A fapi, fapi intermediate, fapi modified lipid nanoparticle encapsulating hsp47 sirna and uses thereof

Abstract

The present application relates to a kind of FAPi, FAPi intermediate, FAPi modified HSP47siRNA loaded lipid nanoparticles and its application, belong to the field of biological medicine.The inventor constructs a kind of HSP47siRNA loaded lipid nanoparticles (FAPi-LNP / siHSP47) of Fibroblast activation protein inhibitor (FAPi) modification in view of the problems of high morbidity of existing technology liver fibrosis, so far no medicine, and ordinary LNP mainly target delivery to liver parenchymal cell after intravenous injection, and cannot enter the activated hepatic stellate cell (HSC) that plays a key role in the development of liver fibrosis.The expression of HSP47 can be effectively knocked down by the mediation of the high expression of Fibroblast activation protein on the surface of activated HSC, and the content of liver fibrosis marker protein a-SMA can be reduced.Compared with LNP without FAPi modification, FAPi-LNP / siHSP47 actively targets activated HSC after entering liver tissue, enters cell under the mediation of FAP, significantly knocks down the expression of HSP47, reduces collagen deposition, and significantly improves liver fibrosis.

Description

Technical Field

[0001] This invention relates to FAPi, FAPi intermediates, FAPi-modified lipid nanoparticles encapsulating HSP47 siRNA, and their applications, belonging to the field of biomedical technology. Background Technology

[0002] Globally, approximately 2 million people die from liver disease each year, including 1 million from complications of cirrhosis and 1 million from viral hepatitis and hepatocellular carcinoma. Chronic liver disease has become a global health burden, and early and effective intervention and treatment are crucial. In the overall progression of liver disease, liver fibrosis is a critical early stage, which can be caused by alcoholism, hepatitis virus infection, genetic abnormalities, fatty liver disease, autoimmune diseases, and other non-communicable diseases such as fatty liver. Long-term, repeated occurrences of these behaviors or diseases lead to liver damage that triggers chronic liver inflammation, resulting in excessive accumulation of extracellular matrix (ECM) proteins, thus forming liver fibrosis. Liver fibrosis distorts the liver structure, further forming regenerative hepatocellular nodules, i.e., cirrhosis. Cirrhosis can lead to hepatocellular dysfunction, progressing to hepatocellular carcinoma (HCC) and liver failure. Therefore, effective treatment at the stage of liver fibrosis to prevent further deterioration and progression is of great significance for the treatment of liver disease.

[0003] Hepatic stellate cells (HSCs) play a crucial role in the development of liver fibrosis, accounting for approximately 5-8% of a normal liver and serving as the primary site of vitamin A storage. In fibrotic livers, quiescent HSCs are activated by long-term stimulation from chronic liver inflammation, transforming into proliferative, migratory, and contractile myofibroblasts. These cells exhibit pro-fibrotic transcriptional and secretory properties, secreting large amounts of extracellular matrix (ECM) molecules, such as collagen and α-smooth muscle actin (α-SMA). These molecules accumulate in the Disse space, forming scar tissue, i.e., liver fibrosis. During the ECM secretion process of activated HSCs, heat shock protein 47 (HSP47) plays a significant promoting role. HSP47 is a collagen-specific chaperone; its high expression promotes collagen synthesis and secretion in HSCs. Therefore, HSP47 could be an attractive therapeutic target for fibrosis treatment. Furthermore, activated HSCs highly express a series of membrane proteins, among which fibroblast activation protein (FAP) has attracted much attention due to its high expression on fibroblasts in many fibrotic tissues, such as liver fibrosis, pulmonary fibrosis, renal fibrosis, solid tumors, and skin scars, but is almost not expressed in normal tissues. Utilizing the high affinity and specificity of FAP inhibitors (FAPi) for FAP, they have been widely used in the non-invasive early diagnosis, staging, differential diagnosis, and prognostic assessment of FAP-positive diseases, becoming an effective target for fibrosis-related diseases.

[0004] siRNA is a double-stranded small RNA that can target and degrade mRNA in cells using the base pairing principle. siRNA therapy has advantages such as high safety, high efficiency, relatively simple synthesis, and the ability to simultaneously silence multiple genes, making it a promising gene therapy drug. However, naked siRNA is unstable and easily degraded by nucleases and autolysis. Encapsulating siRNA in LNPs protects it from degradation by extracellular ribonucleases, enabling efficient delivery. In 2018, the first siRNA drug was approved by the FDA, and its carrier LNP was used clinically for the first time. It has since been widely used in mRNA vaccines, becoming a highly anticipated nanocarrier. After intravenous injection, LNPs target hepatocytes by adsorbing ApoE, providing an important opportunity for hepatocyte-related diseases. However, progress in targeted delivery to other cells and extrahepatic tissues has been slow, limiting its clinical application potential. Therefore, existing LNPs are mainly delivered to hepatocytes after intravenous injection, but cannot enter activated hepatic stellate cells (HSCs), which play a key role in the development of liver fibrosis.

[0005] In view of the above-mentioned problems in the prior art, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies, such as the high incidence of liver fibrosis, the lack of a cure to date, and the fact that ordinary LNPs, after intravenous injection, are mainly delivered to hepatocytes and cannot enter activated hepatic stellate cells (HSCs) that play a key role in the development of liver fibrosis. This invention provides FAPi, FAPi intermediates, FAPi-modified lipid nanoparticles carrying HSP47siRNA, and their applications.

[0007] Based on the promoting effect of HSP47 protein on liver fibrosis and the fact that activated hepatic stellate cells (LNPs) highly express FAP, the inventors synthesized FAPi, which targets and binds to FAP, and modified the surface of LNPs with siRNA targeting HSP47 encapsulated within it. FAPi-LNP / siHSP47 was then prepared using microfluidic technology. The aim is to enable LNPs to be targeted and delivered to activated HSCs under the mediation of FAPi, silencing HSP47 and reducing collagen synthesis and secretion, thereby treating liver fibrosis. This research is expected to provide an effective strategy for overcoming the target cell limitations of LNPs.

[0008] In a first aspect of the present invention, a FAPi is provided, which is DSPE-PEG-FAPi, selected from any one of compounds (1)-(4), and the structural formulas of the four compounds are as follows:

[0009]

[0010] Preferably, the DSPE-PEG-FAPi is DSPE-PEG3400-FAPi, and its structural formula is shown in formula (II):

[0011]

[0012] In a second aspect of the invention, a method for synthesizing DSPE-PEG3400-FAPi with the structural formula shown in formula (Ⅱ) is provided, comprising the following steps:

[0013] 1) Using methyl 6-hydroxyquinoline-4-carboxylic acid (i.e., compound 3-16), acetyl 3-bromopropanethiol (i.e., compound 3-11) and cesium carbonate as raw materials, compound 3-17 was obtained through a substitution reaction. The reaction time was 12 h and the reaction temperature was 60 °C.

[0014] The structural formulas of compounds 3-11, 3-16, and 3-17 are as follows:

[0015]

[0016] 2) Using compound 3-17 as a raw material, compound 3-18 was obtained by hydrolysis with alkali. The reaction time was 1 hour and the reaction temperature was 20℃.

[0017] The structural formula of compound 3-18 is as follows:

[0018]

[0019] 3) Using compound 3-18, HOBT, EDCI and (S)-4,4-difluoro-1-glycylpyrrolidine-2-carboxylonitrile (i.e., compound 3-9) as raw materials, compound 3-19 was obtained by acid-ammonium condensation reaction. The reaction time was 3 h and the reaction temperature was 20 °C.

[0020] The structural formulas of compounds 3-9 and 3-19 are as follows:

[0021]

[0022] 4) Compound 3-20 was obtained by addition reaction using compound 3-19 and DSPE-PEG3400-Mal as raw materials. The reaction time was 1 h and the reaction temperature was 20 °C.

[0023] The structural formulas of DSPE-PEG3400-Mal and compound 3-20 are as follows:

[0024]

[0025]

[0026] Furthermore,

[0027] In step 1), the molar amounts of each raw material are as follows: methyl 6-hydroxyquinoline-4-carboxylic acid ester: acetyl-3-bromopropanethiol: cesium carbonate = 1:2:5.

[0028] In step 2), the molar amounts of each raw material are as follows: compound 3-17: NaOH = 1:2.

[0029] In step 3), the molar amounts of each raw material are as follows: Compound 3-18: HOBT: EDCI: (S)-4,4-difluoro-1-glycylpyrrolidine-2-carboxynitrile = 1:1.5:1.5:1.

[0030] In step 4), the molar amounts of each raw material are as follows: Compound 3-19:DSPE-PEG3400-Mal = 1:1.

[0031] Furthermore,

[0032] In a preferred embodiment of the present invention, the synthesis method comprises the following specific steps:

[0033] 1) Synthesis of compound 3-17

[0034] 6-hydroxyquinoline-4-carboxylic acid methyl ester (i.e., compound 3-16) was dissolved in DMF, and then acetyl-3-bromopropanethiol (i.e., compound 3-11) and cesium carbonate were added. The reaction system was stirred at 60°C for 12 hours. After the reaction was completed, compound 3-17 was obtained by separation and purification. The molar ratio of compound 3-16, compound 3-11 and cesium carbonate was 1:2:5.

[0035] 2) Synthesis of compound 3-18

[0036] Compound 3-17 was dissolved in methanol and water, and then 2M sodium hydroxide aqueous solution was added. The reaction system was stirred at 20°C for 1 hour. After the reaction was completed, the solvent was removed by vacuum concentration, and then methanol was added to dissolve the compound and the salt was removed by filtration to obtain compound 3-18. The molar ratio of compound 3-17 to sodium hydroxide was 1:2.

[0037] 3) Synthesis of compound 3-19

[0038] Compound 3-18 was dissolved in DMF, and then HOBT, EDCI and (S)-4,4-difluoro-1-glycylpyrrolidine-2-carboxylonitrile (i.e., compound 3-9) were added. The reaction system was stirred at 20°C for 3 h. After the reaction was completed, compound 3-19 was obtained by separation and purification. The molar ratio of compound 3-18, HOBT, EDCI and compound 3-9 was 1:1.5:1.5:1.

[0039] 4) Synthesis of compound 3-20, namely DSPE-PEG-FAPi

[0040] Compound DSPE-PEG3400-Mal was dissolved in DMF, and then compound 3-19 was added. The reaction system was stirred at 20°C for 1 h. After the reaction was completed, compound 3-20 was obtained by separation and purification. The molar ratio of DSPE-PEG3400-Mal to compound 3-19 was 1:1.

[0041] In a third aspect of the invention, an intermediate compound for synthesizing FAPi as described in the first aspect is provided, having the following structural formula:

[0042]

[0043] R can be any one of the following groups: SH, COOH, NH2, NHS, or N3.

[0044] Preferably, the FAPi intermediate compound has the structural formula shown in formula (Ⅳ):

[0045]

[0046] In a fourth aspect of the invention, a method for synthesizing a FAPi intermediate with the structural formula shown in formula (Ⅳ) is provided, wherein the method for synthesizing the FAPi intermediate is the same as steps 1)-3) of the synthesis method described in the second aspect of the invention.

[0047] In a fifth aspect of the invention, a FAPi-modified lipid nanoparticle carrying HSP47 siRNA is provided, namely FAPi-LNP / siHSP47, wherein the lipid nanoparticle comprises FAPi as described in the first aspect, and HSP47 siRNA; wherein the HSP47 siRNA comprises the following sequence: CAAGAACAAGGCAGACCTA (SEQ ID No: 1). This sequence is a 5'-3' positive strand.

[0048] Furthermore, the lipid nanoparticles also include auxiliary lipids, which are selected from one or more of phospholipids, steroids, polymer-conjugated lipids, and modifiable lipids. Lipid components such as DlinMC3, PEG-DSPE, DSPC, and Chol can be selected.

[0049] In a sixth aspect of the present invention, a method for preparing lipid nanoparticles FAPi-LNP / siHSP47 as described in the fifth aspect is provided, the method comprising the following steps:

[0050] 1) Weigh out DlinMC3, PEG-DSPE, DSPC, and Chol according to a molar ratio of 50:1.5:10:38.5, and add 1% molar percentage of DSPE-PEG-FAPi (the DSPE-PEG-FAPi used is the same as the DSPE-PEG-FAPi described in the first aspect; in addition, the molar percentage of 1% means that the number of moles of DSPE-PEG-FAPi is in a ratio of 1:100 to the total number of moles of the four materials DlinMC3, PEG-DSPE, DSPC, and Chol). Dissolve the above lipid materials in ethanol as a lipid organic phase solution; prepare a 25mM citric acid solution at pH 4 as an aqueous phase solvent to dissolve HSP47 siRNA as an aqueous phase solution, with a molar ratio of siRNA to lipid materials of 1:10; prepare a 25mM citric acid solution at pH 6 as a buffer phase.

[0051] 2) Set the flow rate of the aqueous phase in the microfluidic device to 9 ml / min and the flow rate of the organic phase to 3 ml / min. Prepare a stable LNP solution using the microfluidic device and add 1.5 times the volume of buffer solution.

[0052] Furthermore, the microfluidic device used is the NEXSTAR Nano3 microfluidic control unit, and the chip is the NEXSTARC5-03 chip. The operation of the microfluidic device is performed in accordance with conventional procedures in the art.

[0053] In a seventh aspect of the invention, the use of FAPi-modified lipid nanoparticles loaded with HSP47 siRNA as described in the fifth aspect, or FAPi-modified lipid nanoparticles loaded with HSP47 siRNA prepared by the method described in the sixth aspect, in the preparation of a drug for treating liver fibrosis is provided.

[0054] In this invention, some of the terms used are explained as follows:

[0055] FAPi—short for Fibroblast Activation Protein Inhibitors—is a class of small molecule enzyme activity inhibitors targeting FAP (fibroblast activating protein).

[0056] LNP—short for Lipid Nanoparticles—is a type of nanoparticle used to deliver therapeutic nucleic acids, such as mRNA and siRNA.

[0057] FAP stands for fibroblast activation protein.

[0058] HSC—hepatic stellate cells, which play a key role in the development of liver fibrosis.

[0059] HSP47—heat shock protein 47—is a collagen-specific partner.

[0060] ECM—Extracellular matrix proteins.

[0061] α-SMA—α-smooth muscle actin—is a marker protein for liver fibrosis.

[0062] DMG-PEG—phospholipid-polyethylene glycol.

[0063] AUC—the area under the curve during drug use—represents the bioavailability of a drug. A larger AUC indicates higher bioavailability, and vice versa.

[0064] siRNA is a type of double-stranded small RNA that can target and degrade mRNA in cells using the principle of complementary base pairing.

[0065] The present invention has the following technical effects:

[0066] 1) Addressing the issues of high incidence and current incurable prevalence of liver fibrosis in existing technologies, and the fact that conventional LNPs, after intravenous injection, primarily target hepatocytes and fail to penetrate activated hepatic stellate cells (HSCs) that play a crucial role in the development of liver fibrosis, the inventors constructed a lipid nanoparticle (FAPi-LNP / siHSP47) modified with Fibroblast activation protein inhibitor (FAPi) and encapsulating HSP47 siRNA. This nanoparticle can be targeted and taken up by cells under the mediation of Fibroblast activation protein, which is highly expressed on the surface of activated HSCs, effectively knocking down HSP47 expression and reducing the content of the liver fibrosis marker protein α-SMA. Compared to unmodified FAPi LNPs, FAPi-LNP / siHSP47 actively targets activated HSCs after entering liver tissue, entering cells under FAP-mediated action, significantly knocking down HSP47 expression, reducing collagen deposition, and significantly improving liver fibrosis.

[0067] 2) The inventors studied the in vivo fate of LNP through pharmacokinetic and protein crown experiments. The results showed that LNP constructed using DSPE-PEG exhibited a higher AUC compared to LNP constructed using DMG-PEG; the AUC further increased after FAPi modification. Among the adsorbed protein crown components, complement-associated protein C3 and apolipoprotein ApoE were significantly reduced, explaining its difficulty in entering hepatocytes. This study, through FAPi modification, enabled LNP to actively target activated hepatic stellate cells, effectively treating liver fibrosis and providing a new approach for LNP targeting cells other than hepatocytes. Attached Figure Description

[0068] Figure 1 To demonstrate the successful synthesis of DSPE-PEG-FAPi. A. 3-20 (DSPE-PEG-FAPi) Synthesis Steps. B. 3-19 (FAPi) 13 C3-19 NMR spectrum. 1 H NMR spectrum. D.3-19 (FAPi) 19 F NMR spectrum. E. DSPE-PEG3400-Mal and 3-20 (DSPE-PEG-FAPi) 1 The disappearance of the characteristic peak of maleimide at 6.7 ppm in the ¹H NMR spectrum indicates that Mal has been completely reacted.

[0069] Figure 2 Design and construction of FAPi LNPs. A. TEM electron microscopy images and DLS particle size distribution of FAPi-LNP / siNC and LNP / siNC. Both LNPs are spherical, with a size of approximately 100 nm. B. Confocal microscopy shows that the nanoparticles (red) encapsulating siRNA linked to Cy5 dye were successfully taken up by HSC cells, and FAPi-LNP / siNC-Cy5 was taken up significantly more by HSC cells than LNP / siNC-Cy5 without a target. C. Flow cytometry analysis of HSC uptake of LNPs among different components. D. Confocal microscopy showing the mean fluorescence intensity (mean ± SD) of HSC uptake of FAPi-LNP / siNC and LNP / siNC. E. Flow cytometry analysis of HSC uptake efficiency (mean ± SD) among different components. (+B+F represents HSCs with high FAP expression stimulated by bleomycin and supplemented with FAPi-LNP / siNC-Cy5; -B+F represents HSCs without bleomycin stimulation and supplemented with FAPi-LNP / siNC-Cy5; +BF represents HSCs with high FAP expression stimulated by bleomycin and supplemented with LNP / siNC-Cy5; -BF represents HSCs without bleomycin stimulation and supplemented with LNP / siNC-Cy5) F. Serum stability test of target-modified LNP. Particle size and PDI trends of LNP(DSPE FAPi) and LNP(DSPE) stored at 4℃ for seven days. *** indicates P<0.001, **** indicates P<0.0001.

[0070] Figure 3FAPi-LNP / siNC-Cy5 successfully targeted HSC cells and FAPi-LNP / siHSP47 knocked down more HSP47. A. In vivo imaging showed that FAPi-LNP / siNC-Cy5 increased LNP accumulation in the liver, while LNP / siNC-Cy5 accumulated to varying degrees in the liver, spleen, and kidney (n=3). ROI values ​​of each organ (mean±SD) are statistically analyzed (right). B. Immunofluorescence section of the liver: top shows co-localization of FAPi-LNP / siNC-Cy5, LNP / siNC-Cy5, and HSC (α-SMA marker); bottom shows co-localization of FAPi-LNP / siNC-Cy5, LNP / siNC-Cy5, and FAP (n=3). C. Immunofluorescence section images of the liver: Top left shows the co-localization of FAPi-LNP / siNC-Cy5 with hepatocytes (CK18 labeled); top right shows the co-localization of FAPi-LNP / siNC-Cy5 with Kupffer cells (F4 / 80 labeled); bottom left shows the co-localization of LNP / siNC-Cy5 with hepatocytes (CK18 labeled) (n=3). D. Statistical analysis (mean±SD) of the mean fluorescence intensity of Cy5 co-localized with FAPi-LNP / siNC-Cy5, LNP / siNC-Cy5, FAP, HSC, hepatocytes, and Kupffer cells in liver fluorescent sections. E.WB imaging results showed that FAPi-LNP / siHSP47 had a higher knockdown effect on HSCs expressing high HSP47 protein (Con represents HSCs without induced high HSP47 expression, F-LNP represents HSCs transfected with FAPi-LNP / siHSP47 for 48 h, LNP represents HSCs transfected with LNP / siHSP47 for 48 h, and PBS represents HSCs induced with high HSP47 expression by bleomycin). * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0071] Figure 4A. Schematic diagram of liver fibrosis model construction and drug administration. B. Immunohistochemical results of liver Masson sections and HSP47 and α-SMA showed that FAPi-LNP / siHSP47 significantly improved liver fibrosis and knocked down more HSP47 than LNP / siHSP47 (scale bar, 1 mm) (n = 6, mean ± SD). (Control group: normal group; PBS: PBS control group; FL: FAPi-LNP / siHSP47 administration group; L: LNP / siHSP47 administration group). C. HE-stained spleen sections showed that FAPi-LNP / siNC-Cy5 improved the pathological condition of the spleen, and HE-stained kidney sections showed no significant toxic effect on the kidneys (scale bar, 1 mm) (n = 6, mean ± SD). D. Immunohistochemical quantitative results of liver collagen deposition, HSP47, and α-SMA in each group of mice, and serum AST, ALT, and AST / ALT detection results (n = 6, mean ± SD). * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0072] Figure 5 Characterization of LNPs with different formulations and target modifications in vitro and in vivo for adsorption of protein crowns. A. Pharmacokinetic curves of LNPs with three different formulations (n=3). B. Comparison of AUC (0-24h) of LNPs with three different formulations. C. Comparison of Ke values ​​of LNPs with three different formulations. D. t-values ​​of LNPs with three different formulations. 1 / 2 Comparison figures. E. Silver staining of proteins separated and collected by SDS-PAGE. F. LC-MS / MS detection of the top 20 proteins adsorbed by the three LNPs. G. GO analysis of molecular functions of LNP(DSPE FAPi) and LNP(DSPE) adsorbed protein crowns. H. KEGG pathway analysis of LNP(DSPE FAPi) and LNP(DSPE) adsorbed protein crowns. (n=3, mean±SD). * indicates P<0.05. Detailed Implementation

[0073] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0074] In the embodiments of the present invention, the materials and reagents involved are described as follows:

[0075] Dlin-MC3-DMA (O02006), DSPE-MPEG200 (B50845), DMG-PEG2000 (O02005), and 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC) (S01005) were purchased from AVT Pharmaceuticals Technology Co., Ltd. (Shanghai, China). Cholesterol was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Citric acid (100071192), oxaloyl chloride (80096326), thionyl chloride (SOCl2) (80128515), and N'N-dimethylformamide (DMF) (81007718) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Boc-4-oxo-L-proline methyl ester (1015166) was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd. (Shanghai, China). Ammonia-methanol solution (7 N / L) (W4201615000) was purchased from Shanghai Titan Technology Co., Ltd. (Shanghai, China). Trifluoroacetic anhydride (TFAA) (W8100300250) and hydrobromic acid (A0103885000) were purchased from Saen Chemical Technology (Shanghai) Co., Ltd. (Shanghai, China). N,N-Diisopropylethylamine (DIEA) (D0006), trifluoroacetic acid (TFA) (W810031), tetrakis(triphenylphosphine)palladium (E060079), cesium carbonate (A66064), 3-(ethyliminomethyleneimino)-N,N-dimethylpropane-1-amine, hydrochloride (EDCI) (A010938), 4-chloro-6-methoxyquinoline (E020798), zinc cyanide (W610490), potassium thioacetate (E060009), and 1,3-dibromopropane (D0202) were purchased from Anhui Zesheng Technology Co., Ltd. (Anhui, China). Fmoc-glycine (1028472) and 1-benzotriazolol (HOBT) (Y14396) were purchased from Shanghai Haohong Biomedical Technology Co., Ltd. (Shanghai, China). DSPE-PEG3400-Mal (H08143) was purchased from Shanghai Pengshuo Biotechnology Co., Ltd. Dichloromethane (CH2Cl2), diethylaminosulfur trifluoride (DAST) (1031315), ethanol (E809061), and olive oil (O815211) were purchased from Maclean Biotech Inc. (Shanghai, China). Carbon tetrachloride (CCl4) (B601416) was purchased from Bohr Chemical Reagents Co., Ltd. (Shanghai, China). Trizol (11668-027) was purchased from Invitrogen (USA). AceQ UniversalSYBR qPCRMaster Mix (Q511-02) and reagents for qPCR were used. IIQ RT Supermix (+g DNAwiper) (R223-01) was purchased from Vazyme (Nanjing, China). Quant-iT RiboGreen RNA Detection Kit (R11490) was purchased from Thermo (USA). Alanine aminotransferase (ALT) Detection Kit (UV-LDH 147 method) was purchased from Changchun Huili Biotechnology Co., Ltd. Aspartate aminotransferase (AST) Detection Kit (GM1103) (aspartate substrate method) was purchased from Leadman Biochemical Co., Ltd. (Beijing, China). Mouse TNF-α ELISA Kit (GEM0004) was purchased from Sewell Biotechnology Co., Ltd. (Wuhan, China). HisSep Ni-NTA agarose 6FF resin (20503ES60) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. (Shanghai, China). Rapid silver staining kit (P0017S) and TMB substrate (P0209) were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Guangzhou Ribobio Biotechnology Co., Ltd. synthesized siRNA targeting mouse HSP47 mRNA. Some experiments utilized the fluorescent dye Cy5 to label the 5' end of the siRNA; the Cy5-labeled siRNA was purchased from Guangzhou Ribobio Biotechnology Co., Ltd. Guangzhou Ribobio Biotechnology Co., Ltd. also synthesized qPCR primers targeting mouse HSP47, fibroblast activating protein (FAP), and GAPDH. Hepatic stellate cells were obtained from Shanghai Youkadi Biomedical Technology Co., Ltd. (Shanghai, China). Six- to eight-week-old male C57BJ / 6 mice were purchased from the Shanghai Laboratory Animal Center and housed under SPF conditions. All animal experiments were conducted in accordance with guidelines approved by the Ethics Committee of East China Normal University.

[0076] Compounds 3-9, 3-11, 3-13, 3-14, 3-15 and 3-16 in Example 1 of this invention are all existing products and can be prepared by referring to the method in Example 1, or they can be purchased directly.

[0077] The CAS registry numbers for each compound are as follows:

[0078] Compounds 3-9, CAS Registry No. 1448440-39-8;

[0079] Compound 3-11, CAS Registry No. 928-46-1;

[0080] Compound 3-13, CAS Registry No. 6443-89-6;

[0081] Compound 3-14, CAS Registry Number 86-68-0;

[0082] Compound 3-15, CAS Registry No. 4312-44-1;

[0083] Compound 3-16, CAS Registry No. 51043-76-6.

[0084] Example 1: Synthesis of DSPE-PEG-FAPi

[0085] The synthetic route for DSPE-PEG-FAPi is shown below. Figure 1 A.

[0086] The synthesis process specifically includes the following steps:

[0087] 1) Synthesis of compound 3-3

[0088] Under nitrogen protection and in an ice-water bath, compound 3-1 (Boc-4-oxo-L-proline methyl ester, 120 g, 493.3 mmol) was dissolved in anhydrous dichloromethane (1200 mL). DAST reagent (3-2) (153.3 g, 951.1 mmol) dissolved in anhydrous dichloromethane was slowly added dropwise, followed by the addition of ethanol (4.5 g, 97.7 mmol). The reaction was carried out at 20 °C for 12 h. TLC analysis confirmed the reaction was complete. Ice-cold sodium bicarbonate aqueous solution was slowly added until no more bubbles were produced. The mixture was then extracted with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to give the target compound 3-3, a yellow liquid, 86 g, with a yield of 65.7%.

[0089] 2) Synthesis of compounds 3-4

[0090] Compound 3-3 (66 g, 248.8 mmol) was dissolved in methanol (200 mL) under ice-water bath conditions. A methanol solution of 7N ammonia (240 mL) was added dropwise. After the addition was complete, the ice-water bath was removed, and the reaction mixture was incubated overnight at 20 °C. The reaction was confirmed to be complete by TLC. The solvent was removed by concentration under reduced pressure to give 59 g of a pale yellow solid, yield: 94.8%.

[0091] 3) Synthesis of compounds 3-5

[0092] Compounds 3-4 (59 g, 13.59 mmol) were dissolved in anhydrous dichloromethane (360 mL) of 20% TFA (90 mL) and stirred at 20 °C for 12 h. The reaction was confirmed to be complete by TLC. The solvent was removed under reduced pressure, and the residue was washed with diethyl ether and filtered to give 61 g of the target compound as a white solid.

[0093] 4) Synthesis of compounds 3-7

[0094] Compound 3-6 (Fmoc-glycine, 64.15 g, 215.8 mmol) was dissolved in anhydrous dichloromethane (500 mL). Oxaloyl chloride (41.12 g, 324 mmol) was added dropwise under an ice-water bath, followed by the addition of a catalytic amount of DMF. The ice-water bath was removed, and the reaction was carried out at 20 °C for 2 h. Compound 3-5 (57 g, 229.8 mmol) and DIEA (59.3 g, 459.6 mmol) in anhydrous dichloromethane were then added dropwise under an ice-water bath. After the addition was complete, the ice-water bath was removed, and the reaction was carried out at 20 °C for 12 h. After the reaction was complete, the mixture was filtered, and the filter cake was purified three times by slurrying with methanol to obtain 75 g of the target compound as a white solid, with a yield of 76%.

[0095] 5) Synthesis of compounds 3-8

[0096] Compound 3-7 (76 g, 177 mmol) was dissolved in anhydrous tetrahydrofuran (800 mL), cooled to -15 °C in an ice-salt bath, and pyridine (28 mL) was slowly added dropwise, followed by dropwise addition of anhydrous tetrahydrofuran solution of TFAA (24.7 mL). After the addition was complete, the ice-salt bath was removed, and the reaction system was allowed to react at 20 °C for 12 h. The reaction was confirmed to be complete by TLC. The solution was washed with 1 M dilute hydrochloric acid, washed with water, and dried over anhydrous sodium sulfate. 71.36 g of a pale yellow, foamy solid was obtained, with a yield of 98%.

[0097] 6) Synthesis of compounds 3-9

[0098] Compound 3-8 (75 g, 182.3 mmol) was dissolved in tetrahydrofuran (360 mL), and piperidine (90 mL) was added. The reaction mixture was reacted at 20 °C for 12 h. After the reaction was complete, the reaction solution was filtered to collect the filter cake, which was then washed with diethyl ether. The collected filter cake was purified twice by slurrying with diethyl ether to obtain 25 g of the target compound, with a yield of 72.5%. 1 H NMR (400MHz, DMSO) δ5.90 (s, 2H), 4.44-4.34 (m, 1H), 4.04-3.90 (m, 1H), 3.81 (dt, J = 3 6.8,11.2Hz,2H),3.63(ddd,J=18.6,13.5,5.3Hz,1H),2.83(td,J=14.0,6.3Hz,1H).

[0099] 7) Synthesis of compound 3-11

[0100] Compound 3-10 (1,3-dibromopropane, 10 g, 49.5 mmol) was dissolved in DMF (50 mL), and then potassium thioacetate (5.6 g, 49 mmol) was added. The reaction mixture was reacted at 20 °C for 12 h. The reaction was confirmed to be complete by TLC. 50 mL of water was added, and the mixture was extracted three times with ethyl acetate (50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. 7.5 g of a yellow oily compound was obtained by column chromatography, yield: 77%. 1 HNMR (400MHz, CDCl3) δ3.47(t,J=6.4Hz,2H),3.03(t,J=6.9Hz,2H),2.36(s,3H),2.15(p,J=6.6Hz,2H).

[0101] 8) Synthesis of compound 3-13

[0102] Compound 3-12 (4-chloro-6-methoxyquinoline, 22 g, 113.6 mmol) was dissolved in DMF (150 mL), followed by the addition of tetratetraphenylphosphine palladium (13.11 g, 11.3 mmol) and zinc cyanide (19.8 g, 168.6 mmol). After purging with nitrogen three times, the reaction mixture was stirred at 100 °C for 12 h. The reaction was confirmed to be complete by TLC. The reaction solution was cooled to room temperature and filtered. The filter cake was washed with dichloromethane, and the filtrate was collected. 200 mL of water was added, and the mixture was extracted with dichloromethane (150 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The product was purified by column chromatography to obtain 18 g of product, yield: 86%.

[0103] 9) Synthesis of compound 3-14

[0104] Compound 3-13 (15.8 g, 85.8 mmol) was dissolved in ethanol (118 mL), and then 20% sodium hydroxide aqueous solution (118 mL) was added. The reaction system was stirred at 20 °C for 3 hours, and the reaction was confirmed to be complete by TLC. Under ice bath conditions, the pH of the reaction solution was adjusted to between 1 and 2 with concentrated hydrochloric acid. After removing the solvent by vacuum concentration, methanol was added to dissolve the product, and the solution was filtered to remove salts. The filtrate was collected and concentrated under vacuum to obtain 16.5 g of crude product.

[0105] 10) Synthesis of compounds 3-15

[0106] The crude compound 3-14 (25 g) was dissolved in 170 mL of hydrogen bromide solution, and the temperature was raised to 130 °C. The reaction system was stirred at 130 °C for 12 h. The reaction was confirmed to be complete by TLC. The hydrogen bromide solution was removed by concentration under reduced pressure, and then the mixture was stirred twice with dichloromethane:methanol = 10:1 to obtain 22 g of yellow solid compound.

[0107] 11) Synthesis of compounds 3-16

[0108] Under ice bath conditions, 42 mL of thionyl chloride was slowly added dropwise to 450 mL of methanol. After stirring the reaction system in an ice bath for 10 min, compound 3-15 (22 g, 116.3 mmol) was added. The reaction system was heated to 80 °C and reacted for 12 h. The reaction was confirmed to be complete by TLC. After cooling the reaction system to room temperature, the solvent was removed by concentration under reduced pressure. The mixture was then slurried with dichloromethane:methanol = 10:1 to obtain 13.2 g of crude yellow solid compound, yield: 55.9%.

[0109] 12) Synthesis of compounds 3-17

[0110] Compound 3-16 (1.6 g, 7.9 mmol) was dissolved in DMF (20 mL), and then compound 3-11 (3.1 g, 15.7 mmol) and cesium carbonate (12.8 g, 39.3 mmol) were added. The reaction mixture was stirred at 60 °C for 12 h. The reaction was confirmed to be complete by TLC. After cooling to room temperature, water (30 mL) was added, and the mixture was extracted three times with ethyl acetate (30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The product was purified by column chromatography to obtain 600 mg of product (yield: 24%). 1 H NMR (400MHz, CDCl3) δ8.89(d,J=4.4Hz,1H),8.25(s,1H),8.10(d,J=9.2Hz,1H),7.98(t,J=7.1Hz,1H),7.45(d,J= 9.2Hz, 1H), 4.50 (t, J = 6.2Hz, 2H), 4.00 (s, 3H), 3.09 (t, J = 7.1Hz, 2H), 2.36 (d, J = 10.2Hz, 3H), 2.20-2.10 (m, 2H).

[0111] 13) Synthesis of compounds 3-18

[0112] Compound 3-17 (600 mg, 1.9 mmol) was dissolved in methanol (6 mL) and water (2 mL), and then 2 M sodium hydroxide aqueous solution (2 mL) was added. The reaction system was stirred at 20 °C for 1 h. The reaction was confirmed to be complete by TLC. After removing the solvent by concentration under reduced pressure, methanol was added to dissolve the compound, and the mixture was filtered to remove the salt, yielding 350 mg of a yellow solid compound (68%). 1H NMR (400MHz, MeOD) δ9.11(t,J=7.9Hz,1H),8.47(d,J=5.4Hz,1H),8.44(s,1H),8.25(t,J=9.7Hz,1H),7.84 (t,J=10.4Hz,1H),7.60-7.19(m,1H),4.40-4.24(m,2H),2.81-2.69(m,2H),2.19(dd,J=12.6,6.1Hz,2H).

[0113] 14) Synthesis of compound 3-19 (FAPi)

[0114] Compound 3-18 (350 mg, 1.3 mmol) was dissolved in DMF (5 mL), and then HOBT (269 mg, 2 mmol), EDCI (382 mg, 2 mmol), and compound 3-9 (251 mg, 1.3 mmol) were added to the reaction mixture. The mixture was stirred at 20 °C for 3 h. The reaction was confirmed to be complete by TLC. 10 mL of water was added, and the mixture was extracted three times with dichloromethane (10 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by concentration under reduced pressure, and the product was purified by column chromatography to obtain 90 mg of product, yield: 15.4%. 1 H NMR (400MHz, CDCl3) δ11.46(s,1H),8.79(d,J=4.4Hz,1H),8.26(s,1H),8.03(d,J =10.1Hz,2H),7.34(d,J=9.1Hz,1H),4.86-4.65(m,1H),4.14(dt,J=23.0,5.7Hz, 4H),4.03(dd,J=14.5,8.0Hz,1H),3.94-3.80(m,1H),3.04-2.78(m,2H),2.73(dd ,J=14.6,7.2Hz,2H),2.10(dt,J=12.6,6.2Hz,2H),1.35(dd,J=19.5,11.4Hz,1H).

[0115] 15) Synthesis of compound 3-20 (DSPE-PEG-FAPi)

[0116] Compound DSPE-PEG3400-Mal (700 mg) was dissolved in DMF (10 mL), and then compound 3-19 (70 mg) was added. The reaction system was stirred at 20 °C for 1 h. The reaction solution was then placed in a dialysis bag and dialyzed with water for 2 h. The water was then replaced and dialyzed again overnight. The water was replaced with tetrahydrofuran, and the dialysis was repeated 3 times, 2 h each time. The solution was then replaced with water and dialyzed twice more, 2 h each time. The solution in the dialysis bag was collected, lyophilized, and 512 mg of a white solid compound was obtained.

[0117] The synthesis steps for DSPE-PEG-FAPi(3-20) are as follows: Figure 1 A. The carbon and proton spectra of 3-19 confirm the successful synthesis of 3-19. Figure 1 BC). By comparing the fluorine spectra of compound 3-19 and compound DSPE-PEG-FAPi, it can be determined that compound 3-19 and compound DSPE-PEG3400-MAL reacted successfully. Figure 1 D). By comparing the proton NMR spectra of compounds DSPE-PEG3400-MAL and DSPE-PEG-FAPi, the successful synthesis of DSPE-PEG-FAPi can be further verified. A characteristic peak of maleimide exists at 6.7 ppm in the NMR spectrum of DSPE-PEG3400-MAL. However, after the reaction of DSPE-PEG3400-MAL with compound FAPi (3-19), this characteristic peak disappears in the NMR spectrum of DSPE-PEG-FAPi, confirming that MAL has been completely reacted. Figure 1 E).

[0118] Example 2 Effect Verification

[0119] I. Experimental Methods:

[0120] Preparation and characterization of FAPi-LNP / siHSP47, LNP / siHSP47, FAPi-LNP / siNC-Cy5, and LNP / siNC-Cy5

[0121] DlinMC3, PEG-DSPE, DSPC, and Chol (molar ratio 50:1.5:10:38.5) were precisely weighed using an analytical balance. When preparing the LNP to connect to the target, 1% DSPE-PEG-FAPi was added. All lipid materials were dissolved in ethanol as the lipid organic phase solution. A 25mM citric acid solution at pH 4 was prepared as the aqueous phase solvent to dissolve siHSP4 or siNC-Cy5 as the aqueous phase solution. The molar ratio of siRNA to lipid materials was 1:10. A 25mM citric acid solution at pH 6 was prepared as the buffer phase. The microfluidic device was set with an aqueous phase flow rate of 9 ml / min and an organic phase flow rate of 3 ml / min. A stable LNP solution was prepared using the microfluidic device, and 1.5 times the volume of the buffer phase solution (NEXSTAR Nano3 microfluidic preparation instrument, NEXSTAR C5-03 chip) was added.

[0122] The sample was pipetteted into a particle size distribution dish, and its hydrodynamic radius and polydispersity index (PDI) were measured using a Malvern dynamic light scattering (DLS) system. Morphology was observed using a transmission electron microscope (TEM). Samples were negatively stained with phosphotungstic acid and observed under a TEM after drying. (Results are shown in...) Figure 2 A)

[0123] The encapsulation efficiency of the prepared LNPs was determined. After treatment with 2% TE-Triton buffer, the siRNA encapsulated by the LNPs was decomposed. The LNPs were then allowed to stand at room temperature for 15 min to allow complete disintegration and release of the encapsulated siRNA. The total siRNA content at this point was determined as C(total). The siRNA encapsulated by the LNPs was determined as C. RiboGreen dye was diluted 200-fold and then added proportionally to 96-well plates containing pre-added samples. After dye addition, the plates were incubated for 5 min. The microplate reader was used to measure the encapsulation efficiency at excitation light of 485 nm and emission light of 528 nm. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency = Encapsulated siRNA / Total siRNA.

[0124] FAPi / cy5-siRNALNP targeting hepatic stellate cells in vitro

[0125] Hepatic stellate cells (HSCs) were cultured at 37°C and 5% CO2 in 1640 complete medium (90% 1640 basal medium + 10% fetal bovine serum + 1% penicillin / streptomycin). Activation of HSCs was achieved by stimulating them with bleomycin (1 μg / mL). The optimal drug concentration was confirmed by qPCR, at which HSCs showed the highest expression of FAP. The uptake of FAP-LNP / siNC-Cy5 and LNP / siNC-Cy5 nanoparticles by HSCs was then assessed using laser confocal microscopy. After co-incubating HSCs with FAP-LNP / siNC-Cy5 and LNP / siNC-Cy5 for 6 hours, the number of Cy5-containing cells was observed by flow cytometry to evaluate in vitro transduction efficiency. (Results are shown in...) Figure 2 BE)

[0126] Model construction of liver fibrosis

[0127] All animal experiments were conducted in accordance with the guidelines approved by the Experimental Animal Welfare and Ethics Committee of East China Normal University (No.: m20240422). Seven- to eight-week-old male C57BL / 6 mice were used. For the first two weeks, they were injected intraperitoneally with 2 ml / kg CCl4 (10% v / v CCl4 in olive oil) every three days. For the next four weeks, they were injected intraperitoneally with 5 ml / kg CCl4 (20% v / v CCl4 in olive oil) every two days, for a total of five weeks to establish a liver fibrosis model. The liver fibrosis model mice were randomly divided into three groups (n=6). Then, they were injected intravenously via tail vein with 0.75 μg / g (calculated as siRNA) of FAPi-LNP / siHSP47 and an equal dose of PBS every three days for a total of four injections. (Results are shown in...) Figure 4 A)

[0128]

[0129] In vivo tissue distribution of FAPi-LNP / siNC-Cy5

[0130] Seven- to eight-week-old male C57BL / 6 mice were randomly divided into two groups (n=3): FAPi-LNP / siNC-Cy5 and a control group (LNP / siNC-Cy5). LNP was then injected via the tail vein, and its in vivo tissue distribution was detected using in vivo imaging. Liver sections were prepared using immunofluorescence, and HSCs were labeled with α-SMA, hepatocytes with CK18, and Kupffer cells with F4 / 80 to observe the co-localization of FAPi-LNP / siNC-Cy5, LNP / siNC-Cy5, and HSCs. (Results are shown in...) Figure 3 AD)

[0131] Treatment efficacy evaluation

[0132] In mice with a liver fibrosis model, 0.1 mL of peripheral blood was collected after nanoparticle injection. The collected peripheral blood was centrifuged at 3000 rpm for 30 min at 4°C to obtain serum, which was then used to detect the levels of AST, ALT, and TNF-α. The heart, liver, spleen, and kidneys of the mice were harvested. Liver sections were subjected to Masson staining and HSP47 and α-SMA immunohistochemical staining, while the remaining sections underwent HE pathological staining. (Results are shown in...) Figure 4 BD)

[0133] RNA preparation and qPCR analysis

[0134] To test HSP47 expression levels, FAPi-LNP / siHSP47 or LNP / siHSP47 was co-incubated with HSCs for 48 hours. The co-incubated cells were then collected, centrifuged, and the supernatant was removed. Total RNA was extracted using a reagent (Cat#15596-018, Life Technologies, Carlsbad, CA, US) according to the manufacturer's instructions. The extracted RNA was then reverse transcribed using a 5×Primescipt RTMaster mix to obtain cDNA. HSP47 expression was detected by qPCR. Primers were provided by Ribobio. The primer sequences used for HSP47 and FAP amplification were:

[0135]

[0136] Protein extraction and Western blot detection

[0137] HSC cells at 5x10 5 HSCs were seeded in six-well plates. After 24 hours, FAPi-LNP / siHSP47 and LNP / siHSP47 containing 150 pmol were added to MEM medium and incubated for 72 hours. The HSC culture medium was removed, and the cells were washed once with PBS. 100-200 μL of lysis buffer was added to each well. The cells were pipetted several times to ensure adequate contact between the lysis buffer and the cells. After complete lysis, the cells were centrifuged at 12000g for 3-5 minutes. The supernatant was collected, and loading buffer was added. The cells were then incubated in a 100°C metal bath for 10 minutes.

[0138] Lysis buffer was added to the frozen liver sample at a ratio of 100-200 μL per 20 mg of tissue (1 mM PMSF was added). The sample was then thoroughly ground in a tissue homogenizer and centrifuged at 12000 g for 3-5 minutes to collect the supernatant. The protein concentration of the supernatant was determined by NanoDrop. Loading buffer was added and the sample was incubated in a 100°C metal bath for 10 minutes.

[0139] Proteins were separated at 15 μg per lane by 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Invitrogen) and then transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The membrane was blocked with 5% skim milk powder for 1 hour at room temperature, and then incubated with primary antibody overnight at 4°C. The membrane was washed three times and then incubated with secondary antibody (1:4000 dilution) for 1 hour at room temperature. Finally, the membrane was washed four times and developed using a chemiluminescence system according to the manufacturer's instructions. (Results are shown in...) Figure 3 E)

[0140] LNP adsorption protein corona analysis detection

[0141] Column packing: Pack a 3 mL empty gravity chromatography column into an N-NTA column (approximately 0.5 cm high, 400 μL Ni-NTA column - 30% EtOH), and wash with 2 mL PBS (5 times, 400 μL each time).

[0142] In vitro protein crown premix: 20 μL of siNC-Cy5-encapsulated LNP (DSPE), LNP (DSPE FAPi), and LNP (DMG) + 60 μL of PEG ScFv (antibody concentration 0.2 mg / mL), incubated at room temperature for 10 min. Then add 20 μL of C57 mouse serum and incubate at 37°C for 30 min, then add to a Ni column and incubate at room temperature for 10 min.

[0143] In vivo protein crown premixing: Seven to eight-week-old male C57BL / 6 mice were injected via tail vein with LNP (DSPE), LNP (DSPE FAPi) and LNP (DMG) encapsulated with siNC-Cy5 (1 mg / kg). 40 μL of serum was collected and incubated with 60 μL of PEG ScFv, then added to a Ni column and incubated at room temperature for 10 min.

[0144] Separation: Column flushing with PBS containing 5 mM imidazole, collecting fractions #1-#10, 200 μL / tube. Starting with #11, flush the column with 100 μL of 100 mg / mL PEG8000. Collect fluorescent fractions approximately at #11, 50 μL per tube, collecting fractions #11-#16. Quantification is performed according to Cy5 concentration; the Cy5 concentrations of the three formulations are consistent (80 μL = 50 μL for #11 + 30 μL for #12).

[0145] Sample loading: 4 μL of each of the three formulations was added to a pre-prepared polyacrylamide gel (8%, epizyme), and 2 μL was loaded with a marker. The gel strips were developed by silver staining, followed by in-gel enzymatic digestion. Peptides extracted from each sample were separated using an Evosep One nano-UPLC system and analyzed by a Tims TOF Pro2 mass spectrometer equipped with a nanoliter ion source. Database searches were performed using SpectroMine (4.2.230428.52329; Biognosys AG) software with the Pulsar search engine. Qualitative analysis was then performed after the search was completed. (Results are shown in...) Figure 5 EH)

[0146] LNP pharmacokinetic assay

[0147] Seven- to eight-week-old male C57BL / 6 mice were randomly divided into three groups and injected via tail vein with LNP (DSPE), LNP (DSPE FAPi), and LNP (DMG) encapsulated with siNC-Cy5 (1 mg / kg) (n=3). Blood samples were collected at 15 min, 30 min, 2 h, 4 h, 8 h, and 24 h. Serum was obtained by centrifugation at 3000 rpm at 4 °C, and the fluorescence intensity of Cy5 was detected. (Results are shown in...) Figure 5 AD)

[0148] LNP stability test

[0149] FAPi-LNP / siNC, LNP / siNC, and serum were stored together at 4°C, and changes in particle size, PDI, and zeta potential of FAPi-LNP / siNC and LNP / siNC were measured for seven consecutive days. (Results are shown in...) Figure 2 F)

[0150] Statistical analysis

[0151] All data are expressed as mean ± standard deviation. Student's t-test (two-tailed) was used to analyze data from two groups. Analysis of variance (ANOVA) was performed on data from three or more groups, followed by Tukey multiple comparisons. Statistical significance was defined as * (p < 0.05), ** (p < 0.01), *** (p < 0.001), or **** (p < 0.0001). All analyses were performed using GraphpadPrism v8.0 software.

[0152] II. Experimental Results:

[0153] 1. Construction and Characterization of FAPi-LNP

[0154] This invention designs and prepares stable LNPs encapsulating Cy5-siRNA and modified with DSPE-PEG-FAPi as a surface targeting ligand to target HSC cells. As shown in the TEM image, the nanoparticles are spherical with a particle size of approximately 100 nm, PDI < 0.2, and ζ potential close to 0 mV. Figure 2 A). When encapsulating different siRNAs, the particle size of LNPs was approximately 100 nm, the PDI was <0.2, and the zeta potential was close to 0 mV (see Table 1).

[0155] To determine the ability of nanoparticles to target HSC cells in vitro, FAPi-LNP / siNC-Cy5, LNP / siNC-Cy5, and HSC cells were incubated for 6 hours, respectively. Confocal microscopy results showed that HSC cells took up more FAPi-LNP / siNC-Cy5 compared to unmodified DSPE-PEG-FAPi LNPs. Figure 2B), quantitative analysis of mean fluorescence intensity (MFI) of HSCs clearly shows that DSPE-PEG-FAPi modification significantly improves the uptake of LNP by cells (see results). Figure 2 D). Flow cytometry results yielded the same conclusion: LNPs modified with DSPE-PEG-FAPi significantly enhanced their targeting of HSC cells (see results). Figure 2 C, 2E).

[0156] The inventors investigated the effect of FAPi target modification on the serum stability of LNPs. Particle size and PDI of FAPi-LNP / siNC and LNP / siNC were measured continuously for 7 days. The results showed that compared with unmodified DSPE-PEG-FAPi LNPs, FAPi-LNP / siNC exhibited smaller changes in particle size, PDI, and zeta potential over 7 days, indicating greater stability (see results). Figure 2 F)

[0157] Table.1.Summary ofLNP size distribution, PDI and zetapotentialresults.

[0158]

[0159]

[0160] 2. FAPi-LNP / siHSP47 targets HSC cells in vivo and knocks down more HSP47 cells.

[0161] To observe the distribution of nanoparticles in mice, liver fibrosis model mice were randomly divided into two groups. After tail vein injection of FAPi-LNP / siNC-Cy5 and LNP / siNC-Cy5 respectively, the mice's organs were removed 24 hours later to obtain in vitro imaging images. The ROI values ​​of each organ were statistically analyzed. The results showed that FAPi modification significantly increased LNP accumulation in the liver, while there was no significant difference in other organs. This may be due to FAPi modification (see results below). Figure 3 A).

[0162] Meanwhile, to investigate the effect of FAPi modification on the distribution of nanoparticles in different liver cells, FAPi-LNP / siNC-Cy5 and LNP / siNC-Cy5 were injected via tail vein. Twenty-four hours after injection, mouse livers were harvested for immunofluorescence sectioning to observe the co-localization of Cy5 (purple)-labeled LNP with α-SMA (green)-labeled HSCs and FAP (green)-labeled HSCs. As shown in the liver immunofluorescence section scans, compared to the unmodified target LNP / siNC-Cy5, FAPi-LNP / siNC-Cy5 entered more HSCs and exhibited significant co-localization with FAP protein. Therefore, FAPi-LNP / siNC-Cy5 has good targeting ability for HSCs, and its entry into HSCs may be mediated by FAP. [Results see...] Figure 3 B, the upper part shows the colocalization of FAPi-LNP / siNC-Cy5, LNP / siNC-Cy5 and HSC (a-SMA marker), and the lower part shows the colocalization of FAPi-LNP / siNC-Cy5, LNP / siNC-Cy5 and FAP).

[0163] Ordinary LNPs, after intravenous injection, are primarily delivered to hepatocytes and phagocytosed by hepatic macrophages. This study further investigated the entry of FAPi-modified nanoparticles into hepatocytes and their phagocytosis by hepatic macrophages (Kupffer cells). Hepatocytes were labeled with CK18 (yellow), and Kupffer cells with F4 / 80 (red). Immunofluorescence scans of liver sections showed that, compared to the FAPi-LNP / siNC-Cy5 modified target, LNP / siNC-Cy5 was taken up by more hepatocytes and Kupffer cells. This further confirms that FAPi-LNP / siNC-Cy5 enters more HSCs under FAP-mediated targeting, demonstrating good targeting of HSCs [Results see...]. Figure 3 C, Top left: co-localization of FAPi-LNP / siNC-Cy5 with hepatocytes (CK18 labeled); Top right: co-localization of FAPi-LNP / siNC-Cy5 with Kupffer cells (F4 / 80 labeled); Bottom left: co-localization of LNP / siNC-Cy5 with hepatocytes (CK18 labeled); Bottom right: co-localization of LNP / siNC-Cy5 with Kupffer cells (F4 / 80 labeled). Statistical analysis of the average fluorescence intensity of Cy5 in each liver fluorescent section showed that, in both α-SMA and FAP protein labeling, FAPi modification significantly introduced LNPs into HSCs, and FAPi modification reduced the phagocytosis of LNPs by both hepatocytes and Kupffer cells (see results). Figure 3 D).

[0164] As shown in the WB results, FAPi-LNP / siHSP47 exhibits a better knockdown effect than LNP / siHSP47, possibly because the modification with FAPi allows for the delivery of more nanoparticles into the HSC, resulting in a better knockdown effect (see results). Figure 3 E).

[0165] 3. FAPi-LNP / siHSP47 significantly improves liver fibrosis

[0166] To demonstrate that FAPi-LNP / siNC-Cy5 has better targeting of HSCs and a higher knockdown rate of HSP47, male C57BL / 6 mice aged seven to eight weeks were used to establish a liver fibrosis model for five weeks. The mouse weight change curve showed that the weight of the model group mice was significantly lower than that of the normal group after intraperitoneal injection on day 11. The successful establishment of the liver fibrosis model was confirmed by the measurement results of AST, ALT and AST / ALT in mouse serum (five mice were randomly selected from the model group for comparison with the normal group).

[0167] To investigate whether target-modified nanoparticles could exert a better therapeutic effect on liver fibrosis, mice with liver fibrosis were randomly divided into three groups (n=6), receiving FAPi-LNP / siHSP47, LNP / siHSP47, or PBS via tail vein injection, respectively. Observation of Masson staining results and HSP47 and α-SMA immunohistochemical results showed that the normal group had a smooth, reddish liver surface; the PBS group had a rough, granular liver surface with a pale yellowish color and severe capsule hyperplasia. All drug-treated groups showed improved liver surface morphology, with the FAPi-LNP / siHSP47 group exhibiting significantly less rough granularity and a smoother surface compared to the LNP / siHSP47 group. Further analysis of Masson staining results revealed that the normal group had intact, neatly arranged liver lobules without obvious blue-stained collagen fibers; while the PBS group showed disordered hepatic cords with abundant blue-stained collagen deposition, forming fibrous septa and pseudolobules between the portal and central vein areas, and between the portal and central vein areas. After administration of FAPi-LNP / siHSP47 and LNP / siHSP47, the blue-stained collagen fibers were reduced in both groups, with a more significant reduction in collagen fibers observed in the FAPi-LNP / siHSP47 group. Simultaneously, immunohistochemical results showed that, compared to the normal group, the PBS group exhibited more brown-stained HSP47 and α-SMA deposition. All administered groups showed varying degrees of reduction in deposition, with FAPi-LNP / siHSP47 showing a more significant knockdown than LNP / siHSP47, resulting in a significant reduction in brown-stained HSP47 and α-SMA deposition (see results). Figure 4B; Control group was the normal group, PBS was the PBS control group, FH was the FAPi-LNP / siHSP47 treatment group, and H was the LNP / siHSP47 treatment group. The above results indicate that FAPi-LNP / siHSP47 shows a better therapeutic effect on liver fibrosis than LNP / siHSP47.

[0168] HE staining of spleen sections revealed that in the normal group, the dorsal membrane was smooth, and the red and white pulp, marginal zone, and other structures were clearly defined. In the PBS group, the white pulp was reduced, the red and white pulp were almost indistinguishable, the number of splenic nodules was reduced, a few fragments were visible, the spleen tissue showed deformation, and even vascular dilation and hemorrhage, with no obvious germinal centers. After intervention with FAPi-LNP / siHSP47 and LNP / siHSP47, the red and white pulp structure of the spleen tissue in mice changed significantly, and the dorsal membrane gradually became smoother. FAPi-LNP / siHSP47 showed a more significant improvement effect on the spleen, therefore, FAPi-LNP / siHSP47 has a potential protective effect against spleen damage in mice with CCl4-induced liver fibrosis. HE staining of the kidneys showed that compared with the normal group, the kidney sections in the PBS group and the treated group were normal, with only a few lymphocyte infiltrations, which are considered normal phenomena. Therefore, FAPi-LNP / siHSP47 and LNP / siHSP47 have no significant toxic effects on the kidneys and are relatively safe (see results). Figure 4 C).

[0169] Figure 4 D represents the immunohistochemical quantitative results of liver collagen deposition, HSP47, and α-SMA in each group of mice, as well as the detection results of serum AST, ALT, and AST / ALT. This indicates that the FAPi-LNP / siHSP47 treatment group had a better therapeutic effect than LNP / siHSP47. Specifically, compared with the normal group, the serum ALT and AST levels in the PBS group were increased (P<0.0001); compared with the PBS group, the serum ALT, AST, and TNF-α levels in each treatment group were decreased (P<0.0001, P<0.001); and compared with the PBS group, the serum AST / ALT levels in each treatment group were decreased (P<0.001, P<0.01). Figure 4 D).

[0170] 4. Characterization of protein corona adsorption in vivo and in vitro by LNPs with different formulations and target modifications.

[0171] To evaluate the effects of different formulations and target modifications on the pharmacokinetics and adsorption of protein crowns of LNPs, onpattro-based LNPs (LNPDMG) were prepared using the same method described above, and pharmacokinetic assessments were performed on three types of LNPs. Figure 5As shown in Table A, the three types of LNPs, LNP(DSPE), LNP(DSPE FAPi) and LNP(DMG), exhibit significantly different pharmacokinetic characteristics. Detailed pharmacokinetic parameters are shown in Table 2.

[0172] When LNPs are injected intravenously, the nanomedicines adsorb endogenous biomolecules on their surface to form protein coronas, which affects the interaction between the nanomedicines and cells, targeting, and therapeutic efficacy. This study investigated the differences in in vivo protein corona adsorption by collecting blood samples 30 minutes after tail vein injection of the three LNPs mentioned above, and compared this with in vitro incubation of protein corona adsorbed in serum.

[0173] Consistent with existing findings, LNP(DMG) was eliminated more rapidly and had a shorter half-life compared to LNP(DSPE). This is likely because the C14 short chain of DMG-PEG is shorter than the C18 long chain of DSPE-PEG, making it more easily detached during systemic circulation, thus facilitating LNP clearance. In contrast, LNP(DSPE FAPi) exhibited significantly different characteristics, reaching peak concentration at 30 min post-dose and then declining at a slower rate, resulting in a longer absorption distribution process. It had the highest area under the curve (AUC) of 0.5004 ± 0.0162, the lowest elimination rate (Ke) of 0.1962 ± 0.0092, and the longest half-life (t). 1 / 2 3.5367±0.1691 Figure 5 AD).

[0174] After intravenous injection of the three types of LNPs, they rapidly exchange with proteins in the blood. Ultimately, the three types of LNPs adsorb different types and amounts of proteins to form LNP-protein crown complexes, which further affect the distribution, efficacy, and clearance of LNPs in the body.

[0175] Figure 5E shows the silver staining image of proteins collected by SDS-PAGE gravity chromatography column. Based on the silver staining results of protein corona adsorption in vivo and in vitro, there was no significant difference in the types and amounts of protein corona adsorbed by the three LNPs in vitro, but the in vivo results showed significant differences. Compared with LNP(DSPE), LNP(DSPE FAPi) adsorbed significantly less protein in terms of both content and types, with fewer proteins adsorbed at 30-40 kDa. This indicates that LNP(DSPE FAPi) adsorbed less apolipoprotein ApoE (34 kDa), allowing LNP(DSPE FAPi) to escape uptake by hepatocytes and enter hepatic stellate cells more readily under FAPi-mediated mobilization. In contrast, both LNP(DSPE) and LNP(DMG) adsorbed more proteins in vivo, and the types of adsorbed proteins differed significantly. LNP(DMG) adsorbed more proteins of 200 kDa (C3), 68-90 kDa, and 30 kDa (ApoE) sizes, exhibiting a greater diversity of adsorbed proteins.

[0176] To further identify and quantify the protein crowns adsorbed in vivo, LC-MS / MS analysis was used to obtain the types and contents of proteins adsorbed by the three LNPs. The results were the same as those obtained by silver staining. Among them, LNP(DMG) was found to adsorb 39 proteins, LNP(DSPE) adsorbed 33 proteins, and LNP(DSPE FAPi) adsorbed 32 proteins.

[0177] Further analysis of the protein corona adsorbed by the three LNPs revealed significant differences in the composition and abundance of the protein corona adsorbed by the three LNPs. Figure 5 F). The lipid chain length of PEG significantly alters the blood circulation and biodistribution of LNP. Compared to LNP (DSPE), LNP (DMG) adsorbs more apolipoproteins, immunoglobulins, and complement proteins. This is because DMG-PEG is a PEG C14 lipid, while DSPE-PEG is a PEG C18 lipid. Shorter PEGs detach from LNPs more quickly, resulting in LNPs adsorbing more protein corona. Complement proteins and other proteins contribute to faster LNP clearance from the blood. In contrast, longer PEG lipid chains anchor more stably to the LNP surface, slowing LNP clearance from the blood by influencing the composition of the protein corona.

[0178] When LNP adsorbs immunoglobulins and complement, it triggers the body's innate immune system to clear the LNP. Compared with the protein crowns adsorbed by LNP(DMG), the content of immunoglobulins and complement in the protein crowns adsorbed by LNP(DSPE) and LNP(DSPE FAPi) is reduced. This allows LNP to further improve its delivery efficiency in the body and reduce non-specific clearance of LNP.

[0179] Following intravenous injection of LNP, LNP rapidly exchanges with proteins in the blood (such as ApoE) and is effectively taken up into hepatocytes via LDL receptors. After FAPi modification, LNP (DSPE FAPi) showed a significantly reduced content of adsorbed protein crowns compared to unmodified LNP. ApoE was not detected among the top 20 most abundant proteins. FAPi modification further targets activated HSCs, reducing LNP uptake by hepatocytes.

[0180] Further analysis of the effect of FAPi modification on the function of proteins adsorbed by LNP revealed significant differences in the biological functions of proteins adsorbed by LNP(DSPE FAPi) and LNP(DSPE) through molecular functional GO and KEGG pathway analysis. LNP(DSPE) exhibited greater cholesterol transfer activity, higher protein binding rate, and greater lipoprotein receptor binding, indicating that LNP(DSPE) enters more hepatocytes under LDL receptor-mediated pathways compared to LNP(DSPE FAPi). Figure 5 GH), which is consistent with the aforementioned Figure 3 The results from BC are consistent.

[0181] Following intravenous injection of the three types of LNPs, they rapidly exchange with proteins in the blood. Ultimately, the three LNPs adsorb different types and quantities of proteins to form LNP-protein corona complexes, which further affect the distribution, efficacy, and clearance of LNPs in the body. Compared with LNP (DMG) and LNP (DSPE), LNP (DSPE FAPi) has a longer distribution phase. After entering the bloodstream, LNP (DSPE FAPi) is transported to various tissues of the body via blood circulation. Under the mediation of FAPi, it is transported to the target site of FAPi, namely HSC cells that highly express FAP protein. Therefore, LNP (DSPE FAPi) can accumulate in liver tissue, serving as a drug reservoir. The liver tissue can slowly release the drug into the blood, avoiding a rapid decrease in blood drug concentration, thereby prolonging the duration of drug action. This pharmacokinetic study result is consistent with the above results. After modification with the FAPi target, LNP increases the drug's blood circulation time, achieves a higher degree of absorption, accumulates more in liver tissue, and has a slower elimination rate. Therefore, LNP modified with the FAPi target adsorbs less protein crown, resulting in better drug delivery efficiency and better therapeutic effect.

[0182] Table 2.Results ofpharmacokinetic parameters of the LNPs.

[0183]

[0184] in conclusion:

[0185] FAP, as a marker of cancer-associated fibroblasts (CAF), participates in the regulation of the extracellular matrix and possesses numerous pro-tumor activities. It is frequently used as a target for various cancers, inhibiting tumor growth by targeting and suppressing FAP activity, and eliminating matrix-tumor cell interactions (especially in solid tumors), increasing intratumoral drug delivery, and promoting the activity of immunotherapeutic drugs. FAP is not only used as a therapeutic target in cancer treatment but has also been applied to design various cancer diagnostic and treatment methods: radiotherapy agents, near-infrared photoimmunotherapy (NIR-PIT), and FAPi-based probes used in positron emission tomography / computed tomography (PET / CT) for diagnosing various malignant tumors. However, the application of FAP in liver disease treatment remains to be explored. In the development and progression of liver fibrosis, activated HSCs highly express FAP. Therefore, FAPi can be used to achieve effective delivery of LNPs to HSCs, thereby inhibiting the central link in liver fibrosis development: HSC activation, and achieving a therapeutic effect.

[0186] Currently, inhibiting the further development of early liver fibrosis and accelerating its regression are feasible strategies for preventing cirrhosis and even liver cancer. However, there are still no effective drugs for treating early liver fibrosis, and there is an urgent need to develop effective anti-liver fibrosis drugs. Hepatocellular carcinoma cells (HSCs) are the main effector cells in the process of liver fibrosis. After liver damage, inflammatory mediators activate HSCs, and activated HSCs produce pro-inflammatory mediators, which perpetuate liver inflammation and lead to the formation of scar tissue, ultimately resulting in further liver failure and damage, developing into cirrhosis and even liver cancer.

[0187] Conventional liver fibrosis nuclei (LNPs) primarily target hepatocytes, making targeted delivery to cells outside the hepatocyte range difficult. This study modifies the surface of LNPs / siHSP47 with FAPi. FAPi specifically binds to FAP on the surface of activated hepatocyte fibrotic cells (HSCs), enabling HSC-specific uptake of LNPs. Knockout of HSP47 expression reduces collagen secretion and deposition, thus effectively treating liver fibrosis. LNP pharmacokinetic studies showed that LNPs constructed using DSPE-PEG exhibited slower blood clearance and higher AUC compared to LNPs constructed using DMG-PEG. FAPi modification of DSPE-PEG-constructed LNPs further slowed blood clearance and increased AUC. In vivo protein corona studies showed that the FAPi-LNP (DSPE) group adsorbed the least amount of complement-associated protein C3 and apolipoprotein ApoE, which may explain its increased AUC and reduced targeted uptake to hepatocytes.

[0188] Pharmacokinetic and coronal assays showed that the carbon chain length of the PEG lipids used in LNPs significantly influenced their in vivo fate. Compared to LNPs constructed using DMG-PEG, LNPs constructed using DSPE-PEG exhibited slower blood clearance and higher AUC, consistent with previous reports. Shorter-chain PEG lipids, such as DMG-PEG (C14), cannot be stably anchored on the LNP surface and tend to detach more quickly after entering the body. This facilitates the rapid adsorption of large amounts of plasma proteins onto the LNP surface, and the presence of cholesterol in the LNP promotes the adsorption of apolipoproteins such as ApoE, thereby mediating greater LNP entry into hepatocytes. Therefore, this study used long-chain PEG lipids (DSPE-PEG), and the results showed that LNPs (DSPE) indeed exhibited slower blood clearance and higher AUC, which may be explained by their lower ApoE adsorption. This provides an effective approach for treating related diseases outside of hepatocytes using LNPs.

[0189] In recent years, the in vivo fate of nanomedicines has received increasing attention, and the adsorption of protein crowns has a significant impact on their in vivo fate. Many studies have shown that protein crowns adsorbed on the surface of actively targeted nanomedicines can mask the target molecules originally modified on the nanomedicine surface, preventing them from binding to their targets and exerting their active targeting effect. This may be one reason why actively targeted nanomedicines cannot achieve better therapeutic effects. However, studies have shown that compared with unmodified FAPi LNPs, FAPi-LNPs exhibit a higher AUC and a significantly reduced content of adsorbed protein crowns, with ApoE not detected in the top 20 most abundant proteins. From in vivo HSC targeting results (… Figure 3 From the results, the adsorption of protein crowns did not significantly affect the in vivo targeted delivery of LNPs. Therefore, the in vivo adsorption of protein crowns for different nanomedicines may be complex and diverse, and the structural characteristics and properties of the target molecule itself may be one of the influencing factors, which warrants in-depth and systematic research.

[0190] Clinical LNP injection is convenient and rapid, and its safety and efficacy have been widely certified. FAPi-LNP / siHSP47 provides a practical treatment for early-stage liver fibrosis. Therefore, the designed and synthesized FAPi-LNP / siHSP47, through FAPi modification, achieves effective delivery to hepatocytes outside the liver parenchyma cells. It not only exhibits reduced ApoE adsorption and increased AUC, but also enhanced targeting of activated HSCs and knockout of HSP47, effectively reducing collagen secretion and deposition. This achieves targeted gene therapy for liver fibrosis, providing a new approach to liver disease treatment.

[0191] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A FAPi, characterized in that, The FAPi is DSPE-PEG3400-FAPi, and a structural formula thereof is shown as formula (II):

2. A FAPi-modified liponanoparticle encapsulating HSP47 siRNA, i.e. FAPi-LNP / siHSP47, according to claim 1, characterized in that, The lipid nanoparticle comprises the FAPi of claim 1, and HSP47 siRNA, wherein the HSP47 siRNA comprises the following sequence: CAAGAACAAGGCAGACCTA; and the lipid nanoparticle further comprises an auxiliary lipid selected from one or more of a phospholipid, a steroid, a polymer conjugated lipid, and a modifiable lipid.

3. The lipid nanoparticle of claim 2, wherein, The preparation method of the lipid nanoparticle comprises the following steps: 1) DlinMC3, PEG-DSPE, DSPC, and Chol are weighed according to a molar ratio of 50:1.5:10:38.5, and DSPE-PEG-FAPi is added in a molar percentage of 1%; the above lipid materials are dissolved in ethanol to form a lipid organic phase solution; a citric acid solution with a pH of 4 and a concentration of 25 mM is prepared as an aqueous phase solvent, and HSP47 siRNA is dissolved as an aqueous phase solution; the molar ratio of siRNA to the lipid material is 1:10; a citric acid solution with a pH of 6 and a concentration of 25 mM is prepared as a buffer phase; 2) a microfluidic device is set, the aqueous phase flow rate is 9 ml / min, and the organic phase flow rate is 3 ml / min; a stable LNP solution is prepared through the microfluidic device, and 1.5 times the volume of the buffer phase solution is added.

4. Use of the FAPi-modified HSP47 siRNA-loaded lipid nanoparticle of any one of claims 2-3 in the preparation of a drug for treating liver fibrosis.

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