Microgel, medicine, preparation method and application

Abstract

The invention discloses microgel, a medicine, a preparation method and application, and relates to the technical field of peritoneal fibrosis. The microgel comprises alginate microgel, and primary human peritoneal mesothelial cells are wrapped in the alginate microgel. The primary human peritoneal mesothelial cells are packaged in the microgel to prevent the primary human peritoneal mesothelial cells from being in direct contact with peritoneal dialysis liquid, the survival rate of the peritoneal mesothelial cells is increased, and the primary human peritoneal mesothelial cells are gradually released by utilizing the slow release effect of the microgel, so that peritoneal mesothelial cell deficiency caused by peritoneal dialysis is supplemented. By inhibiting the POSTN / NF-kB / CXCL8 pathway, the microgel wrapped with the primary human peritoneal mesothelial cells relieves peritoneal fibrosis related to peritoneal dialysis, and the microgel provided by the invention is a promising treatment method for treating and relieving peritoneal fibrosis of peritoneal dialysis patients.

Description

Technical Field

[0001] The present invention relates to the technical field of peritoneal fibrosis, and in particular to a microgel, a medicine, a preparation method and an application thereof. Background Art

[0002] Chronic kidney disease (CKD) is a major global health problem. It is estimated that by 2022, the prevalence of CKD in adults worldwide will be approximately 10-13%, and more than 800 million people will have varying degrees of CKD [1]. Peritoneal dialysis (PD), as a common renal replacement therapy, is widely used in patients with CKD [2-4]. Although peritoneal dialysis is effective in the treatment of CKD, peritoneal fibrosis in dialysis patients has become a concern.

[0003] Peritoneal fibrosis is the most common complication in patients undergoing long-term peritoneal dialysis [5]. Studies have shown that recurrent peritonitis and long-term exposure to nonphysiological peritoneal dialysis fluid can stimulate the structure and function of peritoneal mesothelial cells (PMCs). Peritoneal mesothelial cells participate in inflammatory responses and extracellular matrix accumulation, ultimately leading to peritoneal fibrosis [6]. As peritoneal fibrosis worsens, the permeability of the peritoneum to peritoneal dialysis fluid decreases, resulting in decreased dialysis efficacy. In severe cases, patients need to switch to other treatments [7].

[0004] During peritoneal dialysis, the reduction of peritoneal mesothelial cells is one of the main causes of peritoneal dysfunction. Peritoneal mesothelial cells are the main cells that maintain the structure and function of the peritoneum. During peritoneal dialysis, repeated physical pressure and friction can cause damage and loss of peritoneal mesothelial cells. At the same time, peritoneal dialysis fluid is usually characterized by high glucose concentration, low pH and high osmotic pressure, which can have toxic effects on peritoneal mesothelial cells and stimulate peritoneal mesothelial cells to trigger high glucose metabolism and inflammation, leading to abnormal peritoneal mesothelial cell function and the occurrence of epithelial-mesenchymal transition [8,9].

[0005] Studies have shown that peritoneal inflammation is a key event in the development of peritoneal fibrosis

[10] . CXCL8, as an important chemokine, plays a key role in inflammation and immune response. Recent studies have increasingly focused on the role of CXCL8 in kidney disease. Studies have shown that the expression level of CXCL8 is increased in extracellular vesicles in the peritoneal dialysis waste fluid of peritoneal dialysis patients with high GP96 expression

[11] . In addition, peritoneal dialysis patients with peritoneal inflammation have increased CXCL8 expression levels

[12] . Overexpression of CXCL8 may lead to inflammatory responses and promote the occurrence and development of peritoneal fibrosis.

[0006] NF-κB is a family of transcription factor proteins that plays an important role in cellular inflammation and immune responses

[13] . Studies have shown that activation of the PKCa / IKK signaling pathway can trigger NF-κB activation, thereby promoting the expression and release of IL-8 / CXCL8 in human lung epithelial cells

[14] . Interestingly, in peritoneal dialysis, mitochondrial damage can induce a mild inflammatory response in peritoneal mesothelial cells, making them vulnerable to damage from the peritoneal environment. This process involves the generation of oxidative stress and the activation of NF-κB, and the use of mitoTEMPO (mitochondrial oxidative stress scavenger) and BAY-11708 (NF-κB inhibitor) can alleviate inflammation

[15] .

[0007] Osteopontin (POSTN) is a non-structural extracellular matrix protein that promotes the deposition of extracellular matrix and the proliferation of interstitial cells, and plays an important role in tissue regeneration and fibrosis [16,17]. POSTN is expressed in a variety of tissues, and its expression level is closely related to the occurrence of a variety of diseases, especially in fibrotic diseases

[18] . Studies have found that serum POSTN levels are significantly increased in patients with idiopathic pulmonary fibrosis. During acute attacks, POSTN accumulates significantly in fibrotic lesions

[19] . In addition, POSTN can induce the activation of NF-κB / STAT3, ultimately promoting the occurrence of inflammation

[20] . Importantly, multiple studies have shown that POSTN also plays a role in the progression of renal fibrosis, making it a potential new therapeutic target for chronic kidney disease [21,22]. However, there is currently a lack of reports on whether POSTN induces peritoneal fibrosis.

[0008] Sodium alginate (SA) microgel is a naturally occurring linear polysaccharide extracted from the cell wall of edible seaweed. It has excellent biocompatibility and biodegradability and is widely used in drug delivery, biomedical materials and tissue engineering [23,24]. In addition to being a drug delivery carrier, sodium alginate microgels are increasingly recognized as cell carriers that encapsulate and protect living cells, enhance cell viability, and thus promote cell transplantation and tissue engineering repair [25,26]. Huang et al. demonstrated that chitosan and sodium alginate coated microcapsules filled with maltodextrin can achieve sustained release of astaxanthin and enhance its stability

[27] . Pan et al. used this technology to prepare SA microspheres encapsulating lactic acid bacteria to avoid direct contact with digestive fluid, thereby increasing the number of lactic acid bacteria reaching the small intestine and effectively regulating host health

[28] . Sodium alginate microgels have been shown to successfully encapsulate various types of living cells, such as stem cells and insulin-producing cells, and show broad application prospects in tissue engineering and regenerative medicine [29-31]. Currently, there is a lack of research on the use of sodium alginate microgels to encapsulate primary human peritoneal mesothelial cells for the treatment of peritoneal fibrosis.

[0009] In view of this, the present invention is proposed. Summary of the invention

[0010] The purpose of the present invention is to provide a microgel, a drug, a preparation method and an application to solve the above technical problems.

[0011] The present invention is achieved in that:

[0012] In a first aspect, the present invention provides a microgel, comprising: an alginate microgel, wherein primary human peritoneal mesothelial cells are encapsulated in the alginate microgel.

[0013] In a second aspect, the present invention also provides a method for preparing a microgel, which comprises the following steps: uniformly mixing an aqueous phase and an oil phase obtained by mixing alginate, a chelated exchange ion solution and primary human peritoneal mesothelial cells through a microfluidic chip; and obtaining a chelated exchange ion solution by mixing the exchange ion solution with a chelating agent.

[0014] In a third aspect, the present invention further provides a microgel or a use of the microgel prepared by the above preparation method in preparing a drug for alleviating or treating peritoneal fibrosis;

[0015] In a fourth aspect, the present invention further provides a drug, which comprises the above-mentioned microgel or the microgel prepared by the above-mentioned preparation method.

[0016] The present invention has the following beneficial effects: the present invention encapsulates primary human peritoneal mesothelial cells in microgels to prevent them from direct contact with peritoneal dialysis fluid, improves the survival rate of peritoneal mesothelial cells, and gradually releases primary human peritoneal mesothelial cells by utilizing the sustained-release effect of microgels, thereby replenishing lost cells. By inhibiting the POSTN / NF-κB / CXCL8 pathway, peritoneal fibrosis associated with peritoneal dialysis is alleviated.

[0017] Compared with blank sodium alginate microgel (eSA) treatment, microcapsules encapsulating primary human peritoneal mesothelial cells have significant advantages in inhibiting peritoneal fibrosis and improving cell survival rate. In addition, through biosafety assessment, the microgels provided by the present invention did not cause obvious adverse reactions in peritoneal dialysis model mice, maintained normal liver and kidney function in mice, and showed no histological abnormalities in major organs.

[0018] The sodium alginate microgel encapsulating primary human peritoneal mesothelial cells provided by the present invention is a promising treatment method for alleviating peritoneal fibrosis in peritoneal dialysis patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 The process flow chart of sodium alginate microgel (SA), the microstructure characterization diagram of microgel and the CCK8 assay results (A. The preparation process of sodium alginate microgel; B. The morphology of blank sodium alginate microgel (eSA) and sodium alginate microgel wrapped with primary human peritoneal mesothelial cells (SA / PMC) shown under an optical microscope, scale bar = 200 μm; C. The CCK8 assay results of PMC+eSA and PMC+SA / PMC, showing the absorbance at OD = 450 nm. ▲P<0.05, ***P<0.001, ###P<0.001);

[0021] Figure 2 Immunofluorescence staining of vimentin and cytokeratin 18 (CK18) and growth morphology of primary human peritoneal mesothelial cells (PHPMCs) in patients with maintenance peritoneal dialysis for 1 week (1W-PD), 1 year (1Y-PD) and 3 years (3Y-PD) (A. Immunofluorescence staining of vimentin and CK18; scale bar = 50 μm; B. Growth morphology of PHPMCs in patients with 1W-PD, 1Y-PD and 3Y-PD, scale bar = 100 μm);

[0022] Figure 3Immunofluorescence staining, mRNA expression, and Western blot images of osteopontin (POSTN), nuclear factor-κB p65 (NF-κB P65), phosphorylated nuclear factor-κB p65 (NF-κB p-P65), interleukin-8 (CXCL8), α-smooth muscle actin (α-SMA), type I collagen (Col-1), transforming growth factor-β (TGF-β), and fibronectin (FN) in 1W-PD and 1Y-PD patients (AH. Immunofluorescence staining of POSTN, NF-κB P65, NF-κB p-P65, CXCL8, α-SMA, Col-1, TGF-β, and FN in 1W-PD and 1Y-PD patients; IO. mRNA expression levels of P65, CXCL8, α-SMA, Col-1, TGF-β and FN in 1W-PD and 1Y-PD patients; PU. Western blot and quantitative analysis of POSTN, NF-κBP65, NF-κB p-P65, CXCL8, α-SMA and Col-1 in 1W-PD and 1Y-PD patients. Scale bar = 50 μm. **P<0.01, ***P<0.001);

[0023] Figure 4 The results of the detection of POSTN, NF-κB P65 and CXCL8 expression levels in peritoneal mesothelial cells induced by high glucose medium (A. Immunofluorescence staining of POSTN, NF-κB P65, NF-κB p-P65 and CXCL8 in peritoneal mesothelial cells induced by high glucose medium; B. Immunofluorescence staining of α-SMA, Col-1, TGF-β and FN in peritoneal mesothelial cells induced by high glucose medium; CI. mRNA expression levels of POSTN, NF-κB P65, CXCL8, α-SMA, Col-1, TGF-β and FN in PMCs; JN. Western blot and quantitative analysis of POSTN, NF-κB P65, CXCL8, α-SMA and GAPDH in PMCs; Scale bar = 50 μm. #P<0.05, ##P<0.01, **P<0.01, ***P<0.001);

[0024] Figure 5H&E staining and Masson staining of the parietal peritoneum of peritoneal dialysis model mice, quantification of peritoneal wall thickness, immunohistochemistry, Western blot and quantitative analysis (A. H&E staining and Masson staining of the peritoneum of peritoneal dialysis model mice; B. Quantitative analysis of peritoneal wall thickness of peritoneal dialysis model mice; CJ. Immunohistochemistry and quantitative analysis of POSTN, NF-κBP65, NF-κB p-P65, CXCL8, α-SMA, Col-1 and TGF-β in peritoneal tissue of peritoneal dialysis model mice; KN. Western blot and quantitative analysis of POSTN, NF-κBP65, CXCL8 and GAPDH; O. POSTN, NF-κB P65, NF-κB Immunofluorescence staining of p-P65, CXCL8, α-SMA, Col-1, TGF-β and FN; scale bar = 50 μm. #P<0.05,▲P<0.05,***P<0.001,###P<0.001,▲▲▲P<0.001);

[0025] Figure 6 Figure 1 is the result of the biosafety experiment of sodium alginate microgel (A. Image of hemolysis test; hemolysis rate in BA; C. Weight change of model mice before and after peritoneal dialysis; D. Serum alanine aminotransferase (ALT) level of peritoneal dialysis model mice; E. Serum aspartate aminotransferase (AST) level of peritoneal dialysis model mice; F. Serum urea nitrogen (BUN) level of peritoneal dialysis model mice; G. Creatinine (Scr) level of peritoneal dialysis model mice; H. Glomerular filtration rate (eGFR) level of peritoneal dialysis model mice; IL. H&E staining and Masson staining of liver, heart, lung and kidney of peritoneal dialysis model mice; Scale bar = 50 μm. ns = no statistical difference);

[0026] Figure 7 Schematic diagram of the role of POSTN / NF-κB P65 / CXCL8 in peritoneal dialysis-induced peritoneal fibrosis. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0028] In a first aspect, the present invention provides a microgel, comprising: an alginate microgel, wherein primary human peritoneal mesothelial cells are encapsulated in the alginate microgel.

[0029] The microcapsules provided by the present invention can encapsulate primary human peritoneal mesothelial cells (PHPMCs) to prevent them from direct contact with peritoneal dialysis fluid, thereby improving the survival rate of peritoneal mesothelial cells and gradually releasing the primary human peritoneal mesothelial cells by utilizing the sustained-release effect of the microgel, thereby replenishing the lost cells.

[0030] In a preferred embodiment of the present invention, 1-50 primary human peritoneal mesothelial cells are encapsulated in each alginate microgel. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 30, 35, 37, 40, 45, 48 or 50 primary human peritoneal mesothelial cells are encapsulated in each alginate microgel.

[0031] In a preferred embodiment of the present invention, alginate includes but is not limited to sodium alginate, potassium alginate and calcium alginate. Any alginate is acceptable as long as it can encapsulate primary human peritoneal mesothelial cells.

[0032] In a second aspect, the present invention also provides a method for preparing a microgel, which comprises the following steps: uniformly mixing an aqueous phase and an oil phase obtained by mixing alginate, a chelated exchange ion solution and primary human peritoneal mesothelial cells through a microfluidic chip; and obtaining a chelated exchange ion solution by mixing the exchange ion solution with a chelating agent.

[0033] Using droplet microfluidics technology, the water phase and the oil phase are respectively introduced into the microfluidic chip under the drive of the device. The water phase breaks through the oil phase to disperse the water phase into microdroplets, forming alginate microgels encapsulating primary human peritoneal mesothelial cells.

[0034] In a preferred embodiment of the present invention, the flow rate ratio of the water phase to the oil phase is 1:4-1.5. At the above flow rate, microgels with a high encapsulation capacity of primary human peritoneal mesothelial cells can be obtained.

[0035] In a preferred embodiment of the present invention, the flow rate ratio of the water phase to the oil phase is 1:5.

[0036] In a preferred embodiment of the present invention, the chelate exchange ion solution is obtained by mixing the exchange ion solution and the chelating agent at a molar ratio of 1:1-1:1.1.

[0037] In a preferred embodiment of the present invention, the exchange ion solution is selected from a solution containing Ca 2+ 、Zn 2+ One of the solutions.

[0038] The exchange ion chelating agent is selected from: one of EDTA, PDTA, CDTA and salts thereof, preferably EDTA.

[0039] In a preferred embodiment of the present invention, the alginate is selected from sodium alginate, potassium alginate and calcium alginate.

[0040] In a preferred embodiment of the present invention, the chelate exchange ion solution is selected from calcium disodium ethylenediaminetetraacetate; the alginate is selected from sodium alginate; under the above combination, the sodium ions in the sodium alginate molecules can be more efficiently exchanged with calcium ions to form calcium alginate, and through this exchange reaction, a calcium alginate gel with a three-dimensional network structure is formed.

[0041] In a preferred embodiment of the present invention, in the aqueous phase, the concentration of the ion exchange agent is 38-42 mM; the mass concentration of alginate is 1.8-2.2%. Under the above mixing conditions, a higher ion exchange efficiency is achieved. For example, the concentration of the ion exchange agent is 38 mM, 39 mM, 40 mM, 41 mM, 42 mM; the concentration of alginate is 1.8%, 1.9%, 2.0%, 2.1%, 2.2%.

[0042] In a preferred embodiment of the present invention, in the aqueous phase, 0.5-1 mL of the aqueous phase mixed reagent (alginate and chelate exchange ion solution) and 5×10 6 ~1×10 7 At the above mixing ratio, a higher microcapsule cell encapsulation rate can be obtained.

[0043] In a preferred embodiment of the present invention, the oil phase is selected from: a fluorinated liquid with a fluorinated surfactant added thereto or an alkane oil or other types of oil with a surfactant added thereto.

[0044] The oil phase is a microdroplet generating oil, for example from FluidicLab.

[0045] The surfactant is selected from any one of Span 80, Tween 80, EM 90 or EM 180; the alkane oil or other types of oil is selected from any one of mineral oil, cyclohexane, hexadecane, and isopropyl palmitate.

[0046] In a preferred embodiment of the present invention, the oil phase is selected from a fluorinated liquid to which a fluorinated surfactant is added;

[0047] In a preferred embodiment of the present invention, the fluorinated liquid is selected from any one of 1H, 1H, 2H, 2H-perfluorododecyltrichlorosilane, HFE-7500, HFE-7300, HFE-7200, HFE-7100, FC-40 or FC-70; the fluorinated surfactant is any one of an "ABA" type polymer, "Rf-L-Rh-L-Rf" or "Rh-L-Rf-L-Rh" type perfluoro polymer.

[0048] In a third aspect, the present invention further provides a microgel or use of the microgel prepared by the above preparation method in preparing a drug for alleviating or treating peritoneal fibrosis.

[0049] In a preferred embodiment of the present invention, peritoneal fibrosis is induced by peritoneal dialysis.

[0050] In a preferred embodiment of the present invention, the application includes at least one of the following application modes:

[0051] (1) Reduce POSTN levels;

[0052] (2) Inhibit the phosphorylation and nuclear translocation of NF-κB P65;

[0053] (3) inhibiting the expression of CXCL8;

[0054] (4) suppressing the levels of peritoneal fibrosis-related markers;

[0055] (5) Inhibition of epithelial-mesenchymal transition;

[0056] (6) Relieve peritoneal fibrosis;

[0057] (7) Reduce peritoneal thickness;

[0058] (8) replenish lost peritoneal mesothelial cells;

[0059] (9) Inhibit peritoneal inflammation;

[0060] In a preferred embodiment of the present invention, the fibrosis-related marker is selected from at least one of α-smooth muscle actin (α-SMA), type I collagen (COL1), transforming growth factor β (TGF-β) and fibronectin (FN);

[0061] In a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0062] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipient is selected from at least one of a filler, a disintegrant, a lubricant, a flavoring agent, a binder, a suspending agent and a flavor.

[0063] Pharmaceutically acceptable excipients include but are not limited to: pharmaceutically acceptable carriers, auxiliary substances or solvents. Pharmaceutically acceptable excipients include various organic or inorganic carriers and / or auxiliary materials because they are commonly used for pharmaceutical purposes, especially for solid pharmaceutical preparations. Examples include: excipients, such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders, such as cellulose, methylcellulose, hydroxypropyl cellulose, polypropyl pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch; disintegrants, such as starch, hydrolyzed starch, carboxymethyl cellulose, carboxymethyl cellulose calcium salt, hydroxypropyl starch, ethylene glycol starch sodium, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants, such as magnesium stearate, talc, sodium lauryl sulfate; flavors, such as citric acid, menthol, glycine , orange powder; preservatives, such as sodium benzoate, sodium bisulfite, parabens (such as methyl paraben, ethyl paraben, propyl paraben, butyl paraben); stabilizers, such as citric acid, sodium citrate, acetic acid and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents, such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents, such as water, organic solvents; waxes, fats and oils, such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0064] In a preferred embodiment of the present invention, the dosage form of the drug is tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections or sprays.

[0065] In an optional embodiment, the above-mentioned medicine is a liquid pharmaceutical preparation (such as a kind of injection), such as solution, suspension and gel usually contain liquid carrier, such as water and / or pharmaceutically acceptable organic solvent. In addition, such liquid preparations may also include pH adjusting agents, emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (such as methylcellulose), dyes and / or flavoring agents, such as defined above. The medicine may be isotonic, that is, they may have the same osmotic pressure as blood. The isotonicity of the medicine may be regulated by using sodium chloride and other pharmaceutically acceptable agents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic or organic soluble substances. The viscosity of the liquid composition may be regulated by pharmaceutically acceptable thickeners such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the selected agent.

[0066] In a preferred embodiment of the present invention, the medicament is formulated for administration by injection.

[0067] In a fourth aspect, the present invention further provides a drug, which comprises the above-mentioned microgel or the microgel prepared by the above-mentioned preparation method.

[0068] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0069] Example 1

[0070] This embodiment provides a method for preparing microcapsules.

[0071] Sodium alginate (MACKLIN, Shanghai, China) and Ca-EDTA were dissolved in distilled water to prepare a solution containing 2% sodium alginate and 40 mM Ca-EDTA (1 ml), which was filtered through a 0.22 μm filter (JETBIOFIL, China), followed by the addition of 1×10 7 Primary human peritoneal mesothelial cells (derived from the peritoneum of patients in the First Affiliated Hospital of Wenzhou Medical University) were added to obtain the aqueous phase. 5 mL of droplet generation oil (FluidicLab, China) was filtered through a 0.22 μm filter to obtain the oil phase. The aqueous phase and the oil phase were loaded into syringes respectively and mounted on an injection micropump (LSP01-3A, LONGER, UK). The device was connected to a microfluidic chip (PDMS-FF-100B, FluidicLab, China) to ensure uniform flow of the two liquids through the chip.

[0072] The flow rate of the aqueous phase was set to 7 μL / min, and the flow rate of the oil phase was set to 35 μL / / min.

[0073] After the gel started to form, the material generated in the first 10 min was removed, the product generated after the 10 min was collected, and the material generated in the last 10 min was removed.

[0074] The resulting gel was collected for further studies.

[0075] Example 2

[0076] Compared with Example 1, the only difference is that the flow rates of the water phase and the oil phase are different: the flow rate of the water phase is 1800 μL / min (70 drops / min), the flow rate of the oil phase is 30 μL / min, the particle size of the generated microgel is 1.8-2.4 mm, and the diameter of the microgel is significantly increased. Double staining of live cells and dead cells with Calcein-AM / PI found that cell proliferation and release were slow, but cell activity could be maintained. After freeze-drying, the volume of the microgel was reduced, and cell attachment was visible under an electron microscope.

[0077] Comparative Example 1

[0078] Blank sodium alginate microgel (eSA) was prepared. Compared with Example 1, the only difference was that 1×10 7The remaining steps were the same as those in Example 1.

[0079] Experimental Example 1

[0080] 1. The sodium alginate microgels prepared in Example 1 and Comparative Example 1 were characterized.

[0081] Figure 1 Figure A shows the production process of sodium alginate microgel. Optical microscope photo (see Figure 1 Figure B) shows that the diameter of the blank sodium alginate microgel (eSA) of comparative example 1 is about 60-100 μm. It is worth noting that encapsulating primary human peritoneal mesothelial cells in sodium alginate microgel (ie, prepared in Example 1) (SA / PMC) did not significantly change its diameter, and the encapsulation was successful.

[0082] 2.CCK8 Cell Proliferation Assay

[0083] Culture of peritoneal mesothelial cell lines:

[0084] The peritoneal mesothelial cell line was provided by BLUEFBIO (Shanghai, China). The peritoneal mesothelial cells were cultured in DMEM medium (Gibco, California, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were cultured in a constant temperature cell culture incubator at 37°C and 5% CO2. The culture medium was changed every 2-3 days to remove non-adherent cells and provide nutrients. When the cells reached 80-90% density, they were digested with 0.25% trypsin and re-seeded to expand the cells. The results of the CCK8 cell proliferation assay showed that after the peritoneal mesothelial cells were co-cultured with eSA and encapsulated SA for 48 hours, the proliferation of the peritoneal mesothelial cells in the encapsulated SA group was significantly faster than that in the eSA group (see Figure 1 (C). These findings suggest that alginate microgels are able to encapsulate primary human peritoneal mesothelial cells and promote their release.

[0085] Experimental Example 2

[0086] This experimental example demonstrates that the proliferation capacity of primary human peritoneal mesothelial cells is reduced and epithelial-mesenchymal transition occurs in patients with long-term peritoneal dialysis.

[0087] 1. Extraction, Culture and Identification of Primary Human Peritoneal Mesothelial Cells

[0088] Peritoneal dialysis fluid was collected from patients with different peritoneal dialysis durations of 1 week (1W), 1 year (1Y) and 3 years (3Y). The collected peritoneal dialysis fluid was centrifuged at 1000g for 5 minutes, and the supernatant was discarded. The isolated cells were cultured in DMEM / F12 medium (Gibco, California, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin, and placed in a constant temperature cell culture incubator at 37°C and 5% CO2. The culture medium was replaced every 2-3 days to remove non-adherent cells and provide fresh nutrients.

[0089] Primary human peritoneal mesothelial cells were identified by immunofluorescence staining of cytokeratin 18 (CK18) and vimentin to confirm their mesothelial cell origin. After the cells reached 80-90% density, they were digested with 0.25% trypsin and re-seeded for expansion. Primary human peritoneal mesothelial cells were only allowed to be passaged 2-4 times.

[0090] Co-staining of CK18 and Vimentin confirmed that these cells were primary human peritoneal mesothelial cells ( Figure 2 Figure A). More primary human peritoneal mesothelial cells with stronger proliferation capacity were collected from patients on peritoneal dialysis for 1 week, and these cells showed a cobblestone-like morphology after 11 days of culture. In patients on peritoneal dialysis for 1 year, fewer primary human peritoneal mesothelial cells were collected, and these cells underwent epithelial-mesenchymal transition, changing from a cobblestone-like appearance to a spindle shape, while their proliferation capacity decreased. In patients on peritoneal dialysis for 3 years, the number of primary human peritoneal mesothelial cells collected decreased more significantly, and the cell proliferation capacity decreased significantly ( Figure 2 These studies indicate that long-term peritoneal dialysis reduces the proliferation capacity of peritoneal mesothelial cells and induces epithelial-mesenchymal transition.

[0091] Experimental Example 3

[0092] To investigate the potential mechanisms of peritoneal fibrosis in patients with long-term peritoneal dialysis, primary human peritoneal mesothelial cells were isolated from patients who had undergone dialysis for 1 week and 1 year.

[0093] 1. Immunofluorescence

[0094] For tissue samples, paraffin sections (4 μm) were dewaxed in xylene and hydrated through gradient ethanol solutions. Antigen retrieval was performed using citrate buffer. For cell samples, cells were fixed with 4% paraformaldehyde after three washes with PBS. Both tissue and cell samples were permeabilized with 0.1% Triton X-100 and blocked with goat serum for 1 h. Each step required PBS washing, and then the samples were incubated in the corresponding primary antibody at 4 °C overnight. The next day, after washing the samples with PBS, the corresponding secondary antibodies were added and incubated for 1 h at room temperature. DAPI (Solarbio) was used for visualization of cell nuclei. Images were captured using a KFBIO digital pathology slide scanner (KF-PRO-005, China).

[0095] 2. RNA Extraction and Quantitative Real-time PCR (qRT-PCR)

[0096] Total RNA was extracted from tissue or cell samples using a commercial RNA extraction kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. The concentration and purity of RNA were assessed using a spectrophotometer. cDNA was synthesized from 1 μg of total RNA using a reverse transcription kit (Vazyme, Nanjing, China). Gene-specific primers were designed. qRT-PCR was performed on a real-time PCR instrument using SYBR Green detection chemistry. The relative expression levels of target genes were calculated using the comparative Ct method and normalized with internal reference genes such as GAPDH. All experiments were repeated at least three times.

[0097] 3. Western Blot

[0098] Animal tissue samples were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China) containing 0.1% phenylmethylsulfonyl fluoride. Cells were lysed using RIPA lysis buffer containing 0.1% phenylmethylsulfonyl fluoride. The homogenized cell lysate was sonicated at 30 Hz for 3 seconds, repeated three times. Then, both cells and animal tissue samples were centrifuged at 15,000 rpm for 20 minutes at 4°C. The collected supernatants were subjected to protein quantification analysis. Protein samples were prepared with 5x loading buffer and adjusted with PBS to ensure equal loading. Equal amounts of protein samples were loaded on SDS-PAGE gels for electrophoretic separation and subsequently transferred to PVDF membranes. After blocking with 5% skim milk powder for 2 hours, the membranes were washed with TBST and incubated with primary antibodies overnight at 4°C. The next day, the membranes were washed with TBST and incubated with the corresponding HRP-labeled secondary antibodies for 1 hour at room temperature. The protein bands were visualized using an ECL detection kit. The signals were captured using a chemiluminescent imaging system. The grayscale value of the bands was measured using ImageJ software to quantify the intensity of each band. All experiments were repeated at least three times.

[0099] 4. Statistical Analysis

[0100] Prism 9 software (GraphPad Software, USA) was used to generate bar graphs and perform statistical analysis. Normally distributed data were presented as mean ± standard deviation, and non-normally distributed data were presented as median and interquartile range to represent variability. For comparison of data between groups, independent sample t-test was used when the variance between groups was uniform. P values ​​less than 0.05 were considered statistically significant.

[0101] Immunofluorescence analysis showed that the expression level of POSTN was increased in patients undergoing peritoneal dialysis for 1 year compared with that in patients undergoing peritoneal dialysis for 1 week ( Figure 3 At the same time, POSTN mRNA and protein levels also increased ( Figure 3 I, Q). The downstream target of POSTN, NF-κB P65, was expressed more in 1-year dialysis patients, which was further confirmed by immunofluorescence. The activation and nuclear translocation of p-P65 led to an increase in the level of p-P65 in the nuclei of 1-year dialysis patients ( Figure 3 PCR further confirmed the upregulation of P65 mRNA expression ( Figure 3 Western blot analysis confirmed the increase in p-P65 / P65 levels in the 1-year dialysis group ( Figure 3 In addition, CXCL8, an inflammatory marker associated with peritoneal fibrosis, was increased in 1-year dialysis patients ( Figure 3 D, S), and CXCL8 mRNA levels were also increased ( Figure 3 Importantly, the protein expression levels of peritoneal fibrosis-related markers such as α-SMA, Col-1, TGF-β, and FN were increased in 1-year dialysis patients ( Figure 3 EH, T, U), and the mRNA level increased.

[0102] These findings suggest that elevated POSTN / P65 / CXCL8 levels promote the progression of peritoneal fibrosis in patients undergoing peritoneal dialysis for one year.

[0103] Experimental Example 4

[0104] To verify the increased expression of POSTN under high glucose conditions and its role in fibrosis, in this experimental example, peritoneal mesothelial cell lines were cultured in a 60 mM high glucose concentration medium.

[0105] The experimental method was the same as that of Experimental Example 3, and the peritoneal mesothelial cell culture method was the same as that of Experimental Example 1.

[0106] Immunofluorescence showed that POSTN levels increased in the G group (high glucose stimulation group) and the G+eSA (high glucose stimulation + empty sodium alginate microspheres) group, while POSTN expression decreased in the G+SA / PMC (high glucose stimulation + sodium alginate microspheres wrapped with peritoneal mesothelial cells) group. Similarly, NF-KB P65 expression increased in the G group and the G+eSA group. High glucose stimulation also promoted the phosphorylation and nuclear translocation of NF-KB P65 and increased the expression of CXCL8, while SA / PMC inhibited this process ( Figure 4 In addition, the expression of fibrosis-related markers (α-SMA, Col-1, TGF-β, FN) was increased in the G group and the G+eSA group, while the levels were decreased in the G+SA / PMC group ( Figure 4 Middle B). mRNA analysis confirmed that high glucose stimulation increased the expression of POSTN / P65 / CXCL8, while SA / PMC treatment decreased the expression of these markers ( Figure 4 CI). Western blot results also showed that the expression levels of POSTN, P65, and CXCL8 increased in the G group, but decreased in the G+SA / PMC group. The fibrosis marker α-SMA also increased in the G group and decreased in the G+SA / PMC group ( Figure 4 These results suggest that high glucose induced the expression of POSTN / P65 / CXCL8 and epithelial-mesenchymal transition in peritoneal mesothelial cells, and SA / PMC treatment could alleviate this process.

[0107] Experimental Example 5

[0108] In this experiment, peritoneal dialysis model mice were used to further explore the role of POSTN in peritoneal fibrosis.

[0109] 1. Peritoneal Dialysis Model Mouse

[0110] BALB / c nude mice (male, 8 weeks old) were provided by Charles River Laboratory Animal Technology Co., Ltd. (Zhejiang, China). Mice were housed under standard laboratory conditions with a 12-h light / dark cycle and a temperature of 22±1°C, with free access to food and water. After one week of adaptation, mice were randomly divided into 5 groups (n=8 in each group), labeled as Con (control group), G (high glucose peritoneal dialysis group), G+eSA (high glucose peritoneal dialysis + intraperitoneal injection of empty alginate microspheres group), G+PMC (high glucose peritoneal dialysis + intraperitoneal injection of peritoneal mesothelial cells group) and G+SA / PMC (high glucose peritoneal dialysis + injection of alginate microspheres encapsulated with human primary peritoneal mesothelial cells group). The body weight of mice was recorded at the beginning of the study and throughout the study. Mice in the Con group received intraperitoneal injections of 10 ml / 100 g of normal saline for four consecutive weeks, while mice in the other groups received intraperitoneal injections of 10 ml / 100 g of 4.25% glucose peritoneal dialysis solution for four consecutive weeks. Two weeks after the modeling, mice in the G+eSA group were injected with empty alginate microspheres, and mice in the G+PMC group were directly injected with 5×10 6 Primary human peritoneal mesothelial cells. Mice in the G+SA / PMC group were injected with alginate microspheres encapsulating primary human peritoneal mesothelial cells. Two weeks later, the mice were anesthetized and killed, and the blood, liver, heart, lung, kidney, and peritoneal tissues of the mice were collected and preserved. Some tissues were immediately stored in a -80°C refrigerator, and the other part was fixed with 4% paraformaldehyde.

[0111] 2. H&E staining

[0112] Tissue paraffin sections were dewaxed in xylene and hydrated through a gradient of decreasing ethanol. H&E staining was performed according to the instructions provided with the staining kit (Solarbio, Beijing, China). Sections were stained with hematoxylin for 5 minutes to visualize the nuclei and then rinsed in running tap water. Staining with eosin for 2 minutes was used to stain the cytoplasm and extracellular matrix. The stained sections were dehydrated through increasing ethanol concentrations, transparentized with xylene, and coverslipped with synthetic resin. Images were captured using a Leica microscope. All experiments were repeated at least three times.

[0113] 3.Masson staining

[0114] Masson staining was performed according to the instructions provided by the staining kit (Solarbio, Beijing, China). The sections were left overnight in Bouin solution and then rinsed in running water until the yellow color disappeared. The sections were stained with aniline blue for 2 minutes and rinsed. Mayer hematoxylin was stained for 2 minutes and rinsed. The sections were differentiated in acid ethanol for a few seconds and washed in running water for 10 minutes. Acid fuchsin was stained for 10 minutes and rinsed. Phosphomolybdic acid solution was stained for 10 minutes without washing. Aniline blue solution was stained for 5 minutes without rinsing. Weak acid solution was treated for 2 minutes and quickly dehydrated in 95% ethanol. Then dehydrated three times in anhydrous ethanol for 5-10 seconds each time, and finally transparent in xylene three times for 1-2 minutes each time. Finally, a neutral resin cover glass was used. Images were captured using a Leica microscope. All experiments were repeated at least three times.

[0115] 4. Immunohistochemistry

[0116] 4 μm tissue paraffin sections were dewaxed in xylene and hydrated with graded ethanol solutions. Antigen retrieval was performed with citrate buffer and treated with 3% hydrogen peroxide. The sections were then blocked with goat serum for one hour. Tissue sections were incubated with the corresponding primary antibodies at 4 °C overnight. The next day, HRP-labeled secondary antibodies were incubated after PBS washing, DAB was used to visualize the bound antibodies, and hematoxylin was used to highlight the cell nuclei. Images were captured using a Leica microscope. All experiments were repeated at least three times.

[0117] H&E staining results showed that the infiltration of inflammatory cells increased in the G group and the G+eSA group. Masson staining showed that the peritoneal fibrosis was obvious in the G, G+eSA, and G+PMC groups, while the degree of fibrosis was reduced in the G+SA / PMC group ( Figure 5 In addition, compared with the control group, the peritoneal thickness of the G group and the G+eSA group increased, while the peritoneal thickness of the G+SA / PMC group decreased compared with the G group and the G+eSA group ( Figure 5 In addition, immunohistochemistry results showed that POSTN expression increased in the G group and decreased in the G+SA / PMC group. Importantly, POSTN expression was lower in the G+SA / PMC group than in the G+PMC group. Similarly, the levels of NF-KB P65, NF-KB p-P65, CXCL8, α-SMA, Col-1, and TGF-β increased in the G group and decreased in the G+SA / PMC group ( Figure 5 Western blot results further confirmed that the expression levels of POSTN / P65 / CXCL8 increased in the G group, decreased in the G+SA / PMC group, and were lower than those in the G+PMC group ( Figure 5In addition, immunofluorescence showed that the fluorescence intensity of POSTN, NF-KB P65, NF-KB p-P65, CXCL8, α-SMA, Col-1, and FN increased in the G group, but decreased in the G+SA / PMC group ( Figure 5 These findings suggest that high glucose peritoneal dialysis induces POSTN / P65 / CXCL8 expression and peritoneal fibrosis in mice, and SA / PMC treatment shows superior anti-peritoneal fibrosis effect than eSA.

[0118] Experimental Example 6

[0119] To further evaluate the biosafety of sodium alginate microgels, blood and tissue samples were collected from peritoneal dialysis model mice in this experiment.

[0120] The hemolysis test showed that the hemolysis rate of eSA added to mouse blood was less than 2%, indicating that sodium alginate has good blood compatibility ( Figure 6 In addition, the body weight of mice did not change significantly during peritoneal dialysis ( Figure 6 To evaluate the effects of sodium alginate microgels on liver and kidney function, we measured serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), serum creatinine (Scr), and estimated glomerular filtration rate (eGFR) in mice, and no significant abnormalities were found ( Figure 6 H&E staining and Masson staining showed no obvious abnormalities in liver, heart, lung, and kidney tissues ( Figure 6 These findings suggest that sodium alginate microgels have little adverse effects in mice.

[0121] Schematic diagram of the role of the POSTN / P65 / CXCL8 pathway in peritoneal dialysis-induced peritoneal fibrosis Figure 7 As shown, the ions and glucose contained in the non-physiological peritoneal dialysis fluid will stimulate the peritoneum, activate the POSTN / NF-κBP65 / CXCL8 signaling pathway, lead to epithelial-mesenchymal differentiation and damage and loss of peritoneal mesothelial cells, and ultimately cause peritoneal fibrosis. SA / PMC achieves a therapeutic effect by protecting the embedded primary human peritoneal mesothelial cells and using the characteristics of sustained release to replenish the lost cells.

[0122] In summary, the present invention explores the properties of sodium alginate microgels and their therapeutic potential in long-term peritoneal dialysis-induced peritoneal fibrosis. Optical microscopy confirmed that primary human peritoneal mesothelial cells were encapsulated in SA microgels (SA / PMC), and there was no significant change in the diameter of the microgels. CCK8 cell proliferation experiments showed that both SA / PMC microgels significantly promoted cell proliferation. This shows that SA microgels not only serve as an effective delivery system for primary human peritoneal mesothelial cells, but also maintain their proliferation capacity, which is crucial for tissue regeneration and repair.

[0123] The present invention further investigated the proliferation capacity of primary human peritoneal mesothelial cells and the process of epithelial-mesenchymal transition in different peritoneal dialysis patients. The results showed that with the extension of peritoneal dialysis time, the cell proliferation capacity decreased significantly and epithelial-mesenchymal transition occurred.

[0124] Recent studies have highlighted the increasingly recognized role of high POSTN expression in the development and progression of chronic kidney disease [32,33]. Studies by Mouna Mael-Ainin et al. showed that POSTN mediates TGF-β-induced renal disease, suggesting that blocking POSTN may be an effective therapeutic strategy to prevent the progression of chronic kidney disease. In addition, in ovarian cancer, POSTN mediates the activation of ERK and NF-κB, enhancing M2 macrophages and cancer-associated fibroblasts, thereby promoting ovarian cancer metastasis

[34] . In addition, immunostaining of cartilage in a mouse model of medial femoral ligament instability showed that reduced POSTN expression reduced p65 phosphorylation

[35] . Importantly, POSTN can also trigger a series of inflammatory responses through the NF-κB signaling pathway [36,37]. However, the role of POSTN in peritoneal fibrosis remains unclear. In our study, immunofluorescence, PCR, and Western blot analysis confirmed the elevated expression of POSTN, NF-κB p65, NF-KB p-P65, and CXCL8 in patients with long-term peritoneal dialysis. The increased nuclear translocation of NF-KBp-P65 and the upregulation of fibrosis-related markers such as α-SMA, Col-1, TGF-β, and FN further validated the role of the POSTN / P65 / CXCL8 pathway in peritoneal fibrosis.

[0125] In our study, we demonstrated in vitro and in vivo that alginate microgels encapsulating peritoneal mesothelial cells can alleviate peritoneal dialysis-induced peritoneal fibrosis by activating the POSTN / P65 / CXCL8 pathway. The study showed that peritoneal dialysis led to increased expression of POSTN / P65 / CXCL8 in peritoneal mesothelial cells, while SA / PMC treatment inhibited the above process. This suggests that SA / PMC microgels can effectively inhibit the fibrotic response induced by peritoneal dialysis. Studies in peritoneal dialysis model mice further supported these findings. SA / PMC treatment significantly reduced peritoneal fibrosis, as shown by decreased expression of POSTN, P65, p-P65, and CXCL8, as well as decreased peritoneal membrane thickness. Comparison with eSA+PMC treatment highlighted the superior effect of SA / PMC in reducing fibrosis. In addition, biosafety assessment confirmed that alginate microgels did not cause significant adverse reactions, maintained normal liver and kidney function, and showed no histological abnormalities in major organs.

[0126] In conclusion, our study provides strong evidence that SA / PMC microgels are a promising therapeutic approach to mitigate peritoneal fibrosis in peritoneal dialysis patients. By encapsulating primary human peritoneal mesothelial cells and modulating the POSTN / P65 / CXCL8 pathway, SA / PMC microgels gradually release primary human peritoneal mesothelial cells to replace damaged cells, thereby mitigating epithelial-mesenchymal transition and peritoneal fibrosis. Future studies should focus on clinical trials to further validate these findings and explore the long-term benefits and safety of SA / PMC microgels in peritoneal dialysis patients.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0128] References:

[0129] 1.Si,M.,et al.,Inhibition of hyperglycolysis in mesothelial cellspreventsperitoneal fibrosis.Science translational medicine.11(495):p.eaav5341.

[0130] 2.Mehrotra,R.,et al.,The Current State of Peritoneal Dialysis.Journalof theAmerican Society of Nephrology:JASN.27(11):p.3238-3252.

[0131] 3.Terri,M.,et al.,Mechanisms of Peritoneal Fibrosis:Focus onImmuneCells-Peritoneal St roma Interactions.Frontiers in immunology.12:p.607204.

[0132] 4.Fang,J.,et al.,Glycoprotein 96in Peritoneal Dialysis Effluent-DerivedExtracellular Vesicles:A Tool for Evaluating Peritoneal TransportProperties and Inflammatory Status.Frontiers in immunology.13:p.824278.

[0133] 5.Lee,Y.,et al.,Inflammatory chemokine(C-C motif)ligand8inhibitionameliorates per itoneal fibrosis.FASEB journal:officialpublication of the Federationof American Soc ieties for ExperimentalBiology.37(1):p.e22632.

[0134] 6.Sun,S.-C.,The non-canonical NF-κB pathway in immunityandinflammation.Nature reviews.Immunology.17(9):p.545-558.

[0135] 7.Oh,A.,et al.,NF-κB signaling in neoplastic transition fromepithelial tomesenchyma l phenotype.Cell communication and signaling:CCS.21(1):p.291.

[0136] 8.Lu,R.X.Z.,et al.,Cardiac tissue model of immune-induceddysfunctionreveals the role of free mitochondrial DNA and the therapeuticeffects of exosomes.Science advances.10(13):p.eadk0164.

[0137] 9.Conway,S.J.,et al.,The role of periostin in tissue remodelingacross healthand disease.Cellular and molecular life sciences:CMLS.71(7):p.1279-88.

[0138] 10.Cui,D.,et al.,The multifaceted role of periostin in priming thetumormicroenvironme nts for tumor progression.Cellular and molecular lifesciences:CMLS.74(23):p.4287-4291.

[0139] 11.Chen,C.,et al.,Single-cell and spatial transcriptomics revealPOSTN + cance r-associated fibroblasts correlated with immunesuppression and tumourprogression in non-small cell lung cancer.Clinical andtranslationalmedicine.13(12):p.e1515.

[0140] 12.Drakopanagiotakis,F.,P.Markart,and P.Steiropoulos,AcuteExacerbationsof Interstitial Lung Diseases:Focus on Biomarkers.Internationaljournal of molecularsciences.24(12):p.10196.

[0141] 13.Masuoka,M.,et al.,Periostin promotes chronic allergic inflammationinresponse to Th2 cy tokines.The Journal of clinical investigation.122(7):p.2590-600.

[0142] 14.Yang,Q.,et al.,PFKFB3-Mediated Glycolysis Boosts FibroblastActivationand Subsequent Kidney Fibrosis.Cells.12(16):p.2081.

[0143] 15.Prakoura,N.and C.Chatziantoniou,Periostin in kidneydiseases.Cellularand molecular life sciences:CMLS.74(23):p.4315-4320.

[0144] 16.Pan,T.,et al.,Preparation of bright yellow color sodium alginatesolution.Carbohydrate polymers.337:p.122169.

[0145] 17.Yang,J.,J.Yao,and S.Wang,Electromechanical response performance ofareinforced biomass gel art ificial muscle based on natural polysaccharide ofsodiumalginate dope d with an ionic liquid for micro-nano regulation.Carbohydratepolymers.275:p.118717.

[0146] 18.Dubay,R.,J.N.Urban,and E.M.Darling,Single-Cell MicrogelsforDiagnostics and Therapeutics.Advanced functional materials.31(44):p.2009946.

[0147] 19.Caldwell,A.S.,B.A.Aguado,and K.S.Anseth,Designing MicrogelsforCell Culture and Controlled Assembly of Tissue Microenvironments.Advancedfunctional materials.30(37):p.1907670.

[0148] 20.Huang,J.,et al.,Preparation and characterization of astaxanthin-loadedmicrocapsules s tabilized by lecithin-chitosan-alginate interfaces withlayer-by-layerassembly method.International journal of biologicalmacromolecules.268(Pt 1):p.131909.

[0149] 21.Pan,H.,et al.,Light-Sensitive Lactococcus lactisforMicrobe-Gut-Brain Axis R egulating via Upconversion Optogenetic Micro-NanoSystem.ACSnano.16(4):p.6049-6063.

[0150] 22.Choe,G.,et al.,Anti-oxidant activity reinforced reducedgrapheneoxide / alginate micro gels:Mesenchymal stem cell encapsulation andregeneration ofinfarcte d hearts.Biomaterials.225:p.119513.

[0151] 23.Volpatti,L.R.,et al.,Partially Oxidized Alginate as aBiodegradable Carrierfor Glucose-Res ponsive Insulin Delivery and Islet CellReplacement Therapy.Advanced healthcare materials.12(2):p.e2201822.

[0152] 24.24.Ahn,S.H.,et al.,Single-Step Synthesis of Alginate MicrogelsEnveloped witha Covalent Polymeric Shell:A Simple Way to Protect EncapsulatedCells.ACSapplied materials&interfaces.13(16):p.18432-18442.

[0153] 25.Ching,S.H.,N.Bansal,and B.Bhandari,Alginate gel particles-A reviewofproduction techniques and physical properties.Critical reviews in foodscience andnutrition.57(6):p.1133-1152

[0154] 26.Xu,M.,et al.,Alginate microgels as delivery vehicles for cell-basedtherapies in ti ssue engineering and regenerative medicine.Carbohydratepolymers.266:p.118128.

[0155] 27.Utech,S.,et al.,Microfluidic Generation of Monodisperse,StructurallyHomogeneous Algi nate Microgels for Cell Encapsulation and 3DCell Culture.Advanced healthcare materials.4(11):p.1628-33.

[0156] 28.Zhang,Z.,et al., Lactobacillus rhamnosus Encapsulated inAlginate / Chitosan Micro gels Manipulates the Gut Microbiome to AmeliorateSalt-InducedHepator enal Injury.Frontiers in nutrition.9:p.872808.

[0157] 29.Li,W.,et al.,Encapsulation of Lactobacillus plantarum in cellulosebasedmicrogel w ith controlled release behavior and increased long-termstorage stabil ity.Carbohydrate polymers.223:p.115065.

[0158] 30.Sun,Z.-J.,et al.,Probing the role of microenvironmentformicroencapsulated Sacchromyce s cerevisiae under osmotic stress.Journalofbiotechnology.128(1):p.150-61.

[0159] 31.Kim,P.-H.,et al.,Injectable multifunctional microgelencapsulatingoutgrowth endothelia l cells and growth factors for enhancedneovascularization.Journal of controlled release:official journal of theControlled Rel ease Society.187:p.1-13.

[0160] 32.Wallace,D.P.,Periostin in the Kidney.Advances in experimentalmedicineand biology.1132:p.99-112.

[0161] 33.Bian,X.,et al.,Knockdown of periostin attenuates 5 / 6 nephrectomy-inducedintrarenal r enin-angiotensin system activation,fibrosis,andinflammation in rats.Journal of cellular physiology.234(12):p.22857-22873.

[0162] 34.Lin,S.-C.,et al.,Periostin promotes ovarian cancer metastasisbyenhancing M2 macrophag es and cancer-associated fibroblasts via integrin-mediatedNF-κB and T.Journal of biomedical science.29(1):p.109.

[0163] 35.Duan,X.,et al.,Amelioration of Posttraumatic Osteoarthritis inMice UsingIntraarticu lar Silencing of Periostin via Nanoparticle-Based SmallInterfering RN A.Arthritis&rheumatology(Hoboken,N.J.).73(12):p.2249-2260.

[0164] 36.Koh,S.-J.,et al.,Matricellular Protein Periostin MediatesIntestinalInflammation throu gh the Activation of Nuclear FactorκBSignaling.PloS one.11(2):p.e0149652.

[0165] 37.Zhang,Y.,et al.,Coal dust nanoparticles induced pulmonary fibrosisby promotinginflam mation and epithelial-mesenchymal transition via the NF-κB / NLRP3 pathw ay drivenby IGF1 / ROS-mediated AKT / GSK3βsignals.Cell deathdiscovery.8(1):p.500.

Claims

1. A microgel, characterized in that: It includes: Alginate microgels contain primary human peritoneal mesothelial cells.

2. The microgel according to claim 1, characterized in that Each of the alginate microgels contains 1 to 50 primary human peritoneal mesothelial cells; Preferably, the alginate is selected from sodium alginate, potassium alginate and calcium alginate.

3. A method for preparing a microgel, characterized in that: The method comprises the following steps: uniformly mixing the aqueous phase and the oil phase obtained by mixing alginate, chelated exchange ion solution and primary human peritoneal mesothelial cells through a microfluidic chip; the chelated exchange ion solution is obtained by mixing the exchange ion solution with a chelating agent.

4. The method for preparing microgel according to claim 3, characterized in that: The flow rate ratio of the water phase to the oil phase is 1:4 to 1:5; Preferably, the flow rate ratio of the water phase to the oil phase is 1:

5.

5. The method for preparing microgel according to claim 3, characterized in that: The chelate exchange ion solution is obtained by mixing the exchange ion solution and the chelating agent at a molar ratio of 1:1-1:1.1; Preferably, the exchange ion solution is selected from a 2+ 、Zn 2+ One of the solutions; The exchange ion chelating agent is selected from: EDTA, PDTA, CDTA and salts thereof, preferably EDTA.

6. The method for preparing microgel according to claim 5, characterized in that: The alginate is selected from sodium alginate, potassium alginate and calcium alginate; Preferably, the chelate exchange ion solution is selected from ethylenediaminetetraacetic acid calcium disodium salt; the alginate is selected from sodium alginate; Preferably, in the aqueous phase, the concentration of the ion exchange reagent is 38-42 mM; the mass concentration of the alginate is 1.8-2.2%; Preferably, in the aqueous phase, each 0.5-1 ml of the alginate and chelate exchange ion solution is mixed with 5×10 6 ~1×10 7 Primary human peritoneal mesothelial cells were mixed.

7. The method for preparing microgel according to claim 3, characterized in that: The oil phase is selected from: fluorinated liquid with added fluorinated surfactant or alkane oil or other types of oil with added surfactant; the surfactant is selected from any one of Span80, Tween 80, EM 90 or EM 180; the alkane oil or other types of oil is selected from any one of mineral oil, cyclohexane, hexadecane, isopropyl palmitate; Preferably, the oil phase is selected from a fluorinated liquid to which a fluorinated surfactant is added; Preferably, the fluorinated liquid is selected from any one of 1H, 1H, 2H, 2H-perfluorododecyltrichlorosilane, HFE-7500, HFE-7300, HFE-7200, HFE-7100, FC-40 or FC-70; the fluorinated surfactant is any one of "ABA" type polymer, "Rf-L-Rh-L-Rf" or "Rh-L-Rf-L-Rh" type perfluoro polymer.

8. Use of the microgel according to any one of claims 1 to 2 or the microgel prepared by the preparation method according to any one of claims 3 to 7 in preparing a drug for alleviating or treating peritoneal fibrosis; Preferably, said peritoneal fibrosis occurs by peritoneal dialysis.

9. The application according to claim 8, wherein the application comprises at least one of the following application modes: (1) Reduce POSTN levels; (2) Inhibit the phosphorylation and nuclear translocation of NF-κB P65; (3) inhibiting the expression of CXCL8; (4) suppressing the levels of peritoneal fibrosis-related markers; (5) Inhibition of epithelial-mesenchymal transition; (6) Relieve peritoneal fibrosis; (7) Reduce peritoneal thickness; (8) replenish lost peritoneal mesothelial cells; (9) Inhibit peritoneal inflammation; Preferably, the fibrosis-related marker is selected from at least one of α-smooth muscle actin (α-SMA), type I collagen (COL1), transforming growth factor β (TGF-β) and fibronectin (FN); Preferably, the drug further comprises a pharmaceutically acceptable excipient.

10. A drug, characterized in that The invention comprises the microgel according to any one of claims 1 to 2 or the microgel prepared by the preparation method according to any one of claims 3 to 7.

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