Targeting polymer vesicle modified by cRGD peptide as well as preparation method and application of targeting polymer vesicle

Abstract

The invention relates to a cRGD peptide modified targeting polymer vesicle as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention provides a targeting polymer vesicle modified by cRGD peptide. The targeting polymer vesicle modified by the cRGD peptide is prepared from the following components: a targeting polymer cRGD-PEG-P (TMC-co-DTC), a carrier polymer PEG-P (TMC-co-DTC)-PEI1200 and a siKRAS medicine. The cRGD peptide modified targeted polymer vesicles can efficiently load siKRAS drugs, effective release of the siKRAS drugs in cells is achieved, enrichment and internalization of the vesicles in tumor sites in vivo are effectively improved, tumor growth of a KRAS G12D mutation pancreatic cancer model is well inhibited, the lifetime of model mice is remarkably prolonged, and the cRGD peptide modified targeted polymer vesicles are free of remarkable toxicity and can be applied to preparation of drugs for treating pancreatic cancer. The compound has a great application prospect in preparation of targeted therapeutic drugs for cancers caused by KRAS mutation.

Description

Technical Field

[0001] The present invention relates to a cRGD peptide-modified targeted polymeric vesicle, a preparation method thereof, and an application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Pancreatic cancer is one of the common malignant tumors of the digestive tract. Its clinical manifestations depend on the location of the cancer, the stage of the disease, the presence or absence of metastasis, and the involvement of adjacent organs. Its clinical characteristics are a short overall course, rapid disease progression, and rapid deterioration. The most common clinical symptoms are upper abdominal fullness and discomfort, pain. Currently, gemcitabine is the first-line drug for the clinical treatment of pancreatic cancer. However, drug resistance and easy recurrence seriously affect the clinical effect of using gemcitabine to treat pancreatic cancer. Therefore, it is urgent to find drugs with better effects for treating pancreatic cancer or drugs that can synergistically enhance the sensitivity of gemcitabine to treat pancreatic cancer.

[0003] Research shows that 90% of pancreatic cancers are caused by KRAS mutations. At the same time, research shows that siRNA, as an endogenous regulatory mechanism, can inhibit the expression of mutant KRAS protein by silencing the upstream mRNA. Moreover, siRNA drugs do not have the problems of drug resistance and easy recurrence. Therefore, silencing key genes or drug-resistant genes in cancer growth through siRNA drugs is one of the current research trends in the development of drugs for treating pancreatic cancer. However, the dense fibrotic stroma composed of stromal cells and extracellular matrix in pancreatic cancer will seriously hinder the delivery and penetration of siRNA drugs into pancreatic cancer cells. Therefore, it is urgent to find a method for efficiently delivering siRNA drugs to pancreatic cancer cells so that the siRNA drug treatment of pancreatic cancer can be realized. Summary of the Invention

[0004] To solve the above problems, the present invention provides a cRGD peptide-modified targeted polymeric vesicle, and the components of the cRGD peptide-modified targeted polymeric vesicle include a targeted polymer cRGD-PEG-P(TMC-co-DTC), a carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 and an siKRAS drug.

[0005] In an embodiment of the present invention, in the cRGD peptide-modified targeted polymeric vesicle, the concentration of the targeted polymer cRGD-PEG-P(TMC-co-DTC) is 0.5 - 0.8 mg / mL, and the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 has a concentration of 3 - 5 mg / mL, and the siKRAS drug has a concentration of 0.2 - 1 mg / mL.

[0006] In an embodiment of the present invention, in the cRGD peptide-modified targeted polymeric vesicles, the concentration of the targeted polymer cRGD-PEG-P(TMC-co-DTC) is 0.6 mg / mL, and the carrier polymer is PEG-P(TMC-co-DTC)-PEI 1200 has a concentration of 0.6 mg / mL, and the concentration of the siKRAS drug is 3.4 mg / mL.

[0007] In an embodiment of the present invention, the components of the cRGD peptide-modified targeted polymeric vesicles further include a solvent and a buffer solution; in the cRGD peptide-modified targeted polymeric vesicles, the volume ratio of the solvent to the buffer solution is 1:5 to 10.

[0008] In an embodiment of the present invention, in the cRGD peptide-modified targeted polymeric vesicles, the volume ratio of the solvent to the buffer solution is 1:9.

[0009] In an embodiment of the present invention, the siKRAS drug is a siKRAS-gemcitabine conjugate (siKRAS-n-GEM, where n is an integer greater than or equal to 0); the solvent is N,N-dimethylformamide (DMF); the buffer solution is a PB buffer solution.

[0010] In an embodiment of the present invention, the concentration of the buffer solution is 2 to 10 mM and the pH is 7.4.

[0011] The present invention also provides a method for preparing the above-mentioned cRGD peptide-modified targeted polymeric vesicles, and the method is as follows: cRGD peptide and Mal-PEG-P(TMC-co-DTC) are mixed and then subjected to a Michael addition reaction to obtain the targeted polymer cRGD-PEG-P(TMC-co-DTC); the targeted polymer cRGD-PEG-P(TMC-co-DTC), the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 and the siKRAS drug are mixed to obtain the cRGD peptide-modified targeted polymeric vesicles.

[0012] In one embodiment of the present invention, the method is as follows: Dissolve cRGD peptide in a solvent to obtain a cRGD peptide solution; dissolve cRGD peptide in a solvent to obtain a cRGD peptide solution; dissolve Mal-PEG-P(TMC-co-DTC) in a solvent to obtain a Mal-PEG-P(TMC-co-DTC) solution; after mixing the cRGD peptide solution and the Mal-PEG-P(TMC-co-DTC) solution, carry out a Michael addition reaction under the protection of nitrogen to obtain a reaction product; dialyze the reaction product using the solvent as a dialysis medium, then precipitate it in cold diethyl ether, and finally filter and dry the precipitate to obtain the targeted polymer cRGD-PEG-P(TMC-co-DTC); dissolve the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 in a solvent to obtain a carrier polymer solution; dissolve the targeted polymer cRGD-PEG-P(TMC-co-DTC) in a solvent to obtain a targeted polymer solution; mix the targeted polymer solution and the carrier polymer solution to obtain a polymer mixed solution; dissolve the siKRAS drug in a buffer to obtain a siKRAS drug solution; add the polymer mixed solution to the bottom of the siKRAS drug and let it stand to obtain a mixture A; stir the mixture A to obtain a mixture B; dialyze the mixture B using the buffer as a dialysis medium to obtain the cRGD peptide-modified targeted polymer vesicles.

[0013] The present invention also provides the application of the above cRGD peptide-modified targeted polymer vesicles or the above method in the preparation of targeted therapeutic drugs for cancers caused by KRAS mutations.

[0014] In one embodiment of the present invention, the cancers caused by KRAS mutations are pancreatic cancer, non-small cell lung cancer or colon cancer.

[0015] The technical solution of the present invention has the following advantages:

[0016] The present invention provides a cRGD peptide-modified targeted polymer vesicle, and the components of the cRGD peptide-modified targeted polymer vesicle include the targeted polymer cRGD-PEG-P(TMC-co-DTC), the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 and the siKRAS drug. The cRGD peptide-modified targeted polymer vesicles of the present invention can efficiently load the siKRAS drug, realize the effective release of the siKRAS drug intracellularly, effectively improve the enrichment and internalization of the vesicles at the tumor site in vivo, well inhibit the tumor growth of the KRAS G12D mutant pancreatic cancer model, and significantly prolong the survival period of the model mice, and have no significant toxicity, and have great application prospects in the preparation of targeted therapeutic drugs for cancers caused by KRAS mutations.

[0017] Furthermore, the siKRAS drug is a siKRAS-gemcitabine conjugate (siKRAS-n-GEM, where n is an integer greater than or equal to 0). When the cRGD peptide-modified targeted polymeric vesicles are used to deliver siKRAS-n-GEM for the treatment of pancreatic cancer in the present invention, GEM, as a nucleoside drug, is chemically modified and linked to the siKRAS sequence to obtain a single drug siKRAS-n-GEM with a controllable number of GEM monomers, which can significantly improve the anti-tumor effect and prolong the survival period. Moreover, siKRAS and GEM show a significant synergistic effect. The inhibitory effect of the two drugs co-delivered in the form of a single drug on tumor cells is significantly better than the way of co-administering the two drugs. It has great application prospects in the preparation of targeted therapeutic drugs for cancers caused by KRAS mutations. Brief Description of the Drawings

[0018] Figure 1 : Schematic diagram of the synthesis of PEG-P(TMC-co-DTC).

[0019] Figure 2 : NMR results of the targeted polymer cRGD-PEG-P(TMC-co-DTC) 1 H NMR spectrum (600 MHz, DMSO-d6) of cRGD-PEG-P(TMC-co-DTC)).

[0020] Figure 3 : Particle size distribution and drug loading capacity of cRGD-BCP-siKRAS (15% cRGD density). Figure 3 In, A. Particle size distribution of BCP-siKRAS and cRGD-BCP-siKRAS (15 wt.% siRNA) measured by DLS; B. Particle size distribution of cRGD-BCP-siKRAS for dilution, storage, 10% FBS, and addition or not addition of 10 mM GSH in PBS (pH 7.4, 10 mM); C. Gel electrophoresis pattern of cRGD-BCP-siKRAS treated with (+) or without (-) 10 mM GSH, using free siKRAS at the same concentration as a control; D. Gel electrophoresis pattern of BCP-siKRAS and cRGD-BCP-siKRAS with different drug loading ratios (Drug loading content, DLC).

[0021] Figure 4 : Particle size distribution and drug loading of cRGD-BCP-siKRAS, cRGD-BCP-siKRAS-3G, and cRGD-BCP-siKRAS-5G. Figure 4Among them, A. Particle size distribution of cRGD-BCP-siKRAS, cRGD-BCP-siKRAS-3G and cRGD-BCP-siKRAS-5G determined by DLS; B. Particle size distribution of cRGD-BCP-siKRAS-3G upon dilution and in the presence of 10% FBS; C. Gel electrophoresis patterns of cRGD-BCP-siKRAS, cRGD-BCP-siKRAS-3G and cRGD-BCP-siKRAS-5G with different DLCs.

[0022] Figure 5 : In vivo gene silencing efficacy of tumors in a mouse PANC-1 subcutaneous tumor model on the 13th day after the start of treatment. Figure 5 Among them, A. Silencing of KRAS mRNA in each group; B. Western blot images and semi-quantitative analysis of KRAS protein expression (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0023] Figure 6 : cRGD-BCP-mediated transfection of siKRAS, siKRAS-3G and siKRAS-5G into PANC-1 cells in vitro. Figure 6 Among them, A. Relative KRAS mRNA levels detected by qRT-PCR after 24 hours of incubation (siRNA doses: 50, 100 and 200 nM); B. Relative KRAS mRNA levels detected by qRT-PCR after 48 hours of incubation; C. Relative KRAS mRNA levels detected by qRT-PCR after 72 hours of incubation; D. Relative KRAS mRNA levels detected by qRT-PCR after 4 days and 7 days of incubation (siRNA dose: 200 nM).

[0024] Figure 7 : Cell proliferation, migration and invasion of BCP-siKRAS and cRGD-BCP-siKRAS (15% cRGD density) in vitro. Figure 7 Among them, A. Inhibition of PANC-1 cell proliferation after 48 hours of incubation (siRNA dose: 200 nM), Hoechst was used to stain cell nuclei (blue); B. Cytotoxicity of BCP-siKRAS or cRGD-BCP-siKRAS in PANC-1 cells after 48 hours of incubation; C. Inhibition of PANC-1 cell migration after 24 hours of incubation (siRNA dose: 200 nM); D. Inhibition of PANC-1 cell invasion after 48 hours of incubation (siRNA dose: 200 nM); Scale bar: 100 μm.

[0025] Figure 8:In vitro cell proliferation, migration and invasion of cRGD-BCP-siKRAS, cRGD-BCP-siKRAS-3G and cRGD-BCP-siKRAS-5G. Figure 8 Among them, A. Inhibition of PANC-1 cell proliferation after 48-hour incubation (siRNA dose: 200 nM), and Hoechst was used to stain cell nuclei (blue); B. Inhibition of PANC-1 cell migration after 24-hour incubation (siRNA dose: 200 nM); C. Inhibition of PANC-1 cell invasion after 48-hour incubation (siRNA dose: 200 nM); Scale bar: 100 μm.

[0026] Figure 9 :Intracellular trafficking of cRGD-BCP-siKRAS (15% cRGD density) and BCP-siKRAS in PANC-1 cells. Figure 9 Among them, A. CLSM imaging of PANC-1 cells incubated with cRGD-BCP-siKRAS (Cy5) at the indicated time points after transfection (siRNA dose: 100 nM); B. CLSM images of PANC-1 cells incubated with BCP-siKRAS (Cy5) or cRGD-BCP-siKRAS (Cy5) for 4 hours (siRNA dose: 100 nM); C. Median fluorescence intensity of cRGD-BCP-siKRAS (Cy5) at the indicated time points after transfection; D. Pearson correlation coefficient between siRNA-Cy5 and endosomes / lysosomes at the indicated time points after transfection. Hoechst was used to stain cell nuclei (blue), while lysotracker green was used to stain endosomes / lysosomes (green); Scale bar: 20 μm.

[0027] Figure 10 :In vitro siKRAS transfection mediated by BCP and cRGD-BCP (15% cRGD density) on PANC-1 cells. Figure 10Among them, A. Relative KRAS mRNA levels detected by qRT-PCR after 24-hour incubation, for BCP and cRGD-BCP (siRNA doses: 50, 100, and 200 nM) (*p<0.05; ***p<0.001; ****p<0.0001); B. Relative KRAS mRNA levels detected by qRT-PCR after 48-hour incubation; C. Relative KRAS mRNA levels detected by qRT-PCR after 72-hour incubation; D. Relative KRAS mRNA levels detected by qRT-PCR after 24-, 48-, and 72-hour incubation, cRGD-BCP-siKRAS was freshly prepared (Fresh preparation) or stored for 3 weeks (Storage for 3 weeks) (siRNA dose: 200 nM); E. Relative KRAS mRNA levels detected by qRT-PCR after 4-day and 7-day incubation (siRNA dose: 200 nM); F. Western blot images and semi-quantitative analysis of KRAS protein expression (siRNA dose: 200 nM) (*p<0.05; ***p<0.001).

[0028] Figure 11 : In vivo biodistribution of BCP-siKRAS (Cy5) and cRGD-BCP-siKRAS (Cy5) (15% cRGD density). Figure 11 Among them, A. In vivo fluorescence imaging and semi-quantitative analysis of Cy5 fluorescence in nude mice with subcutaneous PANC-1 tumors at different time points; B. ex vivo fluorescence imaging 48 hours after injection into organs and tumors. C. Semi-quantitative analysis of Cy5 fluorescence (***p<0.001), H-Heart; Li-Liver; S-Spleen; L-Lung; K-Kidney; T-Tumor.

[0029] Figure 12 : Treat the PANC-1 subcutaneous model by tail vein administration (i.v.), administer BCP-siKRAS (3 mg / kg) and cRGD-BCP-siKRAS (1 and 3 mg / kg; 15% cRGD) every 4 days, for a total of 4 injections. Figure 12 Among them, A. Tumor size growth curves of each group after different treatments (n = 5); B. Tumor masses excised from each group on the 13th day; C. Spider diagrams representing the tumor sizes of individual mice in each group; D. Survival rates of tumor-bearing mice (n = 5); E. Body weight changes in each group (survival rates were analyzed using the Kaplan-Meier curve and the log-rank test in GraphPad Prism software, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0030] Figure 13 : Representative HE and TUNEL staining images of tumor sections dissected on day 13 after treatment with BCP-siKRAS (3 mg / kg) and cRGD-BCP-siKRAS (1 and 3 mg / kg; 15% cRGD); solid black arrows indicate apoptotic and necrotic cells; in the TUNEL assay, positive apoptotic cells are stained green (FITC), and cell nuclei are stained with DAPI (blue); scale bar: 50 μm.

[0031] Figure 14 : Representative HE staining images of normal organ sections on day 13 after treatment with BCP-siKRAS (3 mg / kg) and cRGD-BCP-siKRAS (1 and 3 mg / kg; 15% cRGD); scale bar: 50 μm.

[0032] Figure 15 : Treat the PANC-1 subcutaneous model by intravenous (i.v.) administration, and administer siScramble-3G (3 mg / kg), siKRAS (3 mg / kg), and cRGD-BCP-siKRAS-3G (1 and 3 mg / kg; 15% cRGD) every 4 days for a total of 4 injections. Figure 15 In, A. Tumor size growth curves of each group after different treatments (n = 5); B. Spider plots representing the tumor sizes of individual mice in each group; C. Survival rates of tumor-bearing mice (n = 5); D. Changes in body weight of each group; E. Tumor masses excised from each group on day 13; F. Tumor inhibition rates (TIR) of each group (n = 3) (Survival rates were analyzed using Kaplan-Meier curves and analyzed using the log-rank test in GraphPad Prism software, *p < 0.05; **p < 0.01; ***p < 0.001).

[0033] Figure 16 : Representative H&E, TUNEL, PCNA, and Cleaved caspase-3 staining sections of tumors isolated on day 13. Solid white arrows indicate apoptosis and necrosis. Scale bar: 50 μm.

[0034] Figure 17 : Representative H&E staining sections of major organs isolated on day 13. Scale bar: 50 μm.

[0035] Figure 18 : Gene silencing efficiency and RAS downstream signaling pathway protein conditions at the tumor site isolated on day 13. Figure 18A. Relative levels of A.KRAS mRNA (n = 3); B. Western blot of KRAS protein (n = 3); C. Semi-quantitative analysis of KRAS protein (n = 3); D. Western blot of RAS downstream signaling proteins (*p < 0.05; **p < 0.01; ***p < 0.001).

[0036] Figure 19 : Apoptosis assay and quantitative analysis after 48 h of incubation of cRGD-BCP-siKRAS and cRGD-BCP-siKRAS-GEM (number of covalently modified GEM monomers is 1 to 5). (siRNA dose: 200 nM).

[0037] Figure 20 : Cytotoxicity, apoptosis, and synergistic effects of cRGD-BCP-siKRAS-3G. Figure 20 A. Dose-response curve of cell viability after 48 h of incubation (n = 5); B. CI plot of cell viability after 48 h of incubation; C. Apoptosis image after 24 h of incubation (siRNA dose: 100 nM); D. Dose-response curve of apoptosis rate after 24 h of incubation (n = 3); E. CI plot of apoptosis rate after 24 h of incubation (range of Fa is 0.10 to 0.97).

[0038] Figure 21 : Inhibition of KRAS protein and its related signaling pathway proteins in PANC-1 cells after 72 h of incubation with cRGD-BCP-siKRAS-3G. (A) Western blot of RAS downstream signaling pathway proteins (RAF / MEK / ERK and PI3K / AKT / mTOR signaling pathways). (B) Western blot of KRAS protein and its (C) semi-quantitative analysis (n = 3). siRNA concentration: 200 nM. *p < 0.05, **p < 0.01, and ***p < 0.001.

[0039] Figure 22 : RNA-seq analysis after 48 h of transfection of siKRAS and siKRAS-3G by cRGD-BCP in PANC-1 cells. (A) Cluster heatmap of differentially expressed genes (DEGs). Red indicates high gene expression, and blue indicates low gene expression. (B) Volcano plot of DEGs (fold change > 2 and P value < 0.05), including 580 upregulated genes (red) and 316 downregulated genes (green). (C) GO function and (D) KEGG pathway enrichment analysis of downregulated genes between KRAS-3G and KRAS.

[0040] Figure 23 : Effect of cRGD density of cRGD-BCP-siKRAS on in vitro cell uptake and gene silencing efficiency (n = 3). Figure 23A. Flow cytometry plots of PANC-1 cells incubated with A.cRGD-BCP-siKRAS(Cy5) for 1 h; B. Semi-quantification by median fluorescence intensity (MFI); C. KRAS mRNA levels in PANC-1 cells incubated with cRGD-BCP-siKRAS for 48 h (siRNA dose: 200 nM) (*p < 0.05; ***p < 0.001).

[0041] Figure 24 : Endocytosis and intracellular trafficking of cRGD-BCP-siKRAS(Cy5) in PANC-1 cells (n = 3). CLSM images of cRGD-BCP-siKRAS(Cy5) incubated for (A) 4 h and (B) at the indicated time points. (C) MFI and (D) p, with Lysotracker green staining for endo / lysosomes and Hoechst staining for nuclei. siRNA dose: 100 nM. Scale bar: 20 μm.

[0042] Figure 25 : In vitro siKRAS transfection efficiency of cRGD-BCP-siKRAS in PANC-1 cells (n = 3). Relative KRAS mRNA levels measured by qRT-PCR after incubation for (A) 24 h, (B) 48 h, (C) 72 h, and (D) 4 and 7 d. (E) Relative KRAS mRNA levels of cRGD-BCP-siKRAS after storage for 3 weeks. (F) WB images and semi-quantitative analysis of KRAS protein expression after incubation for 72 h. BCP-siKRAS and BCP-siScramble were used as controls. *p < 0.05, ***p < 0.001. For D, E, and F, siRNA dose: 200 nM.

[0043] Figure 26 : In vitro siKRAS transfection efficiency of cRGD-BCP-siKRAS in PANC-1 cells (n = 3). Figure 26 A. Relative KRAS mRNA levels measured by qRT-PCR after incubation for 24 h; B. Relative KRAS mRNA levels measured by qRT-PCR after incubation for 48 h; C. Relative KRAS mRNA levels measured by qRT-PCR after incubation for 72 h; D. Relative KRAS mRNA levels measured by qRT-PCR after incubation for 4 and 7 d (for D, siRNA dose: 200 nM) (*p < 0.05; ***p < 0.001). Detailed implementation

[0044] The following embodiments are provided to better understand the present invention further. It is not limited to the best mode described, and does not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other prior arts falls within the protection scope of the present invention.

[0045] For those not specifying specific experimental steps or conditions in the following embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0046] Example 1: A polymer vesicle and its preparation method

[0047] This example provides a polymer vesicle, and the preparation method of the polymer vesicle includes the following steps:

[0048] 1. Synthesize the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200

[0049] Synthesize the PEG-P(TMC-co-DTC) copolymer: Under the protection of nitrogen, dissolve MeO-PEG-OH (Mn = 5.0 kg / mol, 30 g, 6 mmol, methoxy poly(ethylene glycol)-hydroxyl), TMC (90 g, 891 mmol, trimethylene carbonate), and DTC (12 g, 62.6 mmol, dithiolane trimethylene carbonate) in DCM (380 mL, dichloromethane) to obtain a solution; under the protection of nitrogen, add DPP (15 g, 60 mmol, diphenyl phosphate) to the solution, and then react at 200 rpm and 40 °C for 90 h to obtain a reaction product; add DCM (400 mL) to the reaction product to stop the reaction and dilute the reaction product to obtain a diluted reaction product solution; precipitate the diluted reaction product solution in cold ethanol (4 °C) and then filter, and take the filtered product and dry it under vacuum to obtain the PEG-P(TMC-co-DTC) copolymer.

[0050] Synthesize the PEG-P(TMC-co-DTC)-CDI polymer: Under the protection of nitrogen, dissolve the PEG-P(TMC-co-DTC) copolymer (3.3 g, 0.15 mmol) in DCM (12 mL) to obtain a solution; under the protection of nitrogen, add CDI (73 mg, 0.45 mmol, carbonyldiimidazole) to the solution, and then react at 200 rpm and 30 °C for 4 h to obtain a reaction product; precipitate the reaction product solution in cold ethanol (4 °C) and then filter, and take the filtered product and dry it under vacuum to obtain the PEG-P(TMC-co-DTC)-CDI polymer.

[0051] Synthetic carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 : Transfer branched PEI 1200 (982 mg, 0.82 mmol) to a two-necked round-bottom flask and dissolve it in DCM (19.6 mL) to obtain solution A; dissolve the PEG-P(TMC-co-DTC)-CDI polymer (1.8 g, 82 μmol) in DCM (12 mL) to obtain solution B; under the protection of nitrogen, transfer solution B to a constant-pressure dropping funnel; connect the funnel to the flask, and under the protection of nitrogen, stir at 200 rpm in an ice bath while slowly dropping solution B into solution A (the dropping time is 2 h). After the dropping is completed, continue the reaction at 200 rpm in an ice bath for 4 h to obtain the reaction product; dilute the reaction product solution and precipitate it in cold ethanol / ethanol (4 °C, cold ethanol / ethanol = 1 / 4, v / v), then filter. Take the filtered product and dry it under vacuum to obtain the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 (The synthesis principle is shown in Figure 1 );

[0052] Carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 has the following structural formula:

[0053]

[0054] In the formula, x and y are positive integers.

[0055] 2. Synthesis of non-targeted polymer Mal-PEG-P(TMC-co-DTC)

[0056] Synthesis of non-targeted polymer Mal-PEG-P(TMC-co-DTC): Refer to the synthesis process of the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 and use maleimide polyethylene glycol (Mal-PEG-OH, M n = 7.5 kg / mol, purchased from Beijing KeyGen Biotech Co., Ltd.) to synthesize the non-targeted polymer Mal-PEG-P(TMC-co-DTC).

[0057] 3. Preparation of non-targeted polymer vesicles

[0058] Dissolve the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 in DMF (N,N-dimethylformamide) to a final concentration of 40 mg / mL to obtain the carrier polymer solution; dissolve the non-targeted polymer Mal-PEG-P(TMC-co-DTC) (M n=(7.5-(15.0 - 2.0)), Mw / Mn = 1.1) was dissolved in DMF to a final concentration of 10 mg / mL to obtain a non-targeted polymer solution; the non-targeted polymer solution and the carrier polymer solution were mixed at a mass ratio of 15:85 to obtain a polymer mixed solution (at this time, the targeting density was 15 wt.%). Using PB buffer (2 mM, pH = 6.0) as the initial buffer medium for the solvent exchange method, siKRAS (siKRAS-0-GEM, siKRAS-1-GEM, siKRAS-2-GEM, siKRAS-3-GEM, siKRAS-4-GEM or siKRAS-5-GEM) was dissolved according to the formula in Table 1 using the initial buffer medium to obtain a siKRAS drug solution; 80 μL of the polymer mixed solution was slowly added (over 15 s) to the bottom of 0.72 mL of the siKRAS drug solution and then left standing for 3 s to obtain mixture A; mixture A was stirred at 300 rpm and 30 °C for 10 min to obtain mixture B; mixture B was placed in a dialysis bag (MWCO 7000) and dialyzed for 8 h to obtain non-targeted polymer vesicles BCP-siKRAS (the non-targeted polymer vesicles were stored at 4 °C for later use. In the formula of the non-targeted polymer vesicles, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate and water were provided by the buffer medium, and Mal-PEG-P(TMC-co-DTC), PEG-P(TMC-co-DTC)-PEI 1200 and DMF were provided by the non-targeted polymer mixed solution); during dialysis, the initial buffer medium (PB buffer, 2 mM, pH = 6.0) was first transitioned to the intermediate buffer medium (PB buffer, 10 mM, pH = 7.4), and then to the final buffer medium (PBS buffer, 10 mM, pH = 7.4), and the dialysis medium was changed every 1 h.

[0059] Table 1 Formulation of polymer vesicles

[0060]

[0061] In Table 1, *This number represents the number of GEM (gemcitabine) conjugated to siKRAS. There is no gemcitabine active ingredient in siKRAS-0-GEM. siKRAS-0-GEM to siKRAS-5-GEM were all synthesized by Gemma Gene (solid-phase phosphoramidite method); **This dosage is for a drug loading content (DLC) of 15 wt.%. Similarly, when the drug loading ratios are 5, 10, 20, 25 wt.%, the drug amounts are 0.16, 0.32, 0.64, 0.80 mg respectively;

[0062] The sequence of siKRAS-0-GEM (i.e., Free siKRAS) is:

[0063] Sense strand: 5’-GGAGCUGAUGGCGUAGGCATT-3’ (SEQ ID NO.1);

[0064] Antisense strand: 5’-UGCCUACGCCAUCAGCUCCTT-3’ (SEQ ID NO.2);

[0065] The sequences of siKRAS-1-GEM to siKRAS-5-GEM (i.e., siKRAS-nG) are:

[0066] Sense strand: 5’-GGAGCUGAUGGCGUAGGCATT-(X)n-3’ (SEQ ID NO.1);

[0067] Antisense strand: 5’-UGCCUACGCCAUCAGCUCCTT-3’ (SEQ ID NO.2);

[0068] Wherein, X refers to GEM, and n refers to the number of GEM (gemcitabine) coupled to siKRAS.

[0069] Example 2: A cRGD peptide-modified targeted polymeric vesicle and its preparation method

[0070] This example provides a cRGD peptide-modified targeted polymeric vesicle, and the preparation method of the cRGD peptide-modified targeted polymeric vesicle comprises the following steps:

[0071] 1. Synthesize the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200

[0072] Synthesize the PEG-P(TMC-co-DTC) copolymer: Under the protection of nitrogen, dissolve MeO-PEG-OH (Mn = 5.0 kg / mol, 30 g, 6 mmol, methoxy poly(ethylene glycol)-hydroxyl), TMC (90 g, 891 mmol, trimethylene carbonate), and DTC (12 g, 62.6 mmol, dithiacyclopentane trimethylene carbonate) in DCM (380 mL, dichloromethane) to obtain a solution; Under the protection of nitrogen, add DPP (15 g, 60 mmol, diphenyl phosphate) to the solution, and then react at 200 rpm and 40 °C for 90 h to obtain a reaction product; Add DCM (400 mL) to the reaction product to stop the reaction and dilute the reaction product to obtain a diluted reaction product solution; Precipitate the diluted reaction product solution in cold ethanol (4 °C) and then filter, and take the filtered product and dry it in vacuo to obtain the PEG-P(TMC-co-DTC) copolymer.

[0073] Synthesis of PEG-P(TMC-co-DTC)-CDI polymer: Under the protection of nitrogen, dissolve the PEG-P(TMC-co-DTC) copolymer (3.3 g, 0.15 mmol) in DCM (12 mL) to obtain a solution; under the protection of nitrogen, add CDI (73 mg, 0.45 mmol, carbonyldiimidazole) to the solution, and then react at 200 rpm and 30 °C for 4 h to obtain a reaction product; precipitate the reaction product dilution in cold ethanol (4 °C) and filter, and take the filtered product and dry it under vacuum to obtain the PEG-P(TMC-co-DTC)-CDI polymer.

[0074] Synthesis of the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 : Transfer the branched PEI 1200 (982 mg, 0.82 mmol) to a two-necked round-bottom flask and dissolve it in DCM (19.6 mL) to obtain solution A; dissolve the PEG-P(TMC-co-DTC)-CDI polymer (1.8 g, 82 μmol) in DCM (12 mL) to obtain solution B; under the protection of nitrogen, transfer solution B to a constant-pressure dropping funnel. Connect the funnel to the flask, and under the protection of nitrogen, stir at 200 rpm in an ice bath while slowly dropping solution B (the dropping time is 2 h) into solution A. After the dropping is completed, continue to react at 200 rpm in an ice bath for 4 h to obtain a reaction product; precipitate the reaction product dilution in cold ethanol / ethanol (4 °C, cold ethanol / ethanol = 1 / 4, v / v) and filter, and take the filtered product and dry it under vacuum to obtain the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 (The synthesis principle is shown in Figure 1 ).

[0075] 2. Synthesis of the non-targeted polymer Mal-PEG-P(TMC-co-DTC)

[0076] Synthesis of the non-targeted polymer Mal-PEG-P(TMC-co-DTC): Refer to the synthesis process of the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 and use maleimide polyethylene glycol (Mal-PEG-OH, M n = 7.5 kg / mol, purchased from Beijing KeyGen Biotech Co., Ltd.) to synthesize the non-targeted polymer Mal-PEG-P(TMC-co-DTC).

[0077] 3. Synthesis of the cRGD peptide-modified targeted polymer cRGD-PEG-P(TMC-co-DTC)

[0078] Dissolve cRGD peptide (20 mg, 34.6 μmol, c(RGDfC), purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) in 1.0 mL of DMF (N,N-dimethylformamide) deoxygenated by bubbling with nitrogen to obtain a cRGD peptide solution; dissolve the non-targeting polymer Mal-PEG-P(TMC-co-DTC) (500 mg, 20.1 μmol, M n = 7.5-(15.0-2.0), Mw / Mn = 1.1) in 4.0 mL of DMF (N,N-dimethylformamide) deoxygenated by bubbling with nitrogen to obtain a Mal-PEG-P(TMC-co-DTC) solution; under the protection of nitrogen, mix the cRGD peptide solution and the Mal-PEG-P(TMC-co-DTC) solution, and react at 200 rpm and 30 °C for 48 h to obtain a reaction product; dialyze the reaction product first using DMF as the dialysis medium in a dialysis bag (MWCO 7000), then precipitate it in cold ether (4 °C), and finally filter the precipitate and dry it under vacuum at 30 °C to obtain the cRGD peptide-modified targeting polymer cRGD-PEG-P(TMC-co-DTC) (the structural formula of the cRGD peptide-modified targeting polymer cRGD-PEG-P(TMC-co-DTC) is shown in Figure 2 );

[0079] 4. Preparation of cRGD peptide-modified targeting polymer vesicles

[0080] The carrier polymer PEG-P(TMC-co-DTC)-PEI 1200Dissolve it in DMF to a final concentration of 40 mg / mL to obtain a carrier polymer solution; dissolve the cRGD peptide-modified targeting polymer cRGD-PEG-P(TMC-co-DTC) in DMF to a final concentration of 10 mg / mL to obtain a cRGD peptide-modified targeting polymer solution; mix the cRGD peptide-modified targeting polymer solution and the carrier polymer solution according to a mass ratio of 15:85 to obtain a polymer mixed solution (at this time, the targeting density is 15 wt.%); use PB buffer (2 mM, pH = 6.0) as the initial buffer medium for the solvent exchange method, and dissolve siKRAS-0-GEM according to the formula in Table 1 using the initial buffer medium to obtain a siKRAS drug solution; slowly add 80 μL of the polymer mixed solution (taking 15 s) to the bottom of 0.72 mL of the siKRAS drug solution and let it stand for 3 s to obtain mixture A; stir mixture A at 300 rpm and 30 °C for 10 min to obtain mixture B; place mixture B in a dialysis bag (MWCO 7000) and dialyze for 8 h to obtain cRGD peptide-modified targeting polymer vesicles cRGD-BCP-siKRAS (the cRGD peptide-modified targeting polymer vesicles are stored at 4 °C for later use. In the formula of the cRGD peptide-modified targeting polymer vesicles, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate and water are provided by the buffer medium, and cRGD-PEG-P(TMC-co-DTC), PEG-P(TMC-co-DTC)-PEI 1200 and DMF are provided by the cRGD peptide-modified targeting polymer mixed solution); during dialysis, first transition from the initial buffer medium (PB buffer, 2 mM, pH = 6.0) to the intermediate buffer medium (PB buffer, 10 mM, pH = 7.4), and then to the final buffer medium (PBS buffer, 10 mM, pH = 7.4), and change the dialysis medium every 1 h.

[0081] Detect the substitution degree of the prepared cRGD peptide-modified targeting polymer cRGD-PEG-P(TMC-co-DTC) by the BCA method, and the detection result is 96.2%. Use an Agilent DirectDrive2 600 MHz spectrometer to detect the prepared cRGD peptide-modified targeting polymer cRGD-PEG-P(TMC-co-DTC), and the obtained NMR spectrum is as Figure 2 shown. Determine and calibrate the concentration of the obtained cRGD peptide-modified targeting polymer vesicles by the weighing method and the Nanodrop method, and the calibration result is: 0.40 mg / mL.

[0082] Example 3: A cRGD peptide and gemcitabine co-modified targeting polymer vesicle and its preparation method

[0083] This embodiment provides a targeted polymeric vesicle co-modified with cRGD peptide and gemcitabine. The preparation method of the targeted polymeric vesicle co-modified with cRGD peptide and gemcitabine comprises the following steps:

[0084] 1. Synthesize the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200

[0085] Synthesize the PEG-P(TMC-co-DTC) copolymer: Under the protection of nitrogen, dissolve MeO-PEG-OH (Mn = 5.0 kg / mol, 30 g, 6 mmol, methoxy poly(ethylene glycol)-hydroxyl), TMC (90 g, 891 mmol, trimethylene carbonate), and DTC (12 g, 62.6 mmol, dithiolane trimethylene carbonate) in DCM (380 mL, dichloromethane) to obtain a solution; under the protection of nitrogen, add DPP (15 g, 60 mmol, diphenyl phosphate) to the solution, and then react at 200 rpm and 40 °C for 90 h to obtain a reaction product; add DCM (400 mL) to the reaction product to stop the reaction and dilute the reaction product to obtain a diluted reaction product solution; precipitate the diluted reaction product solution in cold ethanol (4 °C) and then filter, and take the filtered product and dry it in vacuo to obtain the PEG-P(TMC-co-DTC) copolymer.

[0086] Synthesize the PEG-P(TMC-co-DTC)-CDI polymer: Under the protection of nitrogen, dissolve the PEG-P(TMC-co-DTC) copolymer (3.3 g, 0.15 mmol) in DCM (12 mL) to obtain a solution; under the protection of nitrogen, add CDI (73 mg, 0.45 mmol, carbonyldiimidazole) to the solution, and then react at 200 rpm and 30 °C for 4 h to obtain a reaction product; precipitate the reaction product solution in cold ethanol (4 °C) and then filter, and take the filtered product and dry it in vacuo to obtain the PEG-P(TMC-co-DTC)-CDI polymer.

[0087] Synthesize the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 : Add the branched PEI 1200(982 mg, 0.82 mmol) was transferred to a two-necked round-bottom flask and dissolved in DCM (19.6 mL) to obtain solution A; the PEG-P(TMC-co-DTC)-CDI polymer (1.8 g, 82 μmol) was dissolved in DCM (12 mL) to obtain solution B; under the protection of nitrogen, solution B was transferred to a constant-pressure dropping funnel. The funnel was connected to the flask, and under the protection of nitrogen, while stirring at 200 rpm in an ice bath, solution B was slowly added dropwise (the dropping time was 2 h) into solution A. After the addition was completed, the reaction was continued at 200 rpm in an ice bath for 4 h to obtain the reaction product; the diluted reaction product was precipitated in cold ethanol / ethanol (4 °C, cold ethanol / ethanol = 1 / 4, v / v) and then filtered. The filtered product was dried under vacuum to obtain the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 (The synthesis principle is shown in Figure 1 ).

[0088] 2. Synthesis of non-targeted polymer Mal-PEG-P(TMC-co-DTC)

[0089] Synthesis of non-targeted polymer Mal-PEG-P(TMC-co-DTC): Referring to the synthesis process of the carrier polymer

[0090] PEG-P(TMC-co-DTC)-PEI 1200 , maleimide polyethylene glycol (Mal-PEG-OH, M n = 7.5 kg / mol, purchased from Shanghai Biopharm Technology Co., Ltd.) was used to synthesize the non-targeted polymer Mal-PEG-P(TMC-co-DTC).

[0091] 3. Synthesis of cRGD peptide-modified targeted polymer cRGD-PEG-P(TMC-co-DTC)

[0092] The cRGD peptide (20 mg, 34.6 μmol, c(RGDfC), purchased from Shanghai Qiangyao Biotech Co., Ltd.) was dissolved in 1.0 mL of DMF (N,N-dimethylformamide) that had been deoxygenated by bubbling nitrogen to obtain the cRGD peptide solution; the non-targeted polymer Mal-PEG-P(TMC-co-DTC) (500 mg, 20.1 μmol, M n= 7.5 - (15.0 - 2.0), Mw / Mn = 1.1) was dissolved in 4.0 mL of DMF (N,N-dimethylformamide) deoxygenated by nitrogen bubbling to obtain a Mal-PEG-P(TMC-co-DTC) solution; under the protection of nitrogen, the cRGD peptide solution and the Mal-PEG-P(TMC-co-DTC) solution were mixed and reacted at 200 rpm and 30 °C for 48 h to obtain a reaction product; the reaction product was first dialyzed in a dialysis bag (MWCO 7000) using DMF as a dialysis medium, then precipitated in cold ether (4 °C), and finally the precipitate was filtered and dried under vacuum at 30 °C to obtain the cRGD peptide-modified targeted polymer cRGD-PEG-P(TMC-co-DTC) (the structural formula of the cRGD peptide-modified targeted polymer cRGD-PEG-P(TMC-co-DTC) is shown in Figure 2 );

[0093] 4. Preparation of cRGD Peptide and Gemcitabine Co-Modified Targeted Polymer Vesicles

[0094] The carrier polymer PEG-P(TMC-co-DTC)-PEI 1200Dissolve it in DMF to a final concentration of 40 mg / mL to obtain a carrier polymer solution; dissolve the cRGD peptide-modified targeting polymer cRGD-PEG-P(TMC-co-DTC) in DMF to a final concentration of 10 mg / mL to obtain a cRGD peptide-modified targeting polymer solution; mix the cRGD peptide-modified targeting polymer solution and the carrier polymer solution according to a mass ratio of 15:85 to obtain a polymer mixed solution (at this time, the targeting density is 15 wt.%); use PB buffer (2 mM, pH = 6.0) as the initial buffer medium for the solvent exchange method, and dissolve the siKRAS-gemcitabine conjugate (siKRAS-1-GEM, siKRAS-2-GEM, siKRAS-3-GEM, siKRAS-4-GEM or siKRAS-5-GEM) according to the formula in Table 1 using the initial buffer medium to obtain a siKRAS drug solution; slowly add 80 μL of the polymer mixed solution (taking 15 s) to the bottom of 0.72 mL of the siKRAS drug solution and then let it stand for 3 s to obtain a mixture A; stir the mixture A at 300 rpm and 30 °C for 10 min to obtain a mixture B; place the mixture B in a dialysis bag (MWCO 7000) and dialyze for 8 h to obtain cRGD peptide and gemcitabine co-modified targeting polymer vesicles cRGD-BCP-siKRAS-1G to cRGD-BCP-siKRAS-5G (the cRGD peptide and gemcitabine co-modified targeting polymer vesicles are stored at 4 °C for later use. In the formula of the cRGD peptide and gemcitabine co-modified targeting polymer vesicles, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate and water are provided by the buffer medium, and cRGD-PEG-P(TMC-co-DTC), PEG-P(TMC-co-DTC)-PEI 1200 and DMF are provided by the cRGD peptide-modified targeting polymer mixed solution); during dialysis, first transition from the initial buffer medium (PB buffer, 2 mM, pH = 6.0) to the intermediate buffer medium (PB buffer, 10 mM, pH = 7.4), and then to the final buffer medium (PBS buffer, 10 mM, pH = 7.4), and the dialysis medium is changed every 1 h.

[0095] Experimental Example 1: Physicochemical Property Experimental Test of cRGD Peptide-Modified Targeting Polymer Vesicles

[0096] This experimental example provides an experiment for measuring the particle size and Zeta potential of cRGD peptide-modified targeting polymer vesicles. The experimental process is as follows:

[0097] Using siKRAS-0-GEM (Free siKRAS) as a control, the non-targeted polymer vesicles (BCP-siKRAS) prepared in Example 1, the cRGD peptide-modified targeted polymer vesicles (cRGD-BCP-siKRAS) prepared in Example 2, and the cRGD peptide and gemcitabine co-modified targeted polymer vesicles (cRGD-BCP-siKRAS-1G to cRGD-BCP-siKRAS-5G) prepared in Example 3 were characterized by DLS (see the literature "Adv Mater. 2017 Nov; 29(42).") and agarose gel electrophoresis (80V, 15min; see the literature "Adv Mater. 2017 Nov; 29(42)."). The characterization results are as Figures 3 - 4 shown in Table 2 and Table 3.

[0098] Figure 3 The results in Table 2 show that after targeted modification, the particle size of the vesicles was not affected, and it could stably load 15 wt% of siKRAS drug. The vesicles had good colloidal stability and could release siKRAS by reduction-responsive.

[0099] Figure 4 The results in Table 3 show that after covalent modification of the siKRAS sequence with GEM, the loading efficiency and particle size properties of the targeted vesicles cRGD-BCP were not affected. The targeted vesicles could still effectively load 15 wt% of siKRAS-GEM drug, maintaining a small particle size (68 - 69 nm), a good particle size distribution (<0.02), and a nearly neutral Zeta potential, and having good particle size stability. Table 2 Basic properties of BCP-siKRAS and cRGD-BCP-siKRAS (0.40 mg / mL, 15 wt.% siKRAS) with different cRGD densities (i.e., targeting densities)

[0100]

[0101] a) Determined by DLS at 25℃ in PBS (pH 7.4, 10 mM);

[0102] b) Determined by Zetasizer Nano-ZS at 25℃ in PBS (pH 7.4, 10 mM).

[0103] Table 3 Basic properties of cRGD-BCP-siKRAS, cRGD-BCP-siKRAS-3G, and cRGD-BCP-siKRAS-5G (0.40 mg / mL, 15 wt% siKRAS)

[0104] <![CDATA[Size(nm) a > PDIa <![CDATA[Zeta potential(mV) b > cRGD - BCP - siKRAS 68 0.11 -1.1 cRGD - BCP - siKRAS - 3G 68 0.10 1.2 cRGD - BCP - siKRAS - 5G 68 0.17 1.0

[0105] a) Determined by DLS at 25℃ in PBS (pH 7.4, 10mM);

[0106] b) Determined by Zetasizer Nano-ZS at 25℃ in PBS (pH 7.4, 10mM).

[0107] Experimental Example 2: Gene silencing experiments in vitro and in vivo using cRGD peptide-modified targeted polymer vesicles

[0108] This experimental example provides an in vivo and in vitro gene silencing experiment of cRGD peptide-modified targeted polymer vesicles. The experimental process is as follows:

[0109] Experiment 1 (in vivo gene silencing experiment): 5,000,000 PANC-1 cells (purchased from ATCC) were subcutaneously inoculated into the right dorsal abdomen of 4-week-old BABL / c female nude mice (purchased from Genetron Health) to establish a PANC-1 tumor model. When the tumor volume of the PANC-1 tumor model mice grew to 100 mm 3 After that, group administration (tail vein injection) was performed, and the mice were randomly divided into four groups, including a PBS group administered with PBS buffer (dosage 3 mL), a BCP-siKRAS group administered with non-targeted polymer vesicles prepared in Example 1 (dosage 3 mg / kg, dissolved in 3 mL PBS buffer for administration), a low-dose cRGD-BCP-siKRAS group administered with cRGD peptide-modified targeted polymer vesicles prepared in Example 2 (dosage 1 mg / kg, dissolved in 3 mL PBS buffer for administration), and a high-dose cRGD-BCP-siKRAS group administered with cRGD peptide-modified targeted polymer vesicles prepared in Example 2 (dosage 3 mg / kg, dissolved in 3 mL PBS buffer for administration), and this day was designated as day 0. After the grouping was completed, the mice were administered with tail vein administration, one injection every four days, for a total of four injections. The day after the end of drug administration, the mouse tumors were collected to extract mRNA and protein (n=3), and cDNA was obtained by reverse transcription; the PBS group was used as the negative control group, and the cDNA obtained by reverse transcription was used as a template. The GAPDH gene was used as an internal reference gene. The relative expression of the KRAS gene was detected by real-time quantitative fluorescence PCR, and the KRAS gene silencing effect in PANC-1 tumor cells of different groups was analyzed by Western blot. The changes in the silencing efficiency of the siKRAS gene after modifying different GEM monomers were evaluated at the mRNA level. The evaluation results are shown in Figure 5 .

[0110] Experiment 2 (in vitro gene silencing experiment): Take a tube of PANC-1 cells (purchased from ATCC) and inoculate them into 8 mL of DMEM medium (purchased from Gibco) containing 10% (v / v) FBS. Incubate at 37 °C until the cell density reaches 90%. Then add 500 μL of trypsin (purchased from Gibco) and incubate at 37 °C for 2 min to digest into a single-cell suspension; count the cells and inoculate the single-cell suspension into a 24-well plate at an inoculation density of 1×10 5 cells per well. Incubate at 37 °C for 24 h. After incubation, use PBS buffer as a control (PBS), and add the non-targeted polymer vesicles (BCP-siKRAS) prepared in Example 1, the cRGD peptide-modified targeted polymer vesicles (cRGD-BCP-siKRAS) prepared in Example 2, and the cRGD peptide and gemcitabine co-modified targeted polymer vesicles (cRGD-BCP-siKRAS-1G to cRGD-BCP-siKRAS-5G) prepared in Example 3 into the 24-well plate at a final concentration of 50, 100, 200 nM per well and incubate at 37 °C for another 24, 48, 72 h, 4 d, 7 d. During incubation, replace an equal volume of fresh DMEM medium every 2 d. After the second incubation, collect the PANC-1 cells in the 24-well plate, extract the total RNA of PANC-1 cells, and obtain cDNA by reverse transcription. Use the PBS group as a negative control group, use the cDNA obtained by reverse transcription as a template, use the GAPDH gene as an internal reference gene, and detect the relative expression level of the KRAS gene by real-time quantitative fluorescence PCR. Combine Western blot to analyze the KRAS gene silencing effect in PANC1 tumor cells of different groups, and evaluate the change of siKRAS gene silencing efficiency after modifying different GEM monomers at the mRNA level. The evaluation results are shown in Figure 6 .

[0111] The RNA extraction process is as follows: 1. Collect cells by centrifugation, resuspend the obtained cell pellet with 1×PBS, and centrifuge again to discard all the supernatant. 2. Add RNA extraction reagent (RNA-easy, purchased from Vazyme), and add 500 μL of RNA-easy for every 1×10 6 cells. 3. Pipette repeatedly until the cells are fully lysed. 4. Add 2 / 5 volume of RNase-free ddH 2O (purchased from GenePharma, 200 μL for every 500 μL of RNA-easy), invert and mix well, and let it stand at room temperature (25 °C) for 5 min. 5. Centrifuge at 12,000×g at room temperature (25 °C) for 15 min. 6. Take out the centrifuge tube. At this time, the solution is divided into the upper aqueous phase (containing RNA) and the dark lower precipitate (containing impurities such as proteins, DNA, and polysaccharides). Aspirate the upper aqueous phase into a new centrifuge tube. 7. Add an equal volume of isopropanol to the above centrifuge tube, invert and mix well, and let it stand at room temperature (25 °C) for 10 min. 8. After centrifuging at 12,000×g at room temperature (25 °C) for 10 min, carefully discard the supernatant or slowly aspirate the supernatant along the liquid surface, and retain the white precipitate at the bottom. 9. Add 500 μL of 75% (v / v) ethanol (prepared with RNase-free ddH 2 O), flick the bottom of the tube to suspend the precipitate, and invert it several times. 10. Centrifuge at 8,000×g at room temperature (25 °C) for 3 min, and discard the supernatant. 11. Repeat steps 9 and 10 once. After completely discarding the supernatant, let it air dry at room temperature (25 °C). 12. Add an appropriate amount of RNase-free ddH 2 O to dissolve the precipitate, vortex at room temperature (25 °C) for 3 min (or pipette up and down repeatedly) to fully dissolve the RNA precipitate. The extracted RNA product can be aliquoted and stored long-term at -80 °C and short-term at -20 °C.

[0112] When detecting the relative expression level of the KRAS gene by real-time quantitative fluorescence PCR, the upstream primer sequence of the KRAS gene is 5’-AAACTTGTGGTAGTTGGAGCTG-3’ (SEQ ID NO.3), and the downstream primer sequence is

[0113] 5’-CCTCTATTGTTGGATCATATTCG-3’ (SEQ ID NO.4); the upstream primer sequence of GAPDH is 5’-CATGAGAAGTATGACAACAGCCT-3’ (SEQ ID NO.5), and the downstream primer sequence is 5’-AGTCCTTCCACGATACCAAAGT-3’ (SEQ ID NO.6).

[0114] The process of real-time quantitative fluorescence PCR detection is as follows: Take 4 μL of each group of extracted RNA samples and add them to 16 μL of HiScript III RT SuperMix (purchased from Vazyme) and mix well. Reverse transcription is carried out using a gradient PCR instrument to obtain template cDNA. The reverse transcription conditions are: 37 °C for 15 min; 85 °C for 0.05 min; 4 °C forever. The obtained template cDNA is detected by fluorescence quantitative PCR. The reagents used are: Taq Pro Universal SYBR qPCR Master Mix (purchased from Vazyme). The conditions used are: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 12 s; annealing at 62 °C for 30 s; extension at 72 °C for 30 s, for 40 cycles. The results of its expression level are analyzed. Using GAPDH as an internal reference, the relative expression level of the KRAS gene is calculated using the 2 -△△Ct -method. (△△Ct = (Ct 处理组目的基因 - Ct 处理组内参基因 ) - (Ct 对照组目的基因 - Ct 对照组内参基因 )) The value obtained represents the fold change of the target gene in the experimental group relative to the control group.

[0115] Figure 5 The results show that cRGD-BCP-siKRAS can further significantly improve the gene silencing and protein expression inhibition effects on the basis of BCP-siKRAS. And the targeted vesicles can achieve gene silencing and protein inhibition effects equivalent to those of non-targeted vesicles when the dose is reduced by 3 times, indicating that the targeted vesicles increase the accumulation of siKRAS drug at the tumor site and improve the distribution of vesicles at the tumor site.

[0116] Figure 6 The results show that modifying the GEM monomer does not affect the gene silencing effect of the sequence itself. The targeted vesicles reach the best silencing efficiency after incubation for 48 h after sample addition, and can still maintain 60% of the gene silencing efficiency after 7 d.

[0117] Experimental Example 3: Experiment on the effect of cRGD peptide-modified targeted polymer vesicles on cell proliferation and migration

[0118] This experimental example provides an experiment on the effect of cRGD peptide-modified targeted polymer vesicles on cell proliferation and migration. In this experiment, BCP-siSrramble (i.e., siScramble) was used as a control (BCP-siSrramble is based on Free siKRAS, with the sense strand being 5'-GGTGCTGGCGAGCTGAAGATT-3' shown in SEQ ID NO.7 and the antisense strand being 5'-UCUUCAGCUCGCCAGCACCTT-3' shown in SEQ ID NO.8). The experimental procedure is as follows:

[0119] Experiment 1 (Proliferation inhibition experiment): Take a tube of PANC-1 cells (purchased from ATCC) and inoculate them into 8 mL of DMEM medium (purchased from Gibco) containing 5% (v / v) FBS. Incubate at 37 °C until the cell density reaches 90%. Then add 500 μL of trypsin (purchased from Gibco) and incubate at 37 °C for 2 min to digest into a single-cell suspension of PANC-1. Quantify cell proliferation inhibition using Cell Counting Kit-8 (CCK-8, purchased from Dojindo) and Hoechst (purchased from Beyotime). Inoculate the single-cell suspension of PANC-1 into a 96-well plate at a seeding density of 10,000 cells per well and incubate at 37 °C for 24 h. After incubation, using PBS buffer as a control (PBS), add the non-targeted polymer vesicles (BCP-siKRAS) prepared in Example 1 and the cRGD peptide-modified targeted polymer vesicles (cRGD-BCP-siKRAS) prepared in Example 2 to the 96-well plate at a final concentration of 100 nM per well and incubate at 37 °C for 48 h. After incubation, remove the supernatant from the 96-well plate, add 100 μL of fresh DMEM medium containing 10 μL of CCK-8 reagent to each well, and incubate at 37 °C for 1 h again. After the second incubation, measure the absorbance of the 96-well plate at 450 nm using a microplate reader, stain the cell nuclei with Hoechst (purchased from Beyotime), then take pictures and count (for each microscope field). The experimental results are shown in Figure 7 .

[0120] Experiment 2 (Transwell chamber migration and invasion experiment): Take a tube of PANC-1 cells (purchased from ATCC) and inoculate them into 8 mL of DMEM medium (purchased from Gibco) containing 5% (v / v) FBS. Culture at 37 °C until the cell density reaches 90%, then add 500 μL of trypsin (purchased from Gibco) and incubate at 37 °C for 2 min to digest into a single-cell suspension of PANC-1; Place the Transwell chambers (purchased from Corning) into 96-well plates and 6-well plates respectively; Dilute and mix the Matrigel matrix (purchased from Corning) with serum-free medium (purchased from Gibco) to a matrix solution with a concentration of 250 μg / mL; Add 100 μL of the matrix solution to the chambers and incubate at 37 °C for 24 h. After the incubation, add 750 μL of DMEM medium containing 10% (v / v) FBS to the lower chamber; Count, and inoculate the single-cell suspension of PANC-1 into the chambers at an inoculation amount of 3×10 5 cells per well, and use PBS buffer as a control (PBS). Add 250 μL of serum-free medium containing the non-targeted polymer vesicles (BCP-siKRAS) prepared in Example 1 at 200 nM, the cRGD peptide-modified targeted polymer vesicles (cRGD-BCP-siKRAS) prepared in Example 2, and the cRGD peptide and gemcitabine co-modified targeted polymer vesicles (cRGD-BCP-siKRAS-1G to cRGD-BCP-siKRAS-5G) prepared in Example 3 to the chambers. Place the chambers into the lower chamber and incubate at 37 °C for another 24 h (migration experiment) and 48 h (invasion experiment); After the second incubation, first fix with paraformaldehyde and stain with crystal violet, then observe the cells invading the Transwell chamber membrane under a microscope and count and quantify. The experimental results are shown in Figure 8 .

[0121] Figure 7 The results show that after silencing the KRAS gene, it can significantly inhibit the proliferation, migration and invasion of PANC-1 cells, and after the vesicles are targeted-modified, it can further improve the inhibitory effect and reduce the IC 50 value.

[0122] Figure 8 The results show that in the cell proliferation inhibition experiment, compared with the siKRAS group, after the siKRAS sequence is covalently modified with GEM monomers, it can significantly inhibit the cell proliferation rate, and the same results are also shown in the longitudinal migration and invasion experiments of the cells. This indicates that the introduction of GEM can further enhance the impact of siKRAS on cell proliferation, migration and invasion.

[0123] Experimental Example 4: Intracellular transport and drug release experiment of cRGD peptide-modified targeted polymer vesicles

[0124] This experimental example provides an experiment on the intracellular transport and drug release of cRGD peptide-modified targeted polymeric vesicles. The experimental procedure is as follows:

[0125] Take a tube of PANC-1 cells (purchased from ATCC) and inoculate them into 8 mL of DMEM medium (purchased from Gibco) containing 10% (v / v) FBS. Incubate at 37 °C until the cell density reaches 90%. Then add 500 μL of trypsin (purchased from Gibco) and incubate at 37 °C for 2 min to digest into a single-cell suspension. Count the cells and inoculate the single-cell suspension into a glass-bottom dish (20 × 35 mm, Biosharp) at an inoculation density of 200,000 cells per well. Incubate at 37 °C for 24 h. After the incubation, use the non-targeted polymeric vesicles (BCP(Cy5)) prepared in Example 1 as a control. After labeling the cRGD peptide-modified targeted polymeric vesicles prepared in Example 2 with Cy5 (cRGD-BCP(Cy5), Cy5 is labeled at the 5' end of the sense strand), add them to the glass-bottom dish at a final concentration of 100 nM per well and incubate at 37 °C for another 2, 4, 8, 16, and 24 h. After the second incubation, fix the PANC-1 cells with 4% (g / 100 mL) paraformaldehyde, stain the cell lysosomes with Lysotracker green (purchased from Beyotime), and stain the cell nuclei with DAPI (purchased from Beyotime). Observe the difference in the endocytosis amount of BCP-siKRAS and cRGD-BCP-siKRAS under a confocal laser scanning microscope (CLSM, Leica, LSM710), and further observe the intracellular transport and drug release of cRGD-BCP-siKRAS at different times. The observation results are as Figure 9 shown.

[0126] Figure 9 A shows the intracellular transport process of cRGD-BCP-siKRAS modified with the optimal targeting density. Figure 9 B shows that the targeted modified vesicles can effectively improve the internalization efficiency of the siKRAS drug, and with the extension of time, the siKRAS drug is continuously endocytosed. Figure 9 C shows that the drug fluorescence only starts to remain unchanged after 16 h and is equivalent to the fluorescence intensity at 24 h. And through the Pearson correlation coefficient analysis of the drug fluorescence (Cy5) and endosome / lysosome fluorescence (Lysotracker) at different times in Figure 9 A, it is found that ( Figure 9D), at 4 h after the addition of the targeted vesicles, the Pearson correlation coefficient reached its peak, indicating that in the early stage (4 h), the vesicles mainly existed in endosomes / lysosomes. In the subsequent time, the Pearson correlation coefficient began to decline, indicating that the vesicles mediated the escape of the siKRAS drug, and the amount of escape gradually increased with the extension of time, indicating that the targeted vesicles achieved the effective internalization of the siKRAS drug.

[0127] Experimental Example 5: In vitro transfection efficiency experiment of cRGD peptide-modified targeted polymer vesicles

[0128] This experimental example provides an in vitro transfection efficiency experiment of cRGD peptide-modified targeted polymer vesicles. In this experiment, KRAS siRNA (siKRAS), scramble siKRAS (siScramble), and negative control siRNA (siNC) were used as controls (KRAS siRNA used Free siKRAS, scramble siKRAS was based on Free siKRAS, with the sense strand shown in SEQ ID NO.7: 5'-GGTGCTGGCGAGCTGAAGATT-3' and the antisense strand shown in SEQ ID NO.8: 5'-UCUUCAGCUCGCCAGCACCTT-3'; negative control siRNA was based on Free siKRAS, with the sense strand shown in SEQ ID NO.9: 5'-UUCUCCGAACGUGUCACGUTT-3' and the antisense strand shown in SEQ ID NO.10: 5'-ACGUGACACGUUCGGAGAATT-3'). The experimental procedure is as follows:

[0129] Based on the second experiment in Experimental Example 2, qRT-PCR experiment and Western blot analysis were used to evaluate the differences in the in vitro transfection of BCP-siKRAS and cRGD-BCP-siKRAS at the mRNA and protein levels. At the same time, the change in the in vitro transfection efficiency of cRGD-BCP-siKRAS after being stored at 4°C for three weeks was investigated. The evaluation and investigation results are as Figure 10 。

[0130] Western blot analysis was as follows: Equal amounts of proteins measured using a BCA Protein Assay Kit (Pierce, purchased from Thermo Scientific) were added to 10% (g / 100 mL) SDS-PAGE, separated by gel electrophoresis, and then transferred to a polyvinylidene difluoride (PVDF) membrane (purchased from Millipore) and blocked with 5% (g / 100 mL) skim milk. Incubation was carried out with a primary antibody against KRAS (purchased from Proteintech) or a GAPDH antibody (purchased from Abcam) at 4 °C for 16 h. The expression of KRAS was visualized using an enhanced chemiluminescence (ECL) substrate (purchased from Share-Bio), and the bands were quantified using ImageJ software.

[0131] Figure 10 The results showed that the improvement in the internalization efficiency of the siKRAS drug after vesicle targeting modification was characterized at the gene level and protein level by qRT-PCR and Western blot, respectively. The vesicles could still maintain a silencing efficiency of more than 50% after 7 days of co-incubation, and after storage at 4 °C for 3 weeks, the gene silencing efficiency was comparable to that of freshly prepared vesicle preparations. This indicated that cRGD-BCP-siKRAS could improve the in vitro transfection efficiency and effectively protect the siKRAS drug.

[0132] Experimental Example 6: In vivo biodistribution experiment of cRGD peptide-modified targeted polymer vesicles

[0133] This experimental example provided an in vivo biodistribution experiment of cRGD peptide-modified targeted polymer vesicles, and the experimental procedure was as follows:

[0134] 5,000,000 PANC-1 cells (purchased from ATCC) were subcutaneously inoculated into the right dorsal and ventral sides of 4-week-old female BABL / c nude mice (purchased from Gemma) to establish a PANC-1 tumor model. When the tumor volume of the mice with the PANC-1 tumor model grew to 200 mm 3After that, grouped administration (tail vein injection) was carried out. The mice were randomly divided into two groups, including the BCP-siKRAS group administered with the non-targeted polymer vesicles prepared in Example 1 labeled with Cy5 (administration dose: 3 mg / kg, administered after dissolving in 3 mL of PBS buffer) and the cRGD-BCP-siKRAS group administered with the cRGD peptide-modified targeted polymer vesicles prepared in Example 2 labeled with Cy5 (administration dose: 3 mg / kg, administered after dissolving in 3 mL of PBS buffer). After grouping, the mice were administered via the tail vein. At 0, 1, 3.5, 7, 10, 24, and 48 h after the administration ended, an IVIS spectral imaging system (PerkinElmer) was used to image the mice to characterize the in vivo biodistribution of cRGD-BCP-siKRAS and BCP-siKRAS. Then, at 48 h, all major organs and tissues, including the heart, liver, spleen, lungs, kidneys, and tumors, were collected, imaged with the IVIS spectral imaging system to observe the fluorescence signals of the ex vivo organs, and semi-quantitative analysis was performed. The results are shown in Figure 11 .

[0135] Figure 11 The results show that after targeted modification, the vesicles can significantly improve the enrichment of the drug at the tumor site in mice.

[0136] The fluorescence signal of BCP-siKRAS reached the peak at 7 h after administration and then began to slowly decrease. The drug fluorescence signal at the tumor site of cRGD-BCP-siKRAS was significantly higher than that of BCP-siKRAS at 7 h, and the signal continued to increase and reached the peak at the monitoring time point at 10 h. The peak fluorescence signal of cRGD-BCP-siKRAS was about 2.1 times that of the peak of BCP-siKRAS, and within 7 - 48 h, the fluorescence signal of cRGD-BCP-siKRAS was always significantly higher than that of BCP-siKRAS. The change trends of the fluorescence signals of both were similar, and both began to decline after reaching the peak and remained at a certain fluorescence value for a certain period of time.

[0137] Experimental Example 7: In vivo antitumor experiment of cRGD peptide-modified targeted polymer vesicles

[0138] This experimental example provides an in vivo antitumor experiment of cRGD peptide-modified targeted polymer vesicles. The experimental procedure is as follows:

[0139] Experiment 1: A PANC-1 tumor model was established by subcutaneously inoculating 5,000,000 PANC-1 cells (purchased from ATCC) on the right dorsoventral side of 4-week-old female BABL / c nude mice (purchased from Gemma). When the tumor volume of the PANC-1 tumor model mice grew to 100 mm 3After that, group administration (tail vein injection) was carried out. The mice were randomly divided into four groups, including the PBS group administered with PBS buffer (administration dose 3 mL), the BCP-siKRAS group administered with the non-targeted polymer vesicles prepared in Example 1 (administration dose 3 mg / kg, dissolved in 3 mL PBS buffer before administration), the cRGD-BCP-siKRAS low-dose group administered with the cRGD peptide-modified targeted polymer vesicles prepared in Example 2 (administration dose 1 mg / kg, dissolved in 3 mL PBS buffer before administration), and the cRGD-BCP-siKRAS high-dose group administered with the cRGD peptide-modified targeted polymer vesicles prepared in Example 2 (administration dose 3 mg / kg, dissolved in 3 mL PBS buffer before administration), and this day was designated as Day 0. After grouping, the mice were administered via the tail vein, one injection every four days for a total of four injections; during the administration period, the body weight changes of the mice were observed every two days, the tumors of the mice were measured using calipers, and the mouse tumor volume was calculated according to the formula V = 1 / 2 × L × W 2 where L and W are the tumor sizes at the longest and widest points respectively, and the results are as Figure 12 ; on the day after the end of administration, one mouse was randomly selected from each group, ex vivo photos of the tumors were taken, the tumors and the main organs (heart, liver, spleen, lung, kidney) were collected and histological analysis was performed, and the results are shown in Figures 12 - 14 , and the remaining mice were continuously observed for survival after the end of treatment. Among them, the histological analysis was as follows: the excised organs and tumors were sectioned and stained with hematoxylin and eosin (H&E), and the tumor sections were subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL); the H&E images were observed using a digital microscope (Olympus BX41), and the fluorescence images were obtained by LSM710 CLSM.

[0140] Experiment 2: A PANC-1 tumor model was established by subcutaneously inoculating 5,000,000 PANC-1 cells (purchased from ATCC) into the right dorsoventral subcutaneous region of 4-week-old female BABL / c nude mice (purchased from Gemma). When the tumor volume of the mice in the PANC-1 tumor model grew to 100 mm 3After that, grouped administration (tail vein injection) was carried out. The mice were randomly divided into five groups, including the PBS group administered with PBS buffer (administration dose: 3 mL), the cRGD-BCP-siScramble-3G group administered with cRGD-BCP-siScramble-3G (administration dose: 3 mg / kg, dissolved in 3 mL PBS buffer before administration, and cRGD-BCP-siScramble-3G was obtained by replacing siKRAS in siKRAS-5-GEM with siScramble on the basis of cRGD-BCP-siKRAS-3G), the cRGD-BCP-siKRAS group administered with the targeted polymer vesicles modified with cRGD peptide prepared in Example 2 (administration dose: 3 mg / kg, dissolved in 3 mL PBS buffer before administration), the low-dose cRGD-BCP-siKRAS-3G group administered with the targeted polymer vesicles co-modified with cRGD peptide and gemcitabine prepared in Example 3 (administration dose: 1 mg / kg, dissolved in 3 mL PBS buffer before administration), and the high-dose cRGD-BCP-siKRAS-3G group administered with the targeted polymer vesicles co-modified with cRGD peptide and gemcitabine prepared in Example 3 (administration dose: 3 mg / kg, dissolved in 3 mL PBS buffer before administration), and this day was designated as Day 0. After the grouping was completed, the mice were administered via the tail vein, one injection every four days for a total of four injections. During the administration period, the body weight changes of the mice were observed every two days, and the tumors of the mice were measured using calipers. The tumor volume of the mice was calculated according to the formula V = 1 / 2 × L × W 2 where L and W are the tumor sizes at the longest and widest parts respectively. The results are as follows Figure 15 ; One day after the administration ended, one mouse was randomly selected from each group to take in vitro photos of the tumors, and the tumors and major organs (heart, liver, spleen, lung, kidney) were collected for histological analysis. The results are shown in Figures 15 - 17, the remaining mice continued to be observed for survival after the treatment. Among them, histological analysis was as follows: the excised organs and tumors were sectioned and stained with hematoxylin and eosin (H&E), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed on the tumor sections; H&E images were observed using a digital microscope (Olympus BX41), and fluorescence images were obtained by LSM710 CLSM. Several samples were randomly cut from each tumor tissue, cut into pieces without lumps, placed in a mortar, and ground into powder under liquid nitrogen protection. Total RNA and proteins were extracted respectively for qRT-PCR and WB experiments to determine the silencing efficiency of the KRAS gene in the tumor sites of mice and the changes in related pathway proteins (AKT, ERK). To study the phosphorylation of AKT and ERK proteins by extracting pathway proteins, before protein extraction, EGF (50 ng / mL) was added and incubated for 10 min. For the WB experiment of the extracted proteins, phosphatase inhibitors needed to be added additionally to the RIPA protein lysate used. The electrophoresis buffer and transfer membrane solution needed to be freshly prepared and blocked overnight at 4°C with 5% (w / v, g / 100 mL) BSA. p-AKT antibody (Cell Signaling), AKT antibody (Cell Signaling), p-ERK antibody (Cell Signaling), ERK antibody (Beyotime), p-P70S6K antibody (Cell Signaling), P70S6K antibody (Beyotime), and GAPDH antibody (Abcam) were incubated at room temperature for 3 h respectively. The obtained bands were analyzed using ImageJ software, and the results are shown in Figure 18 .

[0141] Figure 12 The results showed that BCP-siKRAS could significantly inhibit the tumor growth of the PANC1 pancreatic cancer model and significantly prolong the survival period of mice, suggesting the feasibility of treating KRAS-mutated pancreatic cancer by siKRAS. After cRGD targeting modification, cRGD-BCP-siKRAS could further improve the tumor inhibitory effect of vesicle drugs and reduce the administration dose of siKRAS drugs. At the administration dose of 3 mg / kg, the median survival period of mice was extended by 3.6 times compared with the PBS group, and there was no obvious change in the body weight of mice during the treatment, indicating that the targeted vesicle cRGD-BCP-siKRAS had no obvious toxic and side effects while effectively inhibiting tumors.

[0142] Figure 13The results showed that compared with the PBS group, obvious tumor cell shrinkage and nuclear chromatin condensation could be observed in the HE sections of tumors in the BCP-siKRAS and cRGD-BCP-siKRAS groups, and tumor cell apoptosis and necrosis occurred. Moreover, the number of apoptotic and necrotic cells in the cRGD-BCP-siKRAS group was significantly more than that in the BCP-siKRAS group. The TUNEL results were similar to the HE results, suggesting that cRGD modification could further improve the tumor inhibitory effect of the vesicles.

[0143] Figure 14 The results showed that no obvious damage to the organs of the mice was caused during the treatment process, indicating that the vesicle preparation had low toxicity and high safety.

[0144] Figure 15 The results showed that at a dose of 3 mg / kg, siKRAS-3G could significantly delay tumor growth compared with siKRAS (**p), and siKRAS-3G could effectively inhibit tumors even at 1 mg / kg, achieving the same median survival time (MST) as 3 mg / kg siKRAS, indicating that the synergistic effect of siKRAS-3G could effectively reduce the treatment dose. And 3 mg / kg siKRAS-3G could significantly prolong the survival time of mice compared with the PBS, siScramble-3G, and siKRAS groups. The MST increased from 24 days in PBS, 28 days in siScramble-3G, and 53 days in siKRAS to 74 days in siKRAS-3G (3 mg / kg).

[0145] Figure 16 The results showed that by performing section analysis on tumor tissues, it was found that compared with PBS and siScramble-3G, obvious tumor cell shrinkage and nuclear chromatin condensation could be observed in the H&E sections of siKRAS and siKRAS-3G, and tumor cell apoptosis and necrosis occurred. Moreover, the number of apoptotic and necrotic cells in siKRAS-3G (3 mg / kg) was significantly more than that in other groups.

[0146] Figure 17 The results showed that the TUNEL staining results were similar to the H&E results. The decrease in PCNA expression and the increase in Cleaved caspase-3 expression in the tumor tissues treated with siKRAS-3G (3 mg / kg), and the absence of any obvious damage in normal organs all indicated that siKRAS-3G could effectively inhibit the proliferation of tumor cells and promote apoptosis, and had no obvious toxic side effects, indicating the feasibility of achieving highly efficient synergistic treatment of pancreatic cancer using cRGD-BCP-siKRAS-3G.

[0147] Figure 18The results showed that the analysis of KRAS gene silencing and the inhibition of pathway protein phosphorylation in tumor tissues found that siKRAS (3 mg / kg) and siKRAS-3G (1, 3 mg / kg) could significantly downregulate KRAS mRNA (***p), and the silencing efficiencies were 91.9%, 84.5% and 98.2% respectively. The tumor WB experiment showed that siKRAS could achieve a 50.1% protein downregulation, while siKRAS-3G could further significantly downregulate the KRAS protein (*p), with a downregulation of 69.9%, which was significantly better than other groups. The phosphorylation analysis of pathway proteins showed a similar inhibitory effect in vitro. siKRAS-3G could enhance the inhibition of the phosphorylation of RAS downstream pathway proteins and achieve a synergistic treatment for pancreatic cancer.

[0148] Experimental Example 8: Experiment on the effect of the number of GEM monomers in cRGD peptide-modified targeted polymer vesicles on apoptosis

[0149] This experimental example provides an experiment on the effect of the number of GEM monomers in cRGD peptide-modified targeted polymer vesicles on apoptosis. The experimental procedure is as follows:

[0150] Take a tube of PANC-1 cells (purchased from ATCC) and inoculate them into 8 mL of DMEM medium (purchased from Gibco) containing 10% (v / v) FBS. After culturing at 37 °C until the cell density reaches 90%, add 500 μL of trypsin (purchased from Gibco) and incubate at 37 °C for 2 min to digest into a single-cell suspension of PANC-1; count, and seed 4×10 5Inoculate the PANC-1 single-cell suspension into a 6-well plate at an inoculum density of [number of cells] and incubate at 37 °C for 24 h. After incubation, use PBS buffer as a control (PBS), and add the non-targeted polymer vesicles (BCP-siKRAS) prepared in Example 1, the cRGD peptide-modified targeted polymer vesicles (cRGD-BCP-siKRAS) prepared in Example 2, and the cRGD peptide- and gemcitabine co-modified targeted polymer vesicles (cRGD-BCP-siKRAS-1G to cRGD-BCP-siKRAS-5G) prepared in Example 3 to the 6-well plate at a final concentration of 200 nM per well and incubate again at 37 °C for 48 h. After the second incubation, collect the cells, wash with PBS buffer and centrifuge for 5 min. Aspirate the supernatant and centrifuge again for 5 min. Select the Annexin-APC / 7-AAD apoptosis detection kit (purchased from Linker Biotechnology), add Annexin-APC and 7-AAD dyes according to the kit instructions and incubate in the dark for 15 min, then detect cell apoptosis by flow cytometry (BD FACS Calibur). Through the apoptosis experiment, determine the optimal number of GEM modifications for the siKRAS sequence, and at the same time compare the difference between co-delivery of the two covalently modified drugs as a single drug and co-administration of the two drugs separately. The comparison results are shown in Figure 19 ( Figure 19 in which, Empty polymersomes are empty vesicles without carrying drugs, siKRAS carries drugs, siKRAS + free 3GEM means siKRAS drug + free 3GEM, and free 27GEM is free 27GEM).

[0151] Figure 19 The results show that when the number of GEM monomers covalently modified on siKRAS increases to 3, the amount of cell apoptosis increases significantly, and even if the amount of GEM introduced into the sequence increases by 3 times, the effect on cell apoptosis is not obvious. This suggests that there may be a certain synergistic effect between the two, and a strong pro-apoptotic effect is produced after reaching a certain ratio. Moreover, this effect requires co-delivery of the two covalently modified drugs as a single drug to occur significantly, and co-administration of the two drugs separately is not obvious.

[0152] Experimental Example 9: Synergistic study experiment on cell proliferation inhibition of cRGD peptide-modified targeted polymer vesicles

[0153] This experimental example provides a synergistic study experiment on cell proliferation inhibition of cRGD peptide-modified targeted polymer vesicles. The experimental procedure is as follows:

[0154] Experiment 1: One tube of PANC-1 cells (purchased from ATCC) was inoculated into 8 mL of DMEM medium (purchased from Gibco) containing 10% (v / v) FBS. After culturing at 37 °C until the cell density reached 90%, 500 μL of trypsin (purchased from Gibco) was added and incubated at 37 °C for 2 min to digest into a PANC-1 single-cell suspension; the cells were counted, and the PANC-1 single-cell suspension was inoculated into a 96-well plate at an inoculation amount of 10,000 cells per well and incubated at 37 °C for 24 h; after the incubation ended, using PBS buffer as a control (PBS), the cRGD peptide-modified targeted polymer vesicles (cRGD-BCP-siKRAS) prepared in Example 2 and the cRGD peptide and gemcitabine co-modified targeted polymer vesicles (cRGD-BCP-siKRAS-1G to cRGD-BCP-siKRAS-5G) prepared in Example 3 were added to the 96-well plate at a final concentration of 0.1, 1, 10, 100, 500, 1000 nM per well and incubated again at 37 °C for 48 h; after the second incubation ended, the same volume of fresh DMEM medium was replaced in each well, and 100 μL of fresh DMEM medium containing 10 μL of CCK-8 reagent (purchased from Dojindo) was added to each well, incubated at 37 °C for 1 h, and the absorbance at 450 nm was measured using a microplate reader. The results are shown in Figure 20 ( Figure 20 In, siKRAS+siScramble-3G refers to the combined use of the siKRAS sequence + siScramble-3G, and siScramble-3G is siScramble conjugated with 3 gemcitabines).

[0155] Experiment 2: One tube of PANC-1 cells (purchased from ATCC) was inoculated into 8 mL of DMEM medium (purchased from Gibco) containing 10% (v / v) FBS and cultured at 37 °C until the cell density reached 90%. Then, 500 μL of trypsin (purchased from Gibco) was added and incubated at 37 °C for 2 min to digest into a PANC-1 single-cell suspension. After counting, the PANC-1 single-cell suspension was inoculated into a 6-well plate at an inoculation amount of 40,000 cells per well and incubated at 37 °C for 24 h. After incubation, using PBS buffer as a control (PBS), the cRGD peptide-modified targeted polymeric vesicles (cRGD-BCP-siKRAS) prepared in Example 2 and the cRGD peptide and gemcitabine co-modified targeted polymeric vesicles (cRGD-BCP-siKRAS-1G to cRGD-BCP-siKRAS-5G) prepared in Example 3 were added to the 6-well plate at a final concentration of 0.1, 1, 10, 100, and 500 nM per well and incubated at 37 °C for another 24 h. After the second incubation, the cells were collected, washed with PBS buffer and centrifuged for 5 min. After aspirating the supernatant, centrifugation was performed again for 5 min. The Annexin-APC / 7-AAD apoptosis detection kit was selected. According to the kit instructions, Annexin-APC and 7-AAD dyes were added respectively and incubated in the dark for 15 min, and then the cell apoptosis was detected by flow cytometry (BD FACS Calibur). The results are shown in Figure 20 .

[0156] Experiment 3: Experimental mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and raised under SPF conditions. The animal research protocol (ASP) was approved by the Institutional Animal Care and Use Committee (IACUC) of Gemma Gene. All in vivo experiments were conducted according to the IACUC guidelines. 5,000,000 PANC-1 cells were subcutaneously inoculated into the right dorsal and ventral sides of 4- to 5-week-old Balb / c nude female mice to establish a PANC-1 tumor model. When the PANC-1 tumor grew to approximately 100 mm 3 , the mice were randomly divided into five groups (n = 8): PBS, cRGD-BCP-siScramble-3G (3 mg / kg), cRGD-BCP-siKRAS (3 mg / kg), cRGD-BCP-siKRAS-3G (1 mg / kg), and cRGD-BCP-siKRAS-3G (3 mg / kg), and this day was designated as day 0. All groups were administered once every 4 days for a total of 4 times. During the treatment period, the body weight and tumor volume (V) of the mice were monitored regularly. The tumor length (L) and width (W) were measured using vernier calipers, and according to the formula V = 1 / 2 × L × W 2Calculate the tumor volume, where L and W are the dimensions at the longest and widest points of the tumor, respectively. On the day after the end of drug administration, three mice in each group were euthanized, and the tumors and major organs (heart, liver, spleen, lungs, kidneys) were collected. After the tumors were photographed and weighed, the tumor inhibition rate (TIR) was calculated according to the following formula: TIR (%) = [(tumor volume in the treatment group - 1) / tumor weight in the PBS group] × 100%. The obtained major organs were subjected to H&E staining to analyze the toxic and side effects of each group during the treatment period. The tumor tissues were observed for cell morphological changes by H&E staining, apoptosis was analyzed by TUNEL and Cleaved Caspase-3, and the cell proliferation status was analyzed by PCNA. The remaining mice (n = 5) were used to observe the survival period. The criteria for judging mouse death were: death, weight loss exceeding 15%, or tumor volume reaching 2000 mm 3 The experimental results are shown in Figure 21 .

[0157] Experiment 4: Using RNA-seq technology, study the genetic differences of PANC-1 cells transfected with siKRAS, siScramble-3G, and siKRAS-3G by cRGD-BCP, and then study the effects of cRGD-BCP-siKRAS-3G at the gene level. PANC-1 cells were seeded in 6-well plates at a density of 400,000 cells per well for 24 h. Then, siKRAS, siScramble-3G, and siKRAS-3G (concentration: 100 nM) loaded with cRGD-BCP were added and incubated for 48 h. The cells in each group were collected, lysed on ice with TRIzol (Invitrogen), and the RNA-seq experiment was completed by Novogene, including RNA extraction and detection, library construction and quality control, and sequencing. The obtained raw data was subjected to differential analysis (cluster heat map, volcano plot) and enrichment analysis (GO function, KEGG pathway) using NovoMagic. The research results are shown in Figure 22 .

[0158] Figure 20The results showed that siKRAS and siScramble-3G, after being loaded by cRGD-BCP and mixed and delivered to PANC-1 cells respectively, could enhance toxicity on the basis of their individual use and produce a synergistic effect of inhibiting cell proliferation (CI < 1.0), but this synergistic enhancement was not obvious. However, when the two drugs were covalently modified to form a single drug siKRAS-3G, a strong synergistic inhibitory effect (CI < 0.5) was produced. This indicated that the inhibitory effect of the two drugs delivered as a single drug on tumor cells was significantly better than the way of co-administering the two drugs. Combining with the previous apoptosis results, it was found that the effect of siKRAS alone on apoptosis was not obvious. Therefore, studying the synergy of the two drugs by means of apoptosis might better show the superiority of the combination. The apoptosis results showed that after incubation for 24 h, neither siKRAS nor siScramble-3G showed obvious apoptosis, while after incubation with siKRAS-3G for 24 h, a large number of early apoptotic cells appeared. Moreover, through the analysis of the synergy index of the number of viable cells at different concentrations in each group, it was also found that siKRAS-3G could produce a very strong synergistic effect.

[0159] Figure 21 The results showed that siKRAS-3G had an enhanced inhibitory effect on the protein phosphorylation of both RAF / MEK / ERK and PI3K / AKT / mTOR pathways. Among them, the ratio of p-AKT / AKT decreased from 0.646 and 0.512 when siScramble-3G and siKRAS were used alone to 0.192 for siKRAS-3G, the ratio of p-P70S6K / P70S6K decreased from 0.770 and 0.519 when siScramble-3G and siKRAS were used alone to 0.331 for siKRAS-3G, and the ratio of p-ERK / ERK decreased from 0.896 and 0.737 when siScramble-3G and siKRAS were used alone to 0.585 for siKRAS-3G. At the same time, siKRAS-3G could also significantly increase the inhibition of KRAS protein (*p), which was also similar to the reported inhibition of KRAS protein and phosphorylation inhibition of pathway proteins in the literature. In pancreatic cancer with KRAS mutation, the mutant activation of KRAS protein will activate downstream effector signaling pathways, such as RAF / MEK / ERK and PI3K / AKT / mTOR pathways, promoting tumor cell proliferation and survival. Among them, the activation of PI3K / AKT / mTOR pathway is related to the resistance of small molecule anti-tumor drugs (such as gemcitabine). The strong synergistic effect of siKRAS-3G may be due to the significant inhibition of KRAS protein and the enhanced inhibition of the phosphorylation levels of RAF / MEK / ERK and PI3K / AKT / mTOR signaling pathways.

[0160] Figure 22The results showed that after treatment with siScramble-3G, siKRAS, and siKRAS-3G, there were differences in genes among the groups compared with the PBS group, and there were also differences between siKRAS-3G and siKRAS. Further differential analysis of siKRAS-3G and siKRAS showed that the volcano plot revealed 580 upregulated genes and 316 downregulated genes in siKRAS-3G compared with siKRAS. GO functional enrichment analysis of the downregulated genes found that the differential genes were enriched in both metabolic processes and ion channel activities. The KEGG pathway enrichment analysis also showed that siKRAS-3G increased the enrichment of glycolysis, receptor interaction, and tumor-related signaling pathways compared with KRAS, which would affect these pathways.

[0161] Overall, the strong synergistic antitumor effect of siKRAS-3G may be due to the significant inhibition of the RAF / MEK / ERK and PI3K / AKT / mTOR signaling pathways. RNA-seq analysis also indicated that there were indeed differences in gene enrichment in these signaling pathways between siKRAS-3G and siKRAS. At the same time, it was found that siKRAS-3G affected metabolic processes, ion channel activities, and the glycolysis pathway, all of which are also related to tumor growth and drug resistance, which may also be the reasons for affecting tumor growth and improving the antitumor effect.

[0162] Experimental Example 10: In vitro Endocytosis and Endosomal Escape Experiments of cRGD Peptide-Modified Targeted Polymer Vesicles

[0163] This experimental example provides in vitro endocytosis and endosomal escape experiments of cRGD peptide-modified targeted polymer vesicles. The experimental procedure is as follows:

[0164] cRGD-BCP-siKRAS (Cy5) and non-targeted BCP-siKRAS (Cy5) were used to visualize the siRNA situation in cells or mice. Vesicle preparations were prepared by mixing 1 wt.% siNC-Cy5 (Cy5 labeled at the 5' end of the sense strand) with siKRAS.

[0165] PANC-1 cells were seeded in 6-well plates (500,000 cells / well) and cultured for 24 h. cRGD-BCP-siKRAS (Cy5) with different cRGD densities was added and incubated for 60 min. After washing and resuspending in PBS, analysis was performed by flow cytometry (n = 3). For confocal experiments, PANC-1 cells were seeded at a density of 200,000 cells / well on glass-bottom dishes (35 mm, Biosharp) and incubated for 24 h. Then, cRGD-BCP-siKRAS (Cy5) with a cRGD density of 15.7% and BCP-siKRAS (Cy5) (siRNA: 200 nM) were added and incubated for 4 h. After washing, the cell nuclei were stained with DAPI. Fluorescence images were observed using CLSM.

[0166] To study the endo / lysosomal escape of siRNA, cRGD-BCP-siKRAS (Cy5) with a cRGD density of 15.7% (siRNA: 200 nM) was incubated with PANC-1 cells for 2, 4, 8, 16, or 24 h. Then, 50 nM Lysotracker green was added to stain the endo / lysosomes for 45 min. The cells were fixed with 4% paraformaldehyde, washed, stained with DAPI, and placed under CLSM for observation, photographing, and analysis of the mean fluorescence intensity (MFI) and Pearson correlation coefficient (p) (n = 3).

[0167] Figure 23 Flow cytometry results showed that the vesicle uptake was significantly dependent on the cRGD density, and the highest endocytosis was achieved at a cRGD density of 15.7%. Gene silencing results also indicated that cRGD-BCP-siKRAS with a cRGD density of 15.7% had a significantly better effect on downregulating KRAS G12D mRNA than other samples, and the gene silencing efficiency was the highest. This suggests that a higher cRGD density may affect its binding to integrin receptors, which is similar to previous reports that the more targeting ligands modified on nanocarriers are not necessarily better, and there needs to be a certain spacing between targeting ligands for better binding to receptors. Unless otherwise stated, the siKRAS DLC of cRGD-BCP-siKRAS is 15 wt.%, and the cRGD density is 15.7%.

[0168] Figure 24The results showed that compared with BCP-siKRAS, cRGD-BCP-siKRAS could effectively improve the internalization of siKRAS. With the prolongation of the incubation time, the endocytosis of cRGD-BCP-siKRAS gradually increased. Whether cRGD-BCP-siKRAS can help siKRAS achieve endosomal escape is crucial for siRNA therapy. We observed the color coincidence (yellow) of siKRAS (Cy5, red) and endo / lysosomes (Lysotracker, green) at different times and analyzed it using the Pearson correlation coefficient (p). The results showed that the co-localization had been increasing (yellow) until 4 h when p reached the peak (1.0), indicating that cRGD-BCP-siKRAS had been endocytosed and was mainly in endo / lysosomes at this time. From 4 to 24 h, p decreased from 1.0 to 0.55, and the red color increased while the yellow color decreased in the merged images, indicating that siKRAS had escaped from endo / lysosomes.

[0169] Experimental Example 11: In vitro gene silencing efficiency experiment of cRGD peptide-modified targeted polymer vesicles

[0170] This experimental example provides an in vitro gene silencing efficiency experiment of cRGD peptide-modified targeted polymer vesicles. The experimental process is as follows:

[0171] To determine the in vitro gene silencing efficiency of cRGD-BCP-siKRAS (unless otherwise specified, 15.7% density cRGD and 15 wt.% drug loading ratio were used subsequently), PANC-1 cells were transfected with BCP-siKRAS, BCP-siScramble, and cRGD-BCP-siKRAS at transfection concentrations of 50, 100, and 200 nM (unless otherwise specified, the sample addition concentration refers to the final concentration of siRNA, and the transfection time was 6 h). After transfection, the incubation times were 1, 2, 3, 4, and 7 d (n = 3). Before transfection, PANC-1 cells were seeded in 12-well plates at a density of 200,000 cells per well and cultured for 24 h. The gene silencing efficiency was evaluated by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and Western blotting (WB). The gene silencing efficiency (%) was calculated by comparing with the value of the PBS group (= 100%). To evaluate the effect of storage time on the gene silencing ability, the relative KRAS mRNA level of cRGD-BCP-siKRAS after storage for 3 weeks was similarly determined (n = 3).

[0172] According to the instructions, siKRAS transfection in PANC-1 cells was carried out using lipofectamine 2000 (Invitrogen) at 200 nM for 4 and 7 d as a positive control. Use RNA-easy TMTotal RNA was isolated from PANC-1 cell samples using Isolation Reagent (R701, Vazyme), and the isolated total RNA was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (R323-01, Vazyme). Subsequently, qRT-PCR experiments were performed using Taq Pro Universal SYBR qPCR Master Mix (Q712-03, Vazyme). The PCR reaction conditions were as follows: 94°C for 3 min, followed by 40 cycles of 94°C for 12 s, 62°C for 30 s, and 72°C for 30 s.

[0173] For WB experiments, total proteins from 200 nM samples incubated for 3 d in each group were extracted using RIPA protein lysate (P0013B) premixed with protease inhibitor, centrifuged at 12,000 rpm at low temperature for 15 min, and the supernatant was taken to calibrate the protein concentration using a BCA detection kit (Pierce / Thermo). Then, bromophenol blue protein loading buffer was added and mixed well, and after heating at 95°C in a water bath for 10 min for denaturation, it was centrifuged at 12,000 rpm for 1 min. The same mass of protein from each group was added to 10% SDS-PAGE for gel electrophoresis separation. The separation conditions were 80 V for 30 min first, and then 120 V for 60 min. After electrophoresis, the protein bands on the gel were transferred to a PVDF membrane (Millipore) by wet transfer (350 mA, 90 min), blocked with 5% skim milk at room temperature for 2 h, then the membrane was incubated with KRAS antibody (Proteintech) or GAPDH antibody (Abcam) overnight at 4°C, incubated with HRP secondary antibody for 2 h at room temperature, and then the expression of KRAS or GAPDH was observed using a chemiluminescent electrophoresis gel imager with ECL substrate (Share-Bio), and the bands were quantitatively analyzed using ImageJ software (n = 3).

[0174] The gene silencing effect of cRGD-BCP-siKRAS was further studied by qRT-PCR and WB in relation to siRNA dose and incubation time. Figure 25It was shown that at 1, 2, and 3 days of incubation, cRGD-BCP-siKRAS could significantly improve the gene silencing efficiency, and the siKRAS mRNA level decreased with the increase in the siKRAS concentration. It should be noted that cRGD-BCP-siKRAS achieved the highest silencing efficiency of approximately 90% at 48 and 72 h at 200 nM and still maintained approximately 60% even after 7 days, which was much better than the KRAS mRNA silencing efficiency of 75% or 67% in the published articles. cRGD-BCP-siKRAS was very robust, and its gene silencing efficiency showed almost no change compared with the fresh samples after being stored at 4 °C for 3 weeks. The WB results showed that the protein inhibition induced by cRGD-BCP-siKRAS was significantly better than that of BCP-siKRAS. These results indicated that cRGD-BCP-siKRAS could effectively protect siKRAS and improve the transfection efficiency in vitro.

[0175] One of the key indicators for evaluating the quality of siRNA drugs is their gene silencing efficiency on the target mRNA, and the modification of the sequence often leads to a decrease in the silencing efficiency, thus affecting the drug efficacy. Figure 26 In this study, by investigating the gene silencing efficiency of siKRAS-3G and siKRAS-5G loaded with cRGD-BCP, it was found that at a low concentration (50 nM) at 24 h of incubation, the increase in the number of modified GEMs would reduce the silencing effect of siKRAS, but there was no significant difference, and it would disappear with the extension of the incubation time and the increase in the transfection concentration, indicating that the modification of GEM monomers did not affect the gene silencing effect of siKRAS itself. The transfection efficiency of cRGD-BCP was not inferior to that of lipofectamine. When the incubation time was extended to 4 and 7 days, a silencing efficiency of 60% could still be achieved, and the modification of GEM monomers did not affect the long-term effectiveness of the siKRAS silencing efficiency.

[0176] Obviously, the above embodiments are only examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A cRGD peptide-modified targeted polymeric vesicle, characterized in that The components of the cRGD peptide-modified targeted polymeric vesicles include the targeted polymer cRGD-PEG-P(TMC-co-DTC), the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 and the siKRAS drug.

2. The cRGD peptide-modified targeted polymeric vesicle according to claim 1, characterized in that In the cRGD peptide-modified targeted polymeric vesicles, the concentration of the targeted polymer cRGD-PEG-P(TMC-co-DTC) is 0.5 - 0.8 mg / mL, and the concentration of the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 is 3 - 5 mg / mL, and the concentration of the siKRAS drug is 0.2 - 1 mg / mL.

3. The cRGD peptide-modified targeted polymeric vesicle according to claim 1 or 2, characterized in that In the cRGD peptide-modified targeted polymeric vesicles, the concentration of the targeted polymer cRGD-PEG-P(TMC-co-DTC) is 0.6 mg / mL, and the concentration of the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 is 0.6 mg / mL, and the concentration of the siKRAS drug is 3.4 mg / mL.

4. The cRGD peptide-modified targeted polymeric vesicle according to any one of claims 1 to 3, characterized in that the composition of the cRGD peptide-modified targeted polymeric vesicle further comprises a solvent and a buffer solution; in the cRGD peptide-modified targeted polymeric vesicle, the volume ratio of the solvent to the buffer solution is 1:5 to 10.

5. The cRGD peptide-modified targeted polymeric vesicle according to claim 4, characterized in that in the cRGD peptide-modified targeted polymeric vesicle, the volume ratio of the solvent to the buffer solution is 1:

9.

6. The cRGD peptide-modified targeted polymeric vesicle according to claim 4 or 5, characterized in that the siKRAS drug is a siKRAS-gemcitabine conjugate; the solvent is N,N-dimethylformamide; the buffer solution is a PB buffer solution.

7. The cRGD peptide-modified targeted polymeric vesicle according to any one of claims 4 to 6, characterized in that the concentration of the buffer solution is 2 to 10 mM and the pH is 7.

4.

8. A method for preparing the cRGD peptide-modified targeted polymeric vesicle according to any one of claims 1 to 7, characterized in that The method is as follows: Mix cRGD peptide and Mal-PEG-P(TMC-co-DTC), and carry out Michael addition reaction to obtain the targeted polymer cRGD-PEG-P(TMC-co-DTC); Mix the targeted polymer cRGD-PEG-P(TMC-co-DTC), the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 and the siKRAS drug to obtain the targeted polymer vesicles modified with cRGD peptide.

9. The method for preparing the cRGD peptide-modified targeted polymeric vesicle according to any one of claims 1 to 7 as claimed in claim 8, characterized in that The method is as follows: dissolve cRGD peptide in a solvent to obtain a cRGD peptide solution; dissolve Mal-PEG-P(TMC-co-DTC) in a solvent to obtain a Mal-PEG-P(TMC-co-DTC) solution; after mixing the cRGD peptide solution and the Mal-PEG-P(TMC-co-DTC) solution, carry out a Michael addition reaction under the protection of nitrogen to obtain a reaction product; dialyze the reaction product using the solvent as a dialysis medium, then precipitate it in cold diethyl ether, and finally filter and dry the precipitate to obtain the targeted polymer cRGD-PEG-P(TMC-co-DTC); dissolve the carrier polymer PEG-P(TMC-co-DTC)-PEI 1200 in a solvent to obtain a carrier polymer solution; dissolve the targeted polymer cRGD-PEG-P(TMC-co-DTC) in a solvent to obtain a targeted polymer solution; mix the targeted polymer solution and the carrier polymer solution to obtain a polymer mixed solution; dissolve the siKRAS drug in a buffer solution to obtain a siKRAS drug solution; adding the polymer mixed solution to the bottom of the siKRAS drug and then standing to obtain a mixed solution A; stirring the mixed solution A to obtain a mixed solution B; dialyzing the mixed solution B using the buffer solution as a dialysis medium to obtain the cRGD peptide-modified targeted polymeric vesicle.

10. Use of the cRGD peptide-modified targeted polymeric vesicle according to any one of claims 1 to 7 or the method for preparing the cRGD peptide-modified targeted polymeric vesicle according to claim 8 or 9 for preparing a targeted therapeutic drug for cancers caused by KRAS mutations.

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