An siRNA drug preparation for pancreatic cancer treatment and a preparation method and application thereof

Abstract

The application discloses an siRNA drug preparation for treating pancreatic cancer and a preparation method and application thereof. G12D The siRNA drug preparation is composed of small interfering RNA (siRNA) targeting KRAS mRNA, a carrier and a solvent, wherein the carrier is composed of nucleotide TPS, TPO, CPS, CPO or DNCA, cationic lipid CLD and auxiliary lipid DSPE-PEG or DSPE-PEG-cRGD. G12D The novel preparation obtained by the small interfering RNA (siG12D) targeting KRAS mRNA can be efficiently targeted to a mouse orthotopic pancreatic tumor site (about 20%) after being injected through a tail vein, significantly inhibits tumor growth, and has no hepatorenal toxicity and immunogenicity. The application lays a foundation for the wide clinical application of the anti-pancreatic cancer siRNA drug, and provides a technical means for conquering difficult drug targets such as KRAS.

Description

Technical Field

[0001] This invention relates to an siRNA drug formulation for the treatment of pancreatic cancer and its preparation method, as well as its application in targeted therapy for pancreatic cancer. This invention belongs to the field of biomedical technology. Background Technology

[0002] Endogenous nucleic acids in organisms carry important genetic information and participate in a wide variety of life processes by encoding functional proteins or directly in the form of oligonucleotides. The rational introduction of exogenous functional oligonucleotides can achieve specific regulation of certain physiological or pathological processes. Currently, the most studied functional oligonucleotides include small interfering RNAs (siRNAs), antisense oligonucleotides (AONs), ribozymes, microRNAs (miRNAs), aptamers, mRNA, and plasmids. They exert biological functions by specifically binding to and silencing specific complementary genes, specifically recognizing targets by forming specific three-dimensional conformations, or expressing sequence-encoded proteins in the cytoplasm.

[0003] Nucleic acid drugs primarily target intracellular targets; therefore, safely and efficiently delivering these drugs to target tissues, overcoming extracellular and intracellular environmental barriers, and achieving successful transfection and binding to targets are critical challenges in their application. siRNA, as a representative nucleic acid drug, treats diseases by binding to mRNA and activating RISC cleavage of the mRNA, thereby inhibiting the expression of disease proteins. In recent years, the US Food and Drug Administration and the European Medicines Agency have approved four siRNA drugs. The first siRNA drug, Onpattro, used an LNP delivery strategy, employing the ionizable cationic liposome DLin-MC3-DMA to bind siRNA via electrochemical interaction. The formulation can reach the liver after intravenous injection and be taken up by hepatocytes. However, cationic liposomes, due to their excessive positive charge, readily adsorb various serum proteins in the blood, reducing delivery efficiency and causing immunogenicity. The other three drugs all use GalNAc conjugation at the 3' end of the positive strand for delivery, but this delivery strategy is only suitable for liver targeting, limiting its application.

[0004] The KRAS proto-oncogene encodes a small GTPase belonging to the RAS superprotein family. Intracellularly, the KRAS protein transitions between inactive and activated states; it is inactive when bound to guanine diphosphate (GDP) and activated when bound to guanine triphosphate (GTP). In most cells, KRAS is inactive. When activated, it can activate multiple downstream signaling pathways, including the MAPK, PI3K, and Ral-GEF signaling pathways. These pathways play crucial roles in promoting cell survival, proliferation, and cytokine release. Therefore, KRAS mutations lead to overactivation of downstream pathways, which is closely related to cancer development and progression. In human cancers, KRAS gene mutations occur in approximately 90% of pancreatic cancers, 30-40% of colon cancers, 15-20% of lung cancers (mostly non-small cell lung cancers), and 17% of endometrial cancers. It also occurs in other cancer types such as bile duct cancer, cervical cancer, bladder cancer, liver cancer, and breast cancer. Of the KRAS gene mutations, 97% involve mutations at amino acid residues 12 or 13, with the most prevalent being G12D, G12V, and G13D mutations. Structural studies have shown that these mutations largely interfere with KRAS's ability to hydrolyze GTP.

[0005] Scientists at the MD Anderson Cancer Center in Texas have successfully delivered siRNA (ss 5'-GUU GGA GCU GAU GGC GUA Gtt-3', as 5'-CUA CGC CAU CAG CUC CAA Ctt-3', tentatively named siG12D; Nature, 2017, 546(7659):498-503) targeting the KRAS G12D mutant mRNA into pancreatic cancer cells using iExosome exosomes. This research is currently in Phase I clinical trials, but the industrial-scale production of exosomes presents significant challenges.

[0006] Based on the above situation, the development of novel delivery strategies is of great significance for the targeted delivery of nucleic acid drugs (especially to tissues outside the liver), and could potentially enable mRNAs such as KRAS to become drug targets. Nucleoside (acid) lipids, as a class of amphiphilic molecules with base, nucleoside, or nucleotide heads, can bind to nucleic acids through secondary bond interactions such as hydrogen bonds and π-π stacking, thereby playing an encapsulation role. These lipids have great application potential and deserve in-depth exploration. Summary of the Invention

[0007] The purpose of this invention is to provide an siRNA drug formulation applicable to targeted therapy of pancreatic cancer and its preparation method.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] This invention discloses an siRNA drug formulation for the treatment of pancreatic cancer, which is derived from a drug targeting KRAS. G12D The vector consists of small interfering RNA (siRNA) for mRNA, a vector, and a solvent. The vector comprises nucleoside lipids TPS, TPO, CPS, CPO, or DNCA, cationic lipid CLD, and auxiliary lipids DSPE-PEG or DSPE-PEG-cRGD. The structural formulas of the lipids TPS, CPS, CLDA, and DSPE-PEG are shown below:

[0010]

[0011] Where B is cytosine or thymine, X is sulfur or oxygen, and R1 is... R2 is C 16 H 33 R3 is C 17 H 35 .

[0012] Preferably, the small interfering RNA (siRNA) is siG12D, with the sequence: ss 5'-GUU GGA GCUGAU GGC GUAGtt-3', as 5'-CUACGC CAU CAG CUC CAACtt-3'.

[0013] Preferably, the molar ratio of the nucleoside lipid material, cationic lipid material and small interfering RNA is 21:31.5:1, 10:5:1 or 30:7.5:1.

[0014] Preferably, the amount of auxiliary lipid material used is 0.7%-3% of the total molar number of nucleoside lipid material and cationic lipid material.

[0015] Preferably, the solvent is a GenOpti solution.

[0016] Furthermore, the present invention also provides a method for preparing the aforementioned oligonucleotide drug formulation, comprising the following steps:

[0017] (1) The target is KRAS G12D Small interfering RNA (siRNA) drugs for mRNA are prepared with enzyme-free water to a stock solution concentration of 0.05 mM to 10 mM. Nucleoside lipids TPS, TPO, CPS, CPO or DNCA and cationic lipids CLD are prepared with anhydrous ethanol to a stock solution concentration of 1 mM to 100 mM. Auxiliary lipids DSPE-PEG or DSPE-PEG-cRGD are prepared with anhydrous ethanol to a stock solution concentration of 0.1 mM to 50 mM.

[0018] (2) Add the stock solution of the small interfering RNA drug to the centrifuge tube, and then add half the volume of GenOpti solution;

[0019] (3) Add the nucleoside lipid material TPS, TPO, CPS, CPO or DNCA, the cationic lipid material CLD, and the auxiliary lipid material DSPE-PEG or DSPE-PEG-cRGD in anhydrous ethanol mother liquor in sequence close to the liquid surface;

[0020] (4) Replenish the remaining half volume of GenOpti solution;

[0021] (5) 70℃, 4KHz ultrasound for 10min.

[0022] Furthermore, the present invention also proposes the application of the aforementioned siRNA drug formulation in the preparation of drugs for treating pancreatic cancer.

[0023] Compared with the prior art, the advantages of the present invention are:

[0024] 1. This invention uses KRAS as the target. G12D Using small interfering RNA (siG12D) as the encapsulation target, and combining nucleoside (acid) lipids (TPS, TPO, CPS, CPO, DNCA) with cationic lipids (CLD), and supplemented by auxiliary lipids (DSPE-PEG or DSPE-PEG-cRGD) as carriers, a breakthrough was achieved in highly efficient extrahepatic delivery of small interfering nucleic acid drugs. Through formulation component optimization, adjustment of lipid ratios, and insertion of tumor-targeting auxiliary lipids, a breakthrough was achieved in efficient extrahepatic delivery of small interfering nucleic acid drugs. After tail vein injection, the targeted formulation accumulated up to ~20% in the orthotopic pancreatic tumor site in mice, with an accumulation time exceeding 176 hours. It significantly silenced the target mRNA within tumor cells, exerting a highly effective inhibitory effect on tumor growth, and the formulation showed high safety. This delivery system can serve as a platform technology for the wide application of extrahepatic targeted delivery of different types of functional oligonucleotides.

[0025] 2. This delivery system carries a KRAS target. G12D The novel formulation of small interfering RNA (siG12D) significantly inhibited tumor growth in an orthotopic pancreatic cancer mouse model, with no hepatotoxicity, nephrotoxicity, or immunogenicity. This lays the foundation for the clinical application of anti-pancreatic cancer siRNA drugs and provides a technical means to overcome difficult-to-drug targets such as KRAS. Attached Figure Description

[0026] Figure 1 To investigate the silencing effect of siG12D formulation on target mRNA in PANC-1 cells under different lipid components and ratios in RT-qPCR experiments;

[0027] Among them, A. the silencing effect of different formulations on target mRNA after administration, Blank was the blank solvent control group, siG12D was the naked siG12D group, and the other 6 groups were 3 formulations with n(TPS / CLD / siG12D) = 30 / 7.5 / 1, 21 / 31.5 / 1, 10 / 5 / 1 and 3 formulations with n(TPS / CLD / DSPE-PEG2000 / siG12D) = 30 / 7.5 / 0.263 / 1, 21 / 31.5 / 0.368 / 1, 10 / 5 / 0.105 / 1; B. the silencing effect of different formulations on target mRNA after administration, Blank was the blank solvent control group, and the other 8 groups were 3 formulations with n(TPS or TPO or CPS or Four formulations with CPO / CLD / DSPE-PEG2000 / siG12D concentrations of 21 / 31.5 / 0.386 / 1 and four formulations with n(TPS or TPO or CPS or CPO / CLD / DSPE-PEG2000 / siG12D) concentrations of 10 / 5 / 0.105 / 1 were used. Cells used: PANC-1; siG12D concentration: 25 nM; Detection time: 24 h post-administration.

[0028] Figure 2 To investigate the uptake of siG12D formulation in PANC-1 cells by flow cytometry under different lipid composition and ratio conditions;

[0029] Blank: blank solvent control; siG12D: naked siG12D administration; the remaining 8 groups consisted of 4 formulations with n(TPS or TPO or CPS or CPO / CLD / DSPE-PEG2000 / siG12D) = 21 / 31.5 / 0.386 / 1 and 4 formulations with n(TPS or TPO or CPS or CPO / CLD / DSPE-PEG2000 / siG12D) = 10 / 5 / 0.105 / 1. A. Cell fluorescence intensity after administration of different formulations; B. Cell uptake rate after administration of different formulations; C. Flow cytometry peak shapes after administration of different formulations. Cells used: PANC-1; siG12D concentration: 25 nM; Detection time: 4 h after administration.

[0030] Figure 3 In vivo imaging and organ accumulation of Cy5.5-siG12D formulation in an orthotopic pancreatic cancer nude mouse model (10h);

[0031] The four administration groups were formulations with n(DNCA / CLD / DSPE-PEG2000-cRGD / Cy5.5-siG12D) = 10 / 5 / 0.105 / 1, 10 / 5 / 0.45 / 1, and n(TPS / CLD / DSPE-PEG2000-cRGD / Cy5.5-siG12D) = 10 / 5 / 0.105 / 1, 10 / 5 / 0.45 / 1, respectively. A. In vivo fluorescence imaging and total fluorescence values ​​of mice at different time points after administration; B. Average fluorescence intensity of various organs in mice 10 hours after administration; C. Fluorescence imaging of various organs in mice 10 hours after administration; D. Accumulation ratio of the drug in various organs of mice 10 hours after administration. Cells used: PANC-1; siG12D dosage: 1 MPk, single dose; Administration route: tail vein injection.

[0032] Figure 4 In vivo imaging and organ accumulation of Cy5.5-siG12D formulation in an orthotopic pancreatic cancer nude mouse model (176 h);

[0033] The study included: A. In vivo fluorescence imaging and total fluorescence values ​​of mice at different time points after drug administration; B. Average fluorescence intensity of various organs in mice 176 hours after drug administration; C. Fluorescence imaging of various organs in mice 176 hours after drug administration; and D. Accumulation ratio of the drug in various organs of mice 176 hours after drug administration. Cells used: PANC-1; siG12D. Dosage: 1 MPa, single dose; Administration route: tail vein injection. Groups and... Figure 3 Consistent.

[0034] Figure 5 The tumor growth inhibition in an orthotopic pancreatic cancer nude mouse model under both siG12D administration and formulation conditions;

[0035] The data includes: A. Mouse tumor seeding, drug administration information, and group settings; B. Bioluminescence intensity of mice in each group at different time points; C. Tumor growth fold of mice in each group at different time points; D. Photographs of dissected mouse tumor tissue; E. Weight of dissected mouse tumor tissue. Cells used: PANC1-luc; siG12D dosage: 1 MPk, administered on days 0, 1, 2, 3, 6, 9, 12, and 15, for a total of 8 administrations; administration method: tail vein injection.

[0036] Figure 6 Living Image image from a pharmacodynamic experiment using a nude mouse model of orthotopic pancreatic cancer;

[0037] Groups and Figure 5 Consistent.

[0038] Figure 7 HE staining results and pathological scores of pancreatic tissue sections from nude mice with in situ pancreatic cancer after siG12D administration in naked form and under formulation conditions;

[0039] A. HE staining images of pancreatic tissue sections from nude mice with orthotopic pancreatic cancer in the Blank group and the siG12D nude mouse group; B. Pathological scores (tumor invasion, inflammation, necrosis, hemorrhage, acinar atrophy) of pancreatic tissue sections from nude mice with orthotopic pancreatic cancer in each group after HE staining. Group and... Figure 5 Consistent.

[0040] Figure 8 KRAS in nude mouse tumor tissues with orthotopic pancreatic cancer under siG12D administration and formulation conditions G12D The silencing effect of mRNA;

[0041] Groups and Figure 5 Consistent.

[0042] Figure 9 The effects of siG12D administration under both naked and formulated conditions on body weight and blood biochemistry in nude mice with orthotopic pancreatic cancer;

[0043] Groups and Figure 5 Consistent.

[0044] Figure 10 The effect of siG12D administration under both naked and formulated conditions on the levels of inflammatory factors in the serum of nude mice with orthotopic pancreatic cancer;

[0045] Groups and Figure 5 Consistent.

[0046] Figure 11 HE staining results of liver and kidney tissue sections from nude mice with in situ pancreatic cancer after siG12D administration in naked form and under formulation conditions;

[0047] Image A: HE-stained mouse liver section; Image B: HE-stained mouse kidney section. Image scale bar: 50 μm. Group and Figure 5 Consistent.

[0048] Figure 12 The particle size and potential of siG12D formulations in each group were analyzed in a pharmacodynamic experiment on nude mice with in situ pancreatic cancer. Detailed Implementation

[0049] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description of the specific embodiments. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0050] Example 1

[0051] This embodiment mainly illustrates the preparation method of the pharmaceutical preparation of the present invention.

[0052] Materials and Methods:

[0053] Depending on the needs of different types of experiments, siRNA was prepared into a stock solution of appropriate concentration using RNase-free water, and each lipid material was prepared into a stock solution of appropriate concentration using anhydrous ethanol. The specific procedures are as follows:

[0054] (1) The target is KRAS G12D Small interfering RNA (siG12D) for mRNA (ss 5'-GUU GGA GCU GAU GGCGUAGtt-3', as 5'-CUACGC CAU CAG CUC CAACtt-3') is prepared as a stock solution with RNase-free water at a concentration of 0.05 mM to 10 mM. Nucleoside lipids (TPS, TPO, CPS, CPO, or DNCA) and cationic lipids (CLD) are prepared as a stock solution with anhydrous ethanol at a concentration of 1 mM to 100 mM. Helper lipids (DSPE-PEG or DSPE-PEG-cRGD) are prepared as a stock solution with anhydrous ethanol at a concentration of 0.1 mM to 50 mM.

[0055] The structural formulas of TPS, CPS, CLDA, and DSPE-PEG are shown below:

[0056]

[0057] Where B is cytosine or thymine, X is sulfur or oxygen, and R1 is... R2 is C 16 H 33 R3 is C 17 H 35 .

[0058] (2) Add the stock solution of the small interfering RNA drug to the centrifuge tube, and then add half the volume of GenOpti solution;

[0059] (3) Add the nucleoside lipid material TPS, TPO, CPS, CPO or DNCA, the cationic lipid material CLD, and the auxiliary lipid material DSPE-PEG or DSPE-PEG-cRGD in anhydrous ethanol mother liquor in sequence close to the liquid surface;

[0060] (4) Replenish the remaining half volume of GenOpti solution;

[0061] (5) 70℃, 4KHz ultrasound for 10min.

[0062] The molar ratio of the nucleoside lipid material, cationic lipid material and small interfering RNA is 30-10:5-35:1.

[0063] The amount of the auxiliary lipid material is 0.5%-3% of the total molar number of nucleoside lipid material and cationic lipid material.

[0064] Example 2

[0065] This example mainly illustrates the effects of different components and ratios of siG12D preparations on KRAS in PANC-1 cells. G12D The silencing effect of mRNA.

[0066] Materials and Methods:

[0067] The small interfering RNA siG12D nucleic acid sequence was ordered from Sangon Biotech (Shanghai) Co., Ltd. and General Biotech (Anhui) Co., Ltd.

[0068] PANC-1 cells were divided into groups of 1.5 × 10⁻⁶. 5 Cells were seeded into 12-well plates with 900 μL of culture medium per well and incubated at 37°C for 16-24 h before transfection. Each formulation was prepared according to the method described in Example 1, with a small interfering RNA siG12D concentration of 250 nM (i.e., a final dosing concentration of 25 nM) and a formulation volume of 100 μL, and administered dropwise.

[0069] Total RNA was extracted using the Trizol method 24 hours after drug administration. 500 μL of Trizol was added to each well of a 12-well plate and mixed thoroughly. After standing at room temperature for 5 min, the mixture was transferred to a 1.5 mL RNase-Free EP tube. 100 μL of chloroform was added and vortexed. The mixture was then centrifuged at 12000 g for 15 min at 4 °C. 200 μL of the aqueous supernatant was collected, and 200 μL of isopropanol was added. The mixture was vortexed and incubated on ice for 15 min. After centrifugation at 12000 g for 15 min at 4 °C, the supernatant was discarded, and RNA precipitate was visible at the bottom of the tube. 1 mL of freshly prepared, pre-chilled 70% ethanol was added to each tube to gently wash the precipitate. The tube was then centrifuged at 12000 g for 15 min at 4 °C, the supernatant was discarded, and the precipitate was dried to near dryness. DEPC water was added to dissolve the precipitate, and the tube was stored at 4 °C or -80 °C.

[0070] After quantification using Nanodrop, 1 μg of total RNA was added to a 0.2 mL RNase-Free EP tube, and enzyme-free water was added to make up to 10 μL. The tube was then placed in a PCR instrument and incubated at 70 °C for 10 min. After removal, the tube was briefly centrifuged and then stored at 4 °C.

[0071] Prepare the reaction solution according to Table 1:

[0072] Table 1. Preparation of Reverse Transcription Reaction Solution

[0073]

[0074] Add reaction solution to each RNA tube, place in a PCR instrument, and incubate at 42°C for 15 min, 95°C for 5 min, and 4°C for 5 min. Store the resulting cDNA at 4°C for later use or at -80°C.

[0075] Dilute 20 μL of the above cDNA with 80 μL of enzyme-free water 5 times, and prepare the reaction solution in an eight-tube strip or a dedicated 96-well plate according to Table 2 and the procedure below for real-time quantitative PCR.

[0076] Table 2. Preparation of Real-Time Quantitative PCR Reaction Solution

[0077]

[0078]

[0079] PCR procedure:

[0080]

[0081] KRAS G12D The upstream and downstream primers for mRNA were (5′-3′): ACT TGT GGT AGT TGG AGC AGA, TTG GAT CATATT CGT CCACAA. The upstream and downstream primers for the internal control (18S) were (5′-3′): GTA ACC CGT TGAACCCCATT, CCATCCAAT CGGTAG TAG CG.

[0082] Results: Using TPS as the representative nucleoside (acid) lipid material, groups were established with TPS / CLD / siRNA ratios of 30 / 7.5 / 1 and 21 / 31.5 / 1, and a smaller lipid material group with a TPS / CLD / siRNA ratio of 10 / 5 / 1 was also established; simultaneously, groups were established with TPS / CLD / DSPE-PEG2000 / siRNA ratios of 30 / 7.5 / 0.263 / 1, 21 / 31.5 / 0.368 / 1, and 10 / 5 / 0.105 / 1. RT-qPCR results showed that ( Figure 1 A) 25 nM siG12D naked administration to the target KRAS in PANC-1 cells G12DThere was no significant silencing effect on mRNA, but all siG12D formulations significantly silenced target mRNA. Formulations with TPS / CLD / siRNA ratios of 30 / 7.5 / 1, 21 / 31.5 / 1, and 10 / 5 / 1 silenced 44%, 80%, and 68% of the target mRNA, respectively. Formulations with TPS / CLD / DSPE-PEG2000 / siRNA ratios of 30 / 7.5 / 0.263 / 1, 21 / 31.5 / 0.368 / 1, and 10 / 5 / 0.105 / 1 silenced 56%, 81%, and 64% of the target mRNA, respectively. Furthermore, the optimal ratios of 21 / 31.5 / 0.368 / 1 and 10 / 5 / 0.105 / 1 were selected to investigate the silencing effects of four nucleoside phospholipids (TPS, TPO, CPS, and CPO) on target mRNA. RT-qPCR results showed that ( Figure 1 B) All formulations showed significant efficacy, with the TPS formulation exhibiting the best efficacy.

[0083] Example 3

[0084] This example mainly illustrates the uptake of Cy5.5-siG12D formulations with different components and ratios in PANC-1 cells.

[0085] Materials and Methods:

[0086] The small interfering RNA siG12D nucleic acid sequence was ordered from General Biotech (Anhui) Co., Ltd.

[0087] PANC-1 cells were divided into groups of 1.5 × 10⁻⁶. 5 Cells were seeded into 12-well plates with 900 μL of culture medium per well and incubated at 37°C for 16–24 h before transfection. siG12D cells with Cy5.5 conjugated to the 5' end of the positive strand were selected and each formulation was prepared according to the method described in Example 1. The nucleic acid concentration in the formulation was 250 nM (i.e., the final dosing concentration was 25 nM), and the formulation volume was 100 μL. The cells were then added dropwise.

[0088] After drug administration, the cells were incubated in the dark for 4 hours. The culture plate was then removed, washed once with PBS, digested with 0.25% trypsin, and the cells were collected into 1.5 mL EP tubes. The cells were centrifuged at 1000 rpm for 3 min at 4°C, the supernatant was discarded, and the cells were washed twice with PBS or serum-free medium. The cells were then resuspended in an appropriate amount of serum-free medium, sieved, and the changes in cell fluorescence value after drug administration were detected by CytoFLEX flow cytometer (BECKMAN COULTER).

[0089] Results: Four hours after drug administration, the fluorescence values ​​of cells in the unencapsulated siG12D group were not significantly different from those in the Blank group, indicating that the drug was difficult to enter the cells without encapsulation. In contrast, cells in the formulation groups showed strong fluorescence with a single peak shape, indicating that under formulation conditions, the drug entered the cells in large quantities with high uniformity. Further analysis of the positive cell ratio showed that when the ratio was 21 / 31.5 / 0.368 (DSPE-PEG2000) / 1, the seroconversion rates of TPS, TPO, CPS, and CPO in PANC-1 cells were 89%, 91%, 93%, and 93%, respectively; and when the ratio was 10 / 5 / 0.105 (DSPE-PEG2000) / 1, the seroconversion rates of TPS, TPO, CPS, and CPO in PANC-1 cells were 83%, 86%, 87%, and 86%, respectively. Figure 2 ).

[0090] Example 4

[0091] This embodiment mainly illustrates the distribution and metabolism of Cy5.5-siG12D targeted agents in a nude mouse model of orthotopic pancreatic cancer.

[0092] Materials and Methods:

[0093] The small interfering RNA siG12D nucleic acid sequence was ordered from General Biotechnology (Anhui) Co., Ltd.

[0094] Six-week-old female BALB / c-nude mice were selected. After intraperitoneal anesthesia with afodin, the abdominal epidermis was disinfected with iodine. A small incision was made on the side of the abdomen, and 10 μL of PANC-1 cell suspension was injected into the pancreatic tail (5 × 10⁻⁶ cells) using an insulin needle. 6(cells / each), sutured. Because the pancreatic cancer cell line PANC-1 highly expresses integrin αvβ3, and cRGD can target integrin αvβ3, to further increase the tumor targeting of the formulation, the auxiliary lipid component DSPE-PEG2000 was replaced with DSPE-PEG2000-cRGD. Four formulations were prepared using siG12D with Cy5.5 conjugated to the 5' end of the positive chain, following the method described in Example 1: two formulations with DNCA / CLD / DSPE-PEG2000-cRGD / siRNA ratios of 10 / 5 / 0.105 / 1 and 10 / 5 / 0.45 / 1, and two formulations with TPS / CLD / DSPE-PEG2000-cRGD / siRNA ratios of 10 / 5 / 0.105 / 1 and 10 / 5 / 0.45 / 1. To determine the optimal incorporation amount of DSPE-PEG2000-cRGD, two incorporation ratios were set: 0.7% and 3% of the total molar amount of nucleoside (acid) ester material to CLD. On day 20 after tumor implantation, the preparations of each group were injected into mice via the tail vein. The nucleic acid dosage in each group was 1 MPk. At different time points after administration, mice were subjected to in vivo fluorescence imaging using a small animal in vivo imaging system (IVIS Spectrum). At the end of the experiment, mice were euthanized by cervical dislocation after isoflurane anesthesia. The brain, heart, lungs, liver, stomach, intestines, pancreas, spleen, and kidneys were collected for fluorescence imaging. The excitation wavelength was 675 nm and the emission wavelength was 720 nm. Quantitative fluorescence was performed using Living Image software.

[0095] Results: In vivo imaging results showed that significant fluorescence accumulation was observed in the pancreas at 6, 8, and 10 hours after a single administration of each formulation. Based on the total fluorescence value in mice, the TPS group with 3% DSPE-PEG2000-cRGD incorporation > the TPS group with 0.7% DSPE-PEG2000-cRGD incorporation > the DNCA group with 3% DSPE-PEG2000-cRGD incorporation > the DNCA group with 0.7% DSPE-PEG2000-cRGD incorporation. This indicates that the overall drug accumulation of the TPS formulation in mice was superior to that of the DNCA formulation, and the formulation with 3% DSPE-PEG2000-cRGD incorporation exhibited a better long-acting circulating effect than the formulation with 0.7% incorporation. Figure 3 A); Ten hours after administration, anatomical imaging and quantitative fluorescence intensity were performed on the organs. Based on the images and average fluorescence intensity values, no significant fluorescence was observed in the nude mouse brain and heart; some fluorescence was observed in the lungs, liver, and spleen; and strong fluorescence was observed in the stomach, intestines, pancreas, and kidneys, with the pancreas exhibiting the highest average fluorescence intensity. Figure 3B, C); The proportion of drug accumulation in each organ was calculated based on the total fluorescence value. The results showed that the formulations with DNCA / CLD / DSPE-PEG2000-cRGD / siRNA ratios of 10 / 5 / 0.105 / 1 and 10 / 5 / 0.45 / 1 accumulated in the pancreas at 19.5% and 23.8% of the total drug accumulation in each organ, respectively. The formulation with TPS / CLD / DSPE-PEG2000-cRGD / siRNA ratio of 10 / 5 / 0.105 / 1 and 10 / 5 / 0.45 / 1 respectively accumulated in the pancreas. The 105 / 1 and 10 / 5 / 0.45 / 1 formulations accumulated 7.75% and 17.6% of the total drug accumulation in the pancreas and other organs, respectively. It is noteworthy that pancreatic tumors in mice grow rapidly, and some tumors infiltrate the stomach and intestines. Therefore, the total drug accumulation ratio in the pancreas, stomach, and intestines is also an important indicator of targeted drug accumulation. The total drug accumulation ratios in the pancreas, stomach, and intestines of the four formulation groups were 69.9%, 75.9%, 78.5%, and 80.9%, respectively. Figure 3 D).

[0096] Further observation of drug distribution and metabolism in mice over a longer period (176 hours) after administration showed that the total fluorescence value of each group of mice decreased over time. Among the four formulations, the formulation with a TPS / CLD / DSPE-PEG2000-cRGD / siRNA ratio of 10 / 5 / 0.45 / 1 consistently exhibited the highest total fluorescence value in mice, indicating that it had the longest-lasting systemic circulation effect. Figure 4 A); 176 hours after a single dose of each formulation, anatomical imaging and quantitative fluorescence intensity were performed on the organs. Based on the images and the average quantitative fluorescence intensity values, the fluorescence intensity of each organ decreased compared to the 10-hour anatomical results. At this time, the stomach, intestines, and pancreas still showed significant fluorescence, while the other organs showed no significant fluorescence. Figure 4 B, C); The proportion of drug accumulation in each organ was calculated based on the total fluorescence value. The results showed that the formulations with DNCA / CLD / DSPE-PEG2000-cRGD / siRNA ratios of 10 / 5 / 0.105 / 1 and 10 / 5 / 0.45 / 1 accumulated 17.4% and 8.72% of the total drug accumulation in each organ, respectively, in the pancreas. The formulations with TPS / CLD / DSPE-PEG2000-cRGD / siRNA ratios of 10 / 5 / 0.105 / 1 and 10 / 5 / 0.45 / 1 accumulated 21.3% and 9.95% of the total drug accumulation in each organ, respectively, in the pancreas. The total drug accumulation proportions in the pancreas, stomach, and intestines of the four formulation groups were 85.9%, 86.5%, 83.8%, and 86.4%, respectively. Figure 4 D).

[0097] Overall, the formulation with a TPS / CLD / DSPE-PEG2000-cRGD / siRNA ratio of 10 / 5 / 0.45 / 1 exhibits a relatively long systemic circulation effect and maintains a superior in vivo drug accumulation level within 25 hours after administration. It also shows ideal accumulation ratios at pancreatic tumor sites and in tumor-infiltrated gastric and intestinal sites. This formulation could be considered for pharmacodynamic evaluation experiments of in situ pancreatic cancer in nude mice.

[0098] Example 5

[0099] This embodiment mainly illustrates the pharmacodynamic effects and safety of siG12D targeted formulations with different components and ratios in an in situ pancreatic cancer nude mouse model.

[0100] Materials and Methods:

[0101] The small interfering RNA siG12D nucleic acid sequence was ordered from General Biotechnology (Anhui) Co., Ltd.

[0102] To facilitate the observation of tumor progression in live mice, an orthotopic pancreatic cancer nude mouse model was constructed using PANC1-luc tumor cells that stably express luciferase. After these tumor cells were inoculated into experimental animals, they produced luminescence within minutes to tens of minutes after intraperitoneal administration of their substrate luciferin. The intensity of the luminescence was linearly correlated with the number of live cells.

[0103] Six-week-old female BALB / c-nude mice were selected. After intraperitoneal anesthesia with afodin, the abdominal epidermis was disinfected with iodine. A small incision was made on the side of the abdomen, and 10 μL of PANC1-luc cell suspension was injected into the pancreatic tail (5 × 10⁻⁶ cells) using an insulin needle. 6 (cells / mouse), sutured. On the 8th day after tumor implantation, mice were randomly divided into 6 groups, with the average total bioluminescence intensity (Total Flux) of each group ranging from 1.7 to 2.6 × 10⁻⁶. 8[p / s], with no significant difference between groups, administration began, recorded as day 0. Mice in the 6 groups were administered blank solvent, siG12D, formulation 1 (TPS / CLD / DSPE-PEG2000-cRGD / siG12D = 10 / 5 / 0.45 / 1), formulation 2 (TPS / CLD / DSPE-PEG2000-cRGD / siG12D = 21 / 31.5 / 1.575 / 1), formulation 3 (TPS / CLD / DSPE-PEG2000-cRGD / siG12D = 21 / 31.5 / 0.368 / 1), and formulation 4 (DNCA / CLD / DSPE-PEG2000-cRGD / siG12D = 10 / 5 / 0.45 / 1), respectively. The dosage of small interfering RNA (siG12D) nucleic acid in each group was 1 MPk. The mice were administered via tail vein injection on days 0, 1, 2, 3, 6, 9, 12, and 15, for a total of 8 administrations. Throughout the experimental period, mice were weighed every other day. In vivo bioluminescence imaging of mice was performed using an IVIS Spectrum small animal imaging system on days 0, 5, 11, and 19, and quantification was performed using LivingImage software.

[0104] On day 20, the experiment reached its endpoint. After blood collection, the mice were euthanized, and the pancreas, liver, and kidneys were dissected. The tumor tissue from the pancreas was weighed, photographed, and placed in an EP tube. 1 mL of Trizol was added, and the tissue was ground and mixed thoroughly. RT-qPCR was then performed (operation as in Example 3). The remaining pancreatic tissue was fixed with 4% paraformaldehyde, trimmed, dehydrated, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), mounted, examined under a microscope, and photographed. Pathological scoring (tumor invasion, inflammation, necrosis, hemorrhage, acinar atrophy) was performed, and the scoring criteria are shown in Table 3 below.

[0105] Table 3. Level 4 Grading System

[0106]

[0107] Blood was incubated in EP tubes at room temperature for at least one hour, then centrifuged at 3500 rpm for 10 minutes to obtain serum. Serum was then used for blood biochemical marker detection (ALT, AST, TBIL, UREA, CREA) and inflammatory factor detection using ELISA (IL-6, IL-1β, IFN-γ). Among the blood biochemical markers, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) indicate liver damage. ALT is mainly distributed in the hepatocyte cytoplasm; elevated ALT reflects damage to the hepatocyte membrane. AST is mainly distributed in the hepatocyte cytoplasm and hepatocyte mitochondria. When hepatocytes are severely damaged, endangering mitochondria, AST will also enter the blood. When liver damage occurs, the efficiency of bilirubin metabolism decreases, and TBIL may increase. Urea (UREA) and creatinine (CREA) indicate kidney damage; simultaneous elevation of Urea and CREA suggests a significant decrease in kidney function. Among inflammatory factors, IL-1β is an important member of the IL-1 family. It has strong pro-inflammatory activity and can induce various pro-inflammatory mediators, such as cytokines and chemokines. IL-1β has multiple functions, acting on various cells and ultimately leading to widespread inflammatory events. Systemically, IL-1β signaling can cause acute phase reactions, hypotension, vasodilation, and fever. IL-6 systemically acts on the liver to produce acute phase proteins, such as all-C-reactive protein (CRP), fibrinogen, and plasminogen activator inhibitors. IFN-γ is a soluble dimeric cytokine and the only member of type II interferon. It is mainly secreted by NK and NKT cells and plays a role in innate immunity. In antigen-specific immunity, it is secreted by CD4 Th1 and CD8 cytotoxic T cells. After fixation with 4% paraformaldehyde, liver and kidney tissues were trimmed, dehydrated, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), mounted, and examined under a microscope. Hematoxylin staining solution is alkaline, which mainly stains the chromatin in the cell nucleus and nucleic acids in the cytoplasm purple-blue; eosin is an acidic dye, which mainly stains the components in the cytoplasm and extracellular matrix red.

[0108] Results: Tumor growth was not significantly inhibited in the siG12D naked group mice, with a mean tumor weight of 17.6 mg, which was not significantly different from the Blank group (mean tumor weight 16.6 mg). The mean Total Flux on day 19 was 8.4 × 10⁻⁶. 8 [p / s], compared to 1.9 × 10 on day 0 8 [p / s] increased by 5.3 times, compared to a Total Flux mean of 1.7 × 10⁻⁶ for the Blank group (day 0). 8 [p / s] Increased 6.2 times to 8.0 x 10 on day 19. 8[p / s]) There was no significant difference, but tumor growth in mice was significantly inhibited in all formulation groups. The mean tumor weights in formulation groups 1-4 were 4.3, 5.0, 3.3, and 3.5 mg, respectively, and the mean Total Flux values ​​on day 19 were 3.1, 2.0, 1.8, and 3.0 × 10⁻⁶, respectively. 8 [p / s], the tumor growth folds were 1.4, 1.5, 1.7, and 1.9 times, respectively. Figure 5-6 The pancreatic histopathological scoring results are as follows: Figure 7 As shown, overall, the pancreatic tissue sections from the No. 3 formulation group had the lowest scores for tumor infiltration, inflammation, necrosis, hemorrhage, and acinar atrophy, indicating that the pancreatic tissue was relatively healthy. Meanwhile, RT-qPCR results showed that the target KRAS in the tumor cells of mice in the siG12D naked group... G12D mRNA was not significantly silenced, while target mRNA in mouse tumor cells of formulations 1-4 was significantly silenced, with silencing efficiencies of 78%, 71%, 93%, and 65%, respectively. Formulation 3 showed the best silencing effect on target mRNA. Figure 8 ).

[0109] Mice in all groups remained in normal condition and had stable weight throughout the experimental period, with no statistically significant differences between groups. Blood biochemistry results showed no significant differences in serum ALT, AST, TBIL, UREA, and CREA levels among the groups. Figure 9 The levels of inflammatory cytokines IL-6, IL-1β, and IFN-γ fluctuated significantly within each group, with no statistically significant differences between groups. However, overall, the inflammatory cytokines in the No. 3 formulation group remained at a low level. Figure 10 No significant pathological changes were observed in liver and kidney tissue sections. Figure 11 The results indicate that the siG12D formulation has good safety and did not cause significant liver or kidney damage.

[0110] Considering both efficacy and safety, formulation No. 3, namely the siG12D formulation with TPS / CLD / DSPE-PEG2000-cRGD / siG12D = 21 / 31.5 / 0.368 / 1, is the preferred in vivo targeted agent against KRASG12D-mutant pancreatic cancer.

[0111] Example 6

[0112] This example mainly illustrates the particle size and zeta potential of each formulation in Example 5.

[0113] Materials and Methods

[0114] The small interfering RNA siG12D nucleic acid sequence was ordered from General Biotech (Anhui) Co., Ltd.

[0115] Each formulation was prepared according to the formulation method in Example 1. The concentration of small interfering RNA (siG12D) in the formulation was 250 nM, and the solution volume was 500 μL. Group 1 was a formulation with TPS / CLD / DSPE-PEG2000-cRGD / siG12D = 10 / 5 / 0.45 / 1; Groups 2 and 3 were formulations with TPS / CLD / DSPE-PEG2000-cRGD / siG12D = 21 / 31.5 / 1.575 / 1 and 21 / 31.5 / 0.368 / 1, respectively; and Group 4 was a formulation with DNCA / CLD / DSPE-PEG2000-cRGD / siG12D = 10 / 5 / 0.45 / 1. Potential particle size distribution was measured using a Malvern Zetasizer Nano-ZS laser scattering particle size analyzer, and the data were analyzed using ELS-8000 software.

[0116] Result: As Figure 12 As shown, the particle sizes of the four formulations are relatively similar, with average values ​​of 153.4 nm, 169.1 nm, 144.3 nm, and 164.3 nm, respectively. Formulations 2 and 3 have lower PDIs, indicating better particle size uniformity, while formulations 1 and 4 have higher PDIs. Formulations 2 and 3 have potentials of -0.4 mV and -1.3 mV, respectively, which are close to neutral, while formulation 1 has a potential of -3.3 mV and formulation 4 has a potential of -8.3 mV. In summary, formulation 3 has an ideal particle size, good particle size uniformity, and a potential close to neutral, making it the preferred formulation.

[0117] The information shown and described in detail herein is sufficient to achieve the above-described objectives of the invention. Therefore, the preferred embodiments of the invention represent the subject matter of the invention, which is broadly covered by the invention. The scope of the invention fully encompasses other embodiments that will be obvious to those skilled in the art, and therefore, the scope of the invention is not limited by anything other than the appended claims, wherein, unless expressly stated otherwise, the singular form of an element does not mean "one and only," but rather "one or more." All structural, compositional, and functional equivalents of the foregoing preferred embodiments and additional embodiments known to those skilled in the art are therefore incorporated herein by reference and are intended to be covered by the claims of the invention.

[0118] Furthermore, no specific device or method is required to express each problem solved by this invention, as they are all included within the scope of the claims. Additionally, regardless of whether all parts, components, or method steps disclosed in this invention are expressly described in the claims, they are not made public. However, it will be apparent to those skilled in the art that various changes and modifications can be made in form, reagents, and synthetic details without departing from the spirit and scope of the invention as set forth in the appended claims.

Claims

1. A siRNA drug formulation for the treatment of pancreatic cancer, characterized in that, The siRNA drug formulation is formulated with a target of KRAS G12D The system consists of small interfering RNA (siRNA) for mRNA, a vector, and a solvent. The vector comprises nucleoside lipid TPS, cationic lipid CLD, and auxiliary lipid DSPE-PEG2000-cRGD. The structural formulas of the lipids are shown below: ； Where B is thymine, X is sulfur, and R1 is... R3 is C 17 H 35 ; The molar ratio of TPS, CLD, DSPE-PEG2000-cRGD, and siG12D is 21:31.5:0.368:

1. The small interfering RNA (siRNA) is siG12D, and its sequence is: ss 5'-GUU GGA GCU GAU GGC GUA Gtt-3' , as 5'-CUA CGC CAU CAG CUC CAA Ctt-3'.

2. The siRNA drug formulation according to claim 1, characterized in that, The solvent is GenOpti solution.

3. A method for preparing the siRNA drug formulation according to claim 1 or 2, characterized in that, Includes the following steps: (1) The target is KRAS G12D Small interfering RNA (siRNA) drugs for mRNA are prepared with enzyme-free water to a stock solution concentration of 0.05 mM to 10 mM; nucleoside lipid material TPS and cationic lipid material CLD are prepared with anhydrous ethanol to a stock solution concentration of 1 mM to 100 mM; and auxiliary lipid material DSPE-PEG2000-cRGD is prepared with anhydrous ethanol to a stock solution concentration of 0.1 mM to 50 mM. (2) Add the stock solution of the small interfering RNA drug to the centrifuge tube, and then add half a volume of GenOpti solution; (3) Add anhydrous ethanol mother liquor of nucleoside lipid material TPS, cationic lipid material CLD and auxiliary lipid material DSPE-PEG2000-cRGD in sequence close to the liquid surface; wherein, the molar ratio of TPS, CLD, DSPE-PEG2000-cRGD and siG12D is 21:31.5:0.368:1; (4) Refill the remaining half volume with GenOpti solution; (5) 70℃, 4KHz ultrasound for 10min.

4. The use of the siRNA drug formulation according to claim 1 or 2 in the preparation of drugs for treating pancreatic cancer.

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