Hydrogel microspheres for modulating senescent microenvironment and preparation method and application thereof

Abstract

The application discloses a hydrogel microsphere for adjusting a senescent microenvironment and a preparation method and application thereof, wherein the hydrogel microsphere is obtained by mixing hollow mesoporous silica IL37@HMS loaded with recombinant protein IL37 and nanoparticles Man-4OI-PLGA modified by mannose and loaded with 4OI when preparing a pH-responsive hydrogel microsphere. The hydrogel can dissociate in an acidic microenvironment of degenerative tissue and release the carried drugs, can effectively protect the biological activity of IL37, realize slow release of the drugs, significantly improve the residence time and treatment efficiency of IL37 at a lesion site, can effectively protect the stability of itaconic acid in the body, and realize precise release of the itaconic acid at the lesion site, and greatly improve the enrichment efficiency of the itaconic acid in macrophages. The hydrogel meets the special requirements of small space, high pressure, mechanical strength, biocompatibility and controllable drug release of an intervertebral disc, and has important significance for treatment of degenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology. It relates to a hydrogel microsphere and its preparation method, and more specifically, to a hydrogel microsphere for regulating the aging microenvironment, its preparation method, and its application. This hydrogel microsphere can be used to treat degenerative diseases such as intervertebral disc degeneration. Background Technology

[0002] The aging microenvironment is a complex system composed of various pro-inflammatory factors, inflammatory mediators, and intricate intercellular signaling networks, playing a crucial role in the development of degenerative diseases, particularly in the field of intervertebral disc degeneration (IVDD). Within the aging microenvironment, nucleus pulposus cells produce senescence-associated secretory phenotypes (SASPs), which include pro-inflammatory cytokines such as IL-6 and IL-8, as well as matrix proteolytic enzymes such as MMPs. These biologically active secretory products induce senescence in neighboring cells through paracrine effects, activate inflammatory pathways, promote extracellular matrix degradation, disrupt the homeostasis of the intervertebral disc, and accelerate the degenerative process. Simultaneously, inflammatory macrophages continuously secrete pro-inflammatory factors such as IL-6, TNFα, and IL-1β, further exacerbating the local inflammatory response, inducing nucleus pulposus cell senescence, and creating a vicious cycle that collectively drives damage to the structure and function of the intervertebral disc. However, the methods for reconstructing the aging microenvironment in IVDD treatment remain unclear and require further investigation.

[0003] In recent years, despite some progress in the treatment of IVDD, single-target therapy has struggled to fundamentally reverse the abnormal state of the aging microenvironment. While recombinant protein IL37 has been preliminarily shown to slow IVDD progression by inhibiting SASP, directly injected IL37 is rapidly degraded in vivo, severely limiting its therapeutic potential. Meanwhile, its metabolite itaconic acid (4OI), as an endogenous metabolic regulator, has attracted considerable attention. Previous studies have confirmed that itaconic acid can regulate macrophage metabolism, promoting the transformation of inflammatory macrophages into anti-inflammatory and repair-oriented macrophages, effectively inhibiting inflammatory responses and improving the local microenvironment. However, itaconic acid is easily and non-specifically taken up in vivo, resulting in low accumulation efficiency in macrophages, significantly limiting its therapeutic effect. Therefore, finding efficient delivery carriers to enhance the therapeutic efficacy of itaconic acid and IL37 has become crucial for overcoming the challenges of IVDD treatment.

[0004] In the field of biomedical materials, although nanocarriers and hydrogel microspheres have shown great potential in the treatment of various diseases, there are still few reports on how to effectively combine the two to build a synergistic treatment system for regulating the aging microenvironment and repairing degenerated intervertebral discs.

[0005] Based on this, this invention explores a novel IVDD treatment strategy that uses responsive hydrogel microsphere carriers to synergistically inhibit senescent nucleus pulposus cells (SASPs) and reprogrammed macrophages. This is of great significance for overcoming the challenges of IVDD treatment and providing innovative solutions for the repair of degenerated tissues. Summary of the Invention

[0006] This invention targets the complex pathological mechanisms of the aging microenvironment in intervertebral disc degeneration (IVDD) by designing responsive hydrogel microspheres. These microspheres synergistically inhibit the senescence-associated secretory phenotype (SASP) of nucleus pulposus cells and reprogram macrophages, thereby reconstructing the aging microenvironment. After improving the intervertebral disc microenvironment, these responsive hydrogel microspheres enable normal nucleus pulposus cells to escape the adverse effects of senescent cells and inflammatory macrophages, allowing them to secrete extracellular matrix normally and restore the weight-bearing and stress-buffering functions of the intervertebral disc, providing a novel strategy for IVDD treatment.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] The first aspect of this invention discloses a method for preparing hydrogel microspheres for regulating the aging microenvironment, comprising:

[0009] S1: Preparation of hollow mesoporous silica (IL37@HMS) encapsulating recombinant protein IL37;

[0010] S2: Preparation of PLGA nanoparticles loaded with 4OI (4OI-PLGA);

[0011] S3: Modify mannose with 4OI-PLGA nanoparticles (Man-4OI-PLGA);

[0012] S4: Prepare pH-responsive hydrogel microspheres, and mix IL37@HMS and Man-4OI-PLGA during the preparation of hydrogel microspheres to obtain pH-responsive hydrogel microspheres (IL37-4OI@MS) loaded with IL37@HMS and Man-4OI-PLGA.

[0013] Further, in S1, ethanol, distilled water, and ammonia were first mixed, and TEOS and anhydrous ethanol were added and stirred to react. After centrifugation and washing, solid silica spheres (sSiO2) were obtained. Then, sSiO2 was dispersed in a solution containing CTAC, distilled water, triethanolamine, and anhydrous ethanol, and 1,3,5-trimethylbenzene and TEOS were added dropwise to form a CTAC-SiO2@sSiO2 composite structure. Then, anhydrous sodium carbonate was added to etch the internal sSiO2 to form cavities. Finally, the template agent CTAC and 1,3,5-trimethylbenzene were removed with hydrochloric acid, and small-particle-size hollow mesoporous silica (HMS) was obtained after centrifugation, washing, and drying. The HMS obtained above was dispersed in PBS, IL37 dissolved in PBS was added, and the mixture was incubated overnight at 4°C on a shaker. After high-speed centrifugation and washing three times with pure water, hollow mesoporous silica (IL37@HMS) loaded with recombinant protein IL37 was obtained.

[0014] Furthermore, in S2, PLGA-COOH and 4OI were fully dissolved in chloroform, then added to a PVA solution. After ultrasonic reaction, mechanical stirring, ultrafiltration washing, and concentration were performed to obtain PLGA nanoparticles loaded with 4OI (4OI-PLGA).

[0015] Furthermore, in S3, 4OI-PLGA was dispersed in MES buffer solution, EDC and NHS were added, and the mixture was reacted in a shaker at 37°C. The solution was adjusted to a weakly alkaline state, mannosamine was added, and the mixture was reacted in a shaker at 37°C overnight. The mixture was then washed by ultrafiltration and finally redissolved in pure water.

[0016] Furthermore, in S4, the preparation of hydrogel microspheres is divided into two parts: microsphere preparation and demulsification and washing. During microsphere preparation, droplet-generating oil and aqueous solution are added to the oil phase and aqueous phase reservoirs, respectively. The air compressor and air source treatment device are turned on to expel air from the pipeline and chip. The flow rate control mode is switched, the flow rate is adjusted, and the uniformity of the droplets is observed. Uniform droplets are collected into centrifuge tubes. During demulsification and washing, the droplet-generating oil at the bottom of the centrifuge tube is removed. A demulsifier is added, the microspheres are shaken to demulsify, and the demulsifier is removed by centrifugation. After repeating the operation, the microspheres are washed multiple times with PBS buffer to obtain solidified transparent microspheres dispersed in PBS buffer. If there is residual oil emulsion, it can be removed by filtration through a cell sieve or by centrifugation and removal of the aqueous phase containing the microspheres from the top.

[0017] The aqueous phase consisted of 30% (w / ml) gelatin, 10 mg / ml IL37@HMS, 10 mg / ml Man-4OI-PLGA, and 20 mg / ml aldehyde-modified hyaluronic acid uniformly dispersed in PBS; the oil phase consisted of 5% (w / ml) Span80 dissolved in paraffin oil. Both the aqueous and oil phases were simultaneously introduced into a microfluidic device, and the flow rates of the aqueous and oil phases were precisely controlled by a syringe pump, set to 10 μL / min and 75 μL / min, respectively. The flow rates were rapidly adjusted to the preset values ​​and maintained at a stable output. A small amount of the emulsion was collected in a hydrophobic culture dish, and the uniformity of the droplets was observed under an optical microscope. After confirming uniformity, the emulsion was collected into a transparent centrifuge tube.

[0018] The second aspect of this invention discloses two types of nanoparticles prepared by the above method, namely IL37@HMS and Man-4OI-PLGA. The particle size of IL37@HMS is typically 80-120 nm, and the particle size of Man-4OI-PLGA is typically 100-150 nm.

[0019] The third aspect of this invention discloses hydrogel microspheres (IL37-4OI@MS) prepared by the above method. Their particle size is typically 50-70 μm.

[0020] The fourth aspect of this invention discloses the application of hydrogel microspheres prepared by the above method in reconstructing the aging microenvironment and treating degenerative diseases such as intervertebral disc degeneration.

[0021] Inspired by the pathological changes induced by the interaction between SASP cells and inflammatory macrophages in the IVDD aging microenvironment, this invention constructs a responsive hydrogel microsphere synergistic therapeutic system. Through a series of cell experiments and animal studies, it was found that these hydrogel microspheres, in the slightly acidic environment of intervertebral disc degeneration, can release internally loaded drugs through responsive cleavage of Schiff base bonds. Specifically, IL37 loaded with hollow mesoporous silica can be slowly released, inhibiting the progression of SASP cells; mannose-modified PLGA targeting macrophages can precisely deliver itaconic acid, thereby achieving macrophage phenotypic reprogramming, effectively blocking the vicious cycle of aging and inflammation, stabilizing the extracellular matrix, and promoting the repair of degenerated tissues.

[0022] The beneficial effects of this invention are as follows:

[0023] (1) This invention designs hydrogel microspheres with pH-responsive Schiff base bonds, which can specifically rupture in the slightly acidic environment of aging intervertebral discs, accurately release the internal drug load, and achieve targeted drug delivery to the lesion site, effectively solving the problem of lack of environmental responsiveness in traditional drug delivery methods.

[0024] (2) To address the problem of short half-life and easy inactivation of recombinant protein IL37 by direct injection, this invention utilizes hollow silica nanoparticles (HMS) as a carrier. With its good biocompatibility, high specific surface area and tunable pore structure, it can not only effectively protect the biological activity of IL37, but also achieve slow drug release, significantly improve the retention time and treatment efficiency of IL37 at the intervertebral disc lesion site, more effectively inhibit SASP of nucleus pulposus cells, and delay the progression of IVDD.

[0025] (3) To address the problem that itaconic acid is easily taken up non-specifically and has low accumulation efficiency, this invention uses a mannose-modified polylactic acid-glycolic acid copolymer (PLGA) nanocarrier system, which can effectively protect the stability of itaconic acid in vivo and achieve its precise release at the lesion site, greatly improve the accumulation efficiency of itaconic acid in macrophages, more effectively induce macrophages to transform from inflammatory type to anti-inflammatory repair type, inhibit inflammatory response, and improve the local aging microenvironment.

[0026] (4) The responsive hydrogel microsphere carrier system constructed in this invention can synergistically inhibit the SASP of senescent nucleus pulposus cells and reprogrammed macrophages, break the vicious cycle of aging and inflammation, and successfully reconstruct the aging microenvironment, providing an innovative and effective solution for the treatment of degenerative diseases such as intervertebral disc degeneration. Attached Figure Description

[0027] Figure 1 Preparation and characterization of responsive microspheres for reconstructing the aging microenvironment. (A) Schematic diagram of responsive microsphere preparation: The process of preparing responsive hydrogel microspheres (IL37-OI@MS) loaded with IL37@HMS and Man-4OI-PLGA using microfluidic technology. (B) Transmission electron microscopy (TEM) images of HMS and IL37@HMS. (CD) Transmission electron microscopy (TEM) images and particle size distribution maps of PLGA, 4OI-PLGA, and Man-4OI-PLGA. (E) Scanning electron microscopy (SEM) images and particle size distribution maps of MS, OI@MS, IL37@MS, and IL37-OI@MS. (F) Elemental distribution map (C, O, N, S, Si, Se) of IL37-OI@MS.

[0028] Figure 2The responsive microspheres inhibited SASP in nucleus pulposus cells by slow-release IL37. Nucleus pulposus cell senescence was promoted in vitro by co-incubation with IL-1β, and their anti-SASP ability was evaluated by co-incubation with different microspheres. (A) Schematic diagram showing the process by which IL37-OI@MS microspheres responsively release IL37 in an acidic environment (decreased pH), thereby inhibiting SASP in nucleus pulposus cells. (BC) Western blot results of senescence-related P21 and P16 proteins in different treatment groups (Control group, IL-1β group, IL-1β+MS group, IL-1β+OI@MS group, IL-1β+IL37@MS group, IL-1β+IL37-OI@MS group), with relative quantification analysis. (DE) Staining of senescence-related SA-β-gal in different treatment groups, with relative quantification analysis.

[0029] (F) The relative levels of mRNA expression of pro-inflammatory cytokines (such as IL6 and IL8) and matrix proteolytic enzymes (such as Mmp3 and Mmp13) secreted by senescent nucleus pulposus cells in different treatment groups.

[0030] Figure 3 The responsive microspheres reprogrammed macrophages by supplementing them with itaconic acid. (A) Schematic diagram showing the process by which IL37-OI@MS microspheres responsively release Man-4OI-PLGA in an acidic environment (decreased pH), supplementing macrophages with itaconic acid and thus reprogramming them. (B) Relative levels of mRNA expression of inflammatory factors (IL1b, Tnfa, Nos2, IL6) in different treatment groups. (C) ELISA results showing the protein level of inflammatory factor (IL6) secreted by macrophages in different treatment groups. (DE) Immunofluorescence staining and quantitative analysis of relative fluorescence intensity of CD86, a marker on the surface of inflammatory macrophages, in different treatment groups.

[0031] Figure 4To delay the progression of intervertebral disc degeneration (IVDD) in rats using responsive microspheres. (A) Schematic diagram of the experimental procedure: Rats were first acclimatized for 3 days, followed by an IVDD puncture model and treatment with different microspheres. Imaging and histological evaluations were performed 8 weeks later. (BC) X-ray images of different treatment groups (Sham as control group, IVDD as model group, IVDD+MS, IVDD+OI@MS, IVDD+IL37@MS, and IVDD+IL37-OI@MS as intervention groups), and the degree of intervertebral disc degeneration in each treatment group was assessed by different intervertebral disc heights. (DE) MRI images of different treatment groups (Sham group, IVDD group, IVDD+MS group, IVDD+OI@MS group, IVDD+IL37@MS group, and IVDD+IL37-OI@MS group), and the degree of intervertebral disc degeneration in each treatment group was assessed by Pfirmann grading. (FG) Histological staining (HE, SO, and alicin blue staining) of different treatment groups to observe morphological changes in intervertebral disc tissue.

[0032] Figure 5 For the in vivo biosafety assessment of responsive microspheres. (AC) CCK8 results of co-incubation of MS, OI@MS, IL37@MS, and IL37-OI@MS with nucleus pulposus cells for 1, 3, and 5 days, respectively. (D) Histological sections (H&E staining) of major organs (heart, liver, spleen, lung, and kidney) in vivo from the Sham group, MS group, OI@MS group, IL37@MS group, and IL37-OI@MS group. Detailed Implementation

[0033] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited to the described embodiments.

[0034] Example 1: Preparation of hollow mesoporous silica (IL37@HMS) encapsulating recombinant protein IL37

[0035] 1. Synthesis of Hollow Mesoporous Silica

[0036] Small-particle-size hollow mesoporous silica (HMS) was synthesized through a multi-step reaction. First, ethanol, distilled water, and ammonia were mixed, followed by the addition of TEOS and anhydrous ethanol. After stirring and centrifugation, solid silica spheres (sSiO2) were obtained. Next, sSiO2 was dispersed in a solution containing CTAC, distilled water, triethanolamine, and anhydrous ethanol. 1,3,5-trimethylbenzene and TEOS were then added dropwise to form a CTAC-SiO2@sSiO2 composite structure. Then, anhydrous sodium carbonate was added to etch the internal sSiO2, creating a cavity structure. Finally, the template agent CTAC and 1,3,5-trimethylbenzene were removed with hydrochloric acid, and the resulting HMS particles were obtained after centrifugation, washing, and drying.

[0037] 2. Carrying IL37

[0038] The synthesized HMS was dispersed in PBS, and IL37 dissolved in PBS was added. The mixture was incubated overnight at 4°C on a shaker. After high-speed centrifugation, the silica was washed three times with pure water to obtain hollow mesoporous silica (IL37@HMS) encapsulating recombinant protein IL37.

[0039] 3. Morphological and structural characterization

[0040] The morphology of IL37@HMS was observed using transmission electron microscopy (TEM), and the results showed that IL37 was successfully loaded into HMS. Figure 1 B).

[0041] Example 2: Preparation of 4OI-PLGA nanoparticles modified mannose (Man-4OI-PLGA)

[0042] 1. PLGA with 4OI

[0043] Dissolve 50 mg PLGA-COOH and 10 mg 4OI in 5 mL chloroform, add 50 mL 1% PVA solution, sonicate for 5 minutes, mechanically stir for 4 hours, wash 3 times with an ultrafiltration tube, and concentrate for later use.

[0044] 2.4OI@PLGA modified mannose

[0045] PLGA was dispersed in MES buffer solution, EDC and NHS were added, and the mixture was reacted at 37°C for 30 minutes. The solution was adjusted to a weakly alkaline state with borax-boric acid buffer solution, and mannosamine was added. The mixture was reacted overnight at 37°C. Finally, the mixture was washed three times with an ultrafiltration tube and reconstituted in pure water.

[0046] 3. Morphological and structural characterization

[0047] The particle size and morphology of PLGA were further observed using transmission electron microscopy (TEM) and dynamic light scattering (DLS), and the results showed that the morphology was relatively uniform. Figure 1 CD).

[0048] Example 3: Preparation of pH-responsive hydrogel microspheres loaded with IL37@HMS and Man-4OI-PLGA

[0049] 1. Synthesis of hydrogel microspheres

[0050] In the microsphere preparation process, the droplet-generating oil and aqueous solution were added to the oil phase and aqueous phase storage tanks, respectively. After air was purged, the flow rate control mode was switched on, the flow rate was adjusted, and the uniformity of the droplets was observed. Finally, uniform droplets were collected. In the demulsification and washing process, the droplet-generating oil was removed, a demulsifier was added, the mixture was shaken to demulsify, and the demulsifier was removed by centrifugation. The microspheres were washed multiple times with PBS buffer to finally obtain solidified transparent microspheres.

[0051] 2. Loaded with IL37@HMS and Man-4OI-PLGA

[0052] The aqueous phase consisted of 30% gelatin, 10 mg / mL IL37@HMS, 10 mg / mL Man-4OI-PLGA, and 20 mg / mL aldehyde-modified hyaluronic acid; the oil phase consisted of 5% Span80. The emulsion was collected in centrifuge tubes by precisely controlling the flow rate using a microfluidic device.

[0053] 3. Morphological and structural characterization

[0054] The morphology of the hydrogel microspheres was observed using field emission scanning electron microscopy (FE-SEM), and the particle size distribution was determined by dynamic light scattering (DLS). The results showed that the microspheres were uniform in size. Figure 1 E). Chemical composition analysis by X-ray diffraction (XRD) confirmed the successful encapsulation of IL37@HMS and Man-4OI-PLGA. Figure 1 F).

[0055] Example 4: Responsive microspheres inhibit SASP in nucleus pulposus cells by sustained release of IL37

[0056] 1. In vitro co-culture

[0057] Nucleus pulposus cells were induced to senescence by co-incubation with IL-1β, followed by co-incubation with different microspheres to evaluate their anti-aging capabilities. Figure 2 A).

[0058] 2. Western blot detection of aging-related proteins

[0059] Total protein was extracted from nucleus pulposus cells, lysed by RIPA, centrifuged, and quantified. After denaturation, protein samples were separated by SDS-PAGE, transferred to PVDF membranes, blocked, and incubated sequentially with primary and secondary antibodies before final detection. Results showed that IL-1β-induced expression of P21 and P16 proteins significantly increased, while the IL37@MS and IL37-OI@MS groups significantly reversed this trend. Figure 2 BC).

[0060] 3. SA-β-gal detection of senescent cells

[0061] Staining nucleus pulposus cells with the SA-β-gal staining kit showed that the proportion of senescent cells in the IL37-OI@MS group was significantly lower than that in the IL-1β group. Figure 2 DE).

[0062] 4. qPCR detection of inflammatory factors and matrix proteolytic enzymes

[0063] Total RNA was extracted from nucleus pulposus cells and cDNA was synthesized. Relative gene levels were detected by qPCR. Results showed that IL-1β-induced expression of inflammatory cytokines and matrix proteolytic enzymes was significantly increased, while the expression levels in the IL37-OI@MS group were significantly decreased. Figure 2 F).

[0064] Example 5: Responsive microspheres reprogram macrophages by supplementing them with itaconic acid. 1. In vitro co-culture

[0065] Macrophages were co-incubated with LPS to induce an inflammatory phenotype, and then co-incubated with different microspheres to evaluate their anti-inflammatory capabilities. Figure 3 A).

[0066] 2. qPCR detection of inflammatory phenotypes

[0067] Total RNA was extracted from macrophages and cDNA was synthesized. Relative gene levels were detected by qPCR. Results showed that LPS-induced expression of inflammation-related genes was significantly increased, while this trend was significantly reversed in the IL37-OI@MS group. Figure 3 B).

[0068] 3. ELISA detection of inflammatory factors

[0069] Cell culture supernatant was collected, and after centrifugation to remove impurities, IL6 levels were detected using an ELISA kit. Results showed that the IL6 protein level in the IL37-OI@MS group was significantly lower than that in the LPS group (…). Figure 3 C).

[0070] 4. Detection of inflammatory phenotypes by cellular immunofluorescence

[0071] Macrophages were fixed, permeabilized, and blocked, then incubated with primary antibody and fluorescently labeled secondary antibody, and finally stained with DAPI. Results showed that the CD86 fluorescence intensity in the IL37-OI@MS group was significantly lower than that in the LPS group (…). Figure 3 DE).

[0072] Example 6: Responsive microspheres delay the progression of intervertebral disc degeneration (IVDD) in rats

[0073] 1. Constructing an animal model

[0074] Forty-eight 12-week-old male Sprague-Dawley rats were randomly divided into six groups. After anesthesia, the location of the Co6 / 7 intervertebral disc was determined by palpation and X-ray. The annulus fibrosus was punctured with a 21G needle and left in place for 1 minute. Finally, 10 μL of material (PBS, MS, OI@MS, IL37@MS, or IL37-OI@MS) was injected. Imaging and histological evaluations were performed 8 weeks later. Figure 4 A).

[0075] 2. X-ray imaging assessment of intervertebral disc degeneration

[0076] Eight weeks post-surgery, X-rays were taken in the prone position of all rats to measure the disc height index (DHI). Results showed a significant decrease in disc height in the IVDD group, while this trend was significantly reversed in the IL37-OI@MS group. Figure 4 BC).

[0077] 3. MRI imaging assessment of intervertebral disc degeneration

[0078] 3.0T MRI was performed on the caudal vertebrae of rats, generating T2-weighted sagittal images. Results showed a significant increase in Pfirrmann's grade in the IVDD group, while a significant decrease was observed in the IL37-OI@MS group. Figure 4 DE).

[0079] 4. Histological staining assessment of intervertebral disc degeneration

[0080] Rat intervertebral disc tissue was fixed, decalcified, and cut into 5 μm thick sections, which were then stained with HE, SO, and alicin blue. The results showed that the IL37-OI@MS group performed best in maintaining the structural integrity of the intervertebral disc tissue. Figure 4 FG).

[0081] Example 7: Biosafety assessment of responsive microspheres in vivo

[0082] 1. In vitro cytotoxicity assessment

[0083] Nucleus pulposus cells were seeded at 5000 cells per well in 96-well plates and treated with different microspheres for 1, 3, and 5 days. CCK-8 assay was then performed. Results showed that the cell viability of all microspheres at 1, 3, and 5 days was similar to the control group, with no significant toxicity. Figure 5 AC).

[0084] 2. In vivo tissue safety assessment

[0085] Rat organs were fixed, sectioned, and stained with hematoxylin and eosin (HE). Results showed that the major organ tissue structures in each microsphere treatment group were normal, with no obvious pathological changes, confirming its good biocompatibility. Figure 5 D).

[0086] like Figure 1-5 As shown in the examples, hydrogel microspheres with different loading conditions were also studied. MS represents unloaded hydrogel microspheres, while OI@MS and IL37@MS represent hydrogel microspheres loaded only with Man-4OI-PLGA and IL37@HMS, respectively. All other preparation conditions were the same.

[0087] As can be seen, this invention utilizes hollow silica nanoparticles (HMS) to load IL37, significantly prolonging the protein's activity time and enhancing its local retention effect, thus providing a strong guarantee for IL37 to exert its therapeutic effect. Simultaneously, the mannose-modified polylactic-co-glycolic acid (PLGA) nanocarrier system, with its unique targeting and excellent protective properties, can specifically bind to mannose receptors on the surface of macrophages, achieving precise release of itaconic acid at the lesion site, effectively improving the accumulation efficiency of itaconic acid within macrophages, and enhancing its anti-inflammatory and pro-repair effects. Furthermore, the hydrogel microspheres with a pH-responsive Schiff base-linked structure can dissociate in the acidic microenvironment of degenerated tissues, releasing the carried drug. This ensures stability during drug transport and achieves precise delivery at the lesion site, meeting the special requirements of the small space and high pressure of the intervertebral disc for carrier mechanical strength, biocompatibility, and controlled drug release.

[0088] The embodiments described above are merely some preferred embodiments of the present invention, and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A hydrogel microsphere for modulating a senescent microenvironment, characterized in that, The hydrogel microspheres are obtained by mixing hollow mesoporous silica IL37@HMS encapsulating recombinant protein IL37 and nanoparticles Man-4OI-PLGA modified by mannose and loaded with 4OI in the preparation of pH-responsive hydrogel microspheres, and specifically, the hydrogel microspheres are prepared by a microfluidic device, wherein the water phase is composed of 30% gelatin by mass fraction, 10 mg / ml IL37@HMS, 10 mg / ml Man-4OI-PLGA and 20 mg / ml aldehyde-modified hyaluronic acid uniformly dispersed in PBS; the oil phase is composed of 5% Span80 by mass fraction dissolved in paraffin oil; the water phase and the oil phase are introduced into the microfluidic device at the same time, the flow rates of the water phase and the oil phase are accurately controlled, the emulsion is collected, and the small particles and uniform hydrogel microspheres are obtained after demulsification and washing.

2. A method for the preparation of hydrogel microspheres for modulating the senescent microenvironment, characterized by, It comprises: Preparation of hollow mesoporous silica IL37@HMS encapsulating recombinant protein IL37; Preparation of 4OI-loaded PLGA nanoparticles 4OI-PLGA; Modification of 4OI-PLGA nanoparticles by mannose to obtain modified particles Man-4OI-PLGA; Preparation of pH-responsive hydrogel microspheres by a microfluidic device, wherein the water phase is composed of 30% gelatin by mass fraction, 10 mg / ml IL37@HMS, 10 mg / ml Man-4OI-PLGA and 20 mg / ml aldehyde-modified hyaluronic acid uniformly dispersed in PBS; the oil phase is composed of 5% Span80 by mass fraction dissolved in paraffin oil, the water phase and the oil phase are introduced into the microfluidic device at the same time, the flow rates of the water phase and the oil phase are accurately controlled, the emulsion is collected, and the small particles and uniform hydrogel microspheres are obtained after demulsification and washing.

3. The method for preparing hydrogel microspheres for modulating a senescent microenvironment according to claim 2, wherein, Preparation of small-particle-size hollow mesoporous silica HMS, dispersion of the obtained HMS in PBS, addition of IL37 dissolved in PBS, 4℃ shaking incubation overnight, high-speed centrifugation and washing with pure water to obtain hollow mesoporous silica IL37@HMS encapsulating recombinant protein IL37.

4. The method for preparing hydrogel microspheres for modulating a senescent microenvironment according to claim 3, wherein, The preparation method of the small-particle-size hollow mesoporous silica HMS is as follows: first, mix ethanol, distilled water and ammonia water, add TEOS and anhydrous ethanol and stir to react, centrifuge and wash to obtain solid silica spheres sSiO2; then disperse sSiO2 in a solution containing CTAC, distilled water, triethanolamine and anhydrous ethanol, drop 1,3,5-trimethylbenzene and TEOS to form a CTAC-SiO2@sSiO2 composite structure; then add anhydrous sodium carbonate to etch the internal sSiO2 to form a cavity; finally, remove the template agent CTAC and 1,3,5-trimethylbenzene with hydrochloric acid, and obtain the small-particle-size hollow mesoporous silica HMS through centrifugation, washing and drying.

5. The method for preparing hydrogel microspheres for regulating the aging microenvironment according to claim 2, characterized in that, PLGA-COOH and 4OI are fully dissolved in chloroform, which is then added to a PVA solution, mechanically stirred after ultrasonic reaction, washed and concentrated to obtain 4OI-loaded PLGA nanoparticles 4OI-PLGA.

6. The method for preparing hydrogel microspheres for modulating a senescent microenvironment according to claim 5, wherein, 4OI-PLGA was dispersed in MES buffer solution, EDC and NHS were added, and the reaction was carried out on a shaking table at 37℃. The solution was adjusted to weak alkaline, and then mannose was added. The reaction was carried out on a shaking table at 37℃ overnight. The solution was ultrafiltrated and washed to obtain Man-4OI-PLGA, which was dissolved in pure water.

7. The method for preparing hydrogel microspheres for modulating a senescent microenvironment according to claim 2, characterized in that, The flow rate of the water phase was set to 10 μL / min, and the flow rate of the oil phase was set to 75 μL / min.

8. Use of the hydrogel microspheres of claim 1 in the preparation of a drug for treating intervertebral disc degeneration.

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