Biocampatible and Biodegradable Anionic Hydrogel System

Abstract

An anionic hydrogel for wound healing comprised of poly(oligoethylene glycol monoacrylate), acrylic, a neutral species and a wild-type fibroblast growth factor 1 (wtFGF1).

Claims

1. A method of producing an anionic hydrogel comprising the steps of: providing a) poly(oligoethylene glycol monoacrylate), acrylic acid and N,N′-methylenebis(acrylamide), and water in a container bubbled with a gas before sealing said container; b) incubating; c) adding an initiator; d) performing one or more evacuate-refill cycles; e) terminating the polymerization by cooling; f) dialyzing against water using dialysis membrane; g) lyophilizing into a form of dry gel; and h) adding a wild-type fibroblast growth factor 1 (wtFGF1) and swelling said gel to form an anionic hydrogel.

2. The method of claim 1 further including the step of adding a neutral species to said poly(oligoethylene glycol monoacrylate) and said acrylic acid, said neutral species spaces out the charge distribution of said anionic hydrogel allowing for the increased release of said a wild-type fibroblast growth factor 1 (wtFGF1).

3. The method of claim 2 wherein said neutral species is NIPAM.

4. The method of claim 3 wherein the weight ratio of said anionic hydrogel is poly(oligoethylene glycol monoacrylate) 850, acrylic acid 25, NIPAM 125.

5. The method of claim 2 wherein said neutral species is gelatin.

6. The method of claim 3 wherein the weight ratio of said anionic hydrogel is poly(oligoethylene glycol monoacrylate) 850, acrylic acid 25, gelatin 160.

7. An anionic hydrogel for wound healing comprising: poly(oligoethylene glycol monoacrylate), acrylic, a neutral species and a wild-type fibroblast growth factor 1 (wtFGF1).

8. The anionic hydrogel of claim 7 wherein said neutral species is NIPAM.

9. The anionic hydrogel of claim 8 wherein the weight ratio of said anionic hydrogel is poly(oligoethylene glycol monoacrylate) 850, acrylic acid 25, NIPAM 125.

10. The anionic hydrogel of claim 7 wherein said neutral species is gelatin.

11. The anionic hydrogel of claim 10 wherein the weight ratio of said anionic hydrogel is poly(oligoethylene glycol monoacrylate) 850, acrylic acid 25, gelatin 125.

12. A method of to enhance cell growth and cell adhesion to treat a wound in a mammal comprising the steps: applying an anionic hydrogel to the wound; said anionic hydrogel comprising: poly(oligoethylene glycol monoacrylate), acrylic, a neutral species and a wild-type fibroblast growth factor 1 (wtFGF1); and said hydrogel configured to controllably release said a wild-type fibroblast growth factor 1 (wtFGF1).

13. The method of claim 12 wherein said hydrogel inhibits the degradation of said a wild-type fibroblast growth factor 1 (wtFGF1).

14. The method of claim 13 further including the step of increasing said neutral species to said poly(oligoethylene glycol monoacrylate) and said acrylic acid, to increase the release of said a wild-type fibroblast growth factor 1 (wtFGF1).

15. The method of claim 14 wherein said neutral species is NIPAM.

16. The method of claim 14 wherein the weight ratio of said anionic hydrogel is poly(oligoethylene glycol monoacrylate) 850, acrylic acid 25, NIPAM 125.

17. The anionic hydrogel of claim 14 wherein said neutral species is gelatin.

18. The anionic hydrogel of claim 7 wherein said neutral species is NIPAM.

19. The anionic hydrogel of claim 8 wherein the weight ratio of said anionic hydrogel is poly(oligoethylene glycol monoacrylate) 850, acrylic acid 25, NIPAM 125.

20. The anionic hydrogel of claim 7 wherein said neutral species is gelatin.

21. The anionic hydrogel of claim 10 wherein the weight ratio of said anionic hydrogel is poly(oligoethylene glycol monoacrylate) 850, acrylic acid 25, gelatin 160.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0054] In the drawings, which are not necessarily drawn to scale, like numerals may describe substantially similar components throughout the several views. Like numerals having different letter suffixes may represent different instances of substantially similar components. The drawings illustrate generally, by way of example, but not by way of limitation, a detailed description of certain embodiments discussed in the present document.

[0055] FIG. 1 shows the synthesis of the injectable hydrogels based on the copolymer networks of PAA-co-POEGA for an embodiment of the present invention.

[0056] FIG. 2 is a plot of the release curve of wtFGF1 and shaped hydrogel of the embodiment of the present invention.

[0057] FIG. 3 shows the lysozyme released profile as a function of time for several embodiments of the present invention.

[0058] FIG. 4 shows the loading capacity of lysozyme in the PAA-co-POEGA for various embodiments of the present invention at various AA concentrations (0, 0.5, 1, 1.5, 2 M) in DPBS (pH 7.4) at room temperature for 3 days.

[0059] FIG. 5A illustrates a loading and release study of the disk hydrogel of PAA-co-POEGA (1:1) with loading capacity of wtFGF1 as a function of time.

[0060] FIG. 5B illustrates a loading and release study of the disk hydrogel of PAA-co-POEGA (1:1) with wtFGF1 released from the hydrogel disk under different temperatures in DPBS (pH 7.4).

[0061] FIG. 6 is an in vivo biocompatibility study using a mouse model for GEL900 (left), 3M Telladerm Gel (middle), and no treatment (right) with the wound size being monitored for 10 days.

[0062] FIG. 7 illustrates the released wtFGF1 as a function of time for GEL900 and GEL800.

[0063] FIG. 8A illustrates a cell proliferation assay of the released wtFGF1 from GEL900 and GEL800 with no wtFGF1 as a control with cell counts of 3T3 fibroblast cells from day 0 to day 3.

[0064] FIG. 8B illustrates a cell proliferation assay of the released wtFGF1 from GEL900 and GEL800 with calculated active released wtFGF1 as a function of time.

[0065] FIG. 9A illustrates the synthesis of the Poly(acrylic acid)-co-poly(oligoethylene glycolmonoacrylate)-co-poly(N-isopropyl acrylamide) (PAA-co-POEGA-co-PNIPAM). The reaction involves an aqueous free radical polymerization of AA, OEGA (M.W. 480), and NIPAM with the cross-linking agent MBAm initiated by APS.

[0066] FIG. 9B shows when after the polymer network is formed, the PAA-co-POEGA-co-PNIPAM polymer will be swollen in buffered saline solution containing 0.1 wt. % Borax (antibacterial agent), forming the injectable hydrogels.

[0067] FIG. 10 shows the controlled release of wtFGF-1 from different formulations of injectable anionic PAA-co-POEGA-co-PNIPAM hydrogels with the release was done in physiological conditions at pH 7.4.

[0068] FIG. 11A is a schematic illustration of the reaction scheme for the synthesis of P(OEGA-co-AA-co-NIPAM) copolymer via a free-radical polymerization reaction initiated by APS for an embodiment of the present invention. The copolymer network is cross-linked by MBAm. The lyophilized copolymer powder is swelled in PBS aqueous to form hydrogel.

[0069] FIG. 11B shows the gels of the embodiments of the present invention indicating the difference in viscosity.

[0070] FIG. 12A is the FTIR absorbance spectra of P(OEGA-co-AA-co-NIPAM) copolymers of 8 different gel formulations.

[0071] FIG. 12B is the FTIR absorbance spectra of Gel 8 showing main representative peaks.

[0072] FIG. 12C is the FTIR absorbance spectra of ester/carbonate C═O peak intensity at 1730 cm.sup.−1 normalized to that of the C—O at 1095 cm.sup.−1.

[0073] FIG. 12D is the FTIR absorbance spectra of the C═O peak intensity at 1645 cm.sup.−1 for amide normalized to ester/carbonate C═O peak intensity at 1730 cm.sup.−1.

[0074] FIG. 13A provides DSC thermograms of the copolymers in the temperature range from 50 to 500° C.

[0075] FIG. 13B is zoomed in for 380-460° C. area.

[0076] FIG. 13C provides TGA curves of the hydrogel formulations in 0-500° C. temperature range.

[0077] FIG. 13D provides derivative plots of TGA curves with zoomed in to 300-500° C. area.

[0078] FIG. 14 provides MTT-assay viability data showing polymer concentration effect on cell viability after treatment with Gel 900 (P(OEGA-co-AA) and Gel 6 P(OEGA-co-AA-co-NIPAM).

[0079] FIG. 15A provides release profiles of hFGF1 as a function of time using fluorescence spectroscopy (λ.sub.ex=280 nm)) from Gel 8 at 25° C. and 37° C. and from Gel 900 at 37° C.

[0080] FIG. 15B shows the controlled release of BSA from Gel 8 (λ.sub.ex=295 nm) for 5 days at 37° C.

[0081] FIG. 15C provides bioactivity of released hFGF1 from Gel 8 at 37° C.

[0082] FIG. 15D shows the stability of hFGF1 in the cell culture media at 37° C. Data in A-C were measured in triplicate (n=3). Error bars represent standard deviation.

[0083] FIG. 16 provides an in vivo study showing normalized wound sizes for wound treated with just gel 8 as a control and Gel 8 loaded with hFGF1 across the study for 10 days.

DETAILED DESCRIPTION OF THE INVENTION

[0084] Detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed method, structure or system. Further, the terms and phrases used herein are not intended to be limiting, but rather to provide an understandable description of the invention.

[0085] In other embodiments, the present involves the synthesis of the injectable hydrogels that consists of a two-step procedure (FIG. 1). The initial step involves an aqueous free radical polymerization of AA and OEGA (M.W. 480) with a cross-linking agent such as MBAm or BAC. The reaction was initiated by APS to form PAA-co-POEGA polymeric network. The second step is to let the PAA-co-POEGA polymeric network swell in a buffered solution such as a saline solution with 0.1 wt. % Borax (antibacterial agent), forming the injectable hydrogels. Five formulations were synthesized as listed in Table 1. It was determined that the fluidity decreased with increased AA component in the polymer chain. The chain length of the copolymer increased with increased weight percent of AA in the polymerization reaction. The longer the polymer chain, the stronger were the intermolecular interactions between the polymer chains leading to the distinctive difference in physical properties of the resulting hydrogels.

[0086] By increasing the concentrations of cross-linker and monomers, the hydrogels gained mechanical strength and became less fluidic. This property of hydrogel helped shaping of the hydrogel as pads or disks as shown in FIG. 2. The pad or disk hydrogel formulation is listed in Table 2.

TABLE-US-00001 TABLE 1 APS Borax POEGA/AA Mon./MBAm MBAm cont. Reaction Reaction Total content POEGA/AA (mol/mol, (mol/mol, cont. (w/v, Temp Time Volume (w/v, Sample (w/w, mg) mmol) mmol) (mM) %) (° C.) (min) (mL) %) GEL1000 1000/0 2.08/0.00 2.08/0.1 10 1.5 70 30 10 0.1 GEL950  950/50 1.98/0.69 2.67/0.1 10 1.5 70 15 10 0.1 GEL900  900/100 1.88/1.39 3.26/0.1 10 1.5 70  5 10 0.1 GEL850  850/150 1.77/2.08 3.85/0.1 10 1.5 70  5 10 0.1 GEL800  800/200 1.67/2.78 4.44/0.1 10 1.5 70  5 10 0.1

TABLE-US-00002 TABLE 2 C.sub.POEGA (M) C.sub.AA (M) C.sub.bis (mM) V (mL) C.sub.APS (mM) T (° C.) 0.5 0 50 1 50 70 0.5 0.5 50 1 50 70 0.5 1 50 1 50 70 0.5 1.5 50 1 50 70 0.5 2 50 1 50 70

[0087] For the injectable hydrogels, a model protein, lysozyme, was mixed with the hydrogels, to demonstrate the withholding capability of the hydrogels. The hydrogel-protein mixtures were placed in PBS and the released amount of lysozyme was monitored by fluorescence spectroscopy up to 48 hours (FIG. 3). Within the first hour, lysozyme was completely released from GEL1000, made from pure POEGA, while only 50% and 35% of lysozyme was released from GEL900 and GEL800, respectively. After 12 hours, the release reached equilibrium wherein 40% and 55% of lysozyme remained in GEL900 and GEL800, respectively suggesting a potential for sustainable release if placed on the wound site. As lysozyme is positively charged at physiological conditions, the increased concentration of AA in the copolymer will enhance the electrostatic interaction(s) between the polymeric network and positively charged proteins and consequently facilitates retention in the hydrogel for prolonged periods of time.

[0088] For the disk hydrogel, the loading capacity was assessed using the positively charged lysozyme (FIG. 4). The amount of lysozyme loaded in the hydrogels increased with increased AA concentration in the copolymer is the result of the electrostatic interaction(s) between negatively charged AA and lysozyme.

[0089] The wtFGF1 was further used to demonstrate the loading and releasing capacity of the disk hydrogel at a 1:1 molar ratio of AA/OEGA (FIG. 5). The loading capacity of disk hydrogels can reach ˜6 mg/g within a week similar to that of lysozyme. The release of wtFGF1 was very slow and temperature dependent. Over the course of 12 days, ˜50% and ˜30% wtFGF1 was released from the hydrogel at 37° C. and 25° C., respectively.

[0090] Since the viscosity of GEL900 is comparable to that of the commercially available 3M Tegaderm injectable hydrogel, it was further considered as a candidate for in vivo studies. The biocompatibility of GEL900 was evaluated using a mouse model of excisional wound healing. FIG. 6 shows the wound size comparison after individual application of GEL900 and the commercially available 3M Tegaderm Gel. On day 1, wounds treated with the GEL900 and 3M Tegaderm Gel increased in size by 20 to 30%. By day 3, the wounds applied with GEL900 began to decrease in size relative to the 3M Tegaderm Gel. By day 10, wounds treated with GEL900 had nearly closed, indicating biocompatibility. Similar to the control group without gel treatment, significant differences were observed between the day 1 or day 3 and the later days (i.e., day 5, 7, or 10) for both groups treated with gels. GEL900 appeared to be comparable to the 3M Tegaderm Gel during healing and neither of the gels was observed to delay the normal healing process.

[0091] In other embodiments, the present invention provides anionic hydrogels based on poly(acrylic acid)-co-poly(oligoethylene glycol monoacrylate) (PAA-co-POEGA) for protein delivery. The hydrogel comprises an injectable hydrogel prepared in two steps, one-pot method which involves an aqueous free radical polymerization, followed by sequential steps to allow the swelling of copolymer into the hydrogels. The hydrogel may then be retained in a number of different predetermined shapes such as a disk, cylinder, sphere and other shapes known to those of skill in the art.

[0092] A disk shape, redox-induced degradable hydrogel may also be prepared from the same monomer but crosslinked by redox-responsive cross-linking reagent. In a preferred embodiment, the injectable hydrogel comprises a copolymer comprising poly(oligoethylene glycol monoacrylate), acrylic acid, N,N′-methylenebis(acrylamide), sodium tetraborate and phosphate-buffered saline wherein a mass percentage of the components comprises: poly(oligoethylene glycol monoacrylate) ranging from 8%-10%; acrylic acid ranging from 2%-0%; N,N′-methylenebis(acrylamide) was fixed to 0.154%; sodium tetraborate was fixed to 0.1%; and phosphate-buffered saline up to 100%. The injectable hydrogel may also have a molecular weight of poly(oligoethylene glycol monoacrylate) of 480 g/mol.

[0093] In other aspects, the present invention provides a method of producing an injectable hydrogel comprising the steps of providing a) Poly(oligoethylene glycol monoacrylate), acrylic acid and N,N′-methylenebis(acrylamide) and water in a Schleck tube or other suitable container which may be bubbled with N.sub.2 gas for 30 min before sealing the container which is followed by one or more and preferably three evacuate-refill cycles with Ar to remove the dissolved oxygen. The reaction solution may then be incubated at 70° C. for 30 min. After incubation, an initiator may be added under Ar flow, followed by another three evacuate-refill cycles with Ar. The reaction was allowed to proceed at 70° C. for 5-30 min under magnetic stirring at a speed of 350 rpm. Immediately after the reaction, the polymerization was terminated by cooling the container. The reaction solution was then dialyzed against water using dialysis membrane. The resulted gel solution was lyophilized into a form of a dry gel. The lyophilized copolymer was swollen PBS containing borax for 24 h to form the injectable anionic hydrogels.

[0094] In other steps, the polymerization may be initiated by ammonium persulphate and the molecular weight cut off of the dialysis membrane is 2 kDa. The viscosity of the injectable hydrogel may be controlled by the molar ratio between the total monomer and crosslink agent. The thermal stability of the injectable hydrogel may be controlled by the molar ratio between the total monomer and crosslink agent. In addition, one or more positively charged proteins may also be bonded to the injectable hydrogel by electrostatic interaction and the protein's capacity controlled by the molar concentration of acrylic acid. The positively charged protein release kinetic of the injectable hydrogel may be controlled by the molar concentration of acrylic acid.

[0095] In other aspects, the present invention provides a disk shape hydrogel comprising a copolymer comprising poly(oligoethylene glycol monoacrylate), acrylic acid, N,N′-methylenebis(acrylamide) and phosphate-buffered saline; wherein a mass percentage of the components may be as follows: poly(oligoethylene glycol monoacrylate) fixed to 24%; acrylic acid ranging from 0%-14.4%; N,N′-methylenebis(acrylamide) fixed to 0.77%; and phosphate-buffered saline up to 100%. The molecular weight of poly(oligoethylene glycol monoacrylate) may be 480 g/mol.

[0096] In other aspects, the present invention provides a method of producing cylindrical shaped hydrogel comprises of the following steps: providing poly(oligoethylene glycol monoacrylate), acrylic acid and N,N′-methylenebis(acrylamide) and water in a container which is bubbled with N.sub.2 gas for 30 min before sealing the container and then adding an imitator. The reaction may be started by heating up to 70° C. for 4 h. The resulting hydrogel is washed preferably with water and allowed to completely swell in PBS for example. When the hydrogel is cylindrical in shape, the swelling ratio may be controlled by the molar ratio between the total monomer and crosslink agent. The polymerization may be initiated by ammonium persulphate.

[0097] A positively charged protein may be bonded to the shaped hydrogel by electrostatic interaction. The protein capacity may be controlled by the molar concentration of acrylic acid and the positively charged protein release kinetic of the disk shape hydrogel may be controlled by a competition between total protein capacity of hydrogel and molar concentration of acrylic acid.

[0098] In other aspects, the present invention provides a biodegradable disk shape hydrogel comprising a copolymer comprising poly(oligoethylene glycol monoacrylate), acrylic acid, N,N′-bis(acryloyl)cystamine and phosphate-buffered saline. The mass percentage of the components is as follows: poly(oligoethylene glycol monoacrylate) fixed to 24%; acrylic acid ranging from 3.6%-14.4%; N,N′-Methylenebis(acrylamide) fixed to 1.3%; and phosphate-buffered saline up to 100%. The molecular weight of poly(oligoethylene glycol monoacrylate) may be 480 g/mol.

[0099] In other aspects, the present invention provides a method of producing a disk shape hydrogel comprising the steps of: poly(oligoethylene glycol monoacrylate), acrylic acid, N,N′-bis(acryloyl)cystamine and water/EtOH mixture in a container was bubbled with N.sub.2 gas for 30 min before sealing the container and adding an imitator with the reaction being started by heating up to 70° C. for 4 h. Next the resulting hydrogels are washed and allowed to swell to completion. In other embodiments, the polymerization may be initiated by ammonium persulphate and the volume ratio of water and EtOH is 3/1. The swelling ratio of the biodegradable disk shape hydrogel can be controlled by the molar ratio between the total monomer and crosslink agent and the positively charged protein may be bonded to the biodegradable disk shape hydrogel.

[0100] Other aspects of controlling aspects of the hydrogel include the following: the protein capacity may be controlled by the molar concentration of acrylic acid; the positively charged protein release kinetic of the disk shape hydrogel may be controlled by a competition between total protein capacity of hydrogel and the molar concentration of acrylic acid; and the biodegrade speed of the hydrogel may be controlled by the free thiol concentration.

[0101] Controlled Release of wtFGF1 from Injectable Anionic Hydrogels.

[0102] In another embodiment, 200 mg of injectable gel was mixed with 100 μL of 3.0 mg/mL wtFGF1 and stored at 4° C. overnight. The mixture was loaded into a transwell membrane plates insert (3 μm pore size) with a polyester membrane and polystyrene plates. The insert was then immersed in 1.5 mL of 1×PBS releasing media for controlled release at room temperature. At desired time points, 1 mL of the released medium was sampled and replaced by an equal volume of fresh medium to maintain a constant volume. The medium was then analyzed using fluorescence spectrophotometer. Then cumulative of released wtFGF1 was calculated from wtFGF1 calibration curve at 309 nm emission wavelength. The process was repeated three times and the results were reported as average values with standard deviations. The concentration of the FGF in each transwell is determined according to the measured fluorescence intensity using a standard curve. Unlike the lysozyme, the results in FIG. 7 indicate that there is little release of the wtFGF1 from the gels. This is possibly due to the strong electrostatic interaction of the wtFGF1 with the PAA component in the gel.

[0103] Cell Proliferation Assay for Bioactivity of Released wtFGF1.

[0104] Bioactivity of FGF was evaluated after release activity from PAA-co-POEGA hydrogels to ensure sustained activity. 3T3 fibroblast cells were grown to 80-90% confluency. Cell proliferation activity of released wtFGF1 was performed by incubating 10,000 cell/well with 50 ng/mL of released wtFGF1 in serum-supplemented medium. Cell proliferation assay was determined by CellTilter-Glo luminescent cell viability assay.

[0105] FIGS. 8A and 8B show the cell proliferation assay of the released wt.FGF1 from GEL900 and GEL800, respectively. Compared to the same amount of freshly prepared wtFGF1, the data shows about 60% of released wtFGF1 was active.

[0106] Poly(acrylic acid)-co-poly(oligoethylene glycolmonoacrylate)-co-poly(N-isopropyl acrylamide) Injectable Anionic Hydrogels for Controlled Release of wtFGF1 release.

[0107] Synthesis of PAA-co-POEGA-co-PNIPAM Injectable Hydrogels.

[0108] Each monomer was purified prior to the polymerization using the following methods. Oligoethylene glycol monoacrylate (M.sub.n=480, OEGA) was purified through a basic Al.sub.2O.sub.3 column. Acrylic acid (AA) was purified through a neutral Al.sub.2O.sub.3 column. N-isopropyl acrylamide (NIPAM) was recrystallized from hexane. The polymer was synthesized by copolymerization of the three monomers, AA, OEGA, and NIPAM in an aqueous solution initiated by ammonium persulfate (APS). During the free radical polymerization, the monomers were cross-linked by N,N′-methylenebis (acrylamide) (MBAm). Typically, 10 mL aqueous solution containing a total amount of 1000 mg of OEGA, PAA and PNIPAM, and 15.4 mg MBAm (10 mM) was added to a 50-mL Schleck tube flask. The weight ratios of the three monomers in different formulation are listed in Table 3. The flask was bubbled with N.sub.2 gas for 30 min before it was sealed. The flask was then subjected to three evacuate-refill cycles with Argon (Ar) to eliminate the dissolved oxygen. The reaction was incubated at 70° C. for 30 min. After incubation, 200 μL aqueous solutions of APS (15 mg) were added to the tube under Ar flow, followed by another three evacuate-refill cycles with Ar. The reaction could proceed at 70° C. for 5 min under magnetic stirring at 350 rpm. Immediately after the reaction, the polymerization was terminated by cooling the tube in an ice-water bath.

TABLE-US-00003 TABLE 3 Different formulations of PAA-co-POEGA- co-PNIPAM injectable hydrogels. Total Sample Volume APS MBAM OEGA/AA/NIPAM Temperature ID (mL) (mg) (mM) w/w/w, mg (° C.) GEL 1 10 15 10 700/200/100 70 GEL 2 10 15 10 900/50/50 70 GEL 3 10 15 10 900/75/25 70 GEL 4 10 15 10 800/150/50 70 GEL 5 10 15 10 800/100/100 70 GEL 6 10 15 10 800/50/150 70

[0109] PAA-co-POEGA-co-PNIPAM Gel.

[0110] The newly developed anionic injectable hydrogels were prepared from the copolymer PAA-co-POEGA-co-PNIPAM, as shown in FIG. 9. The incorporation of the NIPAM in the polymer network may space out the negative charges in the two-component PAA-co-POEGA polymer. This may facilitate the release kinetics of wtFGF1 while maintaining the viscosity of the injectable gels for wound healing applications. In addition, the PNIPAM can response to heat changes that may be used for other applications that would involve with the use of heat as a tuning knob.

[0111] wtFGF1 Control Release Study.

[0112] The release of wtFGF1 was significantly increased using PAA-co-POEGA-co-PNIPAM gels compared to PAA-co-POEGA, as shown in FIG. 10. The decrease of OEGA percentage in the formulation significantly increase the release kinetics of wtFGF1 from the gel. The release of wt-FGF1 could reach nearly 100% at 25 days (data not shown here). In addition, the decrease of the ratio AA/PNIPAM will increase the release kinetics of wtFGF1 from the gel providing a fine modulation of the wtFGF1 release.

[0113] Experimental Methods

[0114] Materials and Chemicals.

[0115] Oligo ethylene glycol monoacrylate (OEGA, M.sub.n=480), Acrylic acid (AA), Dulbecco's Phosphate Buffered Saline (DPBS, 10 mM, pH 7.4) were purchased from Sigma-Aldrich. N-Isopropylacrylamide (NIPAM) was purchased from TCI. N,N′Methylenebisacrylamide (MBAm) and Potassium bromide (KBr) were purchased from Alfa Aesar. Ammonium persulfate (APS) was purchased from VWR. Dialysis membrane (MWCO 2 kDa) was purchased from Spectrum Laboratories. Transwell membrane plates were purchased from Corning.

[0116] Inhibitors were removed OEGA and AA by purification through a basic and neutral aluminum oxide (Al.sub.2O.sub.3) columns for OEGA and AA, respectively. APS was recrystallized from ethanol/water (1:1) while MBAm was recrystallized from acetone. NIPAM was recrystallized from hexane mixed with a few drops of acetone. All other chemicals were used as received and Milli-Q ultrapure water (18 MΩ H.sub.2O) was used in all procedures unless specified.

[0117] Preparation of P(OEGA-co-AA-co-NIPAM) Hydrogels.

[0118] P(OEGA-co-AA-co-NIPAM) copolymers were prepared by a free radical polymerization method. In a typical synthesis, 1 g of monomers (OEGA, AA, and NIPAM at various mass ratios) and 0.1 mmol of MBAm was dissolved in 10 mL of water and purged under N.sub.2 for 5 min. The mixture was then transferred to a 25-mL stopcock, air free, Schlenk flask protected with N.sub.2. Three freeze-pump-thaw cycles with Ar were performed to remove dissolved oxygen from the reaction mixture before it was incubated at 70° C. under Ar. After 30 min, 200 mL of aqueous APS (15 mg) was added to the flask under Ar flow, followed by another three freeze-pump-thaw cycles. The reaction was left to run for 5 min at 70° C. under magnetic stirring at 350 rpm. After another 5 min, the reaction was quenched by cooling the flask in an ice bath for 15 min. The mixture was then dialyzed in MWCO 2 kDa membrane in 1 L of water for a week while refreshing water every day to remove monomer, crosslinker, and initiator residues. The resulting product was lyophilized under vacuum to give a copolymer powder. Each copolymer was swollen in 10 mL of 1×DPBS buffer for 24 h to form a hydrogel.

[0119] Characterization Methods.

[0120] The vibrational spectra of the copolymers were recorded between 400-4000 cm.sup.−1 at a resolution of 4 cm.sup.−1 on Fourier-transform infrared (FTIR) spectrometer (Shimadzu IRAffinity-1S). The samples were prepared by a KBr pellet method using ˜10 mg of each lyophilized copolymer. Thermal properties of the copolymers were investigated using differential scanning calorimeter (DSC, Shimadzu DSC-60). Each copolymer (5 mg) was put in a standard aluminum sample pan and covered with aluminum lid. The pan was crimped with a crimp press SSCP-1 for the DSC measurement with an empty aluminum sample pan as a control. Measurements were carried out at the rate of 10° C./min from 50-500° C. The thermograms were taken using a TGA instrument (TA Q50) where 25 mg of each hydrogel was used. The sample was equilibrated for 10 min at 30° C. and run under N.sub.2 at a flow rate of 60 mL/min from 30 to 500° C. at a rate of 10° C./min.

[0121] Cytotoxicity Studies.

[0122] Cytotoxicity studies of P(OEGA-co-AA-co-NIPAM) were performed. In a typical procedure, polymer was lyophilized for 3 days. Around 200 mg of the lyophilized powder was sterilized under UV light for 2 h. Each of the copolymers tested was mixed in DMEM culture media for 72 h at 37° C. in CO.sub.2 incubator (5%). Fibroblast (NIH3T3) cells were then incubated in culture media to obtain 80% confluency. The copolymers were isolated from the culture media (stock: 0.001 g/μL) and diluted with fresh media to obtain different concentrations ranging from 0-30% polymer. Polymers at different concentrations (mg/mL) were added to the 96-well plate coated with cells to a total volume of 100 μl and cell number of 10,000 cells/well. After 72 h incubation, the viability of the fibroblast cells was measured by MTT assay. The viability of cells grown in the media without polymer treatment was used as a control.

[0123] Proteins Release Studies.

[0124] Release studies of the injectable P(OEGA-co-AA-co-NIPAM) hydrogel were conducted in 12 mm transwell membrane plates. In a typical experiment, around 200 μL of the injectable hydrogel was mixed with a desired amount of protein at the final concentration of 1 mg/mL and the mix was stored at 4° C. overnight. After incubation, the mixture was transferred to a transwell membrane insert (12 mm diameter, 3 μm pore size) then immersed in 1.5 mL of DPBS (pH 7.4). The sample was shaken at 150 rpm throughout the release experiment at 37° C. At different time points, 1.5 mL medium in each well containing the released protein was taken for fluorescence measurement while each well was replaced by fresh medium. The emission spectra were collected at emission wavelengths of 308 nm (with λ.sub.ex=280 nm) and 345 nm (with λ.sub.ex=295 nm) for hFGF1 and BSA, respectively. Percentage of cumulative released proteins was calculated based on the calibration curve corresponding to each protein. After fluorescence measurements, the samples were kept at 4° C. for bioactivity assays if applicable. The bioactivity of released hFGF1 was evaluated by cell proliferation assay. Briefly, NIH3T3 cells were grown to 80% confluency and then incubated with 50 ng/mL of the released protein/hFGF1 in serum-supplemented media at a concentration of 10,000 cell per well. The cell viability was determined by Cell Tilter-Glo luminescent assay.

[0125] Animal Study for Wound Closure.

[0126] C57BL/6J mice (12 weeks old, male, n=11) were anesthetized with 4% isoflurane gas and maintained at 2% isoflurane gas. Each mouse received a subcutaneous injection of an analgesic (Carprofen, 5 mg/kg) before creating a 6 mm full-thickness, excisional wound on the dorsum using a sterile biopsy punch. For each mouse, 100 L of either an hFGF-loaded gel (n=6 mice) or an unloaded control gel (n=5 mice) was applied to each wound. Sterile hydrogels were used to load hFGF at a final concentration of 1 mg/mL. Borax (0.1 w/v %) was used as antiseptic in the PBS for in vivo experiment. Borax (0.1 w/v %) was used as antiseptic in the PBS for in vivo experiment. Wounds were bandaged with Tegaderm and a secondary layer of surgical tape to prevent Tegaderm removal. Wound size was monitored by tracing the wound borders over acetate paper on days 0, 1, 3, 5, 7, and 10. The wound tracings were digitized and average wound area was calculated using Custom MATLAB code that quantified the area within each trace. Wound size measurements were normalized relative to the initial wound size at Day 0.

[0127] Results and Discussion

[0128] Different formulations of P(OEGA-co-AA-co-NIPAM) were synthesized by a free radical polymerization reaction with modifications to copolymerize with NIPAM. FIG. 11A displays the reaction scheme of the copolymerization of three monomers, OEGA, AA and NIPAM in aqueous medium to form P(OEGA-co-AA-co-NIPAM) with APS as an initiator and MBAm as a crosslinker. The copolymer was then swelled in buffer solution to form hydrogel. Eight different formulations were prepared by systematically changing mass ratios among the three monomers to evaluate the effect(s) of each monomer on copolymer properties. Table 4 lists the reaction parameters and conditions. After the synthesis, the copolymers were swelled in aqueous solution to become hydrogels. The viscosity of the gels was qualitatively visualized by their relative fluidity. The fluidity of the P(OEGA-co-AA-co-NIPAM) hydrogels increases with the increased molar percent of OEGA in the formulation. The order of increasing PEOGA molar percent in hydrogel formulations was Gel 1<Gel 3<Gel 8<Gel 4<Gel 7<Gel 5<Gel 6<Gel 2.

[0129] As can been seen in FIG. 11B, Gel 1 having the lowest OEGA content was very viscous, and the viscosity decreased in the order of Gel 1>Gel 3>Gel 2. This result is consistent with previous observation for the P(OEGA-co-AA) hydrogels, wherein AA increased viscosity of the hydrogel due to stronger hydrogen bonding and other intermolecular interactions in the copolymer network. Similar to AA, NIPAM also strengthened the intermolecular interactions in the copolymer compared to POEGA alone.

TABLE-US-00004 TABLE 4 Synthesis parameters and reaction conditions of different P(OEGA-co-AA-co-NIPAM) copolymers to form injectable anionic hydrogel formulations. OEGA/AA/ MBAm/ OEGA/AA/ NIPAM (OEGA+AA+ Sample NIPAM (at/at/at, MBAM NIPAM) APS Time Temp. Volume ID (w/w/w, mg) mmol) (mmol) (at/at, mmol) (mg) (min) (° C.) (mL) Gel 1 700/200/100 1.46/2.78/0.88 0.1 0.1/5.12 15 5 70 10 Gel 2 900/50/50 1.88/0.14/0.80 0.1 0.1/2.81 15 5 70 10 Gel 3 900/75/25 1.67/2.08/0.44 0.1 0.1/4.19 15 5 70 10 Gel 4 850/100/50 1.77/1.39/0.44 0.1 0.1/3.60 15 5 70 10 Gel 5 800/100/100 1.88/1.04/0.22 0.1 0.1/3.14 15 5 70 10 Gel 6 800/150/50 1.88/0.69/0.44 0.1 0.1/3.01 15 5 70 10 Gel 7 900/10/90 1.77/0.35/1.10 0.1 0.1/3.22 15 5 70 10 Gel 8 850/25/125 1.67/1.39/0.88 0.1 0.1/3.94 15 5 70 10

[0130] The copolymers were characterized by FTIR spectra to confirm that their chemical structures contained the functional groups from the monomers. FIG. 12A displays the FTIR spectra of all the copolymers which exhibit similar major peaks representing functional groups of the monomers. The spectrum of P(OEGA-co-AA-co-NIPAM) copolymer for Gel 8 was zoomed in to interpret the peaks observed in FIG. 12B. The broad band around 3500 cm.sup.−1 can be assigned to N—H stretching from PNIPAM and MBAm. The peak at 2870 cm.sup.−1 can be assigned to C—H of the copolymer with a shoulder close to 3000 cm.sup.−1 assigned to O—H stretching from PAA component. The peaks at 1730 cm.sup.−1 and 1645 cm.sup.−1 can be assigned to the carbonyl C═O stretching for ester/carbonate (O═C—O) and amide (O═C—N), respectively. The peak at 1095 cm.sup.−1 can be assigned to C—O from POEGA. FIG. 12C shows the ester/carbonate C═O peak intensity (1730 cm.sup.−1) normalized to that of the C—O at 1095 cm.sup.−1. Likewise, FIG. 12D depicts the C═O peak intensity at 1645 cm.sup.−1 for amide normalized to ester/carbonate C═O peak intensity at 1730 cm.sup.−1. The increase in the peak intensity at 1645 cm.sup.−1 is in accordance with the increase of crosslinking density which is on order of (Gel 8>Gel 1>Gel 7>Gel 5>Gel 4>Gel 6>Gel 2>Gel 3 as it can be seen in Table 4. This can be explained by the fact that more crosslinking density between all monomers will lead to high crosslinker (MBAm) to monomers ratio, thus amide groups in the polymer.

[0131] Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were used to study the thermal properties of the copolymers based on P(OEGA-co-AA-co-NIPAM) and Gel 9 00 (POEGA-co-AA) was added for comparison. DSC thermograms show different behaviors of the copolymers, but mostly similar behaviors were observed between 25-100° C. (FIG. 13A). It is believed that the peaks observed around 100° C. for all the copolymers is the evaporation of water in the copolymers before melting point is reached (endothermic process) at higher temperature. The endothermic peaks were observed between 3900-450° C. range following exothermic peaks around 350° C. for all measured copolymers (FIGS. 13A and 13B). After 350° C., the endothermic process could be an indication of the beginning of melting of some of the monomers. Gel 1, having the highest percentage of AA (54%) of all formulations showed the highest melting point at 433° C. (FIG. 13B). The DSC data suggest that copolymers based on P(OEGA-co-AA-co-NIPAM) melt between 390-430° C. and the melting point is dependent on AA and NIPAM content in the formulation wherein copolymers with low molar percentage of either AA or NIPAM show lower melting temperature. Gel 2 (5% AA) and Gel 3(10% NIPAM) showed the low endothermic peaks at 391 and 394° C., respectively (FIG. 13B) AA give a more stable copolymer because of more hydrogen bonding in the hydrogel network. It is believed that the interactions between NIPAM's hydrophilic groups and AA hydrophilic groups influence thermal stability, so copolymers with more NIPAM and less AA contents like Gel 2 (5% AA and 28% NIPAM) and Gel 7 (11% AA and 34% NIPAM) are instable due to less hydrophilic interactions, therefore, less energy is needed to break the hydrogen bonding causing the phase transition to happen at lower temperature. It is also noted that P(OEGA-co-AA-co-NIPAM) copolymer is stable at the range of temperature where it is intended to be used (physiological conditions) during wound healing. TGA of selected copolymers was performed using a TGA instrument (TA Q50) and TGA curves of P(OEGA-co-AA-co-NIPAM) hydrogels of different molar ratios are presented in FIG. 13C. The peak temperature of water loss was slightly different for each of the hydrogel formulations. The temperature values were obtained from the first derivative plots of the TGA as 112° C. for Gel 9, 108° C. for Gel 3, 115° C. for Gel 8, 130° C. for Gel 900 and 137° C. for GEL 6 (FIG. 13D). As the concentration of AA increased in copolymers, the water evaporation temperature slightly increased (Gel 6 has more AA than Gel 3 and Gel 8) and its water evaporates at higher temperature that the other two formulations. This is due to more hydrogen bonds in the polymer with higher AA content. Further increase in temperature caused the polymers to decompose between 350-380° C. The thermal response of the copolymer is in good agreement with the previous studies on polyvinyl alcohol-copoly (methacrylic acid) hydrogels, suggesting that the copolymers can serve as better carriers for drug delivery. It is worthy to mention that the hydrogel with highest NIPAM content (Gel 9, 43% NIPAM) decomposed at lowest temperature at 323° C. with great mass loss (around 14%) compared to when there is no NIPAM in the hydrogel (Gel 900, 0% NIPAM) or less NIPAM (Gel 3, 10% NIPAM) (FIG. 13D). In addition, high NIPAM content resulted in fast water evaporation and highest decomposition rate at later temperature. The behavior of Gel 9 can be explained by the phase separation at the temperature above PNIPAM′ LCST where it expels water molecules out from the polymer networks due to dominant hydrophobic interactions between its isopropyl groups. Both DSC and TGA suggested that the AA content in the polymer has more effect on thermal response of P(OEGA-co-AA-co-NIPAM) hydrogel linking it to its stability of hydrogel at high temperature.

[0132] Cytotoxicity assays are used to evaluate biocompatibility of materials intended to be used in biomedical applications. The cytotoxicity of the P(OEGA-co-AA-co-NIPAM) copolymer was evaluated at different polymer concentrations. Fibroblast cells were cultured and exposed to copolymers of different concentrations ranging from 0 to 30 mg/mL of NIPAM. NIPAM concentration was considered because NIPAM monomer has been reported by some studies to have toxicity towards some cell lines. Since the biocompatibility of PNIPAM in hydrogels or nanoparticles is still ambiguous because of contradictory results presented throughout the literature, it is necessary to check cytotoxicity of the PNIPAM components to specific cell lines for the applications. MTT assay data show that the (PEG-PAA-PNIPAM) formulation is not toxic to fibroblast cells at the highest concentration used (30 mg/ml). FIG. 14 indicates that the cell viability is nearly 100% and it is comparable to the control (Gel 900) that was previously proven to be biocompatible. It is also believed that when cell viability is above 70% after being exposed to a compound, then that compound is not cytotoxic. This agrees with other studies which found that concentration of PNIPAM between 0.01 to 10 mg/mL did not cause any cytotoxicity where cell viability exceeded 100% in some cases. These results suggest that little cytotoxic effect was found for the copolymers after purification to remove the monomer residues.

[0133] Different formulations of P(OEGA-co-AA-co-NIPAM) hydrogel were tested for their capability for controlled and sustained release of hFGF1 in physiological conditions. Released hFGF1 was monitored by fluorescence spectroscopy for at least 4 days. The released amount of hFGF1 was calculated using a calibration curve and cumulative concentration was obtained. The order of decreasing release is Gel 1>Gel 8>Gel 7>Gel 6>Gel 5>Gel 3>Gel 4>Gel 2. Gel 1 showed the fastest release rate because of the lowest crosslinking density that was easier for proteins to diffuse out of the hydrogel matrix; but the gel was very viscous which makes it hardly injectable. Gel 8 was then chosen as the best formulation in this series of P(OEGA-co-AA-co-NIPAM) study to be further investigated for the controlled release mechanism of proteins and compare with our Gel 900 developed in the previous study. For P(OEGA-co-AA-co-NIPAM), there was a significant difference between release at room temperature (25° C.) versus at 37° C. (FIG. 15A). The release rate was two-fold slower at 25° C. compared to 37° C. This could be explained by the properties of PNIPAM which collapses at temperature higher than its LCST to facilitate release of loaded protein. Compared to Gel 900-P(OEGA-co-AA), incorporating NIPAM in P(OEGA-co-AA) improved the released profile of hFGF1 by 6 times from being less than 7% to 45% in 5 days in physiological conditions (FIG. 15A). The result suggests that PNIPAM in the copolymer reduces the global and local charge density and thus weakens the electrostatic interaction between the protein and the hydrogel to facilitate the release. The electrostatic controlled release mechanism was also evident by a much faster release of BSA (nearly 100% release in 5 days) which is a negatively charged protein (FIG. 15B).

[0134] Following the release study, we evaluated the bioactivity of the released hFGF1. The result from the cell proliferation assay indicated that the released hFGF1 remained 60% active even after 7 days in the hydrogels (FIG. 15C). The protein activity of the released hFGF1 declined overtime because of some denaturation and/or aggregations. Stability of hFGF1 in culture media was also evaluated. hFGF1 is known to be unstable and prone to degradation by proteolytic enzymes in DMEM cell culture media at 37° C. FIG. 15D shows that hFGF1 was completely degraded at 37° C. after 24 hours of incubation in the cell culture medium. hFGF1 is unstable and its instability can limit its applications in physiological conditions. The hFGF1 requires binding to heparin to protect it against denaturation and increase its in vivo half-life. However, heparin does not guarantee stability of hFGF1 in some cases. For instance, hFGF1 may lose its activity after 6 hours at 37° C. even in presence of heparin. As an alternative, hFGF1 can be mutated to increase its stability. It is believed that the sustained release of hFGF1 from P(OEGA-co-AA-co-NIPAM) was made possible by strong electrostatic interactions between the anionic hydrogel and positively charged proteins in physiological conditions. These interactions are thought to stabilize the proteins in the hydrogel network to maintain their biological activity over a period of at least ten days before it starts getting inactive. The sustained bioactivity of other growth factors upon encapsulation and release from hydrogels have been reported in literature where released growth factors enable higher cells' differentiation and proliferation compared to growth factors alone.

[0135] An in vivo study was conducted to evaluate the controlled release of hFGF1 from P(OEGA-co-AA-co-NIPAM) hydrogel (Gel 8) on wound healing. Wound closure data showed that hFGF1 treatment significantly diminished the average wound size (p=0.0047) over the whole data set (FIG. 16). The hFGF1-treated wounds were significantly smaller than the untreated control group on days 3 (p=0.0297) and 5 (p=0.015), which indicate that the effects of hFGF1 were most obvious during the intermediate time points during the proliferative phases of healing (FIG. 16). The significant difference in wound closure is an indication of preserved bioactivity of released hFGF1 that was observed in vitro. The hydrogel was able to prolong the therapeutic effect of loaded hFGF1 to enhance wound healing process compared to control.

[0136] While the foregoing written description enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The disclosure should therefore not be limited by the above described embodiments, methods, and examples, but by all embodiments and methods within the scope and spirit of the disclosure.