A dual gene silencing, crgd modified targeted lnp and its use in the preparation of a colorectal cancer anti-angiogenesis therapeutic drug

Abstract

The application provides a double-gene silencing, cRGD modified targeted LNP preparation and application thereof in colorectal cancer anti-angiogenesis treatment; the LNP is prepared from lipid raw materials and nucleic acid raw materials; wherein the lipid raw materials comprise ionizable cationic lipid SM-102, cholesterol, DOPE phospholipid, DMG-PEG2000 and DMG-PEG2000-cRGD, and the nucleic acid raw materials comprise siEIF3a and siVEGF; in the preparation, the lipids are dissolved in ethanol, the nucleic acids are dissolved in a buffer, and then mixed and placed. The application is realized through cRGD targeted modification combined with double siRNA co-delivery, relies on cRGD to improve the targeting and uptake efficiency of the LNP on colorectal cancer cells, and through the double siRNA acting on the VEGF pathway and the EIF3a-ANG axis respectively, solves the problem that the colorectal cancer anti-angiogenesis treatment drug resistance cannot be effectively broken through in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to tumor-targeted delivery systems and anti-angiogenic therapy for colorectal cancer. In particular, it relates to a targeted lipid nanoparticle (LNP) modified with a dual-gene silencing cyclic arginine-glycine-aspartic acid polypeptide (cRGD) and its application in anti-angiogenic therapy for colorectal cancer. Background Technology

[0002] In the clinical treatment of metastatic colorectal cancer, anti-angiogenic therapy is a core strategy for delaying tumor progression and improving patient survival. Bevacizumab [an anti-vascular endothelial growth factor (VEGF) monoclonal antibody], as a first-line targeted drug against the VEGF / VEGFR pathway, has been widely used clinically for many years. However, this treatment regimen has significant limitations: on the one hand, its clinical efficacy varies greatly among individuals, with an objective response rate of only 20%-50%; on the other hand, even if initial treatment is effective, almost all patients eventually develop drug resistance, leading to treatment failure. The core mechanism of bevacizumab resistance lies in the tumor microenvironment maintaining tumor blood supply by activating VEGF-independent compensatory angiogenesis pathways. This problem has become a key bottleneck restricting the improvement of anti-angiogenic therapy efficacy in colorectal cancer.

[0003] Existing research has preliminarily clarified the common mechanism of drug resistance: tumors maintain blood supply by upregulating VEGF-independent pro-angiogenic signals. Among them, angiopoietin (ANG) is the first potent pro-angiogenic protein found to originate from tumor cells. Its expression level is significantly upregulated after bevacizumab treatment, playing an important role in mediating compensatory tumor angiogenesis and bevacizumab resistance.

[0004] Despite the known role of compensatory factors such as ANG, the current field of anti-angiogenic therapy still faces three major technical shortcomings that prevent effective solutions to the drug resistance problem: First, single-target inhibition strategies dominate clinical practice. Existing drugs (such as bevacizumab and ramucirumab) focus on the VEGF / VEGFR pathway and cannot block the compensatory angiogenesis process mediated by compensatory factors such as ANG, making drug resistance inevitable. Second, combination therapy regimens have significant limitations. Although clinical attempts have been made to combine bevacizumab with chemotherapy and immune checkpoint inhibitors, this can only partially delay the onset of drug resistance and significantly increases the risk of cumulative toxicity, threatening patient safety. Third, gene intervention technologies have significant shortcomings. Although RNA interference (RNAi) technology has shown great potential in tumor targeted therapy, traditional small interfering RNA (siRNA) delivery vectors generally suffer from poor tumor targeting and low cellular endocytosis efficiency. There is a lack of vector systems that can efficiently deliver multi-target siRNAs, hindering the clinical translation of gene therapy.

[0005] To overcome the aforementioned technological bottlenecks, the applicant conducted targeted research and achieved key breakthroughs:

[0006] Eukaryotic translation initiation factor 3a (EIF3a) is the largest functional subunit of EIF3 in eukaryotes, playing a crucial regulatory role in translation initiation. Recent studies have revealed its proto-oncogene characteristics, confirming its close association with tumor development, metastasis, prognosis, and treatment response. EIF3a is gradually emerging as a novel drug target with significant clinical translational potential within the EIF family, particularly demonstrating high application potential in the field of anti-tumor therapy.

[0007] Through a systematic analysis of clinical studies, animal experiments, and molecular mechanism research, the applicant has, for the first time, revealed the core mechanism of EIF3a-ANG-mediated bevacizumab resistance—EIF3a can promote compensatory angiogenesis in tumors and lead to bevacizumab treatment failure by activating the VEGF-independent pathway of the "EIF3a-ANG axis." Simultaneously targeting and inhibiting both EIF3a and VEGF can block angiogenesis from both direct and compensatory pathway perspectives, achieving a synergistic anti-angiogenic effect. Furthermore, the applicant discovered that EIF3a is specifically highly expressed in colorectal cancer tumor tissues, and this high expression is significantly associated with tumor metastasis and poor patient prognosis. Compared to directly targeting ANG, targeting EIF3a not only inhibits ANG-dependent compensatory angiogenesis but also directly inhibits the invasive and metastatic abilities of colorectal cancer cells both in vivo and in vitro, achieving a "two birds with one stone" effect.

[0008] However, while the above research findings have identified a new direction for treatment, current technologies are still unable to support its clinical translation. There is a lack of intervention programs targeting both EIF3a and VEGF, as well as an efficient vector that can deliver dual siRNAs precisely to the tumor site.

[0009] In summary, current technologies are still unable to effectively solve the problem of drug resistance in anti-angiogenic therapy for colorectal cancer. There is an urgent need to develop an siRNA delivery system that can simultaneously target the VEGF pathway and the EIF3a-ANG axis and has high tumor targeting, in order to break through the current treatment bottleneck, improve patient benefit rates and delay the occurrence of drug resistance. Summary of the Invention

[0010] The technical problem to be solved by this invention is to provide a targeted LNP with dual gene silencing and cRGD modification and its application in colorectal cancer anti-angiogenesis. It is achieved by cRGD modification combined with dual siRNA (siEIF3a+siVEGF) co-delivery. It relies on cRGD to improve the targeting and uptake efficiency of LNP on colorectal cancer cells, and the dual siRNAs act on the VEGF pathway and EIF3a-ANG axis respectively. It specifically solves the problem of drug resistance in colorectal cancer anti-angiogenic therapy that cannot be effectively overcome by existing technologies, and fills the gap in siRNA delivery systems that can delay drug resistance and improve treatment benefits.

[0011] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0012] A dual-gene silencing, cRGD-modified targeting LNP is prepared from lipid and nucleic acid raw materials. The lipid raw materials include ionizable cationic lipids SM-102, cholesterol, DOPE phospholipids, DMG-PEG2000, and DMG-PEG2000-cRGD. The nucleic acid raw materials include siRNA targeting EIF3a (siEIF3a) and siRNA targeting VEGF (siVEGF). In preparation, the lipid raw materials are dissolved in ethanol to form a liposome formulation stock solution, and the nucleic acid raw materials are dissolved in buffer to form a dual siRNA mixture. The two are then mixed and incubated on ice (note: the incubation mentioned below refers to ice bath incubation).

[0013] As one of the preferred embodiments of the present invention, the lipid raw materials, in molar ratio, are ionizable cationic lipids SM-102 : cholesterol : DOPE phospholipids : DMG-PEG2000 : DMG-PEG2000-cRGD=50 : 38.5 : 10 : 0.75 : 0.75.

[0014] As one of the preferred embodiments of the present invention, in the nucleic acid raw materials, siRNA targeting EIF3a and siRNA targeting VEGF are mixed at a mass ratio of 1:1.

[0015] As one of the preferred embodiments of the present invention, when preparing the cRGD-modified LNP, the nucleic acid raw material is dissolved in 10mM sodium citrate buffer at pH 4.0.

[0016] As one of the preferred methods of the present invention, when preparing the cRGD modified LNP, the liposome formulation stock solution and the double siRNA mixture are mixed at a volume ratio of 3:1 and allowed to stand for 15 minutes.

[0017] A method for preparing the above-mentioned dual-gene silencing, cRGD-modified targeted LNP includes the following specific steps:

[0018] (1) Raw material pretreatment: Weigh out ionizable cationic lipids SM-102, cholesterol, DOPE phospholipids, DMG-PEG2000 and DMG-PEG2000-cRGD and dissolve them in ethanol solution respectively. After each raw material is completely dissolved, mix the resulting solutions evenly to form the liposome formulation stock solution. At the same time, weigh out siRNA targeting EIF3a and siRNA targeting VEGF, dissolve them together in sodium citrate buffer, shake and mix well to form a dual siRNA mixture.

[0019] (2) Vector assembly: The liposome formulation stock solution obtained in step (1) is mixed with the double siRNA mixture at a volume ratio of 3:1;

[0020] (3) Standing and forming: Place the mixture after step (2) in an ice bath and let it stand for 15 minutes until the lipids self-assemble to form cRGD modified LNPs that encapsulate double siRNA.

[0021] Application of a targeted LNP with dual gene silencing and cRGD modification in the preparation of an anti-angiogenic therapeutic drug for colorectal cancer.

[0022] The advantages of this invention compared to the prior art are:

[0023] (1) Precisely block the core pathway of drug resistance, overcoming the limitations of "single target inhibition".

[0024] Current clinical treatments for colorectal cancer focus solely on the VEGF / VEGFR pathway (e.g., bevacizumab), failing to block the "VEGF-independent compensatory angiogenesis signaling" mediated by compensatory factors such as ANG, leading to inevitable drug resistance. This invention, however, achieves "dual pathway blockade" through synergistic intervention with dual siRNAs.

[0025] siRNA targeting VEGF (siVEGF) can directly inhibit the traditional VEGF-mediated angiogenesis pathway, thus prolonging the core role of anti-angiogenic therapy;

[0026] siRNA targeting EIF3a (siEIF3a) can block the "EIF3a-ANG axis" from upstream: by downregulating EIF3a expression, it reduces ANG secretion (weakening compensatory angiogenesis) and inhibits the invasion and metastasis of colorectal cancer cells, thereby fundamentally breaking the core mechanism of bevacizumab resistance and solving the key shortcoming of existing technologies that "only block a single pathway and cannot deal with compensatory resistance".

[0027] (2) Avoid the toxicity risks of combined therapy and achieve "synergistic effect + safety and controllability".

[0028] Existing combination therapies can only partially delay drug resistance and are prone to cumulative toxicity; however, this invention avoids these problems through a "single-carrier dual-target" design:

[0029] Without the need for additional drugs, dual siRNAs can be delivered via the same cRGD-modified targeting LNP vector to simultaneously target both EIF3a and VEGF, achieving a synergistic effect of "inhibiting metastasis + anti-angiogenesis + anti-drug resistance" while avoiding the risk of cumulative toxicity caused by multiple drug combinations.

[0030] The two siRNAs are precisely matched in a 1:1 mass ratio to avoid the compensatory risk caused by excessive inhibition of a single target, thereby further improving the stability of treatment.

[0031] (3) Solve the siRNA delivery problem to achieve "high targeting + high efficiency delivery".

[0032] Existing siRNA vectors suffer from poor tumor targeting and low endocytosis efficiency, hindering the clinical translation of gene therapy. The cRGD-modified LNP vector of this invention specifically addresses these issues:

[0033] The DMG-PEG2000-cRGD on the surface of the carrier can specifically bind to the integrin receptor highly expressed in colorectal cancer tissue, significantly improving the enrichment efficiency of the carrier at the tumor site, reducing off-target toxicity to normal tissues, and solving the defect of "insufficient targeting" of traditional carriers.

[0034] The lipid raw materials were optimized according to the molar ratio of "SM-102 : cholesterol : DOPE phospholipid : DMG-PEG2000 : DMG-PEG2000-cRGD=50 : 38.5 : 10 : 0.75 : 0.75" and were combined with the preparation process of "ethanol dissolution-sodium citrate buffer mixing-ice bath standing". This can not only ensure the stability of the carrier structure (avoiding siRNA degradation) but also promote the efficient binding and endocytosis of the carrier with cells, improve the intracellular release efficiency of siRNA, and overcome the technical bottleneck of "low delivery efficiency" of existing carriers.

[0035] (4) Focusing on unmet clinical needs, it has high translational value.

[0036] Existing technologies cannot support the clinical translation of "EIF3a+VEGF dual-target intervention," while this invention directly addresses the drug resistance problem of anti-angiogenic therapy in colorectal cancer, providing a complete solution with "clear mechanism, feasible regimen, and compatible vector": its preparation process does not require complex equipment, and batch stability can be ensured by limiting parameters such as volume ratio and settling time, facilitating large-scale production; at the same time, the final product can be made into a sterile injectable for intravenous injection, and the administration method conforms to routine clinical operation. Compared with existing gene therapy regimens, it is easier to achieve clinical translation, and can directly fill the technological gap of "dual-target anti-angiogenic siRNA delivery system", providing a new path for improving the prognosis of colorectal cancer patients and increasing the treatment benefit rate. Attached Figure Description

[0037] Figure 1This is a diagram showing the clinical characteristics of EIF3a in colorectal cancer in Example 1 (Figure A: Survival analysis of 430 colorectal tumor samples included in the TCGA colorectal cancer dataset; Figure B: Compared with normal tissue, eIF3a was significantly highly expressed in COAD and READ of colorectal cancer, and this analysis was performed on the GEPIA platform; Figure C: Expression level of eIF3a messenger RNA (mRNA) in 35 pairs of colorectal tumor tissues and their adjacent normal tissues was detected by real-time quantitative polymerase chain reaction q-PCR; Figure D: Immunohistochemistry of eIF3a was performed on sections of colorectal tumor tissues and their adjacent normal tissues). IHC staining; Figure E: Statistical results of IHC staining scores; Figure F: Analysis of eIF3a mRNA expression in primary tumor tissue (left) and metastatic tissue (right) of colorectal cancer patients based on expression files obtained from the GEO dataset (GSE131418); Figure G: Reporting the percentage of patients with high (blue) and low (red) eIF3a expression in primary tumor tissue (left) and metastatic tissue (right) of colorectal cancer patients; Figure H: Analysis of eIF3a mRNA expression in clinical colorectal tumor samples with and without metastatic disease; Figure I: Percentage of patients with (right) and without (left) metastatic disease, with different eIF3a expression levels (blue, high expression; red, low expression); COAD: Colon Cancer; READ: Rectal Cancer; CRC: Colorectal Cancer; * P <0.05,** P <0.01, *** P <0.001, **** P <0.0001);

[0038] Figure 2This is a graph showing the correlation between eIF3a and colorectal tumor metastasis and tumor angiogenesis in Example 1 (Figure A: Mouse spleen injection liver metastasis model, MC38-Luc-sheIF3a cells and MC38-Luc-shNC cells were injected into the spleen of C57 mice, and in vivo imaging was performed every 3 days after injection; Figure B: Statistical graph of in vivo imaging results; Figure C: Representative images of HE staining results of spleen tumors and liver metastases in each group of mice; Figure D: Representative images of spleen and liver metastases in each group of mice; Figures E and F: Representative images and statistical results of immunohistochemistry of platelet endothelial cell adhesion molecule-1 (PECAM-1 / CD31) in mouse liver metastases; * P <0.05,** P <0.01, *** P <0.001, **** P <0.0001);

[0039] Figure 3 Figures show the results of the intravenous administration of bevacizumab in Example 1 (Figure A: Correlation analysis of EIF3a mRNA expression level and bevacizumab sensitivity in colorectal cancer patients using the GSE60331 dataset; Figure B: Line graph of subcutaneous tumor volume in nude mice; Figure C: Line graph of nude mouse weight; Figure D: Nude mice sacrificed and photographed after administration; Figure E: Subcutaneous tumor dissection and photographed; Figure F: Results of subcutaneous tumor volume measurement in nude mice; Figure G: Results of subcutaneous tumor mass measurement in nude mice; Figures H and I: Immunohistochemical detection and statistical analysis of CD31 expression level in tumor tissue; * P <0.05,** P <0.01, *** P <0.001, **** P <0.0001);

[0040] Figure 4Figure 1 shows the results of EIF3a regulation of angiogenesis in colorectal cancer in Example 1. Figure A: Correlation analysis of EIF3a and angiogenesis signals using colorectal cancer data from TCGA; Figures B and C: Correlation analysis of EIF3a and the number of endothelial cells in colon tumors (B) and rectal tumors (C); Figure D: Correlation analysis of EIF3a and vascular endothelial cell markers in colorectal tumors; Figure E: Effect of eIF3a expression in colorectal cancer cells on the angiogenesis capacity of HUVEC cells in a co-culture system, detected by angiogenesis assay; Figure F: Effect of eIF3a expression in colorectal cancer cells on the migration and invasion capacity of HUVEC cells in a co-culture system, detected by Transwell assay; Figure G: Effect of eIF3a expression in colorectal cancer cells on the proliferation capacity of HUVEC cells in a co-culture system, detected by CCK8 cell proliferation assay; Figures H and I: Effect of eIF3a expression in colorectal cancer cells on the migration capacity of HUVEC cells in a co-culture system, detected by scratch assay and statistical results. P <0.05,** P <0.01, *** P <0.001, **** P <0.0001);

[0041] Figure 5 This is a graph showing the results of eIF3a regulation of ANG secretion in colorectal cancer cells in Example 1 (Figure A: Label-free non-quantitative proteomics screening of eIF3a-regulated secretory proteins in colorectal cancer cells; Figure B: Volcano plot showing the difference in secretory proteins between the eIF3a knockdown group and the control group; Figure C: 96 significantly downregulated and 59 significantly upregulated secretory proteins were found in the proteomic analysis results; Figure D: Heatmap showing the secretory proteins with significant differences; Figure E: Angiogenesis antibody array detection of eIF3a-regulated angiogenesis-related secretory proteins in the supernatant of colorectal cancer cells; Figure F: ELISA experiment verifying the significantly different angiogenesis-related secretory proteins in the label-free proteomic analysis and cytokine antibody array results; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0042] Figure 6 This is a graph showing the screening results of the LNP formulation in Example 3 (Cy3-positive data of cells after delivering Cy3-modified siVEGF to HCT116 cells with different proportions of DMG-PEG2000-cRGD added to the LNP formulation); *** P <0.001);

[0043] Figure 7The figures show the size, particle size, potential, and encapsulation efficiency of the nanoparticles in Example 4 (Figure A: LNP size; Figure B: LNP particle size distribution; Figure C: LNP potential; Figure D: LNP encapsulation efficiency).

[0044] Figure 8 This is a diagram showing the results of the HCT116 cell transfection experiment in Example 4 (Figure A: Fluorescence images of LNP (siE+siV) and cRGD-LNP (siE+siV) after cell uptake; Figure B: Flow cytometry quantification; cell bar = 200µm; siE: siEIF3a; siV: siVEGF).

[0045] Figure 9 This is a diagram showing the results of the HCT116 cell uptake assay in Example 4 (Figures A and B: laser confocal images of LNP (siE+siV) and cRGD-LNP (siE+siV) after cell uptake; LysoTracker labeled lysosomes, Hoechst labeled nuclei, cell bar = 20 μm; siE: siEIF3a; siV: siVEGF).

[0046] Figure 10 Figure 4 shows the results of the in vitro inhibition efficiency verification in Example 4 (Figures A and B: RT-PCR detection of EIF3a and VEGF mRNA expression levels after transfecting colorectal cancer cell lines HCT116 (A) and SW480 (B) with cRGD-LNP(siE), cRGD-LNP(siV), cRGD-LNP(siE+siV), cRGD-LNP(siNC), LNP(siE+siV), or LNP(siNC); Figures C and D: Transfection of colorectal cancer cell lines HCT116 (C) and SW480 (D) with cRGD-LNP(siE), cRGD-LNP(siV), or cRGD-LNP(siE+siV)). After cRGD-LNP (siNC), LNP (siE+siV), or LNP (siNC), Western blotting was used to detect the protein expression levels of EIF3A and VEGF; siE: siEIF3a; siV: siVEGF).

[0047] Figure 11These are the results of the in vivo imaging experiment in mice in Example 4 (Figures A and C: After injecting cRGD-LNP (siE+siV) into the tail vein of the experimental group of tumor-bearing mice, the mice were sacrificed at 12h (A) and 24h (C) after injection, and the heart, liver, spleen, lung, kidney and tumor tissues were isolated for in vivo imaging detection; Figures B and D: After injecting LNP (siE+siV) into the tail vein of the control group of tumor-bearing mice, the mice were sacrificed at 12h (B) and 24h (D) after injection, and the heart, liver, spleen, lung, kidney and tumor tissues were isolated for in vivo imaging detection; siE: siEIF3a; siV: siVEGF).

[0048] Figure 12 This is a diagram showing the results of the in vivo drug administration experiment in Example 4 (Figure A: Experimental flowchart; Figure B: Mouse body weight changes measured and recorded every 3 days; Figure C: Tumor volume changes measured and recorded every 3 days; Figure D: Tumor tissue volume measured; Figure E: Tumor tissue mass measured; Figure F: Immunohistochemical detection of CD31 expression in tumor tissues of each group to reflect the degree of angiogenesis; siE: siEIF3a; siV: siVEGF).

[0049] Figure 13 This is a diagram showing the pathological results of the in vivo toxicity experiment in Example 4 (HE staining results of heart, liver, spleen, lung, and kidney tissue sections from mice in the cRGD-LNP(siNC), cRGD-LNP(sIV), cRGD-LNP(SiE), LNP(siE+siV), and cRGD-LNP(siE+siV) groups; siE: siEIF3a; siV: siVEGF). Detailed Implementation

[0050] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0051] The reagents used in this invention (including lipid raw materials, siRNA, preparation excipients, and experimental reagents) can all be obtained through existing commercial channels, unless otherwise specified in this invention. Specifically, conventional lipid raw materials, preparation excipients, and experimental reagents can be purchased directly from conventional reagent suppliers. The siRNA targeting EIF3a (siEIF3a) and the siRNA targeting VEGF (siVEGF) can be customized by a professional nucleic acid manufacturer according to the sequences shown in Table 1 (this invention was customized by Jintuosi (Wuhan) Biotechnology Co., Ltd.). The experimental conditions and methods involved in this invention, unless otherwise specified in this invention, are all conventional conditions and methods known to those skilled in the art, and will not be described in detail here.

[0052] Table 1. siRNA sequence information used in this invention

[0053]

[0054] Example 1: Preliminary study on the mechanism of resistance to anti-angiogenic therapy in colorectal cancer:

[0055] I. Association between high expression of EIF3a and poor prognosis of colorectal cancer

[0056] First, using mRNA expression data and patient prognostic information from colorectal cancer samples in the TCGA database, survival analysis revealed that colorectal cancer patients with high EIF3a expression had a poorer prognosis. Figure 1 A).

[0057] Subsequently, analysis of colorectal tumor samples from the TCGA database and normal colorectal samples from the GTEx database revealed that the mRNA expression level of EIF3a in colorectal cancer tissue was significantly higher than that in normal colorectal tissue. Figure 1 B); Simultaneously, 35 paired colorectal cancer and adjacent normal tissue samples were collected, and the results were validated at the mRNA and protein levels by RT-PCR and immunohistochemistry, respectively. The results showed that the mRNA expression level of EIF3a in tumor tissues of colorectal cancer patients was significantly higher than that in normal colorectal tissues. Figure 1 C~E);

[0058] Next, to further investigate the role of EIF3a in colorectal cancer metastasis, the GSE131418 dataset, containing 545 primary colorectal cancer lesion samples and 73 metastatic lesion samples, was analyzed. The results showed that the expression level of EIF3a in metastatic colorectal cancer lesions was significantly higher than that in primary lesions. Figure 1 F~G); Subsequently, samples from 44 primary colorectal cancer lesions without metastasis and 79 primary colorectal cancer lesions were collected. The results showed that the mRNA expression level of EIF3a in non-metastatic primary colorectal cancer lesions was significantly lower than that in primary colorectal cancer lesions. Figure 1 H~I).

[0059] The above results indicate that EIF3a is specifically highly expressed in colorectal cancer and is associated with tumor metastasis and poor patient prognosis.

[0060] II. Inhibitory effect of EIF3a knockdown on in vivo metastasis and angiogenesis of colorectal cancer (mouse model)

[0061] The spleen injection liver metastasis model is a classic model for studying colorectal cancer metastasis.

[0062] MC38 cell lines (mouse colon cancer cells) were infected with lentiviruses carrying the luciferase reporter gene (Luc) and shRNA targeting the murine eIF3a gene (shEIF3a). After selection, a stable MC38 cell line (MC38-Luc-shEIF3a) with stable eIF3a gene knockdown and luciferase expression was obtained. Simultaneously, a MC38 cell line infected with lentiviruses carrying luciferase and a non-targeting negative control shRNA (shNC) served as a control stable cell line (MC38-Luc-shNC).

[0063] Two groups of C57 mice were injected into the spleen with MC38-Luc-shEIF3a (EIF3a knockdown group) and MC38-Luc-shNC (negative control group), respectively; in vivo imaging was then performed every 3 days; the results are as follows. Figure 2 As shown in A~B, metastatic lesions were observed in the liver of the control group (shNC) mice as early as day 12, while no obvious liver metastases were found in the shEIF3a group mice until day 21.

[0064] Subsequently, the mice were euthanized and their livers and spleens were removed. The tumors in the spleen of the mice in the shEIF3a group metastasized less to the liver, while the tumors in the control group had significantly stronger invasive and metastatic capabilities (Figures 2C-D).

[0065] New blood vessels are the main channels for tumor cell invasion and metastasis. We detected the vascular endothelial cell marker CD31 by immunohistochemistry and found that the tumor blood vessel density in mice was significantly reduced after EIF3a knockdown (Figure 2E~F).

[0066] The above results indicate that knocking down EIF3a significantly inhibits the in vivo metastatic ability of colorectal cancer cells and the process of tumor angiogenesis.

[0067] III. Downregulation of EIF3a significantly enhances bevacizumab sensitivity.

[0068] The GSE60331 dataset was used to analyze the correlation between EIF3a mRNA expression level and bevacizumab efficacy in colorectal cancer patients. Results showed that approximately 62.5% of patients in the low EIF3a expression group were sensitive to bevacizumab treatment, compared to only 33.3% in the high expression group, suggesting that colorectal cancer patients with low EIF3a expression have better bevacizumab efficacy. Figure 3 A).

[0069] The above results were further verified in animal experiments:

[0070] HCT116 cell lines (human colorectal cancer cell lines) were infected with lentiviruses containing shRNA (shEIF3a) targeting the human EIF3a gene. After selection, stable HCT116 cell lines with EIF3a gene knockdown (HCT116-shEIF3a) were obtained. Simultaneously, HCT116 cell lines infected with lentiviruses carrying negative control shRNA (shNC) served as control cell lines (HCT116-shNC).

[0071] A subcutaneous xenograft model was constructed in nude mice by subcutaneously injecting a stable knockdown colorectal cancer cell line (HCT116-shEIF3a) and a control cell line (HCT116-shNC). The mice were divided into four groups (shEIF3a group, shNC group, shEIF3a + bevacizumab group, and shNC + bevacizumab group), with six mice in each group. The size of the subcutaneous tumors was measured periodically. Figure 3 B) and nude mouse weight ( Figure 3 C); The mice were euthanized approximately one month later. Figure 3 D), tumor removal and photography ( Figure 3 E) After that, the tumor volume was measured ( Figure 3 F) and quality ( Figure 3 G), and CD31 immunohistochemistry was used to detect angiogenesis in the tumor. Figure 3 H).

[0072] The above results, at both clinical and animal levels, indicate that downregulation of EIF3a can significantly improve the sensitivity of colorectal cancer to bevacizumab treatment.

[0073] IV. EIF3a regulates angiogenesis in colorectal cancer

[0074] Analysis of colorectal cancer patient samples showed that EIF3a is associated with intratumoral angiogenesis (… Figure 4 A) Number of endothelial cells ( Figure 4 The mRNA expression levels of vascular endothelial cell markers CD31 (PECAM-I), VE-Cadherin (CDH5), VEGFR2 (KDR), and Vimentin (VIM) were measured (B, C). Figure 4 D) showed a significant positive correlation; the above results suggest at the clinical level that EIF3a is involved in regulating the angiogenesis process in colorectal cancer.

[0075] Further investigation was conducted using in vitro co-culture experiments to determine whether EIF3a in colorectal cancer cells could affect the activity of the vascular endothelial cell line HUVEC in the co-culture system.

[0076] After co-culturing HUVECs with the EIF3a knockdown group and the control group colorectal cancer cell lines HT29 or HCT116 for 48 h, the changes in the angiogenesis, proliferation, migration and invasion abilities of HUVECs were detected by angiogenesis assay, CCK8 cell proliferation assay, Transwell assay and scratch assay.

[0077] The results show that, compared with the control group of colorectal cancer cells, HUVEC cells co-cultured with EIF3a knockout colorectal cancer cells have a significantly higher angiogenesis capacity. Figure 4 E), Migration and Invasion Capabilities ( Figure 4 F, H, I), proliferation capacity (F, H, I), Figure 4 G) all decreased significantly.

[0078] The above results indicate that EIF3a is involved in regulating angiogenesis in colorectal cancer.

[0079] V. EIF3a-ANG axis-mediated drug resistance and synergistic effect of dual-target intervention

[0080] The above studies indicate that EIF3a can significantly affect the angiogenesis capacity of colorectal cancer. Tumor cells mainly regulate angiogenesis in the microenvironment by secreting large amounts of pro-angiogenic factors; therefore, this step further designs experiments to identify secreted proteins that play a key role in this process.

[0081] Label-free proteomics mass spectrometry was used to screen for secreted proteins that showed significant differences between the supernatants of colorectal cancer cells in the EIF3a knockdown group and the control group. Figure 5 (A, B), the results showed that 96 and 59 secreted proteins were significantly upregulated and downregulated, respectively. Figure 5 C, D). Considering that some low-abundance secreted proteins may not be detected by mass spectrometry, a more sensitive angiogenesis antibody array was further used to detect the differences in the secretion levels of pro-angiogenesis-related proteins in the supernatant of colorectal cancer cells. Figure 5 E). Based on the above experimental results and a review of the literature, we selected the secreted proteins (including ANG, PDGF-BB, RANTES, and COX2) that showed the most significant differences and played a key role in angiogenesis, and verified them using an ELISA assay. Figure 5 F). The difference in ANG was the most significant in the results, indicating that EIF3a can significantly promote the secretion of ANG by colorectal cancer cells.

[0082] ANG is the first pro-angiogenic protein discovered to originate from tumor cells, and it is significantly upregulated after bevacizumab treatment. Compared to most angiogenesis factors, ANG has a more potent pro-angiogenic effect. Studies have shown that ANG not only directly participates in processes such as vascular development, vascular maturation, and vascular homeostasis, but it is also essential for the angiogenesis activity of various pro-angiogenic proteins such as VEGF, FGF, and EGF. At the molecular level, ANG promotes angiogenesis by activating a series of signaling cascades in vascular endothelial cells, such as ERK1 / 2, SAPK / JNK, PI3K / AKT, mTOR, NF-κB, and Bcl-2 signaling. Studies have found that serum ANG levels are significantly elevated in colorectal cancer patients, and high ANG levels are associated with higher disease stages, poorer survival, and higher tumor microvessel density in colorectal cancer. After bevacizumab treatment, the expression level of ANG in colon tumor-derived endothelial cells significantly increased. All of these results indicate that ANG plays an important role in compensatory angiogenesis after bevacizumab treatment.

[0083] These results suggest that EIF3a may promote tumor angiogenesis and lead to bevacizumab treatment failure (mediating resistance) by activating the secretion of ANG, a compensatory angiogenesis factor independent of VEGF. At the same time, targeting and inhibiting EIF3a and VEGF can effectively inhibit tumor metastasis and improve the efficacy of anti-angiogenic therapy.

[0084] Example 2: Preparation of cRGD-modified LNPs targeting dual siRNAs:

[0085] Based on the results of Example 1, this example combines the commercially available ionizable cationic lipid SM-102 to construct a lipid nanoparticle delivery system that simultaneously blocks the VEGF pathway (direct target) and the EIF3a-ANG axis (compensatory pathway). Furthermore, it employs cRGD tumor-targeting peptides for modification, further enhancing the nanoparticles' targeting of tumor vascular endothelial cells and tumor cells to achieve synergistic inhibition of tumor angiogenesis through dual pathways. The preparation process is as follows:

[0086] I. Raw material pretreatment

[0087] 1. Preparation of liposome formulation stock solution

[0088] Weigh out the ionizable cationic lipids SM-102, cholesterol, DOPE phospholipids, DMG-PEG2000, and DMG-PEG2000-cRGD separately (in a molar ratio of 50:38.5:10:0.75:0.75). Dissolve each component (SM-102, cholesterol, DOPE phospholipids, DMG-PEG2000, and DMG-PEG2000-cRGD) in anhydrous ethanol at a concentration of 10 mg / mL. Based on a total lipid component to siRNA mass ratio of 20:1, thoroughly mix the lipids of each component to form the liposome formulation stock solution.

[0089] 2. Preparation of dual siRNA mixture

[0090] Weigh out the siRNA targeting EIF3a (siEIF3a) and the siRNA targeting VEGF (siVEGF) (mixed at a mass ratio of 1:1, with a total siRNA mass of 20 μg, the sequences of which are shown in Table 1), and dissolve them together in 40 μL of 10 mM pH 4.0 sodium citrate buffer. Shake to mix well to form a dual siRNA mixture.

[0091] II. Carrier Assembly

[0092] Mix the liposome formulation stock solution and the dual siRNA mixture at a volume ratio of 3:1 until homogeneous.

[0093] III. Static Molding

[0094] Transfer the mixture to an ice bath (0°C) and let it stand in the dark for 15 minutes until the lipids self-assemble to form cRGD-modified LNPs that encapsulate double siRNAs. The target product is then obtained and is denoted as cRGD-LNP (siEIF3a+siVEGF).

[0095] Example 3: Formulation screening and validation of LNP:

[0096] This embodiment is used to verify the effect of the DMG-PEG2000-cRGD ratio in the LNP formulation of the present invention on the transfection efficiency of nanoparticles, so as to confirm the optimal formulation of LNP.

[0097] I. Experimental Materials

[0098] Samples: Ionizable cationic lipid SM-102, cholesterol, dope, DMG-PEG2000, DMG-PEG2000-cRGD.

[0099] Cells: HCT116 colorectal cancer cell line.

[0100] II. Experimental Methods and Results

[0101] Set up the following five LNP formulations:

[0102] Group 1, SM-102: Cholesterol: Dope: DMG-PEG2000: DMG-PEG2000-cRGD=50: 38.5: 10: 1.125: 0.375;

[0103] Group 2, SM-102: Cholesterol: Dope: DMG-PEG2000: DMG-PEG2000-cRGD=50: 38.5: 10: 1: 0.5;

[0104] Group 3, SM-102: Cholesterol: Dope: DMG-PEG2000: DMG-PEG2000-cRGD=50: 38.5: 10: 0.75: 0.75;

[0105] Group 4, SM-102: Cholesterol: Dope: DMG-PEG2000: DMG-PEG2000-cRGD=50: 38.5: 10: 0.5: 1.

[0106] Group 5, SM-102 : Cholesterol : Dope : DMG-PEG2000 = 50 : 38.5 : 10 : 1.5 (without DMG-PEG2000-cRGD, serving as the control group).

[0107] The corresponding liposome formulation stock solution was prepared according to the above formula, and LNPs were prepared. Each group of LNPs was then delivered to HCT116 cells with Cy3-modified siVEGF. The efficiency of intracellular siRNA delivery by LNP formulation was determined by quantifying the CY3 positivity rate using flow cytometry. Results are as follows... Figure 6 As shown.

[0108] The results above show that the optimal LNP formulation after adding tumor-targeting peptides is SM-102 : cholesterol : dope : DMG-PEG2000 : DMG-PEG2000-cRGD=50 : 38.5 : 10 : 0.75 : 0.75, which is the formulation corresponding to Example 2.

[0109] Example 4: Performance validation of cRGD-modified LNPs targeting dual siRNAs:

[0110] This embodiment is used to verify the basic physicochemical properties and application performance of the cRGD-modified LNP targeting dual siRNA prepared in Example 1.

[0111] I. Physicochemical property testing (particle size, potential, encapsulation efficiency)

[0112] 1. Experimental materials

[0113] Samples: cRGD-LNP (siEIF3a+siVEGF) prepared in Example 1, and LNP (siEIF3a+siVEGF) without cRGD modification; among them, LNP (siEIF3a+siVEGF) was used as a control, and its only difference from cRGD-LNP (siEIF3a+siVEGF) was that "DMG-PEG2000" was used instead of "DMG-PEG2000-cRGD" in the preparation process.

[0114] Instruments: Dynamic light scattering instrument, ultracentrifuge, microplate reader, Zeta potential analyzer.

[0115] Reagent: 1×PBS buffer (pH 7.4).

[0116] 2. Experimental Methods and Results

[0117] (1) Particle size and particle size distribution: cRGD-LNP(siEIF3a+siVEGF) / LNP(siEIF3a+siVEGF) was diluted 3 times with 1×PBS buffer, and the particle size was detected using a dynamic light scattering instrument. The results are as follows: Figure 7 As shown in A and B.

[0118] (2) Zeta potential: cRGD-LNP(siEIF3a+siVEGF) / LNP(siEIF3a+siVEGF) was diluted 10-fold with 1×PBS buffer, and the potential was detected using a Zeta potential analyzer. The results are as follows: Figure 7 As shown in C.

[0119] (3) siRNA encapsulation efficiency: The prepared LNP was ultracentrifuged (4 min, 12000 rpm), and the absorbance of the supernatant at 260 nm was measured using a microplate reader. The amount of free siRNA in the solution was counted, and then the amount of siRNA encapsulated in the LNP was obtained, thereby calculating the siRNA encapsulation efficiency. The results are as follows: Figure 7 As shown in D.

[0120] The results above show that the size of LNP (siEIF3a+siVEGF) nanoparticles is around 140 nm, the particle size distribution (PDI) is 0.14, and the zeta potential is -2.2 mV. Furthermore, both LNP (siEIF3a+siVEGF) and cRGD-LNP (siEIF3a+siVEGF) nanoparticles can achieve an encapsulation efficiency of 90% for both siRNAs.

[0121] II. LNP Cell Transfection Assay

[0122] 1. Experimental materials

[0123] Samples: cRGD-LNP(siEIF3a+siVEGF), LNP(siEIF3a+siVEGF).

[0124] Cells: HCT116 colorectal cancer cell line.

[0125] 2. Experimental Methods and Results

[0126] cRGD-LNP (siEIF3a+siVEGF) or LNP (siEIF3a+siVEGF) were transfected into the colorectal cancer cell line HCT116, respectively, and the transfection status of the two nanoparticles in the HCT116 colorectal cancer cell line was observed. The results are as follows: Figure 8 As shown.

[0127] The results above show that both LNP (siEIF3a+siVEGF) and cRGD-LNP (siEIF3a+siVEGF) can deliver the two fluorescently labeled siRNAs into colorectal cancer cells, and the tumor-targeting peptide-modified nanoparticles cRGD-LNP (siEIF3a+siVEGF) have a better delivery effect.

[0128] III. LNP Cell Uptake Assay

[0129] 1. Experimental materials

[0130] Samples: cRGD-LNP(siEIF3a+siVEGF), LNP(siEIF3a+siVEGF).

[0131] Cells: HCT116 colorectal cancer cell line.

[0132] 2. Experimental Methods and Results

[0133] LNPs carrying Cy3-modified siVEGF and FITC-modified siEIF3a were transfected into HCT116 cells. Laser confocal microscopy was used to detect the cellular uptake of both LNP (siEIF3a+siVEGF) and cRGD-LNP (siEIF3a+siVEGF) nanoparticles in the HCT116 rectal cancer cell line. Results are as follows: Figure 9 As shown.

[0134] The results above show that both LNP (siEIF3a+siVEGF) and cRGD-LNP (siEIF3a+siVEGF) can deliver the two fluorescently labeled siRNAs into cells.

[0135] IV. Verification of In vitro Inhibition Efficiency

[0136] 1. Experimental materials

[0137] Samples: cRGD-LNP (siEIF3a), cRGD-LNP (siVEGF), cRGD-LNP (siEIF3a+siVEGF), cRGD-LNP (siNC), LNP (siEIF3a+siVEGF), LNP (siNC); where siNC is the "negative control siRNA".

[0138] Cells: colorectal cancer cell lines HCT116 and SW480.

[0139] 2. Experimental Methods and Results

[0140] After transfecting colorectal cancer cell lines HCT116 and SW480 with cRGD-LNP (siEIF3a), cRGD-LNP (siVEGF), cRGD-LNP (siEIF3a+siVEGF), cRGD-LNP (siNC), LNP (siEIF3a+siVEGF), or LNP (siNC), respectively, the mRNA expression levels of EIF3a and VEGF were detected by RT-PCR, and the protein expression levels of EIF3a and VEGF were detected by Western blotting. Results are as follows: Figure 10 As shown.

[0141] The results above show that after co-delivering dual siRNAs with LNP (siEIF3a+siVEGF) and cRGD-LNP (siEIF3a+siVEGF) nanoparticles, the mRNA and protein expression levels of EIF3a and VEGF in HCT116 and SW480 colorectal cancer cells were significantly reduced; and the cRGD-modified nanoparticles promoted cellular uptake of them. This indicates that the two nanoparticles can significantly downregulate the expression of EIF3a and VEGF in the above cancer cells in vitro through cell transfection.

[0142] V. In vivo targeting verification

[0143] 1. Experimental materials

[0144] Samples: cRGD-LNP(siEIF3a+siVEGF), LNP(siEIF3a+siVEGF).

[0145] Animal: BALB / c Nude mouse.

[0146] 2. Experimental Methods and Results

[0147] A subcutaneous xenograft tumor model was established in nude mice. Mice in the experimental group were injected intravenously with 0.5 mg / kg of cRGD-LNP (siEIF3a+siVEGF), while mice in the control group were injected intravenously with the same dose of LNP (siEIF3a+siVEGF) without a target. Mice were sacrificed at 12 h and 24 h post-injection, and heart, liver, spleen, lung, kidney, and tumor tissues were isolated for in vivo imaging to observe the enrichment of LNPs in the tumor. Results are as follows: Figure 11 As shown.

[0148] The results above show that cRGD-LNP (siEIF3a+siVEGF) promotes the accumulation of nanoparticles at the tumor site and effectively improves the delivery efficiency of siRNA in vivo.

[0149] VI. Verification of in vivo anti-tumor effects

[0150] 1. Experimental materials

[0151] Samples: cRGD-LNP (siEIF3a+siVEGF), LNP (siEIF3a+siVEGF), cRGD-LNP (siEIF3a), cRGD-LNP (siVEGF), cRGD-LNP (siNC).

[0152] Animal: BALB / c Nude mouse.

[0153] 2. Experimental Methods and Results

[0154] To further construct a subcutaneous xenograft model of colorectal cancer and verify its in vivo anti-tumor effect.

[0155] A subcutaneous xenograft tumor model was constructed in nude mice, and the tumor was allowed to grow to 50 mm. 3 Thirty tumor-bearing mice were randomly divided into five groups of six each. Each group was administered cRGD-LNP (siEIF3a+siVEGF), LNP (siEIF3a+siVEGF), cRGD-LNP (siEIF3a), cRGD-LNP (siVEGF), or cRGD-LNP (siNC) via tail vein injection, respectively. Mouse weight and tumor volume were recorded every three days. After administration, the mice were euthanized, and the tumors were dissected to measure volume and weight. Immunohistochemistry was used to detect the expression levels of EIF3a and VEGF in the tumors. Results are as follows: Figure 12 As shown.

[0156] The results above show that the synergistic effect of cRGD-LNP (siEIF3a+siVEGF) on inhibiting tumor growth is significantly better than that of the single-target group. In addition, the effect of cRGD-LNP (siEIF3a+siVEGF) on inhibiting tumor growth is significantly better than that of LNP (siEIF3a+siVEGF) group, which further illustrates that the modification of the tumor-targeting peptide cRGD effectively improves the in vivo targeting of LNPs.

[0157] VII. In vivo toxicity verification

[0158] 1. Experimental materials

[0159] Same as "Verification of in vivo anti-tumor effects".

[0160] 2. Experimental Methods and Results

[0161] HE staining was performed on the heart, liver, spleen, lungs, and kidneys of mice used in the "in vivo antitumor effect verification" study. The results are as follows: Figure 13 As shown, no obvious tissue damage was found.

[0162] The results above indicate that the injection of LNPs in each group did not cause significant in vivo toxicity.

[0163] In summary, the cRGD-modified LNP prepared in this invention can efficiently encapsulate siEIF3a and siVEGF, possessing excellent physicochemical properties such as suitable particle size, low PDI, and high encapsulation efficiency. Furthermore, the cRGD modification enables specific targeted delivery to colorectal cancer cells. Combined with the mechanism of action of EIF3a in regulating tumor angiogenesis and enhancing bevacizumab sensitivity, as confirmed by previous studies, and the advantages of dual-target synergistic inhibition of tumors and reversal of drug resistance, the LNP of this invention can be used to prepare anti-angiogenic therapeutic drugs for colorectal cancer, achieving multiple therapeutic effects of "precise targeting of tumor cells + dual-target synergistic tumor inhibition + reversal of anti-angiogenic drug resistance," providing a new and effective treatment strategy for colorectal cancer (especially bevacizumab resistant patients).

[0164] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A targeted LNP with dual gene silencing and cRGD modification, characterized in that, It is prepared from lipid raw materials and nucleic acid raw materials; The lipid raw materials include ionizable cationic lipid SM-102, cholesterol, DOPE phospholipids, DMG-PEG2000, and DMG-PEG2000-cRGD, and the molar ratio of ionizable cationic lipid SM-102 : cholesterol : DOPE phospholipids : DMG-PEG2000 : DMG-PEG2000-cRGD is 50 : 38.5 : 10 : 0.75 : 0.

75. The nucleic acid raw materials include siRNA targeting EIF3a and siRNA targeting VEGF, which are mixed in a 1:1 mass ratio; the sense strand sequence of the siRNA targeting EIF3a is 5'→3' CGUGCUGAUGAUGAUCGGUUUTT and the antisense strand sequence is 5'→3' AAACCGAUCAUCAUCAGCACGTT; the sense strand sequence of the siRNA targeting VEGF is 5'→3' GGAGUACCCUGAUGAGAUCTT and the antisense strand sequence is 5'→3' GAUCUCAUCAGGGUACUCCTT. During preparation, the lipid raw material is dissolved in anhydrous ethanol. Based on the mass ratio of total lipid components to siRNA of 20:1, the lipids of each component are thoroughly mixed to form a liposome formulation stock solution. The nucleic acid raw material is dissolved in buffer to form a dual siRNA mixture. The two are then mixed and allowed to stand to obtain the final product.

2. The targeted LNP with dual gene silencing and cRGD modification according to claim 1, characterized in that, When preparing LNPs, the nucleic acid raw materials are dissolved in 10mM sodium citrate buffer at pH 4.

0.

3. The targeted LNP with dual gene silencing and cRGD modification according to claim 1, characterized in that, When preparing LNP, the liposome formulation stock solution and the double siRNA mixture were mixed at a volume ratio of 3:1 and allowed to stand for 15 minutes.

4. A method for preparing a targeted LNP with dual gene silencing and cRGD modification as described in any one of claims 1 to 3, characterized in that, The specific steps include the following: (1) Raw material pretreatment: Weigh out ionizable cationic lipids SM-102, cholesterol, DOPE phospholipids, DMG-PEG2000 and DMG-PEG2000-cRGD and dissolve them in ethanol solution respectively. After each raw material is completely dissolved, mix the resulting solutions evenly to form the liposome formulation stock solution. At the same time, take out the siRNA targeting EIF3a and the siRNA targeting VEGF, dissolve them together in sodium citrate buffer, shake and mix well to form a dual siRNA mixture. (2) Vector assembly: The liposome formulation stock solution obtained in step (1) is mixed with the double siRNA mixture at a volume ratio of 3:1; (3) Standing and forming: Place the mixture after step (2) in an ice bath for 15 minutes until the lipids self-assemble to form cRGD modified LNPs that encapsulate double siRNA.

5. The application of a targeted LNP with dual gene silencing and cRGD modification as described in any one of claims 1 to 3 in the preparation of an anti-angiogenic therapeutic drug for colorectal cancer.

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