Functionalized hydrogel microspheres as well as preparation method and application thereof

Abstract

The invention discloses a functional hydrogel microsphere and a preparation method and application thereof, and relates to the technical field of medicines.The preparation method comprises the steps that a hyaluronic acid methacrylic acid precursor solution and an IL-1beta shRNA adenovirus stock solution are mixed to obtain an Ad-atHAMA precursor solution; preparing the Ad (at) HAMA precursor solution through an oil-in-water micro-fluidic technology to obtain an Ad (at) HAMA microsphere; and sequentially adding the Ad (at) HAMA microspheres, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into a 2-morpholinoethanesulfonic acid buffer solution, carrying out activating treatment, adding E selectin, and incubating to obtain the functionalized hydrogel microspheres. The functionalized hydrogel microspheres can inhibit a related mechanism of acute spinal cord injury pathological progress and promote neural function repair by targeting adsorption of neutrophil to regulate Nets-triggered innate immune inflammation cascade.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of functionalized hydrogel microspheres and its preparation method, application, belong to medical technology field. BACKGROUND

[0002] Spinal cord injury (SCI) is a devastating central nervous system (CNS) trauma, leading to loss of sensation and motor function below the level of injury, with more than 700,000 new cases worldwide each year, causing a huge personal and social burden.

[0003] After SCI, damage-associated molecular patterns (DAMPs) initiate monocyte / macrophage activation, and activated macrophages reach a peak in the focal area at 3 and 7 days after injury, and are polarized to "classically activated macrophages (CD86+ macrophages)" at the early stage of inflammation, which secrete more pro-inflammatory factors to exacerbate injury and scarring, and are transformed into "alternatively activated macrophages (CD206+ macrophages)" at the late stage of inflammation, which secrete anti-inflammatory factors to reduce inflammatory response. However, during the innate immune response and disease progression, complex immune responses are closely related to the prognosis outcome, so single regulation of a certain cell is not enough, and a multi-cell interaction exploration model and related treatment strategies need to be established.

[0004] In addition to macrophages, DAMPs first recruit circulating neutrophils to the injury site, and reach a peak within 1 to 3 days, which migrate to the inflammatory site to release pro-inflammatory mediators, myeloperoxidase (MPO), reactive oxygen species (ROS), and other substances to phagocytose and kill bacteria, and participate in inflammatory response. In addition, activated neutrophils release decondensed chromatin (Nets) composed of elastase (NE), MPO and cytoplasmic proteins. In recent years, the role and status of Nets in the inflammatory environment have attracted more and more attention. Previous studies have shown that drugs such as Cl-amidine and DNase1 can intervene in Netosis to some extent or enhance the decomposition of Nets, thereby limiting the amplification effect of Nets on macrophage inflammatory response, and thus promoting the functional recovery of SCI. However, the method of local injection of drugs or transplantation of cells inevitably has the risk of reduced activity and dispersion. Therefore, on the basis of in-depth exploration of the biological mechanism of Nets-macrophages, further construction of biomaterials that can create a protective immune microenvironment, regulation of the cascade effect of innate immune response, and promotion of neural function recovery are expected to break through the current bottleneck of SCI treatment.

[0005] Good biocompatibility is essential for animal experimental research and later clinical conversion of biomaterials. Hyaluronic acid is a natural glycosaminoglycan polymer, as a component of animal tissue extracellular matrix, has the advantages of non-toxicity and low immunogenicity, hyaluronic acid methacrylic acid (HAMA) is introduced into the methacryl group on the hyaluronic acid molecular chain to endow it with photocuring ability, based on its stability and good biocompatibility has been widely used, but the implant material needs secondary surgery to the damage gap, which will undoubtedly increase the pain of the patient and bring the corresponding surgical risk. Although there is a conservative strategy of injecting high-dose methylprednisolone for the treatment of SCI, but hormone therapy also brings lung infection, gastrointestinal bleeding and other complications. Here, we note that Qi et al. prepared nanomicelles modified with E-selectin, a glycoprotein that actively binds to ligands on neutrophils, significantly enhancing the immunospecificity of the drug, and effectively inhibited the growth of primary tumors by systemic administration, taking advantage of leukocytes to increase drug accumulation in tumors. However, systemic administration has problems such as loss of biological information due to liver and kidney metabolism, shortening of biological factor half-life, and insufficient local concentration of damaged tissues.

[0006] Therefore, if a biomaterial treatment strategy based on NETs and macrophage crosstalk mechanism can be designed and constructed to reduce the formation of Nets, it will directly reduce the progression of the innate immune cascade pathology of SCI, maintain the homeostasis of the SCI microenvironment, and be more beneficial to the later recovery of neural function. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings in the prior art, provide a functional hydrogel microsphere and a preparation method and application thereof, and a mechanism for inhibiting the pathological progression of acute spinal cord injury and targeted adsorption of neutrophils to regulate the innate immune inflammatory cascade triggered by Nets to promote neural function repair.

[0008] To achieve the above purpose, the present application is realized by using the following technical solutions: In a first aspect, the present application provides a preparation method of a functional hydrogel microsphere, comprising: dissolving hyaluronic acid methacrylic acid and a photoinitiator in a phosphate buffered saline solution to obtain a hyaluronic acid methacrylic acid precursor solution; mixing the hyaluronic acid methacrylic acid precursor solution with an IL-1β shRNA adenovirus stock solution to obtain an Ad@HAMA precursor solution; preparing Ad@HAMA microspheres from the Ad@HAMA precursor solution by oil-in-water microfluidic technology; The Ad@HAMA microspheres, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide are sequentially added into 2-morpholinoethanesulfonic acid buffer solution, and E-selectin is added after activation treatment, and the functionalized hydrogel microspheres are obtained after incubation.

[0009] Further, the concentration of the hyaluronic acid methacrylic acid and the photoinitiator in the phosphate buffered saline solution ranges from 10:2 to 4 g / L.

[0010] Further, the photoinitiator is 2,4,6-trimethylbenzoyl or Lrgacure 2959.

[0011] Further, the volume ratio of the IL-1β shRNA adenovirus stock solution to the hyaluronic acid methacrylic acid precursor solution ranges from 100 to 150 μL:1 mL.

[0012] Further, the Ad@HAMA precursor solution is prepared into the Ad@HAMA microspheres by the oil-in-water microfluidic technology, which comprises the following steps: The Ad@HAMA precursor solution is introduced into the dispersed phase of the microfluidic chip, the paraffin oil solution is introduced into the continuous phase of the microfluidic chip, and the continuous monodisperse spherical droplets are obtained from the outlet after convergence; The monodisperse spherical droplets are placed in preloaded oil, and are irradiated by ultraviolet light to realize photo-crosslinking reaction, and the Ad@HAMA microspheres are obtained after washing after the photo-crosslinking reaction is completed.

[0013] Further, the flow rate ratio of the continuous phase to the dispersed phase in the microfluidic chip ranges from 2 to 4:30 to 90 μL / min. And / or, the ultraviolet light irradiation is performed every 10 to 20 min until the photo-crosslinking reaction is completed.

[0014] Further, the mass ratio of the Ad@HAMA microspheres, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, 2-morpholinoethanesulfonic acid buffer solution, and E-selectin ranges from 100:16:24:2:3 to 5, and the pH of the 2-morpholinoethanesulfonic acid buffer solution ranges from 5.5 to 6.7.

[0015] Further, the activation treatment comprises activation in a 37°C constant temperature incubator for 10 to 20 min. And / or, the incubation comprises incubation in a 37°C constant temperature incubator overnight.

[0016] In a second aspect, the application further provides a functionalized hydrogel microsphere, which is prepared by the preparation method of the functionalized hydrogel microsphere according to any one of the first aspect.

[0017] In a third aspect, the application further provides a functional hydrogel microsphere for use in regulating the innate immune cascade after spinal cord injury and promoting recovery of nerve function by intrathecal injection within 24-48 hours after the spinal cord injury, wherein the intrathecal injection has a drug concentration of 4-7 muL / g.

[0018] Compared with the prior art, the application has the following beneficial effects: The application takes the vicious positive feedback loop of IL-1beta as the core to activate the proliferation effect of NETs as the guide, takes the HAMA microfluidic technology as the core, and starts from the aspects of internally loading IL-1beta shRNA adenovirus and bridging E-selectin on the surface by amide bonds to construct the gel microspheres that can independently control the "breeding" mode of Nets, inhibit the related mechanisms of the pathological progression of acute spinal cord injury, and target adsorption of neutrophils to regulate the innate immune inflammation cascade triggered by Nets to promote the repair of nerve function, and can be used as an integrated solution with safety, high efficiency and great potential. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A is a preparation method flowchart of the functional hydrogel microspheres, and B is a potential mechanism diagram of the functional hydrogel microspheres for creating a protective immune microenvironment in the treatment of acute spinal cord injury. Figure 2 A is a comparison diagram of green fluorescence showing GFP expression of the Ad@HAMA microspheres and neutrophils after co-culture for 48 hours, B is an analysis diagram of the nuclear and eGFP fluorescence overlap coefficient rate of the Ad@HAMA microspheres and neutrophils after co-culture for 48 hours, and C is a test result diagram of the trypan blue live cell exclusion of the neutrophils after co-culture for 48 hours. Figure 3 A is a comparison diagram of the morphology of the ES-Ad@HAMA functional hydrogel microspheres before and after centrifugation, B is a comparison diagram of the surface morphology of the HAMA microspheres, the Ad@HAMA microspheres and the ES-Ad@HAMA functional hydrogel microspheres in the water phase and the oil phase. Figure 4 SEM images of Ad@HAMA microspheres, ES-Ad@HAMA functionalized hydrogel microspheres and HAMA microspheres prepared in Comparative Example of the present application, wherein A is SEM images of HAMA microspheres, Ad@HAMA microspheres and ES-Ad@HAMA functionalized hydrogel microspheres; B is particle size distribution of ES-Ad@HAMA functionalized hydrogel microspheres; Figure 5 Elemental analysis and infrared FTIR of Ad@HAMA microspheres and ES-Ad@HAMA functionalized hydrogel microspheres prepared in Example 5 of the present application, wherein A is elemental analysis of Ad@HAMA microspheres and ES-Ad@HAMA functionalized hydrogel microspheres; B is elemental mapping of C, O, N and S on ES-Ad@HAMA functionalized hydrogel microspheres; C is infrared FTIR of ES-Ad@HAMA functionalized hydrogel microspheres; Figure 6 Non-targeted metabolomics sequencing of ES-Ad@HAMA group and Control group (NETs and macrophages co-cultured) prepared in Example 5 of the present application, wherein A is principal component analysis of Control group and ES-Ad@HAMA group; B is partial least squares discriminant analysis of Control group and ES-Ad@HAMA group; C is differential gene clustering heat map comparison of Control group and ES-Ad@HAMA group; Figure 7 Differential gene analysis of ES-Ad@HAMA group and Control group (NETs and macrophages co-cultured) prepared in Example 5 of the present application, wherein A is volcano plot of differential genes of ES-Ad@HAMA group and Control group under the condition of |log2foldchange|>1.2; B is KEGG enrichment analysis of differential genes of ES-Ad@HAMA group and Control group under the condition of |log2foldchange|>1.2; Figure 8 Metabolic involvement of ES-Ad@HAMA functionalized hydrogel microspheres prepared in Example 5 of the present application, wherein A is clustering heat of ES-Ad@HAMA in glycolysis and TCA cycle related metabolites; B is target metabolite change of ES-Ad@HAMA in glycolysis and TCA cycle; Figure 9Figure 1 shows the flow cytometry identification of the macrophage subtypes of the Control group (simple macrophages), the Nets group (macrophages / NETs) (Nets group), and the ES-Ad@HAMA group (macrophages / NETs + ES-Ad@HAMA functionalized hydrogel microspheres) in an embodiment of the present application. Figure 1A shows the flow cytometry identification of the macrophage subtypes of the Control group, the Nets group, and the ES-Ad@HAMA group. Figure 1B shows the quantitative analysis of the CD11b+ / CD86+ ratio of the Control group, the Nets group, and the ES-Ad@HAMA group (n = 5). Figure 1C shows the quantitative analysis of the CD11b+ / CD206+ ratio of the Control group, the Nets group, and the ES-Ad@HAMA group. Figure 10 Figure 2 shows the fluorescence staining of the Neu group (simple neutrophils), the NETs group (neutrophils + PMA), and the ES-Ad@HAMA group (neutrophils + PMA + material) in an embodiment of the present application. Figure 11 Figure 3 shows the construction of a mouse spinal cord contusion model and spinal cord specimens in an embodiment of the present application. Figure 3A shows the construction process of the mouse spinal cord contusion model. Figure 3B shows the spinal cord specimens at 7 days, 4 weeks, and 8 weeks after spinal cord injury surgery. Figure 12 Figure 4 shows the BMS score and IPT score of the mouse spinal cord contusion model in an embodiment of the present application. Figure 4A shows the BMS score of the mouse spinal cord contusion model. Figure 4B shows the IPT score of the mouse spinal cord contusion model. Figure 13 Figure 5 shows the tissue (heart, liver, spleen, lung, kidney) compatibility of the mouse spinal cord contusion model at 1 week and 4 weeks after spinal cord injury surgery in an embodiment of the present application. Figure 14 Figure 6 shows the in vivo expression and fluorescence semi-quantitative analysis of IL-1β, CitH3, and MPO of the mouse spinal cord contusion model at 7 days after spinal cord injury surgery in an embodiment of the present application. Figure 6A shows the in vivo expression of IL-1β. Figure 6B shows the in vivo expression of CitH3 and MPO. Figure 6C shows the fluorescence semi-quantitative analysis of IL-1β. Figure 6D shows the fluorescence semi-quantitative analysis of CitH3 and MPO. Figure 15This diagram illustrates the in vivo expression and semi-quantitative fluorescence analysis of Fibronectin, Col-I, and α-SMA in a mouse spinal cord contusion model at 4 and 8 weeks post-surgery, according to one embodiment of the present invention. A represents the in vivo expression of Fibronectin; B represents the in vivo expression of Col-I; C represents the in vivo expression of α-SMA; D represents the semi-quantitative fluorescence analysis of Fibronectin at 4 weeks post-surgery; E represents the semi-quantitative fluorescence analysis of Fibronectin at 8 weeks post-surgery; F represents the semi-quantitative fluorescence analysis of Col-I at 4 weeks post-surgery; G represents the semi-quantitative fluorescence analysis of Col-I at 8 weeks post-surgery; H represents the semi-quantitative fluorescence analysis of α-SMA at 4 weeks post-surgery; and I represents the semi-quantitative fluorescence analysis of α-SMA at 8 weeks post-surgery. Figure 16 This diagram illustrates the in vivo expression and semi-quantitative fluorescence analysis of NG2 and GFAP in a mouse spinal cord contusion model at 4 and 8 weeks post-surgery, according to one embodiment of the present invention. A represents the in vivo expression of NG2 at 4 and 8 weeks post-surgery; B represents the in vivo expression of GFAP at 4 and 8 weeks post-surgery; C represents the semi-quantitative fluorescence analysis of GFAP at 4 weeks post-surgery; D represents the semi-quantitative fluorescence analysis of GFAP at 8 weeks post-surgery; E represents the semi-quantitative fluorescence analysis of NG2 at 4 weeks post-surgery; and F represents the semi-quantitative fluorescence analysis of NG2 at 8 weeks post-surgery. Figure 17 This diagram illustrates the in vivo expression and semi-quantitative fluorescence analysis of Tuj-1 and GAP-43 in a mouse spinal cord contusion model at 4 and 8 weeks post-surgery, according to one embodiment of the present invention. A shows the in vivo expression of Tuj-1 at 4 and 8 weeks post-surgery; B shows the in vivo expression of GAP-43 at 4 and 8 weeks post-surgery; C shows the semi-quantitative fluorescence analysis of Tuj-1 at 4 weeks post-surgery; D shows the semi-quantitative fluorescence analysis of Tuj-1 at 8 weeks post-surgery; E shows the semi-quantitative fluorescence analysis of GAP-43 at 4 weeks post-surgery; and F shows the semi-quantitative fluorescence analysis of GAP-43 at 8 weeks post-surgery. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention. Example 1

[0021] like Figure 1 As shown in Figure A, this embodiment of the invention provides a method for preparing functionalized hydrogel microspheres, comprising the following steps: 1% hyaluronic acid methacrylic acid (HAMA) hydrogel (purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd., China) and 0.25% Lrgacure2959 photoinitiator were dissolved in phosphate buffered saline (PBS solution) to obtain a HAMA precursor solution.

[0022] 5 μL of IL-1β shRNA adenovirus stock solution with eGFP fluorescence (purchased from WZ Biosciences) was mixed with 1 mL of the HAMA precursor solution to obtain different Ad@HAMA precursor solutions.

[0023] Then, the Ad@HAMA microspheres were prepared by using the oil-in-water microfluidic technology, specifically including: The continuous phase, the dispersed phase and the outlet on the microfluidic chip (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., China) were all connected to silica gel tubes, wherein the dispersed phase was connected to the Ad@HAMA precursor solution through a silica gel tube, and the continuous phase was connected to a paraffin oil solution through a silica gel tube.

[0024] The continuous / dispersed flow rate was adjusted to 2:45 μL / min. At this time, due to the shear force generated by the speed difference between the continuous phase and the dispersed phase, continuous monodisperse spherical materials appeared at the convergence. After the droplet size was uniform, it was placed in a beaker containing preloaded oil solution under light-proof conditions. Every 10-20 min, the beaker was taken out, irradiated with ultraviolet light, and then placed back in place. The collected microspheres after crosslinking were washed with 75% ethanol three times and PBS five times, and the PBS was replaced every 3 h to remove the photoinitiator and oil, thereby obtaining the Ad@HAMA microspheres.

[0025] The Ad@HAMA microspheres collected above, 16 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 24 mg of N-hydroxysuccinimide (NHS) were sequentially added to 2 mL of 2-morpholinoethanesulfonic acid (MES) (pH=6.0) buffer. Activation was performed in a 37°C constant temperature shaker for 15 min. Then, 4 mg of E-selectin (R&D, USA) was added to the above system. Incubation was performed at 37°C in a constant temperature shaker overnight to obtain functionalized hydrogel microspheres (ES-Ad@HAMA). Example 2

[0026] The difference between this example and Example 1 is that 10 μL of IL-1β shRNA adenovirus stock solution with eGFP fluorescence was mixed with 1 mL of the HAMA precursor solution to obtain different Ad@HAMA precursor solutions. Example 3

[0027] The difference between this embodiment and embodiment 1 is only that 50 μL of IL-1β shRNA adenovirus stock solution with eGFP fluorescence is mixed with 1 mL of HAMA precursor solution to obtain a different Ad@HAMA precursor solution. Example 4

[0028] The difference between this embodiment and embodiment 1 is only that 100 μL of IL-1β shRNA adenovirus stock solution with eGFP fluorescence is mixed with 1 mL of HAMA precursor solution to obtain a different Ad@HAMA precursor solution. Example 5

[0029] The difference between this embodiment and embodiment 1 is only that 150 μL of IL-1β shRNA adenovirus stock solution with eGFP fluorescence is mixed with 1 mL of HAMA precursor solution to obtain a different Ad@HAMA precursor solution. Example 6

[0030] The difference between this embodiment and embodiment 1 is only that 200 μL of IL-1β shRNA adenovirus stock solution with eGFP fluorescence is mixed with 1 mL of HAMA precursor solution to obtain a different Ad@HAMA precursor solution.

[0031] Comparative Example: The present comparative example provides a preparation method of HAMA microspheres, which comprises the following steps: 0.01 g of lyophilized hyaluronic acid methacrylic acid (HAMA) hydrogel (purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd., China) and 0.0025 g of Lrgacure2959 photoinitiator are dissolved in 1 mL of phosphate buffered saline (PBS solution) to obtain a HAMA precursor solution.

[0032] Then, the HAMA microspheres are prepared by using the oil-in-water microfluidic technology, specifically comprising: The continuous phase, the dispersed phase and the outlet on the microfluidic chip (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., China) are all connected with silica gel tubes, wherein the dispersed phase is connected with the Ad@HAMA precursor solution through the silica gel tube, and the continuous phase is connected with the paraffin oil solution through the silica gel tube.

[0033] The continuous / dispersed flow rate is adjusted to 2:45 μL / min, at this time, due to the shear force generated by the speed difference between the continuous phase and the dispersed phase, continuous monodisperse spherical materials will appear at the convergence. After the droplet size is uniform, it is placed in a beaker containing preloaded oil solution under light-proof conditions, and every 10-20 min, the beaker is taken out, irradiated with ultraviolet light, and then placed back in place. The collected microspheres after crosslinking are washed with 75% ethanol for three times, and with PBS for five times, and the PBS is replaced every 3 h, so as to remove the photoinitiator and oil, thereby obtaining the HAMA microspheres.

[0034] Firstly, to determine the suitable volume ratio of HAMA precursor solution and IL-1β shRNA adenovirus stock solution, the Ad@HAMA microspheres in the processes of Examples 1-6 were co-cultured with neutrophils, and 48 h later, DAPI dye was added to label the cell nucleus, and the cells were stained at 37℃ in the dark for 30 min, and then washed with PBS, and then observed and photographed under a fluorescence microscope, and semi-quantitative analysis was performed by ImageJ software. At the same time, a parallel group was set up, and fresh prepared trypan blue dye (Solarbio) was added, and the trypan blue exclusion cells were identified as living cells, and the swollen and blue-stained cells were dead cells, and after counting, the cell survival rate after infection was calculated, and the results are shown in Figure 2 .

[0035] Figure 2 A is the green fluorescence of GFP expression in neutrophils after 48 h of infection of Ad@HAMA microspheres of different examples, B is an illustration of the analysis of the overlap coefficient rate of the nucleus and eGFP fluorescence, and C is an illustration of the results of the trypan blue live cell exclusion experiment of neutrophils after 48 h of infection of Ad@HAMA microspheres of different examples. It can be seen that the fluorescence overlap coefficients of Example 5 and Example 6 are higher, which are 73.2±5.8% and 74.2±6.9% respectively, which indicates that the transfection efficiency of Example 5 and Example 6 is higher, while the fluorescence overlap coefficients of Examples 1-4 are lower, among which the fluorescence overlap coefficient of Example 4 is the highest, which is only 55.8±6.2%, indicating that the transfection efficiency of Examples 1-4 is weak, but the cell survival rate of Example 6 is the lowest, which is only 70.0±6.6%, so the suitable volume ratio of HAMA precursor solution and IL-1β shRNA adenovirus stock solution is 100-150:1.

[0036] Then, the Ad@HAMA microspheres in Example 5, the ES-Ad@HAMA functionalized hydrogel microspheres and the HAMA microspheres prepared by the comparative example were analyzed.

[0037] The ES-Ad@HAMA functionalized hydrogel microspheres in anhydrous ethanol were subjected to centrifugation (5000 rpm, 3 min), and the comparison before and after centrifugation is shown in Figure 3 A, and then the surface morphology of HAMA microspheres, Ad@HAMA microspheres and ES-Ad@HAMA functionalized hydrogel microspheres in water phase (deionized water) and oil phase (paraffin oil) was observed respectively using an inverted optical microscope, as shown in Figure 3As shown in Figure B, the ES-Ad@HAMA functionalized hydrogel microspheres are nearly transparent in deionized water and have good dispersibility. Only after centrifugation are the microspheres vaguely visible settling at the bottom of the EP tube. Under a bright field inverted optical microscope, the HAMA microspheres, Ad@HAMA microspheres, and ES-Ad@HAMA functionalized hydrogel microspheres are uniform in size, round, and morphologically stable in both aqueous and oil phases.

[0038] SEM scans were performed on HAMA microspheres, Ad@HAMA microspheres, and ES-Ad@HAMA functionalized hydrogel microspheres, and the particle size distribution of the ES-Ad@HAMA functionalized hydrogel microspheres was analyzed. Figure 4 As shown, under SEM, HAMA microspheres and Ad@HAMA microspheres exhibit the characteristic porous structure of hydrogels. The microspheres are generally uniformly elliptical. However, the ES-Ad@HAMA functionalized hydrogel microspheres have a shrunken elliptical shape due to the formation of amide bonds by bridging E-selectin. The average particle size of the ES-Ad@HAMA functionalized hydrogel microspheres is approximately 12.63±1.51µm, indicating that the ES-Ad@HAMA functionalized hydrogel microspheres meet the volume requirements for intrathecal injection.

[0039] EDS analysis was performed on the surfaces of Ad@HAMA microspheres and ES-Ad@HAMA functionalized hydrogel microspheres to analyze their elemental composition. Elemental mapping was also performed to compare the changes in chemical elements on the ES-Ad@HAMA surface. The results are as follows: Figure 5 As shown in A and B. Furthermore, Ad@HAMA microspheres and ES-Ad@HAMA functionalized hydrogel microspheres were mixed with Brk at a mass ratio of 1:100, thoroughly ground, and then pressed into tablets using a tablet press. The samples were then scanned using FTIR (scanning parameters: 128 scans; resolution 4 cm⁻¹). -1 Scanning range: 400~4000cm -1 ), the result is as follows Figure 5 As shown in Figure C, EDS analysis revealed that the proportions of N and S elements in Ad@HAMA microspheres were both 0.00%, while the proportions of N and S elements in ES-Ad@HAMA functionalized hydrogel microspheres were 3.67% and 6.05%, respectively. Furthermore, the mapping diagram of ES-Ad@HAMA functionalized hydrogel microspheres also supports this result. FTIR analysis indicated that the ES-Ad@HAMA functionalized hydrogel microspheres were only at a depth of 1680 cm⁻¹. -1 The peak at the point is slightly broadened, which is due to the formation of amide bonds consuming carboxyl groups. The formation of surface amide bonds effectively demonstrates the successful grafting of E-selectin.

[0040] Next, the in vitro protective immune microenvironment performance of the ES-Ad@HAMA functionalized hydrogel microspheres prepared in Example 5 was analyzed.

[0041] Non-targeted metabolomics sequencing was performed, and the specific operation included: being divided into a Control group (NETs and macrophages were simply co-cultured) and an ES-Ad@HAMA functionalized hydrogel microsphere group (a co-culture system was set: the macrophages were placed in the lower chamber, and the ES-Ad@HAMA functionalized hydrogel microspheres and NETs were placed in the upper chamber) two groups. Metabolomics liquid chromatography-mass spectrometry analysis was performed by BGI Genomics Co. The differential genes with a p value less than 0.05 and a difference fold greater than 1.2 were subjected to gene ontology (KEGG) differential transcript enrichment analysis to determine the biological functions or pathways mainly affected by the differential metabolites, and the results are shown in Figure 6 and Figure 7 .

[0042] In the non-targeted metabolomics results, the differences between groups were obvious, and the repeatability within groups was good ( Figure 6 ), and it can be seen that compared with the Control group, there were 118 down-regulated differential expression metabolites (DEMs) in the ES-Ad@HAMA functionalized hydrogel microsphere group, and 72 up-regulated DEMs ( Figure 7 A). The 190 statistically significant differential metabolites were subjected to KEGG enrichment analysis, and it was found that the central carbon metabolism and tricarboxylic acid (TCA) cycle were enriched ( Figure 7 B). Specifically, it can be seen that Figure 8 compared with the Control group, Glucose 6-Phosphate, AMP, Itaconic were down-regulated, and malate, Oxalosuccinic acid, 6-Phospho-gluconic acid were up-regulated in the ES-Ad@HAMA functionalized hydrogel microsphere group, which effectively explained that the ES-Ad@HAMA functionalized hydrogel microspheres can promote the tricarboxylic acid cycle and inhibit glycolysis metabolism ( Figure 8 B).

[0043] Then, the macrophage subtype of the ES-Ad@HAMA functionalized hydrogel microspheres prepared in Example 5 was identified by flow cytometry.

[0044] The three groups, Control group (simple macrophages), Nets group (macrophages / NETs) (Nets group), and ES-Ad@HAMA group (macrophages / NETs+ES-Ad@HAMA functionalized hydrogel microspheres), were each cultured for 24 hours, after which a cell suspension was prepared using a 0.25% trypsin solution or a cell scraper, transferred to a new EP tube, centrifuged to obtain the supernatant, and the cell fragments were discarded (operated on ice).

[0045] Meanwhile, on the other side, FBS (10%) was added to a PBS solution to prepare a flow antibody diluent / cell washing solution, after which the CD11b / APC and CD86 / FITC flow antibodies were diluted at a ratio of 1:200 to prepare a double-staining working solution (CD11b / APC and CD206 / FITC flow antibodies were also diluted at a ratio of 1:200 to prepare a double-staining working solution), 200 μL / tube of each different staining working solution was added to the above-mentioned EP tube, incubated at 4°C in the dark for 30 minutes, after which the staining was completed, the staining solution was removed by centrifugation at 1500 rpm for 5 minutes and washed 2-3 times with a cell washing solution; finally, the cells were resuspended with PBS and subjected to flow cytometry identification, the obtained data was analyzed and statistically processed using Flow Jo, and the results are shown in Figure 9 As shown in the figure, the expression of CD86+ / CD11b in the Nets group was 18.52±6.77% higher than that in the Control group, while the expression of CD206+ / CD11b was 7.7±2.52% lower, indicating that NETs promoted the M1 inflammatory polarization of macrophages, and with the addition of ES-Ad@HAMA, the expression of CD86+ / CD11b decreased by 24.04±6.35%, and the expression of CD206+ / CD11b increased by 11.32±4.47%, indicating that the functionalized microspheres could effectively inhibit the M1 inflammatory polarization of macrophages mediated by NETs.

[0046] Next, the Neu group (simple neutrophils), the Nets group (neutrophils+PMA), and the ES-Ad@HAMA group (neutrophils+PMA+material) were each cultured for 3 days, washed 3 times with a phosphate buffer, added with SYTOX® Green diluent (1:30000) to cover the cells, incubated in the dark for 15-30 minutes, and then observed under an inverted fluorescence microscope, and the results are shown in Figure 10 As shown in the figure, SYTOX® Green fluorescence staining showed that the ES-Ad@HAMA functionalized hydrogel microspheres had a significant inhibitory effect on NETosis (the process of forming Nets) in vitro, and the Neu group without any intervention had no NETs organization, the Nets group had a large amount of network organization due to the addition of PMA, and the network organization in the ES-Ad@HAMA group was significantly reduced due to the intervention of the material.

[0047] Finally, the in vivo effects of the Ad@HAMA microspheres, ES-Ad@HAMA functionalized hydrogel microspheres in Example 5, and the HAMA microspheres prepared in the comparative example on promoting nerve function repair were tested and analyzed.

[0048] Establishment of a mouse spinal cord contusion animal model C57 mice (100-150 g) were provided by Zhaoyan (Suzhou) New Drug Research Center Co., Ltd., and all surgeries and perioperative treatments were approved by the Ethics Committee of Suzhou University (SUDA20240306A02).

[0049] As shown in Figure 11 A, 8-10-week-old C57 mice were intraperitoneally injected with 0.6% sodium pentobarbital (50 mg·kg −1 ) for anesthesia. After successful anesthesia, all the hair on the back area was removed as much as possible using a shaving razor. The mice were placed in a prone position, and the back was disinfected with iodophor cotton balls 3 times. A longitudinal incision was made on the back centered at T10 (as small as possible to reduce trauma), and the paraspinal muscles were bluntly separated to completely expose the T10 lamina and spinous process. The spinous processes of T9, T10, and T11 and the bilateral lamina of T10 were cut to expose the spinal cord.

[0050] Then the mice were placed on the striking platform of the spinal cord injury striker, with the cannula perpendicular to the spinal cord directly above, a 10g weight was placed in the cannula, and the weight was placed at a height of 80mm from the spinal cord, and the T10 spinal cord area was aligned for free-fall impact. The wound was sutured, the drug was injected intrathecally, and the bladder was manually emptied once every 12 hours after the operation.

[0051] The sham operation group underwent simple laminectomy (Sham group), the blank control group underwent spinal injury surgery without implanting materials (Control group), and 100 μL of HAMA microspheres (HAMA group), Ad@HAMA microspheres (Ad@HAMA group), and ES-Ad@HAMA functionalized hydrogel microspheres (ES-Ad@HAMA group) were injected intrathecally once immediately after spinal cord injury. After anesthesia recovery (pay attention to keep warm), the mice were fed in separate cages (5-6 mice per cage), and 100 μL of ES-Ad@HAMA functionalized hydrogel microspheres were injected intrathecally once 3 days after spinal cord injury (ES-Ad@HAMA (3D) group). The animals were observed for food intake, defecation, and incision conditions daily in the morning and evening, and infection was prevented. Among them, the HAMA group, the Ad@HAMA group, the ES-Ad@HAMA group, and the ES-Ad@HAMA (3D) group are collectively referred to as the treatment group.

[0052] Motor function analysis: Basso Mouse Scale (BMS) motor test and footprint analysis were used to evaluate the recovery of motor function of mice after spinal cord injury. (BMS score: 0 points: complete absence of hind limb movement; 1 point: slight ankle movement. 2 points: obvious ankle movement, but no other joint movement; 3 points: knee and ankle movement, but no coordination; 4 points: mild coordinated movement, occasional foot walking; 5 points: moderate coordinated movement, frequent foot walking; 6 points: coordinated movement, continuous foot walking, but abnormal gait; 7 points: almost normal coordinated movement, gait close to normal; 8 points: normal coordinated movement, normal gait, but abnormal tail posture; 9 points: complete normal motor function), the results are as follows Figure 12 As shown in A, at the 8th week of the BMS score scale, the BMS score of the Control group was 4.7±1.15, the BMS score of the HAMA group was 5.0±1.0, the BMS score of the Ad@HAMA group was 6.7±1.5, and the BMS score of the ES-Ad@HAMA group was 8.3±1.2. The overall trend was Control group≈HAMA group<Ad@HAMA group<ES-Ad@HAMA group.

[0053] IPT experiment: The mouse was placed on a slope covered with a friction pad, the longitudinal axis was parallel to the longitudinal axis of the slope, the head was lifted to one side, and the angle was increased by 5° within 5 s. The highest angle of the mouse was recorded for 5 times. The larger the angle, the stronger the lower limb bearing capacity. The results are as follows Figure 12 As shown in B, in the IPT evaluation, the mouse can still maintain stability when the inclination angle is larger, indicating that the gripping force is improved, and the recovery of the lower limbs is better. The IPT score of the Control group was 41.7±10.4, the IPT score of the HAMA group was 41.7±10.4, the IPT score of the Ad@HAMA group was 40.0±5.0, and the IPT score of the ES-Ad@HAMA group was 58.3±2.9. The overall trend is consistent with the BMS score.

[0054] In summary, through the analysis of motor function, it can be known that the recovery of the lower limbs of the mice treated by ES-Ad@HAMA functional hydrogel microspheres is the best, and basically can achieve normal coordinated movement and normal gait.

[0055] Animal specimen sampling At a specific postoperative time node, 0.6% pentobarbital (50 mg·kg −1After anesthesia of mice, open the abdominal cavity, and cut the sternum with surgical scissors to fully expose the heart; cut off the inferior vena cava, and use 0.9% normal saline to inject the heart for perfusion; cut off the spinal column with a length of 1 cm above and below the center of the spinal cord T10 (or take out the heart, liver, spleen, lung, kidney, and bladder completely), and put it into 4% paraformaldehyde; after 1 week, take out the spinal column, carefully remove the bone and muscle using ophthalmic scissors to obtain a spinal cord specimen with a length of about 5 mm; the spinal cord specimens of mice at 7 days, 4 weeks, and 8 weeks after surgery are as shown in FIG. 2B. Figure 11 B.

[0056] HE staining Postoperative 1-week and 4-week heart, liver, spleen, lung, and kidney specimen processing flow: Fix with 10% formalin for 24 hours; perform gradient alcohol dehydration, and dehydrate the fixed heart, liver, spleen, lung, and kidney specimens using 70%, 80%, 90%, 95%, and 100% ethanol, respectively, for 1 or 2 hours at each level. Then, soak in xylene for 2 times, each for 1 hour, to facilitate paraffin penetration of the specimen.

[0057] Put the specimen after xylene soaking into 60°C molten paraffin for 3 hours until the specimen is cooled and solidified in the paraffin to form a wax block. Take out the embedding box, and use a microtome to perform longitudinal 4-6 μm thick sectioning of the paraffin block, and attach it to a glass slide. After sectioning, place the section attached to the glass slide in deionized water at 45°C, and bake in a 60°C constant-temperature oven for 60 minutes to firmly attach the section.

[0058] Again, deparaffinize the baked section by immersing it in pure xylene solution, and then dehydrate it using 70%, 80%, 90%, 95%, and 100% ethanol, respectively, for 1 or 2 minutes at each level. Then, wash with deionized water for 1-2 minutes to prepare for staining.

[0059] After treating the section with 0.3% hydrogen peroxide methanol solution for 4 minutes, perform non-specific antigen blocking. Add DAB color developing solution and develop for 10 minutes, and then wash with deionized water. Immerse in hematoxylin solution for 5-10 minutes (nuclear staining), and then wash with running water for 1-2 minutes to remove the floating color. Immerse in 1% hydrochloric acid alcohol prepared with 70% alcohol for 10 seconds to remove cytoplasm non-specific staining, and then wash with running water for 15-30 minutes (or an alkaline solution such as ammonia water) to make the cell nucleus blue. Then, immerse in 0.5%-1% eosin Y solution for 30 seconds-2 minutes for cytoplasm staining, and then wash with distilled water for 2 minutes to stop the staining.

[0060] Finally, dehydrate the section using 70%, 80%, 90%, 95%, and 100% ethanol, respectively, for 1 or 2 minutes at each level, xylene for 2 minutes, and then use neutral resin to mount the section. After sufficient drying, observe and take pictures under a bright field microscope to evaluate the biocompatibility of the material and whether there is obvious toxicity. The results are as shown in FIG. 2B.Figure 13 As shown in the figure, the HAMA microspheres, Ad@HAMA microspheres and ES-Ad@HAMA functionalized hydrogel microspheres all have good tissue compatibility and do not cause systemic toxicity.

[0061] Immunofluorescence staining Postoperative 7 days, 4 weeks and 8 weeks spinal cord specimen processing procedure: Fixed with 10% formalin for 24 hours, gradient alcohol dehydration, and the fixed spinal cord specimens were dehydrated with 70%, 80%, 90%, 95% and 100% ethanol for 1 or 2 hours each. Then, the specimens were soaked in xylene for 2 times, 1 hour each time, to facilitate paraffin penetration. The specimens after xylene soaking were placed in 60°C molten paraffin for 3 hours until the specimens were cooled and solidified in paraffin to form a wax block.

[0062] Remove the embedding box and use a microtome to make 4-6 μm thick sections of the paraffin block longitudinally (7 days after surgery, the specimen is transversely taken). Attach the sections to a glass slide. After sectioning, place the sections attached to the glass slide in deionized water at 45°C and bake in a constant temperature oven at 60°C for 60 minutes to firmly attach the sections.

[0063] After baking, the sections are again immersed in pure xylene solution for deparaffinization, and then dehydrated with 70%, 80%, 90%, 95% and 100% ethanol for 1 or 2 minutes each. Then, deionized water is used for 1-2 minutes to prepare for staining.

[0064] After applying the 0.3% hydrogen peroxide methanol solution to the sections, treat with the antigen repair solution for 4 minutes, then perform non-specific antigen blocking. Then, use IL-1β (Abeam, ab254360), CitH3 (Abeam, ab5103), MPO (Abeam, ab208670), GFAP (Abeam, ab7260), NG2 (Abeam, ab275024), Tuj-1 (Proteintech, 66375-1-LG), GAP-43 (Abeam, ab75810) for specific labeling, and then add the corresponding secondary antibody for fluorescent labeling in each specimen. After mounting, observe and take pictures under a fluorescence inverted microscope to evaluate the effects of the materials on the inflammatory microenvironment, fibrous scar, glial scar, neuron differentiation and neovascularization after spinal cord injury.

[0065] The results are shown in Figures 14-17 , wherein Figure 14A is the expression of IL-1β, B is the expression of CitH3, and MPO in vivo within 7 days after spinal cord injury. C and D are the results of fluorescence semi-quantitative analysis of IL-1β and CitH3 at 7 days after spinal cord injury. It can be seen that the silencing effect of ES-Ad@HAMA on IL-1β is the most significant, and the fluorescence semi-quantitative expression is 76.73±7.16. The silencing effect of the ES-Ad@HAMA (3D) group, which is the group injected with the material 3 days after the operation, is stronger than that of the Control group, and the silencing effect is weaker than that of ES-Ad@HAMA. The fluorescence semi-quantitative expression is 120.0±10.75. This is because the activation climax of neutrophils is 24-48h after injury, and ES-Ad@HAMA (3D) is injected 72h after the operation, thus missing the best treatment window. Immunofluorescence staining of CitH3 and MPO shows that the inhibitory effect of ES-Ad@HAMA on NETs is also the most significant, and the fluorescence semi-quantitative expression of CitH3 is 27.51±2.78, which is stronger than that of the Control group. As mentioned earlier, the ES-Ad@HAMA (3D) group also missed the activation climax of neutrophils, and the inhibitory effect on NETs was lacking.

[0066] Figure 15 A is the expression of Fibronectin, B is the expression of Col-I, and C is the expression of α-SMA in vivo at 4 weeks and 8 weeks after spinal cord injury. D-I are the results of fluorescence semi-quantitative analysis of Fibronectin, Col-I, and α-SMA at 4 weeks and 8 weeks after spinal cord injury. Figure 16 A is the expression of NG2, and B is the expression of GFAP in vivo at 4 weeks and 8 weeks after spinal cord injury. C-F are the results of fluorescence semi-quantitative analysis of GFAP and NG2 at 4 weeks and 8 weeks after spinal cord injury. It can be seen that compared with the Control group, the scar proliferation of all treatment groups at 4 weeks and 8 weeks after spinal cord injury decreased to varying degrees, but the scar proliferation of the HAMA group and the Ad@HAMA group was still at a high level. The fluorescence signal of scar markers of the ES-Ad@HAMA group at the two time nodes was the lowest, and the formation of scar tissue was significantly inhibited. There was a significant difference between the Ad@HAMA group and the ES-Ad@HAMA group (statistically significant, P<0.05).

[0067] Figure 17In vivo expression of Tuj-1 and GAP-43 at 4 weeks and 8 weeks after spinal cord injury, and fluorescence semi-quantitative analysis of Tuj-1 and GAP-43 at 4 weeks and 8 weeks after spinal cord injury. Tuj-1 and GAP-43 mark neuron regeneration and axon sprouting, respectively, and are used to evaluate the ability of ES-Ad@HAMA functionalized hydrogel microspheres to promote neural regeneration in the middle and late stages of spinal cord injury repair. At 4 weeks and 8 weeks after spinal cord injury, a large number of Tuj-1 labeled neurons appeared in the ES-Ad@HAMA group, which was significantly better than the control group, the HAMA group and the Ad@HAMA group (p<0.001), which proved that the homing of neural progenitor cells made the local injury in the ES-Ad@HAMA group have more progenitor cells to differentiate into neurons, and had the function of continuously promoting neural regeneration. At the same time, the fluorescence density of GAP-43 in the ES-Ad@HAMA group was significantly higher than that in the other groups, which proved that its regulation of inflammation during acute spinal cord injury can promote neural regeneration for a long time. It is worth mentioning that the expression of Tuj-1 and GAP-43 in the ES-Ad@HAMA group was also stronger than that in the HAMA group and the Ad@HAMA group at the same time point, which showed the advantage of the targeting of functionalized microspheres.

[0068] In summary, the ES-Ad@HAMA functionalized hydrogel microspheres are uniform in size and stable in shape, with a size of about 10 µm, which meets the conditions for intrathecal systemic treatment, and the combination of Figure 1 As shown in B, the ES-Ad@HAMA functionalized hydrogel microspheres can inhibit IL-1β activity and play a role in inhibiting the molecular regulation axis of (IL-1β-MAPK-NETs)-NFκB-IL-1β during the release of NETs. In biological function research, ES-Ad@HAMA can reprogram the metabolic environment of macrophages, inhibit glycolysis, and inhibit Netosis. In in vivo research, the ES-Ad@HAMA treatment group can silence IL-1β in the local injury, inhibit Netosis, and thus reduce astrocyte activation and fibrous scar tissue formation, increase neural progenitor cell homing, promote axon regeneration and blood vessel growth, and thus promote the recovery of neural motor function after implanting into a mouse spinal cord contusion model.

[0069] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and modifications without departing from the technical principles of the present application, and these improvements and modifications should also be considered within the protection scope of the present application.

Claims

1. A method for preparing functionalized hydrogel microspheres, characterized by, The application relates to a functionalized hydrogel microsphere and a preparation method thereof. The hyaluronic acid methacrylic acid and a photoinitiator are dissolved in a phosphate buffered saline solution to obtain a hyaluronic acid methacrylic acid precursor solution; The hyaluronic acid methacrylic acid precursor solution is mixed with an IL-1beta shRNA adenovirus stock solution to obtain an Ad@HAMA precursor solution; The Ad@HAMA precursor solution is prepared into Ad@HAMA microspheres through an oil-in-water microfluidic technique; The Ad@HAMA microspheres, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide are sequentially added into a 2-morpholinoethanesulfonic acid buffer solution, and E-selectin is added after activation treatment, and the functionalized hydrogel microspheres are obtained after incubation.

2. The method for preparing functionalized hydrogel microspheres according to claim 1, characterized in that, The concentration of the hyaluronic acid methacrylic acid and the photoinitiator in the phosphate buffered saline solution ranges from 10:2 to 4 g / L.

3. The method for preparing functionalized hydrogel microspheres according to claim 1, characterized in that, The photoinitiator is 2,4,6-trimethylbenzoyl or Lrgacure2959.

4. The method for preparing functionalized hydrogel microspheres according to claim 1, characterized in that, The volume ratio of the IL-1beta shRNA adenovirus stock solution to the hyaluronic acid methacrylic acid precursor solution is 100-150 muL:1 mL.

5. The method for preparing functionalized hydrogel microspheres according to claim 1, characterized in that, The Ad@HAMA precursor solution is prepared into Ad@HAMA microspheres through an oil-in-water microfluidic technique, and the method comprises the following steps: The Ad@HAMA precursor solution is introduced into a dispersed phase of a microfluidic chip, paraffin oil solution is introduced into a continuous phase of the microfluidic chip, and continuous monodisperse spherical droplets are obtained from an outlet after convergence; The monodisperse spherical droplets are placed in preloaded oil, and are irradiated with ultraviolet light to realize a photo-crosslinking reaction; and the Ad@HAMA microspheres are obtained after washing when the photo-crosslinking reaction is completed.

6. The method of claim 5, wherein the functionalized hydrogel microspheres are prepared by the method comprising the steps of: The flow rate ratio of the continuous phase to the dispersed phase in the microfluidic chip ranges from 2 to 4:30 to 90 muL / min; And / or, the ultraviolet light irradiation is performed every 10-20 min until the photo-crosslinking reaction is completed.

7. The method for preparing functionalized hydrogel microspheres according to claim 1, characterized in that, The mass ratio of the Ad@HAMA microspheres, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, 2-morpholinoethanesulfonic acid buffer solution and E-selectin ranges from 100:16:24:2:3 to 5, wherein the pH of the 2-morpholinoethanesulfonic acid buffer solution ranges from 5.5 to 6.

7.

8. The method for preparing functionalized hydrogel microspheres according to claim 1, characterized in that, The activation treatment comprises activation in a 37 DEG C constant-temperature shaking table for 10-20 min; And / or, the incubation comprises overnight incubation in a 37 DEG C constant-temperature shaking table.

9. Functionalized hydrogel microspheres characterized in that, The functionalized hydrogel microspheres are prepared through the preparation method.

10. Use of functionalized hydrogel microspheres according to claim 9, characterized in that, The functionalized hydrogel microspheres are prepared through the preparation method. The functionalized hydrogel microspheres are prepared through the preparation method.

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