A protein-based co-crystal gel and its preparation method and application

Abstract

The application relates to the field of biological medicine, and discloses a protein-based co-crystal gel as well as a preparation method and application thereof. The protein-based co-crystal gel comprises gelatin, an antibacterial component and a deep eutectic solvent, wherein the components of the Gel-COS-Ed co-crystal gel comprise gelatin, chitosan oligosaccharide, emodin, glycerol and betaine. After the gelatin and the antibacterial component are heated and dissolved, the gelatin is coagulated into a gelatin gel; the gelatin gel is placed in the deep eutectic solvent to obtain the protein-based co-crystal gel. The tensile strength of the obtained Gel-COS-Ed co-crystal gel is 1.01 MPa, the breaking strain can reach 510%, there is no obvious phase change in the temperature range of-80 DEG C to 80 DEG C, the antibacterial rate on staphylococcus aureus reaches 95.7%, and a new solution is provided for the application of wound dressings and biological medical materials.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to a protein-based cocrystal gel, its preparation method, and its applications. Background Technology

[0002] Skin injuries are common clinical conditions, usually caused by acute trauma such as cuts and burns. Hydrogels, due to their excellent biocompatibility, flexibility, and permeability, serve as an effective moisturizing physical barrier, promoting wound healing. Therefore, as a novel wound care material, hydrogels have become a focus of extensive research.

[0003] Hydrogels contain a large amount of water and possess properties similar to biological tissues. However, when used as wound dressings, hydrogels are structurally unstable under normal conditions, and water evaporation leads to gel drying, thus affecting wound healing. To overcome this problem, some studies have employed methods such as increasing the frequency of dressing changes or sealing the hydrogel surface with a thin film. However, increasing the frequency of changes not only increases costs but also labor intensity; while sealing the hydrogel surface can prevent water loss, it reduces gas permeability, which may also inhibit wound healing. Therefore, exploring innovative dressing materials that combine the excellent biocompatibility, wettability, and flexibility of hydrogels with enhanced stability remains a challenge.

[0004] Eutectic gels are prepared by replacing volatile water molecules with non-volatile deep eutectic solvents. Deep eutectic solvents possess excellent biocompatibility, low volatility, and biodegradability. They contain both hydrogen bond acceptors and donors, allowing the resulting eutectic gels to maintain not only hydrogel-like biocompatibility and flexibility but also thermal stability. As a novel gel material, deep eutectic gels have attracted attention in the field of flexible electronics due to their inherent conductivity, but research on their application in wound dressings remains limited.

[0005] Unlike synthetic polymers, naturally derived animal and plant molecules typically exhibit higher biocompatibility. Cocrystal gels prepared by combining biomolecules with deep eutectic solvents can avoid biosafety-related issues. Gelatin, derived from the hydrolysis of animal collagen, possesses excellent biocompatibility and biodegradability, making it an ideal material for tissue engineering scaffolds, drug delivery systems, and wound dressings. Furthermore, due to the disruption of the physiological barrier after skin injury, necrotic tissue at the wound site is prone to bacterial growth and infection. Therefore, as a wound dressing, it not only needs to be biocompatible but also possess excellent antibacterial properties to accelerate wound healing. Gelatin molecules dissolve at high temperatures and form a gel at low temperatures through hydrogen bonding. However, this weak hydrogen bonding makes gelatin hydrogels very fragile and brittle. Moreover, gelatin hydrogels undergo a gel-sol transition near body temperature, changing from a solid colloid to a liquid state, which hinders their use in human applications. To enhance the mechanical properties and stability of gelatin-based hydrogels, researchers have explored various methods, such as producing methacrylated gelatin through methacrylation or using crosslinking agents for chemical crosslinking. However, these methods either significantly increase production costs or alter the properties of the gelatin hydrogel, affecting its degradability and bioactivity.

[0006] Chitosan oligosaccharides are obtained by degrading chitosan into low molecular weight oligosaccharides. They have superior hydrophilicity and high bioavailability and bioactivity compared to chitosan. Emodin is a natural anthraquinone compound extracted from traditional Chinese medicines such as rhubarb. Its poor solubility in water poses challenges to its dispersion in hydrogel networks and the development of high-performance wound dressings, thus limiting its application.

[0007] Existing technology CN202110689116.0 discloses a flexible conductive biopolymer material, which improves the flexibility and ductility of the material and imparts conductivity by plasticizing the biopolymer with a deep eutectic solvent, and is mainly used in flexible electronic devices. However, this prior art does not address antibacterial properties or applications in the biomedical field, especially in wound treatment. Summary of the Invention

[0008] To overcome the defects of traditional gelatin hydrogels described in the prior art, such as fragility, structural instability, insufficient antibacterial properties, and poor solubility of emodin, this invention provides a protein-based cocrystallized gel.

[0009] Another object of the present invention is to provide a method for preparing a protein-based cocrystal gel;

[0010] Another object of the present invention is to provide an application of protein-based cocrystallized gels.

[0011] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0012] A protein-based co-crystal gel comprising gelatin, chitosan oligosaccharide, emodin and deep co-crystal solvent.

[0013] Furthermore, the mass ratio of chitosan oligosaccharide to emodin is 1:0.0075 to 0.0170.

[0014] Preferably, the mass ratio of chitosan oligosaccharide to emodin is 1:0.01.

[0015] Furthermore, in the protein-based cocrystallized gel, the mass fraction of emodin is 0.0027% to 0.0088%.

[0016] Preferably, the mass fraction of emodin is 0.0045%.

[0017] Furthermore, the deep eutectic solvent is composed of polyols and quaternary ammonium salts; the mass fraction of the quaternary ammonium salt is no more than 20%.

[0018] Preferably, the polyols include glycerol and ethylene glycol; the quaternary ammonium salts include betaine and choline chloride.

[0019] A method for preparing the protein-based cocrystal gel includes the following steps: dissolving gelatin, chitosan oligosaccharide and emodin in water and then solidifying them into a gelatin gel; placing the gelatin gel in a deep cocrystal solvent to obtain a protein-based cocrystal gel.

[0020] Preferably, after dissolving in water, the mass concentration of gelatin is 8.5-10%, and the mass concentration of chitosan oligosaccharide is 4-5%.

[0021] Preferably, the gelatin and antibacterial components are dissolved at 40–80°C.

[0022] Preferably, the gelatin and antibacterial components are dissolved at 60°C.

[0023] Upon heating, the Schiff base reaction between chitosan oligosaccharide and emodin promotes the dissolution of emodin, forming a homogeneous solution.

[0024] An application of the protein-based cocrystal gel to prepare dressings for wounds.

[0025] Preferably, the trauma is a bacterial infection wound.

[0026] Preferably, the dressing is an antibacterial dressing.

[0027] Furthermore, dressings that promote wound healing are prepared.

[0028] Furthermore, dressings that promote angiogenesis and collagen regeneration are prepared.

[0029] Furthermore, dressings that promote cell proliferation and migration were prepared.

[0030] This study marks the first time antibacterial properties have been introduced into a cocrystal gel system. Through the synergistic effect of chitosan oligosaccharide, emodin, and betaine, the cocrystal gel exhibits excellent antibacterial properties, effectively preventing wound infection and making it suitable for wound dressings. This not only expands the application range of cocrystal gels but also meets the needs of the biomedical field for antibacterial properties, biocompatibility, and wound healing promotion.

[0031] By using the innovative deep eutectic solvent ratio of this invention, the mechanical properties of the eutectic gel are not reduced by excessive quaternary ammonium salts, and the balance between tensile and antibacterial properties is maintained. This ensures that the gel has good extensibility while being less prone to deformation or breakage, making the eutectic gel of this application more suitable for applications requiring a certain strength and flexibility, such as medical dressings and skin tissue repair.

[0032] This invention is the first to discover and utilize the Schiff base reaction between chitosan oligosaccharide and emodin to improve the solubility of emodin, thereby enabling emodin to be uniformly embedded in the gel network, enhancing its antibacterial activity, and avoiding precipitation caused by insolubility. This invention not only provides a formulation for a co-crystal gel but also offers a novel method for improving the dispersibility of poorly soluble drugs based on chemical reactions, which has broad application value in biopharmaceutical materials and drug delivery systems.

[0033] Compared with the prior art, the beneficial effects of the technical solution of the present invention are:

[0034] The addition of the antibacterial components COS and emodin in this invention has almost no effect on the network strength of the gelatin-based gel. The resulting Gel-COS-Ed eutectic gel has a tensile strength of 1.01 MPa and excellent ductility (fracture strain up to 510%), ensuring structural integrity when used as a dressing. Furthermore, the gel network is entirely composed of reversible physical interactions (such as hydrogen bonds, ionic interactions, and molecular chain entanglement), exhibiting excellent self-healing capabilities. Compared to hydrogels, the non-volatility and low-temperature resistance of the deep eutectic solvent ensure its stability and low-temperature tolerance during long-term use. The Gel-COS-Ed eutectic gel exhibits no significant phase transition in the temperature range of -80°C to 80°C, providing a material basis for wound dressings in complex environments (such as cold or high-temperature conditions). As a wound dressing, the Gel-COS-Ed cocrystal gel not only exhibits excellent biocompatibility but also demonstrates multiple antibacterial activities due to the antibacterial properties of its network components, betaine, COS, and emodin, achieving a 95.7% inhibition rate against Staphylococcus aureus. This cocrystal gel dressing effectively accelerates wound healing through its antibacterial effects and by promoting angiogenesis and collagen regeneration. Particularly in the early stages of treatment, it exhibits accelerated healing efficiency, with healing rates of 65.6% and 89.1% on days 4 and 7, respectively, compared to 166.9% and 136.9% of the control group's healing rates. Attached Figure Description

[0035] Figure 1 (a) Schematic diagram of the preparation process of Gel-COS-Ed eutectic gel; (b) Schematic diagram of the function of eutectic gel dressing.

[0036] Figure 2 (a) FTIR spectra of Gel eutectic gel, Gel-COS eutectic gel and Gel-COS-Ed eutectic gel, (b) XPS spectrum of Gel-COS-Ed eutectic gel, (c) C1s spectrum of Gel-COS eutectic gel, (d) C1s spectrum of Gel-COS-Ed eutectic gel, (e) XRD patterns of Gel eutectic gel, Gel-COS eutectic gel and Gel-COS-Ed eutectic gel, (f) DSC curves of Gel-COS-Ed eutectic gel and Gel-COS-Ed hydrogel before and after solvent exchange;

[0037] Figure 3 (a) Tensile properties of Gel eutectic gel, Gel-COS eutectic gel and Gel-COS-Ed eutectic gel, and corresponding (b) tensile strength and fracture strain, (c) elastic modulus;

[0038] Figure 4(a) Hysteresis loop of Gel-COS-Ed eutectic gel as strain increases from 100% to 500%, and the corresponding (b) dissipated energy and total energy; (c) Cyclic loading and unloading curves of Gel-COS-Ed eutectic gel at 250% strain after waiting times of 1 minute, 5 minutes and 30 minutes; (d) Recovery efficiency at different waiting times; (e) 100-cycle tensile test of Gel-COS-Ed eutectic gel at 250% strain, and the corresponding (f) stress ratio.

[0039] Figure 5 (a) Live and dead cell staining results of different groups of L929 cells at 24 and 48 hours; cell viability of L929 cells at (b) 24 and (c) 48 hours.

[0040] Figure 6 (a) Cell migration of L929 cells in different groups at 0, 12 and 24 hours; scratch healing rate of L929 cells in different groups at (b) 12 hours and (c) 24 hours.

[0041] Figure 7 (a) Antibacterial activity of the three gels against Staphylococcus aureus and SEM images, (b) Antibacterial activity of the three gels against Escherichia coli and SEM images, (c) Survival rate of Staphylococcus aureus, (d) Survival rate of Escherichia coli.

[0042] Figure 8 (a) Infected mouse model with complete skin defect; (b) Wound healing status of mice in the control group, Gel cocrystal gel group, Gel-COS cocrystal gel group and Gel-COS-Ed cocrystal gel group (observed on days 0, 4, 7, 10 and 13, respectively); (c) Wound healing rate on days 4, 7, 10 and 13; (d) HE staining results of different groups of mice on days 4, 7 and 13 after modeling.

[0043] Figure 9 (a) Masson staining results of wounds in different groups on days 4, 7 and 13; (b) Expression of endothelial-specific marker (CD31) and vascular smooth muscle cell marker (α-SMA) detected by immunofluorescence staining on day 13; (c) Quantitative analysis of collagen deposition level; (d) Quantitative analysis of CD31 relative vascular density.

[0044] Figure 10 Live and dead cell staining results of different groups of L929 cells at 24 hours and 48 hours. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0046] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0047] Gelatin, chitosan oligosaccharide, emodin, betaine, and glycerol were all purchased from Aladdin (Shanghai, China). Deionized water was used as the solvent in all experiments. Cell culture media (DuPont modified Eagle Medium, DMEM) and fetal bovine serum (FBS) were purchased from Gibco. The cell counting kit (CCK-8) was provided by MedChemExpress (New Jersey, USA). The live / dead cell staining kit (Calcein / PI Viability / Cytotoxicity Assay Kit) was purchased from Beckman Coulter (Shanghai, China). C57BL / 6J mice were purchased from GemPharmatech Ltd. (Jiangsu, China). All reagents were used as is.

[0048] Example 1

[0049] 1g of gelatin, 0.5g of chitosan oligosaccharide, and 5mg of emodin were dissolved in 10g of deionized water and stirred continuously at 60℃ to form a transparent solution. After defoaming by sonication, the solution was poured into a PMMA mold and refrigerated at 4℃ for 10 minutes to form a brittle gel network. The gel network was removed from the mold and immersed in a deep eutectic solvent (DES) consisting of 20g of betaine and 80g of glycerol for 4 hours for solvent displacement. The resulting eutectic gel was named Gel-COS-Ed eutectic gel.

[0050] Example 2

[0051] 1g of gelatin, 0.6g of chitosan oligosaccharide, and 9mg of emodin were dissolved in 10g of deionized water and stirred continuously at 40°C to form a transparent solution. After defoaming by sonication, the solution was poured into a PMMA mold and refrigerated at 4°C for 10 minutes to form a brittle gel network. The gel network was removed from the mold and immersed in a deep eutectic solvent (DES) consisting of 20g of betaine and 80g of glycerol for solvent displacement over 4 hours. The resulting eutectic gel was named Gel-COS-Ed eutectic gel.

[0052] Example 3

[0053] 1g of gelatin, 0.4g of chitosan oligosaccharide, and 3mg of emodin were dissolved in 10g of deionized water and stirred continuously at 80℃ to form a transparent solution. After defoaming by sonication, the solution was poured into a PMMA mold and refrigerated at 4℃ for 10 minutes to form a brittle gel network. The gel network was removed from the mold and immersed in a deep eutectic solvent (DES) consisting of 20g of betaine and 80g of glycerol for solvent displacement over 4 hours. The resulting eutectic gel was named Gel-COS-Ed eutectic gel.

[0054] Comparative Example 1

[0055] The technical solution of Comparative Example 1 is similar to that of Example 1, except that no chitosan oligosaccharide and emodin are added in Comparative Example 1, and the resulting co-crystal gel is named Gel co-crystal gel.

[0056] Comparative Example 2

[0057] The technical solution of Comparative Example 2 is similar to that of Example 1, except that no emodin is added in Comparative Example 2, and the resulting eutectic gel is named Gel-COS eutectic gel.

[0058] Comparative Example 3

[0059] 1g of gelatin, 0.5g of chitosan oligosaccharide, and 5mg of emodin were dissolved in 10g of deionized water and stirred continuously at 60℃ to form a transparent solution. After defoaming by ultrasonic treatment, the solution was poured into a PMMA mold and transferred to a 4℃ refrigerator for 10 minutes to obtain Gel-COS-Ed hydrogel.

[0060] Comparative Example 4

[0061] 1g of gelatin, 0.5g of chitosan oligosaccharide, and 10mg of emodin were dissolved in 10g of deionized water and stirred continuously at 60℃ to form a transparent solution. After defoaming by sonication, the solution was poured into a PMMA mold and refrigerated at 4℃ for 10 minutes to form a brittle gel network. The gel network was removed from the mold and immersed in a deep eutectic solvent (DES) consisting of 20g of betaine and 80g of glycerol for 4 hours to allow solvent displacement. The resulting eutectic gel was named Gel-COS-Ed eutectic gel.

[0062] Comparative Example 5

[0063] 1 g of gelatin, 1.0 g of chitosan oligosaccharide, and 5 mg of emodin were dissolved in 10 g of deionized water and stirred continuously at 80 °C to form a transparent solution. After defoaming by sonication, the solution was poured into a PMMA mold and refrigerated at 4 °C for 10 minutes to form a brittle gel network. The gel network was removed from the mold and immersed in a deep eutectic solvent (DES) consisting of 20 g of betaine and 80 g of glycerol for solvent displacement over 4 hours. The resulting eutectic gel was named Gel-COS-Ed eutectic gel.

[0064] Detection methods

[0065] 1. Characterization

[0066] The chemical structure of the samples was characterized using Fourier transform infrared spectroscopy (FTIR) on a Thermo Scientific IN 10 instrument equipped with an attenuated total reflectance (ATR) accessory. This technique identifies functional groups and molecular bonds in the samples by measuring infrared absorption at different wavelengths. X-ray diffraction (XRD) analysis was performed using a Bruker D8 Advance diffractometer, generating diffraction patterns using Cu Kα radiation. The samples were analyzed in the 2θ range from 10° to 80°, providing information on the crystalline structure and phase composition of the materials. Differential scanning calorimetry (DSC) was used to investigate the freezing temperature of the eutectic gel. A Mettler DSC3 instrument was used, scanning from -80°C to 80°C at a heating rate of 10°C / min, maintaining a nitrogen flow. This analysis helped determine the thermal properties and phase transitions of the eutectic gel. Furthermore, X-ray photoelectron spectroscopy (XPS) analysis was performed using a Thermo Fisher Scientific K-Alpha system, scanning from 0 to 1350 eV. This technique is used to study the elemental composition and chemical state of a sample, providing valuable insights into surface chemistry.

[0067] 2. Mechanical performance testing

[0068] To evaluate the mechanical properties of the eutectic gel, a series of tensile and cyclic tensile tests were conducted using a general-purpose testing machine (ZHIQU, China) equipped with a 100N load cell. Eutectic gel samples were precisely prepared into dumbbell shapes, 12 mm in length, 2 mm in width, and approximately 1 to 2 mm in thickness, ensuring standardized testing conditions. Tensile tests were performed at a constant rate of 100 mm / min. To ensure statistical validity, at least three samples were tested for each experimental group. In the tensile tests, tensile stress (σ) was calculated by dividing the applied force by the original cross-sectional area of ​​the sample; tensile strain (ε) was determined by dividing the deformed length by the original length of the sample. The elastic modulus (E), representing the stiffness of the gel, was obtained by analyzing the slope of the stress-strain curve within the 20% to 40% strain range.

[0069] Hysteresis behavior was investigated using tensile load-unload tests. The maximum strain values ​​measured were set to 100%, 200%, 300%, 400%, and 500%. Cyclic tensile tests were performed to evaluate energy dissipation characteristics. The dissipated energy (Uhys) was determined by analyzing the area enclosed by the load-unload curves. Furthermore, the total energy was calculated by analyzing the area under the tensile stress-strain curves. The self-healing properties of the gel were evaluated using cyclic loading tests at a maximum strain of 250% and waiting times of 1 minute, 5 minutes, and 30 minutes. This method helps characterize the material's ability to recover its original shape and mechanical properties after deformation. One hundred cycles of tensile testing were conducted at a maximum strain of 250% to verify its stability across multiple cycles.

[0070] 3. In vitro cell compatibility

[0071] CCK-8 assay: L929 cells were seeded at a density of 8000 cells per well in 96-well plates and allowed to attach overnight. Cells were then treated and co-incubated with the gel extract for 24 to 48 hours. CCK-8 solution was then added according to the manufacturer's instructions. After 2 hours of reaction, absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA). Higher optical density (OD) values ​​indicated increased cell proliferation and higher cell viability. The percentage of relative cell viability was calculated using the formula: Cell viability % = [(As - Ab) / (Ac - Ab)] * 100%, where As represents the OD value of the experimental wells (culture medium + cells + extract + CCK-8), Ac represents the OD value of the control wells (culture medium + cells + CCK-8), and Ab represents the OD value of the blank wells (culture medium + CCK-8).

[0072] Live / dead cell staining: L929 cells were seeded at a density of 8000 cells per well in 96-well plates and allowed to attach overnight. After attachment, cells were exposed to gel extract and incubated for 24 to 48 hours. Then, Calcein AM / PI working solution was added at the appropriate ratio. After 40 minutes of incubation, staining was observed using a fluorescence inverted microscope (Carl Zeiss, USA). Live cells showed green fluorescence (due to Calcein AM staining), while dead cells showed red fluorescence (due to propidium iodide staining).

[0073] 4. Scratching and wound healing experiment

[0074] The effect of the gel on the migration ability of L929 cells was evaluated using a scratch wound healing assay. First, cells were seeded in six-well plates and cultured to 80%–90% confluence. Then, the medium was replaced with serum-free medium and incubated overnight. A scratch was made on the cell monolayer in each well using a 200 μL pipette tip, and cell debris was removed with sterile PBS. Then, different concentrations of gel extract were added; the control group used serum-free medium. The scratched areas were observed using an inverted microscope at 0, 12, and 24 hours. The scratch area was measured using ImageJ software, and the scratch healing rate was calculated using the formula: Scratching healing rate (%) = (1 - An / A0) * 100%, where A0 represents the scratch area at time 0, and An represents the scratch area at n hours after gel extract treatment (n = 0, 12, or 24).

[0075] 5. Antibacterial properties

[0076] The antibacterial efficacy of the gel against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus was evaluated using the plate dilution-diffusion method. First, 400 μL of a Staphylococcus aureus or Escherichia coli suspension (concentration 10) was prepared. 6 The CFU / mL extract was mixed with the hydrogel extract. After incubating the mixture at 37°C for 24 hours, it was appropriately diluted and evenly spread onto LB agar plates using sterile swabs. Incubation was then continued at 37°C for another 24 hours. After 24 hours of incubation, the number of colonies was photographed and counted to determine the antibacterial efficacy of the gel.

[0077] 6. Wound healing experiment in mice

[0078] All animal experiments were approved by the Animal Care and Use Committee of the Chinese Academy of Sciences (No.: 202400157). Male C57BL / 6 mice weighing 25g ± 2g were selected for in vivo healing experiments. Mice were fasted for 12 hours before surgery but were allowed free access to water. Under anesthesia, a full-thickness skin defect with a diameter of 8mm was created on the back of each mouse, and 20μL of Staphylococcus aureus suspension (10 μL) was applied to the wound. 6 CFU / mL was used to induce bacterial infection. Once the infection model was established, mice were randomly divided into four groups: control group, gel group, gel-COS group, and gel-COS-Ed group. The control group was treated with sterile gauze soaked in physiological saline, while the other groups were treated with sterile gel dressings fixed with 3M transparent membranes. Wound photographs were taken on days 0, 4, 7, 10, and 13 after modeling to record healing progress and duration. The wound area was analyzed using ImageJ software to calculate the wound healing rate, calculated as follows: Wound healing rate (%) = [(initial wound area - wound area on the day of observation) / initial wound area] * 100%.

[0079] 7. Histopathological analysis and expression levels of α-SMA and CD31 in wound tissue

[0080] On days 4, 7, and 13 post-modeling, mice in each group were humanely sacrificed, and tissue samples were immediately fixed in 4% neutral formalin solution. The fixed tissues were then embedded in paraffin and cut into 4 μm thick sections. Sections were stained with hematoxylin and eosin (HE) and Masson's trichrome staining, respectively, following the protocol provided with the kit. HE staining was used to assess wound pathology and inflammatory response, while Masson's staining was used to assess collagen deposition. In addition, the expression of α-SMA and CD31 in the wound tissue was detected by immunofluorescence staining using anti-α-SMA and anti-CD31 antibodies, performed according to the instructions of the immunostaining kit.

[0081] 8. Statistical Analysis

[0082] Data following a normal distribution are presented as mean ± standard deviation. Analysis of variance (ANOVA) was used for data analysis, followed by Bonferroni post-hoc tests for multiple comparisons. A p-value less than 0.05 was considered statistically significant.

[0083] Analysis and Explanation

[0084] like Figure 1 As shown in Figure a, gelatin, chitosan oligosaccharide, and emodin were first dissolved in water and heated. Emodin combined with chitosan oligosaccharide via a Schiff base reaction, forming a homogeneous solution. At 4°C, hydrogen bonds formed between gelatin molecules, resulting in a brittle gelatin-based hydrogel. Subsequently, it was immersed in a deep eutectic solvent for solvent displacement. Glycerol and betaine molecules interacted with gelatin through hydrogen bonds, increasing the hydrogen bond density and improving the toughness of the eutectic gel. Due to the antibacterial properties of the deep eutectic solvent, chitosan oligosaccharide, and emodin, the final eutectic gel exhibited significant antibacterial effects. Figure 1 As shown in b, this eutectic gel with multiple antibacterial activities can be used as a wound dressing, providing a dual effect of antibacterial and promoting healing, promoting angiogenesis and collagen regeneration, and accelerating the healing of infected wounds.

[0085] 1. Characterization Analysis

[0086] Figure 2 In a, 3295cm -1 The absorption peak is attributed to the stretching vibration of the hydroxyl group (-OH), at 2940 cm⁻¹. -1 Attributable to the stretching vibration of the methylene group (-CH2-), 1640 cm⁻¹ -1 and 1417cm -1The absorption values ​​correspond to C=O and CO groups, respectively. The spectra of the Gel-COS cocrystal gel and the Gel-COS-Ed cocrystal gel are not significantly different from those of the Gel cocrystal gel, indicating that the addition of chitosan oligosaccharide and emodin did not significantly affect the gelatin network structure. Figure 2 The C1s spectrum of the Gel-COS eutectic gel in c shows a CN bond of 286.2 eV. Meanwhile... Figure 2 XPS analysis of b confirmed the formation of C=N bonds in the Gel-COS-Ed eutectic gel, demonstrating that emodin and chitosan oligosaccharides underwent a Schiff base reaction. Figure 2 The C1s spectrum of d showed a C=N bond at 287.2 eV, further supporting the above conclusion. XRD analysis showed that all three gels exhibited an amorphous peak at 2θ = 21°. Figure 2 e) indicates that the main structure of the gel is amorphous. Differential scanning calorimetry (DSC) results show that the Gel-COS-Ed hydrogel exhibits a distinct crystallization peak near 0℃, indicating that water molecules are frozen; in contrast, the Gel-COS-Ed eutectic gel shows no phase transition peak in the range of -80℃ to 80℃, demonstrating its excellent low-temperature resistance. Figure 2 f).

[0087] 2. Mechanical properties and self-healing ability

[0088] Adding other molecules to gelatin-based gels typically affects the molecular structure of the gelatin network, thereby influencing its mechanical properties. For example... Figure 3 As shown in Figure a, the stress-strain curves of the three gels almost completely overlap. Figure 3 In sample b, the fracture strain of the gel-COS-Ed eutectic gel was 510%, and the tensile strength was 1.01 MPa. Compared with the gel eutectic gel, the gel-COS-Ed eutectic gel maintained 91.2% of the fracture strain and 86.2% of the tensile strength, indicating that the addition of COS and emodin molecules had little impact on the structure of the gel-based gel, consistent with the FTIR analysis results. Furthermore, the gel-COS-Ed eutectic gel had an elastic modulus of 74.1 kPa. This moderate flexibility allows the gel to deform in response to skin deformation, making it suitable as a wound dressing while ensuring appropriate fit and compatibility.

[0089] Because the internal cross-linked network of the eutectic gel is composed of hydrogen bonds, these hydrogen bonds effectively dissipate energy during breakage. For example... Figure 4 As shown in figure a, the hysteresis loop of the Gel-COS-Ed eutectic gel increases with strain from 100% to 500%, indicating increased energy consumption. The cyclic curves at different tensile strains partially overlap, attributed to the reversibility of the hydrogen bond network, where broken hydrogen bonds are reconstructed upon unloading. Figure 4 b shows that the dissipated energy is 0.005 MJ / m at 100% strain.3 Increased to 0.80 MJ / m at 500% strain 3 The total energy is 0.039 MJ / m 3 Increased to 1.37 MJ / m 3 The reversibility of hydrogen bonds allows the gel to recover and reorganize after hydrogen bonds break, giving it self-healing capabilities. Figure 4 c shows the cyclic loading and unloading curves of the Gel-COS-Ed eutectic gel at 250% strain and different waiting times. The hysteresis loop area is smallest at 1 minute; however, as the waiting time increases to 5 minutes and 30 minutes, the hysteresis loop increases significantly, indicating the time-dependent process of hydrogen bond network recovery. Figure 4 The results showed that the recovery rate after 1 minute was 42.5%, while the recovery efficiencies after 5 minutes and 30 minutes were 53.7% and 57.9%, respectively, demonstrating the excellent self-healing ability of the Gel-COS-Ed eutectic gel.

[0090] Figure 4 e presents the results of 100-cycle tensile tests on the Gel-COS-Ed eutectic gel. Unlike hydrogels, the eutectic gel does not exhibit reduced mechanical properties due to dehydration during tensile testing. It can be observed that the maximum stress of the gel gradually decreases in the first 20 cycles, and then tends to stabilize in subsequent cycles. Figure 4 f). By comparing the maximum stress during the tensile cycles with the maximum stress of the first cycle, it was found that by the 100th tensile cycle, the maximum stress had reached a stable value, maintaining 77.6% of the initial maximum stress, indicating its excellent stability. In the 100-cycle tensile test, unlike Gel-COS-Ed hydrogels, the eutectic gel did not experience any impact on its mechanical properties due to dehydration during the tensile process. Figure 4 e). Figure 4 The f-value shows that the maximum stress of the gel gradually decreases during the first 20 cycles and then tends to stabilize. Compared with the maximum stress in the first cycle, the maximum stress remains at 77.6% of the initial value at the 100th cycle, indicating its excellent stability.

[0091] 3. Cell compatibility and migration assays of cocrystal gels

[0092] Figure 5 As shown in Figure a, L929 cells in the control group exhibited healthy polygonal or spindle morphology when growth remained normal. The number of dead cells in the Gel co-crystal gel group was increased compared to the control group, possibly due to the influence of the charged deep eutectic solvent in the gel. Conversely, the number of dead cells in the Gel-COS-Ed co-crystal gel group was reduced compared to the control group, indicating that chitosan oligosaccharides and emodin molecules have anti-apoptotic effects. The cell viability rate in the Gel-COS-Ed co-crystal gel group was 97.5% after 24 hours. Figure 5b) The cell survival rate reached 115.4% after 48 hours, indicating that the addition of emodin not only did not impair biocompatibility, but also promoted cell proliferation in the gel.

[0093] The Gel-COS eutectic gel and Gel-COS-Ed eutectic gel groups showed significantly higher scratch healing rates than the Gel eutectic gel group. At 12 hours, the healing rates for Gel-COS eutectic gel and Gel-COS-Ed eutectic gel were 23.9% and 23.6%, respectively. Figure 6 (b) , which increased to 33.5% and 47.9% respectively over 24 hours. Figure 6 c). These results indicate that the addition of chitosan oligosaccharide enhanced the healing ability of the Gel-COS cocrystal gel group. The combined effect of chitosan oligosaccharide and emodin further improved cell migration ability; the scratch healing rate of the Gel-COS-Ed cocrystal gel group at 24 hours was 1.55 times and 1.41 times that of the gelatin cocrystal gel and the Gel-COS cocrystal gel, respectively. Figure 6 a).

[0094] 4. Antibacterial activity

[0095] exist Figure 7 In cases a-b, the bacterial cell membranes in the control group remained intact and smooth, while the addition of gel caused the cell membranes to gradually shrink and deform. After treatment with Gel-COS-Ed eutectic gel, the bacterial membranes severely ruptured. Figure 7 As shown in c-d, at the same bacterial concentration, the bacterial count in all three co-crystal gels decreased compared to the control group, indicating that betaine has antibacterial activity. In the Gel-COS-Ed co-crystal gel group, bacteria were almost eliminated, with a Staphylococcus aureus survival rate of only 4.3% and Escherichia coli survival rate of 17.6%. Compared to the control group, the Gel-COS-Ed co-crystal gel achieved an inhibition rate of 95.7% against Staphylococcus aureus and an inhibition rate of 82.4% against Escherichia coli. This demonstrates that the synergistic effect of betaine, chitosan oligosaccharide, and emodin provides highly effective antibacterial protection against Staphylococcus aureus and Escherichia coli.

[0096] 5. Internal wound healing assessment

[0097] like Figure 8 As shown in Figure a, a gel dressing was used for wound healing in an infected mouse model. Figure 8 As shown in b, the wound size in all four groups began to shrink on day 4, while the Gel-COS-Ed group showed significantly better healing. Figure 8In the gauze group, the healing rate on day 4 was 39.3%, while it was 65.6% in the gel-COS-Ed group. The healing rate of the gel-COS-Ed group was 166.9% of that of the gauze group, validating the significant role of gel-COS-Ed cocrystal gel in promoting wound healing in the early stages. On day 7, the gel-COS-Ed group showed faster granulation tissue growth, more significant wound contraction, and significant epithelialization, with a wound healing rate of 89.1%, compared to 136.9% of the gauze group. By day 13, the wound healing rate of the gel-COS-Ed group reached 99.3%, almost completely healed, while the wounds in the control group and the gel-COS-Ed group still had residual tissue. This indicates that gel-COS-Ed cocrystal gel significantly improved the wound healing rate, especially in the early stages of treatment. Figure 8 As can be seen, on days 4, 7 and 13, the number of inflammatory cells in the wound tissue of the Gel-COS-Ed cocrystal gel group was significantly reduced. Compared with other groups, the growth of granulation tissue was faster, the interstitial edema was relieved more quickly, and the formation of new blood vessels was enhanced and the epidermal regeneration was accelerated.

[0098] like Figure 9 As shown in figure a, the Gel-COS-Ed group showed significantly enhanced collagen production, with relatively orderly collagen fiber arrangement, accompanied by angiogenesis. Figure 9 c indicates that the collagen deposition level in the Gel-COS-Ed group was significantly higher than that in other groups, suggesting that the Gel-COS-Ed group significantly promoted collagen production and maturation, and improved collagen arrangement, thereby accelerating wound re-epithelialization. Figure 9 As shown in b, the fluorescence expression of α-SMA and CD31 in the Gel-COS-Ed group was significantly higher than that in the control group, indicating that the Gel-COS-Ed group significantly promoted angiogenesis and accelerated wound re-epithelialization. Figure 9 The results also showed that the Gel-COS-Ed group significantly promoted angiogenesis.

[0099] like Figure 10 As shown, the cell death rates in Comparative Examples 4 and 5 were significantly higher than those in Example 1. In Comparative Example 4, the excessive addition of emodin caused cytotoxicity to the cells, leading to more cell death. In Comparative Example 5, the excessive proportion of chitosan oligosaccharides altered the physical properties of the gel network, making it overly dense, reducing cell adhesion and proliferation capacity, and affecting the cell's survival environment. Excessive chitosan oligosaccharides also affected nutrient penetration and hindered normal cell metabolism.

[0100] The above results indicate that the Gel-COS-Ed eutectic gel dressing can improve the healing process of infected wounds by inhibiting bacterial growth, promoting wound contraction, enhancing collagen deposition, and promoting angiogenesis. The technical effects of Examples 2 and 3 are similar to those of Example 1.

[0101] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A protein-based co-crystal gel, characterized in that, Its components include gelatin, chitosan oligosaccharide, emodin, and a deep eutectic solvent; the mass ratio of chitosan oligosaccharide to emodin is 1:0.0075~0.0150; the mass fraction of emodin in the protein-based eutectic gel is 0.0027%~0.0080%; the deep eutectic solvent is composed of glycerol and betaine; the preparation method of the protein-based eutectic gel includes the following steps: dissolving gelatin, chitosan oligosaccharide, and emodin in water, and then solidifying them into a gelatin gel; placing the gelatin gel in the deep eutectic solvent for solvent displacement to obtain the protein-based eutectic gel.

2. The protein-based cocrystal gel according to claim 1, characterized in that, The amount of betaine is no more than 20%.

3. A method for preparing the protein-based co-crystal gel according to claim 1, characterized in that, Includes the following steps: Gelatin, chitosan oligosaccharide, and emodin are dissolved in water and then solidified into a gel. The gel is then placed in a deep eutectic solvent for solvent displacement to obtain a protein-based eutectic gel.

4. The method for preparing the protein-based cocrystal gel according to claim 2, characterized in that, Gelatin, chitosan oligosaccharide, and emodin are dissolved at 40-80°C.

5. An application of the protein-based co-crystal gel according to claim 1, characterized in that, Prepare dressings for use in wounds.

6. The application of the protein-based cocrystal gel according to claim 5, characterized in that, Prepare dressings that promote wound healing.

7. The application of the protein-based cocrystal gel according to claim 5, characterized in that, Prepare dressings that promote angiogenesis and collagen regeneration.

8. The application of the protein-based cocrystal gel according to claim 5, characterized in that, Prepare dressings that promote cell proliferation and migration.

Images