A quaternary ammonium salt modified chitosan-based composite hydrogel and a preparation method thereof

Abstract

The application belongs to the technical field of hydrogel preparation, and discloses a quaternary ammonium salt modified chitosan-based composite hydrogel and a preparation method thereof. The composite hydrogel is obtained by mixing an O-quaternary ammonium salt chitosan O-HACC solution and a 3-maleimide propionic acid modified pullulan PL-MA solution according to a certain proportion and standing. The OHACC solution and the PL-MA solution are simply mixed, and the composite hydrogel is quickly prepared by using the reaction between the groups of the two compounds. The hydrogel has a connected porous structure, good self-healing performance, injectable performance, adhesion performance, antibacterial performance and biocompatibility. After the hydrogel in the application in situ encapsulates nanoparticles containing asiaticoside, it is found through a mouse full-thickness skin damage model that the drug-loaded hydrogel in the application can promote the rapid and perfect healing of a wound.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of composite hydrogel preparation, and particularly relates to a quaternary ammonium salt modified chitosan-based composite hydrogel and a preparation method thereof. BACKGROUND

[0002] Skin is the largest organ of the human body, and plays an important role in protecting the body from mechanical damage, maintaining homeostasis, perceiving external stimuli, and participating in immune responses. It is composed of epidermis, dermis and subcutaneous layer. Skin damage can be understood as a condition in which normal skin function is damaged or disordered under the action of external factors such as chemicals, physics or heat. The complete structure of the skin tissue is destroyed, resulting in a wound. Wound healing is a process of recovery and closure after skin damage, and is carried out through a coordinated and orderly time sequence. The whole process is divided into four stages, namely hemostasis, inflammation, proliferation and remodeling. The inflammation stage is a key stage affecting the speed and perfection of wound healing, and long-term inflammation can lead to the formation of scars. The formation of scars not only affects the appearance and function of the skin, but also causes psychological pain to the patient, and excessive scars can even cause loss of normal body function.

[0003] Therefore, it is necessary to develop a new type of composite hydrogel that can achieve scar-free wound repair. SUMMARY

[0004] The purpose of the present application is to provide a quaternary ammonium salt modified chitosan-based composite hydrogel, and to provide a preparation method thereof, which is another purpose of the present application. Based on quaternary ammonium salt chitosan and esterified pullulan, a new type of hydrogel dressing with multiple functions such as self-healing, injectability, adhesion and antibacterial property is prepared. Research shows that after the nanometer ion loaded with asperosaponin is mixed in the preparation of the hydrogel, the drug-loaded composite hydrogel prepared can achieve scar-free wound healing effect.

[0005] In order to achieve the above-mentioned purposes of the application, the technical scheme adopted by the present application is as follows:

[0006] A quaternary ammonium salt modified chitosan-based composite hydrogel, characterized in that the composite hydrogel is obtained by mixing and standing O-quaternary ammonium salt chitosan O-HACC solution and 3-maleimide propionic acid modified pullulan PL-MA solution in a certain proportion.

[0007] The mass fraction of the O-HACC solution and the PL-MA solution is 5w / v% and 4w / v% respectively, and the volume ratio of the mixture of the two is 1:1-4:1.

[0008] Before mixing, the nanoparticles loaded with asperosaponin are mixed with the PL-MA solution, and then mixed with the O-HACC solution.

[0009] The nano-particle loaded with asiaticoside is PDA@Zif-8-xAC, wherein x represents the concentration of added asiaticoside, the unit is mg / mL, and x is 0.5-4.0.

[0010] The preparation method of the quaternary ammonium salt modified chitosan-based composite hydrogel comprises the following steps:

[0011] Step S1, preparing O-quaternary ammonium salt chitosan O-HACC:

[0012] (1) Take 2.0-4.0 g of chitosan and add it to 90-120 mL of deionized water, while adding 10-15 mL of glacial acetic acid solution, stirring at room temperature for 1-2 h, and placing it in a 4℃ refrigerator for 12-20 h. Add 90-120 mL of anhydrous ethanol and stir until a uniform system is obtained; Take 10.0-12.0 g of benzaldehyde solution and dissolve it in 20-30 mL of anhydrous ethanol, and drop it into the chitosan solution within 2-3 h, stirring at high speed while dropping, and after the dropping is completed, the solution is stirred at 60-70℃ for 8-10 h, then 100-120 mL of anhydrous ethanol is added to terminate the reaction, then 100-120 mL of 1 mol / L NaOH solution is added to the solution to obtain a white precipitate, and the precipitate is washed repeatedly with anhydrous ethanol 4-5 times, and the precipitate product is vacuum dried at 50-55℃ for 48-50 h, then the dried product is added to 20-30 mL of 40 w / v% NaOH solution, stirred at 55-65℃ for 1.5-2.5 h, and then filtered, and the product is vacuum dried to obtain alkali-treated N-benzal chitosan Shiff-CS;

[0013] (2) Disperse the dried N-benzal chitosan in 80-100 mL of isopropyl alcohol solution, stir at room temperature for 1.5-2.5 h, then add 10.0-12.0 g of 2,3-epoxypropyltrimethylammonium chloride to the dispersion, continue to stir the system in a 70℃ water bath for 24-30 h, while using a condensation reflux device, after the reaction is completed, naturally cool to room temperature, precipitate the reaction mixture using acetone, filter the precipitate, and wash it with anhydrous ethanol, and vacuum dry the washed product to obtain quaternary ammonium salt grafted N-benzal chitosan (Shiff-HACC);

[0014] (3) The obtained quaternary ammonium salt grafted N-benzylidene chitosan is added to 140-160 mL of 0.25 mol / L hydrochloric acid ethanol solution and stirred for 22-26 h, then the pH of the system is adjusted to neutral with 1 g / mL Na2CO3 aqueous solution, then precipitated with acetone, then washed until the pH of the filtrate is neutral, then filtered and dried to obtain a crude product; the crude product is dialyzed in a dialysis bag with a molecular weight cutoff of 8-14 kD for 2-4 days, and the dialyzed product is freeze-dried to obtain a purified product O-quaternary ammonium salt chitosan O-HACC;

[0015] Step S2, preparation of PL-MA

[0016] First, 10.0-12.0 g of pullulan PL is weighed and added to 100-120 mL of dimethyl sulfoxide solution and stirred for 1-2 h, 3.0-4.0 g of N,N'-dicyclohexyl carbodiimide and 2.0-3.0 g of 1-hydroxybenzotriazole are added to the reaction solution, and stirring is continued for 1-2 h; 6.0-8.0 g of 3-maleimide propionic acid MA is dissolved in 10-20 mL of dimethyl sulfoxide solution, and added dropwise to the above reaction solution, and stirred at 37°C for 35-38 h, after the end, dialysis for 4-6 days in a dialysis bag with a molecular weight cutoff of 3 kD, and the dialyzed product is freeze-dried to obtain a purified product 3-maleimide propionic acid modified pullulan PL-MA;

[0017] Step S3, preparation of PDA@Zif-8-xAC

[0018] (1) 3.94 g of 2-methylimidazole is dissolved in 40 mL of methanol solution, and stirred until completely dissolved, then 1.31 g of zinc acetate dihydrate is added; continue to stir at room temperature for 20-26 h, after the reaction is completed, centrifuge at a speed of 8000-9000 r / min for 8-12 min, the centrifugal precipitate is washed with methanol for 2-4 times, and vacuum dried at 55-65°C for 12-14 h, the obtained product is named as Zif-8;

[0019] (2) 0.2-0.4 g of Zif-8 is added to 40-60 mL of anhydrous ethanol and stirred for 2-4 h, then 0.2-0.4 g of dopamine hydrochloride is added and stirred for 25-35 min, then 20-160 mg of asiaticoside is added and stirred for 1-1.5 h, the pH of the above solution is adjusted to 8.0-9.0 with 0.8-1 mol / L sodium hydroxide solution, and stirring is continued for 22-26 h, after the reaction is completed, the centrifugation is carried out while washing with anhydrous ethanol, the centrifugation time is 8-12 min, the speed is 8000-9000 r / min, and the centrifugal precipitate is vacuum dried, the obtained product is named as PDA@Zif-8-xAC; wherein x represents the concentration of added asiaticoside, unit mg / mL;

[0020] Step S4, preparation of composite hydrogel

[0021] The following method A or method B is selected to prepare the composite hydrogel:

[0022] Method A, first, 0.25g O-HACC and 0.2g PL-MA are dissolved in 5mL deionized water respectively to form 5w / v% O-HACC homogeneous solution and 4w / v% PL-MA homogeneous solution respectively, then the two are mixed according to a certain proportion and left to obtain a composite hydrogel, the obtained hydrogel is named O-HACC / PL-MAxy, hereinafter referred to as HPxy, where x and y represent the volume of O-HACC solution and PL-MA solution added when the gel is formed;

[0023] Method B, 0.1g PDA@Zif-8-xAC powder is added to a total volume of 5mL PLMA solution with a concentration of 4w / v%, then mixed with an equal volume of O-HACC solution with a concentration of 5w / v% to form a gel, the obtained hydrogel is named AA@O-HACC / PL-MA11, and is abbreviated as AA@HP11.

[0024] Compared with the prior art, the present application has the following technical effects:

[0025] 1) The present application quickly prepares a composite hydrogel by simply mixing OHACC solution and PL-MA solution and utilizing the reaction between the groups of the two compounds. The hydrogel has a connected porous structure, good self-healing performance, injectability, adhesion, antibacterial performance and biocompatibility.

[0026] 2) After the hydrogel of the present application in situ encapsulates nanoparticles containing asiaticoside, it is found through a mouse full-thickness skin injury model that the drug-loaded hydrogel of the present application can promote rapid and perfect healing of the wound. Immunohistochemical staining shows that the drug-loaded hydrogel can up-regulate CD31 expression and down-regulate TGF-β1 and α-SMA expression to promote scarless repair of the wound. It provides a new means for subsequent research on the use of hydrogel dressings for scarless wound repair. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Nanoparticle drug loading test. (a) Particle size distribution; (b) asiaticoside release curve; (c) drug loading amount statistics; (d) drug loading efficiency statistics;

[0028] Figure 2 Preparation and morphology characterization of hydrogel. (a) FT-IR spectrum of HP hydrogel; (b) macroscopic picture of HP hydrogel; (c) micro-morphology of hydrogel (the first row scale = 250μm, the second row is an enlarged image scale = 500μm);

[0029] Figure 3Rheological test of composite hydrogel. (a) and (b) Frequency sweep mode and strain sweep mode of HP hydrogel; (c) and (d) Frequency sweep mode and strain sweep mode of AA@HP11 hydrogel;

[0030] Figure 4 Self-healing test of hydrogel. Rheological step cycle mode (a) HP11; (b) AA@HP11; Macroscopic self-healing photo (c) HP11; (d) AA@HP11;

[0031] Figure 5 Adhesion and injectability test of hydrogel. (a) Adhesion photo of HP hydrogel with glass, metal, plastic and human skin; (b) Rheological shear-thinning test, (picture is a schematic diagram of hydrogel injectability);

[0032] Figure 6 Antibacterial performance of hydrogel. (a) Co-culture photo of bacteria and hydrogel in glass test tube (left is E. coli, right is S. aureus); (b) Statistical data of antibacterial rate of E. coli and S. aureus;

[0033] Figure 7 Cell activity evaluation. (a) In vitro cytotoxicity of HP hydrogel extract co-cultured with HUVEC cells for 24h, 48h; (b) Fluorescent staining photo of HUVEC cells cultured for 24h, 48h (scale bar = 200μm);

[0034] Figure 8 Cell scratch healing experiment. (a) Scratch healing photo of HUVEC cells co-cultured with HP hydrogel extract for 6h, 24h (scale bar = 100μm); (b) Quantitative statistics of HUVEC cell healing;

[0035] Figure 9 In vivo animal model healing experiment. (a) Schematic diagram of the process of establishing mouse wound healing model; (b) Photo of mouse wound healing at different time periods; (c) Schematic diagram of wound healing at different time periods; (d) Statistics of wound healing area at different time points;

[0036] Figure 10 Histopathological analysis. (a) H&E (b) Masson staining photo (scale bar: 4mm and 400μm); (c) Statistical data of wound epithelial tissue thickness; (d) Statistical data of wound hair follicle number; (e) Statistical data of wound collagen ratio;

[0037] Figure 11Figure 12d Wound immunohistochemical staining analysis. (a) CD31, (b) TGF-β1, (c) a-SMA staining map (scale bar, 100 pm); (d) CD31, (e) TGF-β1, (f) a-SMA expression level quantitative analysis. DETAILED DESCRIPTION

[0038] The application will be further described in detail below with reference to the accompanying drawings.

[0039] Example 1

[0040] The quaternary ammonium salt modified chitosan-based composite hydrogel of the present embodiment is obtained by mixing and standing an O-quaternary ammonium salt chitosan O-HACC solution and a 3-maleimide propionic acid modified pullulan PL-MA solution in a certain proportion. The preparation method comprises the following steps:

[0041] Step S1, preparation of O-quaternary ammonium salt chitosan (O-HACC):

[0042] (1) Take 2.0 g of chitosan and add it to 90 mL of deionized water, while adding 10 mL of glacial acetic acid solution, stir at room temperature for 1 h, and then place it in a 4°C refrigerator for 18 h. Then add 100 mL of anhydrous ethanol and stir until a uniform system is obtained. Dissolve 10.0 g of benzaldehyde solution in 20 mL of anhydrous ethanol, and add it dropwise into the chitosan solution within 2-3 h, while stirring at high speed with a magnetic stirrer. After the addition is completed, stir the solution at 60°C for 10 h, then add 100 mL of 1 mol / L NaOH solution to obtain a white precipitate, and wash the precipitate with anhydrous ethanol repeatedly for 5 times. Then, vacuum dry the product at 50°C for 48 h, and then add the dried product to 20 mL of 40 w / v% NaOH solution, stir at 60°C for 2 h, and then perform suction filtration. Vacuum dry the obtained product to obtain alkali-treated N-benzylidene chitosan (Shiff-CS);

[0043] (2) Disperse the dried N-benzylidene chitosan in 100 mL of isopropyl alcohol solution, stir at room temperature for 2 h, then add 10.0 g of 2,3-epoxypropyltrimethylammonium chloride to the dispersion, and then place the system in a 70°C water bath and continue to stir for 24-30 h, while using a condensation reflux device. After the reaction is completed, naturally cool to room temperature, precipitate the reaction mixture using acetone, suction filter the precipitate, and wash with anhydrous ethanol. Vacuum dry the washed product to obtain quaternary ammonium salt grafted N-benzylidene chitosan (Shiff-HACC);

[0044] (3) The obtained quaternary ammonium salt grafted N-benzylidene chitosan is added to 150 mL of 0.25 mol / L hydrochloric acid ethanol solution and stirred for 24 h, then the pH of the system is adjusted to neutral with 1 g / mL Na2CO3 aqueous solution, and then precipitated with acetone, and then washed until the pH of the filtrate is neutral, and then filtered and dried to obtain a crude product; the crude product is dialyzed in a dialysis bag with a molecular weight cutoff of 8-14 kD for 3 days, and the dialyzed product is freeze-dried to obtain a purified product O-quaternary ammonium salt chitosan O-HACC;

[0045] Step S2, preparation of PL-MA

[0046] First, 10.0 g of pullulan PL is added to 100 mL of dimethyl sulfoxide solution and stirred for 1 h, 3.0 g of N,N'-dicyclohexyl carbodiimide and 2.0 g of 1-hydroxybenzotriazole are added to the reaction solution, and stirring is continued for 1 h; 6.0 g of 3-maleimide propionic acid MA is dissolved in 10 mL of dimethyl sulfoxide solution, and then added dropwise to the above reaction solution, and stirred at 37°C for 36 h; after completion, dialysis is performed in a dialysis bag with a molecular weight cutoff of 3 kD for 5 days, and the dialyzed product is freeze-dried to obtain a purified product 3-maleimide propionic acid modified pullulan PL-MA;

[0047] Step S3, preparation of composite hydrogel

[0048] First, 0.25 g of O-HACC and 0.2 g of PL-MA are dissolved in 5 mL of deionized water to form 5 w / v% of O-HACC homogeneous solution and 4 w / v% of PL-MA homogeneous solution, respectively, then the two are mixed according to a certain ratio and left to stand to obtain a composite hydrogel, and the obtained hydrogel is named O-HACC / PL-MAxy, which is hereinafter referred to as HPxy, where x and y represent the volume of O-HACC solution and PL-MA solution added when the gel is formed.

[0049] Example 2

[0050] The quaternary ammonium salt modified chitosan-based composite hydrogel of the present embodiment is the same as that of Example 1, except that before mixing, the PDA@Zif-8-xAC nanoparticles loaded with asiaticoside and the PL-MA solution are mixed, and then mixed with the O-HACC solution. Wherein, x represents the concentration of asiaticoside added, unit: mg / mL, x takes a value of 0.5-4.0, and the present embodiment is 2.0.

[0051] The preparation method of the nanoparticles PDA@Zif-8-xAC loaded with asiaticoside is as follows:

[0052] (1) Take 3.94g 2-methylimidazole and dissolve it in 40mL methanol solution, stir until completely dissolved, then add 1.31g zinc acetate dihydrate; continue stirring at room temperature for 24h, after the reaction is completed, centrifuge at 8500r / min for 10min, wash the precipitate with methanol for 3 times, vacuum dry at 60℃ for 12h, the obtained product is named as Zif-8;

[0053] (2) Take 0.2g Zif-8 and add it into 40mL anhydrous ethanol, stir for 3h, then add 0.2g dopamine hydrochloride and stir for 30min, then add 80mg asiaticoside and stir for 1h, adjust the pH of the above solution to 8.5 with 1mol / L sodium hydroxide solution, continue stirring for 24h, after the reaction is completed, centrifuge and wash with anhydrous ethanol, centrifuging and washing with anhydrous ethanol means that after the reaction is completed, centrifuge, wash the precipitate with anhydrous ethanol, then centrifuge again, repeat the washing with anhydrous ethanol, this example is three times, each time the centrifuging time is 10min, the speed is 8500r / min, vacuum dry the centrifuged precipitate, the obtained product is named as PDA@Zif-8-2.0AC; wherein 2.0 represents the concentration of asiaticoside, the unit is mg / mL, which is the mass ratio of asiaticoside to methanol solution.

[0054] When preparing the hydrogel, take 0.1g PDA@Zif-8-xAC powder and add it into 5mL PLMA solution with a concentration of 4w / v%, then mix with O-HACC solution with a concentration of 5w / v% in equal volume to form a gel, the obtained hydrogel is named as AA@O-HACC / PL-MA11, abbreviated as AA@HP11.

[0055] Example 3

[0056] The quaternary ammonium salt modified chitosan-based composite hydrogel of this example is the same as example 1, except that step S1, O-quaternary ammonium salt chitosan O-HACC is prepared:

[0057] (1) Take 2.0 g of chitosan and add it to 90 mL of deionized water, while adding 10 mL of glacial acetic acid solution, stir at room temperature for 1 h, and store in a 4°C refrigerator for 12 h. Add 90 mL of anhydrous ethanol and stir until a uniform system is obtained. Dissolve 10.0 g of benzaldehyde in 20 mL of anhydrous ethanol and add it dropwise to the chitosan solution within 2 h, stirring at high speed with a magnetic stirrer. After the addition is complete, stir the solution at 60°C for 8 h, then add 100 mL of anhydrous ethanol to cool and terminate the reaction. Then add 100 mL of 1 mol / L NaOH solution to the solution to obtain a white precipitate, and wash the precipitate repeatedly with anhydrous ethanol 4 times. Dry the precipitate product under vacuum at 50°C for 48 h, then add the dried product to 20 mL of 40 w / v% NaOH solution, stir at 55-65°C for 1.5 h, and then perform suction filtration. Dry the resulting product under vacuum to obtain alkali N-benzylidene chitosan Shiff-CS;

[0058] (2) Disperse the dried N-benzylidene chitosan in 80 mL of isopropyl alcohol solution, stir at room temperature for 1.5 h, then add 10.0 g of 2,3-epoxypropyltrimethylammonium chloride to the dispersion, and continue stirring in a 70°C water bath for 24 h while using a condensation reflux device. After the reaction is complete, naturally cool to room temperature, precipitate the reaction mixture using acetone, suction filter the precipitate, and wash with anhydrous ethanol. Dry the washed product under vacuum to obtain quaternary ammonium salt grafted N-benzylidene chitosan (Shiff-HACC);

[0059] (3) Add the obtained quaternary ammonium salt grafted N-benzylidene chitosan to 140 mL of 0.25 mol / L hydrochloric acid ethanol solution and stir for 22 h. Then adjust the pH of the system to neutral with 1 g / ml Na2CO3 aqueous solution, precipitate again using acetone, and then wash until the filtrate pH is neutral. Suction filter and dry to obtain a crude product. Dialyze it in a dialysis bag with a molecular weight cutoff of 8 kD for 2-4 d, and freeze-dry the dialyzed product to obtain the purified product O-quaternary ammonium salt chitosan O-HACC;

[0060] Step S2, preparation of PL-MA

[0061] First, take 10.0 g of pullulan PL and add it to 100 mL of dimethyl sulfoxide solution and stir for 1 h. Add 3.0 g of N,N'-dicyclohexyl carbodiimide and 2.0 g of 1-hydroxybenzotriazole to the reaction solution and continue stirring for 1 h. Dissolve 6.0 g of 3-maleimide propionic acid MA in 10 mL of dimethyl sulfoxide solution and add it dropwise to the above reaction solution. Stir at 37°C for 35 h, then dialyze in a dialysis bag with a molecular weight cutoff of 3 kD for 4 d, and freeze-dry the dialyzed product to obtain the purified product 3-maleimide propionic acid modified pullulan PL-MA;

[0062] Example 4

[0063] The quaternary ammonium salt modified chitosan-based composite hydrogel of this example is different from Example 1 in that step S1, preparation of O-quaternary ammonium salt chitosan O-HACC:

[0064] (1) 4.0 g of chitosan was weighed into 120 mL of deionized water, 15 mL of glacial acetic acid solution was added, and it was stirred at room temperature for 2 h, and then it was placed in a 4°C refrigerator for 20 h. 120 mL of anhydrous ethanol was added and stirred until a uniform system was obtained. 12.0 g of benzaldehyde solution was dissolved in 30 mL of anhydrous ethanol, and was added dropwise into the chitosan solution within 3 h, while stirring at high speed. After the addition was completed, the solution was stirred at 70°C for 10 h, and then 120 mL of anhydrous ethanol was added to terminate the reaction. Then 120 mL of 1 mol / L NaOH solution was added to the solution to obtain a white precipitate, which was washed repeatedly with anhydrous ethanol for 5 times. The precipitate was dried in a vacuum oven at 55°C for 50 h, and then the dried product was added to 30 mL of 40 w / v% NaOH solution, and stirred at 65°C for 2.5 h, and then filtered. The product was dried in a vacuum oven to obtain the alkaliized N-benzylidene chitosan Shiff-CS;

[0065] (2) The dried N-benzylidene chitosan was dispersed in 100 mL of isopropanol solution, and stirred at room temperature for 2.5 h. Then 12.0 g of 2,3-epoxypropyltrimethylammonium chloride was added to the dispersion, and the system was placed in a 70°C water bath and continued to stir for 30 h, while using a condensation reflux device. After the reaction was completed, it was naturally cooled to room temperature, and the reaction mixture was precipitated using acetone, and the precipitate was filtered and washed with anhydrous ethanol. The washed product was dried in a vacuum oven to obtain the quaternary ammonium salt grafted N-benzylidene chitosan (Shiff-HACC);

[0066] (3) The obtained quaternary ammonium salt grafted N-benzylidene chitosan was added to 160 mL of 0.25 mol / L hydrochloric acid ethanol solution and stirred for 26 h. Then the pH of the system was adjusted to neutral using 1 g / mL Na2CO3 aqueous solution. The precipitate was precipitated again using acetone, and then washed until the pH of the filtrate was neutral. The precipitate was filtered and dried to obtain a crude product. The crude product was dialyzed in a dialysis bag with a molecular weight cutoff of 8-14 kD for 4 d, and the dialyzed product was freeze-dried to obtain the purified product O-quaternary ammonium salt chitosan O-HACC;

[0067] Step S2, preparation of PL-MA

[0068] Firstly, 12.0 g of pullulan PL is added into 120 mL of dimethyl sulfoxide solution and stirred for 2 h, 4.0 g of N,N'-dicyclohexyl carbodiimide and 2.0-3.0 g of 1-hydroxybenzotriazole are added into the reaction solution, and stirring is continued for 2 h; 8.0 g of 3-maleimide propionic acid MA is dissolved in 20 mL of dimethyl sulfoxide solution, and then added dropwise into the above reaction solution, and stirring is continued at 37 DEG C for 38 h, after which the dialysis product is dialyzed in a dialysis bag with a molecular weight cutoff of 3 kD for 6 d, and the dialysis product is freeze-dried to obtain the purified product 3-maleimide propionic acid modified pullulan PL-MA.

[0069] Preparation of drug-loaded nanoparticles with different concentrations according to Comparative Example 1

[0070] The method is the same as that of Example 2 for preparing PDA@Zif-8-xAC, except that 160 mg, 40 mg and 20 mg of asiaticoside are added respectively under the same conditions to prepare drug-loaded nanoparticles with different concentrations as experimental control groups, and the group without adding asiaticoside is used as a blank control group, and is named as PDA@Zif-8;

[0071] Performance test

[0072] 1. Drug loading test of nanoparticles with different concentrations

[0073] The PDA@Zif-8-xAC nanoparticles of the application are prepared by one-pot method, in which the Zif-8 nanoparticles are first modified with PDA, and then AC is introduced into the surface of PDA@Zif-8. TEM is used to observe the morphology of Zif-8, PDA@Zif-8 and PDA@Zif-8-xAC. On this basis, the size of the nanoparticles is further tested and analyzed Figure 1 a). The average particle size of Zif-8 is about 220 nm, while the average particle size of PDA@Zif-8 increases to about 825 nm due to the loading of PDA. After AC modification, the particle size of PDA@Zif-8-xAC nanoparticles increases with the amount of AC added. In addition, the experimental results show that with the increase of AC content, the release performance Figure 1 b), drug loading capacity Figure 1 c) and drug loading efficiency Figure 1d) also increased. Among them, the drug loading of PDA@Zif-8-4.0AC, PDA@Zif-8-2.0AC, PDA@Zif-8-1.0AC and PDA@Zif-8-0.5AC were 76.85218±4.41532mg, 68.11249±1.72351mg, 34.18581±0.74217mg, 17.99176±0.9818mg, respectively. Through the analysis of the results, it can be seen that increasing the concentration of asiaticoside in the solution in the loading experiment can significantly improve the drug loading of PDA@Zif-8 nanoparticles. However, by comparing the drug loading data of PDA@Zif-8-4.0AC and PDA@Zif-8-2.0AC, it can be seen that when the concentration of asiaticoside increases to a certain level, the MOF structure of PDA@Zif-8 nanoparticles has reached the maximum drug loading capacity. Continue to improve the concentration of asiaticoside in the solution, the improvement of drug loading tends to be not obvious. From the above drug loading results, it can be concluded that there is a significant difference between the drug loading efficiency of asiaticoside and its added amount, so the drug loading efficiency of PDA@Zif-8 nanoparticles is further tested. The drug loading efficiency of PDA@Zif-8-4.0AC, PDA@Zif-8-2.0AC, PDA@Zif-8-1.0AC and PDA@Zif-8-0.5AC were 48.03261±2.75958%, 85.14061±2.15439%, 85.46452±1.85543%, 89.95878±4.90901%, respectively. It can be found that the loading efficiency of PDA@Zif-8-4.0AC is significantly reduced, which is mainly due to the drug loading of PDA@Zif-8 nanoparticles has reached the upper limit. Even if the concentration of the solution is increased, the drug loading is not significantly increased, so it leads to a sharp decrease in its loading efficiency. According to the comparison of all test results, PDA@Zif-8-2.0AC is selected as the best experimental scheme to participate in the preparation of the subsequent drug-loaded hydrogel.

[0074] 2. Preparation and morphology characterization of hydrogel

[0075] The preparation and morphology characterization results of the hydrogel are shown in Figure 2 The O-HACC and PL-MA solutions were mixed at room temperature according to a certain proportion to prepare the HP composite hydrogel. The mechanism of the reaction is that the amino group on the O-HACC and the double bond on the PL-MA occur Michael addition reaction. In the FT-IR spectrum of the HP hydrogel Figure 1 a), the double bond peak of PL-MA at 1700cm -1 -1 disappeared, which may be consumed by the reaction during the gelation; in addition, the peak of the double bond of PL-MA at 1070-1156cm -1The peak stretching vibrations became more pronounced, possibly due to an increase in newly formed carbon-nitrogen single bonds. Meanwhile, after mixing the prepared solutions and inverting them for a period of time, it was observed that the solution did not flow along the test tube wall. Figure 2 b) indicates that the two have completely reacted and formed a new gel-like product. All of these results demonstrate the successful preparation of the HP hydrogel.

[0076] Microscopic observation of the morphology of the hydrogel ( Figure 2 c) It was found that all hydrogels had an interconnected and porous internal structure. As a wound dressing, the porous structure facilitates the flow of oxygen, moisture, and nutrients, and also maintains a moist wound environment. Comparing AA@HP11 hydrogel and HP hydrogel, both are porous structures, but magnified SEM images revealed that the surface of HP hydrogel was smooth while the surface of AA@HP11 hydrogel was rugged, indicating successful drug loading.

[0077] 3. Rheological property analysis of composite hydrogels

[0078] When hydrogels are used in wound dressings, they need to possess a certain degree of rigidity to prevent deformation or breakage under external forces in dynamic environments, thereby better protecting the wound. Two rheological scanning tests were performed on the hydrogels: frequency and strain. In frequency scanning mode (… Figure 3 (a, c) With constant temperature (37℃) and constant strain (1%), the storage modulus (G') of the hydrogels before structural failure was higher than the loss modulus (G”), indicating that all hydrogel materials had relatively stable structures and good mechanical properties. Among them, HP11 hydrogels had the highest G' of approximately 100 Pa, while AA@HP11 hydrogels had a maximum G' of approximately 4200 Pa, much higher than HP hydrogels. This is because the addition of nanoparticles enhanced the mechanical strength of the hydrogels. (In strain scanning mode) Figure 3 (b, d) With constant temperature (37℃) and frequency (1Hz), as the force intensity increased, the hydrogel gradually reached its maximum withstand force, eventually leading to the destruction of the three-dimensional network. HP11 fractured at approximately 650%, HP21 at approximately 400%, HP41 at approximately 140%, and AA@HP11 at approximately 160%. The frequency and strain test results demonstrate the significant advantages of hydrogels in promoting skin wound healing.

[0079] 4. Analysis of the self-healing properties of composite hydrogels

[0080] Self-healing hydrogels possess excellent physical and biological properties, maintaining structural and functional integrity while extending material lifespan. For example... Figure 4a, rheological test results show that when the strain is set to 1%, the storage modulus (G') is significantly higher than the loss modulus (G"), indicating that the sample exhibits typical solid hydrogel properties at this strain, with strong elasticity and stable network structure. However, when the strain increases to 1000%, the G' value decreases, showing that the elasticity of the hydrogel is significantly weakened, and the G" value exceeds G', indicating that the crosslinked network structure of the hydrogel is destroyed at a larger strain, and the hydrogel exhibits typical fluid behavior, losing its original solid form. The same behavior is also observed in the rheological step test of AA@HP11 hydrogel Figure 4 b). These all indicate that the hydrogel has good self-healing properties, and the addition of nanoparticles does not affect this property. The composite hydrogel can also be found to be self-repaired when the hydrogel is cut and reassembled Figure 4 c, d). The self-repairing ability of the hydrogel comes from the reversible hydrogen bonding interaction between the molecules.

[0081] 5. Analysis of the adhesion and injectability of the composite hydrogel

[0082] The adhesion of the hydrogel can help it to be fixed on the surface of the wound, avoiding slipping or displacement, thereby improving the healing effect on the damaged tissue. The adhesion properties of the hydrogel were characterized by macroscopic adhesion experiments, and the results are shown in Figure 5 a, the prepared hydrogel can adhere to objects made of different materials such as glass, metal and plastic, and can also adhere to human skin without falling off. This indicates that the HP composite hydrogel has good adhesion properties. Injectable hydrogel has the advantages of fluidizing under shear stress and then restoring the original mechanical properties, and can be precisely applied to irregularly shaped wound defects in a highly controlled manner. The injectability of HP hydrogel was verified by rheological shear-thinning test Figure 5 b). It was found that at 37°C, the viscosity of the HP hydrogel decreased with increasing shear rate in the range of 0.1-100 s -1 This shear thinning behavior indicates that the HP hydrogel has good injectability. Using a 0.5x24 inch syringe, the hydrogel can be easily drawn into a "five-pointed star" pattern Figure 5 b inset).

[0083] 6. Analysis of the antibacterial properties of the composite hydrogel

[0084] Antibacterial hydrogel is important in wound healing, as it not only provides the necessary moist environment to promote healing, but also effectively prevents bacterial infection and reduces inflammation and delayed healing caused by infection. The co-culture of bacterial solution and sample was used to evaluate the antibacterial ability of the hydrogel. As shown in Figure 6 a, the control group of bacterial solution is more turbid, while the experimental group with hydrogel is clearer. This indicates that the HP hydrogel has good antibacterial ability. According to the bacteriostatic rate statistics,Figure 6 b), HP11 hydrogel showed the best antibacterial effect on both E. coli and S. aureus, with inhibition rates of 69.85 ± 7.33% and 76.60 ± 1.55%, respectively. In addition, the inhibition ability of HP21 hydrogel on the two bacteria was also higher than that of HP41 hydrogel. When co-cultured with E. coli, HP21 was 46.86 ± 16.06%, and HP41 was 29.21 ± 5.54%. When co-cultured with S. aureus, HP21 was 68.60 ± 7.18%, and HP41 was 55.41 ± 3.59%. The antibacterial ability of HP hydrogel is due to the antibacterial ability of chitosan itself and the increase in the antibacterial ability of the material due to the introduction of quaternary ammonium salt ions.

[0085] 6. Biocompatibility analysis of composite hydrogel

[0086] Biocompatibility and safety are indispensable properties of hydrogel used as wound dressings. The cell toxicity of HP hydrogel was verified by co-culturing the hydrogel extract with HUVEC cells. The survival rate of HUVEC cells was evaluated using a CCK-8 cell proliferation assay, and the survival rate of all cells was higher than 100%( Figure 7 a) after co-culturing for 24 h and 48 h, and there was no difference from the control group, indicating that the hydrogel has good biocompatibility. At the same time, the live / dead cell activity of the above cultured cells was detected, and live cells and dead cells were labeled with green and red fluorescent dyes, respectively, as shown in Figure 7 b, most of the cells were dyed green, and almost no dead cells appeared. These experimental results prove that HP hydrogel has excellent biocompatibility and is not dangerous for use as a wound dressing. Cell scratch healing experiments can further verify the cytotoxicity of HP hydrogel. The hydrogel extract was co-cultured with HUVEC cells for 6 h and 24 h, as shown in Figure 8 a, the cells showed a tendency of migration and healing, among which HP11 hydrogel had the best scratch healing effect. Especially after co-culturing for 24 h, the migration number of HP11 was 634( Figure 8 b) HP hydrogel can promote the migration of endothelial cells, which is also crucial for the use of hydrogel for wound healing.

[0087] 7. In vivo wound healing analysis

[0088] The effect of wound scar-free healing of hydrogel dressing loaded with asiaticoside was evaluated by mouse wound healing model experiment. A full-thickness skin wound with a diameter of 1 cm was made on the back of the mouse, and then the control group was treated with normal saline, and the experimental group was treated with HP11 hydrogel and AA@HP11 hydrogel, respectively. The wound healing was recorded by digital camera at 0, 3, 6, 9, and 12 d after operation( Figure 9 a). The wound healing results at different time periods during the experiment are shown inFigure 9 As shown in Figures b and d, the wound area in each group began to decrease on day 3, indicating that the wounds were slowly healing. On day 6, the wound area further decreased, and the trend of the experimental group having a smaller wound area than the control group became more pronounced. On day 9, the wound area of ​​the experimental group using AA@HP11 hydrogel was 13.48 mm². 2 The HP11 hydrogel is 21.51 mm thick. 2 The control group had a diameter of 39.28 mm. 2 This indicates that the drug-loaded hydrogel promotes faster wound healing. On day 12, the control group wounds were not yet fully healed, while the wounds in the AA@HP11 hydrogel group and the HP11 hydrogel group were almost completely healed. More importantly, compared to the HP11 hydrogel group, the AA@HP11 hydrogel group showed almost no scarring. This demonstrates that the AA@HP11 hydrogel loaded with asiaticoside promotes scarless wound healing.

[0089] 8. Histological analysis

[0090] The degree of wound healing was further assessed through histological analysis, including H&E staining and Masson staining. H&E staining was performed on the healed rat skin after different treatments on days 6 and 12. Figure 10 a). The integrity and thickness of the epidermis are important indicators for assessing the degree of wound healing. As shown in the figure, the AA@HP11 group showed the best histological results, with more wound fibrosis observed. Figure 10 c) and the presence of numerous hair follicles in granulation tissue ( Figure 10 d). To further investigate the healing ability of AA@HP11, Masson staining was used to analyze collagen deposition in skin tissue. Figure 10 b displays images and Figure 10 Quantitative statistical results showed that collagen deposition was almost undetectable in the control group. However, the degree of collagen deposition was significantly increased in the HP11 group, while the AA@HP11 group showed the highest level of collagen deposition. This indicates that AA@HP11 achieved ideal wound healing efficiency by accelerating the reconstruction of the extracellular matrix and tissue remodeling.

[0091] 9. Immunohistochemical analysis

[0092] CD31 is a transmembrane protein widely expressed on the surface of vascular endothelial cells, platelets, and immune cells. It participates in cell-cell adhesion, platelet aggregation, vascular endothelial stability, and immune response regulation, playing a crucial role in angiogenesis during wound healing. Therefore, CD31 antibody was used to stain sections of wound healing tissue from mice on day 12 for immunofluorescence staining to assess angiogenesis. The results are as follows: Figure 11a, d, the expression level of CD31 in the tissue of the wound site of the AA@HP11 hydrogel group on the 12th day was the highest compared with the control group and the HP11 hydrogel group, which indicated that more new blood vessels were regenerated, which was beneficial to the rapid repair of damaged tissues and promoted wound healing. TGF-β1 is a key regulatory factor in the process of wound healing and tissue repair, but the balance of its expression is very important. Excessive expression of TGF-β1 can lead to excessive collagen synthesis and fibrosis, and further cause scar formation and fibrotic diseases. The TGF-β1 immunohistochemical staining was performed on the mouse wound group, and the results of the control group, the HP11 hydrogel group and the AA@HP11 hydrogel group were as follows Figure 11 b, e, it is obvious that the expression amount of TGF-β1 of the AA@HP11 hydrogel group is lower than that of the previous two groups, which indicates that the hydrogel loaded with asiaticoside can inhibit the expression of TGF-β1, so as to realize the anti-fibrosis and reduce the effect of scar formation. α-SMA (smooth muscle cell α-actin) is a marker of myofibroblasts. Myofibroblasts play an important role in the process of wound healing and scar formation, can promote the healing of the wound surface by contraction and produce a large amount of collagen to repair damaged tissues. However, excessive myofibroblasts and α-SMA expression will lead to the proliferation of scars. Therefore, the expression of α-SMA is of great significance to the study of scar-free wound healing. It can be found from the results of α-SMA immunohistochemical staining that Figure 11 c, f) the expression amount of α-SMA of the AA@HP11 hydrogel group is the lowest, which indicates that the AA@HP11 hydrogel has great potential in reducing scar formation. In summary, the above results all indicate that the AA@HP11 hydrogel has a positive promoting effect on scar-free wound healing, which may be due to the effect of asiaticoside.

[0093] In summary, the OHACC solution and the PL-MA solution are mixed to quickly prepare the composite hydrogel by the reaction of the groups between the compounds. Subsequently, through a series of related researches, it is found that the hydrogel has a connected porous structure, good self-healing performance, injectable performance, adhesion performance, antibacterial performance and biocompatibility. In addition, after the hydrogel in situ encapsulates nanoparticles containing asiaticoside, through a mouse full-thickness skin damage model, it is found that the drug-loaded hydrogel can promote the rapid and perfect healing of the wound. Immunohistochemical staining finds that the drug-loaded hydrogel can up-regulate the expression of CD31 and down-regulate the expression of TGF-β1 and α-SMA to promote scar-free repair of the wound.

[0094] The above-mentioned embodiments provided by the present application are only the preferred embodiments of the present application, and are not intended to limit the present application in other forms. Any person skilled in the art can make changes or modifications to the equivalent embodiments with the disclosed technical content. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application without departing from the technical solution of the present application still belongs to the protection scope of the technical solution of the present application.

Claims

1. A quaternary ammonium salt modified chitosan-based composite hydrogel, characterized in that, The composite hydrogel was obtained by mixing and allowing to stand a certain ratio of O-quaternary ammonium salt chitosan O-HACC solution and pullulan polysaccharide PL-MA solution modified with 3-maleimide propionic acid; the mass-volume fractions of the O-HACC solution and the PL-MA solution were 5 w / v% and 4 w / v, respectively, and the mixing volume ratio of the two was 1:1-4:

1.

2. The quaternary ammonium salt modified chitosan-based composite hydrogel according to claim 1, characterized in that, Before mixing, the nanoparticles loaded with asiaticoside were mixed with the PL-MA solution, and then mixed with the O-HACC solution.

3. The quaternary ammonium salt modified chitosan-based composite hydrogel according to claim 2, characterized in that, The nanoparticles loaded with asiaticoside are PDA@Zif-8-xAC, where x represents the concentration of asiaticoside added, in mg / mL, and the value of x ranges from 0.5 to 4.

0.

4. The method for preparing the quaternary ammonium salt modified chitosan-based composite hydrogel according to any one of claims 1-3, characterized in that, Includes the following steps: Step S1: Preparation of O-quaternary ammonium salt chitosan O-HACC: (1) Weigh 2.0-4.0g of chitosan and add it to 90-120mL of deionized water, while adding 10-15mL of glacial acetic acid solution. Stir together at room temperature for 1-2 hours, then place in a refrigerator at 4℃ for 12-20 hours. Add 90-120mL of anhydrous ethanol and stir until a homogeneous system is obtained. Dissolve 10.0-12.0g of benzaldehyde solution in 20-30mL of anhydrous ethanol and add it dropwise to the chitosan solution over 2-3 hours while stirring with a high-speed magnetic stirrer. After the addition is complete, stir the solution at 60-70℃ for 8-10 hours. Then add 100-120mL of anhydrous ethanol to cool and terminate the reaction. Next, add 100-120mL of 1mol / L NaOH solution to the solution to obtain a white precipitate. Wash the precipitate repeatedly with anhydrous ethanol 4-5 times. Vacuum dry the precipitate at 50-55℃ for 48-50 hours. Then add the dried product to 20-30mL of anhydrous ethanol. The product was stirred in a 40 w / v% NaOH solution at 55-65℃ for 1.5-2.5 h and then filtered. The product was then dried under vacuum to obtain alkalized N-benzylidene chitosan Shiff-CS. (2) Disperse the dried N-benzylidene chitosan in 80-100 mL of isopropanol solution, stir at room temperature for 1.5-2.5 h, add 10.0-12.0 g of 2,3-epoxypropyltrimethylammonium chloride to the dispersion, place the system in a 70 °C water bath and continue stirring for 24-30 h, while using a reflux condenser. After the reaction is complete, cool naturally to room temperature, precipitate the reaction mixture with acetone, filter the precipitate, wash with anhydrous ethanol, and vacuum dry the washing product to obtain quaternary ammonium salt grafted N-benzylidene chitosan Shiff-HACC; (3) The obtained quaternary ammonium salt grafted N-benzylidene chitosan was added to 140-160 mL of 0.25 mol / L hydrochloric acid ethanol solution and stirred for 22-26 h. The pH of the system was then adjusted to neutral with 1 g / mL Na2CO3 aqueous solution. The system was precipitated with acetone again, and then washed until the pH of the filtrate was neutral. The solution was then filtered and dried to obtain the crude product. Dialyze it in a dialysis bag with a molecular weight cutoff of 8-14 kD for 2-4 days. The dialysis product is freeze-dried to obtain the purified product O-quaternary ammonium salt chitosan O-HACC. Step S2: Preparation of PL-MA First, weigh 10.0-12.0 g of pullulan (PL) and add it to 100-120 mL of dimethyl sulfoxide solution, stirring for 1-2 h. Then, add 3.0-4.0 g of N,N'-dicyclohexylcarbodiimide and 2.0-3.0 g of 1-hydroxybenzotriazole to the reaction solution and continue stirring for 1-2 h. Next, dissolve 6.0-8.0 g of 3-maleimide propionic acid (MA) in 10-20 mL of dimethyl sulfoxide solution and add it dropwise to the above reaction solution. Stir at 37 °C for 35-38 h. After the reaction is complete, dialyze the solution using a dialysis bag with a molecular weight cutoff of 3 kD for 4-6 days. Freeze-dry the dialyzed product to obtain the purified product, 3-maleimide propionic acid-modified pullulan (PL-MA). Step S3: Prepare PDA@Zif-8-xAC (1) Weigh 3.94g of 2-methylimidazole and dissolve it in 40mL of methanol solution. Stir until completely dissolved and then add 1.31g of zinc acetate dihydrate. Continue stirring at room temperature for 20-26h. After the reaction is complete, centrifuge at 8000-9000r / min for 8-12min. Wash the precipitate with methanol 2-4 times and vacuum dry at 55-65℃ for 12-14h. Name the product Zif-8. (2) Take 0.2-0.4g of Zif-8 and add it to 40-60mL of anhydrous ethanol and stir for 2-4h. Then add 0.2-0.4g of dopamine hydrochloride and stir for 25-35min. Then add 20-160mg of asiaticoside and stir for 1-1.5h. Adjust the pH of the above solution to 8.0-9.0 with 0.8-1mol / L sodium hydroxide solution and continue stirring for 22-26h. After the reaction is completed, wash with anhydrous ethanol while centrifuging for 8-12min at a speed of 8000-9000r / min. Dry the centrifuged precipitate under vacuum. Name the product PDA@Zif-8-xAC; where x represents the concentration of asiaticoside added in mg / mL. Step S4: Preparation of composite hydrogel The composite hydrogel was prepared using either method A or method B below: Method A: First, dissolve 0.25g O-HACC and 0.2g PL-MA in 5mL of deionized water to form a 5w / v% homogeneous O-HACC solution and a 4w / v% homogeneous PL-MA solution, respectively. Then, mix the two solutions in a certain ratio and let them stand to obtain a composite hydrogel. The resulting hydrogel is named O-HACC / PL-MAxy, where x and y represent the volumes of O-HACC solution and PL-MA solution added during gel formation, respectively. Method B: Weigh 0.1g of PDA@Zif-8-xAC powder and add it to a total volume of 5mL of PLMA solution with a concentration of 4w / v%. Then mix it with an equal volume of O-HACC solution with a concentration of 5w / v to form a gel. The resulting hydrogel is named AA@O-HACC / PL-MA11, or AA@HP11 for short.

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