Chitosan-gelatin-citric acid gel and its preparation method and application

Abstract

The present invention belongs to the field of drug carrier technology, specifically relating to a chitosan-gelatin-citric acid gel, its preparation method, and its application. The chitosan-gelatin-citric acid gel provided by the present invention uses chitosan and gelatin as cross-linking monomers, citric acid as a cross-linking agent, and is obtained under the condition that the citric acid content is ≥0.4%. It exhibits good swelling properties and bioaccessibility, biocompatibility, biodegradability, and safety, and can achieve effective delivery and sustained release of hydrophobic drug molecules, showing great potential for the delivery of hydrophobic drugs.

Description

Technical Field

[0001] This invention belongs to the field of drug carrier technology, specifically relating to a chitosan-gelatin-citric acid gel and its preparation method and application. Background Technology

[0002] Curcumin (diferoylmethane or 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadien-3,5-dione) is a natural, fat-soluble, low-molecular-weight polyphenol with a unique chemical structure possessing various biological and pharmaceutical properties. Extracted from the root of turmeric, it is insoluble in water and ether, but soluble in methanol, ethanol, and dimethyl sulfoxide. Studies have found that curcumin can effectively act as an antioxidant, primarily due to its oxidation reaction (hydrogen donation reaction). Based on this reaction, curcumin can scavenge most of the most reactive oxygen free radicals (ROS). This property gives curcumin its antioxidant activity in normal cells. Simultaneously, curcumin also possesses anti-cancer effects because it acts as a Michael acceptor in the Michael addition reaction. This structural feature enhances curcumin's potency as an anticancer agent, enabling it to influence the occurrence, promotion, and progression of cancer. Its mechanism of action is related to its effectiveness against various genes and proteins, as well as its antioxidant and free radical scavenging activities. Free radicals can affect a variety of cancers, such as leukemia, lymphoma, melanoma and sarcoma, genitourinary cancers, breast cancer, ovarian cancer, head and neck cancer, lung cancer, and nervous system tumors. Furthermore, curcumin is a highly pleiotropic small molecule that can directly or indirectly affect multiple targets in cellular pathways, causing upregulation or downregulation (depending on the target and cellular environment). Curcumin's molecular targets include molecules that regulate transcription factors, growth factors, receptors, cytokines, kinases, enzymes, and molecules involved in cell survival, metastasis, and apoptosis. Curcumin has been reported to possess pharmacological activities and medicinal value, including anti-inflammatory, antibacterial, antiviral, antifungal, analgesic, anti-neurodegenerative, anti-proliferative, pro-apoptotic, and anti-atherosclerotic effects.

[0003] In medicine, curcumin has therapeutic effects on a variety of diseases, such as neurodegenerative diseases, arthritis, allergies, inflammatory bowel disease, nephrotoxicity, HIV / AIDS, psoriasis, diabetes, multiple sclerosis, cardiovascular diseases, and pulmonary fibrosis. However, regrettably, preclinical studies have shown that curcumin cannot be used to treat any disease due to its low bioavailability. The main reasons for its low bioavailability are its poor hydrophilicity (insufficient absorption capacity), rapid metabolism (short half-life), and rapid excretion by the liver. Summary of the Invention

[0004] The purpose of this invention is to provide a chitosan-gelatin-citric acid gel, its preparation method, and its application. The chitosan-gelatin-citric acid gel provided by this invention has good swelling properties and bioaccessibility. As a carrier for drug-loaded gels, it can achieve effective delivery and sustained release of hydrophobic drug molecules.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] This invention provides a chitosan-gelatin-citric acid gel, which is obtained by crosslinking chitosan, gelatin and citric acid in an organic acid solution; wherein the chitosan-gelatin-citric acid gel contains ≥0.4% by mass of citric acid.

[0007] Preferably, the degree of deacetylation of the chitosan is 80-95%; the molecular weight is 1.5 × 10⁻⁶. 5 Da.

[0008] Preferably, the gelatin has a strength of 100-200g Bloom.

[0009] Preferably, in the chitosan-gelatin-citric acid gel: the mass percentage of chitosan is 1-5%; the mass percentage of gelatin is 1-5%; and the mass percentage of citric acid is 0.4-1%.

[0010] Preferably, the organic acid solution is an aqueous solution of acetic acid, and the organic acid solution has a mass percentage content of 1-1.5%.

[0011] This invention provides a method for preparing the chitosan-gelatin-citric acid gel described in the above technical solution, comprising the following steps:

[0012] Chitosan, citric acid, and gelatin are dissolved in an organic acid solution to obtain a mixed solution;

[0013] The mixed solution was circulated and subjected to a freeze-thaw process to obtain chitosan-gelatin-citric acid gel.

[0014] Preferably, the freeze-thaw process comprises: freezing the mixed solution to obtain frozen material; thawing the frozen material; wherein the freezing temperature is -80 to -70°C, and the thawing temperature is room temperature.

[0015] This invention provides the application of the chitosan-gelatin-citric acid gel described in the above technical solution or the chitosan-gelatin-citric acid gel prepared by the preparation method described in the above technical solution in the preparation of drug-loaded gels.

[0016] This invention provides a drug-loaded gel, comprising a carrier gel and a hydrophobic drug loaded in the carrier gel; the carrier gel is the chitosan-gelatin-citric acid gel described in the above technical solution or the chitosan-gelatin-citric acid gel prepared by the preparation method described in the above technical solution.

[0017] Preferably, the hydrophobic drug includes curcumin, and the mass concentration of the hydrophobic drug in the drug-loaded gel is 2-20 mg / L.

[0018] This invention provides a chitosan-gelatin-citric acid gel, which is obtained by crosslinking chitosan, gelatin and citric acid (CA) in an organic acid solution; wherein the chitosan-gelatin-citric acid gel contains ≥0.4% by mass of citric acid. The chitosan-gelatin-citric acid gel provided by this invention uses chitosan and gelatin as crosslinking monomers and citric acid as a crosslinking agent. Citric acid reacts with chitosan and gelatin respectively through esterification reactions, generating more amide bonds and disrupting the structure of chitosan itself. Thus, this invention combines citric acid, chitosan, and gelatin, effectively altering the mechanical properties of chitosan-gelatin. Simultaneously, under conditions where the citric acid content is ≥0.4%, the resulting chitosan-gelatin-citric acid gel exhibits good swelling properties and bioavailability, making it suitable as a carrier for drug delivery gels, enabling effective delivery and sustained release of hydrophobic drug molecules. Furthermore, the chitosan-gelatin-citric acid gel provided by this invention possesses good biocompatibility and biodegradability, exhibiting high completeness and great potential for delivering hydrophobic drugs. Results from the examples show that the swelling level of the chitosan-gelatin-citric acid gel provided by this invention ranges from 142 g / g to 539 g / g, with the best drug release effect in simulated intestinal fluid.

[0019] This invention provides a drug-loaded gel, comprising a carrier gel and a hydrophobic drug loaded in the carrier gel; the carrier gel is the chitosan-gelatin-citric acid gel described in the above-mentioned technical solution or a chitosan-gelatin-citric acid gel prepared by the preparation method described in the above-mentioned technical solution. This invention uses the chitosan-gelatin-citric acid gel of the above-mentioned technical solution as a drug carrier, which can achieve effective delivery and sustained release of hydrophobic drugs. When the hydrophobic drug in the drug-loaded gel provided by this invention is curcumin (Cur), this invention investigates the sustained-release behavior of the drug-loaded gel in simulated physiological environments such as oral cavity, gastric juice, intestinal juice, and PBS 7.4, and explores its antioxidant activity, antibacterial activity, bioavailability, and degradability. The results of the examples show that when the CA concentration is 0.4%, the swelling index of the gel is 539 g / g, the equilibration time is 40 min, and it exhibits better sustained-release behavior. Cur showed the highest sustained-release efficiency in simulated intestinal fluid, with a release percentage of approximately 42%. The scavenging rate of ABTS free radicals was over 70%, and the inhibition rate against Escherichia coli and Staphylococcus aureus reached over 90%. The bioavailability of gels with CA concentrations of 0.6%, 0.8%, and 1.0% reached 50%, demonstrating the great potential of chitosan / gelatin gels crosslinked with CA for delivering hydrophobic molecules such as Cur. Attached Figure Description

[0020] Figure 1 The infrared spectrum of the chitosan-gelatin-citric acid gel with a concentration of 0.4% prepared in Example 1;

[0021] Figure 2 The infrared spectrum of the drug-loaded gel product with a citric acid concentration of 0.4% prepared in Example 2;

[0022] Figure 3 The graph shows the swelling analysis results of the chitosan-gelatin-citric acid gel product prepared in Example 1 in distilled water.

[0023] Figure 4 The graph shows the swelling analysis results of the chitosan-gelatin-citric acid gel product prepared in Example 1 under different pH conditions.

[0024] Figure 5 The graph shows the swelling analysis results of the chitosan-gelatin-citric acid gel product prepared in Example 1 in NaCl solution;

[0025] Figure 6 The graph shows the swelling analysis results of the chitosan-gelatin-citric acid gel product prepared in Example 1 in CaCl2 solution.

[0026] Figure 7 This is a drug release analysis diagram of the chitosan-gelatin-citric acid gel product prepared in Example 2 in a simulated oral cavity;

[0027] Figure 8 This is a drug release analysis diagram of the chitosan-gelatin-citric acid gel product prepared in Example 2 in simulated gastric juice;

[0028] Figure 9 This is a drug release analysis diagram of the chitosan-gelatin-citric acid gel product prepared in Example 2 in simulated intestinal fluid;

[0029] Figure 10 The image shows the drug release analysis of the chitosan-gelatin-citric acid gel product prepared in Example 2 in PBS at pH 7.4.

[0030] Figure 11 This is a drug release analysis diagram of the chitosan-gelatin-citric acid gel product prepared in Example 2 in physiological saline;

[0031] Figure 12 This is a drug release analysis diagram of the chitosan-gelatin-citric acid gel product prepared in Example 2 in glucose solution;

[0032] Figure 13 The regression curve for the drug release kinetics study of CGAC-0.4 product in Example 2 using the Zero order model;

[0033] Figure 14 The regression curve for the drug release kinetics study of CGAC-0.4 product in Example 2 using the 1st Order model;

[0034] Figure 15 The regression curve for the drug release kinetics study of CGAC-0.4 product in Example 2 using the Higuchi model;

[0035] Figure 16 The regression curve for the drug release kinetics study of CGAC-0.4 product in Example 2 using the Korsmeyer-Peppas model is shown.

[0036] Figure 17 The regression curve for the drug release kinetics study of CGAC-0.4 product in Example 2 using the Baker-Lonsdale model;

[0037] Figure 18 The graph shows the mechanical property test results of the products prepared in Examples 1 and 2;

[0038] Figure 19 The graph shows the experimental results of DPPH free radical scavenging activity of the products prepared in Examples 1 and 2;

[0039] Figure 20The graph shows the experimental results of ABTS scavenging activity of the products prepared in Examples 1 and 2;

[0040] Figure 21 The graph shows the experimental results of FRAP scavenging activity of the products prepared in Examples 1 and 2;

[0041] Figure 22 The graph shows the experimental test results of the products prepared in Examples 1 and 2 against the Gram-negative bacterium Escherichia coli (E. coli);

[0042] Figure 23 The experimental test results of the Gram-positive bacteria Staphylococcus aureus (S. aureus) in the products prepared in Examples 1 and 2 are shown in the figure.

[0043] Figure 24 The graph shows the test results for the bioaccessibility of the products prepared in Examples 1 and 2;

[0044] Figure 25 The graph shows the results of the biodegradability test of the products prepared in Examples 1 and 2 in PBS at pH 7.4;

[0045] Figure 26 The graph shows the results of the biodegradability test of the products prepared in Examples 1 and 2 in the combined solution. Detailed Implementation

[0046] This invention provides a chitosan-gelatin-citric acid gel, which is obtained by crosslinking chitosan, gelatin and citric acid in an organic acid solution; wherein the chitosan-gelatin-citric acid gel contains ≥0.4% by mass of citric acid.

[0047] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0048] In this invention, chitosan serves as the crosslinking monomer of the chitosan-gelatin-citric acid gel, undergoing a crosslinking reaction with gelatin. Chitosan is a linear, semi-crystalline, hydrophobic cationic heteropolysaccharide obtained from chitin through alkaline hydrolysis (deacetylation) or enzymatic reaction. It is typically derived from the shells of crustaceans (such as crabs and shrimp) and is also naturally occurring in the cell walls of some fungi. Chitosan possesses good film-forming properties, barrier properties, antibacterial activity, antioxidant activity, as well as biocompatibility, biodegradability, and safety. Chitosan contains free amino groups and reactive functional groups such as primary amines (NH), hydroxyl groups (OH), and ortho-hydroxyl groups (OH), enabling it to undergo a crosslinking reaction with gelatin without affecting the overall degree of polymerization of the chitosan. In this invention, the degree of deacetylation of the chitosan is preferably 80–95%, more preferably 85–90%; the molecular weight is preferably 1.5 × 10⁻⁶. 5 Da.

[0049] In this invention, the gelatin (CAS: 9000-70-8) serves as the crosslinking monomer for the chitosan-gelatin-citric acid gel, undergoing a crosslinking reaction with chitosan. Gelatin is a hydrolyzed and denatured collagen protein, a major structural protein in mammalian connective tissue. The main amino acids present in gelatin are glycine (27-35%), proline, and hydroxyproline (20-24%). The chemical structure of gelatin is similar to that of collagen. Gelatin also contains a sequence similar to RGD (Arg-Gly-Asp), which is a cell adhesion site that binds to integrins. This invention uses gelatin as a crosslinking monomer to improve cell integration and tissue repair. Simultaneously, in vivo, gelatin can be degraded by proteases such as collagenase and metalloproteinases. In this invention, the preferred strength of the gelatin is 100-200 g Bloom.

[0050] In this invention, citric acid serves as a crosslinking monomer, initiating a crosslinking reaction between chitosan and gelatin.

[0051] In this invention, the organic acid solution serves as the solvent medium for the chitosan-gelatin-citric acid gel. Preferably, the organic acid solution is an aqueous solution of acetic acid, and its mass percentage is preferably 1-1.5%, more preferably 1.5%.

[0052] In this invention, the chitosan-gelatin-citric acid gel contains: the mass percentage of chitosan is preferably 1-5%, more preferably 2%; the mass percentage of gelatin is preferably 1-5%, more preferably 2%; and the mass percentage of citric acid is preferably 0.4-1%, specifically preferably 0.4%, 0.6%, 0.8%, or 1%.

[0053] In this invention, the content of citric acid in the chitosan-gelatin-citric acid gel cannot be too low or too high. If the content of citric acid is too low, the cross-linking reaction between chitosan and gelatin cannot be effectively initiated, affecting the swelling and mechanical properties of the chitosan-gelatin-citric acid gel; if the content of citric acid is too high, it will affect the solubility of chitosan in the solution, making it unable to dissolve completely, forming lumpy insoluble matter, and preventing gel formation.

[0054] The chitosan-gelatin-citric acid gel provided by this invention has excellent swelling properties and can be used as a delivery carrier for hydrophobic drug molecules to achieve effective delivery and sustained release of hydrophobic drugs.

[0055] This invention provides a method for preparing the chitosan-gelatin-citric acid gel described in the above technical solution, comprising the following steps:

[0056] Chitosan, citric acid, and gelatin are dissolved in an organic acid solution to obtain a mixed solution;

[0057] The mixed solution was circulated and subjected to a freeze-thaw process to obtain chitosan-gelatin-citric acid gel.

[0058] This invention dissolves chitosan, citric acid, and gelatin in an organic acid solution to obtain a mixed solution.

[0059] In this invention, the dissolution preferably includes the following steps: firstly, dissolving chitosan in an organic acid solution to obtain a chitosan solution; secondly, dissolving citric acid in the chitosan solution to obtain a citric acid-chitosan solution; and thirdly, dissolving gelatin in the citric acid-chitosan solution to obtain a mixed solution. In this invention, the temperature for the first, second, and third dissolutions is preferably 60°C, and the first, second, and third dissolutions are preferably carried out under stirring conditions. The holding time for the first dissolution is preferably 3 hours.

[0060] After obtaining the mixed solution, the present invention performs a freeze-thaw process on the mixed solution to obtain chitosan-gelatin-citric acid gel.

[0061] In this invention, the freeze-thaw process is preferably performed by freezing the mixed solution to obtain a frozen material, and then thawing the frozen material. In this invention, the freezing temperature is preferably -80 to -70°C, more preferably -80°C; the thawing temperature is room temperature. Before freezing, this invention preferably pre-freezes the mixed solution at -20 to -10°C. This invention does not have specific requirements for the freezing time, as long as the mixed solution is completely frozen into a solid. This invention does not have specific requirements for the thawing time, as long as the frozen material is completely thawed into a liquid. In this invention, the freeze-thaw process is preferably performed twice.

[0062] This invention provides the application of the chitosan-gelatin-citric acid gel described in the above technical solution or the chitosan-gelatin-citric acid gel prepared by the preparation method described in the above technical solution in the preparation of drug-loaded gels.

[0063] This invention provides a drug-loaded gel, comprising a carrier gel and a hydrophobic drug loaded in the carrier gel; the carrier gel is the chitosan-gelatin-citric acid gel described in the above technical solution or the chitosan-gelatin-citric acid gel prepared by the preparation method described in the above technical solution.

[0064] In this invention, the hydrophobic drug is specifically curcumin.

[0065] In this invention, the mass concentration of the hydrophobic drug in the drug-loaded gel is preferably 2 to 20 mg / L, more preferably 2 mg / L.

[0066] This invention provides a method for preparing the above-mentioned drug-loaded gel, comprising the following steps:

[0067] Chitosan, citric acid, and gelatin are dissolved in an organic acid solution to obtain a mixed solution;

[0068] The mixed solution is mixed with a hydrophobic drug to obtain a hydrophobic drug mixed solution;

[0069] The hydrophobic drug mixture was subjected to a freeze-thaw cycle to obtain a drug-loaded gel.

[0070] In this invention, chitosan, citric acid, and gelatin are dissolved in an organic acid solution to obtain a mixed solution. The preparation method of the mixed solution for preparing drug-loaded gels in this invention is the same as the preparation method of the mixed solution for preparing chitosan-gelatin-citric acid gels described above, and will not be repeated here.

[0071] After obtaining the mixed solution, the present invention mixes the mixed solution with a hydrophobic drug to obtain a hydrophobic drug mixed solution. The mixing temperature is room temperature.

[0072] After obtaining the hydrophobic drug mixture solution, the present invention performs a freeze-thaw cycle on the hydrophobic drug mixture solution to obtain chitosan-gelatin-citric acid gel.

[0073] The specific implementation method of the present invention for circulating the hydrophobic drug mixture solution for freezing and thawing is the same as the specific implementation method of circulating the mixture solution for freezing and thawing described above, and will not be repeated here.

[0074] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0075] The raw material used in the embodiments of the present invention is: chitosan (degree of deacetylation: 80.0-95.0%, molecular weight: 1.5 × 10⁻⁶). 5 Da), the strength of gelatin ~100g Bloom, curcumin, citric acid, 2,2-diazodi-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), 2,4,6-triacidyl-S-triazine (TPTZ) and glacial acetic acid were all purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0076] Example 1

[0077] Chitosan (degree of deacetylation: 80.0–95.0%, molecular weight: 1.5 × 10⁻⁶) was used. 5Chitosan (Da) was dissolved in a 1.5 wt% aqueous acetic acid solution. After stirring at 60°C for 3 hours, citric acid was added. Once the citric acid was completely dissolved, gelatin (strength ~100g Bloom, CAS: 9000-70-8) was added. The mixture was heated and stirred until the gelatin dissolved, resulting in a mixed solution. The mixed solution contained 2% chitosan, 2% gelatin, and 0%, 0.4%, 0.6%, 0.8%, and 1.0% citric acid, respectively.

[0078] The mixed solution was then cooled at -20°C and then frozen at -80°C. After freezing, it was thawed at room temperature. This cycle was repeated twice to prepare hydrogels with citric acid mass percentages of 0%, 0.4%, 0.6%, 0.8%, and 1.0%, respectively, denoted as CG, CGA-0.4, CGA-0.6, CGA-0.8, and CGA-1.0. The raw material composition of each product is shown in Table 1.

[0079] Example 2

[0080] Chitosan (degree of deacetylation: 80.0–95.0%, molecular weight: 1.5 × 10⁻⁶) was used. 5 Chitosan (Da) was dissolved in a 1.5 wt% aqueous acetic acid solution. After stirring at 60°C for 3 hours, citric acid was added. Once the citric acid was completely dissolved, gelatin (strength ~100g Bloom, CAS: 9000-70-8) was added. The mixture was heated and stirred until the gelatin dissolved, resulting in a mixed solution. The mixed solution contained 2% chitosan, 2% gelatin, and 0%, 0.4%, 0.6%, 0.8%, and 1.0% citric acid, respectively.

[0081] Curcumin was then added to the mixed solution to obtain a curcumin mixed solution with a curcumin concentration of 2 mg / L. The curcumin mixed solution was first cooled at -20°C, then frozen at -80°C, and thawed at room temperature. This cycle was repeated twice to prepare drug-loaded hydrogels with a curcumin concentration of 2 mg / L and citric acid content of 0%, 0.4%, 0.6%, 0.8%, and 1.0%, respectively, denoted as CGC, CGAC-0.4, CGAC-0.6, CGAC-0.8, and CGAC-1.0. The raw material composition of each product is shown in Table 1.

[0082] Table 1. Content of CA and Cur in different gels of Examples 1 and 2.

[0083] Samplecode CA (%v / v) Cur(w / v, mg / L) CG - - CGA-0.4 0.4 2 CGA-0.6 0.6 2 CGA-0.8 0.8 2 CGA-1.0 1.0 2 CGC - 2 CGAC-0.4 0.4 2 CGAC-0.6 0.6 2 CGAC-0.8 0.8 2 CGAC-1.0 1.0 2

[0084] Test Example 1: Fourier Transform Infrared (FTIR) Spectroscopy of Gel

[0085] The structure of the hydrogel was characterized using an FTIR-ATR spectrometer (Perkin Elmer GX, Shimadzu Corporation, Japan). At a depth of 4000 cm⁻¹, the structure was... -1 Up to 500cm -1 The spectra of the hydrogel were recorded.

[0086] The infrared analysis results of the gel product (CGA-0.4) prepared in Example 1 and the drug-loaded gel product (CGAC-0.4) prepared in Example 2 are as follows: Figure 1 and Figure 2 As shown. In Figure 1 In the middle, 3606cm -1 The peak value is due to the OH tensile vibration in CG, while the peak center of CGA-0.4 is at 3373 cm. -1 Furthermore, the peak of CGA-0.4 is wider, indicating that the addition of CA generates hydrogen bonding. The actual effect is reflected in a significant improvement in the mechanical properties of the CGA-0.4 hydrogel, which is related to… Figure 5 The correspondence is obvious. The characteristic peak of CG is 1714 cm⁻¹. -1 The position of the characteristic peak of the amide bond in CGA-0.4 is significantly shifted, representing the stretching of the CO-NH amide bond. This shift is likely due to the reaction of CA with chitosan and gelatin, resulting in the formation of more amide bonds. At 1255 cm⁻¹... -1 The peak in CG represents the bending vibration of -OH, but this peak disappears in CGA-0.4, possibly due to the addition of CA, which induces an esterification reaction. The CO bond peak appears at 1014 cm⁻¹ in CG. -1 At 1083cm, while in CGA-0.4 it appears at 1083cm. -1 The peak shifted at 1536 cm⁻¹, possibly because CA disrupted the structure of chitosan itself. The carbonyl characteristic peak of CA appeared at 1536 cm⁻¹. -1 In summary, CA is combined with chitosan-gelatin, and the addition of CA significantly alters the mechanical properties of chitosan-gelatin.

[0087] Figure 2 In the sample, the addition of Cur did not cause a significant shift, but the peak position of the -OH group changed, possibly due to hydrogen bonding generated by the addition of Cur. The carbonyl group's peak position was at 1666 cm⁻¹. -1 The peak value is enhanced at 287 cm⁻¹, with the peak value of the CH bond at 287 cm⁻¹. -1 The enhancement at the gel site is likely due to the presence of numerous CH bonds and carbonyl groups in Cur. Results demonstrate that hydrogen bonding exists between Cur and CGA, resulting in Cur being encapsulated within the gel.

[0088] Test Example 2: Swelling Test

[0089] (1) Gel swelling test in deionized water

[0090] To determine the swelling behavior in distilled water, a pre-weighed gel sample was immersed in distilled water. At predetermined time intervals, the gel was removed, blotted dry with filter paper, and the swollen weight of the hydrogel sample was measured. The swelling ratio was calculated using the following formula (1).

[0091]

[0092] In formula (1): M2 and M1 are the weights of the swollen and dried samples, respectively.

[0093] (2) Gel swelling test in buffer solution

[0094] To measure swelling in buffer solutions, solutions with different pH values ​​(2, 4, 6, 7, 8, and 10) were prepared using standard methods. Pre-weighed gel samples were immersed in a prepared phosphate buffer solution at pH 7.4. At predetermined time intervals, the gels were removed, blotted dry with filter paper, and the weight of the swollen gel was recorded. The swelling ratio was calculated using the following formula (2).

[0095]

[0096] In formula (2), M4 and M3 are the weights of the swollen and dried samples, respectively.

[0097] (3) Gel swelling test in electrolyte solution

[0098] To measure the swelling behavior in the electrolyte, solutions of sodium chloride and calcium chloride with different molar concentrations (0.1, 0.3, 0.5, 0.7, and 0.9 M) were prepared using standard methods. Pre-weighed samples were immersed in the electrolyte solutions. The samples were left in the solutions until the equilibrium time. After the equilibrium time interval, the gels were removed, blotted dry with filter paper, and the weight of the swollen gel was recorded. The swelling ratio was calculated using the following formula (3).

[0099]

[0100] In formula (3), M6 and M5 are the weights of the swollen and dried samples, respectively.

[0101] (4) In vitro drug release

[0102] Weigh out the same mass of the Cur-loaded gel prepared in Example 2, and add it to PBS buffer (pH 7.4), simulated oral environment, simulated gastric juice, simulated intestinal juice, glucose solution, and physiological saline for 110 min, respectively. Take samples every 10 minutes and measure the absorbance of Cur at 450 nm. Calculate the drug release percentage according to the method of Zhu Chengzhang et al. (Zhu Chengzhang, Preparation and Performance Study of Hydrogels Loaded with Curcumin / Gelatin / Hyaluronic Acid, 2021, Hefei: Anhui University of Traditional Chinese Medicine, 2021.10.26922 / d.cnki.ganzc.2021.000071).

[0103] (5) Determination of the mechanical properties of gel

[0104] The tensile strength (TS) and elongation at break (EB) of the gel were measured on an electronic universal tensile testing machine (Legend 2345, Instron Ltd., China) according to ASTM standard test method D 882-91 (ASTM, 2003) and the method of Yang et al. (R. Rakhshaei, H. Namazi, H. Hamishehkar, H. S. Kafil, R. Salehi, In situsynthesized chitosan–gelatin / ZnO nanocomposite scaffold with drug delivery properties: Higher antibacterial and lower cytotoxicity effects, Journal of Applied Polymer Science (2019) 47590, https: / / doi.org / 10.1002 / app.47590.).

[0105]

[0106]

[0107] In formulas (5) and (5): Fmax is the maximum load, S is the initial cross-sectional area, L0 is the initial length of the sample, and ΔL is the tensile length of the film.

[0108] (6) Evaluation of the antioxidant activity of the gel

[0109] The antioxidant properties of the gel were evaluated using three methods: ABTS, DPPH, and FRAP. The specific methods are as follows:

[0110]

[0111] In formula (6): A1 is the absorbance of the blank control (70% ethanol) at 734 nm, and A2 is the absorbance of the ABTS solution at 734 nm after the film is added.

[0112]

[0113] In formula (7): A3 is the absorbance of the blank control (95% ethanol) at 517 nm, A2 is the absorbance of 95% ethanol at 517 nm after adding the gel, and A1 is the absorbance of the DPPH solution at 517 nm after adding the gel.

[0114] The method for determining FRAP was based on that of Yang et al. (J.Yang,C.Wang,N.Li,L.Wu,Z.Huang,Z.Hu,X.Li,Z.Qu,Phytochemicals and anti-tyrosinase activities of Paeonia ostiileaves and roots,Plant Physiology and Biochemistry 181(2022)50-60,https: / / doi.org / 10.1016 / j.plaphy.2022.04.001.): The absorbance of the FRAP working solution after adding the gel was measured at 593 nm, and the result was substituted into the regression equation: Y=5.120X+0.1503, R 2 =0.996, where: X represents concentration, Y represents concentration, Fe 2+ The concentration range is 0.01–0.1 mg / mL.

[0115] (7) Evaluation of gel antibacterial activity

[0116] S. aureus and E. coli were diluted using a serial dilution method to an absorbance value (600 nm) of 0.2. The bacteria were then inoculated into 96-well plates, and the gel samples were immersed in the plates and incubated at 37°C for 24 hours. Bacterial cultures were used as controls, and ordinary LB medium was used as a blank. The absorbance value at 600 nm was measured to calculate the antibacterial effect of the gel.

[0117] (8) Assessment of gel bioaccessibility and degradation

[0118] According to the method of Brodkorb et al. (A. Brodkorb, L. Egger, M. Alminger, P. Alvito, R. S.Ballance,T.Bohn,C.Bourlieu-Lacanal,R.Boutrou,F.Carrière,A.Clemente,M.Corredig,D.Dupont,C.Dufour,C.Edwards,M.Golding,S.Karakaya,B.Kirkhus,S.Le Feunteun,U.Lesmes,A.Macierzanka,ARMackie,C.Martins,S.Marze,DJMcClements,O.Ménard,M.Minekus,R.Portmann,CNSantos,I.Souchon,RPSingh,GEVegarud,MSJWickham,W.Weitschies,I.Recio,Infogest static in vitrosimulation of gastrointestinal food digestion,Nature Protocols 14(4)(2019)991-1014, https: / / doi.org / 10.1038 / s41596-018-0119-1.), an INFUGEST 2.0 digestion model was established. 0.5g of sample was taken and simulated oral digestion was performed. The sample was shaken for 2 min (145mg / L α-amylase), followed by simulated gastric digestion (1g / L pepsin, pH=1). The sample was shaken at 37℃ for 2 h, followed by simulated intestinal digestion for 2 h (3g / L pancreatin, 6g / L bile salts, pH=7). The sample was then filtered using a 0.22μm filter membrane.

[0119] Regarding the degradability of the gel, following the method of Saha et al. (R.Saha, P.Tayalia, Clove oil-incorporated antibacterial gelatin-chitosan cryogels for tissue engineering: An in vitro study, ACS Biomaterials Science & Engineering 8(8)(2022)3557-3567), 0.5 g of lyophilized sample was weighed and then added to 10 mL of PBS 7.4 buffer solution or a PBS 7.4 combination solution containing lysozyme (10000 U / mL) and collagenase (20 U / mL) at 37 °C. The solution was changed every 4 days, and the sample was weighed every week. When weighing, the gel was removed, dried in an oven, and then weighed. Bioaccessibility and biodegradation rate were calculated as shown in formulas (8) and (9):

[0120]

[0121]

[0122] In formula (8), C1 is the concentration of Cur in the total digestate, and C2 is the concentration of Cur in the gel;

[0123] In formula (9), W i W is the initial weight of the sample. t This represents the weight after drying for each time period.

[0124] (9) Data analysis of tests (1) to (8) above

[0125] Each experiment was repeated at least three times, and the results are expressed as mean ± standard deviation. All experimental data were analyzed using IBM SPSS 22.0 statistical analysis (P < 0.05, statistically significant). All figures were plotted using Origin software (2018).

[0126] Results and Discussion

[0127] Swelling analysis of gels

[0128] Swelling capacity is a key parameter of gels, indicating their suitability for biomaterial applications such as sensors, drug delivery, tissue engineering, and wound dressings, in which molecular transfer plays a crucial role. Swelling is a vital parameter in drug delivery systems, directly related to drug release characteristics; the gel's water absorption capacity is positively correlated with the amount of drug released—the more water absorbed, the more drug released. Therefore, this invention conducted swelling experiments on CGA gels with different CA concentrations under distilled water, different pH values, and different molar concentrations of NaCl and CaCl2.

[0129] Figure 3The study shows the swelling behavior of CGA in distilled water at different CA concentrations over time. The gels swelled rapidly upon introduction into distilled water. After 10 minutes, the swelling levels of CGA-0.4 and CGA-0.6 quickly reached 300 g / g and 100 g / g, respectively, while the swelling indices of CGA-0.8 and CGA-1.0 were both below 50 g / g. This may be because the addition of CA increased the hydrophilicity of the gel, leading to an increase in the number of hydroxyl groups. However, as the CA concentration continued to increase, the gel structure became more compact, which in turn limited the gel's water absorption. With prolonged time, CGA-0.6 first reached equilibrium at 235 g / g at 30 minutes; CGA-0.4 reached its maximum at 539 g / g at 40 minutes; and CGA-0.8 and CGA-1.0 reached equilibrium last, at 60 minutes and 50 minutes, respectively, with swelling indices of 164 g / g and 142 g / g. CGA-0.4 exhibited the highest swelling level of 539 g / g, which was higher than the highest swelling level (117.23 g / g) shown by the hydrogel prepared by Raza et al., which was made of (3-mercaptopropyl)trimethoxysilane crosslinked chitosan and polyvinylpyrrolidone (MARaza, N. Gull, S.-W. Lee, K.-K. Seralathan, SHPark, Development of stimuli-responsive chitosan based hydrogels with anticancer efficacy, enhanced antibacterial characteristics, and applications for controlled release of benzocaine, Journal of Industrial and Engineering Chemistry 109(2022)210-220, https: / / doi.org / 10.1016 / j.jiec.2022.02.004), indicating better drug release.

[0130] Swelling behavior is affected by the pH of both the gel and the solution. Since the pH varies in different environments within the human body, the swelling behavior of gels under different pH conditions was analyzed. CGA gels in... Figure 4 The pH sensitivity exhibited can be attributed to the presence of amino groups in the chitosan within the gel, making it a pH-sensitive polymer. For example... Figure 4As shown, the swelling levels of all gels were significantly higher under acidic conditions than under neutral and alkaline conditions. This is mainly because chitosan carries –NH2 groups, which protonate and further generate electrostatic interactions. However, in neutral or alkaline media, the relatively lower swelling levels are due to the fact that swelling is mainly driven by solvent diffusion, without the occurrence of a relaxation effect in the gel structure caused by amino protonation. Interestingly, under acidic conditions, the swelling level of the gel initially shows an upward trend, reaching a maximum at pH 2, and then decreases. This is largely due to the positively charged NH2 groups in the more acidic medium (pH=1). 3+ Negatively charged Cl - The counter-shielding effectively suppresses the repulsive force, resulting in reduced swelling.

[0131] The fluid environment within the human body contains a complex array of electrolytes. This invention selects the most common cations in the body, NaCl and CaCl2, to simulate electrolyte solutions for analysis and research. Figure 5 and Figure 6 As shown, the swelling of the gel in electrolyte solutions is reduced compared to that in distilled water. Taking CGA-0.4 as an example, the maximum swelling in NaCl and CaCl2 is 200 g / g and 350 g / g, respectively, far lower than 539 g / g. This is mainly due to reverse osmosis and the imbalance between hydrophobic and hydrophilic ions in the gel structure, which reduces the swelling behavior of the gel in electrolyte solutions. The swelling level of the gel generally decreases with increasing molar concentration of NaCl and CaCl2 in the solution. Taking the swelling behavior in NaCl solution as an example, the decrease in swelling level with increasing ion molar concentration is due to the lower Donnan osmotic pressure and the increase in solution ion osmotic pressure. This phenomenon can be attributed to the increased ion concentration in the electrolyte solution enhancing electrostatic interactions in the gel, thereby increasing cross-linking strength, resulting in a denser gel structure and limiting swelling.

[0132] Drug release analysis

[0133] The release of Cur loaded with CGA under different physiological conditions was explored. Figure 7 and Figure 8 As shown, Cur release is slow in both simulated oral cavity and simulated gastric juice. Regarding release in the oral cavity, α-amylase can only hydrolyze carbohydrates containing α-(1→4) glycosidic bonds, such as starch, and cannot hydrolyze other polymers, hence the slow release of Cur. Similarly, Cur release in simulated gastric juice is not ideal, partly because the low pH limits gel swelling, and partly because pepsin in simulated gastric juice lacks the ability to hydrolyze gelatin. Additionally, Cur's poor solubility in acidic environments may also contribute to its unsatisfactory release.

[0134] Two groups with relatively similar pH values ​​were compared: simulated intestinal fluid (pH 7.0) and PBS 7.4. Figure 9 and Figure 10 As shown, the release percentage of Cur in simulated intestinal fluid exceeded 40% within 150 minutes, with CGAC-0.8 exhibiting the highest release percentage of 45%. In contrast, in PBS 7.4, the highest Cur release percentage from CGAC-0.6 within 150 minutes was only 5%. This is because gelatin has an Arg-Gly-Asp structure, and the trypsin in the simulated intestinal fluid possesses the ability to hydrolyze Arg, thus disrupting the gel structure and allowing for the rapid and substantial release of Cur. Figure 11 and Figure 12 As can be seen from the results, neither physiological saline nor glucose solution is conducive to the release of Cur.

[0135] Drug release kinetics

[0136] According to the method of Raza et al. (MARaza, N. Gull, S.-W. Lee, K.-K. Seralathan, SHPark, Development of stimuli-responsive chitosan based hydrogels with anticancerefficacy, enhanced antibacterial characteristics, and applications for controlled release of benzocaine, Journal of Industrial and Engineering Chemistry 109(2022)210-220, https: / / doi.org / 10.1016 / j.jiec.2022.02.004.), the drug release kinetics of the gel with the highest swelling level (CGAC-0.4) were studied. The Cur release data in intestinal fluid and the mathematical fitting model were combined with specific equations as shown in formulas (10) to (14):

[0137] Zero order: M t =M ∞ +K0t (10);

[0138] 1st Order:

[0139] Higuchi Model: ft = Q = K H ×t 1 / 2 (12);

[0140] Korsmeyer-Peppas:

[0141] Baker-Lonsdale:

[0142] In formulas (10) to (14), K, K o and K H It is the characteristic constant, where M t M represents the cumulative release of Cur at time t. ∞ Let be the cumulative release of Cur at time ∞. A rate constant K0 was obtained by linear fitting of the zero-order model, where K0 = 0.000483669, indicating controlled drug release. For the first-order model, Higuchi model, Korsmeyer-Peppas model, and Baker-Lonsdale model, the regression coefficients are R0, ... 2 =0.95467, 0.98228, 0.98627 and 0.98244, indicating that these models fit the drug release data well. One of the parameters in Equation (13), the diffusion index (n), is used to predict the drug release mechanism. A diffusion index value (n = 0.05688) greater than 0.5 indicates that the drug diffusion pattern follows a non-Fickian release mechanism. These statistical parameter results (Table 2 and Figures 13-17 This indicates that the regression coefficient values ​​are within the acceptable range for model fit.

[0143] Table 2 shows the drug release kinetic parameter equations obtained by fitting various drugs.

[0144] Model Zeroorder 1stOrder HiguchiModel Korsmeyer-Peppas Baker-Lonsdale parameter <![CDATA[K0=0.000483669]]> K＝0.00124 <![CDATA[K H =0.69693]]> n＝0.05688 K＝0.01081 intercept 0.36551 -1.00512 34.28642 -1.15237 0.43941 <![CDATA[R 2 ]]> 0.95902 0.95467 0.98228 0.98627 0.98244

[0145] Mechanical properties of gels

[0146] Mechanical properties are a crucial parameter in both wound dressings and drug delivery systems, effectively ensuring the integrity of the gel's overall structure and protecting it from external forces. Therefore, this invention studies the mechanical properties of drug carriers to verify their feasibility in practical applications. Figure 18 As shown in the left figure, the tensile strength of the gel was significantly improved with the increase of CA addition. The tensile strength of CG without CA was 1.46 MPa, while the tensile strength of CGA with 1% CA concentration was 5.79 MPa, an increase of approximately four times. This is related to the hydrogen bonding between polymers caused by CA, which greatly enhances the gel strength. In addition, the addition of Cur also slightly improved the tensile properties of the gel, possibly due to hydrogen bonding between Cur and the gel matrix, which is corroborated by the infrared spectrum in Test Example 1.

[0147] Figure 18 The right-hand figure reflects the elongation at break of CGA and CGAC. When the CA concentration is 0.4%, 0.6%, and 0.8%, the elongation at break of CGA is around 50% and shows no significant change. However, when the CA concentration reaches 1%, the elongation at break decreases to some extent, by about 15%. This may be because excessive cross-linking reduces the movement of polymer chains to some extent, leading to a decrease in elongation at break. With the addition of Cur, the elongation at break of CGAC decreases, which is related to the hydrophobicity of Cur. This indirectly proves that Cur can be well bound to the carrier. Therefore, the addition of CA allows the gel to better bind Cur, which is beneficial for its use in drug delivery applications.

[0148] Antioxidant and antibacterial activity of gel

[0149] exist Figures 19-21 In this study, the antioxidant activities of CGA and CGAC were tested using three methods: DPPH, ABTS, and FRAP, to assess the antioxidant activity of CGA and CGAC at different CA concentrations. Figure 19 As shown, in the DPPH free radical scavenging activity experiment, compared with CGA, it was found that the antioxidant activity of CGAC was significantly improved due to the addition of Cur. The general scavenging rate of all CGA was around 20%, while the highest scavenging activity of CGAC reached around 70%. This is mainly because Cur (pKa 8.1) exists primarily as a diketone molecule in acidic and neutral (pH 3-7) environments, effectively donating protons and thus exhibiting high antioxidant activity. Furthermore, the free radical scavenging activity experiments using ABTS and FRAP (…) Figures 20-21 In the assay, it was observed that the antioxidant activity of CGA increased with increasing CA concentration. This is likely because CA is a traditional antioxidant that enhances the antioxidant capacity of other substances. CGA also exhibited antioxidant activity in the ABTS and FRAP experiments; for example, in the ABTS experiment, the scavenging rate of CGA generally reached over 40%. This is mainly due to the presence of active amino and hydroxyl groups in chitosan, which can react with free radicals. Chitosan scavenges free radicals through the reaction between superoxide and hydroxyl anion free radicals and their active hydrogen atoms, thereby forming stable free radicals. Contrary to expectations, the antioxidant effect of CGA in DPPH was lower than in the ABTS and FRAP experiments, which may be due to differences in experimental methods.

[0150] The increasing prevalence of drug-resistant microorganisms has made treating infections they cause more challenging. Therefore, the demand for gels with antibacterial properties is rising, and the antibacterial performance of gels is receiving increasing attention. Antibacterial studies were conducted using experiments against both Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus). For example... Figure 22 and 23 As shown, the antibacterial properties of the gels were significantly improved, all reaching over 75%. The gel with a CA concentration of 0.8% exhibited the best antibacterial effect, showing inhibition rates exceeding 95% against both *E. coli* and *S. aureus*. This is because ur has good and broad-spectrum antibacterial activity and plays a crucial role in the antibacterial performance of the gels. Comparing gels without CA and ur in the two antibacterial experiments, CG showed an inhibition rate greater than 50% against *S. aureus*, significantly better than its inhibition rate against *E. coli* (30%).

[0151] Bioaccessibility and degradation of gels

[0152] Bioavailability is defined as the proportion of a compound or phytochemical released from the food matrix into the aqueous phase of the lumen (pre-systemic metabolism) during digestion and available for transport through the jejunum and ileum membranes. This invention defines bioavailability as the proportion of Curl that, after undergoing the entire digestive process, is absorbed, recycled, and utilized by the target tissue (intestinal mucosa). As a hydrophobic polyphenol, Curl forms insoluble complexes with substances such as peptides in gastric and intestinal fluids, resulting in relatively low bioavailability. Therefore, detecting the bioavailability of Curl under simulated oral and gastrointestinal digestion conditions is crucial.

[0153] Compared to the utilization of Cur loaded simply with CG, the utilization of Cur loaded on the cross-linked carrier with added CA (CGA) has a relative advantage. There was no significant difference between CGAC-0.4 and CGC, with utilization rates around 30%, lower than the other three carrier types' approximately 50%. This is mainly because α-amylase, pepsin, trypsin, hydrochloric acid, etc., in the model can to some extent cause the biopolymer to dissociate, which cannot be counteracted by the electrostatic effects of CG formation and the cross-linking effect of 0.4% CA. The utilization rates of CGAC-0.6, CGAC-0.8, and CGAC-1.0 were significantly improved. Figure 24 This may be because the addition of CA makes the gel structure more compact, increasing digestive resistance. The results showed that compared to the control and gels with a lemon concentration of 0.4%, carriers with CA concentrations of 0.6%, 0.8%, and 1.0% had higher bioavailability. Higher bioavailability allows Cur to be absorbed transmembrane more readily, resulting in higher utilization in blood and cells.

[0154] Biodegradability is one of the important parameters for studying natural polymers, and it is also a significant advantage of natural polymers. From... Figure 25 and Figure 26 It can be seen that the drug-loaded gel can be degraded regardless of the addition of lysozyme and collagenase. However, the degradation rate is faster due to the addition of enzymes, mainly because lysozyme can effectively degrade chitosan and collagenase can degrade gelatin. Figure 25 The results showed that the degradation rate of CGC gel without the addition of cross-linking agent CA was about 50% on the first day in PBS 7.4 buffer solution, and continued to increase over time, reaching about 70% on the twenty-first day. In contrast, the degradation rate of gel with added CA was still around 40% on the 21st day, indicating that CA effectively improved the strength of the gel. Figure 25 and Figure 26 Data showed that the degradation rate of most drug-loaded gels (except CGAC-1.0 in a PBS 7.4 combination solution containing lysozyme (10000 U / mL) and collagenase (20 U / mL)) in PBS 7.4 buffer solution exhibited a trend of rapid initial degradation followed by slower degradation. This may be because gelatin lacks amine functional groups, leading to rapid degradation by collagenase in the early stages of degradation, while highly deacetylated chitosan (degree of deacetylation 80.0%–95.0%) generates a large number of amine functional groups, effectively cross-linking with CA, thus undergoing slow degradation. As for... Figure 26 The degradation rate of CGAC-1.0 in the combined solution (PBS 7.4 combined solution containing lysozyme (10000 U / mL) and collagenase (20 U / mL)) showed a trend of slow initial degradation followed by rapid degradation. This may be due to the physical encapsulation of gelatin by chitosan, causing the chitosan in the gel to be degraded by lysozyme first, thus exposing the internal gelatin for degradation by collagenase. Meanwhile, from... Figure 26 The results show that CGAC-1.0 degraded more slowly than the other four groups, indicating that the degradation rate is related to the concentration of the cross-linking agent. Comparing the degradation on the first day, CGAC-0.8 showed the lowest degree of degradation, achieving a better sustained-release effect.

[0155] This invention uses chitosan and gelatin as drug carriers, CA as a cross-linking agent, and Curl as a biomolecular model to explore the swelling equilibrium time, swelling at different pH and ion concentrations, and the release of the drug-loaded gel in simulated oral cavity, simulated gastric juice, simulated intestinal juice, PBS 7.4, glucose, and physiological saline. Experimental results showed that the swelling levels of various carriers ranged from 142 g / g to 539 g / g, with the best drug release effect in simulated intestinal juice. Furthermore, the drug-loaded gel exhibited excellent antioxidant activity, antibacterial activity, bioavailability, and degradability. Comparison of various carriers revealed that a CA concentration of 0.4% resulted in the highest swelling level, enabling more sustained drug release. Therefore, this is beneficial for delivering hydrophobic polyphenols like Curl into the human body.

[0156] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A drug-loaded gel, characterized in that, The invention includes a carrier gel and a hydrophobic drug loaded in the carrier gel; the carrier gel is a chitosan-gelatin-citric acid gel, which is obtained by crosslinking chitosan, gelatin and citric acid in an organic acid solution; in the chitosan-gelatin-citric acid gel: the mass percentage of chitosan is 2%, the mass percentage of gelatin is 2%, the mass percentage of citric acid is 0.4%, and the hydrophobic drug is curcumin.

2. The drug-loaded gel according to claim 1, characterized in that, The degree of deacetylation of the chitosan is 80-95%; the molecular weight is 1.5 × 10⁻⁶. 5 Da.

3. The drug-loaded gel according to claim 1, characterized in that, The gelatin has a strength of 100-200g Bloom.

4. The drug-loaded gel according to claim 1, characterized in that, The organic acid solution is an aqueous solution of acetic acid, and the mass percentage of the organic acid solution is 1-1.5%.

5. The drug-loaded gel according to any one of claims 1 to 4, characterized in that, The preparation method of the chitosan-gelatin-citric acid gel includes the following steps: Chitosan, citric acid, and gelatin are dissolved in an organic acid solution to obtain a mixed solution; The mixed solution was circulated and subjected to a freeze-thaw process to obtain a chitosan-gelatin-citric acid gel.

6. The drug-loaded gel according to claim 5, characterized in that, The freeze-thaw process is as follows: the mixed solution is frozen to obtain frozen material; the frozen material is thawed; the freezing temperature is -80 to -70°C, and the thawing temperature is room temperature.

7. The drug-loaded gel according to claim 1, characterized in that, The hydrophobic drug includes curcumin, and the mass concentration of the hydrophobic drug in the drug-loaded gel is 2–20 mg / L.

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