Bacteria were stained with Live/Dead BacLight.TM. (Invitrogen,
USA), and bacterial suspension was subsequently filtered through a
polycarbonate membrane filter with 0.2 .mu.m pore size (Millipore,
USA), and observed directly with a CCD-CoolSNAP camera (Roper
scientific, Inc., USA) mounted on Nikon Eclipse 80i with 100.times.
oil lens. The number of live and dead cells was determined,
respectively, through FITC and Rhodamine filters with the same
microscope described in Cheng, G.; Zhang, Z.; Chen, S.; Bryers, J.
D.; Jiang, S., "Inhibition of Bacterial Adhesion and Biofilm
Formation on Zwitterionic Surfaces," Biomaterials 28(29):4192-4199,
2007. The results are illustrated in FIG. 6.
[0242] 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.
* * * * *