CN113818244B

CN113818244B - Intramolecular cross-linked self-assembled film modified spinning nanofiber material and preparation method and application thereof

Info

Publication number: CN113818244B
Application number: CN202110884641.8A
Authority: CN
Inventors: 赵名艳; 高翔; 黄瑞; 楚佳奇; 龚帆
Original assignee: Affiliated Hospital of Guangdong Medical University
Current assignee: Affiliated Hospital of Guangdong Medical University
Priority date: 2021-08-03
Filing date: 2021-08-03
Publication date: 2023-07-18
Anticipated expiration: 2041-08-03
Also published as: CN113818244A

Abstract

The invention relates to a spinning nanofiber material modified by an intramolecular cross-linked self-assembled film, and a preparation method and application thereof. The preparation method of the intramolecular cross-linked self-assembled film 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 nanofibers by an electrostatic spinning method; s5, layer-by-layer self-assembly (LBL) surface functionalization of the nanofiber. The intramolecular cross-linked self-assembled film modified spinning nanofiber material provided by the invention has good antibacterial and moisturizing properties and good tissue adhesiveness; the slow-release carrier can be used as a stable and efficient slow-release carrier of water-soluble drugs and bioactive factors, and is suitable for application in tissue regeneration and repair, in particular to the field of skin repair of diabetics.

Description

Intramolecular cross-linked self-assembled film modified spinning nanofiber material and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological materials, in particular to a molecular internal crosslinking self-assembly 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 an in-depth understanding of 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 penetration and proliferation of microorganisms, but should also provide an optimal microenvironment for healing at the wound interface. In particular to chronic wounds such as skin wounds of diabetic foot ulcers with limited epithelial and vascular regeneration capacity, the need for bioactive dressings is increasingly urgent and necessary.

In recent years, electrospun nanofibers are one of the most suitable wound dressings because of their unique structure, such as high specific surface area, high porosity, and microporous structure, similar in structure to the extracellular matrix (ECM). In addition, the open microporous structure provided by the nanofiber matrix facilitates gas exchange and exudate drainage.

To create an ideal wound dressing that mimics the natural extracellular matrix (ECM), we plan to select poly (L-lactic acid) (PLLA), an FDA approved synthetic polyester, for use in preparing a 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 a high hydrophobicity and its surface is detrimental to cell adhesion due to the lack of adhesion ligands, while a suitable surface is critical to support adhesion, stretching and proliferation of skin cells. In addition, poly (L-lactic acid) alone (PLLA) does not possess antibacterial activity and the ability to accelerate tissue repair. Thus, there is a need to functionalize poly (L-lactic acid) (PLLA) nanofiber matrices to render them bioactive and promote wound healing and shorten repair cycles by sustained release of bioactive molecules.

The layer-by-layer self-assembly (LBL) technique, which can alternately deposit natural or synthetic polyelectrolytes with opposite charges, is a simple and effective surface modification method for enhancing the biological activity of nanofibers, and is also an effective strategy for realizing sustained release of drugs. Chitosan (CH) is a natural cationic polymer commonly used in the preparation of polyelectrolyte multilayer films, and in addition, has been widely used as a topical dressing in wound therapy due to its hemostatic, antibacterial, biocompatible and biodegradable properties. Hyaluronic Acid (HA) is a natural polymer existing in various types of connective tissues including 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), with the adhesion of fibroblasts being achieved primarily by this effect and by inducing cell migration and proliferation by initiating the proliferative phase of repair, playing a key role in the wound healing process. In addition, degradation products of Hyaluronic Acid (HA) also have pro-angiogenic effects. Recent studies have shown that Hyaluronic Acid (HA) can also induce lymphangiogenesis via LYVE-1 mediated signaling pathways, thereby promoting wound healing. Thus, surface functionalization modification of nanofibers with Chitosan (CH) and Hyaluronic Acid (HA) by layer-by-layer self-assembly (LBL) techniques may be an effective method to improve the bioactivity of nanofiber-based wound dressings. However, polysaccharide-based biological multilayer films are often unstable under physiological or harsh operating conditions (high salt concentrations, high or low pH values, or mechanical stress). Thus, such coatings may not retain their long-term promoting effect on tissue repair. On the other hand, the chemical and mechanical stability of the layer-by-layer self-assembled (LBL) multilayer film can be improved by covalent crosslinking of the film; however, such crosslinking typically results in a change in the function of the multilayer film.

In contrast to conventional post-crosslinking methods, we have recently developed a unique multilayer film system based primarily on additional intramolecular crosslinking of the polyelectrolyte that forms imine or disulfide bonds between molecules during assembly. We have found that the stability and bioactivity of such multilayer films are greatly improved over polyelectrolyte membranes formed based on ion pairing. Interestingly, leaves and his colleagues reported that complex hydrogels could be formed in situ by Michael addition reactions using maleylated CH (mCH) and thiolated HA (tHA), but it is not clear at present whether intramolecular crosslinked polyelectrolyte multilayer films could be constructed using this system.

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 re-epithelialization of temporary tissues by stimulating keratinocyte migration and growth. Topical application of insulin to diabetic rats can reduce the time required for wound epithelialization and result in thickening of the epidermis layer. In addition, insulin can enhance angiogenesis during wound healing by inducing endothelial cell migration and tube formation. However, topical administration of insulin has problems in that the half-life is short and the bioactivity is lost in a wound environment rich in peptidases. Continuous delivery of insulin using a suitable system is an effective strategy to overcome this problem. In all delivery systems, LBL technology is a common method of preparing films and microcapsules for efficient controlled release of bioactive molecules. Furthermore, crosslinking within LBL films or microcapsules has been reported to greatly affect the release of bioactive molecules, as crosslinking within films or microcapsules can create diffusion barriers to bioactive molecules and protect them from degradation. For example, the intramolecular cross-linked multilayer membrane system showed a longer lasting BMP-2 release effect and enhanced osteogenic differentiation of C2C12 cells compared to the membrane system formed based on ion pairing, indicating that the intramolecular cross-linked multilayer membrane system can more effectively slow release bioactive molecules.

Overall, the intrinsic cross-linking of the multilayer film has a promoting effect on its stability, biological properties and even on the loading and controlled release effects of the bioactive molecules. Thus, we are interested in knowing whether multilayer films based on intramolecular cross-linking can be constructed by LBL technology using mCH and tHA and discussing whether they can be applied to surface functionalization modifications of nanofibers made from PLLA to create a dressing that sustains release of insulin to cure diabetic wounds.

Disclosure of Invention

In order to overcome the defects of the prior art, the primary aim of the invention is to provide a preparation method of a spinning nanofiber material modified by an intramolecular cross-linked self-assembled film.

The second purpose of the invention is to provide the intramolecular cross-linked self-assembled film modified spinning nanofiber material prepared by the preparation method, and the intramolecular cross-linked self-assembled film modified spinning nanofiber material provided by the invention has good antibacterial and moisturizing properties and good tissue adhesiveness; the slow-release carrier can be used as a stable and efficient slow-release carrier of water-soluble drugs and bioactive factors, and is suitable for the field of skin repair, especially for diabetics.

The third object of the invention is to provide the application of the intramolecular cross-linked self-assembled film modified spinning nanofiber material.

The primary purpose of the invention is to adopt the following technical scheme:

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

s1, carrying out sulfhydrylation modification on hyaluronic acid

Thiol modification is carried out on the hyaluronic acid solution, and thiol hyaluronic acid is obtained after dialysis and freeze drying;

s2, maleylation modification of chitosan

Carrying out maleylation modification on the chitosan solution, and obtaining maleylation chitosan after dialysis and freeze drying;

s3, preparation of polyelectrolyte solution

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

s4, preparing nanofiber by electrostatic spinning method

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

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

Repeating the adsorption and elution steps for a plurality of times to obtain the intramolecular cross-linked self-assembled film modified spinning nanofiber material with alternately adsorbed thiolated hyaluronic acid and maleylated chitosan.

Preferably, in the step S1, the (NHS) hyaluronic acid aqueous solution is activated by using 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 the step S1 is performed under hydrochloric acid condition, and the dialysis time is three days.

Preferably, the step S2 molar ratio of chitosan amino groups to Maleic Anhydride (MA) (n-NH) ₂ nMA) is 0.5-2:1.

Preferably, the molar ratio of chitosan amino groups to Maleic Anhydride (MA) in step S2 (n-NH) ₂ /nMA) is 1:1.

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

Preferably, the pH of the thiol hyaluronic acid solution in S4 is 5-7, and the pH of the maleylated chitosan solution is 3-5.

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

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

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

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

The spinning nanofiber prepared by the electrostatic spinning technology has the advantages of larger specific surface area, adjustable porosity, better ductility and adsorptivity, capability of simulating the structure and functions of a natural extracellular matrix and the like, and becomes a new direction for the development of a repairing material, however, the purely artificial synthetic polymer spinning nanofiber often does not have antibacterial property, moisture retention property and biological activity. Proper modification and modification are needed to improve the feasibility of the wound repair. Compared with the modification of CH and HA only, the invention can endow the repairing material with good antibacterial capability after being used for spinning fiber modification by chemical modification of CH and HA without carrying out additional antibacterial drug load.

Although the natural polymer self-assembled modification layer has good biological activity, the stability of the natural polymer self-assembled modification layer in a physiological environment is often insufficient, and the stability of the natural polymer self-assembled 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 bond 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 a small molecular chemical cross-linking agent after the fact is avoided, so that the subsequent steps of a series of small molecular cleaning and the like are reduced, and the potential toxic effect of the small molecular chemical cross-linking agent is avoided to a certain extent.

The self-assembled biofunctionalization modification is adopted to modify the spinning nanofiber, the biological function of the modified repairing material is improved on the basis of maintaining the good performance of the spinning nanofiber matrix material, the modified repairing material has good antibacterial and moisturizing performances, the water retention property of the repairing material is obviously improved, and the repairing material has good tissue adhesiveness. In addition, the spinning nanofiber subjected to biological functionalization modification 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 bracket 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, the subsequent loading of the bioactive factors or medicines is carried out under the condition, the degradation and the denaturation of the material are not caused, and the biological activity of the loading factors is not influenced.

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

the spinning nanofiber material modified by the intramolecular cross-linked self-assembled film is prepared by the preparation method.

The third object of the invention adopts the following technical scheme:

an application of a spinning nanofiber material modified by an intramolecular cross-linked self-assembled film.

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

Preferably, the intramolecular cross-linked self-assembled film modifies the application of the spinning nanofiber material in insulin loading.

Preferably, the intramolecular cross-linked self-assembled film modified spinning nanofiber material is applied to stem cell proliferation promotion.

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

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

the invention provides a molecular internal crosslinking self-assembled film modified spinning nanofiber material with good antibacterial and moisturizing properties 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 water-soluble drugs or bioactive factors, and is suitable for the field of tissue repair, in particular to the field of skin repair of diabetics.

In addition, the method and the system for preparing the intramolecular cross-linked self-assembled film modified spinning nanofiber material are easy to operate and control, mild in experimental conditions and low in cost, avoid high-energy input, and have great popularization and application values.

Drawings

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

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

FIG. 3 shows the inhibitory effect (A) of PLLA nanofiber spinning membranes on E.coli before and after LBL modification and the diameter (B) of the inhibition zone, [ P: spinning the nanofiber by using PLLA alone; P-CH/HA: PLLA spinning nanofiber after modification of natural CH and HA LBL; P-mCH/tHA: PLLA spinning nanofiber modified by mCH and tHA LBL]The method comprises the steps of carrying out a first treatment on the surface of the (C) To utilizeFiber diameters of PLLA, P-PDA, P-CH/HA and P-mCH/tHA obtained by software through a scanning electron microscope; (D) Stress-strain curves for PLLA, P-CH/HA and P-mCH/tHA; (E) Is an infrared spectrogram of PLLA, P-PDA, P-CH/HA and P-mCH/tHA; (F) Full XPS spectrum for PLLA, P-PDA, P-CH/HA and P-mCH/tHA; (G) N1s map of PLLA, P-PDA, P-CH/HA and P-mCH/tHA obtained by XPS detection; (H) S2P spectra of PLLA, P-PDA, P-CH/HA and P-mCH/tHA obtained by XPS detection;

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

FIG. 5 shows the insulin loading efficiency on PLLA nanofiber spinning membranes before and after LBL modification and the in vitro insulin release at different time points; [ P: spinning the nanofiber by using PLLA alone; P-CH/HA: PLLA spinning nanofiber after modification of natural CH and HALML; P-mCH/tHA: PLLA spun nanofibers after mCH and thlbl modification ];

FIG. 6 shows the adhesion, expansion and proliferation behavior of mesenchymal stem cells on PLLA, P-CH/HA, and P-mCH/tHA, wherein (A) and (B) are the SEM morphology and confocal observations of mesenchymal stem cells on PLLA, P-CH/HA, and P-mCH/tHA with or without insulin loading, respectively; boxes are marked as enlarged views of selected pictures, and significant ECM formation is visible; (C) Proliferation behavior of mesenchymal stem cells on PLLA, P-CH/HA, P-mCH/tHA on day 1 and day three in the presence or absence of insulin;

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

FIG. 8 shows the formation, re-epithelialization and collagen deposition of granulation tissue at the wound surface after observation of untreated groups by HE (A) and Mahalanobis trichromatic staining (B) and treatment with PLLA alone, PLLA_IN, P-CH/HA and P-mCH/tHA;

FIG. 9 shows the expression of CD31 (A) and VEGF-R (B) at wound sites observed by immunohistochemical staining IN untreated groups and after 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 embodiments of the present invention are not limited thereto.

Example 1 intramolecular Cross-Linked self-assembled film 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 aqueous solution of Hyaluronic Acid (HA); 25mM of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride and 25mM of N-hydroxysuccinimide were added in this order, and the pH of the system was adjusted to 5.5 with hydrochloric acid. After stirring at room temperature for 2 hours in the dark, adding 0.2g of cysteine hydrochloride into the reaction system, adjusting the pH of the system to 4.75 with hydrochloric acid, stirring at room temperature in the dark, and dialyzing with hydrochloric acid for 3 days; dialysis on the first day, the mixture in the reaction system was dialyzed against 0.2mM hydrochloric acid, with molecular cutoff mw=3.5 kDa; the next day of dialysis, dialysis was performed with 0.2mM hydrochloric acid containing 1% by mass of sodium chloride; and dialyzing for the third day, regulating the reaction system to 3.5 by using 0.2mM hydrochloric acid again, and freeze-drying to obtain the sulfhydrylated HA (tHA).

(2) Maleylation modification of chitosan

Chitosan (CH) was added at 10mg mL ^-1 Concentration dissolution of (2)At 0.5mol L ^-1 And adding maleic anhydride into the chitosan solution according to the feeding ratio of the CH repeating unit to the maleic anhydride of 1:1 in acetic acid, stirring and mixing, stirring at room temperature for 24 hours in a dark place, dialyzing with distilled water for 3 days, removing impurities and unreacted small molecules by using the molecular cutoff Mw=3.5 kDa, and freeze-drying to obtain the maleylated CH (mCH).

(3) Preparation of polyelectrolyte solutions

The polyelectrolyte solutions were formulated as follows:

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

thiol-modified HA (0.5 mg.mL) ^-1 ) Dissolved in aqueous solution, maleylated CH (0.5 mg mL) ^-1 ) Dissolving in 0.05M acetic acid solution, and magnetically stirring overnight to make it fully dissolved; meanwhile, natural HA and natural CH solutions with the same concentration are prepared for comparison.

Before using each solution, the pH value of all the solutions except the dopamine and tHA solutions is adjusted to 4.

(4) Preparation of poly (L-lactic acid) (PLLA) nanofiber by electrostatic spinning method

7wt% of polylactic acid powder was dissolved in DCM/DMF (4:1 v/v) mixture and put into a 10mL syringe and fixed on a microinjection pump, a flattened needle was used as a capillary for injecting a thin stream, the inside diameter of the capillary was 0.4mm, and the extrusion speed was 1 mL.h at 18kv ^-1 . Collecting the fiber obtained by spinning on a rotary drum, vacuum drying for 12 hours, and removing 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

The polylactic acid spinning nanofiber is fully washed after being soaked by 75% ethanol, is firstly transferred into a dopamine solution of 2 mg-mL-1 to be soaked for 30 minutes, and is fully washed, so that a positively charged dopamine base layer is assembled on the surface of the spinning fiber. The dopamine-modified spinning nanofiber is sequentially immersed in tHA and mCH solutions, each layer is adsorbed for 12 minutes and then is accompanied by an elution step (3X 3 min), and the eluent is an aqueous solution; meanwhile, the dopamine-modified spinning nanofiber is sequentially immersed into the HA solution and the CH solution, and the same condition treatment is compared.

And repeating the steps for a plurality of times to finally obtain 9 layers of P-mCH/tHA spinning nanofibers with alternately adsorbed HA and CH (P-CH/HA) and tHA and mCH on the dopamine basic layer.

And detecting physical and chemical properties such as morphological characteristics of the self-assembled modified polylactic acid spinning nanofiber, and the test results are as follows.

Physical and chemical performance test

(1) Identification of reactive groups

FIG. 1A is a schematic diagram of the preparation of tHA by the 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 an mCH synthesized with a vinylcarboxylic acid group by the maleylation reaction of CH with maleic anhydride; chemical structure of tHA and mCH ¹ H NMR characterization. The methylene proton on-CH 2 SH-showed a new peak at 2.81ppm compared to HA (see FIG. 1B), indicating successful introduction of the thiol group. By comparing CH and mCH in FIG. 1B ¹ H NMR spectra, a new peak at 6.17ppm of-ch=ch-was found in mCH, confirming the successful modification of CH with maleic anhydride. The successful thiol-and maleated modifications of HA and CH were further confirmed by standard Ellman testing and maleic standard curve method, wherein the thiol content was 120.4.+ -. 4.8. Mu. Mol g ^-1 While 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

The difference between mCH/tHA multilayer film systems based on intramolecular cross-linking and mCH/HA multilayer film systems based on ion pairing was observed by QCM detection. FIG. 2 shows the variation of adsorption quality (A) and thickness (B) of mCH/tHA and CH/HA membrane systems obtained by QCM detection and calculation using Sauerbey equation in the layer adsorption process, wherein the odd layer is tHA or HA, and the even layer is mCH or CH. As can be seen from FIG. 2, the molecular adsorption quality (i.e., the adsorption capacity 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 those of the CH/HA film system due to the intramolecular covalent crosslinking of the double bond between mCH and tHA and the mercapto group.

(2) Morphology structure of self-assembled modified polylactic acid spinning nanofiber and hydrophilic, moisturizing and water-holding capacities

Contact Angle (WCA) detection is an important means of reactive material surface hydrophilicity, and the WCA value decreases as the hydrophilicity of the material surface increases.

FIG. 3 shows that the intramolecular cross-linked self-assembled film modified spun nanofiber material prepared in example 1 is a total of 9 layers of P-CH/HA and P-mCH/tHA nanofiber alternately adsorbed by HA and CH and tHA and mCH, [ P: pure PLLA spinning membrane, P-PDA: PLLA spinning membrane of adsorption PDA layer, P-CH/HA: PLLA spin-film self-assembled with 9 layers of HA and CH, P-mCH/tHA: PLLA spin-film self-assembled with 9 layers of mCH/tHA ].

In fig. 3A, the Water Contact Angle (WCA) of P-CH/HA and P-mCH/tHA in the process of alternately adsorbing CH (mCH) and HA (tHA) is changed, and it can be seen from the figure that the hydrophilicity of the PLLA film after layer adsorption is significantly improved, and the contact angle shows a trend of alternately changing, which indicates that CH (mCH) and HA (tHA) are successfully alternately adsorbed onto the PLLA film. In addition, with the alternate adsorption of mCH and tHA, the WCA was significantly more varied than CH and HA, further confirming that mCH and tHA had higher adsorption quality than CH and HA; FIG. 3B shows the morphology of PLLA, P-PDA, P-CH/HA and P-mCH/tHA, respectively, and FIG. 3B shows that the surface roughness of PLLA spun fibers is significantly improved after mCH and tHA modification, which indicates that mCH and tHA have been successfully adsorbed onto PLLA spun nanofibers; FIG. 3C is a schematic diagram of utilization ofFiber diameters of PLLA, P-PDA, P-CH/HA and P-mCH/tHA obtained by software through a scanning electron microscope; FIG. 3D is a stress-strain plot of PLLA, P-CH/HA and P-mCH/tHA, from which it can be seen that the tensile strength of PLLA spun fibers modified by LBL is significantly improved, especially the mCH/tHA modified spun fibers have the highest tensile strength, probably because inter-molecular crosslinking in the film enhances the tensile strength; FIG. 3E shows the IR spectra of PLLA, P-PDA, P-CH/HA and P-mCH/tHA, modified by mCH/tHA LBL, P-mCH/tHA at 1640cm ^-1 And 1560cm ^-1 An amide bond (-CONH-) formed by mCH and tHA and an amide II peak on mCH appear, and mCH is originally 864cm ^-1 The peak of the maleic double bond at the position is weakened after self-assembly reaction with tHA; FIG. 3F is a graph of the full XPS spectra of PLLA, P-PDA, P-CH/HA and P-mCH/tHA, wherein FIGS. 3G and 3H are the N1S and S2P spectra of PLLA, P-PDA, P-CH/HA and P-mCH/tHA, respectively, obtained by XPS detection, showing a significant increase in the N content of the PLLA film after LBL modification, and an increase in the S content derived from tHA seen on P-mCH/tHA, further indicating successful LBL modification of PLLA.

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

Sample of	Moisture content (%)	Swelling Rate (%)
			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 can be seen from Table 1, the water content of P-mCH/tHA was increased from 37.5.+ -. 4.17% to 78.59.+ -. 2.10% compared to pure PLLA after modification by mCH/tHA LBL; the P-mCH/tHA swelling ratio was increased from 20.48.+ -. 6.44% to 527.78.+ -. 25.46% compared to 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 the P-CH/HA and P-mCH/tHA after LBL modification increased from 644+ -175 nm (P) to 909+ -185 nm and 906+ -204 nm, respectively, and no difference in fiber diameter was found between the P-CH/HA and P-mCH/tHA; at 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 do not significantly change as compared with PLLA, but the Young's modulus is greatly increased, conversely, the mechanical properties of P-mCH/tHA are greatly improved as compared with that of pure PLLA and P-CH/HA, and the highest tensile stress and Young's modulus at the breaking point are as high as 3.23+ -0.57 MPa and 0.595+ -0.192 MPa.

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

Sample of	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 N content 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.

As can be seen from the graph results, compared with CH/HA, the absorption quality of the mCH/tHA system on PLLA is obviously higher than that of CH and HA due to the intermolecular crosslinking effect, the hydrophilicities of P-CH/HA and P-mCH/tHA modified by CH and HA or mCH and tHA are obviously improved, and the tensile strength of P-mCH/tHA modified by mCH and tHA is also obviously improved, so that the repairing material prepared by the method HAs good water absorption and moisture retention effects and strong mechanical properties.

Comparative example 1

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

Comparative example 2

The same concentration of chitosan and thiol-modified hyaluronic acid (P-CH/HA) solution without maleylation modification as described in example 1 was prepared.

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

The antibacterial effect of the repair material provided in the patent example 1 of the present invention on gram-negative bacteria E.coli was studied by referring to the agar diffusion method in the published literature method, and compared with the PLLA material of comparative example 1 alone and the PLLA material of comparative example 2 after the conventional CH/HA self-assembly modification. The sterilized Luria-Bertani agar medium was first poured into a plate and the bacteria were spread evenly on the surface of the slant medium using an inoculating loop. Only PLLA, P-CH/HA and P-mCH/tHA were each cut into small discs (12 mm) and placed on plates and incubated for 24 hours at 37 ℃. After the completion of the culture, the diameter of the bacterial growth inhibition zone on the plate was measured. The result shows that the pure PLLA HAs no antibacterial property per se, 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. In contrast, the PLLA spinning nanofiber membrane has an obvious antibacterial zone around the spinning fiber after self-assembly modification by mCH/tHA, and the antibacterial zone width is about 6.53 mm+/-1.54, which shows that the repairing material has obvious antibacterial and bacteriostatic capacities, and the specific situation is shown in figure 4.

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

IN our earlier studies, it was found that a certain concentration of Insulin (IN) had a significant ability to promote proliferation of umbilical mesenchymal stem cells under serum-free conditions. On the basis, the self-assembled polymer layer modified by the spinning fiber in the embodiment 1 is used as a carrier, the load of insulin in the spinning fiber is realized by a simple soaking mode, and compared with the PLLA material modified by the self-assembly of the pure PLLA material in the comparative embodiment 1 and the unmodified CH/HA material in the comparative embodiment 2, the load efficiency and the controlled release effect of the repairing material in the embodiment 1 of the invention on insulin are examined.

FIG. 5 shows the loading efficiency and slow release behavior of insulin on three materials, PLLA alone, P-CH/HA and P-mCH/tHA. As can be seen from the results of FIG. 5, the insulin loading in the spun fiber modified by mCH/tHA is significantly improved compared with the other two groups. In addition, the insulin release suddenly appears in the first 3 hours on the three groups of materials, but the release amount of the insulin on the materials after the mCH/tHA modification is obviously reduced, and the materials have obviously better slow release effect on the insulin (the release amount of the insulin in the materials is less than 60% after 9 days).

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

An important factor of the biological material for repairing skin wound surface is that the biological material is required to have the capability of promoting wound surface repair, and early experiments find that insulin with a certain concentration can obviously promote umbilical mesenchymal stem cell proliferation under the serum-free condition under the two-dimensional culture condition. The invention uses an mCH/tHA self-assembled layer as an insulin carrier, discovers that the spinning fiber material modified by mCH and tHA can obviously improve the slow release effect of insulin, and researches the growth and proliferation behaviors of stem cells on PLLA modified by pure PLLA in comparative example 1 and CH/HA in comparative example 2 by comparing and researching the effect of the repairing material in promoting stem cell proliferation. Fig. 6 shows proliferation behaviors of umbilical cord mesenchymal stem cells on the surfaces of different materials, and as can be seen from fig. 6, the spun fiber loaded with insulin can significantly promote cell proliferation, but compared with pure PLLA and PLLA modified by CH/HA, the repair material disclosed by the invention HAs better cell proliferation promoting effect and extracellular matrix secretion promoting effect (partial enlarged graph) through slow release effect on insulin.

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

A full-thickness skin wound model was made on the back of 30 diabetic mice purchased at Nanjing university model animal institute according to literature report methods. The 30 mice were randomly divided into 5 groups, which were untreated (control), PLLA, P_IN, P-CH/HA_IN, and P-mCH/tHA _IN. And (3) in different periods (0, 3 days, 5 days, 9 days and 16 days) of wound surface treatment, taking photos at fixed time to record wound surface healing conditions, taking materials after 16 days of treatment, embedding slices, carrying out histochemical and immunohistochemical staining, and observing the repairing effect of the wound surfaces of different treatment groups.

As shown in fig. 7, there is a significant difference in the healing rate of the wound surface between the different treatment groups, wherein the repair material prepared by the present invention, i.e., the spun fiber material modified by mCH and tHA, significantly promoted the healing of the wound surface. Furthermore, by HE (fig. 7) staining, it was seen that the intact re-epithelialization and distinguishable epithelium characterized by good formation of the epidermis were seen with skin wounds treated with P-mCH/tHA _in, significantly different from the non-treated group of epithelial insufficiency and the unclear epidermis layer of the PLLA treated group. Although signs of complete re-epithelialization were also seen IN the p_in treated group, there was less epidermal delamination and insufficient dermal-epidermal junction. IN addition, while the P-CH/ha_in treated group also seen better wound repair, the P-mCH/tHA _in treated group seen more clearly layered epidermis layers and a complete basal membrane, showing a well developed epidermis-dermis junction. Further, by the marshy trichromatic staining 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 wound surface angiogenesis shows that CD31 and VEGF-R related to angiogenesis are highly expressed IN the P-mCH/tHA _IN group, so that the repair material disclosed by the invention has good angiogenesis promoting effect.

The invention obtains the molecular internal crosslinking self-assembled film modified spinning nanofiber material with good antibacterial and moisturizing properties and suitable tissue adhesiveness by performing biological functional modification on the polylactic acid spinning nanofiber, and the technology can be popularized and applied to other macromolecule spinning nanofiber modification and other tissue engineering application fields.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. The preparation method of the intramolecular cross-linked self-assembled film modified spinning nanofiber material is characterized by comprising the following steps of:

(1) Thiol modification of hyaluronic acid

Under magnetic stirring, 0.2g of hyaluronic acid is dissolved in 50mL of water and stirred uniformly to prepare an aqueous solution of hyaluronic acid; sequentially adding 25mM of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride and 25mM of N-hydroxysuccinimide, and adjusting the pH of the system to 5.5 by using hydrochloric acid; after stirring at room temperature for 2 hours in the dark, adding 0.2g of cysteine hydrochloride into the reaction system, adjusting the pH of the system to 4.75 with hydrochloric acid, stirring at room temperature in the dark, and dialyzing with hydrochloric acid for 3 days; dialysis on the first day, the mixture in the reaction system was dialyzed against 0.2mM hydrochloric acid, with molecular cutoff mw=3.5 kDa; the next day of dialysis, dialysis was performed with 0.2mM hydrochloric acid containing 1% by mass of sodium chloride; dialyzing for the third day, regulating the pH of the reaction system to 3.5 by using 0.2mM hydrochloric acid again, and obtaining the sulfhydrylation HA after the dialysis is completed and freeze-drying is carried out;

(2) Maleylation modification of chitosan

Chitosan was added at 10 mg/mL ^-1 Is dissolved in 0.5 mol.L ^-1 Adding maleic anhydride into chitosan solution according to the feeding ratio of CH repeating units to maleic anhydride of 1:1, stirring and mixing, stirring at room temperature for 24 hours in a dark place, dialyzing with distilled water for 3 days, removing impurities and unreacted small molecules by using molecular cutoff Mw=3.5 kDa, and freeze-drying to obtain maleated CH;

(3) Preparation of polyelectrolyte solutions

The polyelectrolyte solutions were formulated as follows:

dissolving dopamine in Tris-HCl solution with pH of 8.5, wherein the mass concentration of the dopamine in the Tris-HCl solution is 2 mg.mL ^-1 ；

Dissolving sulfhydrylation HA in water solution to prepare the solution with mass concentration of 0.5 mg.mL ^-1 Is a thiol group of HA; dissolving maleylated CH in 0.05M acetic acid solution, and magnetically stirring overnight to make it fully dissolved, wherein the mass concentration of maleylated CH is 0.5 mg.mL ^-1 And the pH value of the maleylation CH solution is adjusted to 4;

(4) Preparation of poly (L-lactic acid) nanofiber by electrostatic spinning method

Dissolving polylactic acid powder into DCM/DMF mixed solution with the volume ratio of 4:1 and filling into a 10mL syringe, wherein the syringe is fixed on a microinjection pump, and the mass fraction of the polylactic acid powder in the mixed solution is 7%; adopting flattened needle head as capillary tube for jetting trickle, the inner diameter of the capillary tube is 0.4mm, and the extrusion speed is 1 mL.h under the voltage of 18kv ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Collecting the fiber obtained by spinning on a rotary drum, vacuum drying for 12 hours, and removing residual organic solvent to obtain poly (L-lactic acid) nanofiber;

(5) Layer-by-layer self-assembled surface functionalization of poly (L-lactic acid) nanofibers

The poly (L-lactic acid) nanofiber is fully washed after being soaked by 75% ethanol, and is firstly transferred to 2 mg.mL ^-1 Soaking in dopamine solution for 30 minutes, then fully washing to enable the dopamine solution to be assembled with a positively charged dopamine base layer on the surface of the spinning fiber, sequentially immersing the spinning nanofiber modified by the dopamine into thiolated HA and maleylated CH solution, and eluting each layer after 12 minutes of absorption, wherein the eluent is an aqueous solution;

and repeating the steps for a plurality of times to finally obtain 9 layers of tHA and mCH alternately adsorbed P-mCH/tHA spinning nanofiber on the dopamine base layer, namely the intramolecular cross-linked self-assembly membrane modified spinning nanofiber material.

2. An intramolecular cross-linked self-assembled film modified spun nanofiber material prepared by the method for preparing an intramolecular cross-linked self-assembled film modified spun nanofiber material according to claim 1.

3. Use of the intramolecular cross-linked self-assembled film modified spinning nanofiber material according to claim 2 for promoting wound repair of diabetic mice.