SiRNA delivery lipid nanoparticle for targeted inhibition of GPNMB expression in macrophage-derived foam cells and application of siRNA delivery lipid nanoparticle

Abstract

The invention discloses siRNA delivery lipid nanoparticles for targeted inhibition of GPNMB expression in macrophage-derived foam cells and application of the siRNA delivery lipid nanoparticles, and belongs to the technical field of biology. According to the siRNA delivery lipid nanoparticle, the lipid nanoparticle serves as a carrier, siGpnmb is loaded on the carrier, monoclonal antibodies of CD36 and TREM2 are coupled to the surface of the carrier, the CD36 monoclonal antibody and the TREM2 monoclonal antibody serve as target heads of an antibody-LNP-siRNA compound, and the lipid nanoparticle loaded with the siGpnmb can be delivered into foam cells in a targeted mode. The siGpnmb released in the foam cells can inhibit the expression of GPNMB, so that the autophagy level in the foam cells is improved, the lysosome degradation capability is enhanced, and the overall foaming level is obviously reduced, thereby reducing the lipid core burden in vascular plaques and realizing the treatment of atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an siRNA delivery lipid nanoparticle for targeted inhibition of GPNMB expression in macrophage-derived foam cells and its application, and particularly relates to an siRNA delivery lipid nanoparticle conjugated with monoclonal antibodies against CD36 and TREM2 on its surface, its preparation method and application, especially its application in the treatment and / or prevention of atherosclerosis. Background Art

[0002] As the chronic disease with the highest mortality rate globally at present, the prevention and treatment of atherosclerosis still face many challenges. The current treatment methods for atherosclerosis mainly include drug treatment, surgical treatment and interventional treatment. Among them, lipid-regulating drugs (the first choice for lipid-lowering drugs - statin drugs) are important treatment means to control risk factors and improve the prognosis of patients.

[0003] Despite these treatment methods, atherosclerosis remains the leading cause of death and disability globally. In addition, many available treatment methods may have adverse side effects. For example, statin drugs have obvious adverse reactions, such as they may cause myopathy, renal failure or even death.

[0004] Therefore, there is still a need for a more effective and comprehensive treatment plan for atherosclerosis. Summary of the Invention

[0005] In view of one or more of the problems existing in the prior art, in the first aspect of the present invention, there is provided an siRNA delivery lipid nanoparticle, which includes a lipid nanoparticle with a core-shell structure, siRNA encapsulated in the core of the lipid nanoparticle, and an antibody conjugated on the surface of the shell of the lipid nanoparticle;

[0006] wherein the siRNA is an siRNA for inhibiting the expression of GPNMB protein in cells; and

[0007] wherein the antibody is an antibody capable of specifically binding to a surface marker protein of macrophages and / or their derived foam cells.

[0008] In some embodiments, the sense strand of the siRNA may include the nucleotide sequence shown in SEQ ID NO: 1 or be composed of it, the antisense strand of the siRNA may include the nucleotide sequence shown in SEQ ID NO: 2 or be composed of it, and TT is overhanging at the 3' ends of both the sense strand and the antisense strand of the siRNA.

[0009] In some embodiments, the antibody may include one or both of a CD36 monoclonal antibody and a TREM2 monoclonal antibody.

[0010] In some embodiments, the CD36 monoclonal antibody may be selected from the CD36 monoclonal antibody sc-7309, and the TREM2 monoclonal antibody may be selected from the TREM2 monoclonal antibody ab305103.

[0011] In some embodiments, the siRNA is an siRNA that inhibits the expression of GPNMB protein in macrophages and / or their derived foam cells.

[0012] The second aspect of the present invention provides an siRNA, the sense strand of which may comprise or consist of the nucleotide sequence shown in SEQ ID NO: 1, the antisense strand of which may comprise or consist of the nucleotide sequence shown in SEQ ID NO: 2, and TT is overhanging at the 3' ends of both the sense strand and the antisense strand of the siRNA.

[0013] The third aspect of the present invention provides the use of a reagent that inhibits the expression of GPNMB protein in macrophages and / or their derived foam cells in the preparation of a drug for the treatment and / or prevention of atherosclerosis. The reagent that inhibits the expression of GPNMB protein in macrophages and / or their derived foam cells may be the siRNA delivery lipid nanoparticles provided by the first aspect of the present invention or the siRNA provided by the second aspect of the present invention.

[0014] The use of the siRNA delivery lipid nanoparticles provided by the first aspect of the present invention or the siRNA provided by the second aspect of the present invention in the following aspects also belongs to the content of the present invention:

[0015] 1) Use in the preparation of a drug for reducing the foaming level of macrophages and / or enhancing the autophagy level and lysosomal function in macrophages;

[0016] 2) Use in the preparation of a drug for reducing the level of macrophage uptake of oxidized low-density lipoprotein (OxLDL), reducing the blood lipid level of patients with atherosclerosis, reducing the atherosclerotic burden on the aortic arch and abdominal aorta of patients with atherosclerosis, and / or reducing the overall aortic atherosclerotic burden of patients with atherosclerosis;

[0017] 3) Use in the preparation of a drug for reducing plaque macrophages and / or their derived foam cells in the aorta of patients with atherosclerosis, reducing the plaque area at the aortic root of patients with atherosclerosis, and / or reducing macrophages and / or their foam cells in the plaque tissue at the aortic root of patients with atherosclerosis; and

[0018] 4) Use in the preparation of a drug for the treatment and / or prevention of atherosclerosis.

[0019] The present invention uses lipid nanoparticles as carriers to deliver siRNA against Gpnmb (siGpnmb), and specifically recognizes macrophage-derived foam cells by conjugating monoclonal antibodies against CD36 and TREM2 on the surface of lipid nanoparticles to achieve targeted inhibition of the expression level of GPNMB in foam cells. In the antibody-LNP-siRNA complex described in the present invention, monoclonal antibodies against CD36 and TREM2 are used as targeting heads, which can bind to the specific surface marker proteins CD36 and TREM2 of macrophage-derived foam cells respectively, so as to deliver the lipid nanoparticles loaded with siGpnmb into foam cells and achieve the targeting effect. Foam cells can phagocytose and degrade the lipid nanoparticles loaded with siGpnmb, releasing siRNA to achieve the inhibitory effect on the mRNA and protein expression levels of GPNMB in foam cells. By inhibiting the expression of GPNMB, the autophagy level in foam cells is increased and the lysosomal degradation ability is enhanced, and the overall foaming level is significantly reduced, thereby reducing the lipid core burden in atherosclerotic plaques and achieving the treatment of atherosclerosis. The results of the examples prove that the antibody-LNP-siRNA complex can be delivered into plaque foam cells in mice, and has a low abundance in other tissues and organs of mice, that is, the targeting effect is reliable. The results of the examples also prove that the antibody-LNP-siRNA complex can effectively reduce atherosclerotic plaques in mice and reduce the levels of total cholesterol and low-density lipoprotein cholesterol in the blood, and has the potential for the treatment of atherosclerosis. The results of the examples also prove that the antibody-LNP-siRNA complex has little toxic effect on other tissues and organs in the mouse body, that is, it has good safety in vivo application. Therefore, it will be of great benefit to prepare drugs for the treatment of atherosclerosis using the antibody-LNP-siRNA complex. Description of the Drawings

[0020] Figure 1 To detect the knockdown efficiency of siRNA on GPNMB in mouse bone marrow-derived macrophages (BMDM) using Western blotting (WB) (panels a and b).

[0021] Figure 2 For the effect of siRNA knockdown on the foaming degree of ApoE - / - mouse bone marrow-derived macrophages. Among them, the anti-GPNMB antibody was labeled with the fluorescent molecule APC, and foam cells were labeled with BODIPY TM 493 / 503 dye, and the intracellular GPNMB expression fluorescence intensity and BODIPY dye fluorescence intensity were detected by flow cytometry. After comparison with BMDM transfected with control siRNA (siNC), the GPNMB expression level and foam cell ratio of each group were obtained.

[0022] Figure 3 For the effect of siRNA knockdown on ApoE- / - Effect on the intracellular lipid accumulation degree of mouse bone marrow-derived macrophages, where panel a shows the Oil Red O staining result, panel b shows the Oil Red O staining degree, and panel c shows the lipid droplet / area.

[0023] Figure 4 To detect ApoE using Western blotting (WB) - / - Effect of siRNA knockdown on the intracellular autophagy level, lysosomes, and autophagy-lysosome function in mouse bone marrow-derived macrophages. The autophagy level in cells is represented by the LC3B expression level, and the lysosome and autophagy-lysosome function levels in cells are represented by the LAMP1 expression level.

[0024] Figure 5 Schematic diagram of the antibody-LNP-siRNA complex of the present invention.

[0025] Figure 6 Particle size of the antibody-LNP-siRNA complex.

[0026] Figure 7 For ApoE - / - Results of the phagocytosis of the antibody-LNP-siRNA complex by mouse bone marrow-derived macrophages. The antibody-LNP-siRNA complex is labeled with red fluorescence, and the phagocytosis of each group of BMDMs at the specified treatment concentration is observed using a fluorescence microscope.

[0027] Figure 8 To observe the lysosomal escape of the antibody-LNP-siRNA complex in ApoE using a laser confocal microscope (Confocal) - / - Lysosomal escape of the antibody-LNP-siRNA complex in mouse bone marrow-derived macrophages. Among them, lysosomes are labeled with the Lysosome Tracker Red fluorescent probe, showing (green) fluorescence, the antibody-LNP-siRNA complex is labeled with the Cy5.5 fluorescent dye, showing (red) fluorescence, and the cell nucleus is labeled with Hoechst 33342, showing (blue) fluorescence.

[0028] Figure 9 To detect ApoE using real-time fluorescence quantitative PCR (qPCR) - / - Knockdown efficiency of the antibody-LNP-siRNA complex on GPNMB in mouse bone marrow-derived macrophages.

[0029] Figure 10 For the antibody-LNP-siRNA complex on ApoE - / - Effect of the antibody-LNP-siRNA complex on the cell viability of mouse bone marrow-derived macrophages.

[0030] Figure 11To observe the effect of antibody-LNP-siRNA complex treatment on the intracellular lipid accumulation in ApoE - / - mouse bone marrow-derived macrophages using Confocal. Intracellular neutral lipids were labeled with Nile Red fluorescent dye, showing (green) fluorescence. The antibody-LNP-siRNA complex was labeled with Cy5.5 fluorescent dye, showing (purple) fluorescence. The cell nuclei were labeled with Hoechst 33342, showing (blue) fluorescence.

[0031] Figure 12 To observe the effect of antibody-LNP-siRNA complex treatment on the uptake level of oxidized low-density lipoprotein (OxLDL) in ApoE - / - mouse bone marrow-derived macrophages using a high-content imaging system. The OxLDL used for the uptake test was labeled with Dil fluorescent dye (Dil-OxLDL), showing (green) fluorescence. The antibody-LNP-siRNA complex was labeled with Cy5.5 fluorescent dye, showing (purple) fluorescence. The cell nuclei were labeled with Hoechst 33342, showing (blue) fluorescence.

[0032] Figure 13 To observe the effect of antibody-LNP-siRNA complex treatment on the uptake level of oxidized low-density lipoprotein (OxLDL) in ApoE - / - mouse bone marrow-derived macrophages using Confocal. The OxLDL used for the uptake test was labeled with Dil fluorescent dye (Dil-OxLDL), showing (red) fluorescence.

[0033] Figure 14 To observe the localization of the antibody-LNP-siRNA complex injected via the tail vein in ApoE - / - mice fed a Western diet (WD) by combining in vivo imaging and small animal CT imaging. The two-dimensional planar fluorescence images were represented by different colors in photon / sec / cm 2 / s.

[0034] Figure 15 To observe the localization of the antibody-LNP-siRNA complex injected via the tail vein in ApoE - / - mice fed a Western diet using a photoacoustic tomography system. Signals of oxyhemoglobin (HbO 2 )、hemoglobin (Hb) and Cy5.5-siRNA-LNP were collected using multi-spectral photoacoustic tomography software.

[0035] Figure 16 To observe the antibody-LNP-siRNA complex in ApoE after 8 tail vein injections using in vivo imaging - / -Localization of atherosclerotic aorta in mice. Two-dimensional planar fluorescence images are represented by different colors in photon / sec / cm 2 / s.

[0036] Figure 17 To observe the localization of antibody-LNP-siRNA complex in other major organs and tissues of ApoE mice fed a Western diet after 8 tail vein injections using in vivo imaging. Two-dimensional planar fluorescence images are represented by different colors in photon / sec / cm - / - / s. 2 / s.

[0037] Figure 18 To determine the proportion of cells containing Cy5.5-labeled antibody-LNP-siRNA complex in the atherosclerotic aorta of ApoE mice fed a Western diet after 8 tail vein injections of the antibody-LNP-siRNA complex. - / -

[0038] Figure 19 To observe the localization of antibody-LNP-siRNA complex in atherosclerotic plaque tissues at the aortic root of ApoE mice using Confocal. Macrophages in plaque tissues are labeled with F4 / 80 antibody, and the anti-F4 / 80 antibody is labeled with fluorescent dye AF488 to show (green) fluorescence. Foam cells are labeled with Nile Red fluorescent dye to show (red) fluorescence. The antibody-LNP-siRNA complex is labeled with Cy5.5 fluorescent dye to show (purple) fluorescence. Nuclei are labeled with Hoechst 33342 to show (blue) fluorescence. - / -

[0039] Figure 20 To observe the overall tissue morphology of major organs of ApoE mice treated with antibody-LNP-siRNA complex using H&E staining. - / -

[0040] Figure 21 To determine the weight gain of ApoE mice fed a Western diet compared to their initial weight after 8 tail vein injections of the antibody-LNP-siRNA complex. - / -

[0041] Figure 22 To determine the liver function indexes of ApoE mice fed a Western diet after 8 tail vein injections of the antibody-LNP-siRNA complex. - / -

[0042] Figure 23 To determine the liver function indexes of ApoE mice fed a Western diet after 8 tail vein injections of the antibody-LNP-siRNA complex.- / - Serum total triglyceride levels in mice.

[0043] Figure 24 After 8 times of tail vein injection of antibody-LNP-siRNA complex, in ApoE mice fed a Western diet - / - Serum total cholesterol levels in mice.

[0044] Figure 25 After 8 times of tail vein injection of antibody-LNP-siRNA complex, in ApoE mice fed a Western diet - / - Serum low-density lipoprotein cholesterol (LDL-C) levels in mice.

[0045] Figure 26 After 8 times of tail vein injection of antibody-LNP-siRNA complex, in ApoE mice fed a Western diet - / - Serum high-density lipoprotein cholesterol (HDL-C) levels in mice.

[0046] Figure 27 After 8 times of tail vein injection of antibody-LNP-siRNA complex, using an animal ultrasound detector to observe plaque formation in the aortic arch and abdominal aorta of male ApoE mice fed a Western diet - / - Plaque formation in the aortic arch and abdominal aorta of mice.

[0047] Figure 28 After 8 times of tail vein injection of antibody-LNP-siRNA complex, using an animal ultrasound detector to observe plaque formation in the aortic arch and abdominal aorta of female ApoE mice fed a Western diet - / - Plaque formation in the aortic arch and abdominal aorta of mice.

[0048] Figure 29 After 8 times of tail vein injection of antibody-LNP-siRNA complex, plaque formation in the entire aorta of male ApoE mice fed a Western diet for 16 weeks. Plaque tissue was labeled with Oil Red O dye. - / - Plaque formation in the entire aorta of mice. Plaque tissue was labeled with Oil Red O dye.

[0049] Figure 30 After 8 times of tail vein injection of antibody-LNP-siRNA complex, plaque formation in the entire aorta of male ApoE mice fed a Western diet for 12 weeks. Plaque tissue was labeled with Oil Red O dye. - / - Plaque formation in the entire aorta of mice. Plaque tissue was labeled with Oil Red O dye.

[0050] Figure 31 After 8 times of tail vein injection of antibody-LNP-siRNA complex, plaque formation in the entire aorta of female ApoE mice fed a Western diet for 12 weeks. Plaque tissue was labeled with Oil Red O dye. - / - Plaque formation in the entire aorta of mice. Plaque tissue was labeled with Oil Red O dye.

[0051] Figure 32 Percentage of macrophages in the plaque-containing aorta of ApoE mice fed a Western diet after 8 intravenous tail vein injections of the antibody-LNP-siRNA complex. - / -

[0052] Figure 33 Percentage of foam cells in macrophages in the plaque-containing aorta of ApoE mice fed a Western diet after 8 intravenous tail vein injections of the antibody-LNP-siRNA complex. - / -

[0053] Figure 34 Peak graph of the fluorescence intensity of BODIPY staining of macrophages in the plaque-containing aorta of ApoE mice fed a Western diet after 8 intravenous tail vein injections of the antibody-LNP-siRNA complex. - / -

[0054] Figure 35 Plaque formation in the aortic root of ApoE mice fed a Western diet for 12 weeks after 8 intravenous tail vein injections of the antibody-LNP-siRNA complex. Plaque tissue was labeled with Oil Red O dye. - / -

[0055] Figure 36 Plaque formation in the aortic root of ApoE mice fed a Western diet for 16 weeks after 8 intravenous tail vein injections of the antibody-LNP-siRNA complex. Plaque tissue was labeled with Oil Red O dye. - / -

[0056] Figure 37 Total number of macrophages and total number of foam cells in the aortic root plaque of male or female ApoE mice fed a Western diet for 12 weeks after 8 intravenous tail vein injections of the antibody-LNP-siRNA complex. Macrophages in plaque tissue were labeled with F4 / 80 antibody, and the anti-F4 / 80 antibody was labeled with the fluorescent dye AF488, showing (green) fluorescence. Foam cells were labeled with Nile Red fluorescent dye, showing (red) fluorescence. Cell nuclei were labeled with Hoechst 33342, showing (blue) fluorescence. - / -

[0057] Figure 38 Total number of macrophages and total number of foam cells in the aortic root plaque of male ApoE mice fed a Western diet for 16 weeks after 8 intravenous tail vein injections of the antibody-LNP-siRNA complex. Macrophages in plaque tissue were labeled with F4 / 80 antibody, and the anti-F4 / 80 antibody was labeled with the fluorescent dye AF488, showing (green) fluorescence. Foam cells were labeled with Nile Red fluorescent dye, showing (red) fluorescence. Cell nuclei were labeled with Hoechst 33342, showing (blue) fluorescence. - / - ​​​​​​Total amount of macrophages and total amount of foam cells in atherosclerotic plaques in the aortic roots of mice. Macrophages in plaque tissues were labeled with F4 / 80 antibody, and the anti-F4 / 80 antibody was labeled with the fluorescent dye AF488, showing (green) fluorescence. Foam cells were labeled with the Nile Red fluorescent dye, showing (red) fluorescence. Cell nuclei were labeled with Hoechst 33342, showing (blue) fluorescence. Detailed implementation mode

[0058] Macrophages play a key role in the development of atherosclerosis. In particular, foam cells derived from macrophage foamification (also referred to as macrophage-derived foam cells in this article) are the main risk factor for atherosclerosis - the main source of the lipid core. Therefore, if the foamification of subendothelial macrophages can be targeted and blocked, it will be of great benefit to the precise treatment of atherosclerosis.

[0059] Multiple omics data indicate that GPNMB (glycoprotein non-metastatic melanoma protein B) is one of the highly expressed proteins in macrophage-derived foam cells. After the inventors successfully knocked down the expression of GPNMB (>50%) in mouse bone marrow-derived macrophages (BMDMs) using siRNA (small interfering RNA), they found that the degree of BMDM foamification was significantly reduced. In addition, the inventors observed an increase in autophagy levels and enhanced lysosomal function in BMDMs in which GPNMB expression was inhibited using siRNA. Both of these can promote intracellular lipid degradation, which may be the reason for the reduced degree of BMDM foamification. Therefore, the inventors believe that GPNMB is a potential therapeutic target for atherosclerosis, and targeted inhibition of GPNMB expression in macrophage-derived foam cells may have therapeutic significance for atherosclerosis.

[0060] Based on this, the present invention provides a lipid nanoparticle that can specifically target and inhibit the expression of GPNMB in macrophage-derived foam cells (also referred to as antibody-LNP-siRNA complex or siRNA delivery lipid nanoparticle herein), which can specifically deliver siRNA (also referred to as siGpnmb herein) that inhibits the expression of GPNMB to macrophage-derived foam cells, thereby inhibiting the expression of GPNMB in macrophage-derived foam cells. Specifically, the antibody-LNP-siRNA complex provided by the present invention uses lipid nanoparticles as a carrier, on which siGpnmb is loaded, and monoclonal antibodies against CD36 and TREM2 are conjugated on the surface of the lipid nanoparticles. The antibody-LNP-siRNA complex uses the monoclonal antibodies against CD36 and TREM2 as targeting heads, which can bind to the specific surface marker proteins CD36 and TREM2 of macrophage-derived foam cells respectively, so as to target and deliver the lipid nanoparticles loaded with siGpnmb into foam cells to achieve the targeting effect. Foam cells can phagocytose and degrade the lipid nanoparticles loaded with siGpnmb, releasing the siGpnmb loaded therein. In foam cells, the released siGpnmb is incorporated into the RNA-induced silencing complex (RISC), and the mature RISC then inhibits the expression of GPNMB by inhibiting mRNA translation, promoting mRNA degradation, and guiding transcriptional gene silencing at the target gene locus, etc. By inhibiting the expression of GPNMB, the autophagy level in foam cells is increased and the lysosomal degradation ability is enhanced, and the overall foaming level is significantly reduced, thereby reducing the lipid core burden in atherosclerotic plaques and achieving the treatment of atherosclerosis.

[0061] When the antibody-LNP-siRNA complex was applied to foam cells induced from mouse bone marrow-derived macrophages, it was found that the foam cells could uptake the complex, and the siRNA entering the cells could successfully escape from lysosomes, and effectively inhibited the expression level of GPNMB in foam cells with basically no effect on cell viability. In addition, the administration of the antibody-LNP-siRNA complex significantly reduced the neutral lipid content in BMDM and the level of BMDM uptake of oxidized low-density lipoprotein. When the antibody-LNP-siRNA complex of the present invention was used to treat atherosclerosis in ApoE - / - mice induced by a Western Diet (WD), the results of the examples showed that the complex could localize to atherosclerotic plaque macrophages and their derived foam cells in the aortic arch and abdominal aorta of mice, achieving a good targeting effect. Without obvious toxicity to other important tissues and organs, the antibody-LNP-siRNA complex reduced ApoE - / -While reducing the body weight, serum total cholesterol, and serum low-density lipoprotein cholesterol of mice, it effectively alleviated the atherosclerotic burden in mice. Therefore, the antibody-LNP-siRNA complex can be used as an effective therapeutic drug for treating atherosclerosis.

[0062] The following details the content of the present invention in conjunction with specific embodiments and the accompanying drawings.

[0063] In the following text, only certain exemplary embodiments are briefly described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present invention. Therefore, the drawings and the description are considered to be exemplary in nature rather than restrictive.

[0064] The methods used in the following embodiments are all conventional methods unless otherwise specified. For specific steps, please refer to: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0065] The acquisition routes of various biological materials described in the embodiments are only provided as a way to obtain through experiments for the purpose of specific disclosure, and should not be a limitation on the source of biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the embodiments.

[0066] Unless otherwise specified, the solvent of the aqueous solution in the following embodiments is a sterile ultrapure aqueous solution.

[0067] Unless otherwise specified, the reagents used in the following embodiments are all analytical pure reagents.

[0068] Unless otherwise specified, the pH of the phosphate buffer solution (PBS) used in the following embodiments is 7.4.

[0069] The sequence of the siRNA targeting Gpnmb (named siGpnmb) in the following embodiments is: sense strand 5'-GCCUGUUUGUCUCCAACAA TT -3' (SEQ ID NO: 1, where the underlined TT indicates that there is a TT overhang at the 3' end, the same below), and antisense strand 5'-UUGUUGGAGACAAACAGGC TT-3’ (SEQ ID NO:2). The following control siRNA (named siNC) sequences were also used in the following examples: sense strand 5’-UUCUCCGAACGUGUCACGU TT -3’ (SEQ ID NO:3), and antisense strand 5’-ACGUGACACGUUCGGAGAA TT -3’ (SEQ ID NO:4).

[0070] In the following examples, the anti-CD36 monoclonal antibody (sc-7309) (also referred to as CD36 monoclonal antibody or CD36 antibody in this article) used was purchased from Santa Cruz, and the anti-TREM2 monoclonal antibody (ab305103) (also referred to as TREM2 monoclonal antibody or TREM2 antibody in this article) used was purchased from Abcam.

[0071] Example 1: siRNA (siGpnmb) effectively inhibits GPNMB expression in mouse bone marrow-derived macrophages

[0072] (1.1) CO 2 The mice were sacrificed, the hind limb muscles of the mice were dissected, and the clean leg bones were taken and soaked in DMEM medium (11965092, Gibco). The two ends of the bones were cut, and the clean medium was aspirated with a syringe and inserted into the bone marrow cavity to wash out the cells until the bone marrow cavity turned white. The cell suspension was pipetted until the cells were dispersed.

[0073] (1.2) The cell suspension was filtered through a 40 nm filter to remove residues, and the cells were collected by centrifugation at 500 g for 5 minutes. After resuspension with red blood cell lysis buffer (Beijing Aoting Biotechnology Co., Ltd.), it was left standing for 2 minutes and then the red blood cell lysis was terminated, and the cells were collected by centrifugation under the same conditions. Subsequently, the cell pellet was resuspended in DMEM complete medium containing 10 ng / mL CSF-1 (colony-stimulating factor-1, AF-315-02, PeproTech), 10% fetal bovine serum (FBS, 16000044, Gibco), and 1% penicillin-streptomycin double antibiotic (C0222, Beyotime), and plated in a 6-well plate, divided into a control group and an experimental group. The medium was changed on the 4th day after plating.

[0074] (1.3) After the BMDMs were fully mature on the 7th day, according to the transfection reagent instructions, siNC and siGpnmb (5 μg / well) were transfected into the cell wells of the control group and the experimental group using Lipofectamine 3000 transfection reagent (L3000001, Invitrogen). After 24 hours, the medium containing the transfection complex was discarded, and fresh DMEM complete medium was added again. Then, after culturing for another 24 hours, the cells were lysed to collect cell proteins. The protein expression levels of the cells in the experimental group and the control group were detected by conventional Western blot (WB) experiments, and the results were asFigure 1 as shown

[0075] From Figure 1 As can be seen from the detection results shown, compared with the control group (transfected with siNC), the intensity of the GPNMB protein band (panel a) and the relative expression level (panel b) in BMDM cells of the experimental group (transfected with siGpnmb) were significantly reduced, and as shown in panel b of Figure 1 , the reduction level exceeded 50%. This result indicates that siGpnmb can successfully and significantly inhibit the expression level of GPNMB in BMDM.

[0076] Example 2: siRNA (siGpnmb) reduces the foaming level of mouse bone marrow-derived macrophages

[0077] (2.1) As described in Example 1, BMDM was isolated and cultured, and siNC and siGpnmb were transfected into BMDM in the control group and the experimental group respectively. After 24 hours of transfection, the medium was replaced with DMEM complete medium containing 50 μg / mL oxidized low-density lipoprotein (OxLDL) and incubated for 48 hours to induce BMDM foaming.

[0078] (2.2) After transfection, the original medium was discarded, and the cells were cultured in fresh DMEM complete medium for another 48 hours. The medium was discarded, and after washing, a part of the cells were digested with trypsin, centrifuged at 500 g for 5 minutes to collect the cells, and an antibody to block the Fc receptor (2.4G2, Thermo Fisher) was added to the cell suspension. After centrifugation and collection, the cells were fixed and permeabilized. After washing with the permeabilization solution, the centrifuged and collected cells were incubated in a mixed dilution solution containing APC-fluorescent dye-labeled GPNMB antibody (CTSREVL, eFluor TM 660, 50-5708-82) and BODIPY fluorescent dye (D3922, Invitrogen) for 30 minutes. After washing with the permeabilization solution, the centrifuged and collected cells were resuspended in PBE buffer, and finally the expression level of GPNMB and the fluorescence signal intensity of the BODIPY dye in the two groups of cells were detected by flow cytometry. The results are as Figure 2 shown.

[0079] (2.3) After another 48 hours of culturing the other part of the transfected cells in fresh DMEM complete medium, the cell culture medium was aspirated, and after washing with PBS, 4% paraformaldehyde fixative was added to the wells and fixed for 10 minutes. After rinsing with PBS, the staining and washing solution in the improved Oil Red O staining kit (C0158S, Beyotime) was added and covered for 20 s. After aspirating the washing solution, the Oil Red O staining solution in the kit was added and stained for 30 minutes. After removing the staining solution, the staining and washing solution was added again and allowed to stand for 30 s. Finally, after rinsing with PBS, an appropriate amount of PBS was added to cover the cells, and the cells were observed and images were collected under a microscope. The results are as Figure 3 shown.

[0080] As can be seen from Figure 2 the results shown in Panel a, the positive expression rate of GPNMB in BMDMs transfected with siNC was 76.5%, while that in BMDMs transfected with siGpnmb was 7.59%, indicating that siGpnmb transfection significantly reduced the expression of GPNMB in BMDMs, which was consistent with the results in Example 1. As can be seen from Figure 2 the results shown in Panel b, the proportion of BODIPY hi Foam Cells in BMDMs transfected with siNC was 80.5%, while that in BMDMs transfected with siGpnmb was 40.0%, indicating that siGpnmb transfection significantly reduced the degree of foam cell formation in BMDMs induced by OxLDL. As can be seen from hi the results shown in Panels a - c, compared with BMDMs transfected with siNC, the content of intracellular neutral lipids labeled by Oil Red O dye and the area of total lipids in BMDMs transfected with siGpnmb were significantly reduced, indicating that the degree of foam cell formation in BMDMs transfected with siGpnmb was significantly lower, which was consistent with Figure 3 the results shown Figure 2 .

[0081] Example 3: siRNA (siGpnmb) increases the autophagy level and lysosome function in mouse bone marrow-derived macrophages Function (3.1) As described in Example 1, BMDMs were isolated and cultured and divided into four groups: control group (siNC), experimental group (siGpnmb), siNC + OxLDL group, and siGpnmb + OxLDL group. The final concentration of OxLDL was 50 μg / mL. Transfection (5 μg siRNA / well) and medium replacement were performed as described above. After 48 hours of OxLDL treatment, the cell proteins of BMDMs in each group were collected. The protein expression levels were detected by conventional Western Blot (WB) assay, and the results were as Figure 4 shown

[0082] As can be seen from Figure 4 the results, the intensity of the GPNMB protein band in BMDMs transfected with siGpnmb was significantly lower, indicating that siGpnmb could successfully silence the expression of Gpnmb in BMDMs, which was consistent with Figure 1 the results in Figure 4The results were consistent. The expression levels of LAMP1 and LC3B in BMDMs of the SiGpnmb+OxLDL group were similar to those in BMDMs of the SiGpnmb group, indicating that OxLDL failed to induce changes in autophagy levels in BMDMs with silenced Gpnmb.

[0083] Example 4: Preparation of antibody-LNP-siRNA complex

[0084] In this example, two antibody-LNP-siRNA complexes were prepared. One loaded siGpnmb (hereinafter referred to as the antibody-LNP-siGpnmb complex, also abbreviated as siGpnmb-LNP or LNP-siGpnmb), and the other loaded siNC (hereinafter referred to as the antibody-LNP-siNC complex, also abbreviated as siNC-LNP or LNP-siNC). The specific preparation methods of the two complexes are described below, both of which respectively include the following operations.

[0085] (4.1) Dissolve 60 mg of liposome material with a molar ratio of DSPE-PEG-Mal (phospholipid polyethylene glycol maleimide, purchased from Carbohydrate Technology), ALC-0315 (((4-hydroxybutyl)azaalkanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (purchased from Carbohydrate Technology), ALC-0159 (polyethylene glycol 2000-N,N-ditetradecylethylacetamide (purchased from Carbohydrate Technology), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine (purchased from Carbohydrate Technology) and cholesterol (cholest-5-en-3β-ol, purchased from Carbohydrate Technology) mixed at a mol ratio of 0.5:50:1:10:38.5 and 1 mg of Cy5.5-COOH (cyanine Cy5.5-carboxyl, purchased from Carbohydrate Technology) in 6 mL of ethanol. Dissolve siRNA (18.5 OD) in 18 mL of citrate buffer (100 mM, pH = 4 - 6). Rapidly mix the buffer and the ethanol solution at a volume ratio of 3:1, incubate at room temperature to allow the LNP (lipid nanoparticles) to fully self-assemble, and purify by dialysis in sterile PBS for 2 hours to remove ethanol and buffer, obtaining a liposome solution.

[0086] (4.2) Disperse the antibody (150 μg of CD36 antibody and 50 μg of TREM2 antibody) in boric acid buffer salt (0.1% EDTA, 0.1 M, pH 8.0), add a certain amount of Traut’s Reagent (purchased from Carbohydrate Technology), incubate and react at room temperature for 1 hour, perform solution replacement through a 10KDa ultrafiltration tube, and disperse the antibody in PBS (0.1% EDTA, 10 mM, pH 7.4) to obtain an antibody solution.

[0087] (4.3) Add the antibody solution obtained in step (4.2) to the liposome solution obtained in step (4.1), vortex, and incubate overnight at 4°C. Then add the mixture to an ultrafiltration tube, wash three times with water in a centrifuge at 4°C, and finally collect the solution. After filtration, an antibody-LNP-siRNA complex (final concentration 0.2 mg / mL, i.e., 20 nmol / mL) is obtained. The schematic diagram of its structure is as shown in Figure 5 shown. This antibody-LNP-siRNA complex uses lipid nanoparticles as a carrier, with siRNA encapsulated in the cavity of the lipid nanoparticles, and two antibodies (CD36 antibody and TREM2 antibody) conjugated to the surface of the lipid nanoparticles (the position of the lipid nanoparticles can be observed in mice through the Cy5.5 labeled on them).

[0088] Perform physicochemical characterization on the assembled complex. The detection indicators include particle size, polydispersity index, zeta potential, carrier concentration, nucleic acid concentration, and drug loading rate. The results of the physicochemical characterization are as shown in Figure 6 and Table 1.

[0089] According to Figure 6 and the results in Table 1, it can be seen that the assembled antibody-LNP-siRNA complex has a small PDI, indicating that its size is uniform; its zeta potential is close to neutral, indicating that it can avoid electrostatic repulsion by negatively charged plasma membranes or cytotoxicity; it has a high encapsulation efficiency and can ensure the stability of the complex during the delivery process and the effectiveness of the delivered siRNA.

[0090] Table 1: Physicochemical characteristics of the antibody-LNP-siRNA complex

[0091]

[0092] Example 5: Endocytosis of antibody-LNP-siRNA complex by mouse bone marrow-derived macrophages and their induced foam cells Complex

[0093] (5.1) According to Example 1, isolate and culture BMDM, which are divided into nine groups: blank control group (0 nM), 6.25 nM-siNC-LNP group (where 6.25 nM refers to the nucleic acid concentration, i.e., treating BMDM with an antibody-LNP-siNC complex with a siNC concentration of 6.25 nM, the same below), 12.5 nM-siNC-LNP group, 25 nM-siNC-LNP group, 50 nM-siNC-LNP group, 6.25 nM-siGpnmb-LNP group, 12.5 nM-siGpnmb-LNP group, 25 nM-siGpnmb-LNP group, and 50 nM-siGpnmb-LNP group.

[0094] (5.2) After the BMDMs matured, they were incubated in complete medium containing 50 μg / mL of oxidized low-density lipoprotein (OxLDL) for 48 hours to induce foam formation. Then, complete medium containing the corresponding antibody-LNP-siRNA complex with the specified nucleic acid concentration was added, and after incubating for 24 hours, the fresh complete medium was replaced and cultured for another 24 hours. Subsequently, the fluorescence intensity of Cy5.5 dye in BMDMs of each group was observed using a fluorescence microscope. The results are as Figure 7 shown.

[0095] According to Figure 7 the results shown, it can be seen that as the concentration gradient of the two antibody-LNP-siRNA complexes increased, the fluorescence signal intensity of Cy5.5 in BMDM cells gradually increased significantly, indicating that macrophages and the foam cells induced by them can normally endocytose the antibody-LNP-siRNA complex.

[0096] Example 6: Lysosomal escape of antibody-LNP-siRNA complex in mouse bone marrow-derived macrophages and their induced foam cells Cells

[0097] (6.1) As described in Example 1, BMDMs were isolated and cultured in a confocal dish. After the BMDMs matured, they were incubated in complete medium containing 50 μg / mL of oxidized low-density lipoprotein (OxLDL) for 48 hours to induce foam formation.

[0098] (6.2) After incubating for 48 hours, the culture medium was aspirated, and a working solution containing Lysosome Tracker Red fluorescent probe (LTR probe, 50 nM, C1046, Beyotime) and Hoechst 33342 staining solution (1:1000, H1399, ThermoFisher Scientific) preheated at 37°C was added. After incubating for 1 hour, fresh complete medium containing the antibody-LNP-siGpnmb complex with a nucleic acid concentration of 50 nM was added. The localization of the antibody-LNP-siGpnmb complex labeled by the Cy5.5 fluorescence signal and the lysosome labeled by the LTR probe fluorescence signal was observed using a laser confocal microscope (Confocal) at 1 hour, 3 hours, and 12 hours after incubation, respectively. The results are as Figure 8 shown.

[0099] From Figure 8As shown in the results, after treatment with the antibody-LNP-siGpnmb complex for 1 hour, the LNP particles phagocytosed in BMDMs showed a high degree of co-localization with lysosomes, indicating that the LNP particles were taken up into the cells via the endocytic pathway and degraded in lysosomes. After 3 hours of treatment, only some of the LNP particles in BMDMs co-localized with lysosomes, indicating that some of the LNP particles escaped from lysosomes and could release the delivered siRNA. After 12 hours of treatment, only very few LNP particles co-localized with lysosomes, indicating that the LNP particles could successfully escape from lysosomes and effectively deliver siRNA.

[0100] Example 7: Antibody-LNP-siGpnmb complex effectively inhibits GPNMB expression in mouse bone marrow-derived macrophages and their induced foam cells Cells

[0101] (7.1) As described in Example 1, BMDMs were isolated and cultured and divided into six groups: 12.5 nM-siNC-LNP group, 25 nM-siNC-LNP group, 50 nM-siNC-LNP group, 12.5 nM-siGpnmb-LNP group, 25 nM-siGpnmb-LNP group, and 50 nM-siGpnmb-LNP group.

[0102] (7.2) After the BMDMs matured, they were incubated in complete medium containing 50 μg / mL of oxidized low-density lipoprotein (OxLDL) for 48 hours to induce foam formation. After changing the medium, the corresponding antibody-LNP-siRNA complexes with the specified nucleic acid concentration were added respectively and incubated for 24 hours, and then fresh medium was replaced and cultured for another 24 hours.

[0103] (7.3) After washing with PBS, Trizol was added to the cells to lyse them sufficiently. Chloroform with a volume of 1 / 5 of that of the cell lysate was added thereto, and the mixture was vigorously shaken for 15 seconds to mix well. Then, it was left standing at room temperature for 5 minutes and centrifuged at 12,000 g for 15 minutes. The upper colorless aqueous phase was carefully aspirated, and an equal volume of isopropanol was added thereto and mixed well. After standing at room temperature for 10 minutes, it was centrifuged at 12,000 g for 10 minutes. After discarding the supernatant, it was washed twice with 75% ethanol. Subsequently, the precipitate collected by centrifugation was air-dried at room temperature for 5 minutes, and finally 50 μL of DEPC water was added to dissolve the RNA precipitate.

[0104] (7.4) The extracted RNA was reverse transcribed into the corresponding cDNA using a reverse transcription kit (PrimeScript RT kit, Takara), and then the mRNA expression level of Gpnmb in BMDMs of each group was detected by real-time fluorescence quantitative PCR (qPCR). The primer sequences used were upstream (5’-CGCCTCCACCTTCAACTC-3’ (SEQ ID NO:5)) and downstream (5’-CAATTGTGATGGTGGCTCTG-3’ (SEQ ID NO:6)). The results were as Figure 9as shown

[0105] According to Figure 9 As shown by the results, it can be seen that when the nucleic acid concentration is 12.5 nM and 25 nM, there is no significant difference in the mRNA expression level of Gpnmb in BMDMs of each siGpnmb-LNP group and siNC-LNP group. However, when the nucleic acid concentration is 50 nM, the mRNA expression level of Gpnmb in BMDMs of the siGpnmb-LNP treatment group is significantly lower than that in BMDMs of the siNC-LNP group (the former is about 50% - 60% of the latter), indicating that the antibody-LNP-siGpnmb complex can effectively inhibit the expression of Gpnmb in BMDMs at this nucleic acid concentration.

[0106] Example 8: Antibody-LNP-siGpnmb complex does not affect the cell viability of mouse bone marrow-derived macrophages and their induced foam cells Cells

[0107] (8.1) As described in Example 1, BMDMs were isolated and cultured, and divided into five groups: blank control group (0 nM), 50 nM-siNC-LNP group, 100 nM-siNC-LNP group, 50 nM-siGpnmb-LNP group, and 100 nM-siGpnmb-LNP group.

[0108] (8.2) After the BMDMs were mature, they were incubated in complete medium containing 50 μg / mL oxidized low-density lipoprotein (OxLDL) for 48 hours to induce foam formation. Then, the corresponding antibody-LNP-siRNA complexes with the specified nucleic acid concentration were added after changing the medium, and after incubating for 24 hours, the fresh medium was replaced and cultured for another 24 hours, 36 hours, or 48 hours.

[0109] (8.3) At the 20th hour, 32nd hour, and 44th hour of culture respectively, the cell medium was changed to fresh cell-free medium added with Alamar Blue reagent (40202ES60, YEASEN) (addition ratio 1:10). The medium containing Alamar Blue was co-incubated with the cells for 4 hours, and the fluorescence intensity of each group of cells was detected using a fluorescence microplate reader at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The results are as Figure 10 as shown

[0110] From Figure 10 As shown by the results, the relative values of the cell viability of BMDMs added with the antibody-LNP-siGpnmb complex and BMDMs without treatment (NC group) detected at three time points (24 h, 36 h, and 48 h) are comparable, indicating that the antibody-LNP-siGpnmb complex does not affect the cell viability of mouse bone marrow-derived macrophages and the foam cells induced by them.

[0111] Example 9: Antibody-LNP-siGpnmb complex reduces the foaming degree of mouse bone marrow-derived macrophages

[0112] (9.1) As described in Example 1, bone marrow-derived macrophages (BMDMs) were isolated and cultured in confocal dishes and divided into two groups: the 50 nM-siNC-LNP group and the 50 nM-siGpnmb-LNP group.

[0113] (9.2) After the BMDMs matured, they were incubated with fresh medium containing an antibody-LNP-siRNA complex with a nucleic acid concentration of 50 nM for 24 hours, and then incubated with complete medium containing 50 μg / mL of oxidized low-density lipoprotein (OxLDL) for 48 hours to induce foam cell formation.

[0114] (9.3) The culture medium was aspirated, and after washing with PBS, a staining solution containing Nile Red fluorescent dye (1:1000, C0009, APPLYGEN) and Hoechst 33342 staining solution (1:1000) was added. After incubating in the dark for 30 minutes, the unbound dye was washed away with PBS, and the fluorescence signal intensities of Cy5.5 and Nile Red dye in BMDMs were observed using a laser confocal microscope. The results are as Figure 11 shown.

[0115] According to Figure 11 the results shown, in the two groups of BMDMs, the fluorescence signal intensity of Cy5.5 was comparable (panel a), but the fluorescence signal of Nile Red dye in BMDMs treated with siGpnmb-LNP was significantly lower than that in BMDMs treated with siNC-LNP (panel b), indicating that when the degree of LNP particle phagocytosis in the two groups of BMDMs was similar, the antibody-LNP-siGpnmb complex significantly reduced the neutral lipid content in BMDMs and the foam cells induced by them.

[0116] Example 10: Antibody-LNP-siGpnmb complex reduces the level of oxidized low-density lipoprotein (OxLDL) uptake by mouse bone marrow-derived macrophages Lipoprotein (OxLDL)

[0117] (10.1) As described in Example 1, bone marrow-derived macrophages (BMDMs) were isolated and cultured in confocal dishes and 96-well plates and divided into two groups: the 50 nM-siNC-LNP group and the 50 nM-siGpnmb-LNP group.

[0118] (10.2) After the BMDMs matured, they were incubated with fresh medium containing an antibody-LNP-siRNA complex with a nucleic acid concentration of 50 nM for 24 hours, and then incubated with complete medium containing OxLDL labeled with Dil fluorescent dye (Dil-OxLDL) at 50 μg / mL for 48 hours.

[0119] (10.3) Discard the culture medium, wash twice with PBS, observe the fluorescence signal intensity of Cy5.5 and Dil-OxLDL in two groups of BMDMs in the 96-well plate using a high-content imaging system, and observe the fluorescence signal intensity of Cy5.5 and Dil-OxLDL in two groups of BMDMs in the confocal dish using a laser confocal microscope. The results are as Figure 12 and Figure 13 shown. Among them, Figure 12 is the result of full-well scanning by the high-content imaging system, Figure 13 is the result of confocal microscope imaging.

[0120] According to Figure 12 the results shown in panels b and c, the fluorescence signal intensity of Cy5.5 and Dil-OxLDL in BMDMs treated with siGpnmb-LNP is significantly lower than that in BMDMs treated with siNC-LNP, indicating that even when less siGpnmb-LNP enters BMDMs, it can significantly reduce the level of Dil-OxLDL uptake by BMDMs. In addition, Figure 12 in panel a of

[0121] compared with other cells in the same field of view, the Cy5.5 fluorescence signal is stronger and the Dil-OxLDL fluorescence signal is weaker in some cells in the area marked by the white square in panel a, suggesting that BMDMs that endocytose more siGpnmb-LNP have a lower level of Dil-OxLDL uptake. Figure 12 Consistent with the Figure 13 results, it can be seen from

[0122] Example 11: Antibody-LNP-siGpnmb complex injected via the tail vein can localize to the atherosclerotic lesions in mice fed a Western diet ApoE - / - on the aorta of mice

[0123] (11.1) Feed 6- to 8-week-old ApoE - / - mice (purchased from Beijing Huafukang Co., Ltd.) with a Western diet feed (XT108C, purchased from Xietong Biotech) for 7 or 11 weeks to establish early or late atherosclerotic mouse models, respectively. In the early atherosclerotic group, male mice were randomly divided into two groups—a control group (injected with siNC-LNP) and a treatment group (injected with siGpnmb-LNP) (3-4 mice in each group), and female mice were randomly divided into the same two groups (3 mice in each group); in the late atherosclerotic group, male mice were randomly divided into two groups—a control group (siNC-LNP) and a treatment group (siGpnmb-LNP) (5 mice in each group).

[0124] (11.2) After establishing an atherosclerotic mouse model, mice were intravenously injected with siNC-LNP or siGpnmb-LNP (1 nmol siRNA per injection, 200 μL) once every three days (for a total of eight times). During the treatment, the mice were continuously fed with a Western diet. Twenty-four hours after the second injection, the abdominal hair of the mice was removed and they were anesthetized with isoflurane. Animal in vivo imaging and small animal CT imaging were used in combination to observe the Cy5.5 fluorescence signal in various tissues and organs of the mice. The results are as Figure 14 shown. Five days after the eighth injection, the mice were completely depilated and anesthetized with isoflurane. A photoacoustic tomography system was used to collect the fluorescence signals of oxyhemoglobin (HbO 2 ), hemoglobin (Hb), and Cy5.5-siRNA-LNP in the mice. The cross-sectional signals were detected layer by layer with a step of 0.3 mm: the spine was used as the reference signal, and oxyhemoglobin was used as the vascular signal to determine the position of the abdominal aorta; the starting point was slightly above the heart (strong pulsation), and the ending point was slightly below the clear abdominal aortic lumen. The results are as Figure 15 shown.

[0125] According to Figure 14 the results shown, the positions of the thoracic cavity and the heart were determined through the CT images of the mice (the intersection of the green line and the blue line), and the longitudinal (Y-axis direction) trend of the abdominal aorta was judged in combination with the position of the xiphoid process. It was found that discontinuous Cy5.5 fluorescence signals could be detected at the aortic position of the mice, and similar signals were observed at the aortic positions of multiple mice. Cy5.5 signals were mainly detected in the aortic arch close to the heart and the abdominal aorta, indicating that the antibody-LNP-siRNA complex could be localized to the aorta of the mice and was very likely to be localized on the aortic plaques of the mice.

[0126] According to Figure 15 the results shown in panel a, extremely strong Cy5.5 fluorescence signals were shown at the positions marked by the white dotted line box in the figure in multiple mice. The arterial position could be determined based on the overall oxyhemoglobin signal ( Figure 15 upper panel of panel c), and the position of the abdominal aorta was determined by the spine and the oxyhemoglobin signal ( Figure 15 blue circle in panel b). Oxyhemoglobin signals were visible in the region of strong Cy5.5 signal ( Figure 15 red circle in panel b) during the scanning of the thoracic cavity, indicating that the region of strong Cy5.5 signal was in the arterial region within the thoracic cavity, but there was a slight distance from the position of the abdominal aorta in the cross-section. Combining the overall Cy5.5 signal ( Figure 15 lower panel of panel c), it was very likely to be the aortic arch part prone to atherosclerosis.

[0127] Example 12: Antibody-LNP-siGpnmb complex injected via the tail vein can specifically localize to atherosclerotic lesions in mice fed a Western diet Fed ApoE - / - on the aortic plaques of mice

[0128] (12.1) As described in Example 11, siNC-LNP or siGpnmb-LNP was injected into the tail vein of mice in the early or late atherosclerosis mouse model according to the grouping, with 1 nmol siRNA (200 μL) injected each time, once every three days for a total of eight times.

[0129] (12.2) After the injection, the mice were continuously fed with a Western diet for 6 days, and then euthanized to collect the aorta, heart, lungs, spleen, liver, kidneys, and fat pads of the mice for ex vivo tissue fluorescence imaging. The results are as Figure 16 and Figure 17 shown. Among them, Figure 16 is the two-dimensional fluorescence imaging of the heart and aorta, Figure 17 and

[0130] is the two-dimensional fluorescence imaging of other tissue organs. Figure 16 As can be seen from the results shown in Figure 16 and Figure 17 , the discontinuous Cy5.5 fluorescence signals on the aorta of the mice indicate that siRNA-LNP can localize to the aortic plaques of the mice. Combining the

[0131] Example 13: Antibody-LNP-siGpnmb complex injected via the tail vein can targetedly deliver siRNA to aortic plaque macrophages and foam cells in mice Cells

[0132] (13.1) As described in Example 11, siNC-LNP or siGpnmb-LNP was injected into the tail vein of mice in the late atherosclerosis mouse model according to the grouping, with 1 nmol siRNA (200 μL) injected each time, once every three days for a total of eight times.

[0133] (13.2) After the injection, the mice were continuously fed with a Western diet for 6 days, and then euthanized to collect the heart and aorta of the mice. The aortic tissue was digested with an enzyme digestion solution, and the cell precipitate was collected by centrifugation after passing through a sieve mesh. The cells were resuspended with a red blood cell lysis solution and allowed to stand for 2 minutes, and then centrifuged again to collect the cells after terminating the red blood cell lysis.

[0134] (13.3) The cells collected by centrifugation were resuspended with PBE buffer, and then the intensity of the Cy5.5 fluorescence signal (PerCP-Cy5.5 channel) in each sample was detected by flow cytometry. The results are as Figure 18 shown.

[0135] (13.4) After fixing the mouse heart in 4% paraformaldehyde for 24 hours, it was embedded in OCT to prepare frozen section samples of the aortic root. The frozen sections were thawed at room temperature within 10 minutes, then dropped with PBS and soaked for 5 minutes to wash away the OCT embedding agent. After soaking in 1.5% Triton X-100 for 20 minutes, they were incubated with 5% goat serum blocking solution for 1 hour. Then, they were incubated overnight at 4°C with the working solution of F4 / 80 antibody (ab300421, Abcam) prepared with 1% goat serum dilution. The unbound primary antibody was washed away with TBST buffer, and then the working solution of secondary antibody containing AF488 fluorescent secondary antibody (A21202, Invitrogen) and Nile Red fluorescent dye was dropped and incubated at room temperature in the dark for 1 hour. After rinsing with TBST buffer, the working solution of Hoechst 33342 was dropped to stain the cell nuclei, and after rinsing with PBS, the slides were sealed. The AF488 fluorescent signal, Nile Red fluorescent signal, and Hoechst fluorescent signal in the plaque tissue of the mouse aortic root were observed using a confocal microscope, and the results are as Figure 19 shown.

[0136] According to Figure 18 the results shown, Cy5.5 fluorescent signal could be detected in the atherosclerotic aorta of mice, indicating the presence of Cy5.5-siRNA-LNP + cells, and the proportion of Cy5.5-LNP + cells in the aorta of the treatment group mice injected with siGpnmb-LNP was lower than that of the total viable cells in the aorta.

[0137] According to Figure 19 the results shown, it can be seen that the Cy5.5 fluorescent signal co-localizes with the AF488-labeled F4 / 80 signal and Nile Red fluorescence, while no obvious Cy5.5 fluorescent signal is seen in the endothelial cells and smooth muscle cells in the plaque tissue, indicating that the antibody-LNP-siRNA complex described in the present invention can target and enter macrophages and foam cells in the plaque tissue of the mouse aortic root.

[0138] Example 14: Antibody-LNP-siGpnmb complex injected via the tail vein causes low organ toxicity in mice

[0139] (14.1) As described in Example 11, in a mouse model of advanced atherosclerosis, siNC-LNP or siGpnmb-LNP was injected into the tail vein according to the grouping, 1 nmol siRNA (200 μL) was injected each time, once every three days, for a total of eight times.

[0140] (14.2) After the injection was completed, the mice were continuously fed with a Western diet for 6 days, and then euthanized to collect the spleen, kidney, lung, and liver of the mice.

[0141] (14.3) After fixing the organs of mice in 4% paraformaldehyde for 24 hours, dehydrate the organs using an automatic dehydrator, and then embed them in paraffin to prepare paraffin section samples of the organs. Bake the sections in an oven at 55 °C for about 30 minutes, then remove the paraffin with xylene solution in turn, and then hydrate them in alcohol with different concentrations, and finally rinse them with distilled water. Drop hematoxylin stain and stain for 2 minutes, then differentiate in hydrochloric acid alcohol solution, and return to blue by rinsing with tap water for 10 minutes. Drop eosin stain and stain for 1 minute, and wash away the floating liquid with distilled water. Dehydrate through alcohol with different concentrations in turn, then clarify with xylene, and finally seal the slices with neutral gum. Figure 20 are the H&E staining results of each organ tissue.

[0142] From Figure 20 the results shown, there is no obvious abnormality in the overall tissue morphology of the above four organs, and no organ abnormality was observed during comprehensive sampling, suggesting that the antibody-LNP-siGpnmb complex injected via the tail vein has low organ toxicity.

[0143] Example 15: Antibody-LNP-siGpnmb complex treatment reduces body weight in Western diet-fed ApoE - / - mice Growth (15.1) As described in Example 11, inject siNC-LNP or siGpnmb-LNP via the tail vein into atherosclerotic mouse models in the early (Early Group) or late (Late Group) stage respectively according to the grouping, inject 1 nmol siRNA (200 μL) each time, once every three days, for a total of eight times.

[0144] (15.2) After the injection is completed, continue to feed the mice with a Western diet for 6 days. Weigh the mice before dissecting them, and subtract the weight of the mice at the beginning of the model establishment to obtain their weight gain. The results are as Figure 21 shown.

[0145] From the results as Figure 21 shown, the weight gain of the mice injected with siGpnmb-LNPs via the tail vein is significantly lower than that of the mice injected with siNC-LNPs, indicating that the treatment with the antibody-LNP-siGpnmb complex reduces the weight gain of ApoE - / - mice fed with a Western diet.

[0146] Example 16: Antibody-LNP-siGpnmb complex treatment alleviates the liver of Western diet-fed ApoE - / - mice Inflammation (16.1) As described in Example 11, inject siNC-LNP or siGpnmb-LNP via the tail vein into atherosclerotic mouse models in the early (Early Group) or late (Late Group) stage respectively according to the grouping, inject 1 nmol siRNA (200 μL) each time, once every three days, for a total of eight times.

[0147] (16.2) After the injection was completed, the mice were continuously fed with a Western diet for 6 days. The mice were anesthetized by intraperitoneal injection of tribromoethanol, and cardiac blood was drawn into EP tubes. The tubes were left standing at room temperature for 2 hours and then centrifuged at 12,000 rpm for 15 minutes. The supernatant was taken as the serum sample. The concentrations of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in the serum of each mouse were detected using an automatic biochemical analyzer. The results are as Figure 22 shown.

[0148] As can be seen from the results Figure 22 shown, in male mice in the early group, the ratio of ALT / AST in the serum of mice injected with siGpnmb-LNPs via the tail vein was significantly lower than that of mice injected with siNC-LNPs. In male mice in the late group, the change trend was the same as that in the early group, suggesting that the antibody-LNP-siGpnmb complex treatment may reduce liver inflammation in ApoE - / - mice fed a Western diet.

[0149] Example 17: Treatment with antibody-LNP-siGpnmb complex reduces blood lipids in Western diet-fed ApoE - / - mice Level

[0150] As described in Example 15, mouse serum samples were taken, and the contents of multiple blood lipid indexes of each mouse were detected using an automatic biochemical analyzer. The results are as Figure 23 - 26 shown (where Early Group represents the early group and Late Group represents the late group). The detection indexes include: total triglyceride (TG, Figure 23 ), total cholesterol (TCHO, Figure 24 ), low-density lipoprotein cholesterol (LDL-C, Figure 25 ), and high-density lipoprotein cholesterol (HDL-C, Figure 26 ).

[0151] As can be seen from Figure 23 , in male mice in the late group, the content of total triglyceride in the serum of mice injected with siGpnmb-LNP via the tail vein was significantly lower than that of mice injected with siNC-LNP. In male mice in the early group, the change trend was the same as that in the late group.

[0152] As can be seen from Figure 24 and Figure 25 , in male mice in the early group, the contents of total cholesterol and low-density lipoprotein cholesterol in the serum of mice injected with siGpnmb-LNP via the tail vein were significantly lower than those of mice injected with siNC-LNP. In male mice in the late group, the change trend was the same as that in the early group.

[0153] As can be seen from Figure 26 , whether in male mice in the early group or the late group, there was little difference in the content of high-density lipoprotein cholesterol in the serum of mice injected with siGpnmb-LNP and siNC-LNP.

[0154] Figure 23 - 26 The combined results indicate that the treatment with the antibody-LNP-siGpnmb complex can overall reduce the blood lipid levels in ApoE - / - mice fed a Western diet.

[0155] Example 18: Treatment with antibody-LNP-siGpnmb complexes alleviates atherosclerosis in Western diet-fed ApoE - / - mice actively Atherosclerotic burden in the aortic arch and abdominal aorta

[0156] (18.1) As described in Example 11, in an early atherosclerotic mouse model, siNC-LNP or siGpnmb-LNP was injected into the tail vein according to the grouping, 1 nmol siRNA (200 μL) was injected each time, once every three days, for a total of eight times.

[0157] (18.2) After the injection, the mice were continuously fed with a Western diet for 6 days, anesthetized with tribromoethanol by intraperitoneal injection, and the plaque formation in the aortic arch and abdominal aorta of the mice was observed using an animal ultrasound detector. The results are as Figure 27 and Figure 28 shown. Among them, Figure 27 is the ultrasound detection result of male mice in the early group, Figure 28 is the ultrasound detection result of female mice in the early group.

[0158] According to Figure 27 the results shown, obvious plaques in the aortic arch and abdominal aorta (marked by the red dotted line box) could be observed in male mice injected with siNC-LNP (also called LNP-siNC in the figure), while obvious plaques were hardly detected in the same parts of mice injected with siGpnmb-LNP (also called LNP-siGpnmb in the figure). As Figure 28 can be seen, the results in female mice were consistent with those in male mice, indicating that the treatment with the antibody-LNP-siGpnmb complex can reduce the atherosclerotic plaque burden in the aortic arch and abdominal aorta of ApoE - / - mice fed a Western diet.

[0159] Example 19: Treatment with antibody-LNP-siGpnmb complex alleviates the overall - / - condition of Western diet-fed ApoE Aortic atherosclerotic burden

[0160] (19.1) As described in Example 11, in early and late atherosclerotic mouse models, siNC-LNP or siGpnmb-LNP was injected into the tail vein according to the grouping, 1 nmol siRNA (200 μL) was injected each time, once every three days, for a total of eight times.

[0161] (19.2) After the injection was completed, the mice were continuously fed with a Western diet for 6 days. The mice were anesthetized with tribromoethanol by intraperitoneal injection. The entire aorta of the mice (including the carotid artery, aortic arch, brachiocephalic trunk, thoracic aorta, abdominal aorta, and iliac artery) was isolated and fixed in 4% paraformaldehyde for 24 hours. Then, Oil Red O dye was used to stain the aortic plaques. The results are as Figure 29 - Figure 31 shown. Among them, Figure 29 is the staining result of male mice in the late group, Figure 30 is the staining result of male mice in the early group, Figure 31 is the staining result of female mice in the early group.

[0162] According to Figure 29 the results shown, obvious Oil Red O dye-labeled plaques could be observed in the aortic arch and trunk aorta of male mice in the late atherosclerotic model injected with siNC-LNP (also called LNP-siNC in the figure), while significantly fewer plaques were visible in the aorta of mice injected with siGpnmb-LNP (also called LNP-siGpnmb in the figure). As Figure 30 can be seen, the results in male mice in the early atherosclerotic model were consistent with those in male mice in the late model. As Figure 31 can be seen, the results in female mice in the early atherosclerotic model were consistent with those in male mice.

[0163] From Figures 29 - 31 the comprehensive results shown, it can be seen that injecting siGpnmb-LNP has a therapeutic effect on early atherosclerosis in mice, and its therapeutic effect on late atherosclerosis in mice is more obvious, indicating that the antibody-LNP-siGpnmb complex treatment reduces the overall aortic atherosclerotic burden in ApoE - / - mice fed with a Western diet.

[0164] Example 20: Antibody-LNP-siGpnmb complex treatment can reduce ApoE in Western diet-fed - / - mice Plaque macrophages and foam cells in the artery

[0165] (20.1) As described in Example 13, aortic cells of control mice and treated mice in the advanced atherosclerosis model were digested and collected. Antibody blocking Fc receptor (clone 2.4G2, ThermoFisher) was added to the cell suspension, and then incubated with the surface antibody working solution prepared with CD45 flow antibody (clone 30-F11, BD Bioscience, 560510), F4 / 80 flow antibody (clone BM8, Invitrogen, MF48004) and CD64 flow antibody (clone x54-5 / 7.1, Biolegend, 139309) at 4 °C for 30 minutes. After centrifuging to collect the cells, they were washed twice with PBS and then labeled with Ghost Dye for 30 minutes.

[0166] (20.2) After centrifuging to collect the cells, they were fixed and permeabilized. After washing with the permeabilization solution, the centrifuged cells were incubated in the antibody dilution solution containing BODIPY fluorescent dye for 30 minutes. After washing with the permeabilization solution, the centrifuged cells were resuspended with PBE buffer. Finally, the expression levels of CD45, F4 / 80 and CD64 in the two groups of cells and the fluorescence signals of Ghost Dye and BODIPY dyes were detected by flow cytometry. The results are as Figures 32 - 34 shown. Among them Figure 32 is the proportion of macrophages in the plaque-containing aorta of mice among the total viable cells. Figure 33 is the proportion of foam cells in the plaque-containing aorta of mice among macrophages, Figure 34 is the fluorescence intensity peak graph of macrophages in the plaque-containing aorta of mice.

[0167] According to Figure 32 the results shown, compared with the control mice injected with siNC-LNP, the proportion of CD45 + F4 / 80 + macrophages among viable cells in the plaque-containing aorta of the treated mice injected with siGpnmb-LNP was slightly lower.

[0168] According to Figure 33 the results shown, compared with the control mice injected with siNC-LNP, the proportion of CD64 + BODIPY hi foam cells among CD45 + F4 / 80 + macrophages in the plaque-containing aorta of the treated mice injected with siGpnmb-LNP was significantly lower. In addition, according to Figure 34 it can be seen that the CD45 + F4 / 80 +The fluorescence intensity of BODIPY in macrophages was significantly lower.

[0169] Comprehensively Figures 32 - 34 Based on the results shown, there were fewer macrophages and foam cells in the aortas with plaques of the mice in the treatment group injected with siGpnmb-LNP, and the intracellular lipid content was lower, indicating that treatment with the antibody-LNP-siGpnmb complex could reduce plaque macrophages and foam cells in the aortas of ApoE - / - mice fed a Western diet and decrease the lipid content in plaque foam cells.

[0170] Example 21: Antibody-LNP-siGpnmb complex treatment can reduce ApoE in Western diet-fed - / - mice main Plaque area at the root of the artery

[0171] As described in Example 13, frozen section samples of the aortic roots were prepared and Oil Red O staining was performed on the plaque tissues of the aortic roots of mice in each group using the same protocol. The results were as Figure 35 and Figure 36 shown. Among them, Figure 35 is the staining result of the mice in the early group, Figure 36 is the staining result of the mice in the late group.

[0172] From Figure 35 the Oil Red O staining results shown, in the early atherosclerotic model mice, the plaque areas at the aortic roots of male or female mice injected with siGpnmb-LNP were smaller than those of the control group mice injected with siNC-LNP.

[0173] From Figure 36 the Oil Red O staining results shown, in the late atherosclerotic model mice, the plaque area at the aortic root of male mice injected with siGpnmb-LNP was also significantly reduced compared with that of the control group mice injected with siNC-LNP, indicating that treatment with the antibody-LNP-siGpnmb complex effectively reduced the plaque area at the aortic roots of ApoE - / - mice fed a Western diet.

[0174] Example 22: Antibody-LNP-siGpnmb complex treatment can reduce ApoE in Western diet-fed - / - mice main Macrophages and foam cells in the plaque tissue at the root of the artery

[0175] As described in Example 13, frozen section samples of the aortic roots were prepared and immunofluorescence staining was performed on the plaque tissues of the aortic roots of mice in each group using the same protocol. The results were as Figures 37 - 38 shown. Among them, Figure 37 is the staining result of the mice in the early group, Figure 38 is the staining result of the female mice in the late group.

[0176] According to Figure 37The immunofluorescence staining results shown indicate that in early atherosclerotic model mice, compared with the control mice injected with siNC-LNP, the lipid content labeled with Nile Red fluorescent dye, F4 / 80 + the number of macrophages, and Nile Red + F4 / 80 + the number of foam cells were all significantly decreased.

[0177] Figure 38 The immunofluorescence staining results shown are consistent with Figure 37 the results shown in. In late atherosclerotic model mice, compared with the control mice injected with siNC-LNP, the lipid content labeled with Nile Red fluorescent dye, F4 / 80 + the number of macrophages, and Nile Red + F4 / 80 + the number of foam cells were all significantly decreased, indicating that through the treatment with the antibody-LNP-siGpnmb complex, the lipid content in macrophages, foam cells, macrophages and foam cells in the aortic root plaque tissue was also significantly decreased.

[0178] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A siRNA delivery lipid nanoparticle, characterized in that The siRNA delivery lipid nanoparticles include lipid nanoparticles with a core-shell structure, siRNA encapsulated in the core of the lipid nanoparticles, and antibodies coupled to the shell surface of the lipid nanoparticles; The siRNA is an siRNA that inhibits the expression of GPNMB protein in cells; and The antibody is an antibody that can specifically bind to a surface marker protein of macrophages and / or foam cells derived therefrom.

2. The siRNA delivery lipid nanoparticle according to claim 1, characterized in that The sense strand of the siRNA comprises or consists of the nucleotide sequence shown in SEQ ID NO: 1, the antisense strand of the siRNA comprises or consists of the nucleotide sequence shown in SEQ ID NO: 2, and TT is overhanging at the 3' end of both the sense strand and the antisense strand of the siRNA.

3. The siRNA delivery lipid nanoparticle according to claim 1 or 2, characterized in that The antibody comprises one or both of CD36 monoclonal antibody and TREM2 monoclonal antibody; Optionally, the CD36 monoclonal antibody is selected from CD36 monoclonal antibody sc-7309, and the TREM2 monoclonal antibody is selected from TREM2 monoclonal antibody ab305103.

4. The siRNA delivery lipid nanoparticle according to any one of claims 1-3, characterized in that The siRNA is an siRNA that inhibits the expression of GPNMB protein in macrophages and / or foam cells derived therefrom.

5. A siRNA, characterized in that The sense strand of the siRNA comprises or consists of the nucleotide sequence shown in SEQ ID NO: 1, the antisense strand of the siRNA comprises or consists of the nucleotide sequence shown in SEQ ID NO: 2, and TT is overhanging at the 3' end of both the sense strand and the antisense strand of the siRNA.

6. Use of an agent for inhibiting the expression of GPNMB protein in macrophages and / or foam cells derived therefrom in the preparation of a drug for treating and / or preventing atherosclerosis.

7. Use of the siRNA delivery lipid nanoparticles according to any one of claims 1 to 4, or the siRNA according to claim 5, in the preparation of a medicament for reducing the foaming level of macrophages and / or increasing the autophagy level and lysosomal function in macrophages.

8. Use of the siRNA delivery lipid nanoparticles according to any one of claims 1 to 4, or the siRNA according to claim 5, in the preparation of a medicament for reducing the level of macrophage uptake of oxidized low-density lipoprotein (OxLDL), reducing the blood lipid level of patients with atherosclerosis, reducing the atherosclerotic burden of the aortic arch and abdominal aorta in patients with atherosclerosis, and / or reducing the overall aortic atherosclerotic burden in patients with atherosclerosis.

9. Use of the siRNA delivery lipid nanoparticles according to any one of claims 1 to 4, or the siRNA according to claim 5, in the preparation of a medicament for reducing plaque macrophages and / or their derived foam cells in the aorta of patients with atherosclerosis, reducing the aortic root plaque area of ​​patients with atherosclerosis, and / or reducing macrophages and / or their foam cells in aortic root plaque tissue of patients with atherosclerosis.

10. Use of the siRNA delivery lipid nanoparticles according to any one of claims 1 to 4, or the siRNA according to claim 5, in the preparation of a medicament for treating and / or preventing atherosclerosis.

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