CN119633170A - A method for preparing composite microspheres for regulating local inflammatory microenvironment and promoting nerve regeneration - Google Patents

Abstract

The invention discloses a preparation method of a composite microsphere for regulating and controlling local inflammation microenvironment and promoting nerve regeneration, which comprises the following steps of preparing liposome loaded with ginsenoside Rg1, preparing GM microsphere loaded with stromal cell derived factor 1 alpha (SDF1α), and preparing the composite microsphere for regulating and controlling inflammation and promoting nerve regeneration. The composite microsphere can reduce inflammatory reaction, reduce scar tissue formation, promote angiogenesis, continuously release SDF1α, and promote endogenous stem cell recruitment and neural differentiation. Therefore, the composite microsphere is a functional biological scaffold which firstly regulates and controls local microenvironment inflammation of spinal cord injury, and then promotes creative sequential inflammation regulation and nerve regeneration of endogenous stem cells for recruitment and differentiation, and provides a new strategy for treating spinal cord injury by tissue engineering.

Description

Preparation method of composite microsphere for regulating and controlling local inflammation microenvironment to promote nerve regeneration

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a preparation method of a nerve regeneration promoting composite hydrogel microsphere for regulating and controlling local inflammation microenvironment.

Background

Spinal cord injury can lead to neuronal death and axonal destruction, leading to permanent sensory and motor dysfunction. SCI places severe physiological, psychological and economic burden on patients and their families. SCI is the result of the combined effects of multiple factors, and intervention against a single factor has limited effect on SCI repair. Exogenous cell transplantation has a powerful potential neuroprotection and has therefore been used for SCI repair. However, the safety, ethical problems, sources, and poor retention and differentiation capabilities of exogenous stem cells after implantation limit their use. Endogenous bone marrow mesenchymal stem cells (BMSCs) provide safer, more convenient treatments for SCI patients than exogenous BMSCs. In the SCI course, endogenous BMSCs are activated and spontaneously migrate to the site of injury, promoting neural network remodeling. However, the number of BMSCs cells recruited to the SCI local lesions is limited. At the same time, acute phase inflammation of SCI reduces BMSCs activity and causes BMSCs to differentiate toward glial cells rather than neurons, severely affecting cell therapy. Therefore, to enhance the effect of in situ BMSCs cell therapy, it is desirable to regulate SCI acute phase inflammation and enhance local BMSCs migration and differentiation capacity.

SCI acute phase inflammation is closely related to the innate immune system, wherein macrophage infiltration induces exacerbation of acute phase inflammation. Macrophage polarization is either a classical activated type 1 (M1) macrophage or a surrogate activated type 2 (M2) macrophage, exhibiting deleterious or protective properties in SCI, respectively. M1 type macrophages induce axon retraction, secondary injury, and neuroinflammation, leading to glial scar formation, impeding axon regeneration, while M2 type macrophages promote anti-inflammatory factor secretion and axon growth. Therefore, the polarization direction of the macrophages is regulated, the proportion of M1 type macrophages is reduced, and the proportion of M2 type macrophages is increased, so that the acute phase inflammation can be effectively relieved, and a proper environment is provided for migration and differentiation of endogenous BMSCs.

Ginseng has been a traditional Chinese herbal medicine for thousands of years of medicinal history. Ginsenoside (Rg 1) is a hydrophobic small molecule, and is one of the main active ingredients of ginseng. Rg1 plays an important role in anti-inflammatory, antioxidant and neuroprotective aspects. Rg1 promotes macrophage polarization to M2 phenotype through NF- κB and PI3K/Akt/mTOR pathways, and reduces SCI acute phase inflammatory response. Meanwhile, rg1 can improve the oxidation resistance of BMSCs, reduce apoptosis, improve survival rate and promote the paracrine of BMSCs. In addition, rg1 promotes differentiation of BMSCs into neurons. Thus, BMSCs in situ cell therapy with Rg1 assist has the potential to treat SCI. However, rg1 has poor water solubility and low drug administration concentration in the conventional manner, which limits the therapeutic effect of Rg 1. In order to fully exert the therapeutic effect of Rg1, it is necessary to introduce an effective drug delivery system.

In recent years, biological materials have provided a breakthrough solution for the treatment of SCI. Hydrogels are the current focus of research due to their strong adjustability, ease of carrying drugs. In nerve tissue engineering, various polymer hydrogels have been explored, including collagen, hyaluronic acid, fibronectin, and the like. These hydrogels can be custom designed to mimic the extracellular matrix of the central nervous system environment, mimic tissue stiffness, and carry cells or drugs. In many areas of tissue engineering research, the binding of hydrogels to drugs or growth factors has become one of the most commonly used systems for achieving sequential transfer and delivery of drugs or growth factors.

Methacryloyl Gelatin (GM) is a photocrosslinked hydrogel material with good biocompatibility, suitable for nerve soft tissue, capable of filling spinal cord defects and providing bridging for spinal cord injury repair. However, GM itself is not prone to carry hydrophobic drugs.

Disclosure of Invention

To solve the above technical problems, an effective drug delivery system is introduced in order to fully exert the therapeutic effect of Rg1. Cationic liposomes (aL) are introduced in the present invention. The aL has a tightly packed double-layer membrane structure, the double-layer lipid shell can be loaded with a hydrophobic drug, and aldehyde groups on the surface can be combined with GM hydrogel through Schiff base reaction. Stromal cell derived factor 1 alpha (sdf1α) can selectively recruit stem cells, and the incorporation of sdf1α into GM hydrogels can enhance the recruitment of BMSCs by biological materials. The invention provides a composite microsphere, a preparation method and application thereof, wherein the composite microsphere is formed by grafting an inflammation regulation carrier aL loaded with Rg1 onto a GM microsphere loaded with SDF1α through a Schiff bond, and the name is GM@SDF1α -aL@Rg1.

According to some technical schemes of the invention, a preparation method of composite microspheres for regulating and controlling local inflammation microenvironment to promote nerve regeneration is provided, and the method comprises the following steps:

step 1, preparing liposome loaded with anti-inflammatory drugs;

Step 2, preparing GM microspheres loaded with cytokines;

Step 3, preparing the inflammation-regulating and nerve-regeneration-promoting composite microsphere, namely grafting the liposome loaded with the anti-inflammatory drug and the GM microsphere loaded with the cytokine.

The invention further provides a preparation method of the composite microsphere for regulating and controlling local inflammation microenvironment and promoting nerve regeneration, wherein the anti-inflammatory drug in the step 1 is ginsenoside Rg1, and the cytokine in the step 2 is stromal cell derived factor 1 alpha.

The invention further provides a preparation method of the composite microsphere for regulating and controlling local inflammation microenvironment and promoting nerve regeneration, wherein the step 1 is prepared by a film dispersion method, and the preparation steps of the liposome loaded with ginsenoside Rg1 are as follows:

Adding 2.5 parts by mass of ginsenoside Rg1, 80 parts by mass of lecithin, 20 parts by mass of cholesterol, 2 parts by mass of DSPE-PEG-CHO and 2.5 parts by mass of octadecylamine into chloroform, mixing to obtain uniform emulsion, removing organic solvent from the emulsion in a rotary evaporator to obtain a colloid product, adding deionized water, hydrating a lipid film on the bottle wall, completely dissolving the lipid film by using ultrasonic treatment to form nano-scale liposome emulsion, and filtering by using a polycarbonate film to obtain uniform single-layer ginsenoside Rg 1-loaded liposome.

The invention further provides a preparation method of the composite microsphere for regulating and controlling local inflammation microenvironment to promote nerve regeneration, wherein the step 2 comprises the following steps:

The preparation method comprises the steps of preparing type A pigskin gelatin into a solution with the concentration of 10% (w/v) and completely dissolving the solution in Phosphate Buffer Saline (PBS) at 60 ℃, then adding Methacrylic Acid (MA) into the gelatin solution, stirring the mixture at 50 ℃ for reaction, diluting the mixture with additional PBS to stop the reaction, then dialyzing the mixture at 40 ℃ to remove unreacted MA and salt, and finally freeze-drying the GM solution to obtain GM.

The invention further provides a preparation method of the composite microsphere for regulating and controlling local inflammation microenvironment to promote nerve regeneration, wherein the step 2 further comprises the step of preparing the GM microsphere by a microfluidic device:

The step 3 prepares GM microsphere through a microfluidic device, and comprises the following steps:

Taking a PBS solution of 7wt% of GM and 0.5wt% of LAP as a water phase, re-suspending SDF1α in a final concentration of 100 mug/mlSDF 1 alpha solution in a 0.1wt% bovine serum albumin solution, adding the SDF1α solution into the water phase to obtain a uniform SDF1α GM solution water phase with the concentration of 100ng/mL, taking a Span 80 of 5wt% as an oil phase, respectively introducing the two fluids of the water phase and the oil phase into an inner port and an outer port of a microfluidic coaxial needle, adjusting the injection rate inside and outside the syringe pump to be 1:30, collecting the generated microspheres, realizing crosslinking by using ultraviolet irradiation, respectively washing by using ethanol and isopropanol so as to remove residual Span 80 and isopropyl myristate, and washing by PBS to obtain the GM microspheres loaded with matrix cell-derived factor 1 alpha.

The invention further provides a preparation method of the composite microsphere for regulating and controlling local inflammation microenvironment to promote nerve regeneration, wherein the step 3 comprises the following steps:

Mixing and grafting the aqueous solution of the GM microsphere loaded with the stromal cell derived factor 1 alpha and the aqueous solution of the liposome loaded with the ginsenoside Rg1, and then washing with deionized water to obtain the GM@SDF1alpha-aL@Rg1 composite microsphere.

The invention further provides a preparation method of the composite microsphere for regulating and controlling local inflammation microenvironment and promoting nerve regeneration, wherein the grafting condition is that the composite microsphere is placed in a 37 ℃ oven, and the grafting time is set to be at least 24 hours.

The invention further provides a composite microsphere for regulating and controlling the local inflammation microenvironment to promote nerve regeneration, which is prepared by using any one of the preparation methods for regulating and controlling the local inflammation microenvironment to promote nerve regeneration.

The invention further provides application of the composite microsphere for regulating and controlling the local inflammation microenvironment and promoting nerve regeneration, and the composite microsphere for regulating and controlling the local inflammation microenvironment and promoting nerve regeneration is used as a medicament for regulating and controlling the inflammation microenvironment and promoting nerve regeneration after spinal cord injury.

The invention aims to provide a preparation method of a composite hydrogel for regulating and controlling inflammatory microenvironment and promoting nerve regeneration, which is used for assembling an amino GM microsphere loaded with SDF1α and an aldehyde cationic liposome loaded with Rg1 to construct a functional composite hydrogel bracket for regulating and controlling spinal cord injury inflammatory microenvironment and promoting nerve regeneration.

The foregoing description is only an overview of the present invention, and is intended to provide a better understanding of the present invention, as it is embodied in the following description, with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 shows the construction and mechanism of action of the GM-aL composite microsphere of the invention, wherein the A construction of the bionic GM-aL microsphere treats spinal cord injury, and the B has the functions of inflammation regulation and nerve regeneration;

FIG. 2 shows the physical properties of a composite GM-aL microgel according to the examples of the present invention, (a) a microscope image of the GM microgel, (b) a SEM image of the GM microgel, (c) a TEM image of aL, (d) a LSCM image of the GM-aL microgel, the aL in the microgel being labeled with DiI dye, few liposomes being released from the microgel being observed in aqueous solution, (e) a SEM image of the liposomes in the GM microgel, (f) the particle size distribution of the liposomes being measured by DLS, (g) the particle size of the GM microgel, (h) the particle size of the GM-aL microgel, (i) the surface charges of the aL, GM microgel and GM-aL microgel, and (j) the microscope image showing the morphological changes of the GM-aL microgel within 5 weeks. (k) Degradation curve of GM-aL microgel, (l) encapsulation rate of Rg1 in liposome, SDF1α in GM and Rg1 in GM-aL microgel, (m) in vitro release of Rg1 from liposome and GM-aL microgel;

FIG. 3 is a representation of a GM-aL microgel according to an embodiment of the present invention, (a) a release profile of Sdf-1a in GM-aL, (b) an FTIR method to evaluate the successful attachment of a chemical bond Schiff base to an aminated GelMA hydrogel microsphere and an aldehyde-based cationic liposome;

FIG. 4 shows the cell compatibility of composite Gelma microspheres in examples of the present invention (a) live/dead images GM@SDF1α -aL, GM-aL@Rg1, GM@SDF1α -aL@Rg1 after incubation with leaching solution, (b) quantification of live cells in live/dead experiments (c) determination of cytotoxicity using CCK-8;

FIG. 5 shows the polarization and inflammatory factors secreted by Raw264.7 in the examples of the present invention, (a) immunofluorescent staining of macrophage markers F4/80 (red) and M1 macrophage marker CD80 (green), (b) immunofluorescent staining of macrophage markers F4/80 (red) and M2 macrophage marker CD206 (green), (c) immunofluorescent staining of M1 macrophage marker CD80, (d) fluorescent semi-quantitative analysis of M2 macrophage marker CD206, (e-h) mRNA expression analysis of TNF- α, IL-1β, IL-10, TGF- β, (i-l) ELISA assay to detect secretion of inflammatory factors TNF- α, IL-1β, IL-10, TGF- β by Raw264.7;

FIG. 6 is a graph showing the promotion of cell migration and recruitment by composite GM microspheres according to an example of the present invention, (a) representative optical microscopy images of scratch tests at 0, 12 and 24 hours, respectively, (b) representative optical microscopy images of Transwell test (c) analysis of percent healing of scratch test by Image J, (d) number of cells migrated by Transwell test;

FIG. 7 shows the expression of neuron-specific markers according to the examples of the present invention (a) immunofluorescent staining of Tuj-1, map-2, NF-200 and Tau stained images of different GelMA microsphere sets, and corresponding semi-quantitative fluorescent analysis. (f-I) mRNA expression analysis of Tuj-1, map-2, NF-200 and Tau;

FIG. 8 is a quantitative analysis of Tuj-1+ cell length in an embodiment of the invention;

FIG. 9 shows H & E staining and motor function scoring of spinal cord specimens according to an embodiment of the invention, (a) H & E staining of rat spinal cord at 4 weeks and 8 weeks, respectively, (b) calculation of void area at 4 weeks and (c) 8 weeks after surgery, (d) assessment of motor function recovery of lower limbs of rats by BBB and (E) IPT scoring.

FIG. 10 shows the phenotypic identification of 7 days post-operative immune cells in the examples of the present invention, (a) immunofluorescent staining of macrophage markers Iba1 (green) and M1 macrophage markers iNOS (red) and quantitative analysis of their optical densities, and (b) immunofluorescent staining of macrophage markers Iba1 (green) and M2 macrophage markers ARG-1 (red) and quantitative analysis of their optical densities;

FIG. 11 shows an assessment of 7 day post-operative inflammatory modulation in examples of the present invention, (a) IL-10, TNF- α and (b) TGF- β, IL-1β immunofluorescence, quantitative analysis of (c) IL-10 and TNF- α immunofluorescence, (d) TGF- β and IL-1β immunofluorescence, assessment of spinal cord samples for levels of TNF- α (e), IL-1β (f), IL-10 (g) and TGF- β (h) gene expression by qRT-PCR, ELISA assay for serum levels of TNF- α (i), IL-1β (j), IL-10 (k) and TGF- β (l);

FIG. 12 shows immunofluorescent staining of activated astrocytes and glial scars in an example of the invention. (a, b) immunofluorescent staining of astrocytes and quantitative analysis with optical density, and (c, d) quantitative analysis;

FIG. 13 shows immunofluorescent staining of neurons in examples of the present invention, (a) immunofluorescent staining of neurons, (b) quantitative analysis;

FIG. 14 shows immunofluorescent staining of neuronal cells and axons according to the examples of the present invention, (a) immunofluorescent staining of neuronal cells and (c) quantitative analysis of optical density of Tuj-1, (b) immunofluorescent staining of axons, and (d) quantitative analysis of optical density of GAP-43;

FIG. 15 shows the vascularization ability of different composite microspheres according to the examples of the present invention, (a) immunofluorescent staining of vascular endothelial cells and (c) quantitative analysis, (b) immunofluorescent staining of neovascular and (d) quantitative analysis

FIG. 16 is an image of H & E staining of rat organs in an embodiment of the invention.

Detailed Description

The following examples are provided to further illustrate some, but not all, of the preferred embodiments of the present invention. Other embodiments of the invention, which are based on the invention, will be apparent to those skilled in the art without undue burden, and are within the scope of the invention. The invention will be further described with reference to the accompanying drawings.

The preparation of materials comprises preparing required chemical reagents and experimental materials, including lecithin, cholesterol, DSPE-PEG-CHO, octadecylamine, chloroform, deionized water, rg1, A-type pigskin gelatin, methacrylic acid, phosphate buffer saline, span 80, isopropyl myristate, SDF1α, bovine serum albumin, LAP and the like.

The basic method of the test is as follows:

Characterization of GM and GM-aL by light field microscope, scanning electron microscope, zeta potential analysis, infrared spectrum analysis, etc., and determining the encapsulation rate of GM to SDF1α, rg1 loading rate, in vitro degradation rate and in vitro sequential drug release rate.

2. In vitro biocompatibility detection of inflammatory control microspheres mice bone marrow mesenchymal stem cells were co-cultured with microspheres and biocompatibility was assessed by live/dead staining and CCK-8 detection.

3. And (3) the characterization of the inflammation regulation function of the composite microsphere, namely, evaluating the inflammation regulation function of the composite microsphere through real-time quantitative PCR detection, ELISA detection and immunofluorescence analysis.

4. Characterization of the recruitment function of composite microspheres for BMSCs was assessed by cell scratch and cell transwell assays.

5. The nerve regeneration promoting function of the composite microsphere is characterized in that the nerve regeneration promoting function of the composite microsphere is evaluated through immunofluorescence staining of a neuron specific marker and qRT-PCR detection of the neuron specific marker gene.

6. The spinal cord injury animal experiment is to establish a spinal cord Allen strike model, randomly group rats, inject different drugs locally into spinal cord, and evaluate animal exercise function and in vivo inflammation regulation function.

7. Histological analysis, namely H & E staining and immunofluorescence staining are carried out on spinal cord specimens, and the conditions of the syringomyelia area, glial scar tissue, endogenous nerve progenitor cells, neurons, angiogenesis and the like are analyzed.

Experimental methods without specific conditions noted in the examples, the present invention constructs new strategies, generally according to conventional conditions, or according to conditions suggested by the manufacturer, with reference to fig. 1.

EXAMPLE 1 preparation of hydroformylation cationic Liposome aL

AL was prepared by a film dispersion method. 80mg of lecithin (Yuanye, shangaai, china), 20mg of cholesterol (Arcos, belgium), 2mg of DSPE-PEG-CHO (Aladin, shangaai, china) and 2.5mg of octadecylamine (Aladin, shangaai, china) were dissolved in 30ml of chloroform (Lingfeng, shangaai, china) and further mixed using ultrasound to give a homogeneous emulsion. Evaporating the emulsion in a rotary evaporator for 1 hour, removing the organic solvent to obtain a colloid product, adding 3mL of deionized water to hydrate the lipid membrane on the bottle wall, and completely dissolving the lipid membrane by ultrasonic treatment to form the nano-scale liposome emulsion. Finally, filtration using 450 and 220nm polycarbonate membranes (Millex-GP, irish) gave a uniform monolayer aL.

EXAMPLE 2 preparation of ginsenoside Rg1 loaded Liposome aL@Rg1

2.5Mg Rg1 (Aladin, shanghai, china), 80mg lecithin and 20mg cholesterol, 2mg DSPE-PEG-CHO and 2.5mg octadecylamine were added to 30mL chloroform, and the other steps were the same as in liposome preparation of example 1.

Example 3 preparation of methacryloyl Gelatin (GM) microspheres;

Type A pigskin gelatin was prepared as a 10% (w/v) solution and completely dissolved in Phosphate Buffered Saline (PBS) at 60 ℃. Subsequently, a certain amount of Methacrylic Acid (MA) was added to the gelatin solution, and the reaction was stirred at 50 ℃ for 1 hour. The mixture was diluted 5-fold with additional PBS to stop the reaction, and then the mixture was dialyzed at 40℃for 1 week to remove unreacted MA and salts (dialysis tubing with a molecular weight cut-off of 12-14 kilodaltons) to obtain a GM solution

Finally, the GM solution was lyophilized for 1 week to produce a white porous foam that could be stored at-80 ℃ for long periods of time.

GM microspheres were prepared by microfluidic device. A PBS solution of 7wt% GM and 0.5wt% LAP (Sigma, USA) was used as the aqueous phase, and a solution of 5wt% Span 80 (Aladin, shanghai, china) in isopropyl myristate (Aladin, shanghai, china) was used as the oil phase. The two fluids are respectively connected into an inner port and an outer port of a microfluidic coaxial needle head (30G/22G), and the injection rate of the injection pump (Lei Fu fluid in China) is adjusted to be 1:30. The resulting microspheres were collected and crosslinked using ultraviolet radiation. Ethanol and isopropanol were used to wash 3 times respectively to remove residual Span 80 and isopropyl myristate. After washing 3 times with PBS, GM microspheres were obtained.

Can be stored at-20deg.C after lyophilization.

Example 4 preparation of GM microsphere gm@sdf1α loaded with stromal cell derived factor 1α (sdf1α);

A PBS solution of 7wt% GM and 0.5wt% LAP was used as the aqueous phase, and then SDF1α (peprotech, USA) was resuspended in 0.1wt% Bovine Serum Albumin (BSA) at a final concentration of 100 μg/mL and added to the above solution in a ratio to give a homogeneous SDF1α GM solution at a concentration of 100 ng/mL. A solution of Span 80 in isopropyl myristate at 5wt% was used as the oil phase. The other steps were the same as in example 4 for the preparation of GM microspheres.

Example 5 preparation of inflammation-controlling nerve regeneration-promoting composite microsphere GM-aL.

2 Ml of GM microsphere solution was mixed with 10ml of liposome solution, placed in an oven at 37℃and set for a grafting time of at least 24 hours. And finally, washing with deionized water for 3 times to obtain the GM-aL composite microsphere.

2 Ml of GM@SDF1α microsphere aqueous solution was mixed with 10ml of aL@Rg1liposome aqueous solution, placed in an oven at 37℃and set for a grafting time of at least 24 hours. And finally, washing for 3 times by using deionized water to obtain the GM@SDF1α -aL@Rg1 composite microsphere.

The aL@Rg1 aqueous solution is obtained by dissolving 3 parts of the whole product of the example 2 in 10ml of aqueous solution, and the GM@SDF1α microsphere aqueous solution is obtained by taking 2ml of the 10ml of aqueous solution of the product of the example 4GM@SDF1α;

example 6 Performance test of composite microspheres

1. Preparation and characterization of GM-aL composite microsphere

GM microspheres are produced from PDMS-based microfluidic devices, which are one of the most widely used devices due to simplicity and practicality. The external channel of the isopropyl myristate solution mixture containing span80 served as the continuous phase, while the central channel containing GM and LAP served as the dispersed phase, and the oily mixture in the continuous phase exerted shear force on GM, resulting in anti-dispersion of GM solution into emulsion droplets which polymerized into microspheres after cross-linking by uv irradiation.

The injection rate was 1:30 by adjusting the inner and outer ports of the coaxial needle. GM microspheres (fig. 2) with average diameters of 107.72 ±4.23 μm are obtained, which are white round spheres (fig. 2 a) under a light microscope, the particle size of the lyophilized GM is reduced, but the spheres are still kept, loose porous structures (fig. 2 b) are shown under a Scanning Electron Microscope (SEM), the porous structures of the microspheres increase the surface area of the microspheres, and conditions are provided for physical adsorption of the drug. GM can be smoothly injected through a needle with the diameter of 29G and is not blocked, and the GM has good injectability and can be used for minimally invasive injection treatment. Liposomes were prepared by modified thin film dispersion to give monodisperse liposomes (FIG. 2 c) with an average particle size of about 163.52.+ -. 13.42nm (FIG. 2 f). The GM-aL composite material with average diameter 126.54 ±6.61 μm (fig. 2 h) was obtained by mixing the GM-aL composite solution prepared above with liposomes in an oven at 37 ℃ with shaking (fig. 2 e), and the freeze-dried GM-aL composite microspheres were observed by SEM, and the liposomes present in the surface cavities of the microspheres were found to be a single dispersion with structural integrity (fig. 2 e). At the same time Zeta potential tests were performed to verify successful adsorption of the liposomes (FIG. 2 i), the average potential of the liposomes was 1.38.+ -. 0.39mV, and the absolute value of the Zeta potential (-13.87.+ -. 0.67 mV) of the GM-aL composite material was significantly lower than that of GM (-18.07.+ -. 1.52 mV) due to the partial charge neutralized by liposome adsorption, which confirmed successful grafting of the liposomes to the GM surface. To further demonstrate successful grafting of liposomes to GM microspheres, using DiL fluorescent-labeled liposomes mixed with GM microspheres, the red fluorescent signal punctate distribution on GM microsphere surface was seen under confocal microscopy (fig. 2 d), indicating that the liposomes were effectively anchored to hydrogel microsphere surface. In addition, fourier infrared spectroscopy (FTIR) observed that the aldehyde liposomes showed a distinct absorption peak around 1730cm "1, a characteristic peak of c=o in the aldehyde group, representing that we successfully modified the liposomes by hydroformylation. After integration of aL with GM hydrogel microspheres, FTIR images showed a decrease in the characteristic peak to peak representing N-H at 3420cm "1, suggesting that the amino groups on the GM microsphere surface were partially consumed during integration, while the peak to peak absorption at 1650 was enhanced, possibly from the newly generated schiff base bond c=n (fig. 3). This suggests that the hydroformylation liposome may be bound to GM microspheres by schiff base reaction.

2. In vitro degradation and release behavior

In order to evaluate the applicability of GM-aL composite microspheres as drug delivery vehicles, their degradation behavior and drug release kinetics were investigated. To simulate the gradual degradation process in vivo, GM-aL composite microspheres were infiltrated in PBS solution containing 0.1U/mL type I collagenase for 4 weeks.

The microspheres showed a substantially uniform slow degradation trend over 4 weeks and substantially degraded at day 28 (fig. 2 j). On day 7, the microspheres were intact in morphology, but some broken particles were observed around the microspheres. On day 14, the morphology of the microspheres began to collapse with irregular edges and cracks. On day 28, the microspheres were substantially degraded. It can be seen that degradation is a slow process from the surface to the inside, facilitating the long-term release of the drug. At the same time, the residual weight of the GM-aL composite microspheres decreased with time, consistent with the morphology change (fig. 2 k).

Molecular diffusion is the primary mechanism of drug release from the hydrogel matrix. The aL is anchored on the surface of the GM microsphere through the Schiff base bond, the GM-aL composite microsphere enhances the stability of the liposome, realizes the slow release effect of aL@Rg1 after injection, and provides an attractive method for controllable and continuous local drug delivery. The released Rg1 was quantified by HPLC, and as shown in FIG. 2l, the encapsulation efficiencies of Rg1 in liposome and GM-aL composite microspheres were (82.17.+ -. 6.32)% and (42 47.+ -. 1.97)%, respectively. The decrease and fluctuation of the encapsulation efficiency of Rg1 in the GM-aL composite microsphere is mainly due to the drug leakage during the process of the dendrite. The encapsulation efficiency of sdf1α in GM was (58.1±3.3)% (fig. 3 a). In addition, the release profile of Rg1 from liposomes and GM-aL complex microspheres was also studied, as shown in FIG. 2m, with Rg1 in the liposomes exhibiting a rapid release behavior with almost complete release in the first week. In contrast, rg1 in the GM-aL composite microsphere had little burst and sustained release for more than 2 weeks. Furthermore, at week 2, the cumulative release of Rg1 in GM-aL composite microspheres was much lower than that of Rg1 in liposomes. On the first day, as shown in fig. 3b, gm@sdf1α -aL had a burst of sdf1α, the release amount of sdf1α was 28.95%, and then the release efficiency was in a slowly decreasing trend, and sdf1α release was on the coming plateau after nearly 2 weeks. Within 28 days, the sdf1α release was approximately 69.37%. These results demonstrate that GM-aL composite microspheres exhibit sustained release of Rg and sdf1α during the degradation period, providing an attractive method for SCI therapy delivery of Rg1 and sdf1α.

3. In vitro biocompatibility detection of GM-aL composite microsphere

To evaluate the potential clinical application of drug-loaded functionalized microspheres, the biocompatibility of GM-aL, gm@sdf1α -aL, GM-al@rg1, gm@sdf1α -al@rg1 in vitro was studied. Each group of material leachates was co-cultured with BMSCs, and the single-cultured BMSCs served as a blank control group, and the biocompatibility of each group was evaluated using live/dead staining and CCK-8 cell counting kit on days 1, 3, and 5 of co-culture. As shown in FIGS. 4a, b, there were essentially no dead cells in the field after live/dead staining with increasing days of culture. Quantitative analysis of CCK-8 showed no significant difference in cell proliferation activity between groups on days 1 and 3. On day 5, however, it was found that the cell proliferation activity of the GM-aL@Rg1, GM@SDF1α -aL@Rg1 group was slightly increased compared to the other three groups (FIG. 4 c). This is probably due to the fact that the slow-release Rg1 in the material starts to act, and the proliferation activity of BMSC is improved. The results show that the functionalized microspheres have good biocompatibility, and the GM@SDF1α -aL@Rg1 has the effect of promoting the proliferation of BMSC.

4. In vitro evaluation of GM-aL composite microsphere-mediated inflammatory microenvironment

To evaluate the ability of GM-aL microspheres to modulate inflammatory responses in vitro, each set of microspheres was co-cultured with LPS-induced raw264.7 cells and related gene expression was determined using real-time quantitative polymerase chain reaction (qRT-PCR). The Raw264.7 cells after LPS induction obviously express inflammatory factor genes IL-1 beta and TNF-alpha, and down regulate the expression of inflammatory factor genes IL-10 and TGF-beta. However, after the intervention of GM-aL@Rg1, GM@SDF1α -aL@Rg1, the expression levels of the pro-inflammatory factors IL-1β, TNF- α and the anti-inflammatory factor genes IL-10 and TGF- β were somewhat reversed and statistically significant (P < 0.05) compared to the other groups (FIG. 5 e-h).

Similarly, the enzyme-linked immunosorbent assay (ELISA) examined the secretion of pro-and anti-inflammatory cytokines, and the results were consistent with the qRT-PCR results (FIGS. 5 i-l).

The Raw264.7 subtype immunofluorescence staining shows that each group of macrophage markers F4/80 (red fluorescence) are highly expressed, which indicates that the Raw264.7 has higher purity. The level of expression of the M1 marker CD86 was significantly increased after LPS treatment compared to the control group, indicating that LPS successfully induced M0 type macrophages to M1 type macrophages. The expression of the M1 marker was significantly lower in the GM@SDF1α -aL@Rg1 group than in the other control group (P < 0.05) (FIG. 5a, c), while the expression of the M2 marker was significantly higher than in the other control group (P < 0.05) (FIG. 5b, d). Similar observations were also observed in the GM-aL@Rg1 group. Taken together, these results indicate that gm@sdf1α -al@rg1 can regulate the polarization direction of macrophages, so that the macrophages are polarized in the M2 direction, promote the secretion of anti-inflammatory cytokines, reduce local inflammatory responses, and create a favorable environment for subsequent nerve regeneration.

5. Characterization of GM-aL composite microsphere cell migration and recruitment function

In the scratch test, BMSCs cells were cultured with material leaches (fig. 6 a). We found that the migration area of the GM@SDF1α -aL, GM@SDF1α -aL@Rg1 group was significantly larger than that of the remaining group (P < 0.05), indicating that GM microspheres increased the migration capacity of stem cells by releasing SDF1α (FIG. 6 c).

In chemotaxis experiments, BMSC cells were loaded into the upper chamber, while each material leach was in the lower chamber, and the cells were expected to migrate down the chamber in response to GM microsphere release sdf1α (fig. 6 b). As shown in FIG. 6d, the number of cells migrated in the GM@SDF1α -aL, GM@SDF1α -aL@Rg1 group was significantly higher than that in the remaining group (p < 0.05), indicating that GM@SDF1α -aL@Rg1 could recruit BMSCs through SDF1α.

6. Characterization of GM-aL composite microsphere for regulating nerve regeneration function

After spinal cord injury, establishing a loose local microenvironment is critical to promote endogenous stem cell differentiation. Since inflammatory cytokines affect differentiation of endogenous stem cells, a BMSCs and microsphere co-culture system was constructed and the effect of GM@SDF1α -aL@Rg1 on differentiation of BMSCs under pathological conditions was further investigated. The induction medium was prepared using low-sugar DMEM and 10% fetal bovine serum (including one of GM-aL, gm@sdf1α -aL, GM-al@rg1, gm@sdf1α -al@rg1). With the aid of a Transwell system, LPS-mediated raw is in the upper chamber, and cell and material leachates are in the lower chamber, and BMSC differentiation behavior into neurons is studied. Immunofluorescent staining and qRT-PCR techniques were used to detect the expression of neuronal specific markers in neuronal-like cells, including neuronal cell specific differentiated tubulin (Tuj-1), microtubule-associated protein 2 (Map-2), neurofilament protein (NF-200), and neuronal cytoskeletal tubulin (Tau). Immunofluorescent staining images (FIG. 7 a), GM-aL@Rg1, GM@SDF1α -aL@Rg1 groups all showed typical neuronal-like changes, such as axonlike and dendritic like dendrites around the cell bodies, and each neuronal-specific marker showed intense green fluorescence after staining (FIG. 7 a). Compared with the weak signals of the control group and the GM@SDF1α -aL group, the expression of the fluorescence of the GM-aL@Rg1 group and the GM@SDF1α -aL@Rg1 group is obviously increased, and the significant statistical difference (p < 0.05) exists. The constructed GM-aL composite scaffold is suggested to be capable of slowly releasing Rg1 and can continuously promote the differentiation of endogenous stem cells to neurons in an inflammatory state (FIGS. 7 b-e). At the same time, similar results appear for different neuronal specific marker gene expression assays (FIG. 7 f-I). The Rg1 group-loaded each nerve-specific marker gene is highly expressed and has statistical significance (p < 0.05). In addition, the length (78.33.+ -. 15.63 μm) of Tuj-1 cells from the GM@SDF1α -aL@Rg1 group was significantly longer than that of Control group (5.33.+ -. 1.53 μm, p < 0.05) (FIG. 8). These results indicate that gm@sdf1α -al@rg1 can promote neuronal differentiation of BMSCs in inflammatory states.

7. Animal athletic function scoring

In vivo treatment of gm@sdf1α -al@rg1 was evaluated using the SD rat T9 spinal cord Allen strike model. Recovery of hind limb motor dysfunction after SCI was assessed weekly by Basso, beattie, bresnahan (BBB) and oblique plane test (IPT) scores. The results showed (FIGS. 9d, e) that the motor function of each group of rats was restored to some extent after surgery. 4 weeks after surgery, the BBB score and IPT score of the gm@sdf1α -al@rg1 group appeared significantly higher than those of the other groups compared to the other groups, and had statistical significance (P < 0.0.5). By 8 weeks after the operation, the BBB score and the IPT score of the GM@SDF1α -aL@Rg1 group reach 13.71+/-1.11,55.86 +/-4.67 degrees respectively, and the difference is statistically significant compared with other groups. In addition, compared with other control groups, the rising rate of BBB score and IPT score curves of the GM@SDF1α -aL@Rg1 group is higher, and the effect of nerve repair can be achieved in a shorter time. Thus, the motor function scoring results suggest that gm@sdf1α -al@rg1 is capable of protecting surviving motor neurons from severe inflammatory response damage during the acute phase of spinal cord injury and promoting nerve repair in a faster time.

8. Inflammatory microenvironment regulation in vivo SCI

Macrophages and microglia reach a peak of recruitment within 7 days after SCI, during which gm@sdf1α -al@rg1 should exert an inflammatory regulatory effect. For this, we evaluated the effect of gm@sdf1α -al@rg1 on SCI early neuroinflammation. On day 7 post-surgery we first examined each group of rat spinal cord specimens by immunofluorescence to assess whether the composite hydrogel microspheres could modulate macrophage subtypes. The results show that gm@sdf1α -al@rg1 group significantly reduced the signal of INOS compared to the other groups (fig. 10 a), increased ARG-1 signal (fig. 10 b), and the difference was statistically significant (P < 0.0.5). Immunofluorescence imaging results show that the GM@SDF1α -aL@Rg1 group not only remarkably reduces the ratio of INOS/Iba-1 macrophages, but also remarkably increases the ratio of ARG-1/Iba-1 macrophages, and the GM@SDF1α -aL@Rg1 can promote local microglial cells/macrophages to be polarized into an M2 phenotype in vivo. Notably, the improvement of inflammation by gm@sdf1α -al@rg1 was more pronounced than GM-al@rg1. This is probably because the SDF1α -recruited BMSCs have a certain immunoregulatory function, 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 group further corroborates the hypothesis.

Local immune cell subtype changes after SCI necessarily lead to changes in inflammatory cell secretion. Through the evaluation of inflammatory gene expression at spinal cord injury, the results show that the expression of pro-inflammatory factors TNF-alpha and IL-1 beta is higher (p < 0.05) in a blank control group and a non-inflammatory regulatory function group (figures 11e and f), while the expression of the inflammatory genes IL-10 and TGF-beta in the GM@SDF1alpha-aL@Rg1 group is obviously increased (figures 11g and h). The results show that GM@SDF1α -aL@Rg1 can inhibit early inflammatory response after SCI, reduce inflammatory factor secretion and create a local microenvironment for inducing spinal cord regeneration.

In addition, the whole body inflammatory reaction of animals is evaluated through serum ELISA detection, and the inflammatory regulation function of the composite microsphere is comprehensively evaluated. As a result, it was found that the serum inflammatory factors TNF-. Alpha.and IL-. 1. Beta.in the control group GM@ -aL were significantly higher than those in the GM@SDF1α -aL@Rg1 group (p < 0.05) (FIG. 11i, j), while the IL-10 and TGF-. Beta.levels in the GM@SDF1α -aL@Rg1 group were higher (p < 0.05) (FIG. 11k, l). The implantation of the inflammation-regulating composite microsphere can improve local immune environment and reduce systemic inflammatory response.

9. Histology and immunohistochemistry for spinal cord repair

Secondary injury to neuroinflammation after SCI results in neuronal death, demyelination of axons, while chronic encapsulation of the injury site by glial cells makes it difficult for regenerated neurons and axons to penetrate and reconstruct damaged spinal cord tissue, a difficulty in spinal cord injury repair. The study incorporates both inflammatory mediators and BMSCs recruitment factors to regulate neuroinflammation, inhibit glial tissue formation and promote nerve regeneration. The H & E stained images are shown (fig. 9 a-c), and the area of the syringomyelia is significantly reduced (P < 0.05) in all material groups compared with the blank group, suggesting that the biological scaffold plays an important role in the spinal cord injury repair process. However, the GM@ -aL and gm@sdf1α -aL groups both seen a large number of parallel red stained collagen fibers and elongated fiber nuclei, indicating glial tissue formation. The GM@SDF1α -aL@Rg1 group had smoother and more uniform distribution of red-stained collagen fibers around the lesions than the other control groups, and the glial tissues were significantly inhibited at 4 weeks. And the area of the spinal cavity is further reduced in 8 weeks of the GM@SDF1alpha-aL@Rg1 group compared with that in 4 weeks, and other material groups have no obvious change, so that the regulation and control of the inflammation in the acute stage of spinal cord injury play a role in the later nerve repair process.

Activated M1 microglia after SCI secrete inflammatory factors TNF-a, IL-1a and C1q, inducing resting astrocytes to polarize towards A1 astrocytes, A1 astrocytes secrete neurotoxins, killing axonally damaged CNS neurons. In addition, glial fibrous acid protein (glial fibrillary acidic protein, GFAP) and glial antigen 2 (neuron GLIAL ANTIGEN, ng 2) which are hypertrophic, proliferated, migrated and expressed by A1 astrocytes promote glial scar formation and prevent nerve repair. To further evaluate the anti-gliosis effect of the inflammation-modulating composite hydrogel microspheres, we fluorescently labeled activated astrocytes and gliosis tissues in spinal cord samples with anti-GFAP antibodies and anti-NG 2 antibodies. The results show (fig. 12a, b) that the material groups (GM-aL, gm@sdf1α -aL, GM-al@rg1, gm@sdf1α -al@rg1) activated significantly less astrocytes and scar tissue than the blank control group (p < 0.05) at both time points 4 weeks and 8 weeks post-surgery, suggesting that the hydrogel can fill the spinal cord injury gap, reducing disordered glial scar formation after nerve injury. Furthermore, the results of the fluorescent semi-quantitative analysis showed that the fluorescence density of activated astrocytes and scar tissue was significantly lower in the gm@sdf1α -al@rg1 group than in the other group (p < 0.05). The result proves that the composite microsphere can regulate and control local inflammation microenvironment in the SCI acute stage, reduce scar tissue formation and lay a good foundation for differentiation of endogenous stem cells into neurons.

Endogenous stem cells are activated following SCI and migrate to the site of injury, but local scar formation of the injured spinal cord tissue impedes cell migration. In addition, the number of endogenous stem cells migrating to the site of injury is insufficient to reestablish the neural network. The above immunohistochemical results demonstrate that the composite microspheres can reduce the formation of activated astrocytes and glial scar tissue, reducing the physical barrier to cell migration. Under physiological conditions, nestin is under-expressed and Nestin-positively labeled cells are greatly increased following spinal cord injury. Therefore, the endogenous nerve progenitor cells in the damaged area are marked by the Nestin antibody fluorescence, and the differentiation of the BMSCs recruited by SDF1α to the nerve progenitor cells under the condition that Rg1 regulates local inflammation microenvironment is indirectly evaluated. The distribution of Nestin-labeled stem cells in injured spinal cord is shown (fig. 13 a). The fluorescent quantitation results showed that the gm@sdf1α -al@rg1 group had significantly more endogenous progenitor cells than the other groups (P < 0.05), whether injured for 4 or 8 weeks (fig. 13 b). The results show that the composite microsphere regulates the local inflammation microenvironment through Rg1, promotes the BMSCs recruited by SDF1α to survive and differentiate towards nerve progenitor cells.

Local loose microenvironment following spinal cord injury is critical to the differentiation of BMSCs. Although BMSCs are capable of differentiating into neurons, astrocytes and oligodendrocytes, the local inflammatory microenvironment after SCI tends to promote differentiation of BMSCs into astrocytes. The Rg1 is reported to regulate the local inflammatory microenvironment after spinal cord injury, improve the oxidation resistance of BMSCs, reduce apoptosis, improve survival rate, and promote BMSC differentiation to neurons. In this study, tuj-1 and GAP-43 were used to label neuronal progenitor, neuronal and axonal sprouting, respectively, and the ability of composite GM microspheres to promote late-stage nerve regeneration in SCI was evaluated. Since gm@sdf1α -al@rg1 can regulate the local inflammatory microenvironment, the physical barrier effect of the glial scar is reduced, and the differentiation of the recruited BMSCs to neurons is promoted, the number of neurons is obviously higher than that of other groups. At 4 and 8 weeks post-surgery, a significant number of Tuj-1-labeled neurons appeared in the material groups (GM-aL, GM@SDF1α -aL, GM-aL@Rg1, GM@SDF1α -aL@Rg1), significantly higher than in the control group (P < 0.05) (FIGS. 14a, c). In addition, the fluorescence intensity of the GM@SDF1α -aL@Rg1 group at 8 weeks after operation is obviously higher than that at 4 weeks, and the difference is statistically significant. These results indicate that gm@sdf1α -al@rg1 can regulate not only SCI local inflammatory microenvironment, but also promote BMSCs homing and differentiation to neurons by sustained release of sdf1α. Growth-related protein 43 (GAP-43), which is a marker of neurite formation, plays an important role in the transduction signal of neurite formation, is expressed low in normal spinal cord, and GAP-43 is activated to be expressed high by cytokines and neurotrophic factors after SCI, promoting neuroprotection and regeneration. As shown in fig. 14b, d, GAP-43 fluorescence intensity was significantly lower in the blank group than in the other groups. While the gm@sdf1α -al@rg1 group has significantly higher fluorescence intensity than the other groups, the difference is statistically significant (P < 0.05), indicating that gm@sdf1α -al@rg1 regulates the inflammatory microenvironment and recruits BMSCs to contribute to neuronal differentiation, which is crucial for neural circuit reconstruction. Furthermore, we noted that the expression of GAP-43 in the GM@SDF1α -aL@Rg1 group was significantly decreased at 8 weeks versus 4 weeks of injury. The results show that the repair time window of SCI is limited, the inflammation regulation is carried out in the SCI acute phase, the inflammatory reaction is reduced, and the nerve regeneration can be better promoted.

The restoration of nerve function depends not only on the regeneration of neurons but also on the generation of spinal cord blood vessels. Angiogenesis is critical for recovery of damaged spinal cord tissue, can improve blood supply, accelerate tissue repair, and support neural cell survival. BMSCs have been reported to promote angiogenesis in injured spinal cord and to significantly enhance neural tissue retention through neurotrophic signaling and immunomodulation. At both time nodes, weeks 4 and 8 of injury, the gm@sdf1α -al@rg1 group CD31 fluorescence intensity was significantly higher than the other control group (fig. 15a, c). In addition, the number of CD 31-labeled vascular endogenous cells in the GM@SDF1α -aL@Rg1 group at 8 weeks of injury was significantly greater than 4 weeks. Similarly, when neovasculature was fluorescently labeled with Von Willebrand Factor (VWF) at 4 and 8 weeks of injury, quantitative results of fluorescent staining showed that VWF fluorescence intensity was significantly higher in the GM@SDF1α -aL@Rg1 group than in the other groups, and that the number of neovasculature labeled with VWF at 4 weeks was significantly less than 8 weeks (FIGS. 15b, d). The result shows that the GM composite microsphere can only induce macrophages and microglia to be polarized into M2 subtype so as to secrete vascular endothelial growth factor, and can also recruit BMSCs to promote angiogenesis together, thus providing a good basis for spinal cord repair. Furthermore, rat organ staining showed that no difference was observed between gm@sdf1α -al@rg1 group and control group, demonstrating that it was not potentially toxic in vivo (fig. 16).

The results show that the composite microsphere has local inflammatory regulation capability and continuous promotion effect on nerve tissue regeneration, and provides a biomaterial strategy which depends on inflammatory microenvironment regulation and BMSCs recruitment coordination for acute SCI repair.

The embodiments described above are intended to further illustrate some, but not all, of the preferred embodiments of the present invention. Other embodiments of the invention, which are based on the invention, will be apparent to those skilled in the art without undue burden, and are within the scope of the invention.

Claims (9)

1. A method for preparing composite microspheres for regulating local inflammatory microenvironment to promote 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 composite microspheres 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 composite microspheres for regulating local inflammatory microenvironment and promoting nerve regeneration according to claim 1, characterized in that 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 above 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 composite microspheres for regulating local inflammatory microenvironment and promoting nerve regeneration according to claim 4, characterized in that step 2 further comprises the step of preparing GM microspheres using 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 composite microspheres for regulating local inflammatory microenvironment and promoting nerve regeneration according to claim 5, characterized in that 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. A method for preparing composite microspheres for regulating local inflammatory microenvironment and promoting nerve regeneration according to claim 6, characterized in that the grafting condition is placed in an oven at 37°C and the grafting time is set to at least 24 hours. 8. A composite microsphere for regulating local inflammatory microenvironment to promote nerve regeneration, characterized in that the composite microsphere is prepared by any one of the methods of claims 1 to 7. 9. An application of composite microspheres that regulate local inflammatory microenvironment to promote nerve regeneration, characterized in that the composite microspheres that regulate local inflammatory microenvironment to promote nerve regeneration are used as drugs for regulating inflammatory microenvironment after spinal cord injury to promote nerve regeneration.