Application of shear force responsive nanoparticle targeted delivery of FBPi in prevention and treatment of calcified aortic valve disease

Abstract

The invention discloses application of shear force responsive nano particle targeted delivery FBPi in prevention and treatment of calcified aortic valve disease, and relates to the technical field of medical bioengineering. According to the method disclosed by the invention, knowledge of how to adjust osteogenesis reprogramming in CAVD by the intercellular burial effect of macrophages is clarified through research; it is clear that FBP1 is inhibited, the interburial effect of macrophages is enhanced, and CAVD is relieved; according to the invention, a shear force responsive nanoparticle targeted delivery drug for effectively targeting the aortic valve FBP1 is constructed for treating CAVD, and the problem of partial CAVD mechanism and the core problem that no effective aortic valve FBP1 targeted drug for treating CAVD exists at present are solved. Experimental data show that the shear force responsive nanoparticles deliver FBPi in a targeted manner to enhance the interment effect of macrophages and relieve aortic valve calcification. The complete strategy not only provides a new drug target treatment method for calcified aortic valve diseases, but also provides a new strategy for treatment of other vascular diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical bioengineering, and particularly relates to the use of shear-responsive nanoparticle targeted delivery of FBPi in preventing and treating calcific aortic valve disease. BACKGROUND

[0002] Calcific Aortic Valve Disease (CAVD) is a kind of degenerative cardiovascular disease characterized by progressive fibro-calcific remodeling of the aortic valve. The pathological core of CAVD is the abnormal deposition of calcium salt in the valve, which leads to valve stenosis or insufficiency, and eventually causes heart failure and even sudden cardiac death.

[0003] Recent studies have found that macrophages play a dual role in the progression of CAVD: on the one hand, as the core immune regulator of the valve microenvironment, it directly drives the osteogenic differentiation of valve interstitial cells (hVICs) by secreting pro-inflammatory factors such as TNFα and IL-6; on the other hand, studies have shown that macrophages maintain tissue homeostasis by phagocytosis of apoptotic cells and neutrophil traps (NETs). Although the protective mechanism of phagocytosis in atherosclerosis and other cardiovascular diseases has been gradually revealed, how it affects the fate of hVICs by regulating the immune metabolic network in CAVD is still unclear.

[0004] FBP1 is a key rate-controlling enzyme in the gluconeogenic pathway, which antagonizes glycolysis through its metabolic activity, but the regulatory mechanism of FBP1 on macrophage phagocytosis is still unknown. Currently, there is no shear-responsive nanoparticle targeted drug delivery for the treatment of CAVD.

[0005] In summary, the prior art has the following defects: (1) the knowledge of how macrophage phagocytosis regulates osteogenic reprogramming in CAVD is still limited; (2) the regulatory mechanism of FBP1 on macrophage phagocytosis is still unknown; (3) currently, there is no shear-responsive nanoparticle targeted drug delivery for the treatment of CAVD. SUMMARY

[0006] To solve the above problems in the prior art, the present application aims to provide a shear-responsive nanoparticle targeted delivery of FBP1 preparation for reducing or inhibiting the expression of FBP1 to promote the phagocytosis of macrophages to maintain the mechanism level of the valve, thereby preventing and alleviating the occurrence of calcific aortic valve disease (CAVD). At the same time, the present application also provides a new animal model for the study of CAVD, which is more convenient for the subsequent study of CAVD, thereby achieving the purpose of preventing or treating CAVD.

[0007] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0008] One of the purposes of the present invention is to provide a product for regulating FBP1 expression for use in the preparation of a drug for preventing and treating calcific aortic valve disease.

[0009] Furthermore, the product for regulating FBP1 expression is a product that reduces or inhibits FBP1 expression.

[0010] Furthermore, the product that reduces or inhibits FBP1 expression promotes macrophage efferocytosis, thereby preventing and treating calcific aortic valve disease.

[0011] Furthermore, the product for reducing or inhibiting FBP1 expression includes targeted delivery of FBPi by responsive nanoparticles.

[0012] A second object of the present invention is to provide use of any of the above products for reducing or inhibiting FBP1 expression in the preparation of a product for promoting macrophage efferocytosis.

[0013] A third object of the present invention is to provide a drug for preventing and treating calcific aortic valve disease, wherein the drug comprises any of the above products for reducing or inhibiting FBP1 expression.

[0014] Furthermore, the drug includes responsive nanoparticles for targeted delivery of FBPi.

[0015] Furthermore, the preparation method of the responsive nanoparticles for targeted delivery of FBPi comprises the following steps:

[0016] S1. A solution containing folic acid (Fl), ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is added to an aqueous solution of sodium alginate (Ag) for reaction activation in a dark environment, and then ethylenediamine (EDA) is added to form a diamine-linked Ag-Fl conjugate;

[0017] S2. Prepare Ag stock solution, Ag-Fl stock solution, calcium chloride stock solution and FBPi stock solution respectively. First, fully mix the Ag stock solution, Ag-Fl stock solution and FBPi stock solution. Then, drop the calcium chloride stock solution into the mixture and mix thoroughly. Finally, centrifuge to obtain Ag@FBPi nanoparticles.

[0018] S3. Using a shear-responsive shell layer to non-covalently bond with Ag@FBPi nanoparticles to synthesize AgCs@FBPi, wherein the AgCs@FBPi is the responsive nanoparticle targeted delivery of FBPi.

[0019] Furthermore, the FBPi content in the FBPi stock solution is 80-120 mmol / L; and the shell material includes chitosan (Cs).

[0020] A fourth object of the present invention is to provide a product that promotes macrophage efferocytosis, wherein the product comprises any of the above products that reduce or inhibit FBP1 expression.

[0021] Furthermore, the product includes the above-mentioned responsive nanoparticles for targeted delivery of FBPi.

[0022] Compared with the prior art, the present invention has the following improvements and beneficial effects:

[0023] 1. Improvement point 1: Dysfunction of macrophage efferocytosis aggravates aortic valve calcification

[0024] ① Technical Plan: A Transwell co-culture system was established, and Western blot and Alizarin Red staining confirmed that dysfunctional macrophage efferocytosis exacerbated aortic valve calcification. ② Experimental Evidence: Expression of bone differentiation markers Runx2 and Osterix was decreased in the efferocytosis group, while upregulated in the apoptosis group. The area of ​​calcified nodules was significantly reduced in the efferocytosis group, while significantly increased in the apoptosis group. ③ Beneficial Effects: It was confirmed that dysfunctional macrophage efferocytosis exacerbates aortic valve calcification.

[0025] 2. Improvement 2: Mechanism of inhibiting FBP1 to enhance macrophage efferocytosis

[0026] ① Technical solution: Through in vivo and in vitro experiments, we explored the mechanism and effect of FBP1 on macrophage efferocytosis, and provided potential therapeutic targets for improving CAVD by driving macrophage efferocytosis. ② Experimental evidence: After inhibiting FBP1, flow cytometry showed that macrophage efferocytosis was enhanced; a mouse model with APOE- / - macrophage-specific knockout of the FBP1 target gene was constructed. M-KO APOE - / - In the mouse model, enhanced macrophage efferocytosis and significantly reduced calcification and fibrosis were observed. ③Beneficial effects: It was confirmed that inhibiting FBP1 enhances macrophage efferocytosis and alleviates CAVD.

[0027] 3. Improvement 3: Targeted delivery of FBPi by shear-responsive nanoparticles enhances macrophage efferocytosis and alleviates aortic valve calcification

[0028] ① Technical Solution: By leveraging the increased shear stress at the CAVD valve, we constructed shear-responsive drug-loaded nanoparticles and investigated their ability to locally release drugs at the CAVD valve to enhance macrophage efferocytosis, ultimately slowing CAVD progression. ② Experimental Evidence: AgCs@FBPi enhanced macrophage efferocytosis of NETs and inhibited CAVD progression in mice. In two mouse models, tail vein injection of AgCs@FBPi hindered the progression of valvular stenosis. ③ Beneficial Effects: We demonstrated that targeted delivery of FBPi via shear-responsive nanoparticles enhances macrophage efferocytosis and reduces aortic valve calcification.

[0029] In summary, this study clarified how macrophage efferocytosis regulates osteogenic reprogramming in CAVD; demonstrated that inhibiting FBP1 enhances macrophage efferocytosis and alleviates CAVD; and constructed a shear-responsive nanoparticle-based drug delivery method that effectively targets FBP1 in the aortic valve for the treatment of CAVD. This approach addresses some of the key issues surrounding the mechanism of CAVD and the current lack of effective drugs targeting FBP1 in the aortic valve for the treatment of CAVD. Experimental data demonstrate that shear-responsive nanoparticle-based drug delivery of FBPi enhances macrophage efferocytosis and alleviates aortic valve calcification. This comprehensive strategy not only provides a new drug target therapy for calcific aortic valve disease but also offers a novel strategy for the treatment of other vascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The analysis results of the RNA sequencing dataset in Example 1 are shown in Figure 1, specifically (a) a heat map of the proportions of 28 aortic valve-infiltrating immune cells in ssGSEA and (b) KEGG enrichment analysis.

[0031] Figure 2 The immunofluorescence staining results in Example 1 are as follows: (a) NAVs and CAVs were fluorescently stained, CD68 + CD86 + Representative images of M1 macrophages, immunofluorescence staining, and statistical analysis of their number (n=10). (b) CD68 + CD206 + Cells represent M2 macrophages. Statistical analysis of the number of M2 macrophages in NAVs and CAVs (n = 10). (c) Statistical analysis of the ratio of M1 to M2 macrophages in NAVs and CAVs (n = 11). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0032] Figure 3For flow cytometry analysis results in Example 1, specifically (a) NAvs and CAVs were detected with CD15 (red) for neutrophils within NAvs and CAVs. (b) Whole blood analysis of NAvs and CAVs by flow cytometry for circulating neutrophils (live cells, CD11b + CD15 + ). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0033] Figure 4 Representative images and statistical graphs for Western blot detection of macrophage iNOS, CD86 (macrophage inflammatory markers) in Example 2.

[0034] Figure 5 Transwell co-culture model of macrophages with hVICs in Example 2, specifically (a) co-culture model of macrophages with hVICs. (b) Western blot detection of hVICs Runx2 and Osterix (osteogenic differentiation marker proteins) expression results. (c) Representative images and statistical graphs of hVICs alizarin red staining. (d) Quantification of hVICs calcium salt deposition. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0035] Figure 6 Flow cytometry analysis results in Example 3, specifically (a) Fructose-1,6-bisphosphate levels were analyzed by LC-MS / MS. (b) Seahorse analysis was performed to measure glycolytic rate, i.e., extracellular acidification rate (ECAR). (c) Flow cytometry detection of macrophage phagocytosis efficiency. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0036] Figure 7 APOE - / - Background Macrophage-specific knockout FBP1 target gene mouse model, Western blot detection of knockout efficiency after BMDMs were isolated.

[0037] Figure 8For the ultrasound evaluation results and H&E staining results of the mouse aortic valve after the construction of the FBP1 target gene knockout mouse model specific to macrophages in the APOE- / - background in Example 3, specifically (a) peak velocity of the mouse aortic valve jet flow was detected by ultrasound. (b) Representative image of H&E staining of the aortic valve and quantitative analysis of the thickness of the valve leaflet. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0038] Figure 9 For the Masson staining and alizarin red staining results after the construction of the FBP1 target gene knockout mouse model specific to macrophages in the APOE- / - background in Example 3, specifically (a) alizarin red staining of the mouse aortic valve and quantitative analysis of the total calcification area. (b) Representative image of Masson staining of the mouse aorta and quantitative analysis of collagen deposition. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0039] Figure 10 For the construction mode diagram of the shear-responsive drug-loaded nanoparticles in Example 4: the shear-responsive shell is composed of Cs, and the Fl-modified Ag nanoparticles loaded with FBPi constitute the core of the nanoparticles, and the Cs and the Ag core are non-covalently bonded.

[0040] Figure 11 For the characterization results of AgCs@FBPi in Example 4, specifically (a) DLS analysis of the effect of chitosan addition amount on particle size. (b) Effect of chitosan addition amount on the surface charge of the nanoparticles. (c) FITR for characterizing the composition of AgCs@FBPi nanoparticles.

[0041] Figure 12 For the optimization results of the shear-responsive nanoparticles AgCs@FBPi in Example 4, specifically (a) statistical analysis of the particle size of the sodium sulfate-enhanced nanoparticles under different shear force treatments. (b) DLS analysis of the particle size of the sodium sulfate-enhanced nanoparticles under different shear force treatments.

[0042] Figure 13 For the evaluation results of the targeting macrophage function of the shear-responsive nanoparticles AgCs@FBPi in Example 4, specifically the targeting ability of the nanoparticles to macrophages before and after being subjected to shear force was evaluated using a laser confocal microscope.

[0043] Figure 14 For the mouse aortic valve ultrasound results in Example 4, specifically (a, b) Representative image and statistical analysis of the peak velocity of the mouse aortic valve jet flow detected by echocardiography of ApoE- / - mice induced by HCD (a) and DWI (b).

[0044] Figure 15 H&E staining results in Example 4, specifically, APOE - / - Two mouse models were treated with PBS, FBPi, and AgCs@FBPi, respectively. (a, b) Representative images of H&E staining of mouse aortic valves and quantitative analysis of valve leaflet thickness. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0045] Figure 16 Alizarin red staining results in Example 4, specifically, APOE - / - Two mouse models were treated with PBS, FBPi, and AgCs@FBPi, respectively. (a, b) Representative images of alizarin red staining of mouse aortic valves and quantitative analysis of total calcified area. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

[0046] The following examples are intended to illustrate the present application but not to limit the scope of the present application. Modifications or replacements of the methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application. The reagents, products and instruments used in the following examples can be obtained from the market, and the methods used in the examples are consistent with the commonly used methods unless otherwise specified.

[0047] The following experimental verification and experimental methods are used in the present application.

[0048] I. Experimental verification

[0049] 1. Macrophage efferocytosis dysfunction aggravates aortic valve calcification

[0050] ①RNA sequencing data set analysis and immunofluorescence staining, systematic comparison of macrophage phenotype characteristics and efferocytosis function differences in NAVs and CAVs samples;

[0051] ②Comparison of normal control and CAVD-derived blood and valve samples by flow cytometry and immunofluorescence staining analysis, neutrophil infiltration and NETs formation difference;

[0052] ③Western blot experiment detection, NETs stimulate macrophages and macrophages phagocytose NETs after efferocytosis, changes of macrophage pro-inflammatory phenotype;

[0053] ④Through Transwell experiment and hVICs co-culture, combined with Western blot, alizarin red staining quantitative analysis, the effect of macrophage phenotype change on hVICs osteogenic differentiation was detected.

[0054] 2. Inhibition of FBP1 enhances macrophage phagocytosis mechanism

[0055] ① Construction of APOE - / - Background: Specific knockout of target genes in macrophages in mouse models, Western blot was used to detect the knockout efficiency after isolation of BMDMs;

[0056] ② Through small animal heart ultrasound, H&E staining, Masson staining and alizarin red staining to detect the hemodynamic changes and aortic valve pathological changes of mice.

[0057] 3. Shear-responsive nanoparticle targeted delivery of FBPi enhances macrophage phagocytosis to reduce aortic valve calcification

[0058] ① Preparation and optimization of shear-responsive nanoparticle AgCs@FBPi;

[0059] ② Evaluation of shear-responsive nanoparticle AgCs@FBPi targeting macrophage function;

[0060] ③ AgCs@FBPi can enhance the phagocytosis of macrophages to NETs and inhibit the progression of CAVD in mice.

[0061] II. Experimental methods

[0062] ① Specimen collection:

[0063] During the collection of valve specimens, strict aseptic operation behavior specifications must be strictly followed. After the aortic valve leaflets were removed during surgery, they were immediately rinsed with ice-preconditioned sterile PBS to remove surface residues. Then the leaflets were roughly divided into three parts: (1) One-third was placed in pre-chilled DMEM medium and quickly sent to the laboratory to extract hVICs using an ice box to maintain low temperature; (2) Another one-third was placed in normal saline, ready to be separated into macrophages by flow cytometry sorting technique; (3) The remaining one-third was cut into complete long-axis full-thickness tissue blocks, wrapped with OCT embedding agent, and then frozen sections were prepared. The remaining tissue samples were fixed with an appropriate amount of 4% paraformaldehyde solution for subsequent paraffin embedding and tissue section preparation.

[0064] ② Flow cytometry:

[0065] Before surgery, a blood sample was collected from the patient and centrifuged at 2500 rpm / min for 10 minutes. Seven milliliters of red blood cell lysis buffer was added and the cells were lysed for 5-10 minutes. The cells were then neutralized with an equal volume of PBS and centrifuged at 2500 rpm / min for 5 minutes. The supernatant was discarded and a single-cell suspension was prepared by pipetting. The prepared single-cell suspension was counted, grouped appropriately, and resuspended in an appropriate amount of PBS, with approximately 1×10 cells / tube in 250 μL of PBS. 6 cells and divided them into 1.5 mL EP tubes; the corresponding flow cytometry antibody was added to each EP tube at a concentration of 0.5 μL / 10 6 Place the cells to be tested and incubate the mixture on ice in the dark for approximately 50 minutes. Set up corresponding control and single-staining groups according to the experimental design. Then, add 1 mL of PBS for neutralization and centrifuge again at 400 g / min for 5 minutes. Discard the supernatant and add 200 μL of 1% FBS in PBS for neutralization. Centrifuge at 400 g / min for 5 minutes. After discarding the supernatant, add 200 μL of PBS to each EP tube. Protect from light, resuspend on a shaker, and analyze.

[0066] ③Construction of ApoE- / - macrophage FBP1 knockout mice:

[0067] The mice used in this experiment were all C57BL6 background mice. - / - Mouse FBP1 loxP (FBP1 fl / fl ) mice and Lyz2-Cre mice were purchased from Alvance Biotechnology Co., Ltd. (Wuhan). - / - FBP1 fl / fl Mice and Lyz2-Cre (Lyz2 cre ) mice were bred to obtain ApoE - / - FBP1 fl / fl Mice and ApoE - / - Lyz2 cre Mice. Then ApoE - / - FBP1 fl / fl Mice and ApoE - / - Lyz2 cre ApoE is acquired by breeding mice - / - Background: Macrophage FBP1-specific knockout mice (ApoE - / - FBP1 fl / fl Lyz2 cre ).

[0068] ④ Preparation of AgCs@FBPi:

[0069] Preparation of the Ag-Fl core: 50 mg of folic acid (Fl) was initially dissolved in 15 ml of dimethyl sulfoxide (DMSO) to form a homogeneous solution. This solution was then slowly added to 20 ml of a 100 mg / ml aqueous solution of sodium alginate (Ag) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The molar ratio of Fl to EDC to NHS was 1:4:2. The mixture was reacted at 25°C in the dark for 1 hour to activate the carboxyl groups on Fl and Ag. To initiate an amidation reaction between the activated carboxyl groups and amino groups, 20 mg of ethylenediamine (EDA) was added to the Fl / Ag mixture. The reaction was carried out at pH 5.5 and 25°C for 24 hours to form the diamine-linked Ag-Fl conjugate. After completion of the reaction, the Ag-Fl conjugate was purified by dialysis to remove unreacted components. The purified conjugate was then freeze-dried to obtain a stable solid form for further characterization and application.

[0070] Preparation of Ag@FBPi: Ag stock solutions (0.5 mg / ml), Ag-Fl stock solutions (0.5 mg / ml), and calcium chloride (CaCl2) stock solutions (18 mmol / L) were prepared in 1% aqueous lactic acid. FBPi stock solutions (100 mmol / L, MCE, HY-136717) were prepared in DMSO. 9 ml of the Ag stock solution, 3 ml of the Ag-Fl stock solution, and 1.4 ml of the FBPi stock solution were uniformly mixed. The mixture was then sonicated for 5 minutes to ensure thorough mixing. 0.63 ml of CaCl2 was added dropwise to the mixture, mixed thoroughly, and sonicated again for 10 minutes. This step promoted the formation of the desired Ag@FBPi nanoparticles. The solution was centrifuged at 10,000 rpm / min to separate and collect the Ag@FBPi nanoparticles.

[0071] Preparation of AgCs@FBPi: The nanoparticles were composed of a core with a shear-responsive shell and folate (Fl) modified sodium alginate (Ag) loaded FBPi. The chitosan shell (Cs) layer was non-covalently bonded to the Ag core, and finally AgCs@FBPi was synthesized, which enabled the high shear force existing in the diseased valve environment to destroy the chitosan and release FBPi with specific binding to macrophages. The specific process is as follows: the influence of the amount of chitosan on the particle size of the nanoparticles was systematically monitored by dynamic light scattering (DLS) technology, the Ag@FBPi prepared in the previous step was mixed with 0.2 mg / ml chitosan at a ratio of 1:1, and then the mixture was ultrasonically treated for 5 minutes to ensure thorough mixing. The formation of the chitosan coating structure was verified by zeta potential characterization, and the presence of the chitosan structure in the composite system was verified from the molecular vibration energy level by detecting the characteristic absorption peak of chitosan at 3000 cm⁻¹ of AgCs nanoparticles by Fourier transform infrared spectroscopy. Finally, in order to improve the shear response threshold of the nanoparticles, 0.5 mg / ml of sodium sulfate was added to the shell layer, and ultrasonic treatment was performed for 5 minutes to ensure thorough mixing, and the stability of the outer shell layer was enhanced by ion coupling.

[0072] The technical solutions of the application will be further described in detail below in conjunction with examples.

[0073] Example 1: Dysfunction of macrophage efferocytosis in calcified aortic valve

[0074] (1) Experimental design

[0075] By comparing the differences in macrophage phenotype characteristics and efferocytosis function between normal aortic valve (NAVs) and calcified valve (CAVs) samples, the effect of macrophage efferocytosis on CAVD was explored.

[0076] (2) Experimental steps

[0077] ① RNA sequencing data set analysis and immunofluorescence staining, systematic comparison of differences in macrophage phenotype characteristics and efferocytosis function between NAVs and CAVs samples.

[0078] ② By flow cytometry and immunofluorescence staining analysis, the differences in neutrophil infiltration and NETs formation between normal control and CAVD derived blood and valve specimens were compared.

[0079] ③ Grouping: normal valve control group: normal valve samples were derived from patients receiving heart transplantation due to dilated cardiomyopathy; calcified valve control group: calcified valve samples were from patients receiving valve replacement due to CAVD.

[0080] (4) Experimental results:

[0081] (3) Experimental results

[0082] ① RNA sequencing dataset analysis: Based on RNA sequencing datasets GSE76717 and GSE15355, this example systematically compared the immune cell differences between NAVs and CAVs samples. The heatmap visualization result showed that the ssGSEA scores of 28 immune cell subsets presented significant heterogeneity between the two groups, among which the macrophage population showed significant differences in subset composition. KEGG enrichment analysis showed that the inflammatory immune response-related pathways (such as NFKB, TNF signal) in CAVs macrophages were significantly activated, while the phagocytosis-related pathways (such as phagosome formation, apoptotic cell clearance) were significantly inhibited (Fig. 2A). Figure 1 ).

[0083] ② Immunofluorescence staining: CAVs infiltrated a large number of M1 macrophages, and the macrophage efferocytosis decreased (Fig. 2B). Figure 2 ).

[0084] ③ Flow cytometry: CAVs increased neutrophil infiltration and accumulated a large number of NETs (Fig. 2C). Figure 3 .

[0085] (4) Experimental results:

[0086] ① Macrophage efferocytosis dysfunction in calcified aortic valve (CAVs);

[0087] ② CAVs increased neutrophil infiltration and accumulated a large number of NETs.

[0088] Example 2 Macrophage efferocytosis dysfunction aggravates aortic valve calcification

[0089] 1. Experimental design

[0090] To clarify the mechanism of macrophage-mediated apoptotic cell clearance and its regulatory effect on valve calcification, this example focuses on the functional analysis of the macrophage-NETs interaction network.

[0091] 2. Experimental steps

[0092] ① Constructing a Transwell co-culture system: co-culture M1 macrophages with hVICs (Fig. 3A). Figures 4-5 .

[0093] ② Western blot: collect cells, extract proteins, specifically, use RIPA cell lysis buffer to lyse VIC cells, then perform ultrasonic cell disruption, ultrasonic power 8%, ultrasonic 1 second, pause 1 second, ultrasonic 10 cycles, then centrifuge at 12,000 rpm / min for 15 min, aspirate the supernatant, add 5x loading buffer and denature the protein at 95°C for 5 min. Detect the expression levels of Runx2 and Osterix proteins.

[0094] ③ Alizarin red staining: after fixing the cells with 4% paraformaldehyde for 10 min, perform alizarin red staining to evaluate the content of late-stage calcium nodules.

[0095] ④ Grouping design: Efferocytosis group: macrophages only undergoing efferocytosis are co-cultured with hVICs; Apoptosis group: macrophages stimulated by NETs are co-cultured with hVICs.

[0096] 3. Experimental results

[0097] ① Western blot ( Figure 5 ): The expression of osteogenic differentiation markers Runx2 and Osterix decreases in the Efferocytosis group; the expression of osteogenic differentiation markers Runx2 and Osterix increases in the Apoptosis group.

[0098] ② Alizarin red staining ( Figure 5 ): The area of calcified nodules significantly decreases in the Efferocytosis group; the area of calcified nodules significantly increases in the Apoptosis group.

[0099] 4. Experimental conclusion

[0100] Macrophage efferocytosis dysfunction exacerbates aortic valve calcification.

[0101] Example 3 Mechanism of inhibiting FBP1 to enhance macrophage efferocytosis

[0102] 1. Experimental design

[0103] By exploring the mechanism and effect of FBP1 on macrophage efferocytosis in vivo and in vitro, potential therapeutic targets for improving CAVD by driving macrophage efferocytosis are provided.

[0104] 2. Experimental steps

[0105] ① After inhibiting FBP1, flow cytometry is used to detect the changes in macrophage efferocytosis function;

[0106] ② Construct APOE - / -Background: Macrophage-specific knockout of the FBP1 target gene in a mouse model. BMDMs were isolated and the knockout efficiency was determined by Western blot.

[0107] ③ The hemodynamics and aortic valve pathological changes of mice were detected by small animal cardiac ultrasound, H&E staining, Masson staining and Alizarin red staining;

[0108] ④Detection indicators: H&E staining was used to evaluate the thickness of the aortic valve; Masson staining and Alizarin red staining were used to evaluate the degree of aortic valve calcification; mouse aortic valve ultrasound was used to evaluate the degree of aortic valve stenosis.

[0109] ⑤CAVD animal model grouping: 4-week-old male FBP1 M-WT APOE - / - and FBP1 M-KO APOE - / - 25 mice were divided into 4 groups:

[0110] (1) ND+FBP1 M-WT APOE - / - :Normal diet feeding (ND)

[0111] (2) HCD+FBP1 M-WT APOE - / - : High-fat diet feeding (ND)

[0112] (3) ND+FBP1 M-KO APOE - / - : Normal diet feeding (HCD)

[0113] (4) HCD+FBP1 M-KO APOE - / - : High-fat diet (HCD)

[0114] The mice were induced with the diet at 8 weeks of age. Ultrasound examination was performed after 24 weeks of feeding, and then the mice were sacrificed to obtain heart and aortic valve specimens for pathological examination.

[0115] 3. Experimental Results

[0116] ① After inhibiting FBP1, flow cytometry showed that macrophage efferocytosis was enhanced ( Figure 6 );

[0117] ②Construct APOE - / - Background: Macrophage-specific knockout of the FBP1 target gene in a mouse model. BMDMs were isolated and Western blot was used to detect knockout efficiency. The results showed that FBP1 knockout was significantly effective ( Figure 7 );

[0118] ③ Ultrasound evaluation of aortic valve function in mice showed that FBP1 M-WT APOE - / - The peak velocity of aortic valve orifice jet in mice was significantly increased, indicating that aortic valve calcification was successfully induced. M-KO APOE - / - In mice, myeloid cell-specific knockout of FBP1 partially restored this change ( Figure 9 ).

[0119] ④H&E staining results showed that HCD-induced FBP1 M-WT APOE - / - Increased aortic valve leaflet thickness and FBP1 M-KO APOE - / - The aortic valve of mice was reduced ( Figure 8 ).

[0120] ⑤Masson staining and Alizarin red staining showed that HCD feeding promoted the expression of FBP1 M-WT APOE - / - Calcium deposition and increased fibrosis in the mouse aortic valve. M-KO APOE - / - Calcification and fibrosis were significantly reduced ( Figure 9 ).

[0121] 4. Experimental Conclusion

[0122] Inhibition of FBP1 enhances macrophage efferocytosis and alleviates CAVD.

[0123] Example 4 Study on the Enhancement of Macrophage Efferocytosis and Reduction of Aortic Valve Calcification by Targeted Delivery of FBPi by Shear-Responsive Nanoparticles (Comparison between Targeted Delivery of FBPi by Shear-Responsive Nanoparticles and Intraperitoneal Injection of FBPi)

[0124] 1. Experimental Design

[0125] (1) By utilizing the increased shear stress at the CAVD valve, shear-responsive drug-loaded nanoparticles were constructed to explore their ability to release drugs locally at the CAVD valve to enhance macrophage efferocytosis, ultimately delaying CAVD progression. (2) The therapeutic effects of targeted delivery of FBPi via shear-responsive nanoparticles and intraperitoneal injection of FBPi in a mouse CAVD model were compared.

[0126] 2. Experimental Procedure

[0127] ① Preparation and Optimization of Shear-Responsive Nanoparticles (AgCs@FBPi): This system utilizes nanoparticles with a shear-responsive shell and a core composed of FBPi-loaded sodium alginate (Ag) modified with folate (Fl). The chitosan (Cs) shell is non-covalently bonded to the Ag core to form the AgCs@FBPi. This allows the high shear forces present in the diseased valve environment to disrupt the chitosan, allowing it to specifically bind to macrophages and release FBPi.

[0128] ② Functional evaluation of shear force responsive nanoparticles AgCs@FBPi targeting macrophages: Macrophages were injected with 230 dyn / cm 2 Shear-treated AgCs@FBPi-230 nanoparticles were incubated together with Ag@FBPi and AgCs@FBPi nanoparticles as controls.

[0129] ③AgCs@FBPi can enhance the efferocytosis of NETs by macrophages and inhibit the progression of CAVD in mice, as evaluated by H&E staining, Alizarin red staining, and mouse aortic valve ultrasound.

[0130] ④AgCs@FBPi / PBS was injected into the tail vein and FBPi / PBS was injected into the peritoneal cavity to observe the therapeutic effect on the CAVD model in mice.

[0131] ⑤ Group design: Two CAVD mouse models were used simultaneously: HCD induced ApoE - / - Mice and guidewire injury (DWI)-induced ApoE - / - In the CAVD model, mice were injected with AgCs@FBPi via the tail vein three times a week starting at week 8, while two other groups of mice were injected intraperitoneally with PBS or FBPi three times a week. Both groups were treated until week 28.

[0132] HCD induces ApoE - / - Mice:

[0133] HCD+PBS group: fed with high-fat diet and intraperitoneally injected with PBS

[0134] HCD+FBPi group: fed with high-fat diet and intraperitoneally injected with FBPi

[0135] HCD+AgCs@FBPi group: fed with high-fat diet, injected with AgCs@FBPi via tail vein

[0136] DWI-induced ApoE - / - Mice:

[0137] HCD+PBS group: fed with high-fat diet and intraperitoneally injected with PBS

[0138] HCD+FBPi group: fed with high-fat diet and intraperitoneally injected with FBPi

[0139] HCD+AgCs@FBPi group: fed with high-fat diet, injected with AgCs@FBPi via tail vein

[0140] 3. Experimental Results

[0141] ① Preparation and optimization of shear-responsive nanoparticles AgCs@FBPi: The sodium sulfate strengthening strategy can precisely regulate the mechanical response threshold of chitosan-based nanocarriers, giving them adaptability to the blood flow environment: maintaining structural integrity within the physiological shear range (<190dyn / cm2), while maintaining stability in pathological high shear environments (>230dyn / cm2). 2 ) triggers a controlled dissociation effect ( Figures 10-12 ).

[0142] ② Evaluation of the macrophage-targeting function of shear-responsive nanoparticles AgCs@FBPi: AgCs@FBPi nanoparticles enhanced with sodium sulfate were able to maintain their original morphology in a normal blood environment and restore their ability to bind to macrophages when exposed to a calcified aortic valve ( Figure 13 ).

[0143] ③AgCs@FBPi can enhance the efferocytosis of macrophages to NETs and inhibit the progression of CAVD in mice: In two mouse models, tail vein injection of AgCs@FBPi treatment hindered the progression of valvular stenosis. Among them, the aortic valve ultrasound of mice: After 28 weeks of feeding, HCD-induced ApoE - / - Mice and DWI-induced ApoE - / - The aortic valve peak flow velocity of mice was significantly increased, while the injection of AgCs@FBPi significantly inhibited the aortic valve peak flow velocity ( Figure 14 ). H&E staining: ApoE induced by HCD after 28 weeks of feeding - / - Mice and DWI-induced ApoE - / - The aortic valve of mice was significantly thickened, and AgCs@FBPi injection significantly inhibited this thickening ( Figure 15 ). Alizarin red staining: ApoE induced by HCD after 28 weeks of feeding - / - Mice and DWI-induced ApoE - / - The aortic valve of mice was significantly calcified, and the injection of AgCs@FBPi significantly inhibited the calcification ( Figure 16 ).

[0144] 4. Experimental Conclusion

[0145] Targeted delivery of FBPi via shear-responsive nanoparticles enhances macrophage efferocytosis and alleviates aortic valve calcification.

[0146] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of products that regulate FBP1 expression in the preparation of drugs for the prevention and treatment of calcific aortic valve disease.

2. The use according to claim 1, characterized in that The product for regulating FBP1 expression is a product for reducing or inhibiting FBP1 expression.

3. The use according to claim 2, characterized in that The product that reduces or inhibits FBP1 expression promotes the efferocytosis of macrophages, thereby playing a role in preventing and treating calcific aortic valve disease.

4. The use according to claim 3, characterized in that The product for reducing or inhibiting FBP1 expression includes responsive nanoparticles for targeted delivery of FBPi.

5. Use of the product for reducing or inhibiting FBP1 expression according to any one of claims 2 to 4 in the preparation of a product for promoting macrophage efferocytosis.

6. A drug for preventing and treating calcific aortic valve disease, characterized in that: The medicine includes the product for reducing or inhibiting FBP1 expression in any one of claims 2 to 4.

7. The drug according to claim 6, characterized in that The drug includes responsive nanoparticles for targeted delivery of FBPi.

8. The drug according to claim 7, characterized in that The preparation method of the responsive nanoparticle targeted delivery FBPi comprises the following steps: S1. A solution containing folic acid (Fl), ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is added to an aqueous solution of sodium alginate (Ag) for reaction activation in a dark environment, and then ethylenediamine (EDA) is added to form a diamine-linked Ag-Fl conjugate; S2. Prepare Ag stock solution, Ag-Fl stock solution, calcium chloride stock solution and FBPi stock solution respectively. First, fully mix the Ag stock solution, Ag-Fl stock solution and FBPi stock solution. Then, drop the calcium chloride stock solution into the mixture and mix thoroughly. Finally, centrifuge to obtain Ag@FBPi nanoparticles. S3. Using a shear-responsive shell to non-covalently bond with Ag@FBPi nanoparticles to synthesize AgCs@FBPi, wherein the AgCs@FBPi is the responsive nanoparticle targeted delivery of FBPi.

9. The drug according to claim 8, characterized in that The FBPi content in the FBPi stock solution is 80-120 mmol / L; the shell layer raw material includes chitosan (Cs).

10. A product for promoting macrophage efferocytosis, characterized in that: The product includes the product for reducing or inhibiting FBP1 expression in any one of claims 2-4.

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