Preparation method of composite microspheres for regulating and controlling local inflammation microenvironment and promoting nerve regeneration

Abstract

The invention discloses a preparation method of composite microspheres for regulating and controlling a local inflammation microenvironment and promoting nerve regeneration. The method comprises the following steps: preparing liposome loaded with ginsenoside Rg1; preparing a GM microsphere loaded with a stromal cell-derived factor 1 alpha (SDF1 alpha); and preparing the inflammation regulation nerve regeneration promoting composite microspheres. The composite microsphere can reduce inflammatory response, reduce scar tissue formation, promote angiogenesis, continuously release SDF1 alpha and promote endogenous stem cell recruitment and neural differentiation. Therefore, the composite microsphere is a functional biological scaffold for firstly regulating and controlling local microenvironment inflammation of spinal cord injury and then promoting collection and differentiation of endogenous stem cells for innovative sequential inflammation regulation and nerve regeneration, and a new strategy is provided for treating spinal cord injury by tissue engineering.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and specifically relates to a method for preparing composite hydrogel microspheres for regulating local inflammatory microenvironment and promoting nerve regeneration. Background Art

[0002] Spinal cord injury can lead to neuronal death and axonal damage, resulting in permanent sensory and motor dysfunction. SCI imposes severe physical, psychological and economic burdens on patients and their families. SCI is the result of the combined effects of multiple factors, and intervention targeting a single factor has limited effect on SCI repair. Exogenous cell transplantation has a strong potential for neuroprotection and has therefore been used in SCI repair. However, the safety, ethical issues, sources, and poor retention and differentiation capacity of exogenous stem cells after implantation limit their application. Compared with exogenous bone marrow mesenchymal stem cells (BMSCs), endogenous BMSCs provide a safer and more convenient treatment for SCI patients. During the course of SCI, endogenous BMSCs are activated and spontaneously migrate to the site of injury to promote neural network reconstruction. However, the number of BMSCs recruited to the local lesion of SCI is limited. At the same time, inflammation in the acute phase of SCI reduces the activity of BMSCs and causes BMSCs to differentiate into glial cells rather than neurons, which seriously affects the effect of cell therapy. Therefore, in order to improve the effect of in situ BMSCs cell therapy, it is necessary to regulate inflammation in the acute phase of SCI and enhance the migration and differentiation ability of local BMSCs.

[0003] Inflammation in the acute phase of SCI is closely related to the innate immune system, among which macrophage infiltration induces aggravated inflammation in the acute phase. Macrophage polarization into classically activated type 1 (M1) macrophages or alternatively activated type 2 (M2) macrophages exhibits harmful or protective properties in SCI, respectively. M1 macrophages induce axon retraction, secondary injury, and neuroinflammation, leading to glial scar formation and hindering axon regeneration, while M2 macrophages promote the secretion of anti-inflammatory factors and axon growth. Therefore, regulating the polarization direction of macrophages, reducing the proportion of M1 macrophages, and increasing the proportion of M2 macrophages can effectively alleviate acute inflammation and provide a suitable environment for the migration and differentiation of endogenous BMSCs.

[0004] As a traditional Chinese herbal medicine, ginseng has a history of medicinal use for thousands of years. Ginsenoside (Rg1) is a hydrophobic small molecule and one of the main active ingredients of ginseng. Rg1 plays an important role in anti-inflammatory, antioxidant and neuroprotective effects. Rg1 promotes the polarization of macrophages to the M2 phenotype through the NF-κB and PI3K / Akt / mTOR pathways, alleviating the inflammatory response in the acute phase of SCI. At the same time, Rg1 can improve the antioxidant capacity of BMSCs, reduce apoptosis, increase survival rate, and promote BMSC paracrine. In addition, Rg1 promotes the differentiation of BMSCs into neurons. Therefore, in situ cell therapy of BMSCs assisted by Rg1 has the potential to treat SCI. However, Rg1 has poor water solubility and low concentration of traditional administration, which limits the therapeutic effect of Rg1. In order to give full play to the therapeutic effect of Rg1, it is necessary to introduce an effective drug delivery system.

[0005] In recent years, biomaterials have provided breakthrough solutions for the treatment of SCI. Due to their high adjustability and ease of drug carrying, hydrogels are currently a hot research topic. In neural tissue engineering, a variety of polymer hydrogels have been explored, including collagen, hyaluronic acid, fibronectin, etc. These hydrogels can be custom designed to mimic the extracellular matrix of the central nervous system environment, simulate tissue hardness, and load cells or drugs. In many areas of tissue engineering research, the combination of hydrogels with drugs or growth factors has become one of the most commonly used systems to achieve sequential transfer and delivery of drugs or growth factors.

[0006] Methacryloyl gelatin (GM) is a photo-crosslinked hydrogel material with good biocompatibility and is suitable for neural soft tissues, which can fill spinal cord defects and provide a bridge for spinal cord injury repair. However, GM itself is not easy to carry hydrophobic drugs. Summary of the invention

[0007] In order to solve the above technical problems and give full play to the therapeutic effect of Rg1, an effective drug delivery system is introduced. The present invention introduces cationic liposomes (aL). aL has a tightly wrapped double-layer membrane structure, the double-layer lipid shell can be loaded with hydrophobic drugs, and the aldehyde groups on the surface can be combined with GM hydrogels through Schiff base reactions. Stromal cell-derived factor 1α (SDF1α) can selectively recruit stem cells, and integrating SDF1α into GM hydrogels can enhance the ability of biomaterials to recruit BMSCs. The present invention provides a composite microsphere, a preparation method and an application thereof. A composite microsphere of the present invention is composed of an inflammation regulating carrier aL loaded with Rg1 grafted to a GM microsphere loaded with SDF1α through a Schiff bond, and is named GM@SDF1α-aL@Rg1.

[0008] According to some technical solutions of the present invention, a method for preparing composite microspheres for regulating local inflammatory microenvironment to promote nerve regeneration is provided, the method comprising the following steps:

[0009] Step 1: Preparation of liposomes loaded with anti-inflammatory drugs;

[0010] Step 2: Preparation of GM microspheres loaded with cytokines;

[0011] Step 3 Preparation of composite microspheres that regulate inflammation and promote nerve regeneration: grafting liposomes loaded with anti-inflammatory drugs and GM microspheres loaded with cytokines.

[0012] The present invention further provides a method for preparing composite microspheres for regulating local inflammatory microenvironment to promote nerve regeneration, wherein the anti-inflammatory drug in step 1 is ginsenoside Rg1, and the cytokine in step 2 is stromal cell-derived factor 1α.

[0013] The present invention further provides a method for preparing composite microspheres that regulate local inflammatory microenvironment and promote nerve regeneration. The above step 1 is prepared by a thin film dispersion method, and the preparation steps of liposomes loaded with ginsenoside Rg1 are as follows:

[0014] 2.5 parts by weight of ginsenoside Rg1, 80 parts of lecithin and 20 parts of cholesterol, 2 parts of DSPE-PEG-CHO and 2.5 parts of octadecylamine were added to chloroform and mixed to obtain a uniform emulsion. The organic solvent was removed from the emulsion in a rotary evaporator to obtain a colloidal product. Deionized water was then added to hydrate the lipid film on the bottle wall, and ultrasonic treatment was used to completely dissolve the lipid film to form a nanoscale liposome emulsion. The polycarbonate membrane was used for filtration to obtain a uniform monolayer liposome loaded with ginsenoside Rg1.

[0015] The present invention further provides a method for preparing composite microspheres that regulate local inflammatory microenvironment to promote nerve regeneration, wherein the above step 2 comprises the following steps:

[0016] Type A pig skin gelatin is prepared into a 10% (w / v) solution and completely dissolved in phosphate buffered saline (PBS) at 60°C; then, methacrylic acid (MA) is added to the gelatin solution and stirred for reaction at 50°C; the mixture is diluted with additional PBS to stop the reaction, and then the mixture is dialyzed at 40°C to remove unreacted MA and salts using a dialysis tube with a molecular weight cutoff of 12-14 kilodaltons; finally, the GM solution is lyophilized to obtain GM.

[0017] The present invention further provides a method for preparing composite microspheres that regulate local inflammatory microenvironment and promote nerve regeneration. The above step 2 also includes the step of preparing GM microspheres using a microfluidic device:

[0018] The above step 3 prepares GM microspheres by a microfluidic device, comprising the following steps:

[0019] A 7wt% GM and 0.5wt% LAP PBS solution was used as the aqueous phase, SDF1α was resuspended in a 0.1wt% bovine serum albumin solution to obtain a final concentration of 100μg / ml SDF1α solution, the above SDF1α solution was added to the aqueous phase to obtain a uniform SDF1αGM solution aqueous phase with a concentration of 100ng / mL, and a 5wt% Span 80 in isopropyl myristate solution was used as the oil phase; the above aqueous and oil phase fluids were respectively connected to the inner and outer ports of the microfluidic coaxial needle, the inner and outer injection rates of the injection pump were adjusted to 1:30, the generated microspheres were collected, and cross-linked by ultraviolet irradiation, and washed with ethanol and isopropanol respectively to remove residual Span 80 and isopropyl myristate, and after washing with PBS, GM microspheres loaded with stromal cell-derived factor 1α were obtained.

[0020] The present invention further provides a method for preparing composite microspheres that regulate local inflammatory microenvironment to promote nerve regeneration, wherein the above step 3 comprises the following steps:

[0021] The aqueous solution of the GM microspheres loaded with stromal cell-derived factor 1α and the aqueous solution of the liposomes loaded with ginsenoside Rg1 were mixed and grafted, and then washed with deionized water to obtain GM@SDF1α-aL@Rg1 composite microspheres.

[0022] The present invention further provides a method for preparing composite microspheres that regulate local inflammatory microenvironment and promote nerve regeneration. The above-mentioned grafting conditions are placing in an oven at 37° C. and setting a grafting time of at least 24 hours.

[0023] The present invention further provides a composite microsphere for regulating local inflammatory microenvironment to promote nerve regeneration. The composite microsphere is prepared by using any one of the above-mentioned preparation methods for composite microsphere for regulating local inflammatory microenvironment to promote nerve regeneration.

[0024] The present invention further provides an application of composite microspheres for regulating local inflammatory microenvironment to promote nerve regeneration. The composite microspheres for regulating local inflammatory microenvironment to promote nerve regeneration are used as drugs for regulating inflammatory microenvironment after spinal cord injury to promote nerve regeneration.

[0025] The purpose of the present invention is to provide a method for preparing a composite hydrogel that regulates the inflammatory microenvironment and promotes nerve regeneration, and to assemble amino GM microspheres loaded with SDF1α and aldehyde-loaded cationic liposomes loaded with Rg1 to construct a functional composite hydrogel scaffold that regulates the inflammatory microenvironment of spinal cord injury and promotes nerve regeneration.

[0026] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The invention relates to the construction of GM-aL composite microspheres and their mechanism of action, wherein A is the construction of bionic GM-aL microspheres for the treatment of spinal cord injury, B is the regulation of inflammation and the effect of nerve regeneration;

[0028] Figure 2 The physical properties of the composite GM-aL microgel in the embodiment of the present invention; (a) Microscope image of GM microgel; (b) SEM image of GM microgel; (c) TEM image of aL; (d) LSCM image of GM-aL microgel; aL in the microgel was labeled with DiI dye; few liposomes were observed to be released from the microgel in aqueous solution, (e) SEM image of liposomes in GM microgel, (f) Particle size distribution of liposomes measured by DLS; (g) Particle size of GM microgel, (h) Particle size of GM-aL microgel; (i) Surface charge of aL, GM microgel and GM-aL microgel; (j) Microscope image showing the morphological changes of GM-aL microgel within 5 weeks. (k) Degradation curve of GM-aL microgel; (l) Encapsulation efficiency of Rg1 in liposomes, SDF1α in GM and Rg1 in GM-aL microgel; (m) In vitro release of Rg1 from liposomes and GM-aL microgel;

[0029] Figure 3 Characterization of GM-aL microgel in the embodiment of the present invention; (a) Release curve of Sdf-1a in GM-aL; (b) FTIR method to evaluate the successful connection of chemical bond Schiff base to amino GelMA hydrogel microspheres and aldehyde-modified cationic liposomes;

[0030] Figure 4 Cell compatibility of composite Gelma microspheres in the embodiments of the present invention (a) Live / dead images of BMSCs after incubation with extracts GM@SDF1α-aL, GM-aL@Rg1, GM@SDF1α-aL@Rg1. (b) Quantification of live cells in live / dead assay; (c) Cytotoxicity determination using CCK-8;

[0031] Figure 5Polarization and inflammatory factors secreted by Raw264.7 in the embodiment of the present invention, (a) immunofluorescence staining of macrophage marker F4 / 80 (red) and M1 macrophage marker CD80 (green), (b) immunofluorescence staining of macrophage marker F4 / 80 (red) and M2 macrophage marker CD206 (green), (c) M1 macrophage marker CD80; (d) fluorescence semi-quantitative analysis of M2 macrophage marker CD206, (eh) mRNA expression analysis of TNF-α, IL-1β, IL-10, and TGF-β, (il) ELISA method to detect the secretion of inflammatory factors TNF-α, IL-1β, IL-10, and TGF-β by Raw264.7;

[0032] Figure 6 Composite GM microspheres promote cell migration and recruitment in the present invention; (a) Representative optical microscopic images of the scratch test at 0, 12 and 24 hours; (b) Representative optical microscopic images of the Transwell test; (c) Analysis of the healing percentage of the scratch test by Image J.; (d) The number of cells migrated in the Transwells test;

[0033] Figure 7 The expression of neuron-specific markers in the present invention; (a) Immunofluorescence staining of Tuj-1, Map-2, NF-200 and Tau staining images of different GelMA microsphere groups, and corresponding fluorescence semi-quantitative analysis. (fI) mRNA expression analysis of Tuj-1, Map-2, NF-200 and Tau;

[0034] Figure 8 It is a quantitative analysis of the length of Tuj-1+ cells in the embodiment of the present invention;

[0035] Fig. 9 H&E staining and motor function scoring of the spinal cord specimens of the embodiment of the present invention; (a) H&E staining of the rat spinal cord at 4 weeks and 8 weeks, respectively; calculation of the cavity area at (b) 4 weeks and (c) 8 weeks after surgery; (d) evaluation of the motor function recovery of the rat lower limbs by BBB and (e) IPT scoring.

[0036] Fig.10 Phenotypic identification of in vivo immune cells 7 days after surgery in the present invention; (a) immunofluorescence staining of macrophage marker Iba1 (green) and M1 macrophage marker iNOS (red), and quantitative analysis of their optical density; (b) immunofluorescence staining of macrophage marker Iba1 (green) and M2 macrophage marker ARG-1 (red), and quantitative analysis of their optical density;

[0037] Fig.11Evaluation of inflammation regulation 7 days after surgery in the present invention; (a) IL-10, TNF-α and (b) TGF-β, IL-1β immunofluorescence double labeling; quantitative analysis of (c) IL-10 and TNF-α immunofluorescence, (d) TGF-β and IL-1β immunofluorescence; evaluation of TNF-α (e), IL-1β (f), IL-10 (g) and TGF-β (h) gene expression levels in spinal cord specimens by qRT-PCR; ELISA to detect serum TNF-α (i), IL-1β (j), IL-10 (k) and TGF-β (l) levels;

[0038] Fig.12 Immunofluorescence staining of activated astrocytes and glial scars in the examples of the present invention. (a, b) Immunofluorescence staining of astrocytes and quantitative analysis by optical density, and (c, d) quantitative analysis;

[0039] Fig.13 The immunofluorescence staining of neurons in the embodiment of the present invention; (a) immunofluorescence staining of neurons, (b) quantitative analysis;

[0040] Fig.14 Immunofluorescence staining of neurons and axons in the embodiments of the present invention; (a) immunofluorescence staining of neurons and (c) quantitative analysis of Tuj-1 optical density; (b) immunofluorescence staining of axons and (d) quantitative analysis of GAP-43 optical density;

[0041] Fig.15 The vascularization ability of different composite microspheres in the embodiments of the present invention; (a) immunofluorescence staining of endothelial cells and (c) quantitative analysis; (b) immunofluorescence staining of new blood vessels and (d) quantitative analysis

[0042] Fig.16 This is an H&E staining image of a rat organ in an embodiment of the present invention. DETAILED DESCRIPTION

[0043] The following embodiments are intended to further illustrate some preferred embodiments of the present invention, but are not all embodiments. Other embodiments based on the present invention made by professionals in the field without creative work all belong to the scope of protection of the present invention. The present invention will be further described below in conjunction with the accompanying drawings.

[0044] Material preparation: prepare the required chemical reagents and experimental materials, including lecithin, cholesterol, DSPE-PEG-CHO, octadecylamine, chloroform, deionized water, Rg1, type A porcine gelatin, methacrylic acid, phosphate-buffered saline, Span 80, isopropyl myristate, SDF1α, bovine serum albumin, LAP, etc.

[0045] The basic test method is as follows:

[0046] 1. Characterization of GM and GM-aL: GM and GM-aL were characterized by light field microscopy, scanning electron microscopy, Zeta potential analysis, infrared spectroscopy, etc., and the encapsulation efficiency of GM for SDF1α, Rg1 loading rate, in vitro degradation rate and in vitro sequential drug release rate were determined.

[0047] 2. In vitro biocompatibility testing of inflammation-modulating microspheres: Mouse bone marrow mesenchymal stem cells were co-cultured with microspheres, and the biocompatibility was evaluated by live / dead staining and CCK-8 assay.

[0048] 3. Characterization of the inflammatory regulation function of composite microspheres: The inflammatory regulation function of composite microspheres was evaluated by real-time quantitative PCR detection, ELISA detection and immunofluorescence analysis.

[0049] 4. Characterization of the BMSCs recruitment function of composite microspheres: The recruitment function of composite microspheres on BMSCs was evaluated by cell scratch test and cell transwell test.

[0050] 5. Characterization of the neuroregeneration-promoting function of composite microspheres: The neuroregeneration-promoting function of composite microspheres was evaluated by immunofluorescence staining of neuron-specific markers and qRT-PCR detection of neuron-specific marker genes.

[0051] 6. Animal experiments on spinal cord injury: A spinal cord Allen impact model was established. Rats were randomly divided into groups and different drugs were injected locally into the spinal cord to evaluate the animal's motor function and in vivo inflammatory regulation function.

[0052] 7. Histological analysis: The spinal cord specimens were subjected to H&E staining and immunofluorescence staining to analyze the spinal cord cavity area, glial scar tissue, endogenous neural progenitor cells, neurons and angiogenesis.

[0053] The experimental methods without specific conditions in the examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Figure 1 .

[0054] Example 1 Preparation of Aldehyde-Formylated Cationic Liposome aL

[0055] aL was prepared by a thin film dispersion method. 80mg lecithin (Yuanye, Shanghai, China), 20mg cholesterol (Arcos, Belgium), 2mg DSPE-PEG-CHO (Aladdin, Shanghai, China) and 2.5mg octadecylamine (Aladdin, Shanghai, China) were dissolved in 30ml chloroform (Lingfeng, Shanghai, China), and further mixed using ultrasound to obtain a uniform emulsion. The emulsion was evaporated in a rotary evaporator for 1 hour, the organic solvent was removed, and a colloidal product was obtained. Then 3mL of deionized water was added to hydrate the lipid film on the bottle wall, and the lipid film was completely dissolved using ultrasound treatment to form a nanoscale liposome emulsion. Finally, a uniform single layer aL was obtained by filtration using 450 and 220nm polycarbonate membranes (Millex-GP, Ireland).

[0056] Example 2 Preparation of liposome aL@Rg1 loaded with ginsenoside Rg1

[0057] 2.5 mg Rg1 (Aladdin, Shanghai, China), 80 mg lecithin, 20 mg cholesterol, 2 mg DSPE-PEG-CHO and 2.5 mg octadecylamine were added to 30 mL chloroform, and the other steps were the same as those in Example 1 for liposome preparation.

[0058] Example 3 Preparation of methacryloyl gelatin (GM) microspheres;

[0059] Type A pig skin gelatin was prepared into a 10% (w / v) solution and completely dissolved in phosphate buffered saline (PBS) at 60°C. Subsequently, a certain amount of methacrylic acid (MA) was added to the gelatin solution and stirred at 50°C for 1 hour. The above mixture was diluted 5 times with additional PBS to stop the reaction, and then the mixture was dialyzed at 40°C for 1 week to remove unreacted MA and salts (dialysis tubing with a molecular weight cutoff of 12-14 kilodaltons) to obtain a GM solution.

[0060] Finally, the GM solution was freeze-dried for 1 week to generate a white porous foam that could be stored at -80 °C for a long time.

[0061] GM microspheres were prepared by a microfluidic device. A PBS solution of 7wt% GM and 0.5wt% LAP (Sigma, USA) was used as the aqueous phase, and a 5wt% Span 80 (Aladdin, Shanghai, China) in an isopropyl myristic acid solution (Aladdin, Shanghai, China) was used as the oil phase. The two fluids were connected to the inner and outer ports of the microfluidic coaxial needle (30G / 22G), respectively, and the injection rate of the syringe pump (Raffer Fluid, China) was adjusted to 1:30. The generated microspheres were collected and cross-linked using ultraviolet irradiation. Ethanol and isopropanol were used to wash 3 times respectively to remove residual Span 80 and isopropyl myristic acid. After washing 3 times with PBS, GM microspheres were obtained.

[0062] It can be freeze-dried and stored at -20°C.

[0063] Example 4 Preparation of GM microspheres GM@SDF1α loaded with stromal cell-derived factor 1α (SDF1α);

[0064] A 7wt% GM and 0.5wt% LAP PBS solution was used as the aqueous phase, and then SDF1α (peprotech, USA) was resuspended in a 0.1wt% bovine serum albumin (BSA) solution to a final concentration of 100 μg / ml, and added to the above solution in a certain proportion to obtain a uniform SDF1α GM solution with a concentration of 100 ng / mL. A 5wt% Span 80 in isopropyl myristate solution was used as the oil phase. The other steps were the same as the GM microsphere preparation method in Example 4.

[0065] Example 5 Preparation of GM-aL composite microspheres that regulate inflammation and promote nerve regeneration.

[0066] 2 ml of GM microsphere solution was mixed with 10 ml of liposome solution, placed in an oven at 37°C, and set for at least 24 hours of grafting time. Finally, it was washed three times with deionized water to obtain GM-aL composite microspheres.

[0067] 2 ml of GM@SDF1α microsphere aqueous solution was mixed with 10 ml of aL@Rg1 liposome aqueous solution, placed in an oven at 37°C, and set for at least 24 hours of grafting time. Finally, it was washed three times with deionized water to obtain GM@SDF1α-aL@Rg1 composite microspheres.

[0068] The aL@Rg1 aqueous solution was obtained by dissolving 3 portions of the entire product of Example 2 in 10 ml of aqueous solution, and the GM@SDF1α microsphere aqueous solution was obtained by taking 2 ml from 10 ml of aqueous solution of the GM@SDF1α product of Example 4;

[0069] Example 6 Performance Test of Composite Microspheres

[0070] 1. Preparation and characterization of GM-aL composite microspheres

[0071] GM microspheres are produced by PDMS-based microfluidic devices, which are one of the most widely used devices due to their simplicity and practicality. The outer channel containing a mixture of span80 isopropyl myristate solution is used as the continuous phase, while the central channel containing GM and LAP is used as the dispersed phase. The oily mixture in the continuous phase exerts shear force on GM, causing the GM solution to de-disperse into emulsion droplets, which polymerize into microspheres after cross-linking under ultraviolet irradiation.

[0072] By adjusting the injection rate of the inner and outer ports of the coaxial needle to 1:30, GM microspheres with an average diameter of 107.72±4.23μm were obtained. Figure 2 ), which appears as a white sphere under a light microscope ( Figure 2 a), the particle size of GM after freeze-drying is reduced, but it still maintains a spherical shape, and the scanning electron microscope (SEM) shows a loose porous structure ( Figure 2 b) The porous structure of the microspheres increases the surface area of ​​the microspheres, providing conditions for the physical adsorption of drugs. GM can be smoothly injected through a 29G diameter needle without being blocked, proving that it has good injectability and can be used for minimally invasive injection therapy. Liposomes were prepared using a modified thin film dispersion method to obtain monodisperse liposomes ( Figure 2 c), the average particle size is about 163.52±13.42nm ( Figure 2 f). The GM microsphere and liposome mixed solution prepared above was placed in a 37°C oven and oscillated for 24 hours to obtain a GM-aL composite material with an average diameter of 126.54±6.61 μm ( Figure 2 h), SEM observation of freeze-dried GM-aL composite microspheres revealed that the liposomes present in the microsphere surface cavity were monodisperse with structural integrity ( Figure 2 e). Zeta potential test was also performed to verify the successful adsorption of liposomes ( Figure 2 i), the average potential of liposomes is 1.38±0.39mV. Since the liposome adsorption neutralizes part of the charge, the absolute value of the Zeta potential of the GM-aL composite material (-13.87±0.67mV) is significantly lower than that of GM (-18.07±1.52mV), which confirms that the liposomes have been successfully grafted onto the GM surface. In order to further prove the successful grafting of liposomes and GM microspheres, DiL fluorescently labeled liposomes were mixed with GM microspheres and observed under a confocal microscope. It can be seen that the red fluorescent signal on the surface of the GM microspheres is distributed in a dot-like manner ( Figure 2d), which indicates that the liposomes are effectively anchored on the surface of the hydrogel microspheres. In addition, Fourier transform infrared spectrometer (FTIR) observed that the aldehyde-modified liposomes showed an obvious absorption peak near 1730 cm-1, which is the characteristic peak of C=O in the aldehyde group, indicating that we have successfully modified the liposomes with aldehyde groups. After integrating aL with GM hydrogel microspheres, the peak value of the characteristic peak representing NH at 3420 cm-1 in the FTIR image decreased, indicating that the amino groups on the surface of the GM microspheres were partially consumed during the integration process. At the same time, the absorption peak at 1650 was enhanced, which may come from the newly generated Schiff base bond C=N ( Figure 3 ). This suggests that the aldehyde-modified liposomes may be combined with GM microspheres through Schiff base reaction.

[0073] 2. In vitro degradation and release behavior

[0074] In order to evaluate the applicability of GM-aL composite microspheres as drug delivery carriers, their degradation behavior and drug release kinetics were studied. In order to simulate the gradual degradation process in vivo, GM-aL composite microspheres were immersed in PBS solution containing 0.1U / mL type I collagenase and incubated for 4 weeks.

[0075] The microspheres showed a trend of slow degradation at a uniform rate within 4 weeks and were basically degraded on the 28th day ( Figure 2 j). On the 7th day, although the microspheres were intact in morphology, some broken particles were observed around the microspheres. On the 14th day, the morphology of the microspheres began to collapse, with irregular edges and cracks. On the 28th day, the microspheres were basically degraded. It can be seen that degradation is a slow process from the surface to the inside, which is conducive to the long-term release of the drug. At the same time, the residual weight of the GM-aL composite microspheres decreased over time, which is consistent with the morphological changes ( Figure 2 k).

[0076] Molecular diffusion is the main mechanism of drug release from the hydrogel matrix. By anchoring aL on the surface of GM microspheres through Schiff base bonds, the GM-aL composite microspheres enhanced the stability of liposomes and achieved a sustained release effect of aL@Rg1 after injection, providing an attractive method for controlled and sustained local drug delivery. The released Rg1 was quantified using HPLC, as shown in Figure 2 As shown in Figure 1, the encapsulation efficiency of Rg1 in liposomes and GM-aL composite microspheres was (82.17±6.32)% and (42.47±1.97)%, respectively. The decrease and fluctuation of the encapsulation efficiency of Rg1 in GM-aL composite microspheres was mainly due to the drug leakage during the branching process. The encapsulation efficiency of SDF1α in GM was (58.1±3.3)% ( Figure 3 a). In addition, the release profiles of Rg1 from liposomes and GM-aL composite microspheres were also studied. Figure 2m showed that Rg1 in liposomes exhibited a rapid release behavior and was almost completely released in the first week. In contrast, Rg1 in GM-aL composite microspheres had almost no burst release and was continuously released for more than 2 weeks. In addition, in the second week, the cumulative release of Rg1 in GM-aL composite microspheres was much lower than that of Rg1 in liposomes. Figure 3 As shown in b, on the first day, GM@SDF1α-aL had a burst release of SDF1α, with an SDF1α release of 28.95%, and then the release efficiency showed a slowly decreasing trend, and the SDF1α release reached a plateau after nearly 2 weeks. Within 28 days, the SDF1α release was about 69.37%. These results indicate that GM-aL composite microspheres exhibited sustained release of Rg and SDF1α during the degradation period, providing an attractive method for delivering Rg1 and SDF1α for SCI treatment.

[0077] 3. In vitro biocompatibility test of GM-aL composite microspheres

[0078] In order to evaluate the potential clinical application of drug-loaded functionalized microspheres, the in vitro biocompatibility of GM-aL, GM@SDF1α-aL, GM-aL@Rg1, and GM@SDF1α-aL@Rg1 was studied. The extracts of each group of materials were co-cultured with BMSCs, and the simple cultured BMSCs were used as the blank control group. Live / dead staining and CCK-8 cell counting kit were used to evaluate the biocompatibility of each group on the 1st, 3rd, and 5th days of co-culture. Figure 4 As shown in a and b, with the increase of culture days, there are basically no dead cells in the field of view after live / dead staining. CCK-8 quantitative analysis showed that there was no significant difference in cell proliferation activity among the groups on days 1 and 3. On day 5, it was found that the cell proliferation activity of the GM-aL@Rg1 and GM@SDF1α-aL@Rg1 groups increased slightly compared with the other three groups ( Figure 4 c). This may be because the sustained-release Rg1 in the material began to play a role and promoted the proliferation activity of BMSCs. The above results show that the functionalized microspheres have good biocompatibility and GM@SDF1α-aL@Rg1 has the effect of promoting the proliferation of BMSCs.

[0079] 4. In vitro evaluation of GM-aL composite microspheres regulating inflammatory microenvironment

[0080] In order to evaluate the ability of GM-aL microspheres to regulate inflammatory response in vitro, each group of microspheres was co-cultured with LPS-induced Raw264.7 cells, and the expression of related genes was determined by real-time quantitative polymerase chain reaction (qRT-PCR). Raw264.7 cells induced by LPS significantly expressed inflammatory factor genes IL-1β and TNF-α, and downregulated the expression of anti-inflammatory factor genes IL-10 and TGF-β. However, after intervention with GM-aL@Rg1 and GM@SDF1α-aL@Rg1, the expression levels of pro-inflammatory factors IL-1β, TNF-α and anti-inflammatory factor genes IL-10 and TGF-β showed a certain reversal, which was statistically significant compared with other groups (P<0.05)( Figure 5 eh).

[0081] Likewise, enzyme-linked immunosorbent assay (ELISA) detected the secretion of pro-inflammatory and anti-inflammatory cytokines, and the results were consistent with the qRT-PCR results ( Figure 5 il).

[0082] Raw264.7 subtype immunofluorescence staining showed that the macrophage marker F4 / 80 (red fluorescence) was highly expressed in all groups, indicating that the purity of Raw264.7 was high. The expression level of M1 marker CD86 after LPS treatment was significantly higher than that in the control group, indicating that LPS successfully induced M0 macrophages into M1 macrophages. The expression of M1 marker in GM@SDF1α-aL@Rg1 group was significantly lower than that in other control groups (p<0.05)( Figure 5 a,c), while the expression of M2 markers was significantly increased, higher than that of other control groups (P<0.05)( Figure 5 b, d). Similar observations were also observed in the GM-aL@Rg1 group. In summary, these results indicate that GM@SDF1α-aL@Rg1 can regulate the polarization direction of macrophages, polarize them toward M2, promote the secretion of anti-inflammatory cytokines, alleviate local inflammatory responses, and create a favorable environment for subsequent nerve regeneration.

[0083] 5. Characterization of cell migration and recruitment function of GM-aL composite microspheres

[0084] In the scratch test, BMSCs cells were cultured using the material leaching solution ( Figure 6 a). We found that the migration area of ​​GM@SDF1α-aL and GM@SDF1α-aL@Rg1 groups was significantly larger than that of the other groups (P<0.05), indicating that GM microspheres enhance the migration ability of stem cells by releasing SDF1α ( Figure 6 c).

[0085] In the chemotaxis assay, BMSC cells were loaded into the upper chamber, while the leaching solution of each material was located in the lower chamber, and the cells were expected to migrate to the lower chamber in response to the release of SDF1α by GM microspheres ( Figure 6 b) If Figure 6 As shown in d, the number of migrated cells in the GM@SDF1α-aL and GM@SDF1α-aL@Rg1 groups was significantly higher than that in the other groups (p < 0.05), indicating that GM@SDF1α-aL@Rg1 could recruit BMSCs via SDF1α.

[0086] 6. Characterization of GM-aL composite microspheres regulating nerve regeneration function

[0087] After spinal cord injury, establishing a relaxed local microenvironment is crucial to promote the differentiation of endogenous stem cells. Since inflammatory cytokines affect the differentiation of endogenous stem cells, a co-culture system of BMSCs and microspheres was constructed and the effect of GM@SDF1α-aL@Rg1 on the differentiation of BMSCs under pathological conditions was further explored. The induction medium was prepared using low-glucose DMEM and 10% fetal bovine serum (including one of GM-aL, GM@SDF1α-aL, GM-aL@Rg1, and GM@SDF1α-aL@Rg1). With the help of the Transwell system, the LPS-intervention raw was in the upper chamber, and the cells and material leaching solution were in the lower chamber to study the differentiation behavior of BMSC into neurons. Immunofluorescence staining and qRT-PCR techniques were used to detect the expression of neuron-specific markers in neuron-like cells, including neuron-specific differentiation tubulin (Tuj-1), microtubule-associated protein 2 (Map-2), neurofilament protein (NF-200), and neural cytoskeleton tubulin (Tau). As shown in the immunofluorescence staining image ( Figure 7 a), GM-aL@Rg1 and GM@SDF1α-aL@Rg1 groups showed typical neuron-like changes, such as axon-like and dendrite-like dendrites around the cell body, and strong green fluorescence after staining with neuron-specific markers ( Figure 7 a). Compared with the weak signals of the control group and GM@SDF1α-aL group, the fluorescence expression of GM-aL@Rg1 and GM@SDF1α-aL@Rg1 groups was significantly increased, with significant statistical differences (p<0.05). This suggests that the constructed GM-aL composite scaffold can slowly release Rg1 and continuously promote the differentiation of endogenous stem cells into neurons under inflammatory conditions ( Figure 7 be). At the same time, similar results were found in the gene expression tests of different neuron-specific markers ( Figure 7fI). The genes of various neural-specific markers in the Rg1-loaded group were highly expressed and statistically significant (p < 0.05). In addition, the length of Tuj-1 cells in the GM@SDF1α-aL@Rg1 group (78.33 ± 15.63 μm) was significantly longer than that in the Control group (5.33 ± 1.53 μm, p < 0.05) ( Figure 8 ). These results indicate that GM@SDF1α-aL@Rg1 can promote the neuronal differentiation of BMSCs under inflammatory conditions.

[0088] 7. Animal motor function scoring

[0089] The in vivo treatment of GM@SDF1α-aL@Rg1 was evaluated using the Allen impact model of the T9 spinal cord in SD rats. The recovery of hindlimb motor dysfunction after SCI was evaluated by Basso, Beattie, Bresnahan (BBB) ​​and inclined plane test (IPT) scores every week after surgery. The results showed that ( Fig. 9 d,e), the motor function of rats in each group was restored to a certain extent after surgery. Four weeks after surgery, compared with other groups, the BBB score and IPT score of the GM@SDF1α-aL@Rg1 group were significantly improved, and the difference was statistically significant (P<0.0.5). At 8 weeks after surgery, the BBB score and IPT score of the GM@SDF1α-aL@Rg1 group reached 13.71±1.11 and 55.86±4.67°, respectively, which were statistically significant compared with other groups. In addition, compared with other control groups, the BBB score and IPT score curves of the GM@SDF1α-aL@Rg1 group rose at a higher rate, which could achieve the effect of nerve repair in a shorter time. Therefore, the motor function score results suggest that GM@SDF1α-aL@Rg1 can protect surviving motor neurons from the severe inflammatory response in the acute phase of spinal cord injury after implantation, and promote nerve repair in a faster time.

[0090] 8. Regulation of the inflammatory microenvironment in SCI in vivo

[0091] Macrophages and microglia reach their peak recruitment within 7 days after SCI, and GM@SDF1α-aL@Rg1 should play a role in regulating inflammation during this period. To this end, we evaluated the effect of GM@SDF1α-aL@Rg1 on early neuroinflammation in SCI. On the 7th day after surgery, we first detected the spinal cord specimens of rats in each group by immunofluorescence to evaluate whether the composite hydrogel microspheres could regulate macrophage subtypes. The results showed that the GM@SDF1α-aL@Rg1 group significantly reduced the INOS signal compared with other groups ( Fig.10 a), increased ARG-1 signal ( Fig.10b), the difference was statistically significant (P<0.0.5). The results of immunofluorescence imaging showed that the GM@SDF1α-aL@Rg1 group not only significantly reduced the proportion of INOS / Iba-1 macrophages, but also significantly increased the proportion of ARG-1 / Iba-1 macrophages, suggesting that GM@SDF1α-aL@Rg1 can promote the polarization of local microglia / macrophages to M2 phenotype in vivo. It is worth noting that the improvement of inflammation by GM@SDF1α-aL@Rg1 is more significant than that by GM-aL@Rg1. This may be because the BMSCs recruited by SDF1α have certain immunomodulatory functions, which can transform M1 macrophages into M2 macrophages and inhibit the expression of inflammatory genes. The difference in the results between the GM@SDF1α-aL group and the GM@aL and blank control groups further confirmed the above hypothesis.

[0092] The subtype change of local immune cells after SCI must cause changes in the secretion of inflammatory cells. The results of evaluating the expression of inflammatory genes in the spinal cord injury showed that the expression of proinflammatory factors TNF-α and IL-1β was higher in the blank control group and the group without inflammatory regulation function (p<0.05)( Fig.11 e,f), while the expression of anti-inflammatory genes IL-10 and TGF-β in the GM@SDF1α-aL@Rg1 group was significantly increased ( Fig.11 g, h). The above results indicate that GM@SDF1α-aL@Rg1 can inhibit the early inflammatory response after SCI, reduce the secretion of inflammatory factors, and create a local microenvironment that induces spinal cord regeneration.

[0093] In addition, the systemic inflammatory response of the animals was evaluated by serum ELISA test to comprehensively evaluate the inflammatory regulation function of the composite microspheres. The results showed that the serum inflammatory factors TNF-α and IL-1β in the blank control group and GM@-aL group were significantly higher than those in the GM@SDF1α-aL@Rg1 group (p<0.05)( Fig.11 i,j), while the IL-10 and TGF-β levels in the GM@SDF1α-aL@Rg1 group were higher (p<0.05)( Fig.11 k, l). These results indicate that implantation of inflammation-regulating composite microspheres can not only improve the local immune environment but also reduce systemic inflammatory responses.

[0094] 9. Histology and immunohistochemistry of spinal cord repair

[0095] Secondary neuroinflammation after SCI leads to neuronal death and axonal demyelination, while the chronic wrapping of the injured area by glial cells makes it difficult for regenerated neurons and axons to penetrate and rebuild the damaged spinal cord tissue, which is a difficulty in repairing spinal cord injury. This study simultaneously introduced inflammatory regulation and BMSCs recruitment factors to regulate neuroinflammation, inhibit glial tissue formation and promote nerve regeneration. H&E staining images are shown in the figure ( Fig. 9 ac), the area of ​​syringomyelia in all material groups was significantly reduced compared with the blank control group (P<0.05), indicating that the biological scaffold plays an important role in the repair of spinal cord injury. However, a large number of parallel red-stained collagen fibers and slender fiber cell nuclei were observed in both GM@-aL and GM@SDF1α-aL groups, indicating the formation of glial tissue. The red-stained collagen fibers around the injury in the GM@SDF1α-aL@Rg1 group were distributed more smoothly and evenly than those in other control groups, and the glial tissue was significantly inhibited at 4 weeks. In addition, the area of ​​syringomyelia in the GM@SDF1α-aL@Rg1 group was further reduced at 8 weeks compared with that at 4 weeks, while there was no significant change in other material groups, proving that the regulation of inflammation in the acute phase of spinal cord injury plays a pivotal role in the later neural repair process.

[0096] Activated M1 microglia after SCI secrete inflammatory factors TNF-a, IL-1a and C1q, inducing the polarization of resting astrocytes to A1 astrocytes. A1 astrocytes secrete neurotoxins to kill CNS neurons with axonal damage. In addition, A1 astrocytes hypertrophy, proliferate, migrate and express glial fibrillary acidic protein (GFAP) and neuron glial antigen 2 (NG2), which promote glial scar formation and hinder neural repair. To further evaluate the anti-glial scar effect of inflammatory-regulated composite hydrogel microspheres, we used anti-GFAP antibodies and anti-NG2 antibodies to fluorescently label activated astrocytes and glial scar tissue in spinal cord samples. The results showed ( Fig.12 a,b), the number of activated astrocytes and scar tissue in the material groups (GM-aL, GM@SDF1α-aL, GM-aL@Rg1, GM@SDF1α-aL@Rg1) was significantly less than that in the blank control group at 4 and 8 weeks after surgery (p<0.05), indicating that the hydrogel can fill the gaps in spinal cord injury and reduce the formation of disordered glial scars after nerve injury. In addition, the results of fluorescence semi-quantitative analysis showed that ( Fig.12 c, d), the fluorescence density of activated astrocytes and scar tissue in the GM@SDF1α-aL@Rg1 group was significantly lower than that in other groups (p<0.05). The results showed that the composite microspheres can regulate the local inflammatory microenvironment in the acute phase of SCI, reduce scar tissue formation, and lay a good foundation for the differentiation of endogenous stem cells into neurons.

[0097] After SCI, endogenous stem cells are activated and migrate to the injury site, but local scar formation in the injured spinal cord tissue hinders cell migration. In addition, the number of endogenous stem cells that migrate to the injury site is insufficient to rebuild the neural network. The above immunohistochemistry results confirm that the composite microspheres can reduce the formation of activated astrocytes and glial scar tissue, and reduce the physical barrier to cell migration. Under physiological conditions, Nestin is lowly expressed, and the number of cells positively labeled with Nestin increases significantly after spinal cord injury. Therefore, the endogenous neural progenitor cells in the injured area are fluorescently labeled with Nestin antibodies to indirectly evaluate the differentiation of BMSCs recruited by SDF1α into neural progenitor cells under the condition of Rg1 regulating the local inflammatory microenvironment. The distribution of Nestin-labeled stem cells in the injured spinal cord is shown in the figure ( Fig.13 a). Fluorescence quantitative results showed that the endogenous progenitor cells in the GM@SDF1α-aL@Rg1 group were significantly more than those in other groups (P<0.05) regardless of whether the injury was 4 or 8 weeks ( Fig.13 b). The results showed that the composite microspheres regulated the local inflammatory microenvironment through Rg1, promoting the survival of BMSCs recruited by SDF1α and their differentiation into neural progenitor cells.

[0098] The local permissive microenvironment after spinal cord injury is crucial for the differentiation of BMSCs. Although BMSCs can differentiate into neurons, astrocytes, and oligodendrocytes, the local inflammatory microenvironment after SCI often promotes the differentiation of BMSCs into astrocytes. It has been reported that Rg1 can regulate the local inflammatory microenvironment after spinal cord injury, improve the antioxidant capacity of BMSCs, reduce apoptosis, increase the survival rate, and promote the differentiation of BMSCs into neurons. In this study, Tuj-1 and GAP-43 were used to mark neural progenitor cells, neurons, and axonal sprouts, respectively, to evaluate the ability of composite GM microspheres to promote neural regeneration in the middle and late stages of SCI. Because GM@SDF1α-aL@Rg1 can regulate the local inflammatory microenvironment, reduce the physical barrier effect of glial scars, and promote the differentiation of recruited BMSCs into neurons, the number of neurons was significantly higher than that of other groups. At 4 and 8 weeks after surgery, a large number of Tuj-1 labeled neurons appeared in the material groups (GM-aL, GM@SDF1α-aL, GM-aL@Rg1, GM@SDF1α-aL@Rg1), which was significantly higher than that in the control group (P<0.05) ( Fig.14a,c). In addition, the fluorescence intensity of Tuj-1 in the GM@SDF1α-aL@Rg1 group at 8 weeks after surgery was significantly higher than that at 4 weeks, and the difference was statistically significant. These results indicate that GM@SDF1α-aL@Rg1 can not only regulate the local inflammatory microenvironment of SCI, but also promote the homing of BMSCs and their differentiation into neurons through the continuous release of SDF1α. Growth-associated protein 43 (GAP-43) plays an important role in the transduction signal of axon formation and is a marker of axon formation. It is lowly expressed in the normal spinal cord. After SCI, GAP-43 is activated by cytokines and neurotrophic factors to achieve high expression, promoting neuroprotection and regeneration. As shown in the figure Fig.14 As shown in b and d, the fluorescence intensity of GAP-43 in the blank control group was significantly lower than that in other groups. The fluorescence intensity in the GM@SDF1α-aL@Rg1 group was significantly higher than that in other groups, and the difference was statistically significant (P<0.05), indicating that GM@SDF1α-aL@Rg1 regulates the inflammatory microenvironment and recruits BMSCs to help neuronal differentiation, which is essential for neural circuit reconstruction. In addition, we noticed that the expression of GAP-43 in the GM@SDF1α-aL@Rg1 group was significantly decreased at 8 weeks compared with 4 weeks of injury. The results show that the repair time window of SCI is limited, and regulating inflammation in the acute phase of SCI and reducing the inflammatory response can better promote nerve regeneration.

[0099] The recovery of neurological function depends not only on the regeneration of neurons, but also on the generation of blood vessels in the spinal cord. Angiogenesis is essential for the recovery of damaged spinal cord tissue, which can improve blood supply, accelerate tissue repair and support the survival of nerve cells. It has been reported that BMSCs can promote angiogenesis in the injured spinal cord and significantly enhance neural tissue retention through neurotrophic signaling and immune regulation. At the two time points of 4 and 8 weeks after injury, the CD31 fluorescence intensity of the GM@SDF1α-aL@Rg1 group was significantly higher than that of the other control groups ( Fig.15 a,c). In addition, the number of CD31-labeled endogenous vascular cells in the GM@SDF1α-aL@Rg1 group at 8 weeks of injury was significantly greater than that at 4 weeks. Similarly, von Willebrand factor (VWF) was used to fluorescently label new blood vessels at 4 and 8 weeks of injury. The quantitative results of fluorescent staining showed that the VWF fluorescence intensity in the GM@SDF1α-aL@Rg1 group was significantly higher than that in the other groups, and the number of new blood vessels labeled by VWF at 4 weeks was significantly less than that at 8 weeks ( Fig.15 b, d). The results showed that GM composite microspheres can only induce macrophages and microglia to polarize to the M2 subtype to secrete vascular endothelial growth factor, and can also recruit BMSCs to promote angiogenesis, providing a good foundation for spinal cord repair. In addition, rat organ staining showed that no difference was observed between the GM@SDF1α-aL@Rg1 group and the control group, proving that it has no potential in vivo toxicity ( Fig.16 ).

[0100] The results showed that the composite microspheres of the present invention have the ability to regulate local inflammation and have a sustained promoting effect on nerve tissue regeneration, providing a biomaterial strategy for the repair of acute SCI that relies on the coordination of inflammatory microenvironment regulation and BMSCs recruitment.

[0101] The above embodiments are intended to further illustrate some preferred embodiments of the present invention, but not all embodiments. Other embodiments based on the present invention made by professionals in this field without creative work are all within the scope of protection of the present invention.

Claims

1. A method for preparing composite microspheres for regulating local inflammatory microenvironment and promoting nerve regeneration, characterized in that: The method comprises the following steps: Step 1: Preparation of liposomes loaded with anti-inflammatory drugs; Step 2: Preparation of GM microspheres loaded with cytokines; Step 3 Preparation of composite microspheres that regulate inflammation and promote nerve regeneration: grafting liposomes loaded with anti-inflammatory drugs and GM microspheres loaded with cytokines.

2. The method for preparing a composite microsphere for regulating local inflammatory microenvironment and promoting nerve regeneration according to claim 1, characterized in that: The anti-inflammatory drug in step 1 is ginsenoside Rg1, and the cytokine in step 2 is stromal cell-derived factor 1α.

3. The method for preparing a composite microsphere for regulating local inflammatory microenvironment and promoting nerve regeneration according to claim 2, characterized in that: The step 1 is prepared by a thin film dispersion method, and the preparation steps of the liposome loaded with ginsenoside Rg1 are as follows: 2.5 parts by mass of ginsenoside Rg1, 80 parts of lecithin and 20 parts of cholesterol, 2 parts of DSPE-PEG-CHO and 2.5 parts of octadecylamine were added to chloroform and mixed to obtain a uniform emulsion. The organic solvent was removed from the emulsion in a rotary evaporator to obtain a colloidal product. Deionized water was then added to hydrate the lipid film on the bottle wall, and the lipid film was completely dissolved by ultrasonic treatment to form a nanoscale liposome emulsion. Liposomes loaded with ginsenoside Rg1 were obtained by filtration using a polycarbonate membrane.

4. The method for preparing a composite microsphere for regulating local inflammatory microenvironment and promoting nerve regeneration according to claim 1, characterized in that: The step 2 comprises the following steps: Type A pig skin gelatin is prepared into a 10% (w / v) solution and completely dissolved in phosphate buffered saline at 60°C; then, methacrylic acid is added to the gelatin solution and stirred for reaction at 50°C; the mixture is diluted with additional PBS to stop the reaction, and then the mixture is dialyzed at 40°C to remove unreacted MA and salts using a dialysis tube with a molecular weight cutoff of 12-14 kilodaltons; GM is obtained.

5. The method for preparing a composite microsphere for regulating local inflammatory microenvironment and promoting nerve regeneration according to claim 4, characterized in that: The step 2 further comprises the step of preparing GM microspheres by a microfluidic device: The step 3 prepares GM microspheres by a microfluidic device, comprising the following steps: A 7wt% GM and 0.5wt% LAP PBS solution is used as the aqueous phase, SDF1α is resuspended in a 0.1wt% bovine serum albumin solution to obtain a final concentration of 100μg / ml SDF1α solution, and the SDF1α solution is added to the aqueous phase to obtain a uniform SDF1αGM solution aqueous phase with a concentration of 100ng / mL, and a 5wt% Span 80 in isopropyl myristate solution is used as the oil phase; the aqueous phase and the oil phase fluids are respectively connected to the inner and outer ports of the microfluidic coaxial needle, and the internal and external injection rates of the injection pump are adjusted to 1:30, the generated microspheres are collected, and cross-linked by ultraviolet irradiation, and washed with ethanol and isopropanol respectively to remove residual Span 80 and isopropyl myristate. After washing with PBS, GM microspheres loaded with stromal cell-derived factor 1α are obtained.

6. The method for preparing a composite microsphere for regulating local inflammatory microenvironment and promoting nerve regeneration according to claim 5, characterized in that: The step 3 comprises the following steps: The aqueous solution of the GM microspheres loaded with stromal cell-derived factor 1α and the aqueous solution of the liposomes loaded with ginsenoside Rg1 were mixed and grafted, and then washed with deionized water to obtain GM@SDF1α-aL@Rg1 composite microspheres.

7. The method for preparing a composite microsphere for regulating local inflammatory microenvironment and promoting nerve regeneration according to claim 6, characterized in that: The grafting conditions are to place the sample in an oven at 37° C. and set the grafting time to at least 24 hours.

8. A composite microsphere for regulating local inflammatory microenvironment to promote nerve regeneration, characterized in that: The composite microspheres are prepared by any one of the methods of claims 1 to 7.

9. An application of composite microspheres for regulating local inflammatory microenvironment to promote nerve regeneration, characterized in that: The composite microspheres that regulate the local inflammatory microenvironment and promote nerve regeneration are used as drugs for regulating the inflammatory microenvironment and promoting nerve regeneration after spinal cord injury.

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