阅读说明：本技术 一种分子内交联自组装膜修饰纺丝纳米纤维材料及其制备方法与应用 (Intramolecular cross-linking self-assembled membrane modified spinning nanofiber material and preparation method and application thereof ) 是由 赵名艳 高翔 黄瑞 楚佳奇 龚帆 于 2021-08-03 设计创作，主要内容包括：本发明涉及一种分子内交联自组装膜修饰纺丝纳米纤维材料及其制备方法与应用。该分子内交联自组装膜修饰纺丝纳米纤维材料的制备方法如下：S1.对透明质酸进行巯基化修饰；S2.壳聚糖的马来酰化改性；S3.聚电解质溶液的制备；S4.静电纺丝法制备纳米纤维；S5.纳米纤维层层自组装(LBL)表面功能化。本发明提供的分子内交联自组装膜修饰纺丝纳米纤维材料具有良好的抗菌和保湿性能,并具有较好的组织粘附性；其可作为水溶性药物及生物活性因子的稳定、高效缓释载体,适用于在组织再生修复中的应用,尤其是糖尿病患者皮肤修复领域。(The invention relates to an intramolecular cross-linking self-assembled membrane modified spinning nanofiber material and a preparation method and application thereof. The preparation method of the intramolecular cross-linking self-assembly membrane modified spinning nanofiber material comprises the following steps: s1, carrying out sulfhydrylation modification on hyaluronic acid; s2, maleylation modification of chitosan; s3, preparing a polyelectrolyte solution; s4, preparing the nano fibers by an electrostatic spinning method; s5, performing layer-by-layer self-assembly (LBL) surface functionalization on the nanofiber. The intramolecular cross-linking self-assembly film modified spinning nanofiber material provided by the invention has good antibacterial and moisturizing performances and good tissue adhesion; it can be used as stable and high-efficiency slow-release carrier of water-soluble medicine and bioactive factor, and is suitable for application in tissue regeneration and repair, especially in the field of skin repair of diabetic patients.)

1. A preparation method of a spinning nanofiber material modified by an intramolecular cross-linking self-assembled film is characterized by comprising the following steps:

s1, carrying out sulfhydrylation modification on hyaluronic acid

Carrying out sulfhydrylation modification on the hyaluronic acid solution, dialyzing, and freeze-drying to obtain sulfhydrylation hyaluronic acid;

s2, maleylation modification of chitosan

Carrying out maleylation modification on the chitosan solution, dialyzing, freezing and drying to obtain maleylation chitosan;

s3. preparation of polyelectrolyte solution

Dissolving dopamine in a Tris-HCl solution to prepare a dopamine solution, and dissolving maleylation modified chitosan in an acetic acid solution; preparing a thiolated hyaluronic acid aqueous solution;

s4, preparing the nano-fiber by an electrostatic spinning method

Soaking the spinning nanofiber in a dopamine solution, then fully washing the spinning nanofiber with water, then sequentially soaking the spinning nanofiber in a thiolated hyaluronic acid solution and a maleylation chitosan solution for adsorption, and eluting the spinning nanofiber after adsorption;

s5, nanofiber layer-by-layer self-assembly surface functionalization

Repeating the steps of adsorption and elution for a plurality of times to obtain the intramolecular cross-linking self-assembly film modified spinning nanofiber material alternately adsorbed by the thiolated hyaluronic acid and the maleylation chitosan.

2. The method for preparing the intramolecular cross-linking self-assembly film modified spinning nanofiber material according to claim 1, wherein in the step S1, N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are selected to activate a hyaluronic acid aqueous solution; cysteine hydrochloride is selected to carry out sulfhydrylation modification on hyaluronic acid.

3. The method for preparing the intramolecular cross-linking self-assembled film modified spinning nanofiber material according to claim 2, wherein the molar ratio of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride to N-hydroxysuccinimide in the step S1 is 1: 1; the mass ratio of the hyaluronic acid to the cysteine hydrochloride is 0.8-1.5: 1; the mass ratio of the hyaluronic acid to the N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride is 0.5-1: 1.

4. The method for preparing the intramolecular cross-linking self-assembly film modified spinning nanofiber material according to claim 1, wherein the molar ratio of chitosan amino groups to maleic anhydride in the step S2 is 0.5-2: 1.

5. The method for preparing the intramolecular cross-linking self-assembly film modified spinning nanofiber material according to claim 1, wherein the mass concentration of the dopamine solution in the step S3 is 1.0-3.0 mg mL^-1The mass concentration of the maleylation modified chitosan is 0.4-0.8 mg mL^-1The concentration of the thiolated hyaluronic acid aqueous solution is 0.4-0.8 mg mL by mass^-1。

6. The method for preparing the intramolecular cross-linking self-assembled film modified spinning nanofiber material according to claim 1, wherein the thiolated hyaluronic acid solution in S4 has a pH of 5 to 7, and the maleylation chitosan solution has a pH of 3 to 5.

7. The method for preparing the intramolecular cross-linking self-assembled film modified spun nanofiber material according to claim 1, wherein the steps of repeatedly adsorbing and eluting thiolated hyaluronic acid (tHA) and maleylated chitosan (mCH) in S5 are carried out for 9-11 times.

8. An intramolecular cross-linked self-assembled film-modified spun nanofiber material prepared according to the preparation method of any one of claims 1 to 7.

9. An application of an intramolecular cross-linking self-assembly membrane modified spinning nanofiber material in promoting stem cell proliferation.

10. An application of an intramolecular cross-linking self-assembly membrane modified spinning nanofiber material in promoting wound repair of diabetic mice.

Technical Field

The invention relates to the technical field of biological materials, in particular to an intramolecular cross-linking self-assembled membrane modified spinning nanofiber material and a preparation method and application thereof.

Background

In Diabetic (DM) patients, poor wound healing is a major problem affecting patient health and is also a critical clinical challenge worldwide. With the insight into the wound healing process, biomaterial-based wound dressings are often used to promote wound healing. Pathophysiologically, wound healing is a very complex process involving cell growth, angiogenesis and extracellular matrix (ECM) deposition. Thus, an ideal wound dressing should not only be able to provide physical protection and resistance to microbial penetration and proliferation, but should also provide an optimal microenvironment for healing at the wound interface. In particular to skin wounds of diabetic foot ulcers with limited epithelial and blood vessel regeneration capacity, and other chronic wounds, the demand for bioactive dressings is increasingly urgent and necessary.

Recently, electrospun nanofibers are one of the most suitable wound dressings due to their unique structure, such as high specific surface area, high porosity and microporous structure, which are structurally similar to extracellular matrix (ECM). In addition, the open microporous structure provided by the nanofiber matrix facilitates the exchange of gases and the drainage of exudates.

To create an ideal wound dressing that mimics the natural extracellular matrix (ECM), we planned to select poly (L-lactic acid) (PLLA), an FDA-approved synthetic polyester, for use in making the nanofiber matrix. Because PLLA has relatively good biomechanical properties and its degradation products are free of toxic side effects. However, many studies have shown that poly (L-lactic acid) (PLLA) has high hydrophobicity and its surface is not favorable for cell adhesion due to lack of adhesion ligands, while an appropriate surface is essential to support adhesion, spreading and proliferation of skin cells. In addition, poly (L-lactic acid) alone (PLLA) has no antibacterial activity and no function of accelerating tissue repair. Therefore, there is a need to functionalize poly (L-lactic acid) (PLLA) nanofiber matrices to make them bioactive and to promote wound healing and shorten the repair cycle by sustained release of bioactive molecules.

The layer-by-layer self-assembly (LBL) technology can alternately deposit natural or synthetic polyelectrolytes with opposite charges, is a simple and effective surface modification method for enhancing the biological activity of the nano fibers, and is an effective strategy for realizing the sustained release of the medicine. Chitosan (CH) is a natural cationic polymer commonly used in the preparation of polyelectrolyte multilayer membranes, and furthermore, has been widely used as a topical dressing in wound treatment due to its hemostatic, antibacterial, biocompatible and biodegradable properties. Hyaluronic Acid (HA) is a natural polymer present in various types of connective tissues including the dermis, and is widely used in medical fields such as beauty and plastic surgery. Studies have shown that the attachment of the CD44 receptor to Hyaluronic Acid (HA) is associated with the adhesion of a large number of cells within the extracellular matrix (ECM), where adhesion of fibroblasts is mainly achieved by this effect and plays a key role in the wound healing process by inducing cell migration and proliferation by initiating the proliferative phase of repair. In addition, degradation products of Hyaluronic Acid (HA) also have a pro-angiogenic effect. Recent studies have shown that Hyaluronic Acid (HA) may also induce lymphangiogenesis through the LYVE-1 mediated signaling pathway, thereby promoting wound healing. Therefore, surface functional modification of nanofibers with Chitosan (CH) and Hyaluronic Acid (HA) by layer-by-layer self-assembly (LBL) technology may become an effective method to improve the bioactivity of nanofiber-based wound dressings. However, polysaccharide-based bio-multilayer films are generally unstable under physiological or harsh operating conditions (high salt concentration, high or low pH, or mechanical stress). Thus, such coatings may not maintain their long-term promoting effect on tissue repair. On the other hand, the chemical and mechanical stability of the multilayer film can be improved by covalent crosslinking of the layer-by-layer self-assembly (LBL) multilayer film; however, such crosslinking often results in a change in the functionality of the multilayer film.

In contrast to the traditional post-crosslinking approach, we have recently developed a unique multi-layer membrane system based primarily on additional intramolecular crosslinking during assembly of the polyelectrolyte with formation of imine or disulfide bonds between molecules. We have found that the stability and bioactivity of such multilayer films is greatly improved over polyelectrolyte films formed based on ion pairing. Interestingly, leaves and colleagues reported that using maleylation of ch (mch) and thiolated ha (tha), complex hydrogels could be formed in situ by the michael addition reaction, but it is not clear at present whether this system could be used to build intramolecular cross-linked polyelectrolyte multilayer films.

More and more recent studies have shown that Insulin (IN) has a great effect on wound healing. Insulin (IN) can increase the expression levels of signaling molecules IN the healing metabolic pathway, such as protein kinase b (akt) and Vascular Endothelial Growth Factor (VEGF), thereby inducing proliferation and differentiation of cells. Insulin has been reported to promote the re-epithelialization of transient tissues by stimulating the migration and growth of keratinocytes. Topical application of insulin to diabetic rats reduces the time required for wound epithelialization and results in thickening of the epidermal layer. In addition, insulin can enhance angiogenesis in the wound healing process by inducing migration and tube formation of endothelial cells. However, topical administration of insulin has the problem of short half-life and loss of biological activity in peptidase-rich wound environments. Continuous delivery of insulin using a suitable system is an effective strategy to overcome this problem. In all delivery systems, the LBL technique is a common method for preparing films and microcapsules for effective controlled release of bioactive molecules. Furthermore, it has been reported that cross-linking within the LBL film or microcapsule greatly affects the release of bioactive molecules, since cross-linking within the film or microcapsule creates a diffusion barrier to bioactive molecules and protects them from degradation. For example, the multi-layer membrane system based on intramolecular cross-linking showed a more durable BMP-2 release effect and enhanced osteogenic differentiation of C2C12 cells, compared to the membrane system based on ion pairing, indicating that the intramolecular cross-linking multi-layer membrane system can more effectively release bioactive molecules.

Overall, the inherent crosslinking of the multilayer film has a promoting effect on its stability, biological performance and even the loading and controlled release effects of bioactive molecules. Therefore, we were interested in understanding whether multi-layer films based on intramolecular cross-linking could be constructed by LBL technology using mCH and tHA and to investigate whether it could be applied to surface functional modification of nanofibers made from PLLA to create a dressing that sustainedly releases insulin to heal diabetic wounds.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide a preparation method of an intramolecular cross-linking self-assembly film modified spinning nanofiber material.

The second purpose of the invention is to provide the intramolecular cross-linking self-assembly film modified spinning nanofiber material prepared by the preparation method, and the intramolecular cross-linking self-assembly film modified spinning nanofiber material provided by the invention has good antibacterial and moisture-retention performances and good tissue adhesion; it can be used as stable and high-efficiency slow release carrier of water-soluble medicine and bioactive factor, and is suitable for skin repair field, especially for skin repair field of diabetic.

The third purpose of the invention is to provide the application of the intramolecular cross-linking self-assembly film modified spinning nanofiber material.

The primary purpose of the invention adopts the following technical scheme:

a preparation method of a spinning nanofiber material modified by an intramolecular cross-linking self-assembled film comprises the following steps,

s1, carrying out sulfhydrylation modification on hyaluronic acid

Carrying out sulfhydrylation modification on the hyaluronic acid solution, dialyzing, and freeze-drying to obtain sulfhydrylation hyaluronic acid;

s2, maleylation modification of chitosan

Carrying out maleylation modification on the chitosan solution, dialyzing, freezing and drying to obtain maleylation chitosan;

s3. preparation of polyelectrolyte solution

Dissolving dopamine in a Tris-HCl solution to prepare a dopamine solution, and dissolving maleylation modified chitosan in an acetic acid solution; preparing a thiolated hyaluronic acid aqueous solution;

s4, preparing the nano-fiber by an electrostatic spinning method

Soaking the spinning nanofiber in a dopamine solution, then fully washing the spinning nanofiber with water, then sequentially soaking the spinning nanofiber in a thiolated hyaluronic acid solution and a maleylation chitosan solution for adsorption, and eluting the spinning nanofiber after adsorption;

s5, nanofiber layer-by-layer self-assembly (LBL) surface functionalization

Repeating the steps of adsorption and elution for a plurality of times to obtain the intramolecular cross-linking self-assembly film modified spinning nanofiber material alternately adsorbed by the thiolated hyaluronic acid and the maleylation chitosan.

Preferably, in the step S1, the (NHS) hyaluronic acid aqueous solution is activated by N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide; cysteine hydrochloride is selected to carry out sulfhydrylation modification on hyaluronic acid.

Preferably, the molar ratio of the N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide in the step S1 is 1: 1; the mass ratio of the hyaluronic acid to the cysteine hydrochloride is 0.8-1.5: 1; the mass ratio of the hyaluronic acid to the N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride is 0.5-1: 1.

Preferably, the dialysis in step S1 is performed under hydrochloric acid condition for three days.

Preferably, the molar ratio (n-NH) of chitosan amino and Maleic Anhydride (MA) in the step S2₂/nMA) is 0.5-2: 1.

Preferably, the molar ratio (n-NH) of chitosan amino and Maleic Anhydride (MA) in the step S2₂/nMA) is 1: 1.

Preferably, the mass concentration of the dopamine solution in the step S3 is 1.0-3.0 mg mL^-1The mass concentration of the maleylation modified chitosan is 0.4-0.8 mg mL^-1The concentration of the thiolated hyaluronic acid aqueous solution is 0.4-0.8 mg mL by mass^-1。

Preferably, the thiolated hyaluronic acid solution in S4 has a pH of 5 to 7, and the maleylated chitosan solution has a pH of 3 to 5.

Preferably, the steps of adsorbing and eluting the thiolated hyaluronic acid (tHA) and the maleylated chitosan (mCH) in the S5 are repeated 9 to 11 times.

In the invention, the modification layer realizes the self-assembly of the modification layer on the surface of the spinning fiber mainly through the reaction of functional groups among macromolecules.

In the invention, the spinning nanofiber in S3 may be a polylactic acid spinning nanofiber, or other polymer spinning nanofibers in the field such as polylactic acid-glycolic acid spinning nanofibers and the like may be selected according to the requirements of the field of repair medicine.

In the invention, the intramolecular cross-linking self-assembly membrane modified spinning nanofiber material can also be prepared by cross-linking thiolated chitosan and thiolated hyaluronic acid through disulfide bonds; the intramolecular cross-linking self-assembly film modified spinning nanofiber material can also be prepared by oxidizing hyaluronic acid and collagen through Schiff bond cross-linking.

The spinning nanofiber prepared by the electrostatic spinning technology has the advantages of large specific surface area, controllable porosity, good ductility and adsorptivity, can simulate the structure and function of natural extracellular matrix and the like, and becomes a new direction for the development of repair materials, however, the pure artificially synthesized high-molecular spinning nanofiber does not have the antibacterial property, the moisture retention property and the bioactivity. It needs to be modified properly to improve the feasibility of using it in wound repair. Compared with the method of simply modifying by CH and HA, the method of the invention can endow the repair material with good antibacterial ability after the CH and HA are chemically modified and used for modifying the spinning fiber, and no additional antibacterial drug load is needed.

Although the natural polymer self-assembly modification layer has good biological activity, the stability of the natural polymer self-assembly modification layer under the physiological environment is often insufficient, and the stability of the natural polymer self-assembly modification layer needs to be improved by a chemical or physical crosslinking means. According to the invention, through chemical modification of HA and CH, sulfydryl and double bonds with reactivity are introduced between molecules, so that intramolecular cross-linking can be formed in situ in the self-assembly modification process of the spinning nanofiber, and the effect of stabilizing the HA and CH modification layer is achieved. Meanwhile, the use of the micromolecule chemical cross-linking agent after the fact is avoided, so that a series of subsequent steps of micromolecule cleaning and the like are reduced, and the potential toxic effect of the micromolecule chemical cross-linking agent is avoided to a certain extent.

The spinning nanofiber is modified through self-assembly biological functional modification, the biological function of the spinning nanofiber is improved on the basis of maintaining the good performance of a spinning nanofiber matrix material, and the repair material obtained through modification has good antibacterial and moisture-retention performances, the water retention property of the repair material is also obviously improved, and the repair material also has good tissue adhesion. In addition, the spinning nanofiber modified by biological functionalization provided by the invention can bear external environmental pressure such as pH, high ionic strength, mechanical force and the like, and can be used as a drug carrier, a tissue engineering scaffold or a wound repair material to be applied to the field of biomedical tissue engineering.

The method adopts a layer-by-layer self-assembly technology to modify the spinning fiber, the reaction medium is aqueous solution, the modification process is carried out at room temperature, and the subsequent loading of the bioactive factor or the medicine is carried out under the condition, so that the degradation and the denaturation of the material can not be caused, and the biological activity of the loading factor can not be influenced.

The second purpose of the invention adopts the following technical scheme:

an intramolecular cross-linking self-assembled membrane modified spinning nanofiber material is prepared by the preparation method.

The third purpose of the invention adopts the following technical proposal:

an application of a nano fiber material modified by an intramolecular cross-linking self-assembly film.

Preferably, the intramolecular cross-linking self-assembly film modifies the application of the spinning nanofiber material in antibiosis.

Preferably, the intramolecular cross-linking self-assembly membrane is used for modifying the spinning nanofiber material in insulin loading.

Preferably, the intramolecular cross-linking self-assembly membrane modified spinning nanofiber material is applied to promoting stem cell proliferation.

Preferably, the intramolecular cross-linking self-assembly membrane modified spinning nanofiber material is applied to promotion of wound repair of diabetic mice.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides an intramolecular cross-linking self-assembly film modified spinning nanofiber material with good antibacterial and moisture retention performances by performing biological functional modification on the existing spinning nanofiber, and the spinning nanofiber material also has good tissue adhesion. The spinning nanofiber material provided by the invention can be used as a stable and efficient slow-release carrier of a water-soluble drug or a bioactive factor, and is suitable for the field of tissue repair, especially the field of skin repair of diabetics.

In addition, the method and the system for preparing the intramolecular cross-linking self-assembly membrane modified spinning nanofiber material are easy to operate and control, the experimental conditions are mild, the cost is low, the investment of high energy is avoided, and the method and the system have great popularization and application values.

Drawings

FIG. 1 shows the synthesis mechanism (A) of thiolated hyaluronic acid (tHA) and maleylated chitosan (mCH) and the mechanism thereof¹H NMR (B) spectrum;

FIG. 2 shows the variation of the adsorption mass (A) and thickness (B) of the membrane molecules during layer adsorption calculated according to the Sauerberry equation after QCM detection; wherein the odd layers are HA or tHA, and the even layers are CH or mCH; [ P: pure PLLA spinning nanofiber; P-PDA: PLLA spinning nanofiber after PDA modification; P-CH/HA: PLLA spinning nanofiber modified by natural CH and HA LBL; P-mCH/tHA: PLLA spun nanofiber after mCH and tHA LBL modification ];

fig. 3 shows the inhibitory effect of PLLA nanofiber spinning membranes on escherichia coli (a) and its inhibitory loop diameter (B) before and after LBL modification, [ P: pure PLLA spinning nanofiber; P-CH/HA: PLLA spinning nanofiber modified by natural CH and HA LBL; P-mCH/tHA: mCH and tHA LBL modified PLLA spinning nanofiber](ii) a (C) To utilizeThe software obtains the fiber diameters of PLLA, P-PDA, P-CH/HA and P-mCH/tHA through a scanning electron microscope picture; (D) stress-strain plots for PLLA, P-CH/HA, and P-mCH/tHA; (E) the infrared spectra of PLLA, P-PDA, P-CH/HA and P-mCH/tHA; (F) full XPS spectra for PLLA, P-PDA, P-CH/HA and P-mCH/tHA; (G) is a N1s diagram of PLLA, P-PDA, P-CH/HA and P-mCH/tHA detected by XPS; (H) is an S2P spectrogram of PLLA, P-PDA, P-CH/HA and P-mCH/tHA obtained by XPS detection;

FIG. 4 is a graph showing the antibacterial activity of gram-negative Escherichia coli on PLLA, P-CH/HA, and P-mCH/tHA, and the diameter of inhibition zone of gram-negative Escherichia coli on PLLA, P-CH/HA, and P-mCH/tHA;

FIG. 5 shows the loading efficiency of insulin on PLLA nanofiber spun membranes before and after LBL modification and the in vitro insulin release at different time points; [ P: pure PLLA spinning nanofiber; P-CH/HA: PLLA spinning nano-fiber modified by natural CH and HALBL; P-mCH/tHA: PLLA spun nanofiber after mCH and tHALBL modification ];

FIG. 6 shows the adhesion, expansion and proliferation behaviors of mesenchymal stem cells on PLLA, P-CH/HA, P-mCH/tHA, wherein (A) and (B) are SEM images and confocal observations of myofibers (red) and nuclei (blue) of mesenchymal stem cells on PLLA, P-CH/HA, P-mCH/tHA with and without insulin loading, respectively; the box is marked as an enlargement of the selected picture, where significant ECM formation is visible; (C) the proliferation behaviors of mesenchymal stem cells on PLLA, P-CH/HA, P-mCH/tHA on the 1 st and the third day under the condition of existence of insulin;

FIG. 7 is a graphical overview of the full thickness skin wound (8mm diameter) of diabetic mice IN different time points IN (A) untreated group and after treatment with PLLA alone, PLLA _ IN, P-CH/HA, and P-mCH/tHA alone; and (B) wound healing rates at different time points as measured by "Image-Pro Plus" software;

FIG. 8 is a photograph of untreated groups observed by HE (A) and Ma's trichrome stain (B) and the formation of granulation tissue, re-epithelialization and collagen deposition on the wound surface after treatment with PLLA alone, PLLA _ IN, P-CH/HA and P-mCH/tHA alone;

FIG. 9 shows the expression of CD31(A) and VEGF-R (B) at wound surface after immunohistochemical staining of untreated group and treatment with PLLA, PLLA _ IN, P-CH/HA and P-mCH/tHA alone.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1 an intramolecular cross-linking self-assembled membrane modified spun nanofiber material

(1) Thiol modification of hyaluronic acid

Under magnetic stirring, 0.2g of Hyaluronic Acid (HA) is dissolved in 50ml of water and stirred uniformly to prepare a Hyaluronic Acid (HA) water solution; 25mM of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride and 25mM of N-hydroxysuccinimide were added in succession, and the system pH was adjusted to 5.5 with hydrochloric acid. Stirring for 2 hours in the dark at room temperature, adding 0.2g of cysteine hydrochloride into the reaction system, adjusting the pH of the system to 4.75 by using hydrochloric acid, stirring in the dark at room temperature, and dialyzing for 3 days by using hydrochloric acid; the first day of dialysis, the mixture in the reaction system was dialyzed against 0.2mM hydrochloric acid, and the molecular cut-off Mw was 3.5 kDa; dialyzing for the next day by using 0.2mM hydrochloric acid containing 1% by mass of sodium chloride; dialyzing for the third day, adjusting the reaction system to 3.5 with 0.2mM hydrochloric acid, and freeze-drying to obtain thiolated HA (tHA).

(2) Maleylation modification of chitosan

Chitosan (CH) was added at 10mg mL^-1Is dissolved in 0.5mol L^-1In acetic acid, adding maleic anhydride into chitosan solution according to a feeding ratio of a CH repeating unit to maleic anhydride of 1:1, stirring and mixing, stirring for 24 hours at room temperature in a dark place, dialyzing for 3 days by using distilled water, removing impurities and unreacted micromolecules by using a molecular cut-off Mw of 3.5kDa, and freeze-drying to obtain maleylation CH (mCH).

(3) Preparation of polyelectrolyte solution

The preparation of each polyelectrolyte solution was as follows:

dopamine (2 mg. mL)^-1) Dissolving in Tris-HCl (pH 8.5) solution;

thiolated modified HA (0.5 mg/mL)^-1) Dissolved in an aqueous solution to maleylate CH (0.5 mg. multidot.mL)^-1) Dissolving in 0.05M acetic acid solution, and magnetically stirring overnight to dissolve completely; meanwhile, natural HA and natural CH solutions with the same concentration are prepared for comparison.

Before each of the above solutions was used, all solutions except dopamine and tHA solutions were adjusted to pH 4.

(4) Preparation of poly (L-lactic acid) (PLLA) nanofibers by electrospinning

Dissolving 7 wt% polylactic acid powder in DCM/DMF (4:1v/v) mixed solution, placing into a 10mL syringe, fixing on a micro-injection pump, using a flattened needle as a capillary for injecting a thin flow, wherein the inner diameter of the capillary is 0.4mm, the extrusion speed is 1mL · h under 18kv^-1. Collecting the fiber obtained by spinning on a rotary drum, drying for 12 hours in vacuum, and removing the residual organic solvent to obtain the poly (L-lactic acid) (PLLA) nanofiber.

(5) Poly (L-lactic acid) (PLLA) nanofiber layer-by-layer self-assembly (LBL) surface functionalization

Soaking the polylactic acid spinning nanofiber with 75% ethanol, then fully washing, firstly transferring the polylactic acid spinning nanofiber into 2 mg/mL-1 dopamine solution for soaking for 30 minutes, and then fully washing to assemble a layer of positively charged dopamine base layer on the surface of the spinning fiber. The spinning nanofiber modified by dopamine is sequentially immersed in tHA and mCH solution, each layer is accompanied with an elution step (3 x 3min) after being adsorbed for 12 minutes, and the eluent is aqueous solution; and meanwhile, the spinning nanofiber modified by dopamine is sequentially immersed in HA and CH solutions, and the processing under the same conditions is compared.

Repeating the steps for multiple times to finally obtain 9 layers of P-mCH/tHA spinning nano fibers with HA and CH (P-CH/HA) and tHA and mCH alternately adsorbed on the dopamine base layer.

The physical and chemical properties such as the morphological characteristics and the like of the polylactic acid spinning nanofiber modified by self-assembly are detected, and the test result is as follows.

Physical and chemical property test

(1) Identification of reactive groups

FIG. 1A is tHA prepared by formation of an amide bond between the carboxyl group of HA and the amine group of cysteine in the presence of a carbodiimide coupling agent, FIG. 1A is mCH synthesized with a vinyl carboxylic acid group by maleylation of CH with maleic anhydride; tHA and mCH by¹And H NMR characterization. The methylene proton on-CH 2 SH-showed a new peak at 2.81ppm (see FIG. 1B) compared to HA, indicating successful introduction of the thiol group. By comparing CH and mCH in FIG. 1B¹H NMR spectrum, a new peak at 6.17 ppm-CH ═ CH-was found in mCH, confirming successful modification of CH with maleic anhydride. The successful thiolation and maleylation modification of HA and CH was further confirmed according to the standard Ellman test and the maleic acid standard curve method, wherein the thiol content is 120.4. + -. 4.8. mu. mol g^-1And the number of acetyl groups bound to CH is 1783.6 + -96.5. mu. mol g^-1。

(2) Quartz Crystal Microbalance (QCM) monitoring growth behavior of multilayer films

Differences between the mCH/tHA multilayer film system based on intramolecular cross-linking and the ion-pairing CH/HA multilayer film system were observed by QCM detection. Fig. 2 shows the variation of the adsorption mass (a) and thickness (B) of mCH/tHA and CH/HA film systems measured by QCM and calculated using Sauerbrey equation during layer adsorption, where the odd layers are tHA or HA and the even layers are mCH or CH. As can be seen from fig. 2, due to the intramolecular covalent crosslinking of the double bond and thiol group between mCH and tHA molecules, the molecular adsorption quality (i.e., the adsorption amount of mCH and tHA is significantly higher than that of CH and HA) and the film thickness of the film system are significantly higher than that of the CH/HA film system.

(2) Morphology structure, hydrophilicity, moisture retention capacity and water holding capacity of self-assembly modified polylactic acid spinning nanofiber

Contact Angle (WCA) detection is an important means for the hydrophilicity and hydrophobicity of the surface of a reaction material, and the WCA value of the reaction material is reduced along with the increase of the hydrophilicity of the surface of the material.

Fig. 3 shows that the intramolecular cross-linking self-assembled film modified spun nanofiber material prepared in example 1 is a P-CH/HA and P-mCH/tHA nano spun fiber with 9 layers of HA and CH in total and tHA and mCH alternately adsorbed, where [ P: pure PLLA spun film, P-PDA: PLLA spun film adsorbing PDA layer, P-CH/HA: PLLA spun film self-assembled with 9 layers of HA and CH, P-mCH/tHA: PLLA spun film self-assembled with 9 layers of mCH/tHA ].

Fig. 3A shows the variation of Water Contact Angle (WCA) of P-CH/HA and P-mCH/tHA during the alternating adsorption of CH (mCH) and HA (tha), and it can be seen that the hydrophilicity of PLLA membrane after layer adsorption is obviously improved, and the trend of alternating contact angle shows that CH (mCH) and HA (tha) are successfully and alternately adsorbed to PLLA membrane. In addition, with the alternate adsorption of mCH and tHA, the change degree of WCA is obviously larger than that of CH and HA, and the adsorption quality of mCH and tHA is further proved to be higher than that of CH and HA; FIG. 3B shows the morphology structures of PLLA, P-PDA, P-CH/HA and P-mCH/tHA, respectively, and it can be seen from FIG. 3B that the surface roughness of PLLA spun fiber is significantly improved after modification by mCH and tHA, which indicates that mCH and tHA have been successfully adsorbed on the PLLA spun nanofiber; FIG. 3C is a schematic representation of the utilizationThe software obtains the fiber diameters of PLLA, P-PDA, P-CH/HA and P-mCH/tHA through a scanning electron microscope picture; FIG. 3D is a graph of stress-strain curves for PLLA, P-CH/HA and P-mCH/tHA, as can be seenThe tensile strength of the PLLA spinning fiber modified by LBL is obviously improved, and particularly the spinning fiber modified by mCH/tHA has the highest tensile strength, which is probably because the tensile strength is enhanced by intermolecular crosslinking in the film; FIG. 3E is the IR spectra of PLLA, P-PDA, P-CH/HA and P-mCH/tHA, modified by mCH/tHA LBL, with P-mCH/tHA at 1640cm^-1And 1560cm^-1The amide bond (-CONH-) formed by mCH and tHA and the amide II band peak on mCH appear, and the mCH is originally 864cm^-1The peak of the maleyl double bond at (A) is weakened after self-assembly reaction with tHA; FIG. 3F is a full XPS spectrum of PLLA, P-PDA, P-CH/HA and P-mCH/tHA, wherein FIGS. 3G and 3H are respectively the N1S and S2P spectra of PLLA, P-PDA, P-CH/HA and P-mCH/tHA obtained by XPS detection, and it is found that the N content of PLLA membrane is increased obviously after LBL modification, while the S content of P-mCH/tHA is increased from tHA, further indicating that LBL modification is successfully performed on PLLA.

Table 1 shows the water content and swelling ratio of PLLA, P-CH/HA and P-mCH/tHA

Sample (I)	Water content (%)	Swelling ratio (%)
			PLLA	37.5±4.17	20.48±6.44
P-CH/HA	70.96±3.64	513.38±49.58
			P-mCH/tHA	78.59±2.10	527.78±25.46

As shown in Table 1, the water content of P-mCH/tHA was increased from 37.5 + -4.17% to 78.59 + -2.10% compared with that of PLLA alone after mCH/tHA LBL modification; the swelling ratio of P-mCH/tHA increased from 20.48 + -6.44% to 527.78 + -25.46% compared to that of PLLA alone.

Table 2 shows the mechanical properties of PLLA, P-PDA, P-CH/HA and P-mCH/tHA

As can be seen from Table 2, the diameters of P-CH/HA and P-mCH/tHA increased from 644. + -. 175 nm (P) to 909. + -. 185nm and 906. + -. 204nm, respectively, after LBL modification, and no difference in fiber diameter was found between P-CH/HA and P-mCH/tHA; under the same size and loading rate, PLLA, P-PDA, P-CHI/HA and P-mCH/tHA have different mechanical properties, and as can be seen from Table 2, PLLA HAs the lowest tensile strength of 1.13 +/-0.09 MPa, the elongation at break and tensile strength of P-CH/HA are not significantly changed compared with PLLA, but Young's modulus is greatly increased, and on the contrary, the mechanical properties of P-mCH/tHA are greatly improved compared with that of pure PLLA and P-CH/HA, and the highest tensile stress and Young's modulus at the break point reach 3.23 +/-0.57 MPa and 0.595 +/-0.192 MPa.

TABLE 3 atomic contents of C, O, N and S for PLLA, P-PDA, P-CH/HA and P-mCH/tHA

Sample (I)	C1s(％)	O1s(％)	N1s(％)	S2p(％)
					PLLA	73.26	25.84	0.73	0.17
P-PDA	65.59	33	1.32	0.09
					P-CH/HA	65.27	32.03	2.66	0.03
P-mCH/tHA	64.62	31.74	3.55	0.28

As can be seen from table 3, the content of N increases with surface modification compared to PLLA. The N content of PLL-PDA, P-CH/HA and P-mCH/tHA increased to 1.32%, 2.66% and 3.55%, respectively. In addition, the S content of P-mCH/tHA was about 0.28%, which is the highest of the four samples.

The results of the above graphs show that compared with CH/HA, the mCH/tHA system HAs significantly higher adsorption quality on PLLA than CH and HA due to the intermolecular crosslinking effect, the hydrophilicity of P-CH/HA and P-mCH/tHA modified by CH and HA or mCH and tHA is obviously improved, and the tensile strength of P-mCH/tHA modified by mCH and tHA is also obviously improved, which indicates that the repair material prepared by the method HAs good water absorption and moisture retention effects and strong mechanical properties.

Comparative example 1

A solution of pure poly (L-lactic acid) (PLLA) was prepared at the same concentration as P-mCH/tHA described in example 1.

Comparative example 2

Chitosan which is not modified by maleylation and hyaluronic acid (P-CH/HA) solution which is modified by thiolation and HAs the same concentration as that of the P-mCH/tHA in example 1 are prepared.

Application test example 1 application of intramolecular cross-linking self-assembled film modified spinning nanofiber material (P-mCH/tHA) provided in example 1 in antibacterial effect

The antibacterial action of the repair material provided in patent example 1 of the present invention on gram-negative bacteria Escherichia coli was studied with reference to the agar diffusion method among the published literature methods, and compared with the PLLA material modified by the conventional CH/HA self-assembly in comparative example 1 and pure PLLA in comparative example 2. First, a sterilized Luria-Bertani agar medium was poured into a plate, and bacteria were spread evenly on the surface of the slant medium using an inoculating loop. Simple PLLA, P-CH/HA and P-mCH/tHA were cut into small discs (12mm) and placed on plates and incubated at 37 ℃ for 24 hours. After the end of the incubation, the diameter of the bacterial growth inhibition zone on the plate was measured. The result shows that the simple PLLA HAs no antibacterial property and no antibacterial zone is found around the PLLA, and no obvious antibacterial zone is found around the PLLA spinning nanofiber modified by unmodified CH and HA, which indicates that the PLLA spinning nanofiber HAs no antibacterial capability. On the contrary, an obvious inhibition zone appears around the spinning fiber after the PLLA spinning nanofiber membrane is modified by mCH/tHA self-assembly, the width of the inhibition zone is about 6.53mm +/-1.54, and the repair material has obvious antibacterial and bacteriostatic capabilities, and the specific situation is shown in figure 4.

Application test example 2 application of intramolecular cross-linked self-assembled film modified spinning nanofiber material (P-mCH/tHA) provided in example 1 in insulin loading

IN our previous studies, it was found that Insulin (IN) at a certain concentration has a significant ability to promote the proliferation of umbilical cord mesenchymal stem cells under serum-free conditions. On the basis, the self-assembled polymer layer modified by the spinning fiber in the example 1 is used as a carrier, the loading of the insulin in the spinning fiber is realized by a simple soaking mode, and compared with the PLLA material which is single-purity in the comparative example 1 and is unmodified in the comparative example 2 and subjected to CH/HA self-assembly modification, the loading efficiency and the controlled release effect of the repair material in the patent example 1 of the invention on the insulin are examined.

FIG. 5 shows the insulin loading efficiency and sustained release behavior on three materials, PLLA alone, P-CH/HA alone and P-mCH/tHA alone. As can be seen from the results in FIG. 5, the loading of insulin in the spinning fiber modified by mCH/tHA is obviously improved compared with that of the other two groups. In addition, the three groups of materials all show burst release of insulin within the first 3 hours, but the release amount of the insulin on the material modified by mCH/tHA is obviously reduced, and the group of materials has obviously better slow release effect on the insulin (the release amount of the insulin in the group of materials is less than 60 percent after 9 days).

Application test example 3 application of intramolecular cross-linked self-assembled membrane modified spinning nanofiber material (P-mCH/tHA) provided in example 1 in promotion of stem cell proliferation

An important factor of the biological material for skin wound repair is that the biological material is required to have wound repair promoting capability, and early experiments show that insulin with a certain concentration can obviously promote proliferation of umbilical cord mesenchymal stem cells under a serum-free condition under a two-dimensional culture condition. The invention discloses a method for improving the effect of a repairing material on the proliferation of stem cells, which is characterized in that a mCH/tHA self-assembly layer is used as an insulin carrier, the slow release effect of insulin can be obviously improved by a spinning fiber material modified by mCH and tHA, and the effect of the repairing material on the promotion of the proliferation of the stem cells is researched by comparing and researching the growth and proliferation behaviors of the stem cells on the PLLA modified by single PLLA in a comparative example 1 and CH/HA in a comparative example 2. Fig. 6 shows the proliferation behavior of umbilical cord mesenchymal stem cells on the surfaces of different materials, and as can be seen from fig. 6, the spinning fibers loaded with insulin can significantly promote cell proliferation, but compared with the PLLA alone and the PLLA modified by CH/HA, the repair material of the present invention HAs a better cell proliferation promotion effect on the sustained release effect of insulin and an effect of promoting the secretion of extracellular matrix (partially enlarged).

Application test example 4 application of intramolecular cross-linked self-assembled membrane modified spinning nanofiber material (P-mCH/tHA) provided in example 1 in promotion of wound repair of diabetic mice

30 diabetic mice were purchased through a model animal institute at Nanjing university and a full-thickness skin wound model was made on the back of the mice according to literature reported methods. 30 mice were randomly divided into 5 groups, which were an untreated group (control group), a PLLA-treated group, a P _ IN-treated group, a P-CH/HA _ IN-treated group, and a P-mCH/tHA _ IN-treated group. Regularly photographing in different periods (0, 3 days, 5 days, 9 days and 16 days) of wound treatment to record the healing condition of the wound, taking materials after 16 days of treatment, embedding the sections, performing histochemistry and immunohistochemical staining, and observing the repairing effect of the wound of different treatment groups.

As shown in fig. 7, the healing speed of the wound surface between different treatment groups has significant difference, wherein the repair material prepared by the invention, namely the spinning fiber material modified by mCH and tHA, significantly promotes the healing of the wound surface. Furthermore, as can be seen by HE (fig. 7) staining, intact re-epithelialization and distinguishable epithelia characterized by formation of a good epidermal layer were seen by skin wounds treated with P-mCH/tHA _ IN, with epithelial insufficiency clearly distinct from the untreated group and unclear epidermal layer of PLLA treated group. Although complete re-epithelialization was also seen IN the P _ IN treated group, there was less epidermal stratification and incomplete dermal-epidermal junction. IN addition, although the P-CH/HA _ IN treatment group also showed good wound repair effect, the P-mCH/tHA _ IN treatment group showed a more distinct layered epidermal layer and an intact basement membrane, indicating a well-developed epidermal-dermal junction. Further, by the mahalanobis trichrome stain shown IN fig. 8, it can be seen that significantly more collagen deposition was seen IN the P-mCH/tHA _ IN treated group, and the deposited collagen formed a basket-like structure similar to normal skin. As shown IN FIG. 9, further observation of the angiogenesis of the wound revealed that both CD31 and VEGF-R associated with angiogenesis were highly expressed IN the P-mCH/tHA _ IN group, indicating that the repair material of the present invention has a good angiogenesis promoting effect.

The invention obtains the intramolecular cross-linking self-assembly membrane modified spinning nanofiber material with good antibacterial property, moisture retention and tissue adhesion property by performing biological functional modification on the polylactic acid spinning nanofiber, and meanwhile, the technology can be popularized and applied to modification of other high-molecular spinning nanofibers and other tissue engineering application fields.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.