U.S. patent application number 14/982631 was filed with the patent office on 2016-08-25 for polymer hydrogel adhesives formed with multiple crosslinking mechanisms at physiologic ph.
The applicant listed for this patent is Northwestern University. Invention is credited to Devin G. Barrett, Phillip B. Messersmith, Iossif A. Strehin.
Application Number | 20160243275 14/982631 |
Document ID | / |
Family ID | 47997936 |
Filed Date | 2016-08-25 |
United States Patent
Application |
20160243275 |
Kind Code |
A1 |
Messersmith; Phillip B. ; et
al. |
August 25, 2016 |
POLYMER HYDROGEL ADHESIVES FORMED WITH MULTIPLE CROSSLINKING
MECHANISMS AT PHYSIOLOGIC PH
Abstract
The present invention encompasses biocompatible reactants,
biocompatible product hydrogels, methods of use thereof, and
methods of synthesis thereof using a novel crosslinking mechanism
between a first reactant compound including an N-Hydroxysuccinimide
(NHS) ester group and a second reactant compound including a
N-terminal cysteine amine group. In certain embodiments, one or
more of the reactant compounds may be a macromonomer.
Inventors: |
Messersmith; Phillip B.;
(Clarendon Hills, IL) ; Barrett; Devin G.;
(Evanston, IL) ; Strehin; Iossif A.; (Evanston,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Northwestern University |
Evanston |
IL |
US |
|
|
Family ID: |
47997936 |
Appl. No.: |
14/982631 |
Filed: |
December 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13798744 |
Mar 13, 2013 |
9259473 |
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14982631 |
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61662563 |
Jun 21, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 2400/06 20130101;
C08J 3/075 20130101; A61L 24/046 20130101; A61L 24/046 20130101;
A61K 47/34 20130101; C08J 3/246 20130101; A61L 24/0031 20130101;
C08J 2371/02 20130101; A61L 24/046 20130101; C08L 101/02 20130101;
C08L 71/02 20130101; A61L 26/008 20130101; A61L 26/0019
20130101 |
International
Class: |
A61L 26/00 20060101
A61L026/00; C08J 3/075 20060101 C08J003/075 |
Goverment Interests
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under R01 DE
021104 awarded by the National Institutes of Health. The government
has certain rights in the invention.
Claims
1. A hydrogel obtained by covalently cross-linking a first
macromonomer comprising an N-Hydroxysuccinimide (NHS) ester group
with a second macromonomer comprising a N-terminal cysteine
group.
2. The hydrogel of claim 1 wherein covalently cross-linking the
first and second macromonomers comprises: (a) forming an amide bond
between the carboxyl carbon of the N-Hydroxysuccinimide (NHS) ester
group of the first macromonomer and the primary amine of the
N-terminal cysteine group of the second macromonomer to form a
third macromonomer, and (b) forming a disulfide bond between
primary thiol groups on two of the third macromonomers produced in
step (a).
3. The hydrogel of claim 1 wherein the hydrogel is
biocompatible.
4. The hydrogel of claim 1 wherein the first macromonomer, the
second macromonomer, or both, comprise polyethylene glycol.
5. The hydrogel of claim 4, wherein the second macromonomer further
comprises a catechol group.
6. The hydrogel of claim 1, wherein the first macromonomer is
selected from the group consisting of: ##STR00013## and wherein the
second macromonomer comprises the chemical structure:
##STR00014##
7. The hydrogel of claim 6, wherein each n has a value in the range
of from 1 to 201, and wherein each R comprises a hexaglycerin core
or a tripentaerythritol core.
8. A method of synthesizing a hydrogel comprising covalently
cross-linking an effective amount of a first macromonomer
comprising an N-Hydroxysuccinimide (NHS) ester group with an
effective amount of a second macromonomer comprising a N-terminal
cysteine group, wherein a hydrogel is formed.
9. The method of claim 8, wherein the step of covalently
cross-linking the first and second macromonomers comprises: (a)
forming an amide bond between the carboxyl carbon of the
N-Hydroxysuccinimide (NHS) ester group of the first macromonomer
and the primary amine of the N-terminal cysteine group of the
second macromonomer to form a third macromonomer, and (b) forming a
disulfide bond between primary thiol groups on two of the third
macromonomers produced in step (a).
10. The method of claim 8, wherein the hydrogel formed is
biocompatible.
11. The method of claim 8, wherein the step of covalently
cross-linking the first and second macromonomers occurs at
physiological pH.
12. The method of claim 8, wherein the first macromonomer, the
second macromonomer, or both, comprise polyethylene glycol.
13. The method of claim 8, wherein the second macromonomer further
comprises a catechol group.
14. The method of claim 8, wherein the first macromonomer is
selected from the group consisting of: ##STR00015## and wherein the
second macromonomer comprises the chemical structure:
##STR00016##
15. The method of claim 14, wherein each n has a value in the range
of from 1 to 201, and wherein each R comprises a hexaglycerin core
or a tripentaerythritol core.
16. A kit for synthesizing a biocompatible hydrogel comprising: (a)
a first macromonomer comprising an N-Hydroxysuccinimide (NHS) ester
group; and (b) a second macromonomer comprising a N-terminal
cysteine group.
17. The kit of claim 16, wherein the first macromonomer, the second
macromonomer, or both, comprise polyethylene glycol.
18. The kit of claim 16, wherein the first macromonomer is selected
from the group consisting of: ##STR00017## and wherein the second
macromonomer is a macromonomer comprising the chemical structure:
##STR00018##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Appn.
No. 61/662,563 filed Jun. 21, 2013, the entirety of which is hereby
incorporated by reference in its entirety for all purposes.
FIELD OF THE INVENTION
[0003] This invention is directed to biocompatible hydrogel
adhesives and methods of synthesizing such adhesives using a novel
crosslinking mechanism.
BACKGROUND OF THE INVENTION
[0004] When first described in 1992,.sup.1 native chemical ligation
(NCL) revolutionized peptide synthesis by providing a facile,
chemoselective synthetic method for preparation of large peptides
and functional proteins from short fragments. In NCL, an
unprotected N-terminal cysteine (Cys) of one peptide reacts with
the thioester-activated C-terminus of another peptide to form a
thioester intermediate that rearranges via an S-to-N acyl migration
to yield an amide bond linking the two fragments together (FIG.
1)..sup.2,3 NCL leads to minimal epimerization and byproduct
formation and is highly selective to the N-terminal Cys, allowing
the use of unprotected, post-translationally modified, and
non-naturally occurring amino acids. NCL has been used to
synthesize ion channel proteins such as KcsA,.sup.4 the plant
protein crambin,.sup.3 glycosylated proteins such as monocyte
chemotactic protein-3,.sup.5 and other difficult or otherwise
impossible protein sequences.
[0005] Several research groups in the biomaterials community have
explored NCL for preparation of functional materials..sup.6-12
These studies focused on the use of NCL for synthesis of collagen
mimetic biomaterials,.sup.9 chemical modification of
polymers.sup.10,11 and self-assembled peptide scaffolds,.sup.8 and
modification of substrate surfaces..sup.6 In principle, the
chemoselectivity of NCL is attractive for in vitro and in vivo use,
allowing chemical reactions to proceed with specificity in a
complex biological milieu, preserving the bioactivity of endogenous
compounds and facilitating the targeting of therapeutic or
diagnostic molecules to specific biomolecular targets such as cell
surface proteins and components of the extracellular matrix. We
have been developing polymer hydrogels cross-linked via
NCL,.sup.7,12 for potential in vitro and in vivo applications. The
general strategy involves the reaction of a thioester-derivatized
polymer with a second polymer containing N-terminal Cys residues.
Mixing of the two polymer precursors under mild aqueous conditions
led to gel network formation via NCL without the need for added
catalysts..sup.7 Later, we extended this strategy to the formation
of gels for in vitro cell encapsulation, incorporating
polymer-bound IL-1 receptor inhibitory peptides that provided an
immunoprotective effect to entrapped insulin secreting
cells..sup.12
[0006] Despite these recent advances, several aspects of the NCL
reaction remain challenging for use in a biological setting. For
example, standard NCL conditions employ the use of strong reducing
agents that may be harmful in living systems. Furthermore, the slow
rate of NCL cross-linking.sup.7, the hydrolytic instability of the
thioester, and the adverse biological effects of the thiol leaving
group.sup.13 remain obstacles to future in vivo applications of
NCL.
[0007] Several modifications of the NCL reaction have been
introduced in an effort to expand the utility of the
method..sup.14-16 Danishefsky and coworkers described the use of
oxo-esters in NCL (FIG. 1), first through an indirect approach
involving o-thiophenolic ester.sup.17 and followed later by a
direct approach utilizing p-nitrophenyl (pNP) activated C-terminal
ester..sup.18 Termed "oxo-ester-mediated NCL" (OMNCL), this
approach enables high efficiency reactions even with bulky
C-terminal amino acids, although disadvantages include hydrolytic
susceptibility of the pNP ester and challenges associated with
direct solid phase synthesis of pNP ester peptides..sup.19
Weissenborn et al. described OMNCL on oxo-ester activated surfaces
and found 2,3,4,5,6-pentafluorophenyl (PFP) to be more efficient
than pNP and N-hydroxysuccinimide (NHS) activating
agents..sup.20
[0008] Here we describe polymer hydrogel formation via OMNCL
between branched polymer precursors containing NHS activated ester
and N-Cys endgroups..sup.21 Mixing of NHS and N-Cys polymer
precursors led to gel formation within seconds, and quantitative
NMR studies revealed the crosslinking mechanism to be OMNCL. In
addition to characterizing the bulk mechanical and adhesive
properties of the hydrogels, we performed the first in vitro and in
vivo studies of OMNCL hydrogels, showing favorable biological
response in cytotoxicity assays and in a subcutaneous implant
model. The OMNCL hydrogel strategy overcomes many of the earlier
limitations of NCL, including cytotoxicity of thiol leaving groups
and slow reaction kinetics, and represents a promising strategy for
chemical cross-linking of hydrogels in a biological context.
[0009] Specifically, hydrogel materials are appealing as their high
water content and efficient mass transfer are similar to that of
native tissue. One current clinical use of hydrogels is as sealants
since they decrease the incidence of reoperation due to surgical
wound leaks and results in the decrease of cost and patient
morbidity..sup.10 In addition, coating tissue surfaces with
exogenous hydrogel materials may lead to the decrease in the
incidence rate of tissue to tissue adhesion thus decreasing
postoperative complications..sup.11,12 Therefore, tissue adhesive
hydrogels are used daily by surgeons to circumvent complications
and establish adequate wound closure. Some limitations attributed
to the existing NCL hydrogels include slow reaction kinetics at
physiological pH and some cytotoxicity associated with the release
of small molecular weight thiol containing molecules.
[0010] Accordingly, there is a need for a tissue adhesive hydrogel
formulation that uses a modification to the NCL chemistry that
results in faster reaction times under physiological conditions and
that produces a strong, non-cytotoxic product.
SUMMARY OF THE INVENTION
[0011] The present invention provides a hydrogel obtained by
covalently cross-linking a first macromonomer comprising an
N-Hydroxysuccinimide (NHS) ester group with a second macromonomer
comprising a N-terminal cysteine group. In one embodiment,
covalently cross-linking the first and second macromonomers
comprises (a) forming an amide bond between the carboxyl carbon of
the N-Hydroxysuccinimide (NHS) ester group of the first
macromonomer and the primary amine of the terminal cysteine group
of the second macromonomer to form a third macromonomer, and (b)
forming a disulfide bond between primary thiol groups on two of the
third macromonomers produced in step (a).
[0012] In one embodiment, the hydrogel is biocompatible and the
first macromonomer, the second macromonomer, or both, comprise
polyethylene glycol.
[0013] In one embodiment, the first macromonomer is selected from
the group consisting of P8G-NHS, P8GG-NHS, P8MG-NHS, P8S-NHS,
P8MS-NHS and T4G-NHS as set forth in FIG. 26.
[0014] In one embodiment, the second macromonomer comprises a
catechol group such as P8Cys.
[0015] In one embodiment, each n has a value in the range of from 1
to 201, and wherein each R comprises a hexaglycerin core or a
tripentaerythritol core.
[0016] The invention also provides a method of synthesizing a
hydrogel comprising covalently cross-linking an effective amount of
a first macromonomer comprising an N-Hydroxysuccinimide (NHS) ester
group with an effective amount of a second macromonomer comprising
a terminal cysteine group, wherein a hydrogel is formed. The step
of covalently cross-linking the first and second macromonomers
comprises: (a) forming an amide bond between the carboxyl carbon of
the N-Hydroxysuccinimide (NHS) ester group of the first
macromonomer and the primary amine of the terminal cysteine group
of the second macromonomer to form a third macromonomer, and (b)
forming a disulfide bond between primary thiol groups on two of the
third macromonomers produced in step (a). The hydrogel formed is
biocompatible and the step of covalently cross-linking the first
and second macromonomers occurs at physiological pH.
[0017] In one embodiment, the first macromonomer, the second
macromonomer, or both, comprise polyethylene glycol.
[0018] In one embodiment the first macromonomer is selected from
the group consisting of P8G-NHS, P8GG-NHS, P8MG-NHS, P8S-NHS,
P8MS-NHS and T4G-NHS as set forth in FIG. 26.
[0019] In one embodiment, the second macromonomer further comprises
a catechol group and is P8Cys.
[0020] In one embodiment, each n has a value in the range of from 1
to 201, and wherein each R comprises a hexaglycerin core or a
tripentaerythritol core.
[0021] The invention also provides a kit for synthesizing a
biocompatible hydrogel comprising: (a) a first macromonomer
comprising an N-Hydroxysuccinimide (NHS) ester group; and (b) a
second macromonomer comprising a N-terminal cysteine group, wherein
the first macromonomer, the second macromonomer, or both, comprise
polyethylene glycol.
[0022] In one embodiment, the first macromonomer, the second
macromonomer, or both, comprise polyethylene glycol.
[0023] In one embodiment the first macromonomer is selected from
the group consisting of P8G-NHS, P8GG-NHS, P8MG-NHS, P8S-NHS,
P8MS-NHS and T4G-NHS as set forth in FIG. 26.
[0024] In one embodiment, the second macromonomer is P8Cys.
[0025] While multiple embodiments are disclosed, still other
embodiments of the present invention will become apparent to those
skilled in the art from the following detailed description. As will
be apparent, the invention is capable of modifications in various
obvious aspects, all without departing from the spirit and scope of
the present invention. Accordingly, the detailed descriptions are
to be regarded as illustrative in nature and not restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1. Generalized reaction schemes for native chemical
ligation (NCL) and oxo-ester mediated native chemical ligation
(OMNCL).
[0027] FIG. 2. 1H NMR spectra and peak assignments for (A) P8G, (B)
P8NHS, (C) P8G-TE-N-acetyl-Cys, and (D) P8G-AM-S-methyl-Cys in
D.sub.2O. The chemical shifts of the C2 and C4 protons of
P8G-TE-N-acetyl-Cys and P8G-AM-S-methyl-Cys were taken as
representative of P8G-TE-Cys and P8G-AM-Cys, respectively, in the
calculation of relative abundance (RA) of species.
[0028] FIG. 3. The two polymer precursors P8NHS and P8Cys react in
aqueous solution via OMNCL to yield polymer hydrogels with network
cross-links as shown at bottom right.
[0029] FIG. 4. Quantitative 1H NMR analysis of the model reaction
between P8NHS and L-Cys in D.sub.2O. Chemical structures (A) and
relative abundance (B) of polymer species observed during the
reaction.
[0030] FIG. 5. Kinetics of the reaction between P8NHS (1.25% (w/v),
5 mM NHS ester) and L-cysteine (29 mM) in unbuffered D.sub.2O. The
two solutions were mixed in a 1:1 v/v ratio and the reaction
followed by 1H NMR. (A) The four polymer species P8NHS, P8G-AM-Cys,
P8G-TE-Cys and P8G were observed throughout the progress of the
reaction. The protons on carbons C2 and C4 were used to quantify
their relative abundances according to Equations 1-4. (B) The
proton chemical shifts were 2.52 ppm for a!, 2.47 ppm for b!, 2.86
ppm for b , 2.61 ppm for a , 2.52 ppm for a*, 2.82 ppm for b*, 2.52
ppm for a and 2.43 ppm for b. (C) The spectra shown correspond to
reaction times ranging from 4.3 minutes to 121 hours. The
disappearance of P8NHS peaks occurs as P8G-TE-Cys and P8G-AM-Cys
peaks emerge.
[0031] FIG. 6. Quantitative 1H NMR analysis of the reaction between
P8NHS and L-Cys in buffered D.sub.2O. (A,B) Relative abundance of
polymer species formed during reaction of P8NHS with L-Cys at (A)
pH 6.0 and (B) pH 7.0, indicating that the reaction proceeds more
quickly at higher pH. (C) Relative abundance of polymer species
formed during the reaction of P8NHS with S-methyl-L-cysteine at pH
7.0, illustrating significantly slower reaction kinetics when the
thiol group is protected.
[0032] FIG. 7. Kinetics of the reaction between P8NHS (10% (w/v),
38 mM NHS ester) and L-cysteine (57 mM) in buffered D.sub.2O, pH
6.0. The two solutions were mixed in a 1:1 v/v ratio and the
reaction followed by 1H NMR. (A) The four polymer species P8NHS,
P8G-AM-Cys, P8G-TE-Cys and P8G were observed throughout the
progress of the reaction. The protons on carbons C2 and C4 were
used to quantify their relative abundances according to Equations
1-4. (B) The proton chemical shifts were 2.52 ppm for a!, 2.47 ppm
for b!, 2.86 ppm for b , 2.61 ppm for a , 2.52 ppm for a*, 2.81 ppm
for b*, 2.52 ppm for a and 2.43 ppm for b. (C) The spectra shown
correspond to reaction times ranging from 3.2 to 85 minutes. The
disappearance of P8NHS peaks occurs as P8G-TE-Cys and P8G-AM-Cys
peaks emerge.
[0033] FIG. 8. Kinetics of NHS hydrolysis for P8NHS in pH 7.0
buffer. Lyophilized 100 mM PBS (pH 7.0) was re-dissolved in
D.sub.2O and used as the solvent for NMR analysis at a
concentration of 10% (w/v) P8NHS (38 mM NHS ester). The relative
abundance of P8G remained below 0.1 during the first 5 minutes of
the reaction.
[0034] FIG. 9. Reaction of P8NHS with L-Cys and L-Gly. (A) Peak
assignment for C2 and C4 protons of P8G-AM-Gly in D.sub.2O. (B)
Reaction between P8NHS (10% (w/v), 38 mM NHS ester) and L-cysteine
(57 mM) plus L-Gly (57 mM) in buffered D.sub.2O, pH 7.0. The two
solutions were mixed in a 1:1 v/v ratio and the 1H NMR spectrum of
the reaction mixture after 8 minutes is shown. Spectral analysis
indicated that 80% of the product was P8G-AM-Cys, 15% was
P8G-AM-Gly and 5% was P8G.
[0035] FIG. 10. P8NHS plus L-Cys form hydrogels. In a
microcentrifuge tube, 19 mM L-Cys in 100 mM PBS (pH 7.0) was mixed
in a 1:1 v/v ratio with 10% (w/v) P8NHS (38 mM NHS ester) in 100 mM
PBS (pH 7.0), forming stable hydrogels within 2 hours. (A)
Incubation of one such hydrogel for 0.75 hours in PBS containing
0.2 M .beta.-mercaptoethanol resulted in partial solubilization of
the gel, and after 1.25 hours the gel was completely solubilized.
(B) Following incubation of another such hydrogel for 0.75 hours in
pure PBS demonstrated that the hydrogel remained intact. The edges
of the hydrogels are marked with dashed lines.
[0036] FIG. 11. The effect of phosphate buffer concentration on
gelation kinetics of 10% w/v hydrogels prepared in PBS (dilutions
of 100 mM PBS) at room temperature with P8Cys and P8NHS (1:1 w/w).
The plot shows gelation time versus phosphate buffer concentration
for an initial buffer pH of 7.3.
[0037] FIG. 12. Change in pH during the reaction of P8NHS with
L-Cys as a function of phosphate buffer concentration (10 to 80 mM,
initial pH 7.3).
[0038] FIG. 13. The effect of initial pH on gelation time of 10%
w/v hydrogels prepared in 100 mM PBS at room temperature with P8Cys
and P8NHS (1:1 w/w).
[0039] FIG. 14. The effect of temperature on gelation time of 10%
w/v hydrogels prepared with P8Cys and P8NHS (1:1 w/w) in 10 mM
phosphate buffered saline (initial pH 7.0).
[0040] FIG. 15. Physical characterization of OMNCL hydrogels formed
by mixing equal volumes of 10% (w/v) P8NHS and 10% (w/v) P8Cys in
PBS. (A) Swelling of OMNCL hydrogels in 10 mM PBS (closed symbols)
or 10 mM PBS substituted with 0.2 M .beta.-ME (open symbols). The
two sets of hydrogels (diamonds and circles, n=5 per set) varied by
the sequence in which they were incubated in PBS or .beta.-ME. In
one case (circles), the hydrogels were incubated in PBS followed by
(3-ME and then PBS again. In the second case (diamonds), the
hydrogels were incubated in PBS for the first few hours and
thereafter in .beta.-ME. (B) Young's moduli (n=4) at various time
points for OMNCL hydrogels incubated in PBS. *p<0.05,
***p<0.001.
[0041] FIG. 16. Analysis of potential hydrolysis for the ester
between glutaric acid and PEG. Lyophilized 100 mM PBS (pH 7.0) was
re-dissolved in D.sub.2O and used as the solvent for NMR analysis.
(A) The 1H NMR spectrum of pure glutaric acid (5 mg/mL) showed that
the protons located on carbons 2 and 4 of glutaric acid appear at
2.19 ppm. (B) 1H NMR spectrum for the reaction mixture of P8NHS and
L-Cys at pH 7.0 at about 5 minutes, revealing no appreciable
hydrolysis of the ester between glutaric acid and PEG.
[0042] FIG. 17. OMNCL hydrogel degradation in PBS. P8Cys and P8NHS
were dissolved in PBS to yield 10% (w/v) solutions, mixed in a 1:1
(v/v) ratio, and allowed to gel for 15 minutes before being
transferred into 2 mL of PBS. The 70 .mu.L hydrogels were incubated
at 37.degree. C., and at various time points, 3 hydrogels were each
washed with ddH.sub.2O before lyophilizing and dry weights
measured. Following 12 weeks of incubation, no significant loss of
dry weight was observed.
[0043] FIG. 18. In vitro cytocompatibility of OMNCL hydrogels. (A)
Quantitative analysis of 3T3 fibroblast viability after 24 hours in
conditioned medium, conducted in accordance with ISO standards
10993-05 and 10993-12. Cell culture medium included either extract
from P8NHS/P8Cys hydrogel or 5% w/v P8NHS. (B) 3T3 fibroblasts
encapsulated in OMNCL hydrogels and stained with calcein AM (green,
live cells) and ethidium homodimer-1 (red, dead cells). Image
analysis indicated 87.+-.7% of cells remained viable after 24 hours
of encapsulation.
[0044] FIG. 19. In vivo subcutaneous characterization of OMNCL
hydrogel. (A) H&E stained tissue section at 20.times.
magnification with gel associated with the outer skin. The gel is
stained blue and surrounding tissue stained blue and red (an
overview of the skin-gel injection area is shown on the right at
4.times. magnification). (B) H&E stained tissue section at
40.times. magnification from sequentially obtained tissue sections,
showing the gel at the bottom, subtle fibrous capsule (B1) and
supra-capsular muscle layer (B2). (C) Picro-Sirius Red stained
section (40.times. magnification) obtained from the same area as
(B). Hydrogel is at the bottom; the capsule surrounding the
hydrogel is a bright-red fibrous structure (C1) and muscle mass
shown in brown-red (C2). The scale bars indicate length in
micrometers (mc).
[0045] FIG. 20. OMNCL crosslinking of P8Cys and P8NHS. Fast
reaction pathways are indicated by solid arrows, slow pathways by
dashed arrows. Thiol capture followed by S-to-N acyl rearrangement
results in polymer cross-linking. Secondary cross-links arise
through the formation of disulfide bonds among network-bound Cys
residues. P.sub.1=P8NHS; P.sub.2=P8Cys.
[0046] FIG. 21. (A) Synthesis of NHS-terminated PEG derivatives. In
step I, the terminal hydroxyl group of PEG is reacted with an
anhydride to form a terminal carboxyl group with variable PEG to
carboxyl spacers (i.e., R.sub.2). In step II, the terminal carboxyl
groups are activated with an NHS ester. Products 2a-e correspond to
examples 2-5 respectively. Products 3a-e correspond to examples
6-10 respectively. (B) Chemical structure of the products described
in examples 6-10.
[0047] FIG. 22. (A) The two components P8NHS and P8Cys are first
dissolved in aqueous buffer. (B) When the two are mixed together,
the solution turns viscous within seconds and forms a stiff
hydrogel (arrowhead) that clogs the pipette tip (arrow). (C) Top
view of a self-standing cylindrical hydrogel (arrowhead). The
hydrogel was prepared using the cap of an Eppendorf tube as a
mold.
[0048] FIG. 23. Supporting data for disulfide bond formation. (A)
Swelling in PBS and (B) young's moduli for 10% (w/v) 1:1
(P8Cys:P8NHS) hydrogels prepared with 11 mM phosphate buffer in
saline. (C) Thioester+amide bonds per glutaric acid over time for
various concentrations of phosphate buffer (mM). (D) Wet weight of
hydrogels at swelling equilibrium that were exposed to a reducing
agent for 23 hours (diamond) and then the reducing agent was
removed (square).
[0049] FIG. 24. A scheme for the synthesis of catechol-containing
PEG precursor for incorporation into a hydrogel through reaction
with PEG-NHS polymers.
[0050] FIG. 25. Hydrogels prepared with T4G-NHS and P8Cys. The
variable R group can be varied in a similar fashion as the PEG
based polymers (i.e., P8G-NHS, P8GM-NHS, etc.).
[0051] FIG. 26. Structures of first macromonomers comprising an
N-Hydroxysuccinimide (NHS) ester group, including A) P8G-NHS, B)
P8GG-NHS, C) P8MG-NHS, D) P8S-NHS, E) P8MS-NHS and F) T4G-NHS.
DETAILED DESCRIPTION OF THE INVENTION
[0052] The present invention provides a new type of oxo-ester
mediated native chemical ligation (OMNCL) for polymer hydrogel
formation, in vitro cell encapsulation, and in vivo implantation.
Multivalent polymer precursors containing N-hydroxysuccinimide
(NHS)-activated oxo-esters and N-cysteine (N-Cys) endgroups were
chemically synthesized from branched poly(ethylene glycol) (PEG).
Hydrogels formed rapidly at physiologic pH upon mixing of aqueous
solutions of NHS and N-Cys functionalized PEGs. the reaction
proceeds through an OMNCL pathway involving thiol capture to form a
thioester intermediate, followed by an S-to-N acyl rearrangement to
yield an amide cross-link. pH and temperature were found to
influence gelation rate, allowing tailoring of gelation times from
a few seconds to a few minutes. OMNCL hydrogels initially swelled
before contracting to reach an equilibrium increase in relative wet
weight of 0%. This unique behavior impacted the gel stiffness and
was attributed to latent formation of disulfide cross-links between
network-bound Cys residues. OMNCL hydrogels were adhesive to
hydrated tissue, generating a lap shear adhesion strength of 46
kPa. Cells encapsulated in OMNCL hydrogels maintained high
viability, and in situ formation of OMNCL hydrogel by subcutaneous
injection in mice generated a minimal acute inflammatory response.
OMNCL represents a promising strategy for chemical cross-linking of
hydrogels in a biological context and is an attractive candidate
for in vivo applications such as wound healing, tissue repair, drug
delivery, and tissue engineering.
[0053] The invention provides biocompatible reactant compounds,
such as macromonomers having an NHS ester or cysteine group,
methods of synthesis of such reactant compounds, methods of
hydrogel formation by reaction of the NHS ester with the cysteine
group, and methods of using such hydrogels.
I. IN GENERAL
[0054] In the specification and in the claims, the terms
"including" and "comprising" are open-ended terms and should be
interpreted to mean "including, but not limited to . . . ." These
terms encompass the more restrictive terms "consisting essentially
of" and "consisting of."
[0055] As used herein and in the appended claims, the singular
forms "a", "an", and the include plural reference unless the
context clearly dictates otherwise. As well, the terms "a" (or
"an"), one or more and at least one can be used interchangeably
herein. It is also to be noted that the terms "comprising",
"including", "characterized by" and "having" can be used
interchangeably.
[0056] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
publications and patents specifically mentioned herein are
incorporated by reference in their entirety for all purposes
including describing and disclosing the chemicals, instruments,
statistical analyses and methodologies which are reported in the
publications which might be used in connection with the invention.
All references cited in this specification are to be taken as
indicative of the level of skill in the art. Nothing herein is to
be construed as an admission that the invention is not entitled to
antedate such disclosure by virtue of prior invention.
[0057] While multiple embodiments are disclosed, still other
embodiments of the present invention will become apparent to those
skilled in the art from the following detailed description. As will
be apparent, the invention is capable of modifications in various
obvious aspects, all without departing from the spirit and scope of
the present invention. Accordingly, the detailed description of the
hydrogels of the present invention are to be regarded as
illustrative in nature and not restrictive.
II. THE INVENTION
[0058] The present invention encompasses biocompatible reactants,
biocompatible hydrogels, methods of use thereof, and methods of
synthesis thereof using a novel crosslinking mechanism between a
first reactant compound including an N-Hydroxysuccinimide (NHS)
ester group and a second reactant compound including a N-terminal
cysteine amine group. In certain embodiments, one or more of the
reactant compounds may be a macromonomer.
[0059] Two distinct crosslink types form between the first and
second reactant compounds. First, upon initial reaction between the
two compounds, a thioester bond is formed between the first and
second compound. The proximity and orientation of the primary amine
in relation to the thioester leads to a rearrangement to form a
more stable and more permanent amide bond. The second crosslink is
a disulfide bond that forms between the resulting products.
Accordingly, the synthetic method encompasses a chemoselective
reaction between a N-Hydroxysuccinimide (NHS) ester structure and a
cysteine structure. The reaction proceed with a mechanism that is
similar to the mechanism of native chemical ligation, where
transthioesterification between the two reactants first gives a
linked thioester-intermediate, and then this intermediate
rearranges irreversibly under the usual reaction conditions to form
a native amide (`peptide`) bond at the ligation site.
[0060] The synthesis and use of specific reactant compounds and
macromonomers that can be used in the synthetic method, as well as
specific hydrogels made using these reactant compounds and
macromonomers, are disclosed herein. At physiological pH, the
disclosed method quickly produces strong, biocompatible and
non-toxic hydrogels. Accordingly, the method can be used in making
biomedical products, such as sutures and tissue replacement
biomaterials, and for encapsulating therapeutic cells and
pharmaceuticals.
[0061] Compared to the conventional native chemical ligation
reaction, the use of an NHS ester in this reaction results in rapid
formation of a conjugate product without the release of soluble
thiol-containing molecules as side products. The resulting
hydrogels have significantly increased biocompatibility compared to
those formed from conventional thioester-conjugated macromonomers
through the native chemical ligation mechanism. We envision that
the disclosed novel conjugation strategy, the disclosed reactant
compounds and macromonomers containing the NHS esters, and methods
of hydrogel formation using NHS esters will have wide-ranging
utility in both basic and applied biomedical applications.
[0062] Accordingly, the invention encompasses the use of the
methods disclosed herein in the development of biomedical products,
such as surgical sutures, tissue replacement materials and
materials for the encapsulation of therapeutic cells and
pharmaceuticals. In one embodiment, this aspect includes a method
of encapsulating a biological sample with biomaterials. The method
is carried out by a) preparing a biocompatible hydrogel according
to the disclosed method, reacting the biocompatible hydrogel with a
biomaterial to form a modified biocompatible hydrogel; and c)
contacting the biological sample with the modified biocompatible
hydrogel, wherein the hydrogel surrounds and encapsulates the
sample. In certain non-limiting exemplary embodiments, the
biomaterial is an anti-inflammatory peptide.
[0063] The cross-linked synthetic polymer hydrogels of the present
invention are synthesized rapidly at physiological conditions
without the production of toxic side products. Because the
cross-linking is sufficiently rapid, the hydrogel can be formed in
situ from a liquid precursor.
A. Biocompatible Macromonomers
[0064] The present invention provides new biocompatible
macromonomers comprising a cyclic NHS ester group or a cysteine
group. By "biocompatible" we mean a macromonomer that does not have
toxic or injurious effects on biological systems and exhibits
minimal local inflammatory response in surrounding tissues. For
instance, the polyethylene glycol core (PEG) of one embodiment of
the disclosed macromonomers is well-recognized as being
biocompatible, as it is non-immunogenic and resistant to
nonspecific protein and cell adhesions. The macromonomers of the
present invention are useful in a wide variety of applications,
including, for instance, tissue repair, wound healing, drug
delivery, preventing surgical adhesions, as coatings on medical
devices, and thin adherent hydrogels on biosensors and chip-based
diagnostic devices for genomic and proteomic assays.
[0065] While PEG comprises the polymeric core in some embodiments,
alternative polymeric cores including but not limited to linear or
branched biocompatible polymers that can be similarly
functionalized may also be used in the macromonomers of the present
invention. By "functionalized" we mean modifying any linear or
branched biocompatible polymer with N-terminal cysteine peptides as
side chain functional groups or endgroups, or similar polymers
functionalized with NHS esters. In a preferred embodiment, where
PEG comprises the polymeric core of the macromonomer, there are
eight arms emanating from the center of the macromonomer of the
present invention. However, in alternative embodiments, the
polymeric core could comprise two to eight or even ten to twenty
different arms emanating from the center of the macromonomer.
B. Biocompatible Hydrogels
[0066] In some embodiments, the present invention provides new
biocompatible hydrogels comprising covalently cross-linked NHS
ester-polymer and N-terminal cysteine-polymer macromonomers. In
previously disclosed NCL cross-linking using straight chain
thioesters, hydrogel formation was inhibited under physiological
conditions. Furthermore, cytotoxic free thiols were released to the
surrounding medium. In contrast, in the present invention, hydrogel
formation occurs in physiological conditions, and no toxic thiols
are released to the surrounding medium.
[0067] By "biocompatible" we mean a hydrogel that does not have
toxic or injurious effects on biological systems. The hydrogels of
the present invention are useful in a wide variety of applications,
including, for instance, medically useful devices or implants that
can release bioactive compounds in a controlled manner for local,
systemic, or targeted drug delivery; medically useful devices or
implants for use as surgical adhesion prevention barriers,
implantable wound dressings, scaffolds for cellular growth for
tissue engineering or as surgical tissue adhesives or sealants;
biomaterials for preventing transplant rejection; and other
medically useful applications such as hydrogel coatings for
preventing bacterial infection of medical device surfaces, and
coatings for chip-based assays of DNA, RNA or proteins.
C. Methods of Synthesis
[0068] The invention also provides novel methods of synthesis of
the biocompatible macromonomers and hydrogels described above. In
general, the inventors first synthesized a first polymer
macromonomer containing an HNS ester and a second polymer
macromonomer containing an N-terminal cysteine residue. The
macromonomers were then covalently cross-linked to form
biocompatible hydrogels. The reaction conditions described herein
lead to rapid hydrogel formation, and cell proliferation studies
confirmed the non-toxic nature of the resulting hydrogels.
[0069] In one non-limiting exemplary embodiment, the methods
comprise synthesizing and cross-linking an 8-armed PEG terminated
with NHS (P8NHS) and an 8-armed PEG terminated with cysteine
(P8Cys) through a mechanism similar to NCL to form a biocompatible
hydrogel. The advantages of using NCL type methods as compared to
other synthetic hydrogel formation techniques are that the reaction
is very specific and the resulting product is biocompatible. The
covalent cross-linking is initially limited to the cysteine and NHS
ester groups on the PEG molecules, whereas in other hydrogel
forming methods cross-linking can also occur between the synthetic
macromonomers and biological components such as cell surface
proteins and agents in the culture media. The hydrogel formation
occurs under mild physiological conditions (pH 7-9), bearing a
minimal toxicity to the cells during encapsulation. Further
cross-linking occurs through the formation of disulfide bonds.
Furthermore, the resultant hydrogel presents thiol groups that
promote cell adhesion inside the hydrogel network and their mild
reductive properties can also be used to protect encapsulated cells
from oxidative stress.
[0070] The methods of macromonomer and hydrogel formation described
herein provide biocompatible macromonomers and hydrogels which are
easily modified with bioactive materials to improve functions of
encapsulated cells such as supporting cell growth, and the
development and secretion of cellular products upon biological
stimulus. By "bioactive" we mean a substance that has or cause an
effect on in biological samples. For example, the macromonomers and
hydrogels may be further functionalized with peptides or other
bioactive materials, such as proteins, growth factors, DNA,
RNA.
D. Methods of Use
[0071] The biocompatible macromonomers and hydrogels of the present
invention are useful in a wide variety of medically useful devices
and implants. For instance, the biocompatible macromonomers of the
present invention are useful in applications ranging from tissue
repair, wound healing, drug delivery, preventing surgical
adhesions, as coatings on medical devices, and thin adherent
hydrogels on biosensors and chip-based diagnostic devices for
genomic and proteomic assays.
[0072] The biocompatible hydrogels of the present invention are
useful in forming medically useful devices or implants that can
release bioactive compounds in a controlled manner for local,
systemic, or targeted drug delivery. Further, the biocompatible
hydrogels are useful in forming medically useful devices or
implants for use as surgical adhesion prevention barriers,
implantable wound dressings, scaffolds for cellular growth for
tissue engineering or as surgical tissue adhesives or sealants.
Further still, the biocompatible hydrogels are useful in forming
peptide-functionalized hydrogels which can protect transplanted
tissue from rejection. As a non-limiting example, such
peptide-functionalized hydrogels could protect pancreatic islet
cells from inflammatory response post-transplantation.
[0073] In one embodiment, the present invention provides a method
of encapsulating a biological sample with biomaterials comprising
preparing a biocompatible hydrogel according to the methods
described above, reacting the biocompatible hydrogel with a
biomaterial to form a modified biocompatible hydrogel, and
contacting the biological sample with the modified biocompatible
hydrogel, wherein the hydrogel surrounds and encapsulates the
sample. By "biological sample" we mean to include a specimen or
culture obtained from any source. Biological samples can be
obtained from animals (including humans) and encompass fluids,
solids, tissues, and gases. Biological samples include blood
products, such as plasma, serum and the like. Such examples are not
however to be construed as limiting the sample types applicable to
the present invention.
[0074] By "biomaterials" we mean materials selected from the group
consisting of anti-inflammatory agents, cell function promoting
agents, various artificial implants, pacemakers, valves, catheters,
and membranes (e.g., a dialyzer), as well as synthetic polymers
such as polypropylene oxide (PPO) and polyethylene glycol (PEG). In
a further preferred embodiment the biomaterial is an
anti-inflammatory peptide such as an inhibitor of cell surface IL-1
receptor.
E. Kits
[0075] In another embodiment of the invention, a kit for preparing
the biocompatible macromonomers and hydrogels of the present
invention is provided. In one embodiment, the kit comprises a
biocompatible macromonomer having an NHS ester group and a
biocompatible macromonomer having a cysteine group, and
instructions for use.
[0076] In a preferred embodiment, the kit comprises a powdered form
of at least one of the biocompatible macromonomers, wherein the
powdered macromonomer is hydrated by the user for immediate use,
such as in a dual syringe device to form a precursor liquid that
rapidly gels. Optionally, the kit may contain a solution for
dissolving the macromonomer.
[0077] In another preferred embodiment, the kit comprises at least
one of the biocompatible hydrogels discussed above and instructions
for use.
[0078] In an alternate embodiment, the kit comprises a
biocompatible hydrogel according to the present invention
formulated, delivered and stored for use in physiologic
conditions.
[0079] By "instructions for use" we mean a publication, a
recording, a diagram, or any other medium of expression which is
used to communicate the usefulness of the invention for one of the
purposes set forth herein. The instructional material of the kit
can, for example, be affixed to a container which contains the
present invention or be shipped together with a container which
contains the invention. Alternatively, the instructional material
can be shipped separately from the container or provided on an
electronically accessible form on a internet website with the
intention that the instructional material and the biocompatible
hydrogel be used cooperatively by the recipient.
III. EXAMPLES
[0080] The following examples are, of course, offered for
illustrative purposes only, and are not intended to limit the scope
of the present invention in any way. Indeed, various modifications
of the invention in addition to those shown and described herein
will become apparent to those skilled in the art from the foregoing
description and the following examples and fall within the scope of
the appended claims.
Example 1
Materials
[0081] PEG-OH (8 Arm, MW 20082) and PEG-NH.sub.2 (8 Arm, MW 19715)
were purchased from JenKem Technology USA Inc. (Allen, Tex.,
USA).
[0082] Glutaric anhydride, N,N-diisopropylethylamine (DIEA),
1,2-ethanedithiol (EDT), triisopropylsilane (TIS), L-cysteine
(L-Cys), S-methyl-L-Cysteine, N-acetyl-L-Cysteine, glutaric acid,
dibasic sodium phosphate (Na.sub.2HPO.sub.4), potassium chloride
(KCl), sodium chloride (NaCl), N-hydroxysuccinimide (NHS) and
ethidium homodimer-1 were purchased from Sigma (St. Louis, Mo.,
USA).
[0083] The coupling reagent
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
was purchased from TCI America (Portland, Oreg., USA).
[0084] Boc-Cys(Trt)-OH and Calcein AM were purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, Calif., USA).
[0085] Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (BOP) was purchased from Advanced ChemTech
(Louisville, Ky., USA).
[0086] Porcine pericardium was purchased from Animal Technologies,
Inc. (Tyler, Tex., USA).
[0087] The 3T3-swiss albino fibroblasts were purchased from the
American Type Culture Collection center (Manassas, Va., USA).
[0088] Trifluoroacetic acid (TFA) and .beta.-mercaptoethanol were
purchased from Merk KGaE (Darmstadt, Germany).
[0089] Hematoxylin and eosin were purchased from Leica Microsystems
(Buffalo Grove, Ill., USA).
[0090] Picro-sirius red was purchased from Poly Scientific (Bay
Shore, N.Y., USA).
[0091] Monobasic potassium phosphate (KH.sub.2PO.sub.4) and
permount mounting medium were purchased from Fisher Scientific
(Waltham, Mass., USA).
[0092] Dulbecco's phosphate buffered saline (PBS) (pH 7.0) without
calcium or magnesium (Life Technologies, Grand Island, N.Y., USA)
was used to prepare hydrogels and included 2.67 mM KCl, 137.93 mM
NaCl, 1.47 mM KH.sub.2PO.sub.4 and 8.06 mM Na.sub.2HPO.sub.4 (PBS).
Hydrogels prepared with this buffer and used in vitro were
incubated in excess PBS (pH 7.0) or culture medium following
gelation (not more than 15 minutes of gelation).
[0093] NMR, pH measurements, and part of the gelation kinetics
experiments were done with phosphate buffer (100 mM PBS) which was
prepared from PBS substituted with additional KH.sub.2PO.sub.4 and
Na.sub.2HPO.sub.4 and included 2.67 mM KCl, 137.93 mM NaCl, 15 mM
KH.sub.2PO.sub.4 and 85 mM Na.sub.2HPO.sub.4. Other concentrations
of PBS were prepared by diluting 100 mM PBS with saline (2.67 mM
KCl, 137.93 mM NaCl).
Example 2
Synthesis of Glutaric Acid-Terminated 8 Arm PEG (P8G)
[0094] Glutaric acid-terminated PEG (P8G) was synthesized by
dissolving 8-arm PEG-OH (20 g, 7.97 mmol OH or 1 equivalent OH) and
glutaric anhydride (4.54 g, 39.84 mmol, or 5 equivalents) in
chloroform (2.5 mL chloroform per 1 mmol OH). Pyridine (5 eq) was
added dropwise, and the reaction mixture was refluxed at 80.degree.
C. for 24 hours under inert air. The product was diluted with MeOH
(240 mL or 12 mL MeOH per gram of PEG), precipitated at -20.degree.
C. for 1 hour, and centrifuged at -5.degree. C. The supernatant was
discarded, and the MeOH wash procedure was repeated twice more.
Following diethyl ether precipitations (180 mL or 9 mL ether per
gram of PEG), the product was dried under high vacuum overnight to
afford a white powder (97.6% yield, 100% conversion). See FIG.
21.
[0095] 1H NMR (500 MHz, CDCl.sub.3), .delta., ppm: .delta. 4.24
(2H, t, terminal PEG CH.sub.2), .delta. 3.64 (1823H, m, backbone
PEG CH.sub.2), .delta. 2.43 (2H, t, C2 of glutaric acid), .delta.
2.39 (2H, t, C4 of glutaric acid), .delta. 1.95 (2H, p, C3 of
glutaric acid).
Example 3
Synthesis of Glycol Glutaric Acid-Terminated 8 Arm PEG (P8GG)
[0096] The same procedure used to synthesize P8G was used to
synthesize P8GG, with the exception that diglycolic anhydride was
used instead of glutaric anhydride (91% yield, 99.9%
conversion).
Example 4
Synthesis of 3-Methyl Glutaric Acid-Terminated 8 Arm PEG (P8MG)
[0097] The same procedure used to synthesize P8G was used to
synthesize P8MG, with the exception that 3-methyl glutaric
anhydride was used instead of glutaric anhydride (87.1% yield,
102.9% conversion).
Example 5
Synthesis of Methyl Succinic Acid-Terminated 8 Arm PEG (P8MS)
[0098] The same procedure used to synthesize P8G was used to
synthesize P8MS, with the exception that methyl succinic anhydride
was used instead of glutaric anhydride (79.5% yield, 99.3%
conversion).
Example 6
Synthesis of N-Hydroxysuccinimide (NHS)-Terminated 8 Arm PEG
(P8G-NHS)
[0099] NHS-terminated 8 arm PEG (P8G-NHS) was synthesized by
dissolving P8G (20 g, 7.62 mmoL or 1 equivalent COOH), NHS (8.77 g,
76.20 mmol or 10 equivalents) and EDC (14.61 g, 76.20 mmol or 10
equivalents) in DMSO (50 mL or 2.5 mL DMSO per gram of PEG). After
30 minutes of stirring at room temperature, the product was diluted
with MeOH (500 mL or 25 mL MeOH per gram of PEG), precipitated at
-20.degree. C. for 1 hour, and spun down at -5.degree. C. The
supernatant was discarded, and the MeOH wash procedure was repeated
twice more. Following diethyl ether precipitations (480 mL or 25 mL
ether per gram of PEG), the product was dried under high vacuum
overnight to afford a white powder (80.6% yield, 97.4% conversion).
See FIG. 21 B.
[0100] 1H NMR (500 MHz, CDCl.sub.3), .delta., ppm: .delta. 4.24
(2H, t, terminal PEG CH.sub.2), .delta. 3.64 (1961H, m, backbone
PEG CH.sub.2), .delta. 2.84 (4H, m, NHS protons), .delta. 2.71 (2H,
t, C4 of glutaric acid), .delta. 2.49 (2H, t, C2 of glutaric acid),
.delta. 2.06 (2H, p, C3 of glutaric acid).
Example 7
Synthesis of N-Hydroxysuccinimide (NHS)-Terminated 8 Arm P8GG
(P8GG-NHS)
[0101] The same procedure used to synthesize P8G-NHS was used to
synthesize P8GG-NHS, with the exception that P8GG was used instead
of P8G (85.1% yield, 68.9% conversion).
Example 8
Synthesis of N-Hydroxysuccinimide (NHS)-Terminated 8 Arm P8MG
(P8MG-NHS)
[0102] The same procedure used to synthesize P8G-NHS was used to
synthesize P8MG-NHS, with the exception that P8MG was used instead
of P8G (92.2% yield, 95% conversion).
Example 9
Synthesis of N-Hydroxysuccinimide (NHS)-Terminated 8 Arm P8S
(P8S-NHS)
[0103] The same procedure used to synthesize P8G-NHS was used to
synthesize P8S-NHS, with the exception that P8S was used instead of
P8G (93.6% yield, 97.1% conversion).
Example 10
Synthesis of N-Hydroxysuccinimide(NHS)-Terminated 8 Arm P8MS
(P8MS-NHS)
[0104] The same procedure used to synthesize P8G-NHS was used to
synthesize P8MS-NHS, with the exception that P8GG was used instead
of P8MS (81.8% yield, 98.5% conversion).
Example 11
Synthesis of Cysteine Terminated 8 Arm PEG (P8Cys)
[0105] Cysteine-terminated 8 arm PEG (P8Cys) was synthesized using
BOP as a coupling reagent. In one reaction vessel, 8 arm PEG amine
(13.4 g, 5.44 mmol NH.sub.2) was dissolved in DMF (25 mL) and DIEA
was added dropwise (947 .mu.L, 5.44 mmol). In a separate reaction
vessel, Boc-Cys(Trt)-OH (10.0 g, 21.75 mmol) and BOP (9.62 g, 21.75
mmol) were dissolved in DMF (25 mL) and DIEA (3.79 mL, 21.75 mmol)
was added dropwise. Five minutes after adding DIEA, the BOP
solution was combined with the PEG solution and the coupling
reaction was allowed to proceed at room temperature for 18 hours.
Following precipitation in cold diethyl ether (2800 mL), the
product was re-dissolved in MeOH (70 mL) and precipitated in cold
diethyl ether once more (280 mL). The cysteine was deprotected with
TFA:TIS:EDT (210 mL, 95:2.5:2.5) cleavage solution at room
temperature for 4 hours. TFA was evaporated under low pressure, and
the product was precipitated in cold diethyl ether (160 mL). P8Cys
was dissolved in MeOH (120 mL), precipitated at -20.degree. C. for
1 hour, and centrifuged at -5.degree. C. The supernatant was
decanted and the MeOH precipitation was repeated twice more.
Following diethyl ether precipitations (30 mL), the product was
dried under high vacuum overnight to afford an extra pure white
powder (50% yield, 86% conversion).
[0106] 1H NMR (500 MHz, Acetic Acid-d.sub.4), .delta., ppm: .delta.
4.42 (1H, t, .delta. --C cysteine), .delta. 3.69 (1872H, m,
backbone PEG CH.sub.2), .delta. 3.13 (2H, d, CH.sub.2
cysteine).
Example 12
Hydrogel Preparation Using P8G-NHS
[0107] PBS was used to prepare 10% (w/v) P8G-NHS and 10% (w/v)
P8Cys and the two solutions were mixed in a 1:1 (v/v) ratio to form
cylindrical 70 .mu.L hydrogels (5 mm diameter.times.3.5 mm
height).
Example 13
Hydrogel Preparation Using P8GG-NHS
[0108] PBS was used to prepare 10% (w/v) P8GG-NHS and 10% (w/v)
P8Cys and the two solutions were mixed in a 1:1 (v/v) ratio to form
cylindrical 70 .mu.L hydrogels (5 mm diameter.times.3.5 mm
height).
Example 14
Hydrogel Preparation Using P8MG-NHS
[0109] PBS was used to prepare 10% (w/v) P8MG-NHS and 10% (w/v)
P8Cys and the two solutions were mixed in a 1:1 (v/v) ratio to form
cylindrical 70 .mu.L hydrogels (5 mm diameter.times.3.5 mm
height).
Example 15
Hydrogel Preparation Using P8S-NHS
[0110] PBS was used to prepare 10% (w/v) P8S-NHS and 10% (w/v)
P8Cys and the two solutions were mixed in a 1:1 (v/v) ratio to form
cylindrical 70 .mu.L hydrogels (5 mm diameter.times.3.5 mm
height).
Example 16
Hydrogel Preparation Using P8MS-NHS
[0111] PBS was used to prepare 10% (w/v) P8MS-NHS and 10% (w/v)
P8Cys and the two solutions were mixed in a 1:1 (v/v) ratio to form
cylindrical 70 .mu.L hydrogels (5 mm diameter.times.3.5 mm
height).
Example 17
NMR Analysis of Reaction Between L-Cys and P8NHS
[0112] All reactions were done at room temperature. First, 100 mM
PBS (pH adjusted to either 6.0 or 7.0) was lyophilized, redissolved
in the same volume of D.sub.2O, and then used to prepare solutions
of L-cysteine (57 mM) and P8NHS (10 w/v %, 38 mM NHS ester). The
two solutions were mixed in a 1:1 v/v ratio and reaction kinetics
followed using 1H NMR in a Varian Inova 500 MHz NMR. Similar
experiments were carried out in pure D.sub.2O as described above by
mixing solutions of L-Cys (29 mM) and P8NHS (1.25% w/v, 5 mM NHS
ester).
[0113] The relative abundance (RA) of polymer species during the
OMNCL reaction between P8NHS and L-Cys was determined by
integrating the triplets associated with the protons bound to the
C2 and C4 carbons of the terminal glutarate linker. In this
reaction four polymer species are possible (P8NHS, PBG, P8G-TE-Cys,
P8G-AM-Cys), each possessing slightly different chemical shifts of
the C2 and C4 protons depending on the composition of the terminal
group (FIG. 2 and Table 1).
TABLE-US-00001 TABLE 1 .sup.1H NMR chemical shift peak assignments
used in calculation of relative abundance of species during
reaction of P8NHS with L-Cys. ##STR00001## Species C2 C4 P8NHS 2.61
2.86 P8G 2.52 2.47 P8G-TE-Cys 2.52 2.81 P8G-AM-Cys 2.52 2.43
[0114] The chemical shifts for the protons associated with the C2
and C4 carbons of glutarate of P8G and P8NHS were determined from
spectra of the synthesized intermediate and final product (see
above), whereas the chemical shifts of thioester (P8G-TE-Cys) and
amide (P8G-AM-Cys) linked products were estimated from spectra
obtained by reaction of P8NHS with N-acetyl-L-cysteine and
S-methyl-L-cysteine, respectively. Integrated peak values were used
in the equations below to calculate the relative abundance of each
species present during the reaction of P8NHS and L-Cys.
RA P 8 G = .delta. 2.47 .delta. 2.61 + .delta. 2.52 ( 1 ) RA P 8
NHS = .delta. 2.61 .delta. 2.61 + .delta. 2.52 ( 2 ) RA P 8 G - TE
- Cys = .delta. 2.52 - .delta. 2.47 - .delta. 2.43 .delta. 2.61 +
.delta. 2.52 ( 3 ) RA P 8 G - AM - Cys = .delta. 2.43 .delta. 2.61
+ .delta. 2.52 ( 4 ) ##EQU00001##
Example 18
pH Measurement
[0115] PBS (100 mM PBS and dilutions of the buffer) was used to
prepare 38 mM L-cysteine and 10% (w/v) P8NHS (38 mM NHS ester). The
two solutions were mixed in a 1:1 v/v ratio and the pH of the
reaction mixture was followed over time using a standard pH meter
(Accumet Titration Controller, Model 150, Fisher Scientific,
Waltham, Mass., USA) and electrode (Beckman 511275-AB Electrode,
Beckman, Pasadena, Calif., USA).
Example 19
Gelation Time
[0116] The time to form a hydrogel was measured using a previously
described protocol..sup.22 Briefly, 10% (w/v) P8NHS and 10% (w/v)
P8Cys were prepared in phosphate buffered saline (PBS, 100 mM PBS,
or dilutions of 100 mM PBS). The two solutions were then mixed in a
1:1 (v/v) ratio and pipetted up and down using a standard 2-200
.mu.L pipette tip. The time at which the material blocked the
pipette tip was designated as the gelation time. Temperature was
controlled within the range 4-60.degree. C. through the use of a
water bath.
Example 20
Hydrogel Swelling
[0117] PBS was used to prepare 10% (w/v) P8NHS and 10% (w/v) P8Cys
and the two solutions were mixed in a 1:1 (v/v) ratio to form
cylindrical 70 .mu.L hydrogels (5 mm diameter.times.3.5 mm height).
The gels were allowed to set for 15 minutes and then transferred to
PBS. The wet weights of the gels were measured at various time
points, and buffer was replaced at least 5 times a week. At 5
hours, half of the hydrogels (n=5) were exposed to reducing agent
(0.2 M .beta.-mercaptoethanol in PBS) and swelling was monitored
until equilibrium was reached for both groups. At 303 hours the
hydrogels equilibrated in PBS were exposed to reducing agent and
swelling was monitored until 447 hours, after which the hydrogels
were transferred back to PBS and swelling was followed until
equilibrium was reached. The hydrogel swelling experiments were all
carried out under room temperature and with five replicates per
group. Relative wet weight was calculated as defined in equation 5,
where w.sub.0 is the initial wet weight and w.sub.t is the wet
weight of the hydrogel at time t.
Relative Wet Weight = w w 0 ( 5 ) ##EQU00002##
Example 21
Mechanical Testing
[0118] The compressive moduli and adhesive strength of the hydrogel
(10% w/v, 1:1 w/w P8NHS:P8Cys, prepared in PBS) were measured at
room temperature with a Syntech Model #20G screw actuation testing
machine equipped with 250 g and 1000 lb load cells. Compressive
moduli were measured using cylindrical hydrogels (6.5 mm
height.times.8.5 mm diameter) that were swollen for 5, 75 or 150
hours in PBS. The hydrogels were compressed along their axes
between two flat plates, and the equilibrium stress was recorded
for strains between 1 and 10%. The Young's modulus was calculated
by measuring the slope of the linear portion of the stress vs.
strain curve.
[0119] The adhesive strength of the hydrogel was measured using a
lap shear test adapted from ASTM standard F2255-05. Unprocessed
porcine pericardium (2.5 cm.times.3 cm) was adhered to aluminum
fixtures using a cyanoacrylate based adhesive and then covered with
a PBS moistened paper. After 1 hour, 100 .mu.L of hydrogel
precursor was placed on one tissue surface and a second tissue
surface brought into contact (2.5 cm.times.1.25 cm overlap) such
that the tissue sections were glued together. After 10 minutes of
curing, the tissue and glue were covered with PBS moistened paper
towel for an additional 50 minutes. The glued tissue was pulled
apart at 5 mm/min in tensile shear, and the peak stress and tissue
overlap area were used to calculate the adhesive strength of the
material.
Example 22
Cytotoxicity
[0120] Cytotoxicity was evaluated using two methods. For the first
assay, the guidelines found in ISO standards 10993-05 and 10993-12
were used. Briefly, 3T3 fibroblasts were exposed to polymer
precursor or hydrogel extract. Extracts were prepared by suspending
a 200 .mu.L hydrogel in 1 mL of culture medium (n=3);
alternatively, P8NHS was dissolved directly in culture medium to
yield a 5% (w/v) solution (n=3). These solutions were then
incubated at 37.degree. C., 5% CO.sub.2 and >90% RH for 24
hours, diluted to various concentrations using fresh medium, and
100 .mu.L of each dilution added to a subconfluent monolayer of 3T3
fibroblasts in 96 well plates (n=3). The cells were incubated in
the presence of the conditioned medium at 37.degree. C., 5%
CO.sub.2 and >90% RH for 24 hours. After washing with PBS the
cells were exposed for 3 hours to 0.4% neutral red prepared in
DMEM. The cells were again washed with PBS and destained using 1%
glacial acetic acid, 50% ethanol and 49% ddH.sub.2O. Following 10
minutes of agitation, absorbance at 540 nm was used to quantify
viability. Culture medium and 0.2% SDS were used as a negative and
positive control and were incubated along with the extracts at
37.degree. C., 5% CO.sub.2 and >90% RH for 24 hours prior to
addition to the subconfluent monolayer of cells. Per the
requirements stated in the ISO standard, IC.sub.50 of the positive
control SDS was found to be within the acceptable range, confirming
the validity of the assay.
[0121] In the second viability assay, cells were suspended in the
P8Cys component dissolved in PBS and mixed with the P8NHS component
dissolved in PBS to yield a 7% (w/v) hydrogel containing 1:1 (w/w)
ratio of P8Cys to P8NHS. The hydrogels were allowed to set for 1 to
2 minutes and then incubated in culture medium at 37.degree. C. and
5% CO.sub.2. After 24 hours, cell viability was quantified using
calcein AM and ethidium homodimer-1. Cells were stained for 15
minutes in culture medium substituted with 4 .mu.M calcein AM and 4
.mu.M ethidium homodimer-1. Images were acquired using a
fluorescent microscope equipped with a 485.+-.10 nm optical filter
for calcein AM (live cells) and a 530.+-.12.5 nm optical filter for
ethidium homodimer-1 (dead cells). The images were merged and
processed using ImageJ (National Institute of Health, Bethesda,
Md.).
Example 23
In Vivo Studies
[0122] B10.BR male mice were obtained from the Jackson Laboratories
(Bar Harbor, Me., USA). The mice were housed in the Animal Facility
of the Wistar Institute and all treatments were approved by the
Wistar IACUC. At 10 weeks of age, mice were injected subcutaneously
at the base of the neck with 100 .mu.L of 10% (w/v) 1:1 (w/w) ratio
of P8Cys to P8NHS hydrogel prepared in PBS. Six weeks later, mice
were euthanized and tissue together with the hydrogel were removed
and fixed in 4% buffered formalin for 24 hours. Tissue was then
washed, dehydrated through serial ethanol washes, cleared with
xylene and embedded in paraffin overnight. Tissue sections of 5-10
.mu.m thickness were cut and mounted on superfrost slides (VWR,
Radnor, Pa., USA).
[0123] Tissue sections were cleared, rehydrated, and then stained
with hematoxylin and eosin or with picro-sirius red, dehydrated,
cleared with xylene and coverslipped with Permount mounting media.
Staining was visualized using an Olympus (AX70) microscope (Olympus
America, Center Valley, Pa., USA) in bright field for H&E and
under polarized light for Picro-Sirius Red. Images were recorded
using a Spot camera with bounded software.
Example 24
Statistical Analysis
[0124] One-way ANOVA was used to detect significant effects among
groups. Tukey's multiple comparison tests were used to detect
significant differences between groups, and a p-value .ltoreq.0.05
was considered significant.
Results.
[0125] Polymer Synthesis and Hydrogel Formation.
[0126] P8NHS was synthesized with an overall yield of approximately
95% through a two-step reaction involving addition of a glutaric
acid linker to an 8-arm PEG followed by activation of terminal acid
groups with NHS. P8Cys was synthesized in one step from amine
terminated 8-arm PEG and N,S-protected Cys amino acid followed by
deprotection and purification for a yield of 50%. Within
approximately 20 seconds of mixing P8Cys and P8HNS the solution
became noticeably viscous and solidified to form a stiff hydrogel
(FIG. 3). Under the same conditions, a hydrogel formed within
approximately 240 seconds when P8Cys was replaced with the 8-arm
PEG-NH.sub.2 polymer used to synthesize P8Cys, suggesting the
reaction mechanism between P8Cys and P8NHS involves thiol capture
followed by a S-to-N acyl rearrangement (OMNCL) rather than a
direct reaction between the activated ester of P8NHS and the
terminal amine of P8Cys.
[0127] Mechanism of Hydrogel Formation.
[0128] In model experiments designed to elucidate the reaction
mechanism, the buffered and unbuffered reaction between P8NHS and
L-Cys were analyzed by 1H NMR. We determined the relative abundance
of four major polymer species with time during the reaction: the
NHS activated polymer precursor (P8NHS), hydrolysis product (P8G),
the thioester formed by thiol capture of L-Cys by P8NHS
(P8G-TE-Cys), and the amide linked product (P8G-AM-Cys) (FIG. 4A).
Analysis of the 1H NMR spectra acquired for up to 120h after mixing
revealed clear chemical shift changes corresponding to the
appearance and disappearance of the reactants, intermediates and
products (FIG. 5). Spectral analysis using peak assignments
obtained from reference spectra (FIG. 2) and calculation of
relative abundance according to equations 1-4 revealed the temporal
progression of the reaction (FIG. 4B).
[0129] Upon mixing P8NHS and L-Cys, rapid disappearance of P8NHS
was accompanied by rapid emergence of the thioester intermediate
P8G-TE-Cys. P8G-TE-Cys reached a maximum value after several
minutes and slowly started to disappear thereafter and was found in
only trace amounts after 75 h (FIG. 4B). Concomitant with
disappearance of P8G-TE-Cys, the amide linked product P8G-AM-Cys
emerged slowly over the first hour and represented over
approximately 80% of species present in the reaction mixture after
75 h. P8G appeared in minor but detectable amounts, present at less
than about 20% at all time points. Similar trends were observed
when the reaction of P8NHS and L-Cys was performed in the presence
of phosphate buffer at pH 6, albeit with significantly faster
kinetics (FIG. 6A and FIG. 7). At pH 7, the reaction was
essentially complete within 5 minutes (FIG. 6B).
[0130] Under the same conditions, control experiments showed that
hydrolysis of P8NHS was insignificant within this timeframe (FIG.
8). At pH 7, when L-Cys was replaced with S-methyl-L-cysteine, the
reaction was significantly slower (FIG. 6C) despite the lower pKa
of the amino group of S-methyl-L-cysteine (8.75.sup.23 vs. 10.78
for L-Cys.sup.23). In a separate experiment at pH 7, P8NHS was
reacted with equal concentrations of both L-Cys and L-Gly (amine
pKa 9.6.sup.23), yielding 80% P8G-AM-Cys, 15% P8G-AM-Gly and 5% P8G
(FIG. 9). Interestingly, mixtures of 10% (w/v) P8NHS (38 mM NHS
ester) and 19 mM L-Cys were observed to form stable gels after 2
hours of incubation (100 mM PBS, pH 7.0), although these gels
liquefied in the presence of .beta.-mercaptoethanol but not PBS
(FIG. 10), implying cross-linking via disulfide bond formation.
[0131] The crosslinking mechanism between P8Cys and P8NHS leads to
the formation of two distinct crosslink types (Table 2). One type
of crosslink is a result of the thiol attacking the NHS ester to
form a thioester. The proximity and orientation of the primary
amine in relation to the thioester leads to a rearrangement to form
a more stable and hence more permanent amide bond. This mechanism
of amide bond formation via a rearrangement is similar to native
chemical ligation (NCL), with the exception that a more reactive
NHS ester is used as opposed to a thioester..sup.14
TABLE-US-00002 TABLE 2 Crosslinking mechanism between P8Cys and
P8NHS: Re- Thiol Attacks First Crosslink arrangement Second
Crosslink NHS Ester NHS Release (Thioester) (Amide) (Disulfide
Bridge) Polymer Inter- action ##STR00002## ##STR00003##
##STR00004## ##STR00005## ##STR00006## ##STR00007## Chemical
Structure Of Cross- link ##STR00008## ##STR00009## ##STR00010##
##STR00011## ##STR00012##
[0132] The second crosslink, a disulfide bond, leads to a decrease
in swelling and improved mechanical properties. Since hydrogels are
hydrophilic materials, once they set they may imbibe physiological
fluid which can lead to a significantly increase in volume. For
example, PEG based hydrogels crosslinked via amide or thioester
bonds alone can swell up to 50%.sup.15 and 400%.sup.16 within the
first 24 hours. In some cases, swelling of implanted materials may
lead to complications such as nerve compression.sup.17-21 or other
serious problems requiring reoperation..sup.22 The consequent
symptoms may range from uncomfortable to life threatening. The
tissue adhesive material presented herein is crosslinked both via
amide and disulfide bonds. The rearrangement step introduces free
thiols that subsequently react with each other to form disulfide
bridges further crosslinking the material (FIG. 23). As a result of
the extra crosslinking, the hydrogels exhibit minimal swelling.
Since the PNC hydrogel shows minimal equilibrium swelling (5%, FIG.
23A) and maintains a high adhesive strength, it may be ideal for
applications where an increase in hydrogel volume can lead to
complications.
[0133] Physical Characterization of Hydrogels.
[0134] Buffer concentration, pH and temperature were found to
strongly influence the rate of gel formation. As the phosphate
buffer concentration (initial pH 7.3) was increased from 10 to 80
mM, gel time decreased from about 28 s to less than 9 s,
respectively (FIG. 11). As we could not measure the pH within the
hydrogel itself, instead we measured the pH of a P8NHS/L-Cys
reaction mixture under equivalent conditions in order to ascertain
pH changes during the reaction. As shown in FIG. 12, the pH was
found to decrease with time during the reaction. Reaction solutions
with higher buffer concentration yielded smaller pH changes, with
buffer concentrations of 80 mM exhibiting a pH drop of 0.8 pH units
during the reaction. Hence, higher buffer concentration led to
faster gelation and a lower drop in pH, implying that the reaction
is accelerated at high pH.
[0135] These findings led us to select a buffer concentration of
100 mM for gel kinetic studies under different pH conditions. The
pH dependence of gelation time for a 10% w/v mixture of P8NHS and
P8Cys in 100 mM PBS is shown in FIG. 13. It can be seen in this
figure that gel time changed significantly within the pH range 6-8,
ranging from about 50 s at pH 6 to <10 s at pH 8. Finally, we
determined the temperature dependence of gelation time, which
showed that gel formation was accelerated by about 9 s when the
temperature was changed from room to body temperature (FIG.
14).
[0136] Several types of mechanical characterizations were
undertaken on hydrogels formed from P8NHS with P8Cys. Swelling
experiments were performed by immersing hydrogel samples in excess
PBS and measuring the weight changes as a function of time. OMNCL
hydrogels increased in relative wet weight by approximately 21% in
the first several hours and then slowly contracted over a period of
many hours to a final increase in relative wet weight of
approximately 0% (FIG. 15A). The young's modulus of the hydrogel
was measured at 5, 75 and 150 h and was found to increase from 128
to 182 kPa during this time (FIG. 15B). The latent modulus increase
is unlikely to be attributed to additional cross-linking by the
OMNCL mechanism, as model 1H NMR studies showed that at pH 7 the
reaction was mostly complete after 5 minutes (FIG. 6B). The
observed swelling and modulus changes are unlikely to reflect mass
changes induced by hydrolytic degradation of the gels, as NMR
analysis of ester hydrolysis (FIG. 16) and a preliminary evaluation
of gel degradation at pH 7.0 showed little mass loss over a 12 week
period (FIG. 17).
[0137] Suspecting therefore that gel shrinkage was the result of
disulfide bond formation, fully equilibrated hydrogels (>300
hours swelling in PBS) were transferred into 0.2 M
.beta.-mercaptoethanol in PBS, whereupon the hydrogels increased in
relative wet weight by approximately 27% (FIG. 15A). After
approximately 140 hours in reducing agent, swollen samples were
transferred back into PBS and swelling was observed to decrease
once again, implying the re-formation of disulfide bonds.
[0138] Finally, the adhesive strength of the OMNCL hydrogel to
unprocessed porcine pericardium was measured in lap shear similar
to the protocol described in ASTM standard F2255-05. Tissue
surfaces were glued together using the OMNCL hydrogel and then
pulled apart after one hour post gelation, yielding a lap shear
adhesive strength of 46.+-.8 kPa.
[0139] Cytotoxicity.
[0140] The cytotoxicity of the hydrogel was tested using two
methods. The first method was conducted in accordance with ISO
standards 10993-5 and 12, from which it was concluded that the
OMNCL hydrogel as well as P8HNS polymer (5% w/v solution) were not
toxic to cells as viability remained well above 90% (FIG. 18A). It
was not possible to analyze P8Cys precursor in this cytotoxicity
assay, as a 5% (w/v) solution of P8Cys in cell culture medium
solidified within 3 hours, presumably via disulfide bond formation.
In the second test, cells were encapsulated in OMNCL hydrogels and
viability was quantified after 24 hours using calcein AM and
ethidium homodimer-1. In this assay we observed that 87.+-.7% of
the encapsulated cells remained viable 1 day following
encapsulation (FIG. 18B).
[0141] In Vivo Studies.
[0142] Subcutaneous injections of OMNCL hydrogel were administered
to B10.BR male mice, and after 6 weeks the implants were extracted
and processed for histological analysis. The results showed the
remnants of an acute inflammatory response typical of PEG based
hydrogels. H&E stained histological sections showed that the
surrounding tissue was intact. A low number of inflammatory cells
were observed in the vicinity of the implant, and a thin fibrous
capsule formed around the hydrogel (FIG. 19A, B), as indicated by
positive staining for collagen fibrils with Picro-Sirius red under
polarized light illumination (FIG. 19C).
[0143] Discussion.
[0144] We have detailed the synthesis, reaction mechanism and
characterization of a two-component hydrogel formulation that sets
rapidly by OMNCL at physiologic pH and ionic strength. The material
is clear (FIG. 22) which makes it an attractive candidate for wound
closure in ophthalmic surgery or other such areas of medicine where
light obstruction is undesirable. The OMNCL reaction involves
covalent capture of the thiol side chain of N-terminal Cys to
initially form a thioester bond. The proximity and orientation of
the primary amine in relation to the thioester leads to a S-to-N
acyl rearrangement to form the more stable amide bond. This
mechanism of amide bond formation via rearrangement is similar to
native chemical ligation (NCL),.sup.24 with the exception that in
OMNCL a oxo-ester is employed as opposed to a thioester (FIG.
1)..sup.18
[0145] Implementation of NCL in a biological context can be
problematic due to the adverse biological effects of the buffers
and reducing agents typically employed in NCL reactions.
Furthermore, we have shown that the small molecule thiol leaving
group liberated during NCL can be toxic to cells..sup.13 OMNCL, on
the other hand, is often practiced in solutions containing
concentrated guanidine (5-6M),.sup.18-20 which would have adverse
effects in a biological system because of its strong denaturing
potential. Our results show that hydrogel formation by OMNCL
proceeds well in phosphate buffer, achieving rapid gel formation
without the use of catalysts or additives. In the pH range of our
experiments (pH 6-7.3), the rate of gel formation by OMNCL was
adjustable through pH control and ranged from several seconds to
less than a minute. This is in contrast to gels formed by NCL
chemistry,.sup.712 which have slower gelation rates at
physiological pH.
[0146] We elucidated the mechanism of cross-linking by following
the reaction of P8NHS and L-Cys by 1H NMR (FIGS. 4 and 6A, B),
taking advantage of chemical shift differences to reveal the
temporal evolution of reactants, intermediates and products.
Several lines of evidence from these studies point to cross-link
formation via OMNCL. First, the rapid increase in thioester
cross-link in parallel with the rapid decrease in P8NHS at the
beginning of the reaction is indicative of thiol capture. The
thioester intermediate reaches as high as >50% relative
abundance within the first few minutes but then decreases until it
is no longer detectable. This decrease cannot be explained by
thioester hydrolysis, as the hydrolysis product (P8G) was never
present at greater than about 10% relative abundance at pH 6 or 7.
Second, the kinetics of thioester cross-link disappearance was
roughly matched with the kinetics of amide cross-link emergence,
which is a strong indicator of S-to-N rearrangement. It should be
noted that at neutral and acidic pH the thiol should be
considerably more reactive than the primary amino group due to the
high pKa of the terminal amine of L-Cys (pKa about 10.78.sup.23).
As evidence of this, in the reaction between P8NHS and L-Cys at
neutral pH, conversion to P8G-AM-Cys and P8G-TE-Cys was 83% within
the first minute. However, reaction of P8NHS with S-methyl-L-Cys
yielded only 20% conversion under the same conditions, despite the
significantly lower pKa of the S-methyl-L-Cys amino group (pKa
about 8.75).sup.23 compared to L-Cys. Finally, further support for
OMNCL pathway was provided by a competitive reaction between P8NHS,
L-Cys and L-Gly, where 80% of the reaction proceeded with L-Cys and
only 15% with L-Gly despite the lower amino pKa of L-Gly (pKa about
9.6.sup.23).
[0147] Taking these findings into consideration, we therefore
propose the OMNCL reaction pathway shown in FIG. 20 for the
gel-forming reaction between P8NHS and P8Cys. For reasons
elaborated above, we consider the hydrolysis of P8NHS and the
thioester intermediate as minor competing reactions under our
conditions. Both of these reactions would produce acid terminated
polymer endgroups that would not be capable of contributing to gel
formation. It is impossible to exclude some contribution from
direct amide bond formation between the terminal amine of P8Cys and
P8NHS. However, it should be noted that the pKa value of the
N-terminal amino of Cys is 10.78 whereas that of the thiol side
chain is 8.33..sup.23 Thus, at the pH values of 6-7.3 employed in
gel formation, most N-terminal amino groups of Cys would be
rendered inactive toward reaction with the NHS activated ester by
protonation. In support of this, mixtures of P8NHS and 8-arm
PEG-amine form gels more than 10 times slower in PBS buffer.
[0148] Most polymer hydrogels typically absorb water under
physiologic conditions, leading to a significant increase in
volume. The results of our gel swelling experiments were notable
for two reasons. First, the increase in relative wet weight (0%)
for OMNCL hydrogels is considerably lower than many experimental
and clinically approved PEG-based hydrogels. For example, studies
of PEG based hydrogels cross-linked via amide or thioester bonds
report increasing relative wet weight values of 50%.sup.25 to
400%.sup.26 within the first 24 hours. In some cases, swelling of
implanted materials may lead to complications such as nerve
compression.sup.27-31 or other serious problems requiring
intervention..sup.32 Secondly, the swelling experiments provided
important evidence of the latent formation of disulfide bonds, and
insight into the mechanical consequences of this secondary
cross-linking mechanism. Exposure of OMNCL gels to reducing agent
resulted in an increase in swelling that was reversible upon
removal of reducing agent, implying the formation of disulfide
bonds within the gel network. This can be understood to be a result
of the S-to-N acyl rearrangement step that releases the thiol side
chain of Cys, which then becomes available for disulfide bond
formation with other network-bound thiols. Through comparison of
the kinetics of the OMNCL reaction (FIG. 6A, B) and the swelling
results (FIG. 15), we surmised that the reduction in swelling
observed after about 5 h results from disulfide bond formation.
Thus, we conclude that the hydrogels form initially by OMNCL and
are later further cross-linked through the formation of disulfide
bonds.
[0149] Our in vitro cell experiments demonstrated low cytotoxicity
of OMNCL hydrogel extracts and high viability of encapsulated cells
(FIG. 18). This led us to undertake an initial in vivo evaluation
of OMNCL hydrogels in a subcutaneous implant model. Gels were
formed by injection of precursor solutions and then evaluated at 6
weeks. Histological sections of explants showed low-level acute
inflammatory response to implanted gels and deposition of a thin
fibrous capsule surrounding the implant (FIG. 19). These findings
confirm the potential of OMNCL hydrogels for in vivo
applications.
[0150] Activated ester PEG polymers are currently approved by the
FDA in the form of the medical sealants DuraSeal.TM. and
COSEAL.TM.. An important distinction between these existing
materials and our OMNCL hydrogels relates to the pH range of the
cross-linking reaction. Both DuraSeal.TM. and COSEAL.TM. are
deployed at highly alkaline pH (typically pH 9-10),.sup.33 whereas
the OMNCL hydrogels reported here are capable of rapid gel
formation at neutral pH. In our studies, PEG was chosen as the
backbone for both components because it is non-cytotoxic and has
demonstrated favorable results in previous in vivo studies. PEG is
also attractive as a simple platform for quantitative NMR studies
and is amenable towards chemical modification. However, it is
important to note that the OMNCL hydrogel chemistry described here
can be easily adapted for use with other suitable polymer
platforms.
[0151] In summary, we have detailed the synthesis and
characterization of a two-component hydrogel formulation that sets
rapidly at physiologic pH and ionic strength in the absence of
catalysts or other additives. The cross-linking mechanism was
revealed by NMR studies to be primarily OMNCL, proceeding by thiol
capture to form a thioester intermediate followed by a S-to-N acyl
rearrangement to generate amide cross-links. OMNCL hydrogels
exhibit attractive mechanical properties that include high
compressive moduli and good adhesion to tissue, and low equilibrium
swelling due to the latent formation of secondary disulfide
cross-links. The biological performance of OMNCL hydrogels was
assessed, showing high in vitro cytocompatibility and low acute
inflammatory response in vivo. OMNCL hydrogels represent attractive
candidates for in vivo applications such as wound repair and
sealing, drug delivery, and tissue engineering.
Example 25
Synthesis of Catechol-Containing Precursors
[0152] This example discloses a reaction scheme for synthesizing
catechol-containing PEG precursors. Such precursors can be
incorporated into hydrogels through reaction with PEG-NHS polymers
through the cross-linking mechanism disclosed herein. The catechol
modification introduces another crosslinking moiety and facilitates
self healing properties in the resulting hydrogel. The reaction
scheme is illustrated in detail in FIG. 24.
[0153] Step (i).
[0154] Dissolve Boc-L-Glu(OFm)-OH (4 mol. eq. relative to
--NH.sub.2) in DMF (2 mL/g PEG). Dissolve BOP (4 mol. eq. relative
to --NH.sub.2). Add DIEA (4 mol. eq. relative to --NH.sub.2)
dropwise and react for 5 min. In a new DMF solution (2 mL/g PEG),
dissolve PEG-NH.sub.2. Combine two solutions of DMF and react
overnight at room temperature. Ether precipitation. Methanol
precipitation. Dry on vacuum line.
[0155] Step (ii).
[0156] Dissolve PEG-Glu(OFm)-NH-Boc in 30% (v/v) TFA in DCM (10
mL/g PEG). Stir for 90 min at room temperature. Rotovap. Ether
precipitation. Methanol precipitation. Dry on vacuum line.
[0157] Step (iii).
[0158] Dissolve Boc-L-Cys(Trt)-OH (4 mol. eq. relative to
--NH.sub.3TFA) in DMF (2 mL/g PEG). Dissolve BOP (4 mol. eq.
relative to --NH.sub.3TFA). Add DIEA (4 mol. eq. relative to
--NH.sub.3TFA) dropwise and react for 5 min. In a new DMF solution
(2 mL/g PEG), dissolve PEG-Glu(OFm)-NH.sub.3TFA. Add DIEA (1 mol.
eq. relative to --NH.sub.3TFA) dropwise. Combine two solutions of
DMF and react overnight at room temperature. Ether precipitation.
Methanol precipitation. Dry on vacuum line.
[0159] Step (iv).
[0160] Dissolve PEG-Glu(OFm)-Cys(Trt)-NH-Boc in 20% (v/v)
piperidine in DCM (10 mL/g PEG). Stir for 2 h at room temperature.
Rotovap. Ether precipitation. Methanol precipitation. Dry on vacuum
line.
[0161] Step (v).
[0162] Dissolve PEG-Glu-Cys(Trt)-NH-Boc in 2:1 (v/v) DMF:DCM (10
mL/g PEG). Dissolve TBDMS-protected dopamine (1.2 mol. eq. relative
to --COOH). Dissolve HBTU (1.2 mol. eq. relative to --COOH). Add
TEA (1.2 mol. eq. relative to --COOH) dropwise. Stir at room
temperature for 2 h. Rotovap. Ether precipitation. Methanol
precipitation. Dry on vacuum line.
[0163] Step (vi).
[0164] Dissolve PEG-Glu(DA-TBDMS)-Cys(Trt)-NH-Boc in a deprotection
solution (20 mL TFA, 1.2 mL triisopropylsilane, 1.2 mL
ethanedithiol per g PEG). Stir for 3 h at room temperature.
Rotovap. Ether precipitation. Methanol precipitation. Dry on vacuum
line. Dialyze against acidic water (pH approximately 3.5) for 24 h.
Dialyze against MilliQ water for approximately 4 h. Freeze and
lyophilize.
Example 26
Synthesis of Glutaric Acid-Terminated Tetronic (T4G)
[0165] The same procedure used to synthesize P8G was used to
synthesize T4G, with the exception that tetronic was used instead
of PEG (100% conversion).
Example 27
Synthesis of N-Hydroxysuccinimide (NHS)-Terminated Tetronic
(T4G-NHS)
[0166] The same procedure used to synthesize P8G-NHS was used to
synthesize T4G-NHS, with the exception that T4G was used instead of
P8G (80% conversion).
Example 28
Hydrogel Preparation Using T4G-NHS
[0167] PBS was used to prepare 15% (w/v) T4G-NHS and 10% (w/v)
P8Cys and the two solutions were mixed in a 1:1 (v/v) ratio to form
cylindrical 70 .mu.L hydrogels (5 mm diameter.times.3.5 mm
height).
Example 29
Drug Delivery with T4G-NHS Hydrogels
[0168] The hydrophobic core of the PPO regions in the T4G-NHS gels
(FIG. 25) can be used to load hydrophobic drugs. This method could
be used for controlled delivery of hydrophobic drugs.
[0169] The above description, attached figures, and below claims
are intended to be illustrative and not limiting of this invention.
Many themes and variations of this invention will be suggested to
one skilled in this and, in light of the disclosure. All such
themes and variations are within the contemplation hereof. For
instance, while this invention has been described in conjunction
with the various exemplary embodiments outlined above and in the
below claims, various alternatives, modifications, variations,
improvements, and/or substantial equivalents, whether known or that
rare or may be presently unforeseen, may become apparent to those
having at least ordinary skill in the art. Various changes may be
made without departing from the spirit and scope of the invention.
Therefore, the invention is intended to embrace all known or
later-developed alternatives, modifications, variations,
improvements, and/or substantial equivalents of these exemplary
embodiments.
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