DOPA modified gelatin for wound healing and methods of making the same

Abstract

To provide a DOPA-Gelatin in which a monophenolic group of tyrosine, a prominent amino acid in porcine gelatin, is converted into a catechol group using an enzyme-based DOPA modification technique.SOLUTION: The resulting DOPA-Gelatin has been discovered to exhibit good mechanical strength and adhesion. Moreover, in vitro studies show that DOPA-Gelatin has no cytotoxicity with HDF and HaCaT cells, two cells typically involved in the skin wound healing process. Further RT-PCR and angiogenesis investigation showed that DOPA-Gelatin can promote the expression of wound-related genes and facilitate neovascularization. In a full-thickness dorsal defect model in mice, DOPA-Gelatin treated groups decreased the wound closure time and enhanced hair follicle growth. These results demonstrate that the DOPA-Gelatin hydrogel compositions disclosed herein are an effective functional biomaterial that can potentiate the wound healing process.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present invention relates generally to 3,4-dihydroxyphenylalanine (DOPA) modified polymers for biomedical applications. More specifically, the present invention relates to a DOPA-gelatin composition and a one-step process for making it using tyrosinase. [Background technology]

[0002] The development of novel and unique wound dressings has been extensively pursued over the past few decades. As regenerative medicine and tissue engineering have advanced, wound dressings have also evolved into state-of-the-art technologies that promote wound healing rather than simply covering the wound with gauze. [1、2] Among the various wound dressings that have been fabricated, bioactive / biocompatible hydrogels have high water content to retain moisture around the wound area. [3~5] , the ability to remove necrotic tissue and absorb wound exudate [6、7] , and a permeable structure for the diffusion of essential gases such as oxygen, carbon dioxide, and water vapor. [8、9] Due to their inherent advantages, they have attracted much attention. However, some wound-covering hydrogels also suffer from poor mechanical strength and adhesive properties.

[10] .

[0003] The need to overcome these obstacles and improve the adhesive capabilities of hydrogels prompted researchers to investigate mussels, which exhibit strong adhesion to a variety of surfaces, even under wet conditions.

[11] Studies have demonstrated that the adhesive ability of mussels is the result of the abundance of 3,4-dihydroxyphenylalanine (DOPA), a special amino acid in proteins secreted by the mussel's foot organ.

[12] Many natural polymers have been modified with DOPA to improve their adhesive properties, and DOPA-chitosan

[13] , DOPA-PEG

[14] , DOPA-HA

[15] , and DOPA-alginate

[16] These include, but are not limited to, DOPA-modified hydrogels. In these studies, DOPA-modified hydrogels not only increase adhesion properties but also improve bioactivity. For example, catechol-modified hyaluronic acid increases cell viability, reduces apoptosis, and enhances the function of two types of cells (human adipose-derived stem cells and hepatocytes). Dopamine-modified alginate hydrogels have better properties for drug adsorption and release.

[0004] The process for making conventional DOPA-containing hydrogels requires multi-step preparation and purification methods, which are time-consuming and complicated. Moreover, most DOPA-related hydrogels incorporate the compound dopamine (DA) as a source of DOPA structure. However, unfortunately, exogenous DA in the synthesis process is usually easily polymerized into polydopamine, which reduces the adhesive properties of the final material.

[17] Therefore, a faster and more direct method of introducing DOPA structures into polymers is highly desirable. In addition, there is a need in the art for new DOPA-gelatin hydrogels that can be used in a variety of therapeutic situations, such as wound healing. Summary of the Invention

[0005] The present invention also includes methods and materials for forming DOPA-gelatin hydrogels from modified porcine gelatin compositions. The present invention also includes DOPA-gelatin adhesive hydrogels made by the methods disclosed herein. The hydrogels formed by the methods disclosed herein have a number of desirable material properties, including enhanced in vivo adhesive and in vivo activity profiles. For example, the hydrogels of the present invention have been found to have the ability to increase biological responses, such as to enhance wound healing.

[0006] As explained below, one way to form DOPA moieties on tyrosine-containing polymers such as gelatin without the use of exogenous DA compounds is by using the enzyme tyrosinase. Tyrosinase, better known as polyphenol oxidase, can directly catalyze the phenolic group in tyrosine to a catechol group, the primary chemical group found on DOPA. The one-step synthesis of modified porcine gelatin using tyrosinase as disclosed herein is a more efficient and environmentally friendly approach to making DOPA-modified hydrogels. The synthesis requires only a few hours, and the procedure results in a less toxic material compared to synthesis using chemical compounds. Furthermore, compared to other materials, gelatin is readily available and exhibits favorable biocompatibility. In addition, the DOPA-gelatin compositions disclosed herein have been found to exhibit hemostatic capabilities, an important aspect of the wound healing process.

[0007] The invention disclosed herein has numerous embodiments. In one methodological embodiment, tyrosinase was used to catalyze the conversion of tyrosine residues to DOPA in a one-step reaction using porcine gelatin. The efficacy of such DOPA-gelatin compositions in promoting in vivo adhesion and wound healing (e.g., at the cellular and gene expression levels) was then evaluated. The results of our study showed that our one-step method using tyrosinase produces porcine gelatin hydrogels that contain the catechol group of DOPA and maintain their adhesive and load-bearing properties required for wound dressings. Our DOPA-gelatin compositions also improve the in vitro proliferation and migration of both fibroblasts and keratinocytes, two key cells involved in the wound healing process. When DOPA-gelatin was applied to the skin wound area of ​​mice, both the healing rate and hair growth were accelerated compared to the control and untreated gelatin groups.

[0008] In one exemplary embodiment of the present invention, a method for making 3,4-dihydroxyphenylalanine (DOPA)-gelatin includes (1) providing a solution containing porcine gelatin, and (2) incubating the solution containing porcine gelatin with tyrosinase to make 3,4-dihydroxyphenylalanine (DOPA)-gelatin. The solution containing porcine gelatin is preferably incubated with tyrosinase for at least 1 hour (e.g., several hours at 37°C, preferably about 3 hours). In one embodiment, the concentration of tyrosinase used is 100-200 U / mL.

[0009] Another embodiment of the present invention is a therapeutic composition comprising porcine gelatin in which substantially all tyrosine residues are converted to 3,4-dihydroxyphenylalanine (DOPA). In some embodiments of the present invention, the composition is sterile and comprises a pharma- ceutically acceptable carrier. Optionally, the composition further comprises at least one additional therapeutic agent, such as an antibiotic, an anti-inflammatory agent, a hemostatic agent, an embolic agent, a chemotherapeutic agent, etc. The therapeutic composition can be used in many situations, for example, to deliver the composition to a wound site (e.g., skin or non-skin) to promote wound healing.

[0010] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating certain embodiments of the present invention, are given by way of illustration and not by way of limitation. Many changes and modifications may be made within the scope of the present invention without departing from the spirit thereof, and the invention includes all such modifications. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1A shows a schematic of the DOPA modification reaction. FIG. 1B shows a photograph showing the tyrosinase concentration-dependent modification of DOPA-gelatin. FIG. 1C shows a UV-VIS measurement of the enzymatic DOPA modification. FIG. 1D shows an FTIR analysis of DOPA-gelatin. FIG. 1E shows a graph showing the quantification of DOPA content by the Arnow method. [Diagram 2] Figure 2A shows graphs of lap shear test (left), maximum load (center) and tensile stress (right) of DOPA-modified gelatin. Figure 2B shows graphs of burst test (left) and maximum burst pressure (right). Figure 2C shows the rheological test results of DOPA-modified gelatin. [Diagram 3] Figure 3A shows representative images of the HDF cell live / dead assay used to demonstrate the in vitro cytotoxicity of DOPA-gelatin. Figure 3B shows the quantitative analysis of HDF cell viability and the proliferation effect of DOPA-gelatin. Figure 3C shows representative images of the HaCaT cell live / dead assay. Figure 3D shows the quantitative analysis of HaCaT cell viability and the proliferation effect of DOPA-gelatin. [Figure 4] FIG. 4A shows a representative image of the HDF cell migration assay of DOPA-gelatin. FIG. 4B shows a quantitative analysis of the HDF cell migration effect of DOPA-gelatin. FIG. 4C shows a representative image of the HaCaT cell migration assay of DOPA-gelatin. FIG. 4D shows a quantitative analysis of the HaCaT cell migration effect of DOPA-gelatin. FIG. 4E shows the gene expression analysis result of the HDF cell migration sample of DOPA-gelatin. FIG. 4F shows the gene expression analysis result of the HaCaT cell migration sample of DOPA-gelatin. [Diagram 5] 5A-5D show the results of angiogenesis assay. FIG. 5A shows a representative image of angiogenesis for the direct method. FIG. 5B is a quantitative analysis of angiogenesis assay for the direct method. FIG. 5C shows a representative image of angiogenesis for the indirect method. FIG. 5D is a quantitative analysis of angiogenesis assay for the indirect method. [Figure 6] 6A-6D show an in vivo study of DOPA-gelatin. Fig. 6A shows wound healing images on a mouse skin model. Fig. 6B shows a quantitative analysis of the wound healing area. Fig. 6C shows histology images. [Figure 7] 1 shows DOPA-gelatin being delivered to a wound site located in dermal tissue. [Figure 8] The calibration curves of absorbance and DOPA concentration by the Arnow method are shown. [Figure 9] Representative images of angiogenesis by direct methods are shown. [Figure 10] Quantitative results of branching points and tube numbers for the direct method are shown. [Figure 11] 1 shows an in vivo degradation study of gelatin and DOPA-gelatin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In describing the embodiments, reference may be made to the accompanying drawings, which form a part of this specification and which show by way of example specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, but the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference to what is commonly understood in the art. Many of the aspects of the techniques and procedures described or referenced herein are well understood and commonly used by those skilled in the art. The following text describes various embodiments of the present invention.

[0013] Embodiments of the present invention include methods of making 3,4-dihydroxyphenylalanine (DOPA)-gelatin. Typically, these methods include providing a solution containing porcine gelatin (which is more desirable than human gelatin due to its commercial availability) and then incubating the solution containing porcine gelatin with tyrosinase for a specific period of time, such as more than 30 minutes or at least 1 hour (e.g., at room temperature or 37°C), thereby making 3,4-dihydroxyphenylalanine (DOPA)-. In certain methodological embodiments, the solution containing porcine gelatin is incubated with tyrosinase at room temperature or 37°C for at least 2 hours or at least 3 hours. Typically, the concentration of tyrosinase is 300 U / mL or less than 200 U / mL, e.g., 100-200 U / mL. Typically, these methods include heating the solution to inactivate tyrosinase (e.g., at which point the appropriate 3,4-dihydroxyphenylalanine (DOPA) is made). In a particular embodiment of the present invention, 3,4-dihydroxyphenylalanine (DOPA)-gelatin is made in a one-step synthetic reaction.

[0014] The present invention includes a 3,4-dihydroxyphenylalanine (DOPA)-gelatine composition made by the method disclosed herein. The present invention includes a therapeutic composition comprising porcine gelatine in which substantially all (e.g., at least 80%, 85%, 90% or 95%) of the tyrosine residues have been converted to 3,4-dihydroxyphenylalanine (DOPA). In certain embodiments of the present invention, the composition is substantially free of metal ions (see, e.g., Y. Chan Choi, JS Choi, YJ Jung, YW Cho, Journal of Materials Chemistry B 2014, 2, 201, the contents of which are incorporated by reference). In some embodiments of the present invention, the composition is sterile and comprises a pharma- ceutically acceptable carrier. Optionally, the composition further comprises at least one additional therapeutic agent selected from an antibiotic, an anti-inflammatory agent, a hemostatic agent, an embolic agent, and a chemotherapeutic agent.

[0015] Choi et al. used tyrosinase to convert phenols in tyrosine residues of gelatin extracted from human adipose tissue and quantified the DOPA content in the formed DOPA-gelatin.

[21] The average tyrosine content in pigskin gelatin is 26 / 1000 residues compared to 10 / 1000 residues in human gelatin.

[33] This significant structural difference (e.g., nearly three times the content of tyrosine residues) results in unpredictable material properties for porcine skin gelatin modified according to the methods disclosed herein (i.e., compared to human gelatin). Surprisingly, the methods disclosed herein have produced modified porcine gelatin compositions with unexpected and highly desirable material properties. In certain embodiments of the invention, the methods for making these compositions are adapted to form compositions with selected material properties. In some embodiments of the invention, 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a shear strength of at least 2 MPa. In certain embodiments of the invention, 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a burst pressure of at least 6 kPa. In certain embodiments of the invention, 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a load force of at least 60 N. In certain embodiments of the invention, 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a tensile stress of at least 3 MPa. In certain embodiments of the present invention, 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a storage modulus of at least 700 Pa. In an exemplary embodiment of the present invention, at least 90% of the tyrosine residues of porcine gelatin are converted to 3,4-dihydroxyphenylalanine (DOPA), the composition is sterile and comprises a pharma- ceutically acceptable carrier, the 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a shear strength of at least 2 MPa, the 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a burst pressure of at least 6 kPa, the 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a load force of at least 60 N, and the 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a tensile stress of at least 3 MPa.

[0016] The embodiments of the present invention include a method of using the present invention, for example, a method of using the therapeutic composition disclosed herein, including delivering the composition to a wound site.Certain embodiments of the present invention include a method of delivering the composition disclosed herein to a preselected site, comprising placing the composition in a tube having a first end and a second end that include an opening, applying a force to the second end of the tube, the force being sufficient to move the composition out of the tube through the opening, and then delivering the composition out of the tube through the opening to a preselected site, for example, an in vivo site (e.g., an in vivo location where an individual has experienced trauma or injury, such as a skin wound).

[0017] Certain embodiments of the composition of the present invention include pharmaceutical excipients, such as those selected from the group consisting of preservatives, tonicity adjusters, surfactants, viscosity adjusters, sugars and pH adjusters.For compositions suitable for human administration, the term "excipients" means to include, but is not limited to, the components described in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2006) (the contents of which are incorporated herein by reference).

[0018] Optionally, the compositions of the invention include one or more therapeutic agents, such as an embolic agent, an anti-inflammatory agent, an agent that modulates coagulation, an antibiotic, a chemotherapeutic agent, and the like. The compositions of the present invention can be formulated for use as carriers or scaffolds for therapeutic agents such as drugs, cells, proteins, and bioactive molecules (e.g., enzymes). As carriers, such compositions may incorporate agents and deliver them to desired sites in the body for the treatment of various pathological conditions.

[0019] In certain embodiments of the present invention, the composition comprises a therapeutic agent selected from an anti-inflammatory agent, an embolic agent, and a chemotherapeutic agent. Exemplary embolic agents include, for example, stainless steel coils, absorbable gelatin pledgets and powders, polyvinyl alcohol foam, ethanol, adhesives, and the like. Exemplary hemostatic agents include, for example, Celox, QuiKClot, and Hemcon. Certain exemplary materials and methods that can be adapted for use in such embodiments of the present invention are found, for example, in Hydrogels: Design, Synthesis and Application in Drug Delivery and Regenerative Medicine 1st Edition, Singh, Laverty and Donnelly Eds, and Hydrogels in Biology and Medicine (Polymer Science and Technology) UK ed. J. Edition by J. Michalek et al. Furthermore, as a scaffold, the composition of the present invention can provide a flexible residency space for cells and other agents for use in tissue repair and regeneration of desired tissues (e.g., skin, cartilage, bone, retina, brain, and nerve tissue repair, vascular regeneration, wound healing, etc.).

[0020] In some embodiments of the invention, the composition is placed into a tube (e.g., a catheter) selected for its ability to facilitate the user to adjust one or more rheological properties of the composition (e.g., by manually applying pressure to a 1.7 mm (5-Fr) outer diameter general catheter or a 0.8 mm (2.4-Fr) outer diameter microcatheter). Certain exemplary materials and methods that can be adapted for use in embodiments of the invention are found, for example, in Biomedical Hydrogels: Biochemistry, Manufacture and Medical Applications (Woodhead Publishing Series in Biomaterials) 1st Edition; Steve Rimmer (Editor).

[0021] FIG. 7 shows the DOPA-gelatin therapeutic material described herein being delivered to a wound site located in skin tissue. The DOPA-gelatin therapeutic material was made by a one-step synthesis reaction that includes enzymatic browning, which uses the enzyme tyrosinase to convert the monophenolic group of tyrosine in gelatin to the catechol group of DOPA. The DOPA-gelatin therapeutic material uses porcine gelatin, which is readily available commercially. In addition, the porcine gelatin is exposed to tyrosinase for several hours, preferably about 3 hours, to ensure complete conversion of all tyrosine residues. The DOPA-gelatin is formed using a 10% (w / w) gelatin solution (Type A, G1890, Sigma, California, USA), which can be prepared by dissolving 1 g of gelatin in 10 g of Milli-Q® water at 80° C. for 1 hour. A stock solution of tyrosinase (10 U / μL) to be added to the gelatin solution is made by adding 50 kU tyrosinase powder (T3824, Sigma, MO, USA) to 5 mL Dulbecco's Phosphate Buffered Saline (DPBS, pH 6.5, Gibco, CA, USA) at room temperature. Various concentrations of tyrosinase can be used (e.g., 0, 50, 100, 200, 500 U / mL). The reaction can be carried out at 37°C in a mixer such as an Eppendorf ThermoMixer® C (Eppendorf, NY, USA) with an oscillation frequency of 2000 rpm. After a certain incubation time (e.g., about 3 hours), the temperature can be increased to 65°C for 1 hour to inactivate the enzyme. The DOPA-gelatin final solution can be used immediately or stored at -80°C for later use. It should be noted that the DOPA-gelatin solution composition is substantially free of metal ions similar to those used in Choi et al.

[0022] It has been experimentally determined that there are optimal conditions for the production of DOPA-gelatin with the desired mechanical properties and biological activity. In one embodiment, tyrosinase is incubated for about 3 hours to ensure that all or substantially all of the tyrosine residues in the porcine gelatin have been converted to DOPA. Furthermore, a tyrosinase concentration of about 100 to about 200 U / mL is desirable from the standpoint of biological activity. Furthermore, the use of porcine gelatin is preferred due to the higher occurrence of tyrosine residues compared to human gelatin. Furthermore, porcine gelatin is commercially available in large quantities.

[0023] FIG. 7 shows DOPA-gelatin delivered to a wound location using a delivery device (e.g., a syringe). DOPA-gelatin can be delivered directly to an application site on tissue (e.g., skin tissue). Skin tissue can be healed using DOPA-gelatin, but other tissue types can also be exposed to therapeutic DOPA-gelatin. Furthermore, DOPA-gelatin can be applied to external wounds as well as internal wounds. In addition, in other embodiments, a delivery device such as a syringe may not be required to apply DOPA-gelatin. DOPA-gelatin can be applied from a container, package, etc.

[0024] Results and Discussion DOPA-gelatin synthesis and characterization DOPA-gelatin was produced using a one-step synthesis reaction (Figure 1A). As one of the polyphenol oxidase (PPO) enzymes involved in the enzymatic browning process, tyrosinase converts the monophenol group of tyrosine in gelatin into the catechol group of DOPA. [23~25] In the experiments disclosed herein, tyrosinase was used at various concentrations to catalyze the synthesis of porcine-derived DOPA-gelatin. The gelatin solution turned brown after 3 hours of reaction (Figure 1B), indicating the formation of DOPA structures.

[21] The brown color deepened with increasing enzyme concentration. The UV absorption peak around 280 nm indicates the catechol chemical structure. [21、26、27]Therefore, the DOPA-gelatin synthesis reaction was monitored over time. In Choi's study, the reaction time was set at 30 min. Here, the effect of reaction time on DOPA formation was investigated. With the extension of the reaction time, the absorption peak around 280 nm gradually increased, suggesting the hydroxylation of monophenols in tyrosine (Figure 1C). The absorbance value did not increase substantially over 3 h of reaction, indicating saturation (Figure 1C, line graph). Thus, the optimized DOPA-gelatin synthesis time for converting monophenol groups to catechol groups was established at approximately 3 h. Compared with similar modification methods used to modify other polymers, the method for modifying gelatin is more effective. For example, the introduction of DOPA into oxidized alginate requires a multi-step 3-day process consisting of incubation, dialysis, and freeze-drying.

[28] DOPA-chitosan hydrogel preparation is also complex, requiring mixing, casting into a mold, overnight storage in a refrigerator, and vacuum drying for 24 hours.

[13] .

[0025] To confirm the enzymatic DOPA conversion, Fourier transform infrared (FTIR) spectroscopy was performed. The FTIR absorption spectrum of pure L-DOPA showed several distinct bands according to the diverse functional groups present in the structure, i.e., amino acid moieties and hydroxylated benzene rings (Figure 1D, Table 1). The most significant absorption band for verifying the DOPA-gelatin synthesis was at 1340 cm due to the OH stretching from the catechol group. -1 and 1252cm due to oxygen attached to the aryl ring. -1 In the spectrum of the DOPA-gelatin sample, a significant increase in absorption corresponding to the o-diphenol rings was observed from the OH and CN groups at 3670–3115 cm. -1 In addition, the peaks were observed in the range of 1786–400 cm -1 From the results, an increase in the absorption band similar to that of pure L-DOPA was observed. [29、30] These results provide strong evidence that hydroxylation of the monophenolic residue in tyrosine occurred satisfactorily.

[0026] [Table 1]

[0027] To assess DOPA modification, we utilized the Arnow assay, which provides a useful colorimetric indicator of the chemical groups present in a compound.

[31] Specifically, the amount of DOPA can be detected without interference in the presence of tyrosine.

[32] Similar to Arnow's protocol, the o-diphenol group in the DOPA structure reacts with nitrite to produce a bright red chromophore in alkaline solution (Figure 8). The absorbance at 520 nm was monitored to quantify the presence of DOPA. The absorbance values ​​and DOPA concentrations appeared to be highly linearly related, indicating a positive correlation between the variables. The DOPA content of each sample was calculated by the standard curve (Figure 1E). The average DOPA concentration in the Tyr100 samples was found to be 24.12 ± 2.40 μg / mL, much higher than that reported in previous studies.

[21] The average tyrosine content in pigskin gelatin is 26 / 1000 residues.

[33] Since the number of residues in gelatin was 10 / 1000 of that in human gelatin, a reaction time of 3 hours was used to approach saturation of the conversion of monophenolic groups to DOPA. In summary, a one-step method for producing DOPA-gelatin compatible with biomedical research has been created. This method is simpler and less toxic than comparable methods for gelatin modification and DOPA conversion to other biopolymers.

[0028] Evaluation of the adhesive properties of DOPA-modified gelatin. Several different methods were used to evaluate the adhesive properties of DOPA-gelatin. The lap shear test is the most commonly used experimental procedure to characterize adhesive behavior due to its simplicity. [34、35]A shear stress is applied to the specimens by applying a tensile load in the axial direction against two overlapping substrates. The maximum load and the point of onset of failure (first drop in load) are shown in Figure 2A. Compared to pure gelatin, all DOPA-gelatins showed a significant increase in shear strength. The tensile strength of pure gelatin was 0.47 ± 0.03 MPa, while the other DOPA-gelatin samples were much higher. Specifically, among the various DOPA-gelatins, the Tyr100 sample showed the highest load force of 84.9 ± 12.4 N and tensile stress of 4.25 ± 0.62 MPa. It was shown that the DOPA structure can increase the adhesive properties of gelatin by improving its strength and resistance to tension. Interestingly, with increasing enzyme concentration, the maximum load force and tensile stress decreased. During the enzymatic reaction, tyrosinase was involved not only in the hydroxylation of monophenols but also in the conversion of o-diphenols to o-quinones.

[36] The oxidation of DOPA to DOPA-quinone then resulted in the formation of a covalent bond that contributes to the adhesive's cohesive strength. [37、38] The unoxidized catechol form of DOPA is primarily responsible for adhesion, and catechol oxidation is detrimental to its adhesive capacity because the o-quinone formed is nonadhesive. [39、40] DOPA-quinone could be monitored by UV-Vis with a peak at around 380 nm. With the extension of the reaction time, the absorbance around 380 nm also increased (Figure 1C), which indicated DOPA-quinone formed with more exposure to tyrosinase. For the Tyr500 sample, due to the higher tyrosinase concentration, more DOPA-quinone was formed, which degraded the adhesive strength of DOPA-gelatin. Production with tyrosinase concentrations of 100-200 U / mL was performed to maximize the adhesive properties of the material.

[0029] Burst pressure testing is another way to investigate the ability of hydrogels to withstand pressure, adhere and seal to tissue, and prevent leakage.

[41] The burst pressure of a material can be influenced by two properties: cohesion (the force within the material that resists pressure) and adhesion (attachment to a surface), with the former having a larger contribution.

[42] Moreover, the burst pressure also increased with DOPA content. For the Tyr500 sample, although it had a lower adhesion force than Tyr100 and Tyr200, it showed a maximum burst pressure of 11.9 kPa due to its strong binding force. Too high a binding force may result in a hard material with no significant affinity to the surface, thus reducing the adhesive force.

[43] Without cross-linking, the burst pressure of DOPA-gelatin had similar performance compared to several commercially available surgical sealants.

[44] Based on these results, further experiments were performed using much less enzyme than reported in previous studies, with the highest adhesion properties obtained by tyrosinase concentrations of 100–200 U / mL.

[21] .

[0030] Rheology is also commonly used to characterize the mechanical properties of hydrogels because it is rapid, sensitive, and requires only small amounts of sample.

[45] It is a powerful tool for characterizing the viscoelastic properties of hydrogels.

[46] To further study the mechanical properties of DOPA-gelatin, rheological tests were used. To be considered a hydrogel, a material must fulfill several requirements according to its rheological behavior: the storage modulus (G') must be relatively independent of the frequency of deformation, and G' must be higher than the loss modulus (G").

[47] An amplitude sweep of shear strain from 0.1% to 10% and a frequency sweep from 0.1 rad / s to 10 rad / s verified that all samples exhibited hydrogel-like behavior (Figure 2C). DOPA-gelatin samples exhibited significantly higher storage moduli than pure gelatin. Although the differences between DOPA-gelatin groups were not evident, the storage moduli of Tyr100 and Tyr200 were 964 Pa and 942 Pa, respectively, which were relatively higher than the 718 Pa of Tyr500. The rheological test results revealed that DOPA-gelatin formed an elastic network with desirable mechanical properties. All samples exhibited excellent mechanical properties at 1–100 s. -1The material also exhibits shear thinning properties, showing a decrease in viscosity with increasing log shear rate scale.

[0031] In vitro cytotoxicity and cytocompatibility of DOPA-gelatin To access the cytotoxicity of DOPA-gelatin, human dermal fibroblast (HDF) and human keratinocyte (HaCaT) cells were used because they are closely related to skin wound healing.

[48] Both of them respond to the inflammatory phase in the skin repair / regeneration process. Inflammatory signals activate their proliferation and maturation, which are essential for wound healing.

[49] Fibroblasts have been shown to deposit matrix-associated proteins such as collagen that constitute the basement membrane separating the epidermis from the dermis, while keratinocytes form tight junctions that form a barrier against pathogens and generate hair follicles. [50,51] The results showed that HDF cells proliferated well on the various DOPA-gelatin samples (Figure 3A). All four concentrations of DOPA-gelatin did not show cytotoxicity as cells proliferated for up to 7 days. On day 7, HDF cell density on DOPA-gelatin samples was significantly higher than on pure gelatin, and cells seeded on DOPA-gelatin had abundant cytoplasm with a more clustered morphology. Cell viability in both gelatin and DOPA-gelatin samples remained above 90% and did not show a statistically significant difference between the groups, indicating that there was no cytotoxicity caused by DOPA-gelatin. The proliferation rate of cells on DOPA-gelatin after 7 days was approximately 13 times that of the original cell concentration, which was consistent with cells seeded on pure gelatin (Figure 1B). The results support the conclusion that the conversion of phenol to a catechol group on tyrosine results in greater fibroblast proliferation that may play a role in dermal restoration. [52、53] .

[0032] Furthermore, both gelatin and DOPA-gelatin samples showed beneficial effects on HaCaT cells, which can be found in the epidermis of the skin (Figure 3C). However, by day 3, the differences between the images showed that the cell density was dependent on the tyrosinase concentration. Higher tyrosinase concentrations resulted in materials achieving higher HaCaT cell densities. As shown in Figure 3D, the proliferation rate after 3 days increased from 930% to 2612% as the tyrosinase concentration increased from 0 to 200 U / mL. After 7 days, the cell viabilities of the DOPA-gelatin samples were all above 90%, and the proliferation rate reached over 3000%, much higher than that of HDF cells.

[0033] According to the experiments carried out herein, DOPA-gelatin has high cytocompatibility and supports both HaCaT and HDF cells in wound healing. While typical sealants may block exposure of the wound site to external pathogens while cells slowly migrate and proliferate, DOPA-gelatin provides bioactive cues that clearly increase the proliferation of two different cell types and accelerate the healing of two skin components.

[0034] Effect of DOPA-gelatin on cell migration in vitro To evaluate cell migration, a scratch wound assay was performed. This assay involves creating a gap in a confluent cell monolayer to mimic a wound and monitor the subsequent cell movement. HDF cells showed faster migration on DOPA-gelatin samples than on uncoated or pure gelatin-coated plates (Figure 4A). At 24 h, the wound contraction rates in Tyr100 and Tyr200 samples were almost identical, reaching over 90%, which was significantly higher than the other groups with rates of about 60%. Most notably, Tyr100 facilitated a wound closure rate of 97% (Figure 4B). It indicated that DOPA-gelatin could facilitate HDF migration and promote the deposition of extracellular matrix (ECM) components to form the dermal layer. HaCaT cells showed similar migration behavior (Figure 4C, 4D). Tyr100 and Tyr200 samples had the maximum migration speed at 6 h, while the other groups were relatively low. However, after 24 hours, unlike HDF, the wound contraction rates between the groups showed no difference, all exceeding 95%.These results showed that both keratinocytes and fibroblasts migrated well on DOPA-gelatin compared to unmodified gelatin and the control group, especially for the Tyr100 and Tyr200 samples.

[0035] Cell migration is essential for wound healing and tissue remodeling. During wound healing, keratinocytes migrate from the basal population around the wound edge to cover the lesion and restore the skin barrier function.

[55] Dermal fibroblasts can also migrate to the wound site, where they synthesize the provisional ECM required for cutaneous wound contraction. [56,57] DOPA-gelatin was demonstrated to be a good candidate for its potential healing effects in epidermal and dermal layer healing as well as in vivo wound closure experiments.

[0036] Quantitative real-time polymerase chain reaction (RT-PCR) assay Apart from cell migration, gene expression is constantly changing to coordinate the repair response to prevent lasting damage to the wound site. Wound healing involves a variety of processes, interactions between cells and their surrounding microenvironment, including cell migration, proliferation, differentiation, angiogenesis, epithelialization, matrix deposition, and remodeling.

[58] Therefore, we analyzed the gene expression that contributes to these processes. The results showed that both vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) had the highest expression levels in the DOPA-gelatin group. HDFs showed more expression of MMP2 on DOPA-gelatin compared with other groups (Figure 4E). It is well known that VEGF stimulates wound healing through multiple mechanisms, including collagen deposition, angiogenesis, and epithelialization.

[59] Another well-characterized growth factor, EGF, is synthesized by keratinocytes that can stimulate re-epithelialization and increase the tensile strength of skin incisions. [60、61] In addition, vimentin directly regulates fibroblast proliferation, collagen accumulation, keratinocyte transdifferentiation, and re-epithelialization in wound healing. It is an intermediate filament involved in cell anchoring as well as epithelial-mesenchymal migration processes. Loss of vimentin is also known to contribute to severe deficiencies in fibroblast proliferation.

[62] Matrix metalloproteinase 2 (MMP2) has been reported to be involved in interstitial remodeling and basement membrane remodeling. [63、64] During the anagen phase of hair growth, mature melanocytes synthesize melanin from tyrosine, which is regulated by tyrosinase-related protein 1 (TRP1).

[65] The results showed that the DOPA-gelatin group had the highest expression of TRP1, which could promote hair growth during wound healing. In summary, DOPA-gelatin was proven to promote many important processes in wound healing, demonstrating its potential for biological activation.

[0037] Angiogenesis assay Based on gene expression analysis, the effect of DOPA-gelatin on angiogenesis was also investigated. The effect of DOPA-gelatin on tube formation was directly monitored by adding gelatin or DOPA-gelatin samples to dishes of human umbilical vein endothelial cells (HUVECs). In HUVEC growth medium, angiogenesis was observed as early as 4 hours after seeding and confirmed at 6 hours (Figure 9). Specifically, tube formation in the DOPA-gelatin group appeared 2 hours earlier than the control and gelatin groups. Furthermore, quantification showed that the DOPA-gelatin group had higher results in both branch points and number of tubes (Figure 10). At 6 hours, the number of tubes in the DOPA-gelatin group was about 1.7 times that of the other two groups. These results indicate that DOPA modification affects the bioactive properties of vascular endothelial cells and aids in the re-endothelialization of blood vessels, thus promoting wound healing.

[68] Alternatively, the indirect method involves the application of conditioned medium collected from dishes of HDF and HaCaT cells to culture HUVECs. Using this method, similar trends were observed for both the HDF and HaCaT groups. At the 6-h time point, DOPA-gelatin showed significantly better results for each parameter compared to the control and gelatin groups (Figure 5). Although the differences between the control and gelatin groups were not dramatic, they corresponded to VEGF expression in these two cell incubation systems. As shown in Figure 4E, both HDF and HaCaT cells showed the greatest relative expression of VEGF in the DOPA-gelatin group, while the gelatin group was close to that seen in the control group. It was proven that the increased secretion of VEGF induced by DOPA facilitates angiogenesis.

[69] Compared with the control group, VEGF expression was greater in HaCaT than in HDF. The results shown in Figures 5B and 5D show that at 6 hours, the number of vessels was greater in the DOPA-gelatin group cultured with HaCaT culture supernatant.

[0038] The remodeling and establishment of new blood vessels is one of the key factors in wound healing, as blood vessels supply nutrients and oxygen to cells at the wound site, and these angiogenic activities proceed simultaneously during all stages of the repair process. [66、67] Both the tube formation images and quantification results here demonstrate the superiority of DOPA-gelatin in the angiogenesis process. This material can accelerate angiogenesis and shorten the overall wound healing period.

[0039] in vivo studies In vivo degradation test To assess in vivo degradation, pure gelatin and DOPA-gelatin, both labeled with fluorescein isothiocyanate (FITC), were implanted into the dorsal subcutaneous tissue of mice and monitored over a 14-day period. As shown in the fluorescence images in Figure 11, both gelatin and DOPA-gelatin remained subcutaneous for 14 days. However, over time, the fluorescence intensity decreased due to degradation. At day 14, hematoxylin and eosin (H&E) staining showed that both gelatin and DOPA-gelatin implants caused minor inflammation during degradation, but there was no sign of infection, specific cellular infiltration (neutrophils and lymphocytes), or progression to chronic inflammation.

[0040] In vivo skin wound healing research Full-thickness wounds were created on the dorsal skin of mice. Pure gelatin and DOPA-gelatin were applied to the wounds, and the macroscopic morphology was evaluated on days 0, 7, and 14 (Figure 6A). On day 14, the injury group showed a central scab on the back, whereas most of the wounds had healed in the DOPA-gelatin group. Notably, hair regrowth was not observed around the wound in the injury group, whereas all treatment groups (gelatin, DOPA-gelatin) showed hair regrowth around the wound area. Interestingly, the DOPA-gelatin group was found to increase hair regrowth more than the other groups. Quantitative data of total wound contraction in Figure 6B showed that the injury group had a maximum wound area of ​​71.79 ± 12.25% on day 7. Among the treatment groups, the DOPA-gelatin group showed the fastest wound contraction rate, with a wound area of ​​41.69 ± 11.78%.

[0041] Histological analysis We also utilized histological studies to further investigate the efficacy of DOPA-gelatin on the skin wound healing process. Keratinocyte proliferation and migration are key features of re-epithelialization during wound healing.

[54] Epithelialization with the same structure in vivo is accompanied by angiogenesis, collagen deposition, and granulation tissue formation, which greatly promote tissue growth and healing. H&E staining at 7 days after injury (Figure 6C) shows that re-epithelialization was more prominent in wounds treated with DOPA-gelatin compared to the open injury and gelatin groups. The re-epithelialization rates in the control, gelatin, and DOPA-gelatin groups were 25.6±7.2%, 41.0±5.9%, and 55.4±13.7%, respectively (Figure 6D). At 14 days, all groups had healed wounds, while the DOPA-gelatin group also increased hair growth. Masson's trichrome staining of sections at 14 days after injury showed enhanced collagen deposition in DOPA-gelatin-treated mice, revealing higher levels of collagen maturation compared to controls. This is speculated to be a result of greater fibroblast infiltration and proliferation in the DOPA-gelatin treated group.

[0042] Histological studies revealed faster wound healing in wound sites treated with DOPA-gelatin in terms of increased wound contraction, enhanced collagen synthesis, more hair follicles, and higher re-epithelialization. These results support the ability of DOPA-gelatin therapeutic material to be used as a skin-healing functional material.

[0043] conclusion DOPA-gelatin hydrogels conceived from mussels are synthesized by a tyrosinase-catalyzed one-step reaction. The desired mechanical strength and adhesive power of DOPA-gelatin form the basis of its application in skin wound healing. In vitro studies proved that DOPA-gelatin possessed the desired biocompatibility and enhanced regenerative activities such as cell proliferation, migration, angiogenesis, and upregulation of wound healing-related genes. In vivo experiments proved that DOPA-gelatin could facilitate more rapid skin healing. These findings characterize the role of DOPA-gelatin conceived from mussels in promoting the repair process of skin wounds. The remarkable functional DOPA-gelatin hydrogels should be further explored and studied in other tissues as a mechanism to promote tissue repair and regeneration.

[0044] Experimental Section Enzyme-mediated synthesis of DOPA-gelatin: A 10% (w / w) gelatin solution (Type A, G1890, Sigma, CA, USA) was prepared by dissolving 1 g of gelatin in 10 g of Milli-Q® water at 80° C. for 1 h. A stock solution of tyrosinase (10 U / μL) was made by adding 50 kU tyrosinase powder (T3824, Sigma, MO, USA) to 5 mL Dulbecco's phosphate buffered saline (DPBS, pH 6.5, Gibco, CA, USA) at room temperature. Amounts of 0, 5, 10, 20, and 50 μL of tyrosinase stock solution were added to 1 mL of gelatin solution, respectively, to make gelatin solutions with various tyrosinase concentrations (0, 50, 100, 200, 500 U / mL). The corresponding samples were named Tyr0, Tyr50, Tyr100, Tyr200, Tyr500. Reactions were carried out at 37°C in an Eppendorf ThermoMixer® C (Eppendorf, NY, USA) with a shaking frequency of 2000 rpm. After the specified incubation time, the temperature was increased to 65°C for 1 h to inactivate the enzyme. The DOPA-gelatin solution was used immediately or stored at -80°C for later use.

[0045] UV-Visible Spectroscopy: The gelatin solution catalyzed by tyrosinase was monitored by a spectrophotometer DeNovix® DS11-FX (DeNovix, Delaware, USA). Two microliters of the reaction mixture was collected and scanned at wavelengths from 220 nm to 500 nm. DOPA content was analyzed at a wavelength of 280 nm.

[21] .

[0046] FTIR analysis: Samples of Tyr0 to Tyr500 and pure L-dopamine were analyzed by Fourier transform infrared spectroscopy (JASCO, FT / IR-420, Maryland, USA) at 400–4000 cm -1 Over a range of 1 cm -1 The samples were characterized by 128 scans at 100 nm resolution. All samples were freeze-dried and ground to a fine powder with a mortar and pestle. Potassium bromide (KBr) pellets were prepared at 1% sample weight content.

[0047] Quantification of DOPA content: The Arnow method was used to determine the content of DOPA and its further oxidized derivatives.

[31] Three reagents were prepared to quantify the DOPA content: Reagent A: 0.5M HCl solution, Reagent B: Nitrite-molybdate solution (10 g NaNO 2 and 10 g Na 2 MoO 4 Reagent A: 1M NaOH solution (4 g sodium hydroxide dissolved in 100 mL water), and Reagent B: 1M NaOH solution (4 g sodium hydroxide dissolved in 100 mL water). Pure DOPA was used to make standard solutions with various concentrations of 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL and 0.1 mg / mL. 1 mL of water was used as a blank control. One milliliter of each standard solution and each measurement sample was placed in a tube, followed by the addition of 1 mL of Reagent A, followed by vortexing. Reagents B and C (1 mL each) were then added in rapid succession at room temperature, and each tube was mixed briefly on a vortexer. Each sample was immediately analyzed with a spectrophotometer (DeNovix® DS11-FX) to characterize the absorbance at 520 nm.

[0048] Lap shear and burst pressure tests: Samples were strained to failure in lap shear using an Instron® 5943 mechanical testing machine (Massachusetts, USA) equipped with a 100-N load cell at a crosshead speed of 1 mm / min. 20 μL of sample was applied to a 10 mm × 20 mm area of ​​one glass slide, after which another glass slide was placed on top of this area and then left at 4° C. for 1 h. Each sample was tested at least three times. To investigate the burst pressure of DOPA-modified gelatin, the sealing ability was measured according to the modified ASTM standard F2392-04 for burst pressure as previously described. [70、71]Briefly, a circular collagen sheet with a diameter of 30 mm was immersed in DPBS before sample preparation. A circular defect area with a diameter of 3 mm was punched in the center of the collagen sheet. 20 μL of sample was pipetted onto the defect area and placed at 4° C. for 5 min before testing. The collagen sheet with the sample was then fixed in the center of two stainless steel rings using a custom-made burst pressure device in which the upper ring contained a hole with a diameter of 10 mm. Air was then applied to the system at a rate of 20 mm / min by a syringe (50 mL) pump. The maximum burst pressure was recorded (n≧3) by SPARKvue™ (PASCO Scientific, CA, USA) software.

[0049] Rheological analysis: The rheological properties of DOPA-gelatin hydrogels were evaluated by a rheometer (AR-G2, TA instruments protocol). Storage modulus, loss modulus and viscosity were measured using a parallel stainless metal plate configuration with a diameter of 25 mm. All samples were equilibrated at 37°C for 1 hour before testing. Mineral oil was added around the plate after loading the sample to prevent water evaporation. Storage modulus, loss modulus and viscosity were recorded by Anton Paar Rheocompass™ software.

[0050] Live / dead assay: HDF cells and HaCaT cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, CA, USA) and 1% penicillin / streptomycin (Gibco, CA, USA) in a humidified incubator (37 °C, 5% CO 2 ). Cell viability was assessed using a Live / Dead Viability Kit (LIVE / DEAD™ Viability / Cytotoxicity Kit, Invitrogen, USA). The samples were coated on the bottom of the chamber. Cells (10 per chamber) were then plated on the bottom of the chamber. 51000 cells (passage 4) were seeded on the coated samples and incubated at 37°C for 1, 3, and 7 days. Stained cells were imaged by fluorescence microscopy (Zeiss Axio Observer; Carl Zeiss, Jena, Germany). For each time point, samples were analyzed in triplicate. The number of live and dead cells was counted using Image J software (NIH, MD, USA). Cell viability (%) was expressed as the ratio of live cells to the total cell number at the mean ± SD (standard deviation). Proliferation (%) was calculated by the ratio of cell concentration at 1, 3, and 7 days to the initial seeding concentration at the mean ± SD.

[0051] Cell migration assay: Samples were uniformly coated on a 150 mm diameter Petri dish and incubated for 10 6 HDF cells and HaCaT cells were seeded and subsequently incubated (37°C, 5% CO 2 ). When cells grew to full confluence on the dish, they were scratched with a scratch tip. After scratching, the dish was gently washed to remove detached cells. The medium was then supplemented with fresh medium without serum to inhibit cell proliferation. The dishes were placed in an incubator for 0, 6, 12 and 24 h. Cells were imaged using an inverted microscope (Zeiss Axio Observer; Carl Zeiss, Jena, Germany) before harvesting for RT-PCR gene expression analysis. Wound contraction was defined as Equation 1:

[0052]

number

[0053] In the formula, A 0 is the area of ​​the wound measured immediately after scratching (0 h), and A t is the area of ​​the wound measured at time t (t = 6, 12, 24 h) after scratching. The wound area was calculated by manually tracing the acellular areas in the images and counted by Image J software (NIH, MD, USA).

[0054] RT-PCR assay: Total RNA was isolated from HDF and HaCaT cells using Qiazol lysis reagent (Qiagen, CA, USA) according to the manufacturer's instructions. One microgram of total RNA was transcribed into cDNA using QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR (initial denaturation at 95°C for 5 min, 45 cycles of denaturation at 95°C for 5 s and amplification at 60°C for 10 s) was performed using the Rotor-Gene SYBR Green PCR Kit (Qiagen).

[0055] Angiogenesis assay: In the direct method, 250 μL of Matrigel (Corning Inc, NY, USA) was placed into each well (24-well plate). The plate was then incubated in a humid chamber for 30 min to allow the gel structure to form. HUVECs (passages 4–6) were seeded (approximately 1.5 × 10 4 Cells / well). Each condition was supplemented with 100 μL of conditioned medium (Promocell, Heidelberg, Germany) containing 60 μL of gelatin or DOPA-gelatin. The assay was performed for 6 h in a humidified chamber. HUVEC angiogenesis was imaged by an inverted fluorescent microscope (Zeiss Axio Observer; Carl Zeiss, Jena, Germany). In the indirect method, 250 μL of Matrigel and 100 μL of cell culture supernatant + HUVEC cells were added to the wells. Other conditions were the same as in the direct method.

[0056] In vivo biodegradation and wound healing studies: All animal experiments were approved by the UCLA Animal Research Committee. Animal experiments were performed in accordance with the relevant guidelines. Seven-week-old male mice weighing approximately 20 grams were purchased from Jackson Laboratory (Sacramento, CA), fed, and housed in clean cages maintained at 25°C. At the start of the experiment, mice were pretreated with isoflurane (100% O 2The animals were anesthetized by inhalation of 1.5% in 100 mL of ethanol. Anesthesia was maintained throughout the survival surgery. The dorsal skin was shaved and washed with iodophor (0.2% w / v). The dorsal skin was then surgically excised to create a full-thickness circular skin defect wound area (approximately 1 cm diameter). Three groups were prepared, including no treatment (injury), pure gelatin (gelatin), and Tyr100 DOPA-modified gelatin (DOPA-gelatin). Each wound in the treatment group was covered evenly with 200 μL of the corresponding sample. Wound healing was assessed by measuring the wound area size by digital caliper and taking pictures on specific days (days 0, 7, and 14). The wound area was calculated according to formula 2:

[0057]

number

[0058] In the formula, A 0 and A t are the wound areas on day 0 and day t, respectively. For the degradability test, pure gelatin and DOPA-gelatin labeled with fluorescein isothiocyanate (FTIC, Sigma) were applied to the wound site for 0, 7, and 14 days, and the fluorescent images were taken by a fluorescence microscope (Zeiss Axio Observer; Carl Zeiss, Jena, Germany).

[0059] Histological analysis: CO on specific days (days 0, 7, and 14) 2 The mice were sacrificed using a 10% neutral buffered formalin (Leica The tissues were fixed in 100% ethanol (DeepL Biosystems, IL, USA). Fixed tissues were processed and embedded in paraffin blocks using standard methods. Blocks were cut at 4 μm thickness, and sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome (MT) stains. Histological images were acquired with a Nikon inverted microscope.

[0060] Statistical analysis: Data are presented as mean ± standard deviation (SD). All statistical analyses and graphs were performed by SPSS Statistics software (IBM, IL, USA) and GraphPad Prism 8.0 (GraphPad Software, CA, USA). Multiple comparisons were analyzed using one-way ANOVA with Tukey post-hoc test for data sets with 3 or more groups. P<0.05 was considered significant.

[0061] While embodiments of the present invention have been shown and described, various modifications can be made without departing from the scope of the invention. Accordingly, the invention should not be limited, except in the following claims and their equivalents.

[0062] References

[0063] [Table 2-1]

[0064] [Table 2-2]

[0065] [Table 2-3]

[0066] [Table 2-4]

[0067] All publications mentioned herein (eg, the references listed above as numbers) are incorporated herein by reference to disclose and describe the aspects, methods, and / or materials in connection with which the publications are cited.

Claims

1. A method for making 3,4-dihydroxyphenylalanine (DOPA)-gelatin, comprising: Providing a solution containing porcine gelatin; and incubating a solution containing said porcine gelatin with tyrosinase to produce 3,4-dihydroxyphenylalanine (DOPA)-gelatin.

2. A method of incubating a solution containing said porcine gelatin with tyrosinase to form 3,4-dihydroxyphenylalanine (DOPA)-gelatin, comprising the steps of: The 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a shear strength of at least 2 MPa; said 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibiting a burst pressure of at least 6 kPa; The 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a loading force of at least 60 N; and 2. The method of claim 1, wherein the 3,4-dihydroxyphenylalanine (DOPA)-gelatin is selected to exhibit a tensile stress of at least 3 MPa.

3. 3. The method of making (DOPA)-gelatin according to claim 2, wherein the solution containing the porcine gelatin is incubated with tyrosinase for more than 30 minutes or more than 60 minutes.

4. A method for producing (DOPA)-gelatin according to any one of claims 1 to 3, wherein the concentration of tyrosinase is 100-200 U / mL.

5. 2. The method of making the (DOPA)-gelatin of claim 1, further comprising heating said solution to inactivate said tyrosinase.

6. 2. The method of making 3,4-dihydroxyphenylalanine (DOPA)-gelatin according to claim 1, wherein the (DOPA)-gelatin is made in a one-step synthesis reaction.

7. 2. A 3,4-dihydroxyphenylalanine (DOPA)-gelatin composition made by the method of claim 1.

8. A therapeutic composition comprising porcine gelatin in which substantially all tyrosine residues have been converted to 3,4-dihydroxyphenylalanine (DOPA).

9. The therapeutic composition of claim 8 , wherein the composition is substantially free of metal ions.

10. The therapeutic composition of claim 8, wherein the composition is sterile and comprises a pharma- ceutically acceptable carrier.

11. 9. The therapeutic composition of claim 8, wherein the 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a shear strength of at least 2 MPa.

12. 9. The therapeutic composition of claim 8, wherein the 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a burst pressure of at least 6 kPa.

13. 9. The therapeutic composition of claim 8, wherein the 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a loading force of at least 60N.

14. 9. The therapeutic composition of claim 8, wherein the 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a tensile stress of at least 3 MPa.

15. 9. The therapeutic composition of claim 8, wherein the 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a storage modulus of at least 700 Pa.

16. 10. The therapeutic composition of claim 8, further comprising at least one additional therapeutic agent selected from an antibiotic, an anti-inflammatory agent, a hemostatic agent, an embolic agent, and a chemotherapeutic agent.

17. At least 90% of the tyrosine residues of the porcine gelatin are converted to 3,4-dihydroxyphenylalanine (DOPA); The composition is sterile and comprises a pharma- ceutically acceptable carrier; The 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a shear strength of at least 2 MPa; said 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibiting a burst pressure of at least 6 kPa; The 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a loading force of at least 60 N; and 9. The therapeutic composition of claim 8, wherein the 3,4-dihydroxyphenylalanine (DOPA)-gelatin exhibits a tensile stress of at least 3 MPa.

18. 10. A method of delivering the composition of claim 8 to a preselected site, comprising: disposing the composition within a tube having a first end and a second end including an opening; applying a force to the second end of the tube, the force being sufficient to displace the composition from the tube through the opening; and delivering said composition from said tube through said opening to said preselected site.

19. The method of claim 18, wherein the site is an in vivo site.

20. The method of claim 1 , wherein the site is an in vivo location where an individual experienced trauma or injury.

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