Chitosan and recombinant collagen composite nanomaterial as well as preparation method and application thereof

Abstract

The invention relates to the field of nano materials, in particular to a chitosan and recombinant collagen composite nano material as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing chitosan with an acid solution to obtain a chitosan acid solution; mixing the recombinant collagen with a buffer solution to obtain a recombinant collagen buffer solution; dropwise adding the chitosan acid solution into the recombinant collagen buffer solution, so that the chitosan particles and the recombinant collagen particles are self-assembled into composite nanoparticles; the particle size of the composite nanoparticles is smaller than or equal to 300 nm. The chitosan acid solution and the recombinant collagen buffer solution form a stable nanoparticle structure through the self-assembly effect of positive and negative charges, and the problem of toxicity introduced by traditional chemical crosslinking is avoided. The particle size of the composite nanoparticles is less than or equal to 300nm, so that the transdermal efficiency of the collagen is improved; due to the structural characteristics of the nanoparticles, the permeability and the local bioavailability of the medicine are remarkably improved.

Description

Technical Field

[0001] The present application relates to the field of nanomaterials, and specifically to a chitosan and recombinant collagen composite nanomaterial and its preparation method and application. Background Art

[0002] Recombinant collagen is a protein with a triple helical structure. Its excellent cell adhesion and ability to promote tissue regeneration make it a core material for soft tissue repair and drug delivery.

[0003] However, the transdermal efficiency and bioavailability of recombinant collagen still need to be improved. Summary of the Invention

[0004] The purpose of the embodiments of the present application is to provide a chitosan and recombinant collagen composite nanomaterial and its preparation method and application.

[0005] In a first aspect, the present application provides a method for preparing a chitosan and recombinant collagen composite nanomaterial, comprising:

[0006] mixing chitosan with an acid solution to obtain a chitosan acid solution;

[0007] mixing the recombinant collagen with a buffer to obtain a recombinant collagen buffer solution;

[0008] The chitosan acid solution is added dropwise to the recombinant collagen buffer solution to allow the chitosan particles and the recombinant collagen particles to self-assemble into composite nanoparticles.

[0009] In the above technical solution, chitosan acid solution and recombinant collagen buffer solution form a stable nanoparticle structure through the self-assembly of positive and negative charges, avoiding the toxicity problems introduced by traditional chemical cross-linking and improving the transdermal efficiency of collagen; the structure of the formed nanoparticles has a high specific surface area and unique physicochemical properties (Din Darwin effect), which significantly improves the permeability and local bioavailability of the drug.

[0010] Furthermore, compared with recombinant collagen, this composite nanomaterial can further accelerate tissue repair.

[0011] Furthermore, the composite nanomaterial has good biosafety; further, compared with simple recombinant collagen, the composite nanomaterial has better antibacterial properties.

[0012] In other embodiments of the present application, the above-mentioned acid solution includes at least one of a lactic acid solution or an acetic acid solution.

[0013] In other embodiments of the present application, the concentration of the acid solution is 1% w / v-5% w / v.

[0014] In other embodiments of the present application, the pH of the chitosan acid solution is 2-3.

[0015] In other embodiments of the present application, the pH of the recombinant collagen buffer solution is 7-8.

[0016] In other embodiments of the present application, the method further includes:

[0017] After the chitosan acid solution is added dropwise into the recombinant collagen buffer solution to obtain a composite solution, the pH of the composite solution is adjusted to 4-4.5.

[0018] In other embodiments of the present application, adjusting the pH of the composite solution to 4-4.5 includes:

[0019] An alkaline solution is added to the composite nanoparticle solution for adjustment.

[0020] In other embodiments of the present application, the alkaline solution is selected from at least one of a sodium bicarbonate solution and a sodium hydroxide solution.

[0021] In a second aspect, the present application provides a chitosan and recombinant collagen composite nanomaterial, which is prepared using the preparation method of the chitosan and recombinant collagen composite nanomaterial provided in any one of the first aspects.

[0022] In other embodiments of the present application, the particle size of the composite nanoparticles is less than or equal to 300 nm.

[0023] In a third aspect, the present application provides an application of a chitosan and recombinant collagen composite nanomaterial in the preparation of medicines or cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is a diagram showing the antibacterial effects of Staphylococcus aureus and Escherichia coli in the examples and comparative examples of the present application;

[0026] Figure 2 These are the appearance renderings of wound healing after surgery in the examples and comparative examples of this application (Day 4 and Day 7). DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments.

[0028] Therefore, the following detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0029] Chitosan is a natural polysaccharide extracted from the shells of crustaceans. It possesses excellent biocompatibility, antibacterial properties, and biodegradability. Despite its widespread application in medical materials, chitosan alone suffers from weak mechanical properties and limited stability, hindering its full application in complex biological environments.

[0030] Recombinant collagen, a triple-helical protein, has been shown to possess excellent cell adhesion and the ability to promote tissue regeneration, making it a key material for soft tissue repair and drug delivery. However, deficiencies in the mechanical properties and degradation rate of natural collagen limit its application in high-load tissue repair.

[0031] Research has found that combining the two opens up new possibilities for the design of novel materials. Chitosan, which is positively charged when dissolved in acid, and recombinant collagen, which is also positively charged, form a stable composite material through electrostatic interaction. This composite nanoparticle material not only exhibits excellent mechanical properties and bioactivity but also significantly improves drug transdermal absorption and wound healing. For example, in Franz diffusion cell experiments, the transdermal absorption efficiency of a model drug was measured to be approximately 50% higher than that of recombinant collagen alone. In vitro cell experiments also demonstrated a significant effect in accelerating fibroblast proliferation. These data demonstrate that composite nanoparticles can significantly optimize transdermal delivery and tissue repair performance.

[0032] Furthermore, nanoparticles have a high specific surface area and unique physicochemical properties, which can effectively improve the solubility and bioavailability of drugs. In transdermal drug delivery systems, nanoparticles can diffuse through the stratum corneum of the skin barrier into the dermis, enabling more precise drug delivery and significantly enhancing therapeutic efficacy.

[0033] Based on the above research, the present invention provides a method for preparing a chitosan and recombinant collagen composite nanomaterial, comprising:

[0034] mixing chitosan with an acid solution to obtain a chitosan acid solution;

[0035] mixing the recombinant collagen with a buffer to obtain a recombinant collagen buffer solution;

[0036] The chitosan acid solution is added dropwise to the recombinant collagen buffer solution to allow the chitosan particles and the recombinant collagen particles to self-assemble into composite nanoparticles.

[0037] In the above technical solution, chitosan acid solution and recombinant collagen buffer solution form a stable nanoparticle structure through the self-assembly of positive and negative charges, avoiding the toxicity problems introduced by traditional chemical cross-linking and improving the transdermal efficiency of collagen; the structure of the formed nanoparticles has a high specific surface area and unique physicochemical properties (Din Darwin effect), which significantly improves the permeability and local bioavailability of the drug.

[0038] Furthermore, compared with recombinant collagen, the composite nanomaterial can further accelerate tissue repair; further, the composite nanomaterial has good biosafety; further, compared with simple recombinant collagen, the composite nanomaterial has better antibacterial properties.

[0039] Further optionally, illustratively, in some embodiments of the present application, the particle size of the composite nanoparticles is 300nm, 290nm, 280nm, 270nm, 260nm, 250nm, 240nm, 230nm, 220nm, 210nm, 200nm, 190nm, 180nm, 170nm, 160nm, 150nm, 140nm, 130nm, 120nm, 110nm, 100nm or a range between any two of the foregoing values.

[0040] Furthermore, in some embodiments of the present application, the acid solution includes at least one of a lactic acid solution or an acetic acid solution.

[0041] For example, in some embodiments of the present application, the acid solution is selected from either a lactic acid solution or an acetic acid solution. Alternatively, in some embodiments of the present application, the acid solution is selected from a mixture of a lactic acid solution and an acetic acid solution. Alternatively, in some embodiments of the present application, the lactic acid solution and the acetic acid solution in the mixture can be mixed in any ratio.

[0042] Furthermore, in some embodiments of the present application, the concentration of the acid solution is 1% w / v-5% w / v.

[0043] Illustratively, in some embodiments of the present application, the concentration of the acid solution is 1% w / v, 1.1% w / v, 1.2% w / v, 1.3% w / v, 1.4% w / v, 1.5% w / v, 1.6% w / v, 1.7% w / v, 1.8% w / v, 2.0% w / v, 2.2% w / v, 2.5% w / v, 2.8% w / v, 3.0% w / v, 2.8% w / v, 2.8% w / v, 3.0% w / v, 3.2% w / v, 3.5% w / v, 3.6% w / v, 3.8% w / v, 4.0% w / v, 4.2% w / v, 4.5% w / v, 4.8% w / v, 5% w / v or a range between any two of the foregoing values.

[0044] Furthermore, in some embodiments of the present application, the pH of the chitosan acid solution is 1-5.

[0045] Illustratively, in some embodiments of the present application, the pH of the chitosan acid solution is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, or a range between any two of the foregoing values.

[0046] Furthermore, in some embodiments of the present application, the pH of the recombinant collagen buffer solution is 7-8.

[0047] Illustratively, in some embodiments of the present application, the pH of the recombinant collagen buffer solution is 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 8, or a range between any two of the foregoing values.

[0048] Furthermore, in some embodiments of the present application, the above preparation method further comprises:

[0049] After the chitosan acid solution is added dropwise into the recombinant collagen buffer solution to obtain a composite solution, the pH of the composite solution is adjusted to 4-4.5.

[0050] Illustratively, in some embodiments of the present application, after chitosan acid solution is added dropwise to recombinant collagen buffer solution to obtain a composite solution, the pH of the composite solution is adjusted to 4, 4.1, 4.2, 4.3, 4.4, 4.5 or a range between any two of the foregoing values.

[0051] After adding the chitosan acid solution dropwise to the recombinant collagen buffer solution to obtain a composite solution, the pH of the composite solution is adjusted to 4-4.5, which can greatly improve the antibacterial effect.

[0052] Furthermore, in some embodiments of the present application, adjusting the pH of the composite solution to 4 to 4.5 includes:

[0053] An alkaline solution is added to the composite nanoparticle solution for adjustment.

[0054] Further optionally, in some embodiments of the present application, the alkaline solution is selected from at least one of a sodium bicarbonate solution or a sodium hydroxide solution.

[0055] In some optional embodiments of the present application, the alkaline solution is selected from either sodium bicarbonate solution or sodium hydroxide solution. Alternatively, in some optional embodiments of the present application, the alkaline solution is selected from a mixed solution of sodium bicarbonate solution or sodium hydroxide solution.

[0056] Further optionally, in some embodiments of the present application, the alkaline solution is selected from at least one of a saturated solution of sodium bicarbonate or a saturated solution of sodium hydroxide.

[0057] Further optionally, in some embodiments of the present application, in the mixed solution of the sodium bicarbonate solution and the sodium hydroxide solution, the sodium bicarbonate solution and the sodium hydroxide solution can be mixed in any proportion.

[0058] In other optional embodiments of the present application, the preparation method of the above-mentioned chitosan and recombinant collagen composite nanomaterial further includes functionalization treatment.

[0059] The functionalization treatment mentioned above includes coating the surface of nanoparticles with drugs or functional coatings to improve the performance in specific applications, such as antioxidant properties or targeting capabilities.

[0060] Some embodiments of the present application also provide a chitosan and recombinant collagen composite nanomaterial, which is prepared using the preparation method of the chitosan and recombinant collagen composite nanomaterial provided by any of the aforementioned embodiments.

[0061] Furthermore, in some embodiments of the present application, the particle size of the composite nanoparticles is less than or equal to 300 nm.

[0062] Further optionally, illustratively, in some embodiments of the present application, the particle size of the composite nanoparticles is 300nm, 290nm, 280nm, 270nm, 260nm, 250nm, 240nm, 230nm, 220nm, 210nm, 200nm, 190nm, 180nm, 170nm, 160nm, 150nm, 140nm, 130nm, 120nm, 110nm, 100nm or a range between any two of the foregoing values.

[0063] Some embodiments of the present application provide an application of a chitosan and recombinant collagen composite nanomaterial in the preparation of medicines or cosmetics.

[0064] For example, in some embodiments of the present application, the above-mentioned medicines or cosmetics may be wound healing medicines or cosmetics, or antibacterial medicines or cosmetics.

[0065] The features and performance of the present invention are further described in detail below with reference to the embodiments:

[0066] Example 1

[0067] Provided is a chitosan and recombinant collagen composite nanomaterial, which is prepared according to the following steps:

[0068] (1) Preparation of lactic acid solution: Ultrapure water was measured using a graduated cylinder, followed by a 80% w / v lactic acid solution. The lactic acid solution was slowly added dropwise to the ultrapure water under a magnetic stirrer at 200 rpm to prepare a 1% w / v lactic acid solution.

[0069] (2) Preparation of chitosan acid solution: Chitosan (CS) was weighed and added to the 1% w / v lactic acid solution prepared in step (1) above. The mixture was stirred at room temperature for 30 minutes to obtain a lactic acid chitosan solution; the pH of the chitosan acid solution was 2.

[0070] (3) Preparation of recombinant collagen buffer solution: Dissolve recombinant collagen in PBS buffer (pH = 7.4) and adjust the concentration to 2% (w / v). Ultrasonic treatment is performed to remove bubbles and ensure solution homogeneity; the recombinant collagen buffer solution is obtained; the pH of the recombinant collagen buffer solution is 7.

[0071] (4) Self-assembly of lactic acid chitosan and recombinant collagen (RC): The recombinant collagen solution was slowly added dropwise to the lactic acid chitosan solution and stirred slowly for 1 h to obtain a composite solution;

[0072] (4) pH adjustment: Saturated sodium bicarbonate solution was slowly added dropwise to the composite solution prepared in step (3) until the pH reached 4.12, and then the adjustment was stopped. The saturated sodium bicarbonate solution NaHCO3 was prepared by weighing an excess of NaHCO3 solid (about 300-350 g), adding an appropriate amount of distilled water (about 500 mL), and stirring at room temperature until the solution no longer dissolved, thereby forming a saturated solution (the room temperature solubility of NaHCO3 is 9.6 g / 100 mL). Subsequently, the solution was filtered to remove undissolved solids, transferred to a volumetric flask, fixed to the required volume, and sealed to prevent carbon dioxide from escaping.

[0073] Example 2

[0074] Provided is a chitosan and recombinant collagen composite nanomaterial, which is prepared according to the following steps:

[0075] (1) Preparation of lactic acid solution: Ultrapure water was measured using a graduated cylinder, followed by a 80% w / v lactic acid solution. The lactic acid solution was slowly added dropwise to the ultrapure water under a magnetic stirrer at 200 rpm to prepare a 5% w / v lactic acid solution.

[0076] (2) Preparation of chitosan acid solution: Chitosan (CS) was weighed and added to the 5% w / v lactic acid solution prepared in step (1) above. The mixture was stirred at room temperature for 30 minutes to obtain a lactic acid chitosan solution. The pH of the chitosan acid solution was 2.

[0077] (3) Preparation of recombinant collagen buffer solution: Dissolve recombinant collagen in PBS buffer (pH = 7.4) and adjust the concentration to 2% (w / v). Ultrasonic treatment is performed to remove bubbles and ensure solution homogeneity; the recombinant collagen buffer solution is obtained; the pH of the recombinant collagen buffer solution is 8.

[0078] (4) Self-assembly of lactic acid chitosan and recombinant collagen (RC): The recombinant collagen solution was slowly added dropwise to the lactic acid chitosan solution and stirred slowly for 1 h to obtain a composite solution;

[0079] (4) pH adjustment: Saturated sodium hydroxide solution is slowly added dropwise to the composite solution prepared in step (3), and the pH is adjusted to 4.28, and then the adjustment is stopped. The preparation of saturated sodium hydroxide solution is as follows: excess NaOH solid (about 500-600g) is weighed, and distilled water (about 500mL) is slowly added, stirring while adding to prevent splashing or container rupture caused by strong exothermic reaction (the solubility of NaOH at room temperature is 111g / 100mL). After stirring until the solution is saturated, if there are impurities, they can be filtered, and finally transferred to a volumetric flask to be fixed to the required volume, and stored in a sealed alkali-resistant container to avoid moisture absorption or reaction with carbon dioxide in the air to form sodium carbonate.

[0080] Example 3

[0081] Provided is a chitosan and recombinant collagen composite nanomaterial, which is prepared according to the following steps:

[0082] (1) Preparation of lactic acid solution: Ultrapure water was measured using a graduated cylinder, followed by a 80% w / v lactic acid solution. The lactic acid solution was slowly added dropwise to the ultrapure water under a magnetic stirrer at 200 rpm to prepare a 3% w / v lactic acid solution.

[0083] (2) Preparation of chitosan acid solution: Chitosan (CS) was weighed and added to the 3% w / v lactic acid solution prepared in step (1) above. The mixture was stirred at room temperature for 30 minutes to obtain a lactic acid chitosan solution; the pH of the chitosan acid solution was 3.

[0084] (3) Preparation of recombinant collagen buffer solution: Dissolve recombinant collagen in PBS buffer (pH = 7.4) and adjust the concentration to 1% (w / v). Ultrasonic treatment is performed to remove bubbles and ensure solution homogeneity; the recombinant collagen buffer solution is obtained; the pH of the recombinant collagen buffer solution is 7.5.

[0085] (4) Self-assembly of lactic acid chitosan and recombinant collagen (RC): The recombinant collagen solution was slowly added dropwise to the lactic acid chitosan solution and stirred slowly for 1 h to obtain a composite solution;

[0086] (4) pH adjustment: Saturated sodium bicarbonate solution was slowly added dropwise to the composite solution prepared in step (3) until the pH reached 4.45, and then the adjustment was stopped. The saturated sodium bicarbonate solution, NaHCO3, was prepared by weighing an excess of NaHCO solid (approximately 300-350 g), adding an appropriate amount of distilled water (approximately 500 mL), and stirring at room temperature until the solution no longer dissolved, thereby forming a saturated solution (the solubility of NaHCO at room temperature is 9.6 g / 100 mL). The solution was then filtered to remove undissolved solids, transferred to a volumetric flask, fixed to the required volume, and sealed to prevent carbon dioxide from escaping.

[0087] Example 4

[0088] Provided is a chitosan and recombinant collagen composite nanomaterial, which is prepared according to the following steps:

[0089] (1) Preparation of lactic acid solution: Ultrapure water was measured using a graduated cylinder, followed by a 80% w / v lactic acid solution. The lactic acid solution was slowly added dropwise to the ultrapure water under a magnetic stirrer at 200 rpm to prepare a 3% w / v lactic acid solution.

[0090] (2) Preparation of chitosan acid solution: Chitosan (CS) was weighed and added to the 3% w / v lactic acid solution prepared in step (1) above. The mixture was stirred at room temperature for 30 minutes to obtain a lactic acid chitosan solution; the pH of the chitosan acid solution was 3.

[0091] (3) Preparation of recombinant collagen buffer solution: Dissolve recombinant collagen in PBS buffer (pH = 7.4) and adjust the concentration to 1% (w / v). Ultrasonic treatment is performed to remove bubbles and ensure solution homogeneity; the recombinant collagen buffer solution is obtained; the pH of the recombinant collagen buffer solution is 7.5.

[0092] (4) Self-assembly of lactic acid chitosan and recombinant collagen (RC): The recombinant collagen solution was slowly added dropwise to the lactic acid chitosan solution and stirred slowly for 1 h to obtain a composite solution;

[0093] (4) pH adjustment: A mixed solution of sodium bicarbonate solution and sodium hydroxide solution is slowly added dropwise to the composite solution prepared in step (3), and the adjustment is stopped after the pH is adjusted to 4.05. The preparation of saturated sodium bicarbonate NaHCO3 solution is as follows: an excess of NaHCO solid (about 300-350g) is weighed, an appropriate amount of distilled water (about 500mL) is added, and the solution is stirred at room temperature until the solution no longer dissolves, forming a saturated solution (the room temperature solubility of NaHCO is 9.6g / 100mL). Subsequently, the solution is filtered to remove undissolved solids, transferred to a volumetric flask, fixed to the required volume, and sealed to prevent carbon dioxide from escaping. The preparation of saturated sodium hydroxide NaOH solution is as follows: an excess of NaOH solid (about 500-600g) is weighed, distilled water (about 500mL) is slowly added, and stirring is performed while adding to prevent splashing or container rupture caused by a strong exothermic reaction (the room temperature solubility of NaOH is 111g / 100mL). After stirring until the solution is saturated, if it contains impurities, filter it and finally transfer it to a volumetric flask to make up to the required volume. Store it in a sealed alkali-resistant container to avoid moisture absorption or reaction with carbon dioxide in the air to form sodium carbonate.

[0094] Example 5

[0095] Provided is a chitosan and recombinant collagen composite nanomaterial, which is prepared according to the following steps:

[0096] (1) Preparation of acetic acid solution: Ultrapure water was measured using a graduated cylinder, followed by aspiration of 80% acetic acid solution. Under a magnetic stirrer at 200 rpm, the acetic acid solution was slowly added dropwise to the ultrapure water to prepare a 3% w / v acetic acid solution.

[0097] (2) Preparation of chitosan acid solution: Chitosan (CS) was weighed and added to the 3% w / v acetic acid solution prepared in step (1) above. The mixture was stirred at room temperature for 30 minutes to obtain chitosan acetate solution; the pH of the chitosan acid solution was 3.

[0098] (3) Preparation of recombinant collagen buffer solution: Dissolve recombinant collagen in PBS buffer (pH = 7.4) and adjust the concentration to 1% (w / v). Ultrasonic treatment is performed to remove bubbles and ensure solution homogeneity; the recombinant collagen buffer solution is obtained; the pH of the recombinant collagen buffer solution is 7.5.

[0099] (4) Self-assembly of chitosan acetate and recombinant collagen (RC): The recombinant collagen solution was slowly added dropwise to the chitosan acetate solution and stirred slowly for 1 h to obtain a composite solution;

[0100] (4) pH adjustment: A mixed solution of saturated sodium bicarbonate solution and saturated sodium hydroxide solution is slowly added dropwise to the composite solution prepared in step (3), and the adjustment is stopped after the pH is adjusted to 4.34. The saturated sodium bicarbonate solution NaHCO3 is prepared by weighing an excess of NaHCO solid (about 300-350g), adding an appropriate amount of distilled water (about 500mL), and stirring at room temperature until the solution no longer dissolves to form a saturated solution (the room temperature solubility of NaHCO is 9.6g / 100mL). Subsequently, the solution is filtered to remove undissolved solids, transferred to a volumetric flask, fixed to the required volume, and sealed to prevent carbon dioxide from escaping. The saturated sodium hydroxide solution NaOH is prepared by weighing an excess of NaOH solid (about 500-600g), slowly adding distilled water (about 500mL), and stirring while adding to prevent splashing or container rupture caused by a strong exothermic reaction (the room temperature solubility of NaOH is 111g / 100mL). After stirring until the solution is saturated, if it contains impurities, filter it and finally transfer it to a volumetric flask to make up to the required volume. Store it in a sealed alkali-resistant container to avoid moisture absorption or reaction with carbon dioxide in the air to form sodium carbonate.

[0101] Example 6

[0102] Provided is a chitosan and recombinant collagen composite nanomaterial, which is prepared according to the following steps:

[0103] (1) Preparation of acetic acid solution: Ultrapure water was measured using a graduated cylinder, followed by aspiration of 80% acetic acid solution. Under a magnetic stirrer at 200 rpm, the acetic acid solution was slowly added dropwise to the ultrapure water to prepare a 3% w / v acetic acid solution.

[0104] (2) Preparation of chitosan acid solution: Chitosan (CS) was weighed and added to the 3% w / v acetic acid solution prepared in step (1) above. The mixture was stirred at room temperature for 30 minutes to obtain chitosan acetate solution; the pH of the chitosan acid solution was 3.

[0105] (3) Preparation of recombinant collagen buffer solution: Dissolve recombinant collagen in PBS buffer (pH = 7.4) and adjust the concentration to 1% (w / v). Ultrasonic treatment is performed to remove bubbles and ensure solution homogeneity; the recombinant collagen buffer solution is obtained; the pH of the recombinant collagen buffer solution is 7.5.

[0106] (4) Self-assembly of chitosan acetate and recombinant collagen (RC): The recombinant collagen solution was slowly added dropwise to the chitosan acetate solution and stirred slowly for 1 h to obtain a composite solution;

[0107] (4) pH adjustment: A mixed solution of saturated sodium bicarbonate solution and saturated sodium hydroxide solution is slowly added dropwise to the composite solution prepared in step (3), and the pH is adjusted to 4.10, and then the adjustment is stopped. The preparation of saturated sodium bicarbonate NaHCO3 solution is as follows: an excess of NaHCO solid (about 300-350g) is weighed, an appropriate amount of distilled water (about 500mL) is added, and the solution is stirred at room temperature until the solution no longer dissolves, forming a saturated solution (the room temperature solubility of NaHCO is 9.6g / 100mL). Subsequently, the solution is filtered to remove undissolved solids, transferred to a volumetric flask, fixed to the required volume, and sealed to prevent carbon dioxide from escaping. The preparation of saturated sodium hydroxide NaOH solution is as follows: an excess of NaOH solid (about 500-600g) is weighed, distilled water (about 500mL) is slowly added, and stirring is performed while adding to prevent splashing or container rupture caused by a strong exothermic reaction (the room temperature solubility of NaOH is 111g / 100mL). After stirring until the solution is saturated, if it contains impurities, filter it and finally transfer it to a volumetric flask to make up to the required volume. Store it in a sealed alkali-resistant container to avoid moisture absorption or reaction with carbon dioxide in the air to form sodium carbonate.

[0108] Example 7

[0109] Provided is a chitosan and recombinant collagen composite nanomaterial, which is prepared according to the following steps:

[0110] (1) Preparation of acetic acid solution: Ultrapure water was measured using a graduated cylinder, followed by aspiration of 80% acetic acid solution. Under a magnetic stirrer at 200 rpm, the acetic acid solution was slowly added dropwise to the ultrapure water to prepare a 3% w / v acetic acid solution.

[0111] (2) Preparation of chitosan acid solution: Chitosan (CS) was weighed and added to the 3% w / v acetic acid solution prepared in step (1) above. The mixture was stirred at room temperature for 30 minutes to obtain chitosan acetate solution; the pH of the chitosan acid solution was 3.

[0112] (3) Preparation of recombinant collagen buffer solution: Dissolve recombinant collagen in PBS buffer (pH = 7.4) and adjust the concentration to 1% (w / v). Ultrasonic treatment is performed to remove bubbles and ensure solution homogeneity; the recombinant collagen buffer solution is obtained; the pH of the recombinant collagen buffer solution is 7.5.

[0113] (4) Self-assembly of chitosan acetate and recombinant collagen (RC): The recombinant collagen solution was slowly added dropwise to the chitosan acetate solution and stirred slowly for 1 h to obtain a composite solution;

[0114] (4) pH adjustment: A mixed solution of saturated sodium bicarbonate solution and saturated sodium hydroxide solution is slowly added dropwise to the composite solution prepared in step (3), and the pH is adjusted to 4.47 before stopping the adjustment. The preparation of saturated sodium bicarbonate NaHCO3 solution is as follows: an excess of NaHCO solid (about 300-350g) is weighed, an appropriate amount of distilled water (about 500mL) is added, and the solution is stirred at room temperature until the solution no longer dissolves, thereby forming a saturated solution (the room temperature solubility of NaHCO is 9.6g / 100mL). Subsequently, the solution is filtered to remove undissolved solids, transferred to a volumetric flask, fixed to the required volume, and sealed to prevent carbon dioxide from escaping. The preparation of saturated sodium hydroxide NaOH solution is as follows: an excess of NaOH solid (about 500-600g) is weighed, distilled water (about 500mL) is slowly added, and stirring is performed while adding to prevent splashing or container rupture caused by a strong exothermic reaction (the room temperature solubility of NaOH is 111g / 100mL). After stirring until the solution is saturated, if it contains impurities, filter it and finally transfer it to a volumetric flask to make up to the required volume. Store it in a sealed alkali-resistant container to avoid moisture absorption or reaction with carbon dioxide in the air to form sodium carbonate.

[0115] Example 8

[0116] Provided is a chitosan and recombinant collagen composite nanomaterial, which is prepared according to the following steps:

[0117] (1) Preparation of acetic acid solution: Ultrapure water was measured using a graduated cylinder, followed by aspiration of 80% acetic acid solution. Under a magnetic stirrer at 200 rpm, the acetic acid solution was slowly added dropwise to the ultrapure water to prepare a 3% w / v acetic acid solution.

[0118] (2) Preparation of chitosan acid solution: Chitosan (CS) was weighed and added to the 3% w / v acetic acid solution prepared in step (1) above. The mixture was stirred at room temperature for 30 minutes to obtain chitosan acetate solution; the pH of the chitosan acid solution was 3.

[0119] (3) Preparation of recombinant collagen buffer solution: Dissolve recombinant collagen in PBS buffer (pH = 7.4) and adjust the concentration to 1% (w / v). Ultrasonic treatment is performed to remove bubbles and ensure solution homogeneity; the recombinant collagen buffer solution is obtained; the pH of the recombinant collagen buffer solution is 7.5.

[0120] (4) Self-assembly of chitosan acetate and recombinant collagen (RC): The recombinant collagen solution was slowly added dropwise to the chitosan acetate solution and stirred slowly for 1 h to obtain a composite solution;

[0121] (4) pH adjustment: A mixed solution of saturated sodium bicarbonate solution and saturated sodium hydroxide solution is slowly added dropwise to the composite solution prepared in step (3), and the pH is adjusted to 4.18, and then the adjustment is stopped. The preparation of saturated sodium bicarbonate NaHCO3 solution is as follows: an excess of NaHCO solid (about 300-350g) is weighed, an appropriate amount of distilled water (about 500mL) is added, and the solution is stirred at room temperature until the solution no longer dissolves, forming a saturated solution (the room temperature solubility of NaHCO is 9.6g / 100mL). Subsequently, the solution is filtered to remove undissolved solids, transferred to a volumetric flask, fixed to the required volume, and sealed to prevent carbon dioxide from escaping. The preparation of saturated sodium hydroxide NaOH solution is as follows: an excess of NaOH solid (about 500-600g) is weighed, distilled water (about 500mL) is slowly added, and stirring is performed while adding to prevent splashing or container rupture caused by a strong exothermic reaction (the room temperature solubility of NaOH is 111g / 100mL). After stirring until the solution is saturated, if it contains impurities, filter it and finally transfer it to a volumetric flask to make up to the required volume. Store it in a sealed alkali-resistant container to avoid moisture absorption or reaction with carbon dioxide in the air to form sodium carbonate.

[0122] Example 9

[0123] A chitosan and recombinant collagen composite nanomaterial (final pH not adjusted) is provided, which is prepared according to the following steps:

[0124] (1) Preparation of lactic acid solution: Ultrapure water was measured using a graduated cylinder, followed by a 80% w / v lactic acid solution. The lactic acid solution was slowly added dropwise to the ultrapure water under a magnetic stirrer at 200 rpm to prepare a 1% w / v lactic acid solution.

[0125] (2) Preparation of chitosan acid solution: Chitosan (CS) was weighed and added to the 1% w / v lactic acid solution prepared in step (1) above. The mixture was stirred at room temperature for 30 minutes to obtain a lactic acid chitosan solution; the pH of the chitosan acid solution was 2.

[0126] (3) Preparation of recombinant collagen buffer solution: Dissolve recombinant collagen in PBS buffer (pH = 7.4) and adjust the concentration to 2% (w / v). Ultrasonic treatment is performed to remove bubbles and ensure solution homogeneity; the recombinant collagen buffer solution is obtained; the pH of the recombinant collagen buffer solution is 7.

[0127] (4) Self-assembly of lactic acid chitosan and recombinant collagen (RC): The recombinant collagen solution was slowly added dropwise to the lactic acid chitosan solution and stirred slowly for 1 h to obtain a composite nanomaterial (pH = 2.62);

[0128] Comparative Example 1

[0129] A chitosan and recombinant collagen mixed solution is provided, which is prepared according to the following steps:

[0130] Weigh chitosan (CS);

[0131] The recombinant collagen was dissolved in PBS buffer (pH=7.4) and the concentration was adjusted to 1% (w / v). The bubbles were removed by ultrasonic treatment to ensure the homogeneity of the solution, thereby obtaining a recombinant collagen buffer solution. The pH of the recombinant collagen buffer solution was 7.5.

[0132] Chitosan (CS) was added into recombinant collagen buffer solution (RC); (pH=7.21).

[0133] 1. Material performance test

[0134] 1. Particle size test of composite nanomaterials

[0135] The test method is as follows:

[0136] The prepared nanoparticles were measured for particle size using a laser particle size analyzer. A 2.0 mL sample of each prepared nanoparticle group was injected into a sample cell (a four-way cuvette), ensuring that the sample level occupied two-thirds of the cuvette volume. The cuvette was then placed in the instrument's sample cell for measurement. Each sample was measured three times to ensure the reliability and accuracy of the results. The mean and standard deviation were calculated to characterize the particle size distribution of each nanoparticle group.

[0137] 2. Dispersion test method

[0138] The test method is as follows:

[0139] The prepared nanoparticles were dispersible using a laser particle size analyzer. A 2.0 mL sample of each prepared nanoparticle group was injected into a sample cell (a four-way cuvette), ensuring that the sample level occupied two-thirds of the cuvette volume. The cuvette was then placed in the instrument's sample cell for measurement. Each sample was measured three times to ensure the reliability and accuracy of the results. The mean and standard deviation were calculated to characterize the particle size distribution characteristics of each nanoparticle group.

[0140] 3. Stability test method

[0141] The test method is as follows:

[0142] The prepared nanoparticle samples were placed in an environment at 4°C to evaluate their stability under normal storage conditions. Using a dynamic light scattering (DLS) instrument, the particle size and polymer dispersity index (PDI) of the samples were measured every two days for 14 days, and the changing trends of particle size and PDI were recorded to explore the stability of the nanoparticles.

[0143] 4. Tyndall effect test:

[0144] The test method is as follows:

[0145] When conducting a Tyndall effect test, first place the liquid to be tested in a transparent glass container, such as a test tube or beaker; then operate in a darkroom or a darker environment to reduce interference from ambient light and enhance the observation of scattered light; finally, use a laser pen or flashlight to illuminate the liquid from the side or bottom of the container, allowing the light beam to pass through the liquid, and observe from the side to see whether a clearly visible bright light band appears to determine whether the Tyndall effect exists.

[0146] The test results are shown in Table 1.

[0147] Table 1

[0148]

[0149] As can be seen from the results of Examples 1-9, the nanoparticles prepared by the present invention have a good particle size, all less than 300nm, and the nanoparticle size is within the range of 198.34-248.10nm; and the PDI is stable in the range of 0.2-0.3, and the stability is good, and all have a Tyndall effect; while Comparative Example 1 is unable to form nanoparticles normally, the materials cannot be combined together, the stability is poor, and after standing and stratification, no Tyndall effect is formed. In Comparative Example 1, the chitosan is only suspended in the buffer solution and has almost no antibacterial properties.

[0150] 2. Application Performance Testing

[0151] 1. Antibacterial effect

[0152] The obtained nanoparticles were applied to antibacterial (common Gram-positive and Gram-negative bacteria) and the experimental method for evaluating their antibacterial effect was as follows:

[0153] Experimental steps:

[0154] (1) Preparation of culture medium: Prepare sufficient amounts of liquid culture medium and solid culture medium according to the LB culture medium formula: 5 g of tryptone, 2.5 g of yeast extract, 5 g of sodium chloride, 5 g of agar powder, and 500 mL of ultrapure water. Place the culture medium, culture dish, and PBS in an autoclave at 121°C for 20 min.

[0155] (2) Cultivation of Escherichia coli and Staphylococcus aureus: Pour the solid culture medium into a culture dish, use an inoculation loop to pick up Escherichia coli and Staphylococcus aureus respectively and draw a "Z" shape on the culture dish, and place it in a 37℃ incubator for 24 hours. After 24 hours, separate the single colony into the liquid culture medium, place it in a 37℃ shaking incubator for culturing, and use a UV spectrophotometer to detect whether the OD value of the bacterial solution is in the logarithmic phase (0.6-0.8) at 600nm. Take 1mL each of Escherichia coli and Staphylococcus aureus, centrifuge at 5000rpm for 6 minutes, discard the supernatant, add PBS to resuspend and dilute to a bacterial solution concentration of 10 5 CFU / mL;

[0156] (3) Escherichia coli, Staphylococcus aureus and nanoparticles were co-cultured with each sample: 100 μL of 10 5 CFU / mL of Escherichia coli and Staphylococcus aureus were added to the control group, RC group, and each example nanoparticle group, respectively. The concentration of RC and the RC in the example nanoparticle group was kept consistent. The control group was 100 μL of pure water (i.e., blank control group), the RC group was 100 μL of pure collagen liquid solution (i.e., recombinant collagen group), and each example group was added with 100 μL of the corresponding example nanoparticle solution, gently pipetted to mix, and placed in a 37°C incubator for 24 h;

[0157] (4) Plate spreading: Each group took 100 μL of bacterial solution after 24 h of culture and spread it on the plate. Use a spreading rod to spread it evenly, invert and culture at 37°C for 24 h, observe the results, take pictures, and use Image J software to count the colonies.

[0158] The experimental results are shown in Table 2.

[0159] Table 2

[0160]

[0161]

[0162] From the experimental data in the above table, we can see that:

[0163] Three different treatment methods were used to study the antibacterial effects on Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli): Control group, RC group and example nanoparticle (1-9) group. In the Staphylococcus aureus (S.aureus) experiment, the number of colony forming units (CFU) in the Control group was higher, indicating that the bacteria grew more vigorously without treatment. The number of CFU in the RC treatment group and the Example 9 group was significantly lower than that in the Control group, indicating that RC and the Example 9 group had an inhibitory effect on Staphylococcus aureus. In contrast, the number of CFU in the Example nanoparticle (1-8) group was significantly reduced, far lower than that in the other groups, showing a better antibacterial effect. In the Escherichia coli (E.coli) experiment, the Control group also showed a higher number of CFU, and although the number of CFU in the RC treatment group and the Example 9 group decreased, it was still significantly higher than that in the Example nanoparticle (1-8) group.

[0164] Figure 1 Comparison of the blank control group (Control group), the recombinant collagen group (RC group) and Example 1 also clearly shows that the sample of Example 1 exhibits a very excellent antibacterial effect.

[0165] In summary, the nanoparticles (1-8) group exhibited strong antibacterial activity against both Staphylococcus aureus and Escherichia coli, significantly outperforming the RC treatment alone, the unadjusted pH group in Example 9, and the control group. This demonstrates that the nanoparticles require a pH greater than 4 to exhibit effective antibacterial properties, and offer superior antibacterial efficacy compared to RC alone. Comparative Example 1, however, showed little antibacterial activity due to the lack of nanoparticle formation and was not further tested.

[0166] 2. Transdermal absorption efficiency test

[0167] Experimental Procedure: Ex vivo transdermal experiments are a classic method for studying transdermal transdermal efficiency, often using pig skin, rat skin, or mouse skin as skin models. In this experiment, the abdominal skin of SD rats was used for transdermal testing using a Franz diffusion cell system. Specifically, the skin was positioned between a donor cell and a receiver cell, ensuring that the stratum corneum faced upward. The sample was placed on the stratum corneum surface, with the underside of the skin in contact with the buffer solution in the receiver cell. After the test substance came into contact with the stratum corneum, liquid samples were collected from the receiver cell at different time points. The amount or rate of penetration of the test substance was analyzed to assess its transdermal performance.

[0168] (1) Treatment of ex vivo transdermal skin sources:

[0169] After healthy SD rats were sacrificed, their abdominal skin was taken for in vitro transdermal experiments. First, use a hair removal tool to remove abdominal hair, and wash the skin with warm water and an appropriate amount of detergent to remove surface dirt and impurities. After cleaning, gently wipe the skin surface dry with a dry towel to keep the skin dry. Subsequently, remove the full layer of abdominal skin and rinse thoroughly with normal saline to remove attached blood and other impurities. Carefully wipe the skin surface with a moistened cotton cloth to remove excess tissue, and repeatedly rinse with normal saline to ensure that the skin is clean and intact. The treated skin is used for subsequent transdermal experiments.

[0170] (2) Preparation of experimental equipment:

[0171] The equipment required for transdermal experiments was cleaned with ultrapure water to ensure that the experimental conditions were contamination-free and the equipment was clean and suitable for the experimental requirements.

[0172] (3) Pretreatment of Franz diffusion cell system:

[0173] Add an appropriate amount of water to the drug transdermal diffusion tester (TPY-2), set the temperature to 37±1°C, and adjust the stirring speed to 300 r / min to ensure an experimental environment that simulates human physiological conditions.

[0174] (4) Placement on SD rat skin:

[0175] Place the stirring bar into the receiving chamber of the vertical diffusion cell and fill it with physiological saline so that the liquid surface can fully contact the dermis of the skin. Lay the treated SD rat skin on the bottle mouth of the diffusion cell receiving chamber, ensuring that the skin is flat and fits without any gaps. The stratum corneum of the skin faces the supply chamber and the dermis faces the receiving chamber. The effective penetration area (S) is about 3.14cm 2 The receiving cell volume is approximately 7.0 mL. Maintain a constant temperature water bath at 37 ± 1°C and ensure there are no bubbles in the water bath to ensure experimental stability. To ensure experimental reproducibility, configure three independent vertical diffusion cells for each sample for transdermal experiments.

[0176] (5) Sample administration and experiment initiation:

[0177] Prepare samples of the example nanoparticle group with the same RC concentration, as well as pure RC solution with the same RC concentration. Place the receiving chamber of the vertical diffusion cell in a TPY-2 transdermal drug diffusion tester. Add 1 mL of the corresponding independent sample to the dosing chamber of each group and its replicates. Quickly close the transdermal diffusion tester to ensure the system is airtight, then start the experiment and begin the timer to record the experimental progress.

[0178] The formula for calculating the transdermal amount Q is as follows: Q = [Cn×V+ΣCi×V0] / S (i=1, 2, 3) Q: cumulative permeation amount; S: effective diffusion area; V: volume of receiving fluid in the receiving chamber; V0: volume of each sampling; Ci: drug concentration in the receiving fluid from the first to the last sampling; Cn: drug concentration in the receiving fluid at the nh sampling.

[0179] The results of the 24-hour transdermal transdermal test in the in vitro transdermal model are shown in Table 3.

[0180] Table 3

[0181]

[0182]

[0183] From the experimental results in the above table, it can be seen that in the 24-hour transdermal transdermal experiment in the ex vivo transdermal model, the average total transdermal amount of the RC group and the example nanoparticle (1-9) group were significantly different. Quantitative analysis showed that the transdermal efficiency of the example nanoparticle (1-9) group was significantly improved compared with the RC group. In addition, pH adjustment had no significant effect on the transdermal performance of the nanoparticle system, and there was no significant difference between the Example 9 group and the Example (1-8) group. The above data confirm that the nanoparticle composite system exhibits better drug delivery efficiency in the ex vivo transdermal model, and its transdermal performance remains stable under pH 4.5 conditions.

[0184] 3. Irritation and sensitization test

[0185] Test method:

[0186] Irritation and sensitization tests were conducted on a zebrafish model using the following methods:

[0187] 3.1 Sensitization experiment (zebrafish)

[0188] 3.1.1 Zebrafish strains

[0189] This study used wild-type zebrafish (AB) strains at 2 days post fertilization (dpf). Ten zebrafish were used for each experiment, and three biological replicates (n=3) were performed to ensure data reliability and reproducibility.

[0190] 3.1.2 Sensitization test

[0191] The reared zebrafish were randomly divided into 12-well plates (10 fish / well). The experimental group was given the test sample in a water-soluble manner, ensuring that the volume of the drug-containing culture medium in each well was maintained at 1 mL.

[0192] Each group was incubated with BAPNA substrate for 24 hours at 28°C in the dark. The OD value at 405 nm was then measured using a microplate reader to assess the expression level of tryptase (T). The final value was calculated using the formula to assess its sensitization.

[0193]

[0194] 3.2 Stimulation experiment (zebrafish)

[0195] 3.2.1 Zebrafish strains

[0196] This study used transgenic neutrophil green fluorescent zebrafish (MPX) as an experimental model. The fish were 2 days old (2 dpf) at the time of the experiment. Each experiment included 15 zebrafish, and three biological replicates (n=3) were performed.

[0197] 3.2.2 Sensitization test

[0198] The reared zebrafish were randomly divided into 6-well plates (15 per well). The experimental group was given each sample to be tested in a water-soluble manner to ensure that the volume of the drug-containing culture medium in each well was maintained at 3 mL. After incubation at 28°C in the dark for 18 hours, 10 zebrafish were randomly selected from each group and images were taken under a fluorescence microscope. Subsequently, the data were analyzed using processing software, and the number of neutrophils (n) in the zebrafish skin was counted. The final value was calculated after entering the calculation formula to evaluate its irritation.

[0199]

[0200] The changes in tryptase expression levels and neutrophil counts produced by zebrafish after addition are shown in Table 4.

[0201] Table 4

[0202]

[0203]

[0204] From the test results in Table 4 above, we can see that

[0205] There was no significant difference in the tryptase expression level between each example group and the control group, indicating that each example did not induce a significant allergic reaction, thereby preliminarily confirming that it was not allergenic in the zebrafish model.

[0206] The number of neutrophils in each example group was not significantly increased compared with the control group, indicating that each example did not induce a significant inflammatory response, thereby preliminarily confirming that it had no significant skin irritation in the zebrafish model.

[0207] 4. Wound healing effect test

[0208] A postoperative wound healing model was used to test and evaluate the effect of nanoparticles on wound healing.

[0209] The test method is as follows:

[0210] This experiment used SD rats (weight range: 160-180 grams) and randomly divided them into four groups: a blank control group, a recombinant collagen group, and each example group, with a sample size of 6 rats in each group. Before the experiment began, the rats were anesthetized by intraperitoneal injection of 5% chloral hydrate. After they were fully anesthetized, the backs were shaved and a full-thickness skin defect model with a diameter of 1.2 cm was established on both sides of the midline using a punch. After the operation, the wound was immediately disinfected with iodine and closed using conventional clinical cosmetic suture techniques to simulate the clinical treatment of wound healing.

[0211] During the experiment, drug treatment was administered on the first day (1d) and the fourth day (4d) after surgery. In addition, wound tissue samples were collected on the fourth day (4d) and the seventh day (7d) after surgery and fixed with 4% paraformaldehyde for subsequent histological analysis to further evaluate the wound repair.

[0212] The test results are shown in Table 5.

[0213] Table 5

[0214]

[0215] From the above table data we can see that:

[0216] On the 4th day after surgery, the nanoparticle groups of each embodiment showed a more significant healing effect than the other groups, while the wounds of the other groups were still not completely closed. The results on the 7th day showed that the wounds of all groups were basically healed, but from the perspective of the appearance of wound healing, the healing effect of the example nanoparticle group was the best. This shows that chitosan and recombinant collagen composite nanoparticles can further enhance the effect of RC in wound healing. Combining the observations on the 4th and 7th days, the example nanoparticle group not only made the wound that originally required 7 days to heal basically heal on the 4th day, but also significantly improved the appearance of the wound after healing.

[0217] Figure 2 Comparison of the blank control group, the recombinant collagen group, and Example 1 also clearly shows that the sample of Example 1 exhibits a very excellent wound healing effect.

[0218] In summary, the composite nanomaterials provided in the embodiments of the present application do not require any chemical crosslinking agents, thus avoiding toxicity issues. Furthermore, precise control of self-assembly conditions allows for the formation of nanoparticles with smaller particle sizes and greater stability.

[0219] The composite nanomaterials and nanoparticles provided in the embodiments of the present application have antibacterial and biocompatibility properties, which are crucial for ensuring the safety of treatment.

[0220] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for preparing a chitosan and recombinant collagen composite nanomaterial, characterized in that: include: mixing chitosan with an acid solution to obtain a chitosan acid solution; mixing the recombinant collagen with a buffer solution to obtain a recombinant collagen buffer solution; The chitosan acid solution is added dropwise to the recombinant collagen buffer solution to allow the chitosan particles and the recombinant collagen particles to self-assemble into composite nanoparticles.

2. The method for preparing the chitosan and recombinant collagen composite nanomaterial according to claim 1, characterized in that: The acid solution includes at least one of a lactic acid solution and an acetic acid solution.

3. The method for preparing the chitosan and recombinant collagen composite nanomaterial according to claim 1, characterized in that: The concentration of the acid solution is 1% w / v to 5% w / v.

4. The method for preparing the chitosan and recombinant collagen composite nanomaterial according to claim 1, wherein: The pH of the chitosan acid solution is 2-3.

5. The method for preparing the chitosan and recombinant collagen composite nanomaterial according to claim 1, characterized in that: The pH of the recombinant collagen buffer solution is 7-8.

6. The method for preparing the chitosan and recombinant collagen composite nanomaterial according to claim 1, characterized in that: The method further comprises: After the chitosan acid solution is added dropwise to the recombinant collagen buffer solution to obtain a composite solution, the pH of the composite solution is adjusted to 4-4.

5.

7. The method for preparing the chitosan and recombinant collagen composite nanomaterial according to claim 6, characterized in that: The step of adjusting the pH of the composite solution to 4 to 4.5 comprises: An alkaline solution is added to the composite nanoparticle solution for adjustment.

8. The method for preparing the chitosan and recombinant collagen composite nanomaterial according to claim 7, characterized in that: The alkaline solution is selected from at least one of a sodium bicarbonate solution and a sodium hydroxide solution.

9. A chitosan and recombinant collagen composite nanomaterial, characterized in that: The composite nanomaterial is prepared by the method for preparing the chitosan and recombinant collagen according to any one of claims 1 to 8; Optionally, the particle size of the composite nanoparticles is less than or equal to 300 nm.

10. Use of the chitosan and recombinant collagen composite nanomaterial according to claim 9 in the preparation of medicines or cosmetics.

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