Double-nucleic-acid nano-drug based on membrane fusion liposome as well as construction method and application of double-nucleic-acid nano-drug

Abstract

The invention discloses a dual-nucleic acid nano-drug based on membrane fusion liposome as well as a construction method and application of the dual-nucleic acid nano-drug, and belongs to the technical field of biomedical engineering. The invention constructs a dual-nucleic acid nano-drug siKRAS (at) FL (at) ApM based on membrane fusion liposome, wherein the dual-nucleic acid nano-drug siKRAS (at) FL (at) ApM is loaded with a nucleic acid aptamer ApM and a siRNA drug siKRAS. Experimental results prove that siKRAS (at) FL (at) ApM has relatively high stability, delivery efficiency and targeting property, and toxicity to normal cells is avoided; and nucleic acid drugs can be accurately delivered to corresponding subcellular spaces, so that efficient multi-space multi-target inhibition is realized, and the anti-cancer activity is synergistically enhanced. In-vivo experiment results prove that siKRAS (at) FL (at) ApM can efficiently inhibit proliferation and migration of tumor cells and has good biological safety. The invention provides a new strategy for efficient delivery of cancer-inhibiting nucleic acid drugs, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and particularly to a dual-nucleic acid nanomedicine based on membrane-fusion liposomes, a construction method thereof, and an application thereof. Background Art

[0002] The HGF / Met signal transduction pathway is closely related to the occurrence and development of various diseases. The HGF / Met signaling pathway is abnormally activated in a variety of tumors. Common causes include mutations and amplifications of the Met gene, abnormal autocrine or paracrine of HGF, activation of other receptor tyrosine kinase bypasses, etc. The abnormally activated HGF / Met pathway leads to tumor growth, migration, and invasion through multiple mechanisms. For example, activation of the HGF / Met pathway can cause changes in key cell cycle proteins (such as p27, phospho-Rb, E2F1, and c-Myc) to enable cells to escape from cell cycle arrest, can mediate cell survival by activating the PI3K and AKT signaling pathways, protect cells from the drugs commonly used in chemotherapy and radiotherapy, and further promote cancer cell proliferation, survival, and migration by triggering RAS-dependent Erk1 / 2 activation and STAT2 signaling. The abnormal activation of the HGF / Met pathway is not only a carcinogenic driving factor but also related to poor prognosis. Inhibiting the HGF / Met signaling pathway is an important target for the research and development of anti-tumor drugs.

[0003] However, tumors have multiple escape and drug resistance mechanisms that allow them to resist the inhibition of a single target. Although currently common HGF / Met inhibitors in clinical practice (small molecule Met kinase inhibitors and anti-HGF / Met antibody drugs) can significantly prolong the survival period of tumor patients, most tumor patients rapidly develop drug resistance after 3-5 months of targeted therapy. Among them, the abnormal activation of the Met downstream signal RAS / MAPK signaling pathway is one of the important factors leading to Met inhibitor resistance. Compared with single-target inhibition, simultaneous multi-target inhibition can better overcome the drug resistance caused by single-target inhibition and block the progression of tumors. However, as a once "undruggable" target KARS, there are few currently approved drugs available. Currently, there are mainly two small molecule inhibitors, Sotorasib and adagrasib. Existing studies have found that tumors can develop drug resistance to KRAS small molecule inhibitors by compensatorily increasing mutant KRAS proteins. Therefore, there are few drugs available for Met drug-resistant patients with KRAS mutations. In addition, simply combining the two drugs has no association between them, and they inhibit multiple targets in a "fighting alone" manner, with limited effects. Therefore, developing novel and effective intervention molecules targeting Met and KRAS to achieve efficient multi-target synergistic inhibition is still an ideal goal for drug research and development.

[0004] With the continuous development of molecular biology technology, nucleic acid drugs have attracted much attention due to their great application potential in disease diagnosis and treatment, including nucleic acid aptamers, small interfering nucleic acids (siRNAs), antisense nucleic acids, etc. Among them, nucleic acid aptamers are a new type of molecular recognition tool, also known as "chemical antibodies", which are screened by a systematic evolution of ligands by exponential enrichment (SELEX) technology. Due to their high specificity, non-immunogenicity, easy synthesis and modification, low cost and other advantages, nucleic acid aptamers have successfully been widely used as alternatives to antibodies for therapeutic drugs, imaging probes or as targeted delivery media. Currently, nucleic acid aptamers of hepatocyte growth factor receptor (Met) have been used in signal pathway regulation and anti-tumor research. Different from nucleic acid aptamers, siRNAs mainly act on intracellular mRNA through base complementary pairing, and then regulate protein expression to achieve therapeutic effects. siRNA drugs have many advantages such as short R & D cycle, high gene silencing effect, low in vivo toxicity, and a wide range of drug targets. Currently, a variety of siRNAs targeting different KRAS mutant subtypes have been designed and synthesized for tumor treatment research. Although these nucleic acid drugs can play a good inhibitory role, there are still some problems that limit their further clinical application: 1. The action time of nucleic acid aptamers is short: Nucleic acid aptamers targeting the RTK receptor protein on the cell membrane cannot continuously exert the inhibitory function of the membrane receptor-mediated signal pathway due to the endocytosis of cells. 2. Difficult delivery of siRNAs: Naked siRNAs have problems such as lack of targeting ability, difficulty in passing through the cell membrane barrier and lysosome barrier, which affect the exertion of their activity. 3. Both nucleic acid aptamers and siRNAs have the problem of short in vivo half-life: Free nucleic acid drugs are easily degraded by nucleases, and due to the small size of free nucleic acid drugs, they are easily filtered and cleared by the glomerulus in the body and excreted out of the body.

[0005] Currently, there is no report on a drug that integrates two nucleic acid drugs (a nucleic acid aptamer that targets and inhibits Met and an siRNA that targets and inhibits KRAS) into one based on Met and KRAS as targets, precisely targets and delivers them to their corresponding action sites (membrane / cytoplasm) in different spaces, realizes efficient multi-space multi-target inhibition, and exerts an efficient anti-cancer effect. Summary of the Invention

[0006] The purpose of the present invention is to provide a dual-nucleic acid nanodrug based on membrane-fusion liposomes and its construction method and application to solve the problems existing in the above-mentioned prior art. The present invention constructs a targeted anti-tumor drug from membrane-fusion liposomes, nucleic acid aptamers and siKRAS, realizes efficient multi-space multi-target inhibition, and synergistically enhances anti-cancer activity.

[0007] To achieve the above purpose, the present invention provides the following solutions:

[0008] The present invention provides a construction method of a dual-nucleic acid nanodrug based on membrane-fusion liposomes, including the following steps:

[0009] Construct membrane-fusion liposomes (named FL) using DMPC, DSPE-PEG2000, and DOTAP;

[0010] Mix the membrane-fusion liposomes with the siKRAS solution to prepare siKRAS membrane-fusion liposome particles (named siKRAS@FL);

[0011] Denature ApM-chol, mix it with the siKRAS membrane-fusion liposome particles, rotate, and centrifuge to collect the precipitate, thereby obtaining the dual-nucleic acid nanomedicine based on membrane-fusion liposomes (named siKRAS@FL@ApM);

[0012] Wherein, the nucleotide sequence of the siKRAS is as shown in SEQ ID NO.2; in the ApM-chol, chol represents cholesterol modification, and the nucleotide sequence of the ApM is as shown in SEQ ID NO.1.

[0013] Preferably, the ApM-chol is a cholesterol-modified Met nucleic acid aptamer; the siKRAS is an siRNA targeting and inhibiting mutant KRAS.

[0014] Preferably, the construction of the membrane-fusion liposomes includes the following steps: dissolve the DMPC, the DSPE-PEG2000, and the DOTAP in chloroform respectively, mix them, and perform rotary evaporation at a temperature of 45°C and a rotation speed of 45 rpm until the air pressure drops to 30 Pa, then introduce nitrogen gas to obtain the membrane-fusion liposomes in the form of a thin film;

[0015] Wherein, the molar ratio of the DMPC, the DSPE-PEG2000, and the DOTAP is 76.2:3.8:20.

[0016] Preferably, the preparation of the siKRAS membrane-fusion liposome particles includes the following steps: perform rotary evaporation of the siKRAS solution and the membrane-fusion liposomes for 1 h under normal pressure, and extrude back and forth through a polycarbonate membrane using a liposome extruder to obtain the siKRAS membrane-fusion liposome particles.

[0017] Preferably, the temperature of the rotation is 45°C, the rotation speed is 10 rpm, and the time is 1 h.

[0018] The present invention also provides a dual-nucleic acid nanomedicine based on membrane-fusion liposomes prepared by the above construction method.

[0019] The present invention also provides an application of the above dual-nucleic acid nanomedicine based on membrane-fusion liposomes in the preparation of a drug for treating cancer.

[0020] Preferably, the cancer treatment includes inhibiting cancer cell proliferation and inhibiting cancer cell migration.

[0021] Preferably, the cancer is non-small cell lung cancer

[0022] The present invention also provides a drug for treating cancer, and the drug takes the above-mentioned membrane-fusion liposome-based dual nucleic acid nanodrug as the main active ingredient.

[0023] Preferably, it further includes pharmaceutically acceptable excipients.

[0024] The present invention discloses the following technical effects:

[0025] The present invention provides a membrane-fusion liposome-based dual nucleic acid nanodrug, and its membrane-fusion liposome has good fusion performance with cell membranes and can efficiently deliver two nucleic acid drugs to their corresponding sites in different spaces (ApM is delivered to the cell membrane, and siKRAS is delivered to the cytoplasm), which can maximize the inhibition efficiency of nucleic acid drugs and ensure the stability of nucleic acid drugs during the delivery process to avoid degradation.

[0026] The nucleic acid drug system constructed by the present invention only needs to change the nucleic acid aptamer and siRNA sequences therein to inhibit specific targets with different spatial distributions, which is a general and customizable drug design strategy and can be used for personalized design of synergistic treatment strategies for different molecular subtypes and different cancer types. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings without creative efforts based on these drawings.

[0028] Figure 1 It is a figure showing the results of FL characterization experiments; among them, A is the particle size distribution; B is the surface potential; C is the transmission electron microscope image, and the scale bar is 200 nm; D is the result of the change in particle size over time;

[0029] Figure 2Figure for verifying the membrane fusion of different groups with cell membranes by the sulforhodamine B (SRB) colorimetric assay; A shows the relative fluorescence intensity analysis of different mixed systems (SRB@FL and A549-CM, SRB@L and A549-CM) at different reaction times; B shows the expression of Met protein in A549 cells and NCL-H661 cells; C shows the relative fluorescence intensity analysis of different mixed systems (SRB@FL@ApM and A549-CM, SRB@FL@ApM and H661-CM) at different reaction times.

[0030] Figure 3 Figure for the characterization experiment results of siKRAS@FL@ApM; among them, A shows the particle size distribution of FL, siKRAS@FL, FL@ApM, and siKRAS@FL@ApM; B shows the surface potential distribution of FL, siKRAS@FL, FL@ApM, and siKRAS@FL@ApM; C shows the transmission electron microscope images of FL and siKRAS@FL@ApM, and the scale bar is 200 nm;

[0031] Figure 4 Figure for the observation by laser scanning confocal microscope of siKRAS@FL@ApM; among them, FL is labeled with DiO dye (green), ApM is labeled with Cy5 (red); siKRAS is labeled with Cy3 (blue); Merged is the overlapping field of view; the scale bar is 1 μm;

[0032] Figure 5 Figure for the stability experiment results of free siKRAS and siKRAS@FL in 10% FBS;

[0033] Figure 6 Figure for the intracellular distribution of siKRAS in siKRAS@FL@ApM; among them, H33342 is the nucleus stained with Hoechst 33342 (blue); Lyso is the lysosome labeled with LysoTraCker Green DND-26 dye (green), and Cy3 is the labeled siKRAS (red); Merged is the overlapping field of view; Enlarged is the enlarged image of the part marked by the dotted box in the Merged figure; the scale bar is 1 μm;

[0034] Figure 7Uptake of siKRAS@FL and siKRAS@FL@ApM by A549 and NCL-H661 cells; where H33342 is the nucleus (blue) stained with Hoechst 33342; Lyso is the lysosome (green) stained with LysoTraCker Green DND-26 dye, and Cy3 is the labeled siKRAS (red); Merged is the overlapping field of view; Enlarged is the enlarged image of the part marked by the dashed box in the Merged figure; the scale bar is 1 μm; **** indicates P < 0.0001, and n.s. indicates P > 0.05;

[0035] Figure 8 Results of the effects of siKRAS@FL@ApM and each control group (HGF, siKRAS, siKRAS@FL, and FL@ApM) on the expression levels of p-Met and KRAS in A549 cells; where A is the protein expression levels of p-Met and KRAS analyzed by WB; B is the transcriptional level of the KRAS gene analyzed by RT-qPCR; * indicates P < 0.05, ** indicates P < 0.01, and **** indicates P < 0.0001;

[0036] Figure 9 Results of the effects of siKRAS@FL@ApM and each control group (HGF, siKRAS, siKRAS@FL, and FL@ApM) on the phosphorylation levels of Akt and Erk1 / 2 proteins in A549 cells;

[0037] Figure 10 Results of the effects of siKRAS@FL@ApM and different treatment groups (siKRAS@FL and FL@ApM) on the activities of different cell lines; where A is the analysis of the KRAS expression levels and mutation types in A549 cells and NCL-H661 cells by bioinformatics analysis; B is the analysis of the effects of siKRAS@FL@ApM on the activities of A549 cells and NCL-H661 cells by CCK-8 assay; * indicates P < 0.05, ** indicates P < 0.01, and n.s. indicates P > 0.05;

[0038] Figure 11 Results of the CCK8 assay analyzing the survival rates of A549 cells after treatment with different concentrations of siKRAS@FL@ApM, FL@ApM, and siKRAS@FL;

[0039] Figure 12 Growth curve of A549 cells under serum-containing conditions after different treatments (negative control, siKRAS@FL, FL@siKRAS, and siKRAS@FL@ApM) analyzed by RTCA;

[0040] Figure 13 Results of scratch assay examining the effects of siKRAS@FL, FL@ApM, and siKRAS@FL@ApM on cell migration ability; * indicates P < 0.05;

[0041] Figure 14 Results of in vivo targeting performance evaluation experiment of siKRAS@FL@ApM;

[0042] Figure 15 Results of anti-tumor activity evaluation experiment (tumor appearance, tumor mass, and tumor volume) of siKRAS@FL@ApM; * indicates P < 0.05, ** indicates P < 0.01, **** indicates P < 0.0001, n.s. indicates P > 0.05;

[0043] Figure 16 Results of HE and IHC experiments for anti-tumor activity evaluation of siKRAS@FL@ApM; scale bar is 50 μm; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, n.s. indicates P > 0.05;

[0044] Figure 17 Results of the effect of siKRAS@FL@ApM on mouse body weight;

[0045] Figure 18 Results of biosafety evaluation of siKRAS@FL@ApM; among them, the scale bar is 50 μm. Detailed implementation manners

[0046] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0047] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0049] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which will be obvious to those skilled in the art. Other embodiments obtained from the specification of this invention will be obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0050] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0051] The DMPC of this invention is dimyristoylphosphatidylcholine, with a CAS number of 18194-24-6;

[0052] DSPE-PEG2000 is 1,2-distearoyl-sn-glycero-3-phosphocholine-polyethylene glycol 2000, with a CAS number of 474922-22-0;

[0053] DOTAP is 1,2-dioleoyl-3-trimethylammonium propionate, with a CAS number of 132172-61-3.

[0054] Example 1 Preparation of Dual-Nucleic Acid Nanodrugs Based on Membrane-Fusion Liposomes

[0055] 1. Design and Synthesis of Monovalent Nucleic Acid Aptamers and siRNA

[0056] The nucleic acid aptamer ApM-chol that targets and inhibits the function of Met, where chol represents cholesterol-modified, and the nucleotide sequence of ApM is:

[0057] 5’-ATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTGATTTTT-3’(SEQ ID NO.1);

[0058] [[ID=3]]The nucleotide sequence of the siRNA (siKRAS) that targets and inhibits the KRAS G12S site is:

[0059] 5'-AGAAUAUCCAAGAGACAGGTT-3’(SEQ ID NO.2).

[0060] Preparation of FL

[0061] First, take 3.7 mg of DMPC powder, 0.23 mg of DSPE-PEG2000 powder and 1 mg of DOTAP powder according to the molar ratio of DMPC, DSPE-PEG2000 and DOTAP as: DMPC:DSPE-PEG2000:DOTAP = 76.2:3.8:20, and dissolve them in chloroform respectively. Mix the three solutions evenly and make the total volume up to 1 mL. Next, pour 1 mL of the chloroform solution into a round-bottom flask. Set the water bath temperature of the rotary evaporator to 45 °C and the rotation speed to 45 rpm. Start the rotary evaporator and gradually reduce the air pressure from 1000 Pa to 30 Pa evenly. Then seal the bottle mouth with a sealing film and introduce nitrogen into the round-bottom flask to promote more complete volatilization of chloroform, so that the FL film is more evenly dispersed. Subsequently, add 1.6 mL of DEPC water into the round-bottom flask. Under normal pressure, use the rotary evaporator to fully mix the DEPC water and the FL film. After 1 h, turn off the rotary evaporator and collect the solution. Then, extrude the solution back and forth 20 times through a polycarbonate membrane of a certain size using a liposome extruder to obtain uniform FL particles.

[0062] Preparation of siKRAS@FL@ApM

[0063] Synthesize a uniform FL film according to the synthesis method of FL. Then add 1.6 mL of siKRAS solution with a final concentration of 1 μM into the round-bottom flask. Under normal pressure, use the rotary evaporator to fully mix the DEPC-treated water and the FL film. After 1 h, turn off the rotary evaporator and collect the solution. Extrude the solution back and forth 20 times through a polycarbonate membrane of a certain size using a liposome extruder to obtain uniform siKRAS@FL particles. After denaturing ApM-chol into a stable secondary structure, mix ApM-chol with siKRAS@FL particles in a certain proportion, place them on a rotator (rotation speed set to 10 rpm), and synthesize for 1 h in an environment of 45 °C. Then centrifuge at 10000 g and 4 °C for 8 min to separate the supernatant and the precipitate, and resuspend the precipitate to obtain siKRAS@FL@ApM.

[0064] Characterization of the nanomaterials in Example 2

[0065] 1. Experimental method

[0066] 1.1 Particle stability distribution of FL and siKRAS@FL@ApM

[0067] The FL was extruded back and forth 20 times through polycarbonate membranes with pore sizes of 100 nm, 200 nm, and 400 nm using a liposome extruder, and the FL was diluted 10-fold with serum-free RPMI164021 medium and DEPC water respectively. The particle size distribution of FL was measured using a Malvern particle size analyzer at different time points. After synthesizing siKRAS@FL@ApM, siKRAS@FL@ApM was diluted with DEPC water to a concentration of 0.05 μM for ApM-chol, and the particle size distribution of siKRAS@FL@ApM was measured using a Malvern particle size analyzer at different time points.

[0068] 1.2 Quantification of siKRAS@FL@ApM

[0069] First, prepare standard products of Cy3-siKRAS and Cy5-ApM-chol with different concentrations, detect the fluorescence intensities of the standard products with different concentrations using an Agilent fluorescence spectrometer, and draw the standard curves of Cy3-siKRAS and Cy5-ApM-chol according to the fluorescence intensity-concentration relationship. Measure the fluorescence intensities of FL@ApM and siKRAS@FL@ApM, and calculate the content of Cy5-ApM-chol in FL@ApM and the contents of Cy3-siKRAS and Cy5-ApM-chol in siKRAS@FL@ApM according to the standard curves. According to the measured contents, substitute them into the following calculation formula to calculate the number of ApM-chol molecules conjugated to each FL:

[0070] DNA loading = N(DNA) / N(liposomes);

[0071] N(total) = 8π(d / 2) 2 ÷α;

[0072] N(liposomes) = C(lipid) × N A ×V÷N(total);

[0073] In the above formulas, N A is the Avogadro constant, N(total) is the number of lipids in each liposome, N(DNA) is the measured DNA content, N(liposomes) is the liposome content calculated according to the formula, d is the diameter of the material measured in the particle size analysis experiment, α is the apparent area of the lipid head group, and C is the concentration of the substance.

[0074] 1.3 Transmission Electron Microscope (TEM)

[0075] After appropriately diluting FL, FL@ApM, and siKRAS@FL@ApM with DEPC water, 10 μL of the sample was dropped onto a copper grid. After 5 min, the excess liquid was aspirated. Then, a mixture of DEPC water and 1% phosphotungstic acid negative staining solution with a volume ratio of 1:1 was dropped onto the copper grid. After negative staining for 30 min, the excess negative staining solution was aspirated and air-dried. The morphologies, sizes, and dispersibilities of FL, FL@ApM, and siKRAS@FL@ApM were observed using TEM.

[0076] 1.4 Investigation of the serum stability of siKRAS@FL@ApM

[0077] Free Cy3-siKRAS and Cy3-siKRAS@FL containing an equal concentration of Cy3-siKRAS were co-incubated with 5% FBS for 0, 1, 6, 12, and 24 h, respectively. Subsequently, Triton-x 100 with a final concentration of 1% was added to the solution, and the mixture was incubated at 37 °C for 30 min. Then, the siKRAS content in the mixed solution was detected by agarose gel electrophoresis.

[0078] 2. Experimental results

[0079] As Figure 1 shown in A, the average particle size of FL was approximately 105 nm, and the Polymer Dispersity Index (PDI) was 0.015, indicating good homogeneity of FL; as Figure 1 shown in D, the average particle size of FL remained at about 105 nm within 16 d, indicating good stability of FL. As Figure 1 shown in B, the surface potential of FL was 38.7 mV.

[0080] Subsequently, the present invention characterized the morphology of FL using a transmission electron microscope (TEM). As Figure 1 shown in C, FL was hollow spherical particles with a particle size of about 40 - 60 nm.

[0081] As Figure 3 shown in A, the particle size of siKRAS@FL@ApM was 276 nm; while the particle sizes of siKRAS@FL and FL@ApM incorporated with a single nucleic acid drug were both about 150 nm. This may be due to the fact that siKRAS@FL@ApM incorporated two nucleic acid drugs simultaneously, resulting in a larger particle size.

[0082] From Figure 3 shown in B, the surface potential of siKRAS@FL@ApM was -24.7 mV, lower than that of siKRAS@FL and FL@ApM. This may be because it contains two negatively charged nucleic acid drugs simultaneously.

[0083] TEM results showed that siKRAS@FL@ApM was spherical solid particles with uniform particle size, about 200 nm( Figure 3 as shown in C).

[0084] To more intuitively characterize the composition of siKRAS@FL@ApM, we imaged siKRAS@FL@ApM by CLSM. Limited by the resolution of CLSM, we synthesized siKRAS@FL@ApM with a particle size of about 500 nm, labeled siKRAS with Cy3 (blue), ApM-chol with Cy5 (red), and FL with DiO dye (green). From Figure 4 it can be seen that the red, blue, and green lights highly overlapped, indicating the binding of siKRAS, ApM, and FL, further demonstrating the successful construction of siKRAS@FL@ApM.

[0085] As Figure 5 shown, within 24 h of incubation, the content of free Cy3-siKRAS gradually decreased, while the content of Cy3-siKRAS@FL remained at a high level, indicating that the membrane-fusion liposomes effectively slowed down the degradation of siKRAS.

[0086] Example 3 Study on membrane fusion effect by sulforhodamine B (SRB) colorimetric method

[0087] Prepare membrane-fusion liposomes FL according to the method in Example 1.

[0088] First, prepare FL and liposomes L by the thin-film hydration method. Take 100 μL of SRB dye and mix it with 100 μL of FL / L solution, and extrude it back and forth through a 100-nm polycarbonate membrane 20 times using a liposome extruder, and dialyze it for 24 h using a microdialysis bag to remove the SRB dye not encapsulated in FL and L, and synthesize SRB@FL and SRB@L. Next, mix ApM-chol with SRB@FL and SRB@L respectively, place it on a rotator (rotation speed set to 10 rpm), and synthesize it in an environment of 45 °C for 1 h, then centrifuge at 10,000 g and 4 °C for 8 min to separate the supernatant and precipitate, and resuspend the precipitate to obtain SRB@FL@ApM and SRB@L@ApM. Then, we lysed the cells with a hypotonic lysis solution, extracted the cell membranes of A549 and NCL-H661 cells with a homogenizer, and extruded the cell membranes back and forth through a 100-nm polycarbonate membrane 30 times to prepare cell membrane vesicles (CM vesicle).

[0089] Subsequently, SRB@FL, SRB@L, SRB@FL@ApM, and SRB@L@ApM were mixed with the cell membrane vesicle solution at a mass ratio of 1:5. The fluorescence intensity in the mixed solution was measured every 5 min for a duration of 40 - 60 min. Finally, 1 μL of 1% (w / v) Triton-x 100 was added to generate the maximum fluorescence signal in the solution.

[0090] And the content mixing efficiency was calculated using the following formula:

[0091] Content mixing efficiency (%) = (SRB t - SRB min ) / (SRB max - SRB min ) × 100%;

[0092] In the above formula, SRB t is the fluorescence intensity of SRB at time t, SRB max is the maximum fluorescence intensity after adding Triton-x 100, and SRB min is the fluorescence intensity of SRB at t = 0.

[0093] Experimental results

[0094] As Figure 2 shown in A of, the fluorescence recovery of the SRB dye was only observed in the co-incubation group of SRB@FL and CM vesicle, while no fluorescence recovery of the SRB dye occurred in the co-incubation group of SRB@FL and FL and the co-incubation group of SRB@L and CM vesicle. This result confirmed that FL could undergo membrane fusion with the cell membrane.

[0095] Furthermore, the effect of ApM modification on the membrane fusion performance of FL was investigated. First, through literature research, we selected human non-small cell lung cancer A549 cells with high expression of Met protein and human large cell lung cancer NCL-H661 cells with low expression of Met protein for the experiment, and verified the expression of Met protein in the two cell lines by protein immunoblotting (Western blotting, WB) experiment ( Figure 2 shown in B of). Then, we separately extracted A549 cell membrane (A549-CM) and NCL-H661 cell membrane (H661-CM), and co-incubated them with the spherical nucleic acid nanodrug mixed with SRB (SRB@FL@ApM). The effect of ApM modification on the membrane fusion performance of FL was investigated by monitoring the fluorescence recovery of the SRB dye. From Figure 2As shown by the results of C, obvious fluorescence recovery was observed in the co-incubation group of SRB@FL@ApM with A549-CM with high expression of Met protein, while the fluorescence recovery degree in the co-incubation group of SRB@FL@ApM with H661-CM with low expression of Met protein was low. The above results indicate that the modification of ApM can indeed prevent FL from undergoing membrane fusion with cells with low expression of Met protein through electrostatic interaction. However, when Met receptors exist on the cell membrane surface, due to the interaction between ApM and the receptors, the cell membrane can closely contact with the liposome, thereby promoting the membrane fusion of the liposome and realizing the target-mediated membrane fusion ability.

[0096] Example 4 Targeting Performance Analysis and Escape Ability Exploration of siKRAS@FL@ApM

[0097] Prepare siKRAS@FL@ApM according to the method in Example 1.

[0098] 1. Targeting Performance Analysis

[0099] 1.1 Experimental Method

[0100] Respectively inoculate A549 cells (1.5×10 5 cells / dish) and NCL-H661 cells (1.5×10 5 cells / dish) into confocal culture dishes. After culturing at 37°C and 5% CO2 for 12 h, replace the culture medium with a culture medium containing ApM-chol, FL@ApM, siKRAS@FL or siKRAS@FL@ApM (the concentration of siKRAS is 50 nM, and the concentration of ApM-chol is 200 nM), and co-incubate with the cells for 2 h. Then, remove the culture medium, wash 3 times with PBS, and label the cell nuclei with H 33342 staining solution (co-incubate at 37°C for 10 min).

[0101] 1.2 Experimental Results

[0102] The present invention selects the cell line A549 with high expression of Met and the cell line NCL-H661 with low expression of Met, and observes the uptake behaviors of the two cell lines for siKRAS@FL and siKRAS@FL@ApM by CLSM to investigate whether siKRAS@FL@ApM can target and deliver siKRAS into the cytoplasm of cells with high expression of Met.

[0103] The results are as Figure 7As shown, compared with the NCL-H661 group, the A549 group treated with siKRAS@FL@ApM had stronger Cy3-siKRAS fluorescence (red); after siKRAS@FL was co-incubated with NCL-H661 cells and A549 cells respectively, the red fluorescence intensities of Cy3-siKRAS in the two groups of cells were close, and both fluorescence intensities were significantly lower than those in the co-incubation group of siKRAS@FL@ApM and A549.

[0104] The above results indicate that siKRAS@FL@ApM can not only target and deliver siKRAS into the cytoplasm of cells with high Met expression, but also, due to the modification of ApM, further promote the uptake of siKRAS@FL@ApM by cells and facilitate the cytoplasmic delivery of siKRAS.

[0105] 2. Investigation of escape ability

[0106] 2.1 Experimental method

[0107] A549 cells (2×10 5 cells / dish) were seeded in confocal dishes and cultured in a 37°C, 5% CO2 incubator for 12 h. Then, siKRAS@FL and siKRAS@FL@ApM (siKRAS concentration was 50 nM, ApM-chol concentration was 200 nM) were added to co-incubate with the cells for 2 h. Next, the culture medium was removed, fresh culture medium was replaced and the cells were cultured for another 1 h, washed 3 times with PBS, and the nuclei were labeled with H 33342 dye (37°C, 30 min), and the cell lysosomes were labeled with LysoTracker Green DND-26 lysosome dye (37°C, 20 min).

[0108] 2.2 Experimental results

[0109] As Figure 6 shown, after siKRAS@FL@ApM was incubated with A549 cells, a large amount of the red light of siKRAS was distributed in the cytoplasm and hardly overlapped with the green light of lysosomes, indicating that siKRAS@FL@ApM can achieve the cytoplasmic delivery of siKRAS, and this property will help improve the gene silencing ability of siKRAS.

[0110] Example 5 Investigation of the application of siKRAS@FL@ApM in anti-tumor activity at the cellular level

[0111] siKRAS@FL@ApM was prepared according to the method in Example 1.

[0112] 1. Protein immunoblotting experiment (Western Blotting, WB)

[0113] 1.1 Experimental method

[0114] A549 cells (1×10 6 cells / dish) were seeded in 6-cm dishes and cultured in an incubator at 37 °C with 5% CO2 for 12 h. Then the culture medium was replaced with serum-free medium containing FL, different concentrations of FL@ApM, siKRAS@FL, and siKRAS@FL@ApM. After co-incubation for 2 h, HGF was added at a final concentration of 1 nM, and the cells were further cultured for 30 min.

[0115] To investigate the effects of ApM-chol and FL@ApM on the HGF / Met signaling pathway under serum-containing conditions, A549 cells were seeded in 6-cm dishes at a seeding density of 1×10 6 cells / dish and cultured in an incubator at 37 °C with 5% CO2 for 12 h. Then the culture medium was replaced with complete medium containing different concentrations of ApM-chol and FL@ApM, and the cells were co-incubated for 12 h. Cells from each group were collected, and proteins were lysed and extracted using Western&IP cell lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor on a shaker at room temperature for 45 min. The protein concentration of each sample was quantified using a BCA protein assay kit. Proteins from different treatment groups were separated by 8% and 12% SDS-PAGE electrophoresis. Subsequently, the proteins were transferred onto PVDF membranes. After blocking with milk, antibodies against phosphor-Met (Tyr1234 / 5), Met, phosphor-AKT (S473), AKT, phosphor-ERK1 / 2 (Thr202 / Tyr204), ERK1 / 2, KRAS, and Tubulin were added and incubated at room temperature for 1 h and then overnight at 4 °C. The next day, after the primary antibodies were rewarmed for 30 min, the corresponding secondary antibodies were added and incubated at room temperature for 1 h. Finally, imaging was performed using a chemiluminescence imaging system (ChemiDocTXRS+).

[0116] 1.2 Experimental results

[0117] As Figure 8 shown in Figure A, FL@ApM could inhibit Met phosphorylation induced by HGF, but had no obvious effect on the expression level of KRAS. siKRAS@FL could significantly inhibit the expression of KRAS, but had no obvious effect on Met phosphorylation. In contrast, siKRAS@FL@ApM could simultaneously inhibit Met phosphorylation induced by HGF and KRAS expression, and the inhibitory effects of siKRAS@FL@ApM on the phosphorylation of KRAS protein and Met protein were better than those of single nucleic acid nanodrugs (FL@ApM and siKRAS@FL).

[0118] As Figure 9As shown, compared with the HGF group, the expression levels of p-Akt and p-Erk1 / 2 were downregulated in both the siKRAS@FL@ApM and siKRAS@FL treatment groups, with the siKRAS@FL@ApM group being the most obvious. In contrast, FL@ApM had a weaker inhibitory effect on the expression level of p-Akt and almost no inhibitory effect on the expression level of p-Erk1 / 2.

[0119] 2. Real-time fluorescence quantitative reverse transcription PCR (RT-qPCR)

[0120] 2.1 Experimental method

[0121] A549 (1×10 6 cells / dish) cells were seeded in 6-cm dishes and cultured in a 37°C, 5% CO2 incubator for 12 h, and then the culture medium was replaced with a medium containing HGF, siKRAS, FL@ApM, siKRAS@FL, and siKRAS@FL@ApM (siKRAS concentration was 4 nM, ApM-chol concentration was 72 nM). After culturing in serum-free medium for 24 h, HGF with a final concentration of 1 nM was added to act on the cells for 30 min, and the cells in each group were collected. RNA extraction and reverse transcription of each group were performed according to the operation manuals of the Novizan RNA extraction kit and reverse transcription kit, and the mRNA level of KRAS in cells of different treatment groups was detected by real-time fluorescence quantitative reverse transcription PCR technology.

[0122] Experimental results: As Figure 8 shown in B, the silencing effect of different treatment groups (Control, HGF, siKRAS@FL@ApM, FL@ApM, and siKRAS@FL) on the KRAS gene at the mRNA level was analyzed by real-time fluorescence quantitative PCR (RT-qPCR) experiments. The expression of KRAS was significantly downregulated in the siKRAS@FL@ApM and siKRAS@FL groups, and the downregulation amount of KRAS expression by siKRAS@FL@ApM was higher than that of the siKRAS@FL group, further confirming that siKRAS@FL@ApM can directly silence the expression of the KRAS gene at the RNA level, thereby inhibiting the activation of the RAS / MAPK pathway. And due to the presence of ApM-chol, the uptake of siKRAS@FL@ApM by cells was increased, and further improved its ability to silence the target gene.

[0123] 3. CCK8 cell viability assay

[0124] A549 cells were seeded in 96-well plates (1500 cells / well) and cultured in an incubator at 37 °C with 5% CO2 for 24 h to allow them to adhere. Subsequently, different concentrations of FL, ApM-chol, and FL@ApM were added, and the cells were co-incubated with them in a serum-free environment containing 1 nM HGF for 48 h. According to the instructions of the CCK8 kit, CCK8 reagent was added to each group, and a microplate reader was used to detect the cell viability of A549 cells in different treatment groups. A549 cells were seeded in 96-well plates (1500 cells / well) and cultured in an incubator at 37 °C with 5% CO2 for 24 h to allow them to adhere. Subsequently, different concentrations of FL, ApM-chol, and FL@ApM were added, and the cells were incubated with them in a serum-containing environment for 24 h and co-incubated for 48 h. According to the instructions of the CCK8 kit, CCK8 reagent was added to each group, and a microplate reader was used to detect the cell viability of A549 cells in different treatment groups. A549 and NCL-H661 cells (1500 cells / well) were seeded in 96-well plates and cultured in an incubator at 37 °C with 5% CO2 for 24 h to allow them to adhere. Subsequently, different concentrations of FL@ApM, siRNA@FL, and siKRAS@FL@ApM were added, and the cells were co-incubated with them in a serum-free environment containing 1 nM HGF for 48 h. According to the instructions of the CCK8 kit, CCK8 reagent was added to each group, and a microplate reader was used to detect the cell viability of A549 cells and NCL-H661 cells in different treatment groups.

[0125] Experimental results: As Figure 10 shown, we investigated the anti-tumor activity of siKRAS@FL@ApM at the cellular level. First, through the Human Protein Atlas website and the Depmap Portal database, we analyzed the gene backgrounds of A549 and NCL-H661 cells and examined the inhibitory performance of siKRAS@FL@ApM on different cells through CCK8 experiments. Normalized Transcripts Per Million (nTPM) represents the number of transcripts of a given gene quantified from RNA sequencing results in one million people. As Figure 10 shown in A below, the nTPM of the KRAS gene in the A549 cell line was 20.2; the nTPM of the KRAS gene in the NCI-H661 cell line was 32.6, both confirming the expression of the KRAS gene in these two cell lines. Further, we analyzed the mutation status of the KRAS gene in these two cell lines through the Cancer Cell Line Encyclopedia (CCLE) database and found that the A549 cell line had a KRAS G12S site mutation, while the NCL-H661 cell line had a KRAS G12C site mutation. As Figure 10As shown in B, the inhibitory performance of siKRAS@FL on A549 cells and NCL-H661 cells is similar. FL@ApM significantly inhibits the proliferation of A549 cells, and its inhibitory performance increases with increasing concentration. However, there is no obvious inhibitory effect on H661 cells with low Met expression. This is because H661 cells have low Met expression, so there is no obvious cell proliferation induced by HGF. Similarly, there is no cell activity inhibition caused by the inhibition of the HGF / Met signaling pathway. The dual-nucleic acid nanodrug siKRAS@FL@ApM has a stronger inhibitory effect on A549 cells than on H661 cells due to the targeting effect of ApM. The above results indicate that whether siKRAS@FL@ApM can exert multi-target inhibition depends on whether the cells have a genomic background with specific tumor suppressor gene mutations and oncoprotein expressions. This selectively targeted nanodrug has better biocompatibility with other cells that do not express oncogenes and oncoproteins.

[0126] As Figure 11 shown, by comparing the IC 50 values, it can be seen that among different treatment groups, the IC 50 of the dual-nucleic acid nanodrug (siKRAS@FL@ApM) for A549 cells is significantly lower than that of the single-nucleic acid nanodrugs (siKRAS@FL, FL@ApM), indicating that the dual-nucleic acid nanodrug has the best inhibitory effect on A549 cells. It should be noted that when the siKRAS / ApM concentration of siKRAS@FL@ApM reaches 16 / 288 nM, the inhibition rate of the siKRAS@FL@ApM group on A549 cells is close to 98%, which almost kills all A549 cells rather than simply inhibiting their proliferation.

[0127] 4. Real-Time Cell Analysis (RTCA)

[0128] A549 cells were seeded into an RTCA e-plate (2×10 3 cells / well). Then the e-plate was placed in an RTCA analyzer and incubated at 37°C and 5% CO2. After 24 h of incubation, blank control, ApM, FL@ApM, siKRAS@FL, and siKRAS@FL@ApM (concentration: siKRAS = 5 nM, ApM = 90 nM) were added to co-incubate with the cells. After 48 h of adding the drugs, the drugs were added again, and the RTCA analyzer recorded cell data in real time for a total of 90 h.

[0129] Experimental results: As Figure 12It can be seen that when A549 cells were co-incubated with these treatment groups for 90 h respectively, in the siKRAS@FL and FL@ApM treatment groups, A549 cells showed 41% and 50% proliferation inhibition effects respectively, while in the siKRAS@FL@ApM treatment group, the growth of 70.18% of A549 tumor cells was significantly inhibited. This may be because siKRAS@FL@ApM simultaneously inhibits the Met signaling pathway and mutant KRAS, achieving dual-target simultaneous inhibition, and delivering the two nucleic acid drugs to their targets (membrane / cytoplasm) located in different spaces through membrane-fused liposomes. Therefore, it can exert a stronger target inhibition ability, thereby efficiently inhibiting the proliferation of A cells.

[0130] 5. Cell scratch assay

[0131] A549 cells (7×10 5 cells / well) were seeded in 12-well plates, and subsequent experiments could be carried out when the cell confluence reached over 95%. A 10 μL pipette tip was perpendicular to the bottom of the culture dish for scratching, and then washed 3 times with PBS to wash away the scratched cells. Then, serum-free medium mixed with 0.5% BSA was added to co-incubate with the cells, and blank control group, HGF group, different concentrations of ApM-chol, FL@ApM, siKRAS@FL and siKRAS@FL@ApM groups were set to treat the cells. The cells were placed in an incubator at 37 °C and 5% CO2 for culture, and the healing of the scratches was photographed and recorded at 0 h, 12 h, 24 h, and 36 h time points respectively. The photographed images were analyzed using Image J software.

[0132] Experimental results: We explored whether siKRAS@FL@ApM could inhibit the migration ability of A549 tumor cells through scratch assay and compared it with siKRAS@FL and FL@ApM. From the scratch assay results ( Figure 13 ), we could observe that at the 0 h measurement starting point, the scratch areas of cells in each group were basically the same. After 12 h, the group added with HGF significantly promoted the scratch healing of A549 cells compared with other groups. At the 24 h measurement end point, the scratch area of cells in the HGF group had a healing degree of 72%, but the healing degree of the scratch areas of A549 cell lines in the siKRAS@FL, FL@ApM and siKRAS@FL@ApM treatment groups was significantly inhibited. And the scratch healing area of the siKRAS@FL@ApM group was the smallest, only 9%. This indicates that compared with other groups, siKRAS@FL@ApM can more efficiently inhibit the migration ability of cells through dual-target inhibition.

[0133] Example 6 Investigation of the application of siKRAS@FL@ApM in anti-tumor therapy at the in vivo level

[0134] Prepare siKRAS@FL@ApM according to the method in Example 1. In the SFM group, siKRAS / ApM = 1 / 9 nM / 20 μg, and in the FM group, ApM = 9 nM / 20 μg, with a volume of 150 μL.

[0135] 1. Experimental method

[0136] 1.1 Establishment of mouse subcutaneous tumor model and administration

[0137] BALB / C male nude mice were purchased from Jiangsu Genscript Biotech Co., Ltd. and raised in an SPF-class animal room at a constant temperature (24 °C) and constant light time (12 h light, 12 h dark). The number of mice in each cage was maintained at 2 - 4, and water and feed were added regularly to ensure normal drinking and eating of the mice. Six-week-old BALB / C male nude mice (about 20 g) were selected for surgery. First, after culturing the cells to the logarithmic growth phase, the cells were digested with trypsin, collected, centrifuged, and the cell pellet was resuspended in PBS. The cell suspension was placed on ice for later use. The cell suspension (the number of injected cells was 1.5×10 7 , with a volume of 200 μL, including 160 μL PBS + 40 μL Matrigel) was injected at the bilateral axillary positions of the mice.

[0138] After successful model establishment, we administered Cy3-siKRAS@FL@Cy5-ApM and Cy5-FL@ApM to the mice by tail vein injection respectively. The distribution of Cy3-siKRAS@FL@Cy5-ApM and Cy5-FL@ApM in the mice at different time periods was studied by in vivo fluorescence imaging of mice and fluorescence imaging of organ tissues. During the treatment process, the fluorescence intensity of the tumors in the mice was detected by a small animal in vivo imager to evaluate the drug treatment effect.

[0139] After the administration was completed, the mice were sacrificed by cervical dislocation, and the tumors of each group of mice were taken out for photographing and weighing.

[0140] 1.2 HE experiment

[0141] Stain the cytoplasm and nucleus of the paraffin tissue sections of the mouse tumor after dewaxing according to the operation steps of the hematoxylin-eosin staining kit. After sealing with neutral resin, observe the histopathological changes with an optical microscope and take pictures (40×).

[0142] Stain the cytoplasm and nucleus of the paraffin tissue sections of the mouse heart, liver, spleen, lung, and kidney organs after dewaxing according to the operation steps of the hematoxylin-eosin staining kit. After sealing with neutral resin, observe the histopathological changes with an optical microscope and take pictures (200×).

[0143] 1.3 IHC experiment

[0144] The collected tumor tissues were fixed in 4% paraformaldehyde for 24 h, and dehydrated successively in ethanol with different concentrations (50%, 75%, 85%, 95%, 100%) and xylene. Then, they were embedded in paraffin. The embedded tissue sections (with a thickness of 4 μm) were dewaxed. The dewaxed sections were put into boiling sodium citrate solution for antigen retrieval for 20 min. Then, the sections were incubated with 3% hydrogen peroxide solution at room temperature for 10 min to reduce the activity of endogenous catalase, and blocked with goat serum at room temperature for 60 min. p-Met, Kras, Ki-67, and Caspase-3 antibodies were added and incubated with the sections overnight at 4°C. Then, the corresponding secondary antibodies were incubated. The sections were stained according to the operation steps of the DAB staining kit and eosin staining kit. After sealing with neutral resin, the sections were observed and photographed (40×) under an inverted fluorescence microscope.

[0145] 2. Experimental results

[0146] 2.1 Fluorescence detection results

[0147] As Figure 14 can be seen, the present invention uses the constructed mouse subcutaneous tumor model to evaluate the distribution of the nano-drug siKRAS@FL@ApM in mice. siKRAS+ApM-Chol and siKRAS@FL@ApM were injected via the tail vein, where ApM was labeled with Cy5 fluorescence and siKRAS was labeled with Cy3 fluorescence. The distribution and metabolism of the drugs within 0 h, 0.5 h, 2 h, 4 h, 6 h, 8 h, and 24 h were observed using a small animal in vivo imager.

[0148] As Figure 14 can be seen, the fluorescence signals of Cy3-siKRAS and Cy5-ApM could be detected in the tumor site at 2 h, 4 h, 6 h, and 8 h for siKRAS@FL@ApM, while the fluorescence intensity of the siKRAS+ApM-Chol group at the tumor mass was significantly lower than that of the siKRAS@FL@ApM group. The above results indicate that the dual nucleic acid nano-drug siKRAS@FL@ApM based on membrane fusion liposomes can achieve targeted delivery to the tumor site at the in vivo level.

[0149] 2.2 Tumor growth experiment results

[0150] As Figure 15 can be seen, by monitoring the tumor volume of tumor-bearing nude mice at different times and measuring the final tumor weight, the present invention can preliminarily evaluate the difference in the inhibitory activity of siKRAS@FL@ApM and the corresponding control groups against transplanted tumors in vivo. The results show that with the passage of time, the tumors grew rapidly, and neither the control group FL@ApM nor the FL@ApM+siKRAS@FL group could inhibit the growth of tumors.

[0151] In contrast, significant tumor growth inhibition was observed in the siKRAS@FL@ApM group.

[0152] 2.3 Microscopic observation results of tumor tissues

[0153] As shown by the H&E results ( Figure 16 ), tumor tissues treated with the FL@ApM and FL@ApM+siKRAS@FL mixtures showed histological features of malignant tumors similar to those of the control group, such as enlarged cell nuclei, deepened cytoplasmic color, and increased nucleus / cytoplasm ratio. In contrast, significant changes were observed in the morphology of tumor tissues treated with siKRAS@FL@ApM, including karyopyknosis and lightened cytoplasmic color, indicating tumor suppression. The anti-cancer ability of siKRAS@FL@ApM was further investigated by analyzing the proliferation-related protein Ki67 and apoptosis-related protein Caspase3 in tumor tissues through IHC experiments and detecting apoptotic cells through TUNEL experiments. The results showed that compared with the control group, the positive cells of Ki67, Kras, and p-Met in tumor tissues of the siKRAS@FL@ApM treatment group were significantly downregulated, and the proportion of Caspase-3 positive cells was significantly increased, indicating that siKRAS@FL@ApM could inhibit the proliferation of tumor cells in in vivo anti-tumor therapy.

[0154] 2.4 Results of biosafety evaluation

[0155] As Figure 17 and Figure 18 shown, during the treatment period, the experimental mice were weighed every three days, and no significant changes were observed in the body weights of the mice in each group. By performing H&E staining analysis on the major organs (heart, liver, spleen, lung, kidney) of the mice, the results showed that compared with the Control group, no obvious morphological features such as cell inflammation and necrosis were observed in the major organs of each treatment group (FL@ApM, FL@ApM+siKRAS@FL, and siKRAS@FL@ApM). These results indicate that the constructed nucleic acid drug siKRAS@FL@ApM has good biosafety.

[0156] The embodiments described above are only for describing the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A construction method of a dual-nucleic acid nanodrug based on membrane-fusion liposomes, characterized in that, It includes the following steps: Construct membrane-fusion liposomes using DMPC, DSPE-PEG2000, and DOTAP; Mix the membrane-fusion liposomes with the siKRAS solution to prepare siKRAS membrane-fusion liposome particles; Denature ApM-chol, mix it with the siKRAS membrane-fusion liposome particles, rotate, and centrifuge to collect the precipitate, thus obtaining the dual-nucleic acid nanodrug based on membrane-fusion liposomes; Among them, the nucleotide sequence of the siKRAS is shown in SEQ ID NO.2; In the ApM-chol, chol represents cholesterol modification, and the nucleotide sequence of the ApM is shown in SEQ ID NO.

1.

2. The construction method according to claim 1, characterized in that, The construction of the membrane-fusion liposomes includes the following steps: Dissolve the DMPC, the DSPE-PEG2000, and the DOTAP separately in chloroform, mix them, and perform rotary evaporation at a temperature of 45°C and a rotation speed of 45 rpm until the air pressure drops to 30 Pa, then introduce nitrogen gas to obtain the membrane-fusion liposomes in the form of a thin film; Among them, the molar ratio of the DMPC, the DSPE-PEG2000, and the DOTAP is 76.2:3.8:

20.

3. The construction method according to claim 1, wherein The preparation of the siKRAS membrane-fusion liposome particles includes the following steps: Under normal pressure, rotary evaporate the siKRAS solution and the membrane-fusion liposomes for 1 h, and extrude them back and forth through a polycarbonate membrane using a liposome extruder to obtain the siKRAS membrane-fusion liposome particles.

4. The construction method according to claim 1, wherein The temperature of the rotation is 45°C, the rotation speed is 10 rpm, and the time is 1 h.

5. A dual-nucleic acid nanodrug based on membrane-fusion liposomes prepared by the construction method according to any one of claims 1-4.

6. Use of a dual-nucleic acid nanodrug based on membrane-fusion liposomes as claimed in claim 5 in the preparation of a drug for treating cancer.

7. The application according to claim 6, wherein, The treatment of cancer includes inhibiting cancer cell proliferation and inhibiting cancer cell migration.

8. The application according to claim 6 or 7, characterized in that, The cancer is non-small cell lung cancer.

9. A drug for treating cancer, characterized in that, The drug takes the dual-nucleic acid nanodrug based on membrane-fusion liposomes as claimed in claim 5 as the main active ingredient.

10. The drug according to claim 9, characterized in that, It also includes pharmaceutically acceptable excipients.

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