Medical use of gelled cells loaded with sulfonated chitosan in the treatment of atherosclerosis

Abstract

The application discloses medical use of gelated cells loaded with sulfonated chitosan in treatment of atherosclerosis, and belongs to the technical field of biological medicine. The gelated cells loaded with sulfonated chitosan take macrophages as host cells, and intracellular cross-linking forms a photocrosslinking hydrogel network composed of methacrylated hyaluronic acid and sulfonated chitosan. The gelated cells completely retain cell membrane structure and membrane surface protein expression, lose the proliferation ability, and have excellent blood lipid selective adsorption capacity and foam cell lipid metabolism regulation capacity. In-vivo and in-vitro experiments prove that the gelated cells can significantly reduce blood lipid levels of atherosclerosis model animals, reduce aortic plaque area, and have good biological safety, thereby providing a new strategy and candidate drug for prevention and treatment of atherosclerosis, and having a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the medicinal use of gelled cells loaded with sulfonated chitosan in the treatment of atherosclerosis. Background Technology

[0002] Atherosclerosis (AS) is a chronic inflammatory disease of the arterial wall caused by abnormal lipid metabolism, and is a major cause of coronary heart disease, cerebral infarction, and peripheral vascular disease. AS is a complex, multifactorial process involving multiple biochemical pathways, including inflammation, lipid metabolism, cell proliferation, and apoptosis. When endothelial cells are damaged, an inflammatory response is triggered, attracting monocytes and macrophages into the blood vessel wall. These cells take up excessive amounts of oxidized low-density lipoprotein (ox-LDL), forming foam cells. These foam cells accumulate in the blood vessel wall, forming fatty streaks, and eventually developing into fibrous plaques.

[0003] Foam cell formation is a core component in the development and progression of atherosclerosis. Its functions extend beyond lipid accumulation, encompassing inflammatory responses, oxidative stress, and plaque stability. The massive aggregation of foam cells within plaques leads to vascular stenosis and blood flow restriction. Simultaneously, the release of inflammatory factors recruits more monocytes into the plaque area, exacerbating local inflammation. Foam cell apoptosis and necrosis result in significant lipid deposition, forming a necrotic lipid core. This process further disrupts the fibrous cap structure, reducing plaque stability and significantly increasing the risk of plaque rupture and thromboembolic events. Therefore, exploring new integrated diagnostic and therapeutic approaches for atherosclerosis by breaking the vicious cycle of inflammation and lipid phagocytosis has become a crucial research direction.

[0004] Intracellular gelation, also known as in situ intracellular gelation, involves introducing gel-forming precursors into living cells. Under specific intracellular microenvironment or external triggering conditions, polymerization or cross-linking reactions occur, forming a soft, three-dimensional polymer network within the cytoplasm. Intracellular gelation is characterized by proliferation inhibition and long-term stability. Gelated cells lose their ability to divide, avoiding the risk of uncontrolled proliferation that may occur during in vivo application. Notably, despite gelation, the structural integrity of the cell membrane, protein content and integration, membrane fluidity, and lipid order are well preserved. Gelated cells act as highly efficient "cellular sponges," specifically binding to and neutralizing pathogens or inflammatory factors through receptor-ligand interactions. According to Gao C et al., intracellular gelation technology has applications in osteoarthritis and viral myocarditis caused by Coxsackievirus B3.

[0005] Lipid purification therapy refers to a class of methods that use extracorporeal blood circulation to remove lipid components from the blood. It is commonly used for lipid management in patients who do not respond to or are intolerant of lipid-lowering drugs. Among these methods, dextran sulfate adsorption (DSA) and heparin-mediated low-density lipoprotein (LDL-C) precipitation (HELP) are commonly used lipid adsorption techniques. Their mechanism is attributed to the electrostatic binding between negatively charged dextran sulfate or heparin and positively charged LDL-C. Chitosan, a natural and renewable alkaline polysaccharide, exhibits high sulfation and a high negative charge density after sulfonation modification, and its structural characteristics are highly similar to heparin. However, whether sulfonated chitosan possesses lipid adsorption capabilities remains unclear. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention aims to construct gelled cells loaded with sulfonated chitosan for medicinal use in the prevention or treatment of ankylosing spondylitis (AS). The HSCS disclosed in this invention can inhibit the progression of AS disease and is expected to become a new strategy for AS prevention and intervention.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0008] This invention discloses a gelled cell loaded with sulfonated chitosan, characterized in that the gelled cell is prepared by blending methacrylamide hyaluronic acid with sulfonated chitosan to form a hydrogel precursor, followed by in-situ gelation within the cell.

[0009] Furthermore, the gelled cells use macrophages as host cells.

[0010] The present invention also discloses the use of gelled cells loaded with sulfonated chitosan as described in any of the above claims in the preparation of drugs for the prevention or treatment of atherosclerosis.

[0011] Furthermore, the drug is used to reduce serum triglyceride, total cholesterol, low-density lipoprotein levels, and / or increase serum high-density lipoprotein levels.

[0012] Furthermore, the drug is used to inhibit the formation of atherosclerotic plaques, reduce the area of ​​atherosclerotic plaques, and / or inhibit the formation of macrophage-derived foam cells.

[0013] The present invention also discloses a method for preparing gelled cells loaded with sulfonated chitosan as described in any one of the above claims, characterized by comprising the following steps: S1 Preparation of methacrylamide hyaluronic acid powder and sulfonated chitosan powder; S2. Methacrylamide hyaluronic acid powder and sulfonated chitosan powder were dissolved in sterile water at a mass ratio of 1:2 to prepare a 1% (w / v) hydrogel precursor solution. A photoinitiator was added and the mixture was stirred to obtain the hydrogel working solution. S3 Macrophages in the logarithmic growth phase were collected, washed with PBS, resuspended in the hydrogel working solution, and collected as cell pellets by centrifugation after freeze-thaw treatment. S4 After washing the cell pellet with PBS, cross-linking reaction was carried out using 365nm ultraviolet light to obtain the gelled cells loaded with sulfonated chitosan.

[0014] Further, in step S2, the photoinitiator is I2959, and the amount added is 0.5% of the mass of the hydrogel precursor solution.

[0015] Furthermore, in step S3, the freeze-thaw treatment involves freezing at -80°C for 10 minutes followed by complete thawing at room temperature; in step S4, the ultraviolet crosslinking reaction takes 10 minutes.

[0016] Furthermore, the use of the gelled cells loaded with sulfonated chitosan described in any of the above claims in the preparation of medicaments for the prevention and / or treatment of atherosclerosis.

[0017] The present invention also discloses a pharmaceutical preparation for the prevention or treatment of atherosclerosis, characterized in that it comprises gelled cells loaded with sulfonated chitosan as described in any one of the above-mentioned claims and a pharmaceutically acceptable carrier.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0019] This invention is the first to combine sulfonated chitosan with intracellular gelation technology to construct gelled cells loaded with sulfonated chitosan (HCSCS), filling the gap in the application of sulfonated chitosan in the field of atherosclerotic cell therapy. At the same time, it breaks through the functional limitation of existing intracellular gelled cells that can only target and neutralize inflammatory factors, endowing gelled cells with dual anti-AS functions of "lipid adsorption + lipid metabolism regulation".

[0020] The HSCS constructed in this invention completely preserves the structure of macrophage membrane-specific proteins (CD11b, F4 / 80) while completely inhibiting cell proliferation through an intracellular hydrogel network, eliminating the safety risks of uncontrolled proliferation in live cell therapy. At the same time, a two-step freeze-thaw-photocrosslinking method is used to achieve stable intracellular loading of sulfonated chitosan, avoiding burst release and degradation of active ingredients and ensuring long-lasting in vivo effects.

[0021] The HSCS of this invention has excellent selective adsorption performance for blood lipids. It can adsorb triglycerides, cholesterol, and low-density lipoprotein (LDL-C) in a concentration-dependent manner, while having a very weak adsorption effect on high-density lipoprotein (HDL-C), which has a vascular protective effect. It can reduce harmful blood lipids while retaining the physiological functions of beneficial blood lipids, which has significant advantages over traditional non-selective blood lipid purification methods.

[0022] The HSCS of this invention can break the vicious cycle of "inflammation-lipid phagocytosis" in the progression of AS from the root: on the one hand, it can directly adsorb ox-LDL in the environment through intracellular sulfonated chitosan, reducing the phagocytosis of oxidized lipids by macrophages; on the other hand, it can significantly downregulate the expression of the lipid phagocytosis-related gene CD36 in foam cells, while upregulating the expression of lipid efflux-related genes ABCA1 and ABCG1, reducing intracellular lipid deposition, inhibiting the formation and activation of foam cells, and has a more comprehensive anti-AS effect compared with single lipid-lowering therapy.

[0023] This invention demonstrates through in vivo animal experiments that HSCS can significantly reduce body weight, serum triglyceride, total cholesterol, and LDL-C levels in AS model mice, while upregulating HDL-C levels; it can reduce the proportion of aortic plaque area from 26.35±3.04% to 5.07%, and reduce the aortic root plaque area by more than 70%, exhibiting extremely strong in vivo anti-AS activity; at the same time, it has no obvious toxic side effects on major organs such as heart, liver, spleen, lung, and kidney in mice, with excellent biosafety and extremely high clinical translational value. Attached Figure Description

[0024] Figure 1 Construction and characterization of gelled cells loaded with sulfonated chitosan. Figures a and b are 1H NMR spectra of hyaluronic acid and methacrylamide hyaluronic acid; Figure c is Fourier transform infrared spectrum of chitosan and sulfonated chitosan; Figure d is scanning electron micrograph of Raw264.7 cells and HSCS; Figures e and f are flow cytometry images of Raw264.7 cells and HSCS; Figure g is inverted fluorescence image of Raw264.7 cells and HSCS; Figure h is particle size distribution of Raw264.7 cells and HSCS.

[0025] Figure 2 The adsorption effects of SCS and HSCS on different blood lipid components. Figure ad shows the adsorption effect of SCS on triglyceride, cholesterol, low-density lipoprotein, and high-density lipoprotein standards; Figure eh shows the adsorption effect of HSCS on triglyceride, cholesterol, low-density lipoprotein, and high-density lipoprotein standards.

[0026] Figure 3HCSCS regulates lipid metabolism in foam cells. Figure ac is a statistical graph of RT-PCR results; Figure d shows the effect of BODIPY staining on HCS inhibiting lipid uptake in Raw264.7 cells; Figure ef is a flow cytometry graph.

[0027] Figure 4 The therapeutic effect of HCSCS on mice with atherosclerosis. Figure a is a statistical graph of body weight after HCS treatment and that of the Ctrl group; Figure be is the blood lipid content of AS mice; Figure fg is the gross oil red staining image of the aorta and its quantitative statistical graph; Figure hi is the oil red staining image of the aortic valve section and its quantitative statistical graph; Figure j is the HE staining image of the major organs of the mice. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0029] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0030] Example 1: Construction and characterization of gelled cells loaded with sulfonated chitosan.

[0031] 1. Construction of gelled cells loaded with sulfonated chitosan.

[0032] 1.1 Dissolve 1 g of hyaluronic acid (HA, Macklin, H909936, molecular weight: 200,000-400,000) in 100 mL of ultrapure water, add 5 mL of methacrylic anhydride (Macklin, M812900), adjust the pH to 8, and stir at 0 °C for 24 h. Then, precipitate the mixture with ice-cold ethanol and collect the precipitate by centrifugation. Dialyze the precipitate in deionized water for 3 days. Finally, freeze the resulting solution overnight at -80 °C, then freeze-dry for 3 days to obtain methacryloyl-modified hyaluronic acid (HAMA) powder.

[0033] 1.2 Chitosan (CS, Macklin, C804726, degree of deacetylation ≥95%, viscosity 100-200 mPa·s) was dissolved in formamide (Macklin, F6287) solution. A chlorosulfonic acid (RHAWN, R049949) / dimethylformamide (Macklin, N6259) mixed solution was thoroughly mixed at a volume ratio of 4:1 and then added dropwise to the chitosan solution. The reaction was carried out at 50℃ for 2 h. The resulting solution was dialyzed against deionized water for 4 days. Finally, the resulting solution was stored at -80℃ overnight and freeze-dried for 3 days to obtain sulfonated chitosan (SCS) powder.

[0034] 1.3 Prepare a 1% solution by mixing HAMA powder and SCS powder with sterile water at a weight ratio of 1:2, and add I2959 photoinitiator (Sigma, 410896) at a weight ratio of 0.5% to obtain the HAMA / SCS hydrogel solution.

[0035] 1.4 The mouse macrophage cell line (Raw264.7) was cultured in Dulbecco modified Eagle high-glucose medium (DMEM, BasalMedia, China). All media contained 10% fetal bovine serum (VivaCell, China) and 1% penicillin / streptomycin (P / S, Sangon Biotech, China). All cells were cultured in a sterile incubator at 37°C and 5% CO2.

[0036] 1.5 After discarding the culture medium from the logarithmic growth phase of Raw264.7 cells, wash twice with PBS, and then uniformly disperse the Raw264.7 cells in 1% HAMA / SCS hydrogel solution at a cell-to-hydrogel ratio of 102. 8 Cells were dispersed in 100 μL of hydrogel solution. The resulting mixture was frozen at -80 °C for 10 min and then completely thawed at room temperature. Subsequently, the precipitate was collected by centrifugation at 1200 rpm for 5 min. The precipitate was washed twice with PBS and crosslinked under ultraviolet light (UV) at 365 nm for 10 min. The resulting precipitate was named sulfonated chitosan-loaded gelled cells (HCSCS).

[0037] 1.6 To characterize the loading and release of sulfonated chitosan by gelled cells, FITC-labeled chitosan saccharides (SCS) were constructed, and FITC-labeled HCSCS were then constructed using these saccharides. The SCS were dissolved in Tris buffer at pH 8.5 to prepare a solution of 1... wt % SCS solution. Then, 5 mg FITC (MCE, HY-66019) was added to the SCS solution, and the reaction was carried out in the dark for 24 hours. The resulting solution was dialyzed against deionized water for 4 days and then freeze-dried to obtain FITC-SCS. FITC-labeled gelled cells loaded with sulfonated chitosan (FITC-HCSCS) were constructed using the same method as in 1.5.

[0038] 2. Characterization of gelled cells loaded with sulfonated chitosan.

[0039] 2.1 To investigate the successful synthesis of SCS and HAMA, Fourier transform infrared spectroscopy and proton NMR were used for characterization. The results were obtained in the mid-infrared range (400-4000 cm⁻¹). -1FTIR spectra of CS and SCS were collected, and proton NMR spectra of SCS, HA, and HAMA were measured in a deuterium oxide (D2O) environment. 1 (H NMR), and the results were analyzed using MestReNova software.

[0040] 2.2 The morphology of Raw264.7 cells and HSCS was observed using a JEM-1200 scanning electron microscope. Raw264.7 cells were cultured on sterile silicon wafers for one day; simultaneously, HSCS collected by centrifugation was dropped onto the surface of sterile silicon wafers. All samples were then fixed with 2.5% glutaraldehyde for 3 hours, dehydrated in a gradient of 30%, 50%, 90%, and 100% ethanol solutions, and finally soaked in tert-butanol solution for 24 hours and freeze-dried for 48 hours. Samples were observed at 3 kV, and images were randomly captured to obtain SEM images. The particle size was measured by randomly selecting 100 cells using Nanomeasure software.

[0041] 2.3 To observe the morphology of Raw264.7 cells and HSCS, 1×10⁻⁶ cells were prepared. 4 Cells were seeded in 24-well plates and observed using an inverted fluorescence microscope, with photographs taken randomly.

[0042] 2.4 To investigate whether the engineered gelled cells retained their cell membrane proteins intact, flow cytometry was used for analysis. Cells were collected by centrifugation at 1200 rpm for 5 min, yielding at least 1 × 10⁻⁶ cells. 6 Raw264.7 cells and HSCS were washed once with PBS. The cells were labeled with flow cytometry antibodies CD11b, F4 / 80 (Lianke Biotechnology Anti-Human / Mouse CD11b, APC; Lianke Biotechnology Anti-Human / Mouse F4 / 80, PE) for 30 min in the dark before being analyzed by flow cytometry.

[0043] The results show that: 1 HNMR spectroscopy confirmed the success of MA modification of HA. Figure 1 ab). With HA 1 Compared to the HNMR spectrum, new characteristic absorption peaks of 5.73 ppm and 6.17 ppm were formed in HAMA, attributed to carbon-carbon double bonds, and new characteristic absorption peaks at 1.856 ppm, 1.917 ppm, and 1.936 ppm were attributed to carbon-carbon triple bonds. FITR spectroscopy confirmed the successful synthesis of SCS, with a wavelength of 1245 cm⁻¹. -1 The tensile vibration at this point is the S=O absorption peak and 810 cm⁻¹. -1 The tensile vibration at that point is the CO absorption peak ( Figure 1c). The cells adhered uniformly to the silicon wafer, exhibiting a spherical or ellipsoidal morphology. Abundant extracellular matrix was present on the cell surface, with occasional pseudopodia and microfilaments. Compared to the SEM images of the cells, the extracellular matrix and pseudopodia of HSCS cells were almost completely absent, resulting in a spherical structure. Furthermore, the cell size of HSCS cells was significantly larger than that of Raw264.7 cells (9.37±2.53 mm). vs 7.69±1.23μm) Figure 1 d, 1h). Flow cytometry showed that the specific proteins CD11b and F4 / 80 on the surface of macrophages were almost completely preserved. Figure 1 e, 1f). Compared to Raw264.7 cells, HSCS cells exhibited a dead cell morphology, and the FITC-SCS filling the cells emitted green fluorescence (e, 1f). Figure 1 g).

[0044] The above results demonstrate the successful preparation of gelled cells and the successful loading of SCS.

[0045] Example 2: Adsorption of different blood lipid components by gelled cells loaded with sulfonated chitosan.

[0046] 1. To observe the ability of SCS to adsorb blood lipids, 1 mg of SCS was added to 1 mL of low-density lipoprotein (LDL-C) standard solutions at concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L. EP tubes were incubated at 37°C for 1 h at 90 rpm in a constant-temperature shaker, and the supernatant was collected by centrifugation at 12000 rpm for 10 min. The concentration of remaining blood lipids in the supernatant was tested using a low-density lipoprotein cholesterol assay kit. The concentrations of other blood lipid component standard solutions are shown in the table below.

[0047] 2. Using a hemocytometer, draw at least 1 × 10⁻⁶ blood cells. 5 One HSCCS was tested using the same method as in section 1.

[0048] The results showed that as the concentration of the blood lipid standard increased, the amount of blood lipids adsorbed by SCS also gradually increased. Figure 2 The adsorption of HDL-C by SCS reaches saturation at an HDL-C concentration of 0.4 mmol / L. Figure 2 d). Like SCS, HCSCS has a good adsorption effect on lipid components such as LDL-c. Figure 2 eh).

[0049] Example 3: HSCS regulates lipid metabolism in foam cells.

[0050] 1. Real-time quantitative PCR was used to detect the lipid regulation effect of HSCS on foam cells.

[0051] 1.1 Raw264.7 cells were cultured at a rate of 3 × 10⁻⁶. 5 The cells were seeded at a density of [missing information] into 6-well plates, and a foam cell model was established by co-incubating Raw264.7 cells with 100 μg / mL ox-LDL for 1 day. Subsequently, 1×10 [missing information] 4 After co-culturing HSCS with Raw264.7 cells and foam cells for 1 day, the lipid-regulating effect of HSCS on foam cells was detected by PCR.

[0052] 1.2 Extraction of cellular RNA.

[0053] A. Discard the cell supernatant, wash once with PBS, and add 1 mL of Trizol to each well.

[0054] B. Let it stand at room temperature for 5 minutes, add 1 / 5 of the chloroform, then invert it to mix well, and let it stand for 15 minutes.

[0055] Centrifuge at 10000 rpm and 4℃ for 10 min.

[0056] D. Remove the supernatant, add an equal volume of isopropanol, invert and mix well, then let stand at room temperature for 10 min.

[0057] Centrifuge at 10000 rpm and 4℃ for 10 min, then discard the supernatant.

[0058] F. Add 1 mL of 75% ethanol, gently shake the centrifuge tube, and suspend the precipitate.

[0059] G. Centrifuge at 10000 rpm, 4℃ for 10 min, and discard the supernatant.

[0060] H. Let it air dry at room temperature until it becomes transparent.

[0061] I. Add 25 μL of enzyme-free water to dissolve the RNA.

[0062] 1.3. RNA was reverse transcribed into cDNA. The Takara reverse transcription kit was used.

[0063] A. Remove genomic DNA.

[0064] B. Reverse transcription reaction.

[0065] Reaction conditions: 37℃ for 15 min; 85℃ for 5 s; 4℃ for ∞.

[0066] 1.4 Primer sequences.

[0067] 1.5. Quantitative Real-Time PCR.

[0068] Prepare 20 μL qPCR reaction mixtures according to the volumes shown in the table above, with 3 replicates per sample group. Reaction conditions: 95℃ for 5 min, (95℃ for 5 s, 60℃ for 30 s, 72℃ for 30 s) × 40 cycles, followed by 72℃ for 5 s and 95℃ for 15 s. (Using 2...) －△△Ct The method analyzes the expression of each sample group.

[0069] 2. Bodipy staining to detect the lipid-regulating effect of HSCS on foam cells.

[0070] Raw264.7 cells were grown at a rate of 1×10⁻⁶. 5 The cells were seeded at a density of [missing information - likely a specific density] onto a glass slide in the lower chamber of a Transwell 24-well plate. After cell adhesion, 100 μg / mL ox-LDL was added to the lower chamber, and 5 × 10 [missing information - likely a specific concentration] ox-LDL was added to the upper chamber (3 μm pore size). 3 HSCS cells were co-cultured for one day, then the culture medium in both the upper and lower chambers was discarded, and the cells were fixed with 4% paraformaldehyde for 15 min. Bodipy dye was prepared as a 1 μM working solution using PBS and incubated for 30 min. The dye working solution was removed, and the cells were washed twice with PBS, 5 min each time. The above process was repeated to stain the cell nuclei with Dapi. After satisfactory staining, the cells were observed using a fluorescence microscope, and photographs were taken randomly.

[0071] 3. Dil-oxLDL assay to detect the lipid regulation effect of HSCS on foam cells.

[0072] Raw264.7 cells were grown at a rate of 1×10⁻⁶. 6 The cells were seeded at a density suitable for the lower chamber of a Transwell 6-well plate. After cell attachment, 40 μg / mL Dil-ox-LDL was added to the lower chamber, and 1 × 10⁻⁶ Dil-ox-LDL was added to the upper chamber (3 μm wells). 5 After co-culturing HSCS cells for 2 hours, HSCS cells from the upper chamber and Raw264.7 cells from the lower chamber were collected separately. Fluorescence in the samples was detected using the PE channel of a flow cytometer.

[0073] The results showed that PCR results indicated that after co-culturing Raw264.7 with oxLDL for 1 day, the expression of the scavenger receptor CD36 gene was significantly increased, indicating the successful establishment of the foam cell model. After co-culturing HCSCS with foam cells for 1 day, the expression of the CD36 gene was significantly decreased, while the expression of lipid efflux genes ABCA1 and ABCG1 was increased. Figure 3 Bodipy staining and flow cytometry results showed that HSCS could reduce intracellular lipid deposition (ac). Figure 3 de), and HSCS can adsorb oxLDL in the culture medium (de), and HSCS can adsorb oxLDL in the culture medium ( Figure 3 f).

[0074] Example 4: The therapeutic effect of HSCS on mice with atherosclerosis.

[0075] 1. Laboratory animals and their care.

[0076] Laboratory animal species, sex, age, and origin: ApoE - / - Male mice, 8 weeks old. ApoE - / - Mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Mice were housed in a specific pathogen-free (SPF) animal facility at a temperature of (22±2)℃ and a humidity of 45%–70%, with a 12-hour light cycle and free access to food and water.

[0077] 2. ApoE - / - Establishment of a mouse model of atherosclerosis.

[0078] ApoE - / - Mice were randomly divided into a control group and an experimental group, with 12 mice in each group. After being fed a high-fat diet for 11 weeks, the experimental group and the control group were injected with 1×10⁻⁶ mg / L via the tail vein. 6 Mice were given an equal volume of HSCS suspension and physiological saline for 4 weeks, twice a week, with weekly weight recording. Aorta, serum, and major organs were collected from the mice to evaluate the therapeutic effect of HSCS.

[0079] 3. Aortic dissection and Oil Red staining.

[0080] After deep anesthesia, the mice were euthanized, and excess organs were removed. The thymus was excised to expose the auricle and aortic arch. The brachiocephalic trunk was dissected, and the right subclavian artery was transected from its origin. The right common carotid artery was then dissected distally to a suitable length. Subsequently, the left common carotid artery and left subclavian artery were dissected sequentially to a suitable length. Following this, the descending aorta, thoracic aorta, and abdominal aorta were dissected, with intercostal arteries, bronchial branches, and other branch vessels transected from their origin. Only the left and right renal arteries and left and right common iliac arteries were dissected and preserved to a suitable length. Finally, the completely freed vessels were transected sequentially, starting from the right common carotid artery, to obtain the complete aorta.

[0081] Under a stereomicroscope, carefully dissect the intact aortic tissue, completely removing any attached adipose and connective tissue. The aortic tissue is then stored on ice. Oil Red O stock solution is mixed with distilled water at a 3:2 ratio, thoroughly mixed, and allowed to stand for 10 minutes before filtering through a 0.45 μm filter. The aortic tissue is then immersed in Oil Red working solution and stained at room temperature in the dark for 15-30 minutes. Non-specific staining is washed away with 60% isopropanol, followed by gentle rinsing with distilled water until the background is clear. The tissue is observed and photographed using a bright-field microscope, and the plaque area is quantitatively analyzed using ImageJ software.

[0082] 4. Aortic valve sections and Oil Red O staining.

[0083] The heart was transversely transversely along the lower edge of the left and right atrial appendages. The aortic root was placed upright in the center of the mold, and the tissue was completely covered with OCT. The tissue was then rapidly frozen on a freezing stage. The cryostat chamber temperature was pre-cooled to -20°C to -22°C, and the sample head temperature was set to -18°C to -20°C. Careful trimming was performed until three symmetrically distributed, slightly convex "crescent-shaped" or "hill-like" structures were visible around the lumen. The section thickness was adjusted to 5 μm. The section was gently flattened with a brush or glass slide and attached to a room-temperature slide. Oil Red staining of the aortic valve was performed using the same method as described in section 3.

[0084] 5. Detect the blood lipid content in mouse serum. The specific reagent kit and method are the same as in Example 2.

[0085] 6. Sections of major organs and HE staining. Specific steps are as follows.

[0086] 1) Preparation of paraffin sections.

[0087] A. Sample collection: The main organs of mice, such as the heart and liver, were placed in a 4% paraformaldehyde solution overnight.

[0088] B. Dehydration: Dehydration was carried out according to different alcohol concentrations: 70% alcohol for 2 hours, 80% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol I for 4 hours, 95% alcohol II overnight, 100% alcohol I for 1.5 hours, and 100% alcohol II for 1.5 hours.

[0089] C. Transparency: Immerse the tissue block in xylene I solution for 1 hour, then remove it and immerse it in xylene II solution for 1 hour.

[0090] D. Wax impregnation: overnight in paraffin I, 1 hour in paraffin II, and 1 hour in paraffin III.

[0091] E. Embedding: The tissue block was embedded in paraffin and left at room temperature.

[0092] F. Sectioning: The tissue block was sectioned using a paraffin microtome to a thickness of 3 μm, and the sections were then mounted on glass slides.

[0093] G. Slide drying and baking: After placing the slides on a 60°C slide dryer for 1 hour, place the slides in a 65°C oven for 48 hours.

[0094] 2) Dewaxing the sections: Place the sections in the following reagents according to the steps: xylene I for 20 min, xylene II for 20 min, 95% ethanol I for 15 min, 95% ethanol II for 15 min, 90% ethanol for 10 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and finally place them in distilled water for 30 min.

[0095] 3) Cell nuclear staining.

[0096] A. Immerse the slices in a hematoxylin solution for 20 minutes.

[0097] B. Place the paraffin sections in 1% hydrochloric acid for 30 seconds to differentiate, then rinse with running water.

[0098] C. Place the paraffin sections in ammonia water for 30 seconds to perform nucleus blueing, then rinse with running water.

[0099] 4) Cytoplasmic staining: Place the paraffin sections in a water-soluble eosin solution for 5 minutes to stain, and then rinse with running water.

[0100] 5) Transparency: Place the paraffin sections in the following reagents according to the steps: 80% alcohol for 5 min, 90% alcohol for 5 min, 100% alcohol I for 5 min, 100% alcohol II for 5 min, xylene I for 5 min, and xylene II for 5 min.

[0101] 6) Mounting: Place the paraffin sections in a fume hood to dry, and then mount the sections with neutral resin.

[0102] 7) Observe the HE staining results under a microscope and take pictures.

[0103] The results showed that mice in the HSCS group experienced significant weight loss one week after administration, and this trend continued until the end of administration. Figure 4a) In the HSCCS group, triglycerides, total cholesterol, and LDL-C were significantly decreased, while HDL-C was significantly upregulated. Figure 4 The Ctrl group showed significant plaque formation, covering 26.35 ± 3.04% of the entire aorta, while the HSCCS group showed a significantly smaller plaque area, accounting for 5.07% of the aorta. Figure 4 fg). Oil Red staining and quantitative analysis of aortic root sections showed a reduction in plaque area of ​​more than 70% ( Figure 4 Hi). No significant tissue necrosis was observed in the major organs of all groups after HE staining, indicating that HSCS has good biocompatibility and no obvious organ toxicity. Figure 4 j).

[0104] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A gelled cell loaded with sulfonated chitosan, characterized in that, The gelled cells are prepared by blending methacrylamide hyaluronic acid with sulfonated chitosan to form a hydrogel precursor, followed by in-situ gelation within the cells.

2. The gelled cells loaded with sulfonated chitosan according to claim 1, characterized in that, The gelled cells use macrophages as host cells.

3. The use of the gelled cells loaded with sulfonated chitosan as described in any one of claims 1-2 in the preparation of a drug for the prevention or treatment of atherosclerosis.

4. The application according to claim 3, characterized in that, The drug is used to lower serum triglyceride, total cholesterol, and low-density lipoprotein levels, and / or upregulate serum high-density lipoprotein levels.

5. The application according to claim 3, characterized in that, The drug is used to inhibit the formation of atherosclerotic plaques, reduce the area of ​​atherosclerotic plaques, and / or inhibit the formation of macrophage-derived foam cells.

6. A method for preparing gelled cells loaded with sulfonated chitosan according to any one of claims 1-2, characterized in that, Includes the following steps: S1 Preparation of methacrylamide hyaluronic acid powder and sulfonated chitosan powder; S2. Methacrylamide hyaluronic acid powder and sulfonated chitosan powder were dissolved in sterile water at a mass ratio of 1:2 to prepare a 1% (w / v) hydrogel precursor solution. A photoinitiator was added and the mixture was stirred to obtain the hydrogel working solution. S3 Macrophages in the logarithmic growth phase were collected, washed with PBS, resuspended in the hydrogel working solution, and collected as cell pellets by centrifugation after freeze-thaw treatment. S4 After washing the cell pellet with PBS, cross-linking reaction was carried out using 365nm ultraviolet light to obtain the gelled cells loaded with sulfonated chitosan.

7. The preparation method according to claim 6, characterized in that, In step S2, the photoinitiator is I2959, and the amount added is 0.5% of the mass of the hydrogel precursor solution.

8. The preparation method according to claim 6, characterized in that, In step S3, the freeze-thaw treatment involves freezing at -80°C for 10 minutes followed by complete thawing at room temperature; in step S4, the ultraviolet crosslinking reaction takes 10 minutes.

9. The use of the gelled cells prepared by the method for preparing sulfonated chitosan-loaded gelled cells according to claims 6-8 in the preparation of medicaments for the prevention and / or treatment of atherosclerosis.

10. A pharmaceutical preparation for the prevention or treatment of atherosclerosis, characterized in that... Gel-forming cells containing sulfonated chitosan as described in any one of claims 1-2 and a pharmaceutically acceptable carrier.

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