Stem cell-loaded drug-loaded nanovesicle-silk gel-based hydrogel and applications thereof

Abstract

The present application relates to the technical field of medicine, especially to drug-loaded nanovesicle-silk sericin-based hydrogel loaded with stem cells and application thereof.The present application uses silk sericin as a base material, obtains photo-crosslinking silk sericin (SerMA) through chemical grafting of methyl acrylate anhydride on silk sericin (sericin), and then grafts cell membrane nanovesicles modified by propenoic acid ester-polyethylene glycol-succinimide ester (AC-PEG-NHS) loaded with procyanidins (PC) onto the molecular skeleton through a photo-crosslinking reaction, thereby successfully preparing SerMA-PC@PNVs hydrogel.The in-vitro and in-vivo experimental researches of the hydrogel show that the SerMA-PC@PNVs / PDLSCs hydrogel can promote survival of damaged retinal ganglion cells (RGCs) by resisting oxidation and regulating polarization state of microglia cells, and can be used in treatment of eye diseases such as glaucoma.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to drug-loaded nanovesicles-silicone-based hydrogels loaded with stem cells and their applications. Background Technology

[0002] Glaucoma is a neurodegenerative disease characterized primarily by damage to retinal ganglion cells (RGCs). The main treatments for glaucoma involve lowering intraocular pressure (IOP) through medication, laser therapy, or surgery. However, these methods only slow disease progression and cannot effectively prevent the progressive death of RGCs and the continuous loss of visual function, which is the root cause of blindness in glaucoma. RGCs are terminally differentiated cells with extremely poor regenerative capacity, especially in adults where they cannot self-renew. RGC apoptosis is a challenge faced by many diseases, including glaucoma, diabetic retinopathy, and optic neuritis. Inflammation is one of the key factors leading to RGC apoptosis. Inflammatory responses at the site of injury lead to the excessive accumulation of reactive oxygen species (ROS). ROS, in synergy with pro-inflammatory cytokines, exacerbate retinal inflammation, further damaging RGCs and harming the optic nerve. Therefore, developing new strategies that can inhibit inflammation and clear ROS to promote RGC survival has become a hot and challenging research topic.

[0003] In recent years, stem cell transplantation has offered a new solution for glaucoma treatment due to its ability to repair or replace damaged retinopathy cells (RGCs). However, current methods for intraocular stem cell transplantation mainly involve intravitreal or subretinal injection. Intravitreal injection faces challenges such as poor cell homing ability to the retina, easy cell loss, and low survival rates; subretinal injection carries potential risks such as retinal detachment and subsequent neuronal damage. Therefore, developing novel stem cell transplantation systems is of significant clinical importance. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide drug-loaded nanovesicles-silicone-based hydrogels loaded with stem cells and their applications.

[0005] This invention provides drug-loaded nanovesicles comprising: AC-PEG-NHS modified cell membrane vesicles and glaucoma treatment drugs;

[0006] The drug-loaded nanovesicles of this invention are specifically prepared by mixing AC-PEG-NHS modified cell membrane vesicles (obtained by modifying cell membrane vesicles with AC-PEG-NHS) with an ophthalmic disease treatment drug (in a specific embodiment of this invention, the modified cell membrane vesicles are named PNVs, and the modified cell membrane vesicles are mixed with the ophthalmic disease treatment drug to obtain PC@PNVs); the mixing step involves sonication at 0-4°C followed by repeated extrusion through porous membranes at 800nm, 400nm, 200nm, and 100nm.

[0007] The cell membrane is derived from human periodontal ligament stem cells;

[0008] The mass ratio of the product to the ophthalmic disease treatment drug is 1:2 to 2:1.

[0009] In this invention, the mass ratio of the product to the ophthalmic disease treatment drug in the drug-loaded nanovesicles was optimized to be 1:2 to 2:1. The experimental results show that the physical stability is best when the mass ratio of the product to the ophthalmic disease treatment drug is 1:1.

[0010] Furthermore, in this invention, the eye disease treatment drug includes a glaucoma treatment drug; the glaucoma treatment drug includes: proanthocyanidins, bilberry anthocyanins, ginkgo biloba extract, lutein, zeaxanthin, methylcobalamin and / or nicotinamide; in a specific embodiment of this invention, the eye disease treatment drug is proanthocyanidin PC;

[0011] In the drug-loaded nanovesicles of the present invention, the concentration of the glaucoma treatment drug is 50 μg / mL to 800 μg / mL.

[0012] In this invention, the concentration of the glaucoma treatment drug was optimized (always maintaining a 1:1 mass ratio of the product to the ocular disease treatment drug). The results showed that in the hydrogel (containing PC@PNVs and methacrylamide-modified sericin SerMA), the compressive strength initially increased with the increase of PC@PNVs content, but then decreased. When the PC content was 400 μg / mL, SerMA-PC@PNVs exhibited the best compressive strength and no cytotoxicity.

[0013] Furthermore, the cell membranes in the AC-PEG-NHS modified cell membrane vesicles are derived from human periodontal ligament stem cells;

[0014] The AC-PEG-NHS modified cell membrane vesicles are obtained by modifying cell membrane vesicles with AC-PEG-NHS.

[0015] The mass ratio of the cell membrane vesicles to the AC-PEG-NHS is 1:2;

[0016] The photoinitiator includes LAP and / or Irgacure 2959; in a specific embodiment of the present invention, the photoinitiator LAP has a concentration of 0.5 wt%.

[0017] In a specific embodiment of the present invention, the method for preparing the drug-loaded nanovesicles includes the following steps:

[0018] Step 1: Human periodontal ligament stem cells (PDLSCs) were lysed to remove cell debris, and cell membrane vesicles (NVs) were obtained.

[0019] Step 2: The cell membrane NVs are reacted with AC-PEG-NHS under an amide reaction to obtain AC-PEG-NHS modified cell membrane vesicles (PNVs).

[0020] Step 3: The AC-PEG-NHS modified PNVs are mixed with proanthocyanidin PC to obtain drug-loaded nanovesicles PC@PNVs.

[0021] In step 1, the disruption method is ultrasonic disruption, and the ultrasonic disruption conditions are 40% power, 1.5 min; 1 second on / 2 seconds off; the method for removing cell debris is centrifugation; the centrifugation conditions are 4,000×g for 30 min; the method for obtaining cell membrane vesicles is centrifugation, and the centrifugation conditions are 4℃, 150,000×g for 70 min; by varying the centrifugation force, after removing other unwanted cell debris, the desired cell membrane vesicles are obtained.

[0022] In step 2, the mass ratio of cell membrane vesicles (NVs) to AC-PEG-NHS is 1:2; in a specific embodiment of the present invention, the mass of the NVs used is 5 mg, and the corresponding mass of AC-PEG-NHS is 10 mg; the photoinitiator is LAP, and its concentration is 0.5 wt%.

[0023] In step 3, the AC-PEG-NHS modified cell membrane vesicles (PNVs) are mixed with proanthocyanidins (PC) and repeatedly extruded. Then, the product drug-loaded nanovesicles (PC@PNVs) are collected by centrifugation.

[0024] This invention provides a hydrogel composition comprising methacrylamide-modified sericin, a photoinitiator, and the drug-loaded nanovesicles described herein. The hydrogel composition transforms from a mobile phase to a hydrogel under 405 nm blue light irradiation. In specific embodiments of this invention, the hydrogel composition is named a hydrogel precursor solution. In practical use, it can be directly irradiated with 405 nm blue light to form a hydrogel, or it can be mixed with other substances and then irradiated with blue light, or it can be injected into certain sites and then irradiated with blue light.

[0025] In the hydrogel composition of the present invention, the photoinitiator includes LAP and / or Irgacure2959; specifically, the photoinitiator is LAP with a concentration of 0.5 wt%; the concentration of the methacrylamide-modified sericin (SerMA) is 140–160 mg / mL, specifically 150 mg / mL; the concentration of the glaucoma treatment drug in the drug-loaded nanovesicles is 50–800 μg / mL, preferably 400 μg / mL; the mass ratio of the AC-PEG-NHS modified cell membrane to the glaucoma treatment drug is 1:2–2:1, preferably 1:1.

[0026] This invention provides a composition containing stem cells, comprising the hydrogel described herein and stem cells. The stem cells include mesenchymal stem cells, neural stem cells, deciduous tooth pulp stem cells, and / or human periodontal ligament stem cells. A specific embodiment of this invention uses human periodontal ligament stem cells.

[0027] This invention provides the use of at least one of the following (I) to (III) in the preparation of products for the treatment and / or prevention of eye diseases:

[0028] I) The drug-loaded nanovesicles described in this invention;

[0029] II) The hydrogel composition of the present invention;

[0030] III) The mixture described in this invention.

[0031] This invention provides a product for the treatment and / or prevention of eye diseases, characterized in that the raw materials include at least one of those shown in i) to iii):

[0032] i) The drug-loaded nanovesicles described in this invention;

[0033] ii) The hydrogel composition of the present invention;

[0034] iii) The mixture described in this invention.

[0035] The eye diseases mentioned include glaucoma.

[0036] In this invention, drug-loaded nanovesicles are represented by PC@PNVs, methacrylic anhydride-modified sericin is represented by SerMA, hydrogels prepared from methacrylic anhydride-modified sericin and drug-loaded nanovesicles are represented by SerMA-PC@PNVs, and hydrogels loaded with stem cells are represented by SerMA-PC@PNVs / PDLSCs.

[0037] This invention prepares drug-loaded nanovesicles PC@PNVs; uses drug-loaded nanovesicles PC@PNVs and SerMA as raw materials to prepare SerMA-PC@PNVs; uses PDLSCs and SerMA-PC@PNVs as raw materials to prepare SerMA-PC@PNVs / PDLSCs; this invention gradually studies the specific properties of SerMA-PC@PNVs through experiments;

[0038] In some embodiments of the in vitro experiments of the present invention, compared with SerMA, SerMA-PC@PNVs significantly improved the free radical scavenging rate at PC concentrations of 200 μg / mL, 400 μg / mL, and 800 μg / mL, with the highest free radical scavenging rate at a PC concentration of 800 μg / mL. However, at a PC concentration of 800 μg / mL, the mixture of SerMA-PC@PNVs and PDLSCs inhibited cell proliferation, leading to a decrease in PDLSC cell activity. Furthermore, compared with other concentrations, SerMA-PC@PNVs exhibited the best stress resistance properties at a PC concentration of 400 μg / mL. Therefore, a PC concentration of 400 μg / mL is optimal, and the stability is best when the mass ratio of PC to PNVs is 1:1.

[0039] In some embodiments of the in vitro tests of the present invention, compared with direct doping of SerMA and PC (SerMA / PC hydrogel), SerMA-PC@PNVs have a sustained-release effect of PC, and the drug release rate at 30 days is significantly lower than that of SerMA / PC hydrogel.

[0040] In some embodiments of the in vitro experiments of the present invention, the SerMA-PC@PNVs hydrogel can promote the secretion of multiple neurotrophic factors such as BDNF, IGF-1, CNT and NGF;

[0041] In some embodiments of the in vitro experiments of the present invention, the SerMA-PC@PNVs and SerMA-PC@PNVs / PDLSCs can inhibit H2O2-induced oxidative stress-induced apoptosis of R28 cells. Compared with the control (R28 cell apoptosis rate of 44.8%), the apoptosis rate of R28 cells treated with SerMA-PC@PNVs is about 22.4%, and the apoptosis rate of R28 cells treated with SerMA-PC@PNVs / PDLSCs is about 14.1%; and it also has good antioxidant function.

[0042] In some embodiments of the in vitro experiments of the present invention, the SerMA-PC@PNVs hydrogel can promote the conversion of pro-inflammatory M1 type HAPI to anti-inflammatory M2 type HAPI, and after loading stem cells, SerMA-PC@PNVs / PDLSCs have better anti-inflammatory effects.

[0043] In some embodiments of the in vivo animal experiments of the present invention, compared with the model group, it was shown that SerMA-PC@PNVs / PDLSCs have excellent optic nerve protection and can promote RGC survival.

[0044] In the rat retinal RNA-Seq sequencing analysis of this invention, the experimental results showed that the SerMA-PC@PNVs / PDLSCs hydrogel may promote RGC survival by influencing inflammation-related pathways and regulating the state of microglia.

[0045] Glaucoma retinal ganglion cell (RGC) damage is often characterized by accumulated inflammation. Existing naturally derived biomaterials have failed to adequately integrate anti-inflammatory and anti-inflammatory properties to promote the survival of glaucoma-damaged RGCs. This study designed and constructed a stem cell-loaded nanovesicle sericin-based hydrogel, SerMA-PC@PNVs, to promote the survival of glaucoma-damaged RGCs. Cell membrane nanovesicles (NVs, or cell membranes) derived from natural periodontal ligament stem cells (PDLSCs) were prepared using ultracentrifugation and liposome extrusion. Methacrylamide sericin (SerMA) was obtained through chemical grafting modification using sericin as a substrate. Then, NVs (PNVs) modified with acrylate-polyethylene glycol-succinimide (AC-PEG-NHS) loaded with proanthocyanidins (PC) were grafted onto its molecular backbone via photocrosslinking, successfully preparing the SerMA-PC@PNVs hydrogel. This hydrogel can promote the secretion of more neurotrophic factors by PDLSCs. In vitro studies have shown that this hydrogel exerts antioxidant and anti-apoptotic effects. In vitro and in vivo studies have shown that this hydrogel can inhibit M1 polarization of microglia and promote M2 polarization, thereby exerting an anti-inflammatory effect and promoting RGC survival. Rat retinal RNA-Seq sequencing indicates that this hydrogel can affect pathways related to "inflammatory response" and "apoptosis." Rat retinal single-cell sequencing shows that this hydrogel can increase the number of RGCs and reduce the number of inflammation-related neutrophils and microglia. Further analysis of the Microglia cell population, including GO analysis, indicates that this hydrogel can affect pathways related to "inflammatory response." In summary, this SerMA-PC@PNVs hydrogel can promote the survival of RGCs damaged by glaucoma by regulating microglia polarization and influencing the inflammatory response.

[0046] This invention uses sericin as a substrate, obtaining photocrosslinked sericin (SerMA) through chemical grafting of methacrylic anhydride with sericin, and then grafting PC-loaded acrylate-polyethylene glycol-succinimide (AC-PEG-NHS) modified cell membrane nanovesicles onto its molecular backbone via a photocrosslinking reaction, successfully preparing SerMA-PC@PNVs hydrogel. In vitro and in vivo experimental studies of this hydrogel show that the SerMA-PC@PNVs / PDLSCs hydrogel can promote the survival of damaged RGCs through antioxidant effects and regulation of microglia polarization, and can be used in the treatment of eye diseases such as glaucoma. Attached Figure Description

[0047] Figure 1 A schematic diagram showing how SerMA-PC@PNVs / PDLSCs hydrogel promotes the survival of retinal ganglion cells (RGCs);

[0048] Figure 2 The images show the 1H NMR spectrum, DLS and TEM images of PNVs; where A is the 1H NMR spectrum of PNVs; and B is the DLS and TEM image of PNVs.

[0049] Figure 3 The standard calibration curve showing PC concentration;

[0050] Figure 4 The images show PNVs and PC@PNVs and their performance tests; where A is DLS and TEM images of PC@PNVs; B is Zeta potential analysis of PNVs and PC@PNVs; C is NTA analysis of PC@PNVs; and D is stability assessment of PC@PNVs under storage conditions.

[0051] Figure 5 The following are the performance tests of SerMA-PC@PNVs hydrogel: A shows the 1H NMR spectrum of Serricin and SerMA; B shows the sol-gel transition behavior of SerMA; C shows the transparency effect of SerMA-PC@PNVs hydrogel; D shows the SEM image of SerMA and SerMA-PC@PNVs hydrogel; E shows the FTIR spectrum of Serricin, SerMA, and SerMA-PC@PNVs; F shows the compressibility of SerMA and SerMA-PC@PNVs hydrogel; and G shows the DPPH free radical scavenging activity of SerMA and SerMA-PC@PNVs hydrogel.

[0052] Figure 6The in vitro cytological evaluation of SerMA-PC@PNVs hydrogel is shown; where A is the immunofluorescence staining of CD146 and STRO-1 expression in PDLSCs; B is the cytotoxicity analysis of SerMA and SerMA-PC@PNVs hydrogel extracts on PDLSCs; C-F are the ELISA quantification of neurotrophic factors in the culture medium, including BDNF (C), IGF-1 (D), CNTF (E) and NGF (F);

[0053] Figure 7 The in vitro drug release profiles of SerMA / PC and SerMA-PC@PNVs hydrogels are shown at 1, 3, 7, 14 and 30 days.

[0054] Figure 8 The effects of H2O2 or hydrogel on R28 cells are shown; where A represents the cytotoxicity of R28 cells after H2O2 treatment; B represents the live / dead staining of R28 cells; C represents the flow cytometry analysis of R28 cell apoptosis; and D represents the quantitative analysis of the apoptotic cell population.

[0055] Figure 9 This study presents in vitro studies demonstrating the antioxidant and anti-apoptotic effects. A shows the assessment of intracellular reactive oxygen species (ROS) levels using DCFH-DA and DHE immunofluorescence staining; B shows the expression of CD86 (M1 polarization marker) in HAPI microglia by flow cytometry; C shows the expression of CD206 (M2 polarization marker) in HAPI cells by flow cytometry; D shows the quantitative analysis of M1 polarized HAPI cells; and E shows the quantitative analysis of M2 polarized HAPI cells.

[0056] Figure 10 Statistical analysis of intraocular pressure in the right eye of rats in different treatment groups;

[0057] Figure 11 This study evaluates the in vivo therapeutic efficacy of SerMA-PC@PNVs hydrogel. A shows Brn3a immunofluorescence staining of retinal ganglion cells (RGCs) in rat retina; B shows quantitative analysis of RGCs based on Brn3a-positive cells; C shows H&E staining and RBPMS immunofluorescence staining of rat retinal tissue; D shows quantitative analysis of RGCs based on RBPMS-positive cells; and E shows immunofluorescence staining of CD86 (M1 marker) and CD206 (M2 marker) in rat retina to assess microglial polarization status.

[0058] Figure 12RNA-Seq analysis of gene expression in rat retinal tissue was performed. A is a Venn diagram showing the overlap of expressed genes between the COH (modeling) group and the Test (SerMA-PC@PNVs / PDLSCs treatment) group; B is a PCA diagram showing the gene expression profiles of the COH and Test groups; C is a violin diagram showing the distribution of gene expression between the COH and Test groups; and D is a volcano diagram showing differentially expressed genes between the COH and Test groups.

[0059] Figure 13 Statistical analysis of differentially expressed genes between the COH group and the Test group;

[0060] Figure 14 A heatmap showing differentially expressed genes related to inflammation between the COH and Test groups;

[0061] Figure 15 GO enrichment analysis of differentially expressed genes between the COH group and the Test group;

[0062] Figure 16 KEGG enrichment analysis of differentially expressed genes between the COH group and the Test group;

[0063] Figure 17 Single-cell transcriptomic analysis of retinal cell populations is shown, where A represents the 10 major retinal cell types identified in the COH (modeling) group; and B represents the 10 major retinal cell types identified in the Test (treatment) group.

[0064] Figure 18 The distribution of retinal cell types and differential gene expression in the COH and Test groups are shown. A shows the proportional distribution of 10 identified retinal cell types in the COH and Test groups; B is a volcano plot showing the differentially expressed genes in microglia in the COH and Test groups.

[0065] Figure 19 Subpopulation analysis of RGCs was performed, and nine different subpopulations were identified based on transcriptome characteristics using the [UMAP / t-SNE] dimensionality reduction method.

[0066] Figure 20 KEGG pathway enrichment analysis of differentially expressed genes in microglia;

[0067] Figure 21 GO enrichment analysis of differentially expressed genes in microglia;

[0068] Figure 22 H&E staining of heart, liver, spleen, lung, and kidney tissues from rats in different treatment groups was used for histopathological evaluation of systemic biocompatibility.

[0069] Figure 23 This study demonstrates the stability of PC@PNVs mixed with PNVs and PC at mass ratios of 1:1, 1:2, and 2:1 in PBS for one week. Detailed Implementation

[0070] This invention provides drug-loaded nanovesicles-silicone-based hydrogels containing stem cells and their applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0071] In this invention, the hydrogel, which may also be referred to as a hydrogel scaffold, is formed by the hydrogel composition changing from a mobile phase to the hydrogel under 405nm blue light irradiation.

[0072] Procyanidine (PC)

[0073] Periodontal ligament stem cells (PDLSCs);

[0074] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0075] Example 1: Construction of stem cell-loaded nanovesicle-silk gel

[0076] This study developed a novel in-situ engineered SerMA-PC@PNVs / PDLSCs scaffold based on the combination of SerMA, PC-loaded nanovesicles, and PDLSCs. By exerting anti-inflammatory and antioxidant functions, it promotes the survival of RGCs in glaucoma-damaged tissues. The specific working principle is as follows: Figure 1 As shown.

[0077] I. Materials and Methods

[0078] 1. Materials

[0079] Silkworm cocoons were purchased from Suzhou Silebao Biotechnology Co., Ltd. (China); lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP) and proanthocyanidins (PC) were purchased from Aladdin Company (China); ELISA kits were purchased from Sangon Biotech Co., Ltd. (China); calcein-AM / PI assay kits were provided by Shanghai Yeasen Biotechnology Co., Ltd. (China); MA and LPS were purchased from Sigma-Aldrich Corporation (USA); and DAPI was purchased from Dalian Meilun Biotechnology Co., Ltd. (China). PDLSCs were purchased from iCell Bioscience Inc. (China); AC-PEG-NHS from Suzhou Yongqinquan Intelligent Equipment Co., Ltd. (China); DCFH-DA from Beyotime Biotechnology Co., Ltd. (China); DPPH from Shanghai Solarbio Bioscience & Technology Co., Ltd. (China); other common chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (China); Sprague Dawley rats (male, 6-8 weeks old) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. (China).

[0080] 2. Preparation and characterization of PC@PNVs

[0081] PDLSCs were cultured in DMEM / F12 medium (Gibco BRL) supplemented with 10 vt% heat-inactivated fetal bovine serum (FBS) and 1 vt% penicillin-streptomycin (P / S). Cells from passages 3 to 5 were used for experiments. After large-scale expansion, PDLSCs were washed three times with PBS, resuspended in PBS, and subjected to intermittent sonication (40% power, 1.5 min; 1 second on / 2 seconds off) under ice-water bath conditions to obtain lysate. The lysate was centrifuged at 4,000 × g for 30 min to remove debris, and the supernatant was ultracentrifuged at 150,000 × g for 70 min (4°C) to obtain cell membrane NVs (the cell membrane mentioned here actually includes the cell membrane and cell membrane derivatives, including cell membrane vesicles, which play a role in encapsulation and transport in actual operation).

[0082] 5 mg of NVs were dissolved in PBS containing LAP, and an appropriate amount of AC-PEG-NHS was added. The mass ratio of NVs to AC-PEG-NHS was 1:2. The solution was dissolved by vortexing in the dark for 5 min, and then incubated at 4 °C in the dark for 24 h to obtain AC-PEG-NHS modified NVs, i.e., PNVs. After lyophilizing the prepared PNVs, 5 mg was dissolved in 0.5 mL of D2O and placed in an NMR tube for 1H NMR analysis to evaluate the modification results of PNVs. The morphology was observed using DLS (Dynamic Light Scattering) and TEM.

[0083] PNVs and PC were mixed in PBS at a 1:1 mass ratio, and the mixture was ultrasonically dispersed in an ice-water bath. The mixture was then repeatedly extruded through porous membranes at 800 nm, 400 nm, 200 nm, and 100 nm using a liposome extruder. Finally, the drug-loaded nanovesicles PC@PNVs were collected by ultracentrifugation at 150,000 × g for 70 min at 4 °C.

[0084] Morphological characteristics were characterized by transmission electron microscopy (TEM, HT7800, Japan), hydrodynamic diameter was determined by dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern, UK), and particle size distribution was analyzed by nanoparticle tracking analyzer (NTA, NanoSight NS300, Malvern, UK). The encapsulation efficiency (EE) of PC was calculated by high performance liquid chromatography (HPLC). The chromatographic column was C18 (250 mm × 4.6 mm, 5 μm), the mobile phase was 95% acetonitrile solution, and the UV detector was 280 nm. The sample injection volume was 20 μL, and the operating flow rate was 1 mL / min. EE was calculated according to the following formula (1):

[0085]

[0086] Where Wa is the total mass of the drug added, and Wb is the mass of the unencapsulated drug in the supernatant.

[0087] 3. Preparation and Characterization of SerMA

[0088] 20g of natural silkworm cocoons were washed with double-distilled water and then immersed in 800mL of boiling 0.02M Na2CO3 solution for 1 hour. Insoluble residues were removed by centrifugation (3000rpm, 10min) and filtration to obtain a crude sericin extract. The crude sericin extract was dialyzed in double-distilled water for 3 days using a dialysis bag with a molecular weight cutoff of 3.5kDa. After lyophilization, purified sericin was obtained.

[0089] 10 g of lyophilized purified sericin was dissolved in 40 mL of pH 8.5 PBS buffer and stirred thoroughly for 3 h to obtain a sericin solution. 2.93 g of methacrylic anhydride (MA) was dissolved in 100 mL of pH 8.5 PBS to prepare an MA stock solution. The MA stock solution and sericin solution were reacted overnight at room temperature, then dialyzed in double-distilled water for 3 days using a dialysis bag with a 3.0 kDa molecular weight cutoff. The resulting SerMA was then lyophilized for later use.

[0090] The methacrylation modification of SerMA was confirmed by detection in D2O using a 1H NMR spectrometer (Bruker 400MHz Advance, Switzerland).

[0091] 4. Preparation and characterization of SerMA-PC@PNVs

[0092] A hydrogel precursor solution was prepared by dissolving 150 mg SerMA and different masses of PC@PNVs in 1 mL of PBS containing LAP (LAP concentration of 0.5 wt%, pH 7.4) (where the concentration of PC after dissolution of PC@PNVs was 0, 50, 100, 200, 400, and 800 μg / mL, and the mass ratio of PNVs to PC was 1:1). Then, the precursor solution was irradiated with 405 nm visible blue light for 60 s to form a SerMA-PC@PNVs hydrogel (or hydrogel scaffold).

[0093] In addition, a hydrogel precursor solution was prepared by dissolving 150 mg SerMA in 1 mL of PBS containing LAP (LAP concentration of 0.5 wt%, pH 7.4). Then, the precursor solution was irradiated with 405 nm visible blue light for 60 s to form a SerMA hydrogel as a control.

[0094] The microstructure of the freeze-dried hydrogel was observed using scanning electron microscopy (SEM) (FEI Quanta 200, FEI, Czech Republic). The functional groups of the hydrogel were characterized using infrared spectroscopy (Thermo Nicolet Nexus 470, Nicolet). The compressive strain of the hydrogel was tested using a tensile-compression testing machine (Shimadzu AGS-V universal testing machine, equipped with a 20N sensor) in compression mode.

[0095] 5. DPPH scavenging rate determination

[0096] 0.5 g of SerMA and SerMA-PC@PNVs hydrogels were dispersed in 1 mL of ethanol containing 100 μM DPPH, respectively. SerMA hydrogel was used as a control sample. The SerMA and SerMA-PC@PNVs hydrogels were then shaken in the dark for 30 min, and the remaining DPPH in the solution was detected using UV-Vis spectroscopy. The DPPH scavenging rate was calculated according to formula (2):

[0097]

[0098] Ac and An are the absorbance values ​​of DPPH / ethanol solution and DPPH / ethanol / sample solution at 515 nm, respectively.

[0099] 6. Immunofluorescence assay of CD146 and STRO-1

[0100] In the immunofluorescence assay of PDLSCs, the primary antibodies were mouse anti-human antibodies, including STRO-1 (Invitrogen) and CD146 (Invitrogen), and the secondary antibodies included goat anti-mouse IgG1 Alexa Fluor 488 and IgG1 Alexa Fluor 594.

[0101] 7. Cell proliferation test

[0102] First, SerMA and SerMA-PC@PNVs hydrogels (containing different masses of PC@PNVs (PC: 50, 100, 200, 400, 800 μg / mL, PNVs to PC mass ratio 1:1) were lyophilized. Then, the cytotoxicity of their extracts was detected using AlarmaBlue. 1 g of lyophilized hydrogel was mixed with 10 mL of culture medium and soaked for 48 h. The supernatant was then stored at 4 °C. Ten plants were then seeded. 5 PDLSCs were cultured in 48-well cell culture plates, with 0.5 mL of supernatant added to each well, and incubated at 37°C for 24 h. Finally, AlarmaBlue assay solution was added, and the absorbance was measured using a microplate reader.

[0103] 8. PC drug release

[0104] One mL of SerMA-PC@PNVs hydrogel and one mL of SerMA / PC (PC is physically doped, with SerMA at 150 mg / mL and PC at 400 μg / mL) hydrogel were each immersed in 1 mL of ddH2O (PC concentration 400 μg / mL). The samples were then transferred to centrifuge tubes and stored in a 37°C constant-temperature shaker. After incubation for 1, 3, 7, 14, and 30 days, the solutions were centrifuged. High-performance liquid chromatography (HPLC) was used to quantitatively analyze the PC released from the separated solutions. The chromatographic column was C18 (250 mm × 4.6 mm, 5 μm), the mobile phase was 95% acetonitrile solution, and the UV detector was 280 nm. The sample injection volume was 20 μL, and the flow rate was 1 mL / min.

[0105] 9. Effects of hydrogels on the secretion of neurotrophic factors by PDLSCs

[0106] The experiment was divided into two groups, one group planted with 1×10 5 One group of PDLSCs was cultured in 48-well cell culture plates, with 0.5 mL of supernatant added to each well. Another group first mixed PDLSCs with a SerMA-PC@PNVs hydrogel precursor solution, then irradiated with 405 nm visible blue light to form a SerMA-PC@PNVs / PDLSCs hydrogel, which was then cultured in 48-well cell culture plates with a consistent cell concentration at 37°C for 24 h. The secretion levels of various neurotrophic factors, including BDNF, CNTF, NGF, and IGF-1, in the culture medium were then measured using an ELISA kit, and differences between the different groups were compared.

[0107] 10. Establish an oxidative stress H2O2-R28 cell model

[0108] The viability of R28 cells after H2O2 treatment was determined using the CCK-8 assay. R28 cells were incubated at 5 × 10⁶ cells / year. 3 R28 cells were seeded at a density of 10 cells / well in 96-well plates. R28 cells were treated with different concentrations of H2O2 (0, 20, 40, 80, 160, 320, 640 μM) for 24 h, and then the viability of R28 cells was assessed using the CCK-8 kit according to the manufacturer's instructions.

[0109] 11. In vitro cell experiments

[0110] In vitro live / dead cell staining study. R28 cells were stained at 1×10⁻⁶ cells / cells. 5Cells were seeded at a density of 100 cells / well in 48-well cell culture plates. After incubation for a period of time, the experiment was divided into three groups: H2O2, SerMA-PC@PNVs, and SerMA-PC@PNVs / PDLSCs. The H2O2 group was treated with only 80 μM H2O2; the SerMA-PC@PNVs group used a Transwell contactless co-culture system, placing the hydrogel in the upper chamber and then treating it with 80 μM H2O2; the SerMA-PC@PNVs / PDLSCs group used a Transwell contactless co-culture system, placing the stem cell-containing hydrogel in the upper chamber and then treating it with 80 μM H2O2. After 24 h, the cells were incubated with Calcin-AM and PI for 15 min, and finally, the fluorescence signal was observed using an inverted fluorescence microscope.

[0111] In vitro apoptosis experiments were conducted. The three groups of cells mentioned above were collected and stained with Annexin V-FITC and PI according to the manufacturer's instructions. The stained cells were then analyzed using flow cytometry, and the apoptosis results were analyzed using FlowJo software (Stanford University).

[0112] In vitro cellular reactive oxygen species (ROS) assay. The experiment was divided into three groups: H2O2, SerMA-PC@PNVs, and SerMA-PC@PNVs / PDLSCs (pre-staining procedures were the same as above). After treating the cells with different groups, the cells were stained with the ROS fluorescent indicator DCFH-DA / DHE. The generation of ROS resulted in fluorescence, and the fluorescence signal was observed using an inverted fluorescence microscope.

[0113] In vitro microglial cell polarization assay. To simulate inflammation-mediated activation, cells with a density of 1×10⁻⁶ were used. 5 Rat HAPI cells were seeded at 100 cells / well in 6-well cell culture plates and cultured overnight to promote cell attachment. The normal medium was then replaced with serum-free medium, and lipopolysaccharide (LPS) was added to a final concentration of 1 μg / mL to stimulate HAPI cell transformation to an inflammatory phenotype, followed by incubation for 12 h. SerMA-PC@PNVs hydrogel (300 μL) and SerMA-PC@PNVs / PDLSCs hydrogel (300 μL) encapsulated with PDLSCs were placed in the upper Transwell chamber, and then 1 mL of serum-free α-MEM medium was added. Serum-free medium was used here because serum may bind to LPS, reducing the stimulatory effect. After 24 h of culture, cells were collected, and the polarization status of HAPI cell M1 (CD86) and M2 (CD206) cells was analyzed by flow cytometry.

[0114] 12. In vivo animal experimental studies

[0115] First, a glaucoma model was established in SD rats by intravenous injection of hypertonic saline into the sclera. This method involved creating an intraocular pressure elevation model lasting more than 4 weeks through endothelial cell damage and venous sinus fibrosis. Hypertonic saline was injected intravenously into the sclera of the rats using a microinfusion pump. After injection, the needle was removed, and antibiotic eye drops were administered to prevent infection. Subsequently, intraocular pressure was monitored regularly to ensure the successful establishment of the glaucoma model.

[0116] After successful modeling, the experiment was divided into three groups: normal group (NC), model group (COH), and experimental group (Test, treated with SerMA-PC@PNVs / PDLSCs). The NC group received no treatment, the COH group was the glaucoma model group, and the Test group, after glaucoma modeling, received SerMA-PC@PNVs / PDLSCs hydrogel precursor solution injected into the vitreous cavity, followed by irradiation with 405nm visible blue light (1 min) through the pupil to form the hydrogel. Postoperative IOP of the rats was measured periodically. Four weeks after treatment, the rats were sacrificed, and retinal tissue was sampled and prepared for slide examination. Immunofluorescence staining with Brn3a was performed to analyze the survival of retinoid globulins (RGCs). Simultaneously, the eyeballs were fixed, and RGC survival was assessed using H&E staining and PBPMS immunofluorescence staining. Furthermore, CD86 and CD206 immunofluorescence staining was performed on the eyeball tissues of different groups to evaluate the polarization transition of microglia in the retina.

[0117] 13. Rat retinal sequencing analysis

[0118] First, RNA-Seq sequencing was performed on rat retinas. To systematically elucidate the molecular mechanism of SerMA-PC@PNVs / PDLSCs treatment, retinal tissues from the glaucoma model group (COH group) and the SerMA-PC@PNVs / PDLSCs treatment group (Test group) were collected one month after treatment. The tissues were rapidly transferred to pre-cryopreservative tubes and flash-frozen in liquid nitrogen for 5 min, then stored at -80℃ to maintain RNA integrity. Gene expression profiling analysis was performed by Guangzhou Codio Biotechnology Co., Ltd. Differentially expressed genes between the two groups were analyzed using the R language "edgeR" package, with a p-value <0.05 and an absolute fold change >1.2 as the selection criteria. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the R language "clusterProfiler" package to analyze the biological processes, molecular functions, cellular components, and potential signaling pathways involved in the differentially expressed genes.

[0119] Furthermore, single-cell sequencing analysis was performed on the rat retina. After tissue collection, tissue digestion was performed using the SeekMate Tissue Digestion Kit A Pro (SeekGene, K01801301) from SeekGene, according to the manufacturer's instructions. DNase I (Sigma, 9003-98-9) was added based on the viscosity of the homogenate. After red blood cell removal (Solarbio, R1010), the tissue was analyzed using a fluorescence cell analyzer. Rigel (S2) was used in conjunction with AO / PI reagents for cell counting and viability assessment.

[0120] Fresh cells were washed twice with RPMI 1640 medium (Gibco) and resuspended in RPMI 1640 + 2 wt% FBS (Gibco) at a concentration of 1×10⁻⁶. 6 cells / mL. The single-cell RNA-Seq library was provided by SeekGene. The digital droplet single-cell 3' library was constructed using a kit. A simplified procedure is as follows: An appropriate amount of cells were mixed with the reverse transcription reagent and then added... In the sample wells of chip S3, barcoded hydrogel beads (BHBs) and phase-separating oil were simultaneously added to their respective wells. After forming emulsion droplets, a reverse transcription reaction was performed at 42°C for 90 min, followed by inactivation at 85°C for 5 min. After emulsion breaking, cDNA was extracted and PCR amplified. The amplified cDNA product was sequentially purified, fragmented, end-repaired, A-tailed, and adapter-ligated. Finally, an indexed PCR reaction was performed to amplify the 3' polyA-tail expression sequence containing the cell barcode and unique molecular identifier (UMI). The library was purified using VAHTSDNA CleanBeads (Vazyme, N411-01) and then quantified and quality-assessed using a Qubit instrument (Thermo Fisher Scientific, Q33226) and a Bioptic fragment analyzer (Bioptic, Qsep400). The final library was sequenced on an Illumina NovaSeq6000 or DNBSEQ-T7 platform with a PE150 read length.

[0121] 14. Statistical Analysis

[0122] Data are expressed as mean ± standard deviation (mean ± SD), with a sample size ≥ 3. Unpaired two-tailed t-tests were used for comparisons between two groups, and one-way (or two-way) ANOVA was used for comparisons among multiple groups. Significance markers: *P < 0.05; **P < 0.01; ***P < 0.001.

[0123] II. Results and Discussion

[0124] 1. Synthesis and Characterization of PC@PNVs

[0125] First, PDLSCs were expanded on a large scale, and cells from passages 3 to 5 were used for further experiments. Then, the obtained PDLSCs were disrupted using sonication to obtain cell membrane NVs. Further modification of the NVs was performed using acrylate-polyethylene glycol-succinimide AC-PEG-NHS. The succinimide (NHS) groups react with the amino groups (-NH2) on the NV membrane proteins to form stable amide bonds, while the carbon-carbon double bonds on the acrylate (AC) groups are retained, thus obtaining modified NVs, i.e., PNVs. Figure 2 As shown in A, 1 The 1H NMR spectrum showed three new characteristic peaks at chemical shifts of 6.1 ppm, 6.4 ppm, and 6.6 ppm, which are characteristic peaks of the AC group, indicating that the PNVs were successfully prepared. Figure 2 As shown in B, the DLS results show that the hydrated particle size of PNVs is 115.24±5.33, and the TEM electron microscopy shows that PNVs exhibit a typical cup-shaped structure.

[0126] Furthermore, a mixture of PNVs and PC was repeatedly extruded using a liposome extruder to prepare PC@PNVs. Different concentrations of PC were detected by HPLC, and a standard curve was plotted. The results are as follows: Figure 3 As shown, the encapsulation efficiency of PC is approximately 56.1% according to the standard curve and formula (1). Figure 4 As shown in Figure A, DLS results indicate that the hydrated particle size of PC@PNVs is 128.92 ± 6.08 μm, and TEM electron microscopy shows that PC@PNVs have a similar particle size. Figure 4 As shown in Figure B, the zeta potential changes of PNVs and PC@PNVs are small, indicating that the PC loading has little effect on the zeta potential of PNVs. Nanoparticle tracking analysis (NTA) results are as follows... Figure 4 The C-values ​​in the data show that the particle size distribution is most widespread at 111 nm. Furthermore, the stability of the PC@PNVs nanoparticles was characterized on days 1, 3, 5, and 7. Figure 4 As shown in D, no significant changes in the hydrodynamic size of PC@PNVs nanoparticles in PBS were observed, indicating that PC@PNVs nanoparticles can remain stable in PBS.

[0127] 2. Synthesis and Characterization of SerMA-PC@PNVs

[0128] Serin was extracted from natural silkworm cocoons using a hot alkali degumming method, and then sericin was chemically modified with methyl methacrylate (MA) to prepare SerMA. The modification effect of MA on sericin was verified using proton nuclear magnetic resonance (HMR) spectroscopy. Figure 5 The example shown in section A 1 The appearance of new C=C peaks at δ = 5.7 ppm and 6.1 ppm in the ¹H NMR spectrum confirmed the successful coupling of MA and SerMA. SerMA and LAP were mixed in PBS (SerMA concentration: 150 mg / mL, LAP concentration: 5 mg / mL), and the gelation process of SerMA was visually tested using reaction flasks. The experimental results are as follows... Figure 5 As shown in Figure B, SerMA was the mobile phase before irradiation with 405 nm blue light, exhibiting good fluidity in the container. When the reaction flask was placed upright and irradiated with 405 nm blue light for 1 minute, the SerMA inside the flask transformed into a gel, demonstrating that SerMA possesses the ability to transform from a mobile phase into a hydrogel under photocuring conditions. Figure 5 The C-type display shows that SerMA and SerMA-PC@PNVs have good light transmittance, making them suitable for intraocular implantation. Figure 5 In, Ⅰ: sericin; Ⅱ: SerMA; Ⅲ: SerMA-PC@PNVs (PC: 50μg / mL); Ⅳ: SerMA-PC@PNVs (PC: 100μg / mL); Ⅴ: Ser MA-PC@PNVs (PC: 200μg / mL); VI: SerMA-PC@PNVs (PC: 400μg / mL); VII: SerMA-PC@PNVs (PC: 800μg / mL)).

[0129] To investigate the effect of PC@PNVs doping on the microstructure of SerMA hydrogels, the porous morphology of the hydrogels was characterized using SEM. Figure 5 As shown in Figure D, both SerMA and SerMA-PC@PNVs lyophilized hydrogels exhibit a distinct three-dimensional porous morphology, with pore sizes mainly distributed in the range of 80–150 nm, sufficient to support cell survival. Figure 5 As shown in Figure E, functional group analysis of Sericin, SerMA, and SerMA-PC@PNVs was performed using FTIR. Experimental results show that the doping of PC@PNVs affects the amide I band (1643 cm⁻¹). -1 ), Amide II band (1514cm) -1 ) and amide III band (1069cm) -1 The absorption peak of PC@PNVs has little effect, indicating that doping with PC@PNVs does not affect the protein structure of SerMA.

[0130] like Figure 5As shown in F, the compressive properties of SerMA and SerMA-PC@PNVs hydrogels were tested. The results showed that the compressive strength initially increased with increasing PC@PNVs content, but then decreased. SerMA-PC@PNVs exhibited the best compressive strength, approximately 127.7 kPa, when the PC content was 400 μg / mL.

[0131] Furthermore, since PC is a natural antioxidant, antioxidant experiments were conducted on SerMA-PC@PNVs. For example... Figure 5 As shown in G, based on the absorbance of the solution at 515 nm after the reaction, the DPPH scavenging efficiency of the control group SerMA hydrogel was approximately 24.9%, the DPPH scavenging efficiency of the SerMA-PC@PNVs hydrogel with a PC concentration of 50 μg / mL was approximately 46.0%, and the DPPH scavenging efficiency of the SerMA-PC@PNVs hydrogel with a PC concentration of 100 μg / mL was approximately 57.4%. Furthermore, the DPPH scavenging efficiencies of the SerMA-PC@PNVs hydrogels with PC concentrations of 200 μg / mL, 400 μg / mL, and 800 μg / mL were approximately 67.1%, 70.7%, and 73.0%, respectively. The experimental results indicate that the DPPH scavenging efficiency gradually increases with increasing PC concentration.

[0132] 3. In vitro cell experiments

[0133] To determine the stem cell characteristics of the obtained PDLSCs, immunofluorescence staining was first performed. The results showed significant expression of two major stem cell surface markers, CD146 and STRO-1. Figure 6 (A) This confirmed the stemness of the cells. SerMA and SerMA-PC@PNVs hydrogels were lyophilized, and the extract was then extracted to test its effect on the cell viability of PDLSCs. Figure 6 As shown in Figure B (where II: SerMA; III: SerMA-PC@PNVs (PC: 50 μg / mL); IV: SerMA-PC@PNVs (PC: 100 μg / mL); V: SerMA-PC@PNVs (PC: 200 μg / mL); VI: SerMA-PC@PNVs (PC: 400 μg / mL); VII: SerMA-PC@PNVs (PC: 800 μg / mL)), on day 3, cell viability decreased when the PC concentration was 800 μg / mL. Based on the combined results of the stress resistance and DPPH scavenging experiments, a PC@PNVs concentration of 400 μg / mL was ultimately selected for covalent cross-linking with the SerMA hydrogel to prepare the SerMA-PC@PNVs hydrogel.

[0134] Furthermore, drug release studies were conducted on covalently cross-linked SerMA-PC@PNVs hydrogels and physically doped SerMA / PC. For example... Figure 7 As shown, on day 1, the release rate of PC in the SerMA / PC hydrogel was approximately 20.2%, and the release rate of PC in the SerMA-PC@PNVs hydrogel was approximately 10.8%, indicating that covalent cross-linking prevented the burst release of PC. Meanwhile, on day 30, the release rate of PC in the SerMA / PC hydrogel was approximately 72.9%, and the release rate of PC in the SerMA-PC@PNVs hydrogel was approximately 53.7%, indicating that covalent cross-linking is more conducive to the sustained release of the drug.

[0135] The effect of SerMA-PC@PNVs hydrogel on the secretion level of neurotrophic factors in PDLSCs was determined using an ELISA kit. The results showed that SerMA-PC@PNVs hydrogel could promote BDNF (diuretic factor-dependent neurotrophic factor secretion). Figure 6 C) IGF-1 Figure 6 D in (C) CNTF ( Figure 6 E) and NGF( Figure 6 It secretes various neurotrophic factors such as F.

[0136] R28 cells were treated with H2O2 to establish a cell model of oxidative stress. Figure 8 As shown in Figure A, when the H2O2 concentration was 80 μM, the survival rate of R28 cells was approximately 46.2%. Therefore, an 80 μM H2O2 concentration was chosen to establish a cell model of oxidative stress. Furthermore, the protective effects of SerMA-PC@PNVs and SerMA-PC@PNVs / PDLSCs against oxidative stress in R28 cells were investigated. Figure 8 As shown in Figure B, H2O2 stimulation promotes R28 cell death, while SerMA-PC@PNVs promote R28 cell survival through their antioxidant effects. SerMA-PC@PNVs / PDLSCs exhibit the best effect in promoting R28 cell survival, possibly because the hydrogel promotes the secretion of neurotrophic factors by PDLSCs, thereby enhancing R28 cell survival. Subsequently, flow cytometry analysis was used to investigate the effects of SerMA-PC@PNVs and SerMA-PC@PNVs / PDLSCs on R28 cell apoptosis. Figure 8 As shown in C and D, the apoptosis rate of R28 cells after H2O2 stimulation was approximately 44.8%, the apoptosis rate of R28 cells after SerMA-PC@PNVs treatment was approximately 22.4%, and the apoptosis rate of R28 cells after SerMA-PC@PNVs / PDLSCs treatment was approximately 14.1%.

[0137] Furthermore, reactive oxygen species (ROS) assays were performed on R28 cells using DCFH-DA and DHE. Figure 9 As shown in Figure A, H2O2 treatment of R28 cells produces a large amount of reactive oxygen species and emits fluorescence. When treated with SerMA-PC@PNVs or SerMA-PC@PNVs / PDLSCs, the fluorescence of R28 cells is significantly reduced, and the ROS is greatly decreased, indicating that the hydrogel has good antioxidant properties.

[0138] To simulate inflammation-mediated activation, HAPI cells were stimulated with 1 μg / mL LPS to promote their transformation into the M1 inflammatory phenotype. SerMA-PC@PNVs or SerMA-PC@PNVs / PDLSCs hydrogels were then incubated in the upper Transwell chambers for 24 h. Results showed that the proportion of M1-type HAPI cells was approximately 31.4% in the Control group (LPS stimulation), approximately 16.4% in the SerMA-PC@PNVs group, and approximately 7.81% in the SerMA-PC@PNVs / PDLSCs group. Figure 9 (B and D in the text). Furthermore, the proportion of M2 type HAPI in the Control group was approximately 1.44%, in the SerMA-PC@PNVs group approximately 10.9%, and in the SerMA-PC@PNVs / PDLSCs group approximately 18.3% (…). Figure 9 (C and E in the text). In summary, SerMA-PC@PNVs hydrogel can promote the conversion of pro-inflammatory M1 HAPI to anti-inflammatory M2 HAPI, and SerMA-PC@PNVs / PDLSCs have better anti-inflammatory effects after being loaded with stem cells.

[0139] 4. In vivo animal experimental studies

[0140] The main characteristics of glaucoma are selective death of rhabdomyocardial cells (RGCs) and optic nerve atrophy, as well as visual field defects, caused by pathologically high intraocular pressure. Currently, commonly used clinical treatments such as laser therapy, medication, and surgery primarily control pathologically high intraocular pressure, but they cannot reverse the already damaged optic nerve and visual function. Furthermore, even after intraocular pressure is controlled within the normal range, some patients experience further damage to their optic nerve and visual function, ultimately leading to complete visual loss. This study established a glaucoma animal model in SD rats by intravenous injection of hypertonic saline into the sclera. This method constructs an elevated intraocular pressure model through endothelial cell damage and venous sinus fibrosis.

[0141] After successful glaucoma model establishment, the experiment was divided into three groups: NC, COH, and Test (the Test group consisted of SerMA-PC@PNVs / PDLSCs). Figure 10As shown, compared with the NC group, the intraocular pressure in the COH group and the Test group remained elevated, indicating that the chronic high intraocular pressure model of glaucoma was successfully established. Eight weeks after modeling, ocular tissues from different groups were harvested for retinal smears and RGCs counting. Figure 11 As shown in A and B, the RGC density in the normal group is approximately 264 cells / 0.184 mm. 2 The RGC density in the COH group was approximately 117 per 0.184 mm. 2 The RGC density in the Test group was approximately 221 per 0.184 mm. 2 Experimental results show that SerMA-PC@PNVs / PDLSCs have excellent optic nerve protection effects and can promote the survival of RGCs.

[0142] Furthermore, ocular tissues from different groups were fixed, followed by H&E staining and RBPMS immunofluorescence staining. Figure 11 As shown in C and D, the density of RGCs in the Test group was significantly increased compared to the COH group, consistent with the results of retinal patch analysis, indicating that SerMA-PC@PNVs / PDLSCs can promote the survival of damaged RGCs. Furthermore, the polarization status of microglia in rat retinal tissue was analyzed. Figure 11 As shown in Figure E, CD86 fluorescence was enhanced in the COH group, while it decreased in the Test group after treatment with SerMA-PC@PNVs / PDLSCs. This indicates a reduction in M1 microglia in the Test group, suggesting that SerMA-PC@PNVs / PDLSCs treatment can alleviate inflammation. Furthermore, compared to the COH group, the Test group showed stronger CD206 fluorescence, indicating that SerMA-PC@PNVs / PDLSCs promoted the transformation of microglia to M2 type, demonstrating excellent anti-inflammatory effects. In summary, SerMA-PC@PNVs / PDLSCs may exert their anti-inflammatory effect by promoting the transformation of pro-inflammatory M1 microglia to anti-inflammatory M2 microglia, thereby promoting RGC survival.

[0143] 5. Rat retinal RNA-Seq sequencing analysis

[0144] To systematically elucidate the molecular mechanism by which SerMA-PC@PNVs / PDLSCs exert their effects, RNA-Seq sequencing analysis was first performed on rat retinal tissue. One month after SerMA-PC@PNVs / PDLSCs injection, retinal tissues from the COH and Test treatment groups were collected, rapidly transferred to pre-frozen tubes, flash-frozen in liquid nitrogen for 5 min, and then stored at -80°C to maintain RNA integrity. A total of 12,352 co-expressed genes were detected in the retinal tissues of both groups, with 749 and 111 specifically expressed genes in the COH and Test groups, respectively. Figure 12 (A) Principal component analysis (PCA) plots showed significant separation between the COH and Test groups at the transcriptomic level, indicating a clear difference in gene expression profiles between the two groups. Figure 12 (B) Violin Diagram ( Figure 12 The C) analysis showed consistent sequencing depth, ensuring the reliability of subsequent data analysis. Further differential gene expression analysis revealed that, compared to the COH group, 488 genes were significantly upregulated and 2955 genes were significantly downregulated in the Test group. Figure 12 D and Figure 13 The focus was on analyzing changes in the expression of inflammation-related genes, particularly those associated with the inflammatory response. Figure 14 The results showed that the expression levels of several key genes closely related to the inflammatory response were significantly increased in the Test group, indicating that SerMA-PC@PNVs / PDLSCs may play an anti-inflammatory role. To systematically analyze the signaling pathways involved in these differentially expressed genes, further GO enrichment analysis and KEGG pathway enrichment analysis were performed. Figure 15 and Figure 16 GO analysis showed that the pathways significantly enriched in the Test group included key biological processes such as "Immune system process," "Regulation of cytokine production," "Inflammatory response," and "Apoptotic process." Figure 15Meanwhile, KEGG functional enrichment analysis showed that differentially expressed genes in the Test group were involved in signaling pathways closely related to inflammatory responses and apoptosis, including the "Chemokine signaling pathway," "MAPK signaling pathway," "TNF signaling pathway," and "Apoptosis." Figure 16 In summary, the RNA sequencing results further confirm that the SerMA-PC@PNVs / PDLSCs hydrogel can affect inflammation-related pathways, consistent with in vivo experiments, thereby promoting RGC survival.

[0145] Furthermore, single-cell sequencing analysis was performed on the retinal tissues of the COH and Test groups. First, we performed cell population analysis on the retinal tissues. For example... Figure 17 As shown, the cells were divided into 10 groups, including bipolar cells, cone cells, endothelial cells, horizontal cells, microglia, Müller cells, neutrophils, retinal ganglion cells (RGCs), rod cells, and retinal pigment epithelial cells (RPEs). Figure 18 As shown, compared with the COH group, the Test group showed an increased proportion of RGC cells and a decreased proportion of Microglia and Neutrophils cells. These results indicate that injection of SerMA-PC@PNVs / PDLSCs hydrogel can promote RGC survival while reducing the proportion of inflammation-related cells, Microglia and Neutrophils.

[0146] The next step is to further analyze the RGC and Microglia cell populations. For example... Figure 19As shown, RGCs were divided into 9 cell subpopulations. Further analysis of differentially expressed genes in Microglia from the COH and Test groups revealed changes in several inflammation-related genes. To systematically investigate the signaling pathways involved in these differentially expressed genes, GO enrichment and KEGG pathway enrichment analyses were performed. KEGG analysis showed that significantly enriched pathways in the Test group included the "TNF signaling pathway," "apoptosis," "Toll-like receptor signaling pathway," and "NF-κB signaling pathway," all closely related to inflammation and apoptosis. Figure 20 Meanwhile, GO functional enrichment analysis showed that differentially expressed genes in the Test group were involved in key biological processes such as "inflammatory response," "negative regulation of neuron apoptotic process," "response to oxidative stress," and "apoptotic signaling pathway." Figure 21 In addition, biosafety studies were conducted, and HE staining was performed on the major organs (heart, liver, spleen, lung, and kidney) of different groups of rats. Figure 22 As shown, there was no difference in HE staining among the NC, COH, and Test groups, indicating that the hydrogel has good biocompatibility. In summary, the retinal single-cell sequencing results suggest that the SerMA-PC@PNVs / PDLSCs hydrogel may promote RGC survival by influencing inflammation-related pathways and regulating the state of microglia.

[0147] III. Conclusion

[0148] This study successfully constructed a stem cell-loaded nanovesicle hydrogel, SerMA-PC@PNVs / PDLSCs, which exerts anti-inflammatory effects through antioxidant activity and microglia regulation, thereby promoting the repair of glaucoma rhabdomyosarcoma (RGC) damage. This hydrogel can promote the secretion of more neurotrophic factors by PDLSCs. In vitro and in vivo experimental studies showed that this hydrogel can inhibit M1 microglia polarization and promote M2 microglia polarization, thus exerting anti-inflammatory effects and promoting RGC survival. Rat retinal RNA-Seq sequencing showed that this hydrogel can affect pathways related to "inflammatory response" and "apoptotic process." Rat retinal single-cell sequencing showed that this hydrogel can increase the number of RGC cells and reduce the number of inflammation-related Neutrophils and Microglia cells. Further analysis of Microglia cell subsets, GO analysis, showed that this hydrogel can affect pathways related to "inflammatory response." In summary, the stem cell-loaded SerMA-PC@PNVs hydrogel can exert a neuroprotective effect on glaucoma by regulating microglia polarization and influencing the inflammatory response.

[0149] Example 2: Optimization of PNVs and PC Ratio

[0150] PNVs and PC were mixed in PBS at mass ratios of 1:1, 1:2, and 2:1, and the mixture was ultrasonically dispersed in an ice-water bath. The mixture was then repeatedly extruded through porous membranes at 800 nm, 400 nm, 200 nm, and 100 nm using a liposome extruder. Finally, drug-loaded nanovesicles were collected by ultracentrifugation at 150,000 × g for 70 min at 4 °C to obtain PC@PNVs. The hydrodynamic diameter was determined using a dynamic light scattering (DLS) system (Zetasizer Nano ZS90, Malvern, UK). Results are as follows: Figure 23 As shown, continuous monitoring of particle size changes in PBS buffer for 7 days at a 1:1 mass ratio revealed a small range of particle size fluctuations, indicating that PC@PNVs at a 1:1 mass ratio have excellent physical stability. A 1:1 mass ratio is preferred for further experimental studies.

[0151] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hydrogel composition, characterized in that, It was prepared by irradiation with 405 nm light in the presence of a photoinitiator by methacrylamide-modified sericin and drug-loaded nanovesicles. The drug-loaded nanovesicles include: AC-PEG-NHS modified cell membrane vesicles and glaucoma treatment drugs; The mass ratio of the AC-PEG-NHS modified cell membrane vesicles to the glaucoma treatment drug is 1:2 to 2:

1. The glaucoma treatment drug is proanthocyanidins; The cell membrane vesicles in the AC-PEG-NHS modified cell membrane vesicles are derived from human periodontal ligament stem cells. The AC-PEG-NHS modified cell membrane vesicles are obtained by modifying cell membrane vesicles with AC-PEG-NHS. The photoinitiator is LAP.

2. The hydrogel composition according to claim 1, characterized in that, The concentration of the glaucoma treatment drug is 50 μg / mL to 800 μg / mL.

3. A mixture containing stem cells, characterized in that, The product is prepared by irradiation with 405 nm light in the presence of a photoinitiator, consisting of stem cells, methacrylamide-modified sericin in the hydrogel composition of claim 1 or 2, and drug-loaded nanovesicles. The photoinitiator is LAP; The stem cells mentioned are periodontal ligament stem cells.

4. Use of at least one of the following shown in I) to II) in the preparation of products for the treatment and / or prevention of eye diseases: I) The hydrogel composition according to claim 1 or 2; II) The mixture according to claim 3; The eye disease mentioned is glaucoma.

5. A product for the treatment and / or prevention of eye diseases, characterized in that, Including at least one of the following: i) to ii) i) The hydrogel composition according to claim 1 or 2; ii) The mixture according to claim 3; The eye disease mentioned is glaucoma.

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