CN-121041241-B - Double-gene silencing cRGD modified targeting LNP and application thereof in preparation of colorectal cancer anti-angiogenesis therapeutic drugs

Abstract

The invention provides preparation of double-gene silencing and cRGD modified targeting LNP and application thereof in colorectal cancer anti-angiogenesis treatment, wherein the LNP is prepared from lipid raw materials and nucleic acid raw materials, the lipid raw materials comprise ionizable cationic lipids SM-102, cholesterol, DOPE phospholipid, DMG-PEG2000 and DMG-PEG2000-cRGD, the nucleic acid raw materials comprise siEIF a and siVEGF, and when the LNP is prepared, the lipid is dissolved in ethanol, and the nucleic acid is dissolved in buffer solution and then mixed uniformly and kept stand. The invention is realized by combining cRGD targeting modification and double siRNA co-delivery, not only promotes targeting and uptake efficiency of LNP on colorectal cancer cells by depending on cRGD, but also solves the problem of anti-angiogenesis treatment drug resistance of colorectal cancer which cannot be effectively broken through in the prior art by acting on VEGF channel and EIF3a-ANG axis through double siRNA respectively.

Inventors

• MEI CHAO
• GUO JIAJING
• WU SANLAN
• GONG WEIJING
• ZHU XINXIN

Assignees

• 华中科技大学同济医学院附属协和医院

Dates

Publication Date

20260508

Application Date

20251009

Claims (5)

1. 1. The double-gene silencing cRGD modified targeting LNP is characterized by being prepared from a lipid raw material and a nucleic acid raw material; Wherein the lipid raw materials comprise ionizable cationic lipid SM-102, cholesterol, DOPE phospholipid, DMG-PEG2000 and DMG-PEG2000-cRGD, and the ionizable cationic lipid SM-102 comprises cholesterol, DOPE phospholipid, DMG-PEG 2000-cRGD=50, 38.5, 10, 0.75; The nucleic acid raw material comprises siRNA targeting EIF3a and siRNA targeting VEGF, wherein the siRNA targeting EIF3a and the siRNA are mixed according to a mass ratio of 1:1, the sequence of the sense strand of the siRNA targeting EIF3a is 5 '. Fwdarw.3' CGUGCUGAUGAUGAUCGGUUUTT, the sequence of the antisense strand is 5 '. Fwdarw.3' AAACCGAUCAUCAUCAGCACGTT, and the sequence of the sense strand of the siRNA targeting VEGF is 5 '. Fwdarw.3' GGAGUACCCUGAUGAGAUCTT, and the sequence of the antisense strand is 5 '. Fwdarw.3' GAUCUCAUCAGGGUACUCCTT; The preparation method comprises the steps of dissolving lipid raw materials in absolute ethyl alcohol, respectively sucking lipid of each component according to the mass ratio of total lipid components to siRNA being 20:1, fully mixing to form liposome formula stock solution, dissolving nucleic acid raw materials in buffer solution to form double siRNA mixed solution, uniformly mixing the two, and standing to obtain the liposome.
2. 2. The dual gene silencing, cRGD modified, targeted LNP of claim 1 wherein the LNP is prepared by dissolving the nucleic acid starting material in 10mM sodium citrate buffer at pH 4.0.
3. 3. The dual gene silencing, cRGD modified targeting LNP of claim 1 wherein the liposome formulation stock solution and the dual siRNA mixture are mixed in a volume ratio of 3:1 for a resting time of 15 minutes when preparing LNP.
4. 4. A method for preparing the double-gene silencing cRGD modified targeting LNP according to any one of claims 1 to 3, comprising the following steps: (1) Raw material pretreatment, namely weighing ionizable cationic lipid SM-102, cholesterol, DOPE phospholipid, DMG-PEG2000 and DMG-PEG2000-cRGD, respectively dissolving in ethanol solution, uniformly mixing the obtained solutions after the raw materials are completely dissolved to form liposome formula stock solution, simultaneously sucking siRNA targeting EIF3a and siRNA targeting VEGF, dissolving the siRNA and the siRNA together in sodium citrate buffer solution, and uniformly oscillating to form double siRNA mixed solution; (2) Carrier assembly, namely uniformly mixing the liposome formula stock solution obtained in the step (1) with the double siRNA mixed solution according to the volume ratio of 3:1; (3) And (3) standing and forming, namely standing the mixed solution obtained after the step (2) is uniformly mixed under ice bath condition for 15 minutes, and forming the cRGD modified LNP wrapping the double siRNA after lipid self-assembly.
5. 5. Use of a dual gene silencing, cRGD modified, targeted LNP as described in any one of claims 1-3 in the preparation of a medicament for anti-angiogenic treatment of colorectal cancer.

Description

Double-gene silencing cRGD modified targeting LNP and application thereof in preparation of colorectal cancer anti-angiogenesis therapeutic drugs Technical Field The invention belongs to the technical field of biological medicines, relates to a tumor targeted delivery system and colorectal cancer anti-angiogenesis treatment technology, and particularly relates to a double-gene silencing, cyclic arginine-Glycine-aspartic acid polypeptide (CYCLIC ARGININE-Glycine-ASPARTIC ACID, CRGD) modified targeted lipid nanoparticle (Lipid Nanoparticle, LNP) and application thereof in colorectal cancer anti-angiogenesis treatment. Background In clinical treatment of metastatic colorectal cancer, anti-angiogenesis therapy is a core strategy to delay tumor progression, improve patient survival, wherein bevacizumab [ anti-vascular endothelial growth factor (Vascular Endothelial Growth Factor, VEGF) monoclonal antibody ] is used as a first-line targeting drug for the VEGF/VEGFR pathway, which has been widely used clinically for many years. However, the treatment scheme has the remarkable limitations that on one hand, the clinical curative effect of the treatment scheme has great individual difference, the objective remission rate is only 20% -50%, and on the other hand, even if the initial treatment is effective, almost all patients can eventually have drug resistance, so that the treatment failure is caused. The core mechanism of bevacizumab resistance is that the tumor microenvironment maintains tumor blood supply by activating a VEGF independent compensatory angiogenesis pathway, and the problem has become a key bottleneck for restricting the improvement of anti-angiogenesis therapeutic effect of colorectal cancer. The prior study has preliminarily clarified the common mechanism of drug resistance that tumors can maintain blood supply by up-regulating angiogenesis promoting signals independent of VEGF, wherein Angiogenin (ANG) is a first potent angiogenesis promoting protein which is found to be derived from tumor cells, the expression level of which is significantly up-regulated after bevacizumab treatment, and plays an important role in mediating tumor compensatory angiogenesis and bevacizumab drug resistance. However, although the effects of compensatory factors such as ANG are known, three technical shortboards still exist in the current anti-angiogenesis treatment field, the problem that the drug resistance cannot be effectively overcome is that firstly, a single target inhibition strategy dominates clinical practice, existing drugs (such as bevacizumab, ramucirumab and the like) are focused on VEGF/VEGFR channels and cannot block compensatory factor-mediated compensatory angiogenesis processes such as ANG and the like, so that the drug resistance cannot be avoided, secondly, the combined treatment scheme has remarkable limitations, although the bevacizumab is used with chemotherapy and immune checkpoint inhibitors in clinical trial, the drug resistance can only be partially delayed, toxicity superposition risk can be remarkably increased, and drug safety of patients is threatened, thirdly, the gene intervention technology has obvious defects, the RNA interference (RNA INTERFERENCE, RNAI) technology shows huge potential in tumor targeted treatment, the problems of poor tumor targeting, low endocytosis efficiency and the like in the traditional small interfering RNA ‌ (SMALL INTERFERING RNA, SIRNA) delivery carrier generally exist, and the clinical transformation of gene therapy is hindered due to the lack of a carrier system capable of efficiently delivering siRNA with multiple targets. In order to break through the technical bottleneck, the applicant carries out targeted research and makes key breakthroughs: eukaryotic translation initiation factor 3a (Eukaryotic translation initiation factor a, EIF3 a) is the largest functional subunit of eukaryotic translation initiation factor 3 (EIF 3), playing a key regulatory role in translation initiation. Recent studies reveal their proto-oncogene properties, confirming that they are closely related to tumorigenic development, metastasis, prognosis and therapeutic response. EIF3a is gradually becoming a novel drug action target with clinical transformation potential in EIF family, and particularly shows extremely high application potential in the field of anti-tumor. Through systematic analysis of clinical researches, animal experiments and molecular mechanism researches, the applicant firstly reveals that EIF3a can promote tumor compensatory angiogenesis and cause bevacizumab treatment failure by activating an 'EIF 3a-ANG axis' VEGF independent pathway, which is a core mechanism of an EIF3a-ANG mediated bevacizumab drug resistance mechanism. Meanwhile, the EIF3a and VEGF are targeted to inhibit, so that angiogenesis can be blocked from the double dimensions of a direct pathway and a compensation pathway, and a synergistic anti-angiogenesis effect is realized. Furthermore, th