Patent Application
20230241228
Not applicable.
Not applicable.
Hydrogels are three-dimensional, cross-linked polymer networks that can retain a large amount of water. They allow biomolecules to be trapped in their porous structures for delivery in applications such as tissue engineering and wound healing. In general, hydrogels can be made from both natural and synthetic polymers. Although natural polymers are often biocompatible and biodegradable, hydrogels made of natural polymers are difficult to be chemically functionalized for sustained release and/or potentially immunogenic in the host body. In contrast, hydrogels made from synthetic polymers have several advantages including modifiable chemical properties, tunable mechanical properties, controllable porosity and transport properties. Poly(ethylene glycol) (PEG) is commonly used to form hydrogels due to its good biocompatibility and non-immunogenity; however, PEG hydrogels typically possess minimal or no intrinsic biological activity due to the antifouling nature of the PEG polymers.
PEG is also known for its biocompatibility, neutrality, and anti-biofouling properties that are desired for the delivery systems. For this reason, PEG-hydrogels have shown an important property in providing a controlled and sustained release of active therapeutics for important cellular functions. Furthermore, PEG has been used to modify non-biocompatible and non-biodegradable polymers to have biocompatibility and biodegradability desired in biomedical applications. Despite their excellent properties, PEG hydrogels suffer from weak mechanical properties which can potentially limit their applications. Studies have shown that PEG hydrogels copolymerized with other monomers could enhance their chemical, physical, and mechanical properties. For instance, poly(acrylic acid) (PAA) was incorporated in PEG to make a copolymer network of PEG-PAA hydrogels with improved mechanical properties. Furthermore, PAA introduces negative charges to the neutral PEG for enhancement of gel functionalities for protein delivery; however, the charge distribution is difficult to control and often forms pocket containing high density of negatives charges in the gel that binds to positively-charged protein too tight to prevent their release. Here we develop a method to introduce neutral species to space out the negative charges in the gel and demonstrate the enhancement of protein release.
In other embodiments, the present invention provides a method, system, approach and solution that provide biocompatible and biodegradable hydrogels for sustained delivery of biological therapeutic agents.
In other embodiments, the present invention provides a system, method, approach and solution that provide biocompatible and biodegradable hydrogels for regenerative medicine applications.
In other embodiments, the present invention provides a system, method, approach and solution that provide a method to synthesize biocompatible and biodegradable anionic hydrogels based on poly(acrylic acid)-co-poly(oligoethylene glycol monoacrylate) for protein delivery.
In other embodiments, the present invention provides a system, method, approach and solution that provide a synthesis that involves an aqueous free radical polymerization.
In other embodiments, the present invention provides a system, method, approach and solution that provide a synthesis that involves an aqueous free radical polymerization, followed by sequential steps to allow the swelling of copolymer into the hydrogels.
In other embodiments, the present invention provides a system, method, approach, and solution wherein the introduction of an anionic group-containing acrylic acid into the copolymers changes the thermal properties and viscosity of the hydrogels due to the alternation of intermolecular interactions in the polymer networks. In these embodiments, electrostatic interaction(s) between the anionic hydrogels and positively charged proteins render the hydrogels capable of a sustainable release of the proteins.
In other embodiments, the present invention provides a system, method, approach and solution that provide an ionizable component, acrylic acid (AA), into the PEG-based hydrogel to form a biocompatible and biodegradable, anionic hydrogel of poly(acrylic acid)-co-poly(oligoethylene glycol monoacrylate) (PAA-co-POEGA).
In other embodiments, the present invention provides a system, method, approach and solution that provide a synthesis that involve an aqueous free radical polymerization, followed by sequential steps to allow the swelling of copolymer into the hydrogels. The presence of AA provides a negative charge in the hydrogel at physiological pH that improves the retention of the entrapped, positively charged proteins through electrostatic interactions for sustained delivery. PAA mimics heparin which possesses antithrombin-activating properties and may promote anti-inflammatory processes.
In other embodiments, the present invention provides a system, method, approach and solution wherein monomers AA and PEG acrylate (OEGA) may be polymerized by a cross-linker such as N,N′-methylenebis(acrylamide) (MBAm) or N,N′-bis(acryloyl)cystamine (BAC). Depending on the ratio of AA/OEGA/cross-linker, the chemical and physical properties of the resulting hydrogel may be tuned to facilitate the protein delivery for different applications.
In other embodiments, the present invention provides a system, method, approach and solution wherein the sustained release of proteins from the hydrogel may be attained by using both the model protein lysozyme and wild-type fibroblast growth factor 1 (wtFGF1) that are positively charged under the physiological condition.
In other embodiments, the present invention provides a system, method, approach and solution wherein the injectable PAA-co-POEGA hydrogel is biocompatible. The biodegradability of the hydrogel may be attained by hydrogel cross-linking BAC.
In other embodiments, the present invention provides a method, system, approach and solution that provide biocompatible and biodegradable hydrogels for sustained delivery of biological therapeutic agents.
In other embodiments, the present invention provides a method, system, approach and solution that provide biocompatible and biodegradable hydrogels for regenerative medicine applications.
In other embodiments, the present invention provides a method, system, approach and solution that provide a method to synthesize biocompatible and biodegradable anionic hydrogels based on poly(acrylic acid)-co-poly(oligoethylene glycol monoacrylate) for protein delivery.
In other embodiments, the present invention provides a method, system, approach and solution that involves an aqueous free radical polymerization, followed by steps to allow the swelling of copolymer into the hydrogels.
In other embodiments, the present invention provides a method, system, approach and solution wherein the introduction of an anionic group-containing acrylic acid into the copolymers changes the thermal properties and viscosity of the hydrogels due to the alternation of intermolecular interactions in the polymer networks.
In other embodiments, the present invention provides a method, system, approach and wherein there is an electrostatic interaction(s) between the anionic hydrogels and positively charged proteins, thus, rendering the hydrogels capable of a sustainable release of the proteins.
In other embodiments, the present invention provides a method, system, approach and solution that provide an ionizable component, acrylic acid (AA), into the PEG-based hydrogel to form a biocompatible and biodegradable, anionic hydrogel of poly(acrylic acid)-co-poly(oligoethylene glycol monoacrylate) (PAA-co-POEGA).
In other embodiments, the present invention provides a method, system, approach and solution that involve an aqueous free radical polymerization, followed by sequential steps to allow the swelling of copolymer into the hydrogels.
In other embodiments, the present invention provides a method, system, approach and solution wherein the presence of AA provides a negative charge in the hydrogel at physiological pH that improves the retention of the entrapped, positively charged proteins through electrostatic interactions for sustained delivery.
In other embodiments, the present invention provides a method, system, approach and solution wherein PAA mimics heparin which possesses antithrombin-activating properties and may promote anti-inflammatory processes.
In other embodiments, the present invention provides a method, system, approach and solution wherein the hydrogel synthesis consists of a two-step procedure involving a free radical polymerization to form a copolymer network followed by allowing the copolymer network swelling into hydrogel.
In other embodiments, the present invention provides a method, system, approach and solution wherein monomers AA and PEG acrylate (OEGA) may be polymerized by a cross-linker such as N,N′-methylenebis(acrylamide) (MBAm) or N,N′-bis(acryloyl)cystamine (BAC).
In other embodiments, the present invention provides a method, system, approach and solution wherein, depending on the ratio of AA/OEGA/cross-linker, the chemical and physical properties of the resulting hydrogel may be tuned to facilitate the protein delivery for different applications.
In other embodiments, the present invention provides a method, system, approach and solution wherein the sustained release of proteins from the hydrogel may be attained by using both the model protein lysozyme and wild-type fibroblast growth factor 1 (wtFGF1) that are positively charged under the physiological condition.
In other embodiments, the present invention provides a method, system, approach and solution wherein the injectable PAA-co-POEGA hydrogel is biocompatible. The biodegradability of the hydrogel may be attained by hydrogel cross-linking BAC.
In other embodiments, the present invention provides a method, system, approach and solution wherein the synthesis of the injectable hydrogels consists of a two-step procedure, 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 and the second step is to let the PAA-co-POEGA polymeric network swell in buffered solution such as a saline solution with 0.1 wt. % Borax (antibacterial agent), forming the injectable hydrogels.
In other embodiments, the present invention provides a method, system, approach and solution wherein the reaction may be initiated by APS to form PAA-co-POEGA polymeric network.
In other embodiments, the present invention provides a fast and simple synthesis method for the preparation of anionic hydrogels based on poly(oligo ethylene glycol monoacrylate-co-acrylic acid) P(OEGA-co-AA) using a free polymerization reaction.
In other embodiments, the present invention provides a preparation of anionic hydrogels based on poly(oligo ethylene glycol monoacrylate-co-acrylic acid) P(OEGA-co-AA) for controlled release of positively charged proteins such as lysozyme.
In other embodiments, the present invention provides an anionic injectable hydrogel developed for protein delivery based on poly(oligoethylene glycol monoacrylate-co-acrylic acid-co-N-isopropylacrylamide) or P(OEGA-co-AA-co-NIPAM) that is synthesized via a free-radical polymerization reaction.
In other embodiments, the present invention provides an anionic injectable hydrogel developed for protein delivery based on poly(oligoethylene glycol monoacrylate-co-acrylic acid-co-N-isopropylacrylamide) or P(OEGA-co-AA-co-NIPAM) that is synthesized via a free-radical polymerization reaction and incorporating PNIPAM to the P(OEGA-co-AA) spaces out the charge distribution of the anionic gel allowing for the increased release of positively-charged proteins.
In other embodiments, rather than incorporating PNIPAM to the P(OEGA-co-AA), other neutral species such as gelatin, may be used.
For the embodiments of the present invention, a release study was carried out using human acidic fibroblast growth factor (hFGF1) under physiological conditions. The results indicated that the release kinetics from the P(OEGA-co-AA-co-NIPAM) gel is temperature-dependent and release rate was increased by six times than the P(OEGA-co-AA) gel due to weaker electrostatic interactions between hFGF1 and the P(OEGA-co-AA-co-NIPAM) gel. This charge-driven release mechanism was also evidenced by much faster and sustained release kinetics of negatively-charged proteins from the P(OEGA-co-AA-co-NIPAM) gel compared to that of hFGF1. The released hFGF1 remained bioactive and promoted fibroblast's proliferation in vitro and skin wound healing in vivo. This study demonstrates a delivery system that enables sustained release of bioactive positively charged proteins for enhanced wound healing.
In other embodiments, the present invention provides an additional component poly (N-isopropylacrylamide), PNIPAM, to the copolymer to tune the charge density of the P(OEGA-co-AA) anionic hydrogel to control the release of active hFGF1 for wound healing. PNIPAM is a charge-neutral polymer and copolymerizing with P(OEGA-co-AA) provides an avenue for adjusting the charge distribution of the anionic hydrogel thereby reducing the strength of electrostatic interactions to facilitate the release of hFGF1. Additionally, the hydrophobic (propyl group) and hydrophilic (amide group) moieties of the repeating unit render PNIPAM a thermo-responsive polymer known to have a lower critical solution temperature (LCST) close to human body temperature. PNIPAM-based hydrogels have been used as drug delivery systems as they can release drugs due to the change of their structural conformation at their LCST. These hydrophobic and hydrophilic moieties can provide additional interactions to regulate the protein control release of the anionic hydrogels.
In other embodiments, the present invention synthesized P(OEGA-co-AA-co-NIPAM) via a free radical polymerization and formulated the anionic hydrogels intending to improve the release of active hFGF1 for enhanced wound healing. Vibrational spectroscopy was used to monitor the formation of the copolymer P(OEGA-co-AA-co-NIPAM). The water content of the hydrogel and thermal stability of the copolymers were studied by thermogravimetric analysis (TGA). No cytotoxic effect of the hydrogels was observed for the copolymer P(OEGA-co-AA-co-NIPAM).
In other embodiments, the present invention synthesized an injectable anionic biocompatible hydrogel system based on P(OEGA-co-AA-co-NIPAM) using a simple radical polymerization reaction. AA was added to the hydrogel to introduce negative charges in the hydrogel and NIPAM was used to space out the negative charges within the hydrogel to control the release rate of positively charged proteins. FTIR and Raman spectroscopy verified the chemical structure of the copolymers. DSC and TGA data shows increasing molar percentage of AA and NIPAM in the hydrogel increases thermal stability of the hydrogel. It was also observed that more NIPAM in the hydrogel cause the hydrogel to lose its water much faster and have a greater weight loss. P(OEGA-co-AA-co-NIPAM) hydrogel showed the capacity for a sustained release of hFGF1 with preserved bioactivity in physiological conditions. The sustained release was based on electrostatic interactions that held the proteins in the hydrogel and sustained its bioactivity overtime. Incorporating NIPAM in P(OEGA-co-AA) hydrogel system increased release rate of hFGF1 which may be explained by the neutral PNIPAM spacing out and/or lessening of negative charges on the anionic hydrogel, thus, reducing the interactions between the anionic hydrogels and the positively charged proteins. PNIPAM thermo-responsive properties also enhanced release profile at 37° C. compared to 25° C. because of the change in hydrogel structure to repel loaded protein upon increase in temperature above PNIPAM's LCST. Most of the encapsulated BSA was completely released from P(OEGA-co-AA-co-NIPAM) hydrogel because of its negative net charges that repel with similarly charged hydrogel and led to much faster release rate than hFGF1. The release profile of BSA confirm the importance of electrostatic interactions between the hydrogel and the protein resulting in a sustained release of the protein.
In other embodiments of the present invention, hFGF1 remains active after encapsulation, that the rate of sustained delivery of FGFs can be tuned by hydrogel synthesis, and that the hydrogel can be used as a delivery vehicle for positively charged proteins such as FGFs in wound healing applications.
In another embodiment, the present invention provides 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 the 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 the gel to form an anionic hydrogel. The method may further include the step of adding a neutral species to the poly(oligoethylene glycol monoacrylate) and the acrylic acid, the neutral species spaces out the charge distribution of the anionic hydrogel allowing for the increased release of the a wild-type fibroblast growth factor 1 (wtFGF1).
In another embodiment of the present invention, the neutral species may be NIPAM.
In another embodiment of the present invention, the weight ratio of the anionic hydrogel is poly(oligoethylene glycol monoacrylate) 850, acrylic acid 25, NIPAM 125.
In another embodiment of the present invention, the neutral species is gelatin.
In another embodiment of the present invention, the weight ratio of the anionic hydrogel is poly(oligoethylene glycol monoacrylate) 850, acrylic acid 25, gelatin 160.
In another embodiment, the present invention provides an anionic hydrogel for wound healing comprising: poly(oligoethylene glycol monoacrylate), acrylic, a neutral species and a wild-type fibroblast growth factor 1 (wtFGF1).
In another embodiment, the present invention provides 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; the anionic hydrogel comprising: poly(oligoethylene glycol monoacrylate), acrylic, a neutral species and a wild-type fibroblast growth factor 1 (wtFGF1); and the hydrogel configured to controllably release the a wild-type fibroblast growth factor 1 (wtFGF1).
In another embodiment of the present invention, the hydrogel inhibits the degradation of the wild-type fibroblast growth factor 1 (wtFGF1).
In another embodiment, the present invention further includes the step of increasing the neutral species to the poly(oligoethylene glycol monoacrylate) and the acrylic acid, to increase the release of the a wild-type fibroblast growth factor 1 (wtFGF1).
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
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.
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.
In other embodiments, the present involves the synthesis of the injectable hydrogels that consists of a two-step procedure (
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
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 (
For the disk hydrogel, the loading capacity was assessed using the positively charged lysozyme (
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 (
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.
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.
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.
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 N2 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.
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.
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.
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 N2 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.
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.
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.
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 N2 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.
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.
Controlled Release of wtFGF1 from Injectable Anionic Hydrogels.
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
Cell Proliferation Assay for Bioactivity of Released wtFGF1.
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.
Poly(acrylic acid)-co-poly(oligoethylene glycolmonoacrylate)-co-poly(N-isopropyl acrylamide) Injectable Anionic Hydrogels for Controlled Release of wtFGF1 release.
Synthesis of PAA-co-POEGA-co-PNIPAM Injectable Hydrogels.
Each monomer was purified prior to the polymerization using the following methods. Oligoethylene glycol monoacrylate (Mn=480, OEGA) was purified through a basic Al2O3 column. Acrylic acid (AA) was purified through a neutral Al2O3 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 N2 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.
PAA-co-POEGA-co-PNIPAM Gel.
The newly developed anionic injectable hydrogels were prepared from the copolymer PAA-co-POEGA-co-PNIPAM, as shown in
wtFGF1 Control Release Study.
The release of wtFGF1 was significantly increased using PAA-co-POEGA-co-PNIPAM gels compared to PAA-co-POEGA, as shown in
Experimental Methods
Materials and Chemicals.
Oligo ethylene glycol monoacrylate (OEGA, Mn=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.
Inhibitors were removed OEGA and AA by purification through a basic and neutral aluminum oxide (Al2O3) 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 H2O) was used in all procedures unless specified.
Preparation of P(OEGA-co-AA-co-NIPAM) Hydrogels.
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 N2 for 5 min. The mixture was then transferred to a 25-mL stopcock, air free, Schlenk flask protected with N2. 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.
Characterization Methods.
The vibrational spectra of the copolymers were recorded between 400-4000 cm−1 at a resolution of 4 cm−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 N2 at a flow rate of 60 mL/min from 30 to 500° C. at a rate of 10° C./min.
Cytotoxicity Studies.
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 CO2 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.
Proteins Release Studies.
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 λex=280 nm) and 345 nm (with λex=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.
Animal Study for Wound Closure.
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.
Results and Discussion
Different formulations of P(OEGA-co-AA-co-NIPAM) were synthesized by a free radical polymerization reaction with modifications to copolymerize with NIPAM.
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The copolymers were characterized by FTIR spectra to confirm that their chemical structures contained the functional groups from the monomers.
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. (
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).
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. (
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 (
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 (
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.
This application is a continuation in part of U.S. Ser. No. 17/183,083 filed on Feb. 23, 2021, which is a divisional of U.S. Ser. No. 16/409,495 filed on May 10, 2019, which claims priority to U.S. Provisional Application Ser. No. 62/670,578 filed on May 11, 2018, both of which are hereby incorporated in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62670578 | May 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16409495 | May 2019 | US |
| Child | 17183083 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17183083 | Feb 2021 | US |
| Child | 18298348 | US |
