SiTGF-beta1 / siGAS6 co-delivery system for aerosol inhalation synergistic treatment of idiopathic pulmonary fibrosis

Abstract

The invention relates to the technical field of molecular biology and biological medicine, in particular to a siTGF-beta1 / siGAS6 co-delivery system capable of being used for aerosol inhalation synergistic treatment of idiopathic pulmonary fibrosis. Specific siTGF-beta1 is designed aiming at mRNA (messenger ribonucleic acid) of a transforming growth factor TGF-beta1, and the siTGF-beta1, siGAS6 capable of effectively inhibiting GAS6 gene expression and a mannan-polypeptide conjugate carrier are further compounded to form a mannan-polypeptide conjugate co-delivery system of the siTGF-beta1 and the siGAS6, namely a mannan-polypeptide / siTGF-beta1 / siGAS6 co-delivery system. The system can be used for preparing a medicine for treating idiopathic pulmonary fibrosis, especially for preparing a medicine capable of being inhaled by atomization. The idiopathic pulmonary fibrosis can be synergistically treated in an aerosol inhalation mode. According to the technical scheme, lung macrophages can be efficiently targeted, and a synergistic treatment effect is generated to realize efficient treatment on idiopathic pulmonary fibrosis.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and biomedicine, and in particular to an siRNA pharmaceutical composition for the synergistic treatment of idiopathic pulmonary fibrosis. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease characterized by inflammation and fibrosis of the alveolar walls, leading to a gradual decline in lung function. It is characterized by widespread fibrotic lesions and has a higher mortality rate than many cancers. IPF typically occurs during the abnormal repair process following epithelial cell damage, resulting in scarring in the gas exchange areas of the lungs. Several factors are known to contribute to epithelial cell damage, including viral infections, smoking, environmental exposure, gastroesophageal reflux, and aging. Currently, the only treatments for IPF are pirfenidone and nintedanib, which can slow disease progression but cannot reverse fibrosis. Several other drugs are currently in clinical trials, including PRM-151, treprostone (Tyvaso), and acetylcysteine, offering new hope for future treatments.

[0003] In recent years, nucleic acid therapy has emerged as a potential strategy for treating idiopathic pulmonary fibrosis (IPF) due to its high specificity and well-defined mechanism of action. Despite its theoretically significant potential, the delivery and stability of nucleic acid therapy in vivo remain major challenges, particularly pulmonary delivery. Inhalation administration allows for maximum drug deposition in the lungs, improves patient compliance, promotes rapid drug absorption, avoids first-pass metabolism, and reduces systemic exposure, making it considered an ideal approach for treating respiratory diseases. However, currently, there is a lack of effective nucleic acid delivery drugs specifically for IPF. Summary of the Invention

[0004] Given the current lack of effective nucleic acid delivery drugs for idiopathic pulmonary fibrosis (IPF) in the existing technology, this invention provides a siTGF-β1 / siGAS6 co-delivery system that can be used for nebulized inhalation in conjunction with the treatment of IPF.

[0005] More specifically, this invention first provides an interfering RNA (siTGF-β1) that effectively inhibits the expression of the transforming growth factor TGF-β1 gene. This invention designs a specific siRNA (siTGF-β1) targeting the mRNA of transforming growth factor TGF-β1, and its delivery to target cells can achieve inhibition of transforming growth factor TGF-β1 expression.

[0006] The present invention further combines the siTGF-β1 with siRNA (siGAS6) that effectively inhibits the expression of the GAS6 gene to form an siRNA drug composition for the synergistic treatment of idiopathic pulmonary fibrosis.

[0007] This invention also provides a mannan-peptide coupler and its preparation method.

[0008] The present invention further combines the siRNA drug composition for the synergistic treatment of idiopathic pulmonary fibrosis with a mannan-peptide coupler to form a mannan-peptide coupler co-delivery system of siTGF-β1 and siGAS6 (i.e., mannan-peptide / siTGF-β1 / siGAS6 co-delivery system).

[0009] The siTGF-β1 and siGAS6 mannan-peptide co-delivery system provided by the present invention can be used to prepare therapeutic drugs for idiopathic pulmonary fibrosis. In particular, the therapeutic drug for idiopathic pulmonary fibrosis is a nebulized inhalation drug, which can be co-delivered by nebulization during use.

[0010] When used, the siTGF-β1 and siGAS6 mannan-peptide co-delivery system achieves co-delivery via nebulized inhalation, which can efficiently target lung macrophages and produce a synergistic therapeutic effect to achieve highly effective treatment of idiopathic pulmonary fibrosis.

[0011] The objective of this invention can be achieved through the following technical solutions:

[0012] The present invention first provides an interfering RNA that effectively inhibits the expression of transforming growth factor TGF-β1 gene, namely, an interfering RNA that targets the TGF-β1 encoding gene, referred to as siTGF-β1, wherein the interfering RNA contains any one or more of the nucleotide sequences shown in SEQ ID NO: 1-14.

[0013] TGF-β1 gene overexpression is common in various diseases, including chronic thromboembolic pulmonary hypertension (CTEPH), keloids, diabetic nephropathy, and various tissue fibrosis diseases. Studies have also shown that elevated TGF-β1 levels are associated with the formation of chemotherapy resistance in tumors. The mechanism of action of TGF-β1 in fibrotic diseases mainly includes two aspects: (1) Activation of fibroblasts and promotion of extracellular matrix deposition: TGF-β1 regulates its downstream signaling pathways, such as promoting the proliferation and activation of myofibroblasts through the PI3K / AKT / mTOR pathway, which stimulates the activation and proliferation of fibroblasts, leading to extracellular matrix (ECM) deposition. This deposition is a key feature of fibrotic diseases, in which a large number of activated fibroblasts and excessive ECM deposition lead to the loss of normal function of the affected organs. (2) Epithelial-mesenchymal transition (EMT): TGF-β1-mediated EMT is an important step in the fibrotic process. It activates mesenchymal cells, prompting them to synthesize ECM, providing a favorable microenvironment for the occurrence and development of fibrosis. TGF-β1 has therefore become an important target for the treatment of fibrotic diseases.

[0014] Currently, interfering RNA (RNA) is rapidly developing as a highly efficient and sequence-specific technique for silencing or reducing gene expression in multiple disease areas. However, there are currently no marketed RNA interfering RNA drugs targeting transforming growth factor TGF-β1. Only Sirnaomics has related drugs in clinical trials. Based on this, this invention provides an RNA interfering RNA that effectively inhibits the expression of the transforming growth factor TGF-β1 gene, thereby inducing the downregulation of TGF-β1 expression.

[0015] Preferably, the interfering RNA consists of any one or more nucleotide sequences shown in SEQ ID NO: 1-14 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14).

[0016] In one embodiment of the present invention, the interfering RNA comprises a sense strand and / or an antisense strand;

[0017] The positive chain contains one or more nucleotide sequences (e.g., 1, 2, 3, 4, 5, 6, 7) of any one of SEQ ID NO: 1-7;

[0018] The antisense strand contains one or more nucleotide sequences (e.g., 1, 2, 3, 4, 5, 6, 7) of any one of SEQ ID NO: 8-14.

[0019] Preferably, the justice chain and the antisense chain are complementary pairs.

[0020] Preferably, the positive strand of the interfering RNA is as shown in any one or more of SEQ ID NO: 1-7 (e.g., 1, 2, 3, 4, 5, 6, 7).

[0021] Preferably, the antisense strands of the interfering RNA are as shown in any one or more of SEQ ID NO: 8-14 (e.g., 1, 2, 3, 4, 5, 6, 7).

[0022] In one embodiment of the present invention, the interfering RNA is selected from any one of the following groups:

[0023] A) The sense strand contains the nucleotide sequence shown in SEQ ID NO: 1, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 8;

[0024] B) The sense strand contains the nucleotide sequence shown in SEQ ID NO: 2, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 9;

[0025] C) The sense strand contains the nucleotide sequence shown in SEQ ID NO: 3, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 10;

[0026] D) The sense strand contains the nucleotide sequence shown in SEQ ID NO: 4, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 11;

[0027] E) The sense strand contains the nucleotide sequence shown in SEQ ID NO: 5, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 12;

[0028] F) The sense strand contains the nucleotide sequence shown in SEQ ID NO: 6, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 13;

[0029] G) The sense strand contains the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 14.

[0030] The specific pairing information for the justice chain and antithesis chain is shown in Table 1.

[0031] In one specific embodiment of the present invention, the interfering RNA includes one or more combinations of (A), (B), (C), (D), (E), (F) or (G) above.

[0032] In one embodiment of the present invention, the interfering RNA further comprises a hanging base;

[0033] Preferably, the interfering RNA contains 1-10 dangling bases, more preferably 2-4 dangling bases;

[0034] Preferably, the dangling base is located at the end (preferably the 3' end) of the sense and / or antisense strands of the interfering RNA; there may also be n dangling bases to increase the activity of the interfering RNA. The dangling bases can be the same or different deoxyribonucleotides (e.g., deoxythymidine (dT), deoxycytidine (dC), deoxyuridine (dM), etc.), and n is an integer from 1 to 10.

[0035] In one specific embodiment of the present invention, the ends of the sense and / or antisense strands of the interfering RNA contain two dangling bases.

[0036] In one specific embodiment of the present invention, the ends of the sense and / or antisense strands of the interfering RNA contain two identical or different dangling bases.

[0037] In one specific embodiment of the present invention, the ends of the sense and / or antisense strands of the interfering RNA contain two identical or different dangling bases, wherein the dangling bases are deoxythymidine (dT) or deoxyuridine (dM), preferably, the dangling bases are deoxynucleosides.

[0038] More preferably, the suspended base is dTdT, dTdC, or dMdM.

[0039] In one embodiment of the present invention, the interfering RNA comprises one or more of siRNA, dsRNA, shRNA, aiRNA, or miRNA.

[0040] Preferably, the interfering RNA is siRNA. The siRNA is a double-stranded RNA of 19-23 nt in length. After entering the cell, it undergoes a series of enzymatic processes and forms a complex with RISC. Through base pairing, it specifically targets and silences the target gene.

[0041] In one embodiment of the present invention, the interfering RNA further includes at least one modification; the modification includes modifications on bases, sugar rings and / or phosphate backbones;

[0042] Preferably, the modification of the base includes, but is not limited to, 5-position pyrimidine modification, 8-position purine modification, pseudouracil modification and / or 5-bromoguanine substitution;

[0043] Preferably, the modification of the sugar ring includes, but is not limited to, the substitution of 2'-OH with groups such as H, OZ, halo, SH, SZ, NH2, NHZ, NZ2 or CN, wherein Z is an alkyl group;

[0044] Preferably, the modification of the phosphate skeleton includes, but is not limited to, thiophosphate modification.

[0045] Preferably, the alkyl group represents a straight-chain or branched hydrocarbon chain radical that does not contain unsaturated bonds, and the hydrocarbon chain radical is connected to other parts of the molecule by a single bond. Typical alkyl groups contain 1 to 20 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, 1-methylundecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, and eicosyl, etc.

[0046] In one embodiment of the invention, the modification further includes nucleotides having inosine, piracetamine, xanthine, 2'-methylribose, non-natural phosphodiester bonds and / or peptides.

[0047] In one specific embodiment of the present invention, the modification may be methylation, fluorination, thiophosphorylation, ethylene glycol nucleotides, etc.

[0048] Modified interfering RNA has better properties than its unmodified counterpart, such as higher stability and lower immunogenicity.

[0049] Preferably, the interfering RNA can be obtained by any method in the prior art, such as chemical synthesis, in vitro transcription, enzymatic digestion, or in vivo transcription.

[0050] In one specific embodiment of the present invention, the interfering RNA is chemically synthesized.

[0051] The present invention also provides a pharmaceutical composition comprising the above-described interfering RNA and a pharmaceutically acceptable carrier or excipient. In one specific embodiment of the present invention, the pharmaceutical composition is used to inhibit TGF-β1 gene expression.

[0052] In one specific embodiment of the present invention, the carrier in the pharmaceutical composition is a mannan-polysaccharide coupling compound.

[0053] The present invention further provides the use of the interfering RNA or the above-described pharmaceutical composition in the preparation of medicaments for the treatment and / or prevention of diseases related to transforming growth factor TGF-β1 gene expression; said diseases include, but are not limited to, liver fibrosis, pulmonary fibrosis, osteoarthritis, and diabetic nephropathy.

[0054] The present invention further provides an siRNA pharmaceutical composition for the synergistic treatment of idiopathic pulmonary fibrosis, the siRNA pharmaceutical composition for the synergistic treatment of idiopathic pulmonary fibrosis comprising interfering RNA that effectively inhibits the expression of transforming growth factor TGF-β1 gene and siRNA (siGAS6) that effectively inhibits the expression of GAS6 gene.

[0055] The siRNA (siGAS6) that effectively inhibits GAS6 gene expression can effectively suppress the expression of the Gas6 gene. GAS6 (Growth Arrest-Specific Protein 6) is a secreted 75kDa glycoprotein whose function depends on vitamin K. It is expressed in various cell types and tissues, such as the heart, lungs, stomach, kidneys, pancreas, bone marrow, central nervous system, and intestines. The involvement of the Gas6 / TAM signaling pathway has been associated with the progression of fibrosis in multiple organ regions, including the lungs, kidneys, intestines, and liver. In addition, some literature reports that Gas6 inhibits the induction of TNF-α through Mer and Axl receptors on microglia and promotes the expression of repair-related factors IL-10 and TGF-β. Currently, a strategy of synergistically treating idiopathic pulmonary fibrosis by co-delivering two interfering RNAs, siTGF-β1 and siGAS6, via nebulized inhalation has not been reported.

[0056] In one embodiment of the present invention, the siRNA drug composition for synergistic treatment of idiopathic pulmonary fibrosis contains siTGF-β1 and siGAS6 in an equimolar ratio.

[0057] In one embodiment of the invention, the siRNA pharmaceutical composition for synergistic treatment of idiopathic pulmonary fibrosis further includes a pharmaceutically acceptable carrier or excipient.

[0058] In one specific embodiment of the present invention, the carrier in the siRNA drug composition for synergistic treatment of idiopathic pulmonary fibrosis is a mannan-polysaccharide coupling compound.

[0059] Mannan is a polysaccharide composed of D-mannose molecules linked by β-1,4 or β-1,6 glycosidic bonds. Mannan can achieve targeted delivery to specific cell types by recognizing specific receptors on the surface of macrophages (such as mannose receptors). In IPF, macrophages play a crucial role in inflammation and fibrosis; therefore, mannan-modified nanoparticles can effectively deliver nucleic acids to these cells, enhancing therapeutic efficacy. Furthermore, as a natural polysaccharide, mannan exhibits good biocompatibility and is unlikely to induce immune responses or toxicity, making it an ideal biomaterial. Its physicochemical properties can be modulated by altering its molecular weight, structure, or binding with other substances to meet diverse drug delivery requirements. Based on this, this application provides a mannan-polysaccharide coupling compound as a carrier.

[0060] In one specific embodiment of the present invention, the grafting degree of the polypeptide in the mannan-peptide coupler is 5%-80%.

[0061] In one specific embodiment of the present invention, the polypeptide in the mannan-peptide coupler comprises one or more of the following structures:

[0062]

[0063]

[0064] Where x, y, and z are selected from integers from 1 to 10.

[0065] In one specific embodiment of the present invention, the mannan in the mannan-peptide coupler has the following structure:

[0066] Where n is an integer between 1 and 500.

[0067] In one specific embodiment of the present invention, the method for preparing the mannan-peptide coupler includes the following steps:

[0068] Mannan modified with double bonds is prepared by reacting mannan with molecules containing double bonds, and then mannan-peptides are prepared by the addition reaction of double bond-modified mannan with peptides or the Thiol-ene reaction.

[0069] In one specific embodiment of the present invention, the molecule with the double bond includes the following molecular structure:

[0070]

[0071] The double-bond modified mannans described herein include the following structures:

[0072]

[0073] The present invention further provides a siTGF-β1 / siGAS6 co-delivery system for nebulized inhalation in the synergistic treatment of idiopathic pulmonary fibrosis, namely, a mannan-peptide coupling system of siTGF-β1 and siGAS6, which can also be expressed as a mannan-peptide coupling system of siTGF-β1 and siGAS6, or a mannan-peptide / siTGF-β1 / siGAS6 co-delivery system, which is a combination of siRNA drug composition and mannan-peptide coupling for the synergistic treatment of idiopathic pulmonary fibrosis.

[0074] This invention further provides a method for preparing a co-delivery system of siTGF-β1 and siGAS6 mannan-peptide coupling, comprising the following steps: mixing siTGF-β1 and siGAS6 in equimolar amounts to prepare a nucleic acid storage solution with a certain phosphorus (P) concentration; simultaneously preparing a mannan-peptide storage solution with a certain nitrogen (N) concentration; vortex mixing the two storage solutions at a certain N / P ratio and incubating at room temperature for 30-60 min; the resulting nanocomposite is the siTGF-β1 and siGAS6 mannan-peptide coupling co-delivery system. The P and N concentrations can be the same or different.

[0075] This invention also provides the application of a mannan-peptide co-delivery system for siTGF-β1 and siGAS6 in the preparation of a medicament. The medicament is, but is not limited to, a treatment for idiopathic pulmonary fibrosis, and may also be used to treat other diseases characterized by TGF-β1 and GAS6 overexpression.

[0076] Preferably, the drug for treating idiopathic pulmonary fibrosis is a drug that can be inhaled via nebulization.

[0077] The innovation of this application lies in:

[0078] This invention designs a specific siRNA (siTGF-β1) targeting the mRNA of transforming growth factor TGF-β1, and its delivery to target cells can inhibit the expression of transforming growth factor TGF-β1.

[0079] The present invention further combines the siTGF-β1 with siRNA (siGAS6) that effectively inhibits the expression of the GAS6 gene to form an siRNA drug composition for the synergistic treatment of idiopathic pulmonary fibrosis.

[0080] This invention also provides a mannan-peptide coupler and its preparation method.

[0081] The present invention further combines the siRNA drug composition for the synergistic treatment of idiopathic pulmonary fibrosis with a mannan-peptide coupler to form a mannan-peptide coupler co-delivery system of siTGF-β1 and siGAS6 (i.e., mannan-peptide / siTGF-β1 / siGAS6 co-delivery system).

[0082] The siTGF-β1 and siGAS6 mannan-peptide co-delivery system provided by the present invention can be used to prepare therapeutic drugs for idiopathic pulmonary fibrosis, especially the therapeutic drug for idiopathic pulmonary fibrosis is a nebulized inhalation drug.

[0083] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0084] The siRNA drug composition for synergistic treatment of idiopathic pulmonary fibrosis provided by this invention has excellent TGF-β1 gene expression inhibition activity and GAS6 gene expression inhibition activity, and has good potential for treating diseases related to TGF-β1 and GAS6 gene expression. The mannan-peptide / siTGF-β1 / siGAS6 co-delivery system provided by this invention can achieve synergistic treatment of idiopathic pulmonary fibrosis through nebulized inhalation.

[0085] This technical solution can efficiently target lung macrophages and produce a synergistic therapeutic effect, achieving highly effective treatment for idiopathic pulmonary fibrosis. After siTGF-β1 is delivered to the target cells, it can effectively inhibit the expression of the TGF-β1 gene, which can be used to regulate the inflammatory process in the target tissue. This is of great significance for the preparation of drugs to treat diseases characterized by increased expression of transforming growth factor TGF-β1. The siGAS6 can effectively inhibit the expression of the Gas6 gene, which can be used to regulate inflammation in the target tissue. This is also of great significance for the preparation of drugs to treat diseases characterized by increased Gas6. The mannan-peptide co-delivery system can co-deliver siTGF-β1 and siGAS6 via nebulized inhalation, efficiently targeting lung macrophages and achieving synergistic treatment for idiopathic pulmonary fibrosis. Attached Figure Description

[0086] Figure 1 Mass spectra of transforming growth factor siRNA (siTGF-T1);

[0087] Figure 2 Mass spectra of transforming growth factor siRNA (siTGF-T2);

[0088] Figure 3 Mass spectrometry of transforming growth factor siRNA (siTGF-T3);

[0089] Figure 4 Mass spectra of transforming growth factor siRNA (siTGF-T4);

[0090] Figure 5 Mass spectrometry of transforming growth factor siRNA (siTGF-T5);

[0091] Figure 6 Mass spectra of transforming growth factor siRNA (siTGF-T6);

[0092] Figure 7 Mass spectra of transforming growth factor siRNA (siTGF-T7);

[0093] Figure 8Mass spectrometry of negative control siRNA (siNC);

[0094] Figure 9 The relative expression level of the TGF-β1 encoding gene in cells after using TGF-β1 siRNA in each group.

[0095] Figure 10 Cell survival rate after using TGF-β1 siRNA in each group.

[0096] Figure 11 The relative expression level of the TGF-β1 encoding gene in cells after using chemically modified transforming growth factor TGF-β1 siRNA.

[0097] Figure 12 NMR spectrum of mannan-peptide coupler.

[0098] Figure 13 : Particle size potential before and after atomization of the co-delivery system.

[0099] Figure 14 : Cytotoxicity of the delivery system.

[0100] Figure 15 The knockdown efficiency of TGF-β1 and GAS6 genes in the co-delivery system.

[0101] Figure 16 : In vivo biological distribution of the delivery system.

[0102] Figure 17 : The residence time of the delivery system in the lungs. Detailed Implementation

[0103] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0104] The described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the protection scope of the present invention.

[0105] Reagents or instruments used in the examples:

[0106] Human mononuclear cells THP-1 were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee, catalog number: SCSP-567;

[0107] Lipofectamine RNAi MAX TM The transfection reagent was purchased from Invitrogen (Shanghai) Trading Co., Ltd., item number: 13778075;

[0108] The RNA rapid extraction kit was purchased from Tianweng Biotechnology (Guangzhou) Co., Ltd., item number: 400-100;

[0109] The HiScript II One Step qRT-PCR SYBR Green Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: Q221-01.

[0110] PKC activator PMA / TPA was purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number: S1819;

[0111] In the examples, siTGF represents the siRNA sequence targeting transforming growth factor TGF-β1; siNC represents the negative control sequence.

[0112] Example 1: siRNA Design and Synthesis

[0113] Seven pairs of siRNA sequences were designed based on the mRNA sequence encoding transforming growth factor TGF-β1 (GenBank: NM_000660.7). The sequence information is shown in Table 1. In actual use, dT / dT dangling bases were added to the 3' end of the sense and / or antisense strands of the siRNA. Mass spectrometry detection results of the siRNA are shown in Table 1. Figure 1-7 .

[0114] from Figure 1-7 The following conclusions can be drawn: the spectrum shows a single, sharp peak, with no obvious impurities or degradation products, indicating that the target siRNA was successfully synthesized with high purity.

[0115] Table 1: Designed TGF-β1 siRNA sequences

[0116]

[0117] The above sequences were synthesized by Nanjing Genscript Biotech Co., Ltd. During synthesis, a dTdT dangling base was added to the 3' end. A negative control siRNA sequence (siNC) was also synthesized. The sense strand was UUCUCCGAACGUGUCACGUdTdT (SEQ ID NO:15), and the antisense strand was ACGUGACACGUUCGGAGAAdTdT (SEQ ID NO:16). The siNC mass spectrometry results are as follows: Figure 8 .from Figure 8 The following conclusions can be drawn: the spectrum shows a single, sharp peak shape, which matches the molecular weight of the target siNC and has high purity, proving the successful synthesis of siNC.

[0118] Example 2: Inhibitory effect of interfering RNA on the gene encoding transforming growth factor TGF-β1

[0119] To detect the inhibitory effect of siRNAs targeting transforming growth factor TGF-β1 mentioned in this invention, seven siRNAs were prepared targeting TGF-β1 (Example 1). In cell experiments, the prepared siRNAs were first delivered to cultured cells. Cell samples were collected and total RNA was extracted at a predetermined time after transfection. Ct values ​​were obtained using RT-qPCR. By standardizing the data from each group, the relative expression of TGF-β1 mRNA was obtained, thereby comparing the inhibitory effect of each siRNA on TGF-β1 mRNA expression.

[0120] 1. Cell culture and transfection

[0121] The inhibitory effect of siRNA was verified by using THP-1 cells (human monocytes) induced by phorbol ester to form macrophages.

[0122] (1) THP-1 cells were cultured in RPMI 1640 medium (Gibco, catalog number C11875500BT) containing 10% FBS (Gibco, catalog number A5669701), with 100 M / mL Penicillin and 100 μg / mL Streptomycin added; cultured at 37℃ in a 5% CO2 saturated humidity incubator. 48 h before transfection, 100 ng / mL phorbol ester (also known as PMA or TPA) was added to induce macrophage formation. Cells were cultured at a ratio of 1.5 x 10⁻⁶ cells / mL. 5 / well inoculated into a 24-well plate.

[0123] (2) Take 50 μL of opti-MEM medium (Gibco, catalog number 31985070) and dilute 1 μL of each group of siTGFβ1 (the concentration after dilution is 0.2 μM) or siNC (the concentration after dilution is 0.2 μM). Take 50 μL of opti-MEM medium and dilute 3 μL of Lipofectamine RNAiMAX transfection reagent. Mix the diluted siRNA and transfection reagent of each group separately, incubate for 5 min before use, and set up a blank group.

[0124] (3) Two hours before the experiment, the cells in the 24-well plate were changed to antibiotic-free 1640 complete medium, 450 μL per well. During the experiment, 50 μL of each mixture from step (2) was added to each well, so that the final volume of each well was 500 μL. The final concentration of siRNA transfection in each well was 10 nM.

[0125] (4) 24 hours after transfection, the well plate was removed from the 37°C, 5% CO2 saturated humidity incubator, and the cells were collected to extract RNA for subsequent detection of the expression level of the target gene.

[0126] 2. Extraction of total RNA from cells

[0127] (1) Cell RNA was extracted according to the standard operating instructions provided with the Cell RNA Rapid Extraction Kit manufactured by Albatross Biotechnology (Guangzhou) Co., Ltd. In short, after washing with PBS, 500 μL of the lysis buffer provided in the kit was added to each well, and the cells were thoroughly lysed by pipetting 10 times. Then, 500 μL of anhydrous ethanol was added, and the cells were mixed by pipetting 10 times. The mixed liquid was then transferred to an RNA purification column and centrifuged at 12000 x g for 1 min. After discarding the filtrate, 500 μL of washing buffer was added, and the cells were centrifuged at 12000 x g for 1 min. After discarding the filtrate, the empty tube was centrifuged at 12000 x g for 1 min. Finally, 30 μL of elution buffer was added, and the tube was allowed to stand for 2 min before centrifuging in a new EP tube to collect the purified RNA.

[0128] (2) RNA quality control, using NanoQuant Plate TM RNA purity was determined by 1% agarose denaturing gel electrophoresis to assess RNA integrity.

[0129] 3. RT-qPCR detection process

[0130] Using total RNA extracted from the samples as a template, the reaction system shown in Table 2 was established using the Novizan RT-qPCR one-step kit:

[0131] Table 2: One-step RNA RT-qPCR system

[0132] reagents Added amount 2x One Step Sybr Green Mix 10μL One Step SYBR Green Enzyme Mix 1μL Gene Specific Primer Forward(10μM) 0.4μL Gene Specific Primer Reverse(10μM) 0.4μL Template RNA 2μL Rnase-Free Water 6.2μL total 20μL

[0133] The primer sequences used in RT-qPCR are shown in Table 3. The primer sequences were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0134] Table 3: Primer sequences

[0135]

[0136] Perform PCR reverse transcription and amplification according to the procedure in Table 4.

[0137] Table 4: PCR Reverse Transcription and Amplification Procedures

[0138]

[0139] 4. Inhibition effect

[0140] Using GAPDH as the housekeeping gene, the relative expression level of TGF-β1 mRNA was calculated using the ΔΔCt method; the mRNA expression level of each group was standardized with the Blank group expression level as 100%.

[0141] The relative expression levels of mRNA in each group are shown in Table 5. Figure 9 .

[0142] Table 5: Relative expression levels of TGF-β1 mRNA

[0143]

[0144] Note: siNC in the table is the negative control.

[0145] Table 5 Figure 9 The results showed that, compared with the blank and siNC groups, the relative expression level of TGF-β1 mRNA in cells treated with the siRNA involved in this invention decreased significantly at 24 h after transfection, indicating that the screened TGF-β1 siRNA has a significant and sustained inhibitory effect on TGF-β1 gene expression. Among them, the siTGF-T3 group showed the best inhibitory effect.

[0146] Example 3: Cytotoxicity experiment of interfering RNA.

[0147] The THP-1 cell culture procedure is the same as in Example 2 (1), with PMA / PTA-induced THP-1 cells cultured at a density of 2*10 cells per well. 4 Each plate was seeded and incubated in a 37°C, 5% CO2, saturated humidity incubator. Two hours before transfection, the culture medium in each well was replaced with 90 μL of antibiotic-free medium. The transfection reagents for each group were prepared according to the Lipofectamine RNAiMAX instructions, as described in Example 2 (2). 10 μL of the prepared transfection reagent was added to each well, and the plate was placed in a 37°C, 5% CO2, saturated humidity incubator. 24 hours after transfection, 10 μL of CCK-8 solution was added to each well, and the plate was incubated for another hour. Infinite E Plex multi-functional microplate reader for absorbance (A) detection 450 -A 630 Using the Blank group as the standard and cell-free blank wells as the background, the cell viability of each group was calculated.

[0148] Cell viability% = 100 * (A 实验 -A 空白 ) / (A Blank -A 空白 )

[0149] The results are shown in Table 6. Figure 10 .

[0150] Table 6: Cell viability of each group after TGF-βsiRNA treatment

[0151]

[0152] Note: siNC in the table is the negative control.

[0153] Differences were analyzed using one-way ANOVA, Table 6. Figure 10 The results showed that, compared with siNC, the siRNA involved in this invention did not show a significant difference in cell survival rate, and it can be considered that the siRNA involved in this invention is non-cytotoxic.

[0154] Example 4: siRNA sequence modification and evaluation of biological activity

[0155] 1. siTGF-T3 sequence modification

[0156] The siTGF-T3 sequence was selected, and three sequences with different modifications were designed by adding methylation, fluorination, and ethylene glycol nucleic acid substitution, as shown in Table 7. The modified sequences were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0157] Table 7: Chemical modifications of the siTGF-T3 sequence.

[0158]

[0159] Note: In the table, f represents 2'-fluorination modification, m represents 2'-O methylation modification, and iGNA represents ethylene glycol nucleic acid.

[0160] 2. Evaluation of the biological activity of the modified nucleic acid

[0161] The biological activity of the modified nucleic acid was detected by RT-qPCR, following the same experimental protocol as in Examples 2 and 3. The results are shown in Table 8. Figure 11 Table 8 Figure 11 The results showed that the modified siTGF-T3 sequence exhibited similar or better inhibition of transforming growth factor TGF-β1 mRNA expression as the unmodified siTGF-T3 sequence.

[0162] Table 8: mRNA expression level of TGF-β1 in the chemically modified siTGF-T3 group.

[0163]

[0164] Example 5: Preparation of a mannan-peptide / siTGF-β1 / siGAS6 co-delivery system

[0165] Using mannan (70 kDa) as the backbone and a 17% grafting degree of peptide R3H7C (Man-A-R3) as an example (structure shown below), a stock solution with a certain nitrogen concentration was prepared, where N comes from the amino group of the guanidine group on arginine. On the other hand, siTGF-β1 (sequence shown in Table 9, siGAS6 positive strand as shown in SEQ ID NO. 21, siGAS6 antisense strand as shown in SEQ ID NO. 22) with the sequence siTGF-T3R1 were prepared into a stock solution with a phosphorus concentration of 7 pmol / μl, where P comes from the phosphate group in the nucleic acid. The mannan-peptide stock solution and the two nucleic acids (molar ratio of siTGF-β1 and siGAS6 of 1:1) were mixed at a certain N / P ratio (5 or 10), vortexed, and incubated at room temperature for at least 30 min to obtain mannan-peptide / siTGF-β1 / siGAS6 nanocomposites with uniform particle size.

[0166] The mannan-peptide structure is shown below:

[0167]

[0168] NMR spectra of mannan-peptide couples are as follows Figure 12 As shown, it can be seen that the mannose-peptide Man-A-R3 with a grafting degree of 17% was successfully synthesized.

[0169] Table 9: siGAS6 sequence.

[0170]

[0171] Example 6: Physicochemical properties of the mannan-peptide / siTGF-β1 / siGAS6 co-delivery system were characterized by using dynamic light scattering instruments to characterize the particle size and potential of the mannan-peptide / siTGF-β1 / siGAS6 co-delivery system (the molar ratio of mannan-peptide (N), siTGF-β1 (P), and siGAS6 (P) was 5 / 0.5 / 0.5), as well as the stability of the nanocomposite before and after atomization.

[0172] In this embodiment, the selection of mannan-peptide, siTGF-β1, and siGAS6 is the same as in Example 5.

[0173] Particle size potentials of the co-delivery system before and after atomization, as follows Figure 13 As shown, Figure 13In Example 5, the particle size of the co-delivery system with N / P = 5 before and after atomization is shown. "Prenebμlzed" refers to the particle size before atomization, and "Postnebμlzed" refers to the particle size after atomization. M-AI-R3H7 is a mannan-peptide coupler; where Man refers to mannan, AI refers to the linker compound 3-propenyl isocyanate between mannan and the peptide, and R3H7 refers to the peptide.

[0174] from Figure 13 The following conclusions can be drawn: the particle size, distribution and potential of the Man-AI-R3H7 / siRNA delivery system remained basically unchanged before and after physicochemical treatment, which indicates the excellent anti-shear properties of the mannan-peptide coupling delivery system and its potential for atomized drug delivery.

[0175] Example 7: Cytotoxicity experiment of mannan-peptide / siTGF-β1 / siGAS6 co-delivery system.

[0176] The THP-1 cell culture procedure is the same as in Example 2 (1), with PMA / PTA-induced THP-1 cells cultured at a density of 2*10 cells per well. 4 Each plate was seeded and incubated in a 37°C, 5% CO2, saturated humidity incubator. Two hours before transfection, the medium in each well was replaced with 90 μL of antibiotic-free medium. Mannan-peptide / siTGF-β1 / siGAS6 nanocomplex and positive control RNAiMAX / siTGF-β1 / siGAS6 were added to each well, and the plates were placed in a 37°C, 5% CO2, saturated humidity incubator. The positive control RNAiMAX / siTGF-β1 / siGAS6 was prepared according to the protocol provided by RNAiMAX. 50 μL of opti-MEM medium (Gibco, catalog number 31985070) was diluted with 1 μL of diluted siTGF (siTGF-T1, siTGF-T2, siTGF-T3, siTGF-T4, siTGF-T5, siTGF-T6, siTGF-T7 from Example 1) or siGAS6 (stock solution concentration 10 μM). 50 μL of opti-MEM medium was diluted with 3 μL of Lipofectamine RNAiMAX transfection reagent. The diluted siRNA and transfection reagent were mixed thoroughly, and the mixture was incubated for 5 min before use. A blank control group was also included. 24 h after transfection, 10 μL of CCK-8 solution was added to each well, and the mixture was incubated for another 1 h. The absorbance (A450-A630) was measured using the Infinite E Plex multi-plate reader. Cell viability was calculated for each group using the Blank group as the standard and cell-free blank wells as the background.

[0177] Cell viability % = 100 * (Experiment A - Blank A) / (A) Blank -A blank)

[0178] In preparing the mannan-peptide / siTGF-β1 / siGAS6 nanocomplex and the positive control RNAiMAX / siTGF-β1 / siGAS6, the selection and preparation method of mannan-peptide in Example 5 were followed, and the N / P ratios were prepared as 2, 5, 10, 15, 20, 25, and 30, respectively.

[0179] Figure 14 In this context, Man-AI-R3H7C refers to a mannan-peptide coupler, where Man refers to mannan, AI refers to the linker compound 3-propenyl isocyanate between mannan and the peptide, and R3H7 refers to the peptide.

[0180] The results are as follows Figure 14 As shown, this co-delivery system exhibits low cytotoxicity.

[0181] Example 8: Characterization of the release performance of the mannan-peptide / siTGF-β1 / siGAS6 co-delivery system

[0182] The release performance of the mannan-peptide / siTGF-β1 / siGAS6 co-delivery system was characterized by qRT-PCR. Mouse alveolar macrophages (MHS) were used at a concentration of 1*10-1 5 The samples were seeded at a density of 1000 μL in 24-well plates and incubated at 37°C for 24 hours. Then, mannan-peptide / siTGF-β1 / siGAS6 nanocomplex (prepared by the method in Example 5 with an N / P ratio of 5) and positive control RNAiMAX / siTGF-β1 / siGAS6 (prepared by the method in Example 7) were added. After 8 hours of incubation, the culture medium was replaced with fresh medium and the samples were incubated for another 16 hours. The expression of TGF-β1 and GAS6 was detected according to the above RT-qPCR detection procedure.

[0183] The results of TGF-β1 and GAS6 knockdown are shown in Figure 15 , Figure 15In the results, M-AI-R3H7 / siNC refers to the complex formed by M-AI-R3H7 and the control siRNA (siNC) at N / P = 5 / 1; M-AI-R3H7 / siTGF-β1 refers to the complex formed by M-AI-R3H7 and siTGF-β1 at N / P = 5 / 1; MA-R3H7 / siGAS6 refers to the complex formed by M-AI-R3H7 and siGAS6 at N / P = 5 / 1; and M-AI-R3H7 / siTGF-β1 / siGAS6 refers to the complex formed by M-AI-R3H7, siTGF-β1, and siGAS6 at N / P / P = 5 / 0.5 / 0.5. The results indicate that co-delivery of TGF-β1 and GAS6 has a good synergistic effect.

[0184] Example 9: Biodistribution of Mannan-Peptide Co-delivery System in Vivo

[0185] To determine whether the mannan-peptide delivery system can protect nucleic acids from rapid lung degradation after nebulized drug administration, a co-delivery system containing Cy5.5 labeling, Man-AI-R5H5 / siNC-Cy5.5 / siNC, was prepared using mannan-peptide M-AI-R5H5 as an example. M-AI-R5H5 refers to a mannan-grafted peptide with the amino acid sequence R5H5. The Cy5-labeled M-AI-R5H5 / siNC-Cy5.5 / siNC was nebulized into the lungs of mice, and organs were harvested and immediately imaged at 2, 4, 8, 24, 48, and 72 hours. The preparation method of the M-AI-R5H5 / siNC-Cy5.5 / siNC complex is as follows: M-AI-R5H5 was prepared into a solution containing 10 mM nitrogen, and siNC-Cy5.5 was prepared into a solution containing 300 μM phosphorus (P). Then, 3.75 μL of the M-AI-R5H5 / siNC-Cy5.5 solution was mixed with 25 μL of the siNC-Cy5.5 solution at an N / P ratio of 5, and incubated at room temperature for 30 min. Figure 16 As can be seen, the lungs exhibit the strongest fluorescence signal, while other tissues show almost no fluorescence signal, indicating that M-AI-R5H5 / siNC-Cy5.5 / siNC is mainly distributed in the lungs, demonstrating good targeting ability. Figure 17 As shown, the M-AI-R5H5 / siNC-Cy5.5 / siNC complex still exhibited uniform fluorescence signals in the lung lobes 72 hours after nebulized administration, while the fluorescence signal of siRNA began to decay 6 hours after nebulized administration and completely disappeared after 24 hours. Experimental results ( Figure 17 This indicates that the M-AI-R5H5 material can effectively protect siRNA from rapid degradation in the lungs, thereby maintaining its therapeutic effect.

[0186] Example 10: Mannan-peptide / siTGF-β1 / siGAS6 co-delivery system for in vivo treatment of idiopathic pulmonary fibrosis

[0187] To investigate the effect of the mannan-peptide / TGF-β1 / siGAS6 complex (prepared as in Example 5) on the treatment of pulmonary fibrosis in mice, a bleomycin-induced pulmonary fibrosis model was constructed (3.0 mg / kg). Seven days after bleomycin administration, mice were randomly divided into 5 groups. Mice were administered the drug twice daily (every 3 days) via inhalation, with each dose containing 0.5 mg / kg of siRNA. The mannan-peptide / siTGF-β1 / siGAS6 complex significantly reduced TGF-β expression in the lung tissue of pulmonary fibrosis mice.

[0188] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An interfering RNA effective to inhibit expression of a transforming growth factor TGF-β1 gene, characterized by, The interfering RNA comprises a nucleotide sequence as shown in any one or more than two of SEQ ID NO: 1-14.

2. The interfering RNA according to claim 1, which is effective in inhibiting the expression of a transforming growth factor β1 (TGF-β1) gene, wherein the interfering RNA comprises a nucleotide sequence of SEQ ID NO:

1. The interfering RNA comprises a sense strand and / or an antisense strand; The sense strand comprises a nucleotide sequence as shown in any one or more than two of SEQ ID NO: 1-7; The antisense strand comprises a nucleotide sequence as shown in any one or more than two of SEQ ID NO: 8-14.

3. The interfering RNA according to claim 1, which is effective in inhibiting the expression of a transforming growth factor β1 (TGF-β1) gene, wherein the interfering RNA comprises a nucleotide sequence of SEQ ID NO:

1. The interfering RNA is selected from any one of the following groups: A) the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 1, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 8; B) the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 2, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 9; C) the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 3, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 10; D) the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 4, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 11; E) the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 5, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 12; F) the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 6, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 13; G) the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 7, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO:

14.

4. The interfering RNA for effectively inhibiting the expression of transforming growth factor TGF-β1 gene according to claim 1, characterized in that, The interfering RNA further comprises overhang bases; Preferably, the interfering RNA comprises 1-10 overhang bases, and more preferably 2-4 overhang bases; Preferably, the overhang bases are located at the end of the sense strand and / or the antisense strand of the interfering RNA; Preferably, the overhang bases are deoxynucleosides; More preferably, the overhang bases are dTdT, dTdC or dUdU.

5. The interfering RNA for effectively inhibiting the expression of transforming growth factor TGF-β1 gene according to claim 1, characterized in that, The interfering RNA further comprises at least one modification; The modification comprises a modification on the base, sugar ring and / or phosphate backbone, or the modification further comprises a nucleotide with inosine, queosine, xanthine, 2'-methylribose, non-natural phosphodiester bond and / or peptide; Preferably, the modification on the base comprises a 5-position pyrimidine modification, 8-position purine modification, pseudouridine modification and / or 5-bromouracil substitution; Preferably, the modification on the sugar ring is selected from the group consisting of 2'-OH being substituted by H, OZ, halo, SH, SZ, NH2, NHZ, NZ2 or CN, wherein Z is an alkyl group; Preferably, the modification on the phosphate backbone is selected from the group consisting of phosphorothioate modification; The alkyl group represents a linear or branched hydrocarbon chain radical, which does not contain unsaturated bonds, and which is connected to the rest of the molecule by a single bond.

6. The interfering RNA for effectively inhibiting the expression of transforming growth factor TGF-β1 gene according to claim 1, characterized in that, The interfering RNA comprises one or more than two combinations of siRNA, dsRNA, shRNA, aiRNA or miRNA; Preferably, the interfering RNA is siRNA.

7. A pharmaceutical composition, characterized by, It comprises the interfering RNA of any one of claims 1-6, and a pharmaceutically acceptable carrier or adjuvant.

8. Use of the interfering RNA of any one of claims 1-6 or the pharmaceutical composition of claim 7 in the preparation of a medicament for treating and or / preventing a disease associated with the expression of the transforming growth factor TGF-β1 gene; the disease is selected from liver fibrosis, pulmonary fibrosis, osteoarthritis or diabetic nephropathy.

9. An siRNA pharmaceutical composition for use in the co-treatment of idiopathic pulmonary fibrosis, characterized in that, The siRNA pharmaceutical composition for synergistically treating idiopathic pulmonary fibrosis comprises the interfering RNA of any one of claims 1-6, and an siRNA effective for inhibiting the expression of the GAS6 gene.

10. The siRNA pharmaceutical composition for use in the co-treatment of idiopathic pulmonary fibrosis according to claim 9, characterized in that, One or more of the following situations are selected: (1) In the siRNA pharmaceutical composition for synergistically treating idiopathic pulmonary fibrosis, the interfering RNA is in equimolar ratio with siGAS6. (2) The siRNA pharmaceutical composition for synergistically treating idiopathic pulmonary fibrosis further comprises a pharmaceutically acceptable carrier or adjuvant.

11. The siRNA pharmaceutical composition for use in the co-treatment of idiopathic pulmonary fibrosis according to claim 9 or 10, characterized in that, The carrier in the siRNA pharmaceutical composition for synergistically treating idiopathic pulmonary fibrosis is a mannan-polysaccharide conjugate.

12. The siRNA pharmaceutical composition for use in the co-treatment of idiopathic pulmonary fibrosis according to claim 11, characterized in that, The mannan-polysaccharide conjugate is selected from one or more of the following situations: (1) The grafting degree of the polypeptide in the mannan-polypeptide conjugate is 5%-80%. (2) The polypeptide in the mannan-polypeptide conjugate comprises one or more of the following structures: wherein x, y, z are independently selected from integers of 1-10. (3) The mannan in the mannan-polypeptide conjugate is the following structure: wherein n is an integer between 1 and 500.

13. The siRNA pharmaceutical composition for use in the co-treatment of idiopathic pulmonary fibrosis according to claim 11, characterized in that, The preparation method of the mannan-polysaccharide conjugate comprises the following steps: Preparation of double bond modified mannan by reaction of mannan with a double bond containing molecule, followed by Michael addition reaction or Thiol-ene reaction of the double bond modified mannan with a polypeptide to prepare a mannan-polypeptide; The double bond containing molecule comprises the following molecular structure: The double bond modified mannan comprises the following structure:

14. A system for co-delivery of siTGF-β1 and siGAS6 with a mannan-polypeptide conjugate, characterized in that, It is the combination of the siRNA pharmaceutical composition for synergistically treating idiopathic pulmonary fibrosis and the mannan-polypeptide conjugate.

15. The method of producing a mannan-polypeptide conjugate co-delivery system of siTGF-βl and siGAS6 of claim 14, characterized in that, Comprising the following steps: Mix siTGF-β1 and siGAS6 in equimolar ratio to prepare a nucleic acid storage solution with a certain P concentration, and prepare a mannan-polypeptide storage solution with a certain N concentration, vortex mix the above two storage solutions according to a certain N / P and incubate at room temperature for 30-60 min to form a nanocomposite, which is a mannan-polypeptide conjugate co-delivery system of siTGF-β1 and siGAS6, wherein the P concentration and the N concentration can be the same or different.

16. Use of the mannose-polypeptide conjugate co-delivery system of siTGF-βl and siGAS6 of claim 14 for the preparation of a medicament, characterized in that, The drug is a therapeutic drug for diseases characterized by overexpression of TGF-β1 and GAS6.

17. Use of the siTGF-βl and siGAS6 mannan-polypeptide conjugate co-delivery system according to claim 16 for the preparation of a medicament, characterized in that, The drug is a drug for treating idiopathic pulmonary fibrosis.

18. Use of the siTGF-βl and siGAS6 mannan-polypeptide conjugate co-delivery system according to claim 17 for the preparation of a medicament, characterized in that, The drug for treating idiopathic pulmonary fibrosis is an aerosol inhalation drug.

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