Gene nano-drug for inducing autodeath of tumor cells as well as preparation method and application of gene nano-drug

Abstract

The invention discloses a gene nano-drug for inducing autodeath of tumor cells as well as a preparation method and application of the gene nano-drug. The gene nano-drug comprises plasmids and polymer nano-particles, the plasmids are plasmids for inducing the expression of related genes of autodeath of tumor cells; the polymer nanoparticles consist of ionizable lipids, cationic lipids and polymers, and can be used for in-vivo external loading and delivery of nucleic acid drugs. The cationic lipid and ionizable lipid assisted polymer nanoparticles are utilized to deliver plasmids, oil-in-water nanoparticles are formed through hydrophilic and hydrophobic interaction to carry out plasmid loading, excellent stability is achieved, a polyethylene glycol shell layer can achieve long circulation in vivo, and the stability is good. The nano-scale (about 100 nm) size can increase the enrichment of particles in tumors through the high permeability and retention effect (EPR effect) of solid tumors, the cationic lipid can assist the nano-particles in being ingested by cells, and the ionizable lipid can assist the nano-particles in lysosome escape.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a gene nanomedicine for inducing tumor cell apoptosis, its preparation method, and its application. Background Technology

[0002] Herpes simplex virus thymidine kinase (HSV-TK) is a key enzyme in herpesvirus infection of host cells. It converts thymidine (a nucleoside) into thymidine triphosphate (a bioactive form that can be utilized by the virus). Through this process, the herpesvirus replicates its DNA and infects host cells. Thymidine kinase can bind to the prodrug ganciclovir (GCV), inducing phosphorylation of ganciclovir to form ganciclovir triphosphate. This triphosphate compound is toxic and competitively inhibits DNA replication, leading to cell death. The resulting triphosphate compound can diffuse into surrounding cells via the intercellular pathway, achieving a widespread killing effect.

[0003] Herpes simplex virus thymidine kinase combined with ganciclovir therapy can effectively kill target cells, but the toxic substances produced can also non-specifically kill normal cells, resulting in toxic side effects. To address this issue, studies (Santo D, Cordeiro RA, ...) have been conducted... PV, Serra AC, Coelho JFJ, Faneca H. Glycopolymers Mediate Suicide Gene Therapy in ASGPR-Expressing Hepatocellular Carcinoma Cells in Tandem with Docetaxel. Biomacromolecules. 2023 Mar 13; 24(3):1274-1286.) By modifying the carrier and adding targeting groups, thymidine kinase can be specifically expressed in tumor cells, reducing toxic side effects. However, although carrier-targeted methods can effectively induce specific expression of thymidine kinase in tumor cells, problems such as target group detachment and insufficient modification exist during carrier delivery, thus limiting the application of this therapy in tumor treatment.

[0004] In summary, further breakthroughs are needed to find ways to induce tumor cells to express thymidine kinase efficiently, specifically, and safely, thereby effectively catalyzing the prodrug ganciclovir to be specifically expressed in tumor cells and enhancing the tumor-killing effect. Summary of the Invention

[0005] The purpose of this invention is to provide a gene nanomedicine for inducing tumor cell apoptosis, its preparation method, and its application. It aims to solve the technical problem in the prior art of how to induce tumor cells to express thymidine kinase efficiently, specifically, and safely, thereby effectively catalyzing prodrugs (such as ganciclovir GCV) and improving tumor killing effects.

[0006] To achieve the above objectives, the technical solution of this invention is as follows:

[0007] A first aspect of the present invention provides a gene nanomedicine for inducing tumor cell apoptosis, comprising: a plasmid and polymer nanoparticles; wherein the plasmid is a plasmid for expressing genes related to inducing tumor cell apoptosis; and the polymer nanoparticles are composed of ionizable lipids, cationic lipids and polymers, and can be used for in vivo and in vitro encapsulation and delivery of nucleic acid drugs.

[0008] Preferably, in conjunction with the first aspect, the plasmid is a plasmid expressing the herpes simplex virus thymidine kinase mutant HSV-SR39TK.

[0009] Preferably, in conjunction with the first aspect, the plasmid is at least one of human and mouse genes; and / or, the promoter of the plasmid is a tumor-specific promoter; the tumor-specific promoter is at least one of melanoma-specific tyrosinase Tyr promoter and tumor-universal survivin Sur promoter; and / or, the terminator of the plasmid is bGHpolyA.

[0010] Preferably, in conjunction with the first aspect, the ionizable lipid is at least one of Dlin-MC3-DMA, SM-102, ALC-0315, Dlin-KC2-DMA, BHEM-DBA, BHEM-APMP, BHEM-EAA, and BHEM-AEA.

[0011] Preferably, in conjunction with the first aspect, the cationic lipid is at least one of trimethyl-2,3-diolenoyloxypropylammonium chloride, trimethyl-2,3-dioleoyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleoyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-bisoctadecyloxypropylammonium bromide, N-(2-speramidyl)-N',N'-bisoctadecylglycineamide, and 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester.

[0012] Preferably, in conjunction with the first aspect, the molar ratio of the cationic lipid to the ionizable lipid is 1:1-4; and / or, the molar ratio of the cationic lipid to the plasmid is 1:1-4.

[0013] Preferably, in conjunction with the first aspect, the average particle size of the polymer nanoparticles is 50 nm to 350 nm; and / or, the polymer in the polymer nanoparticles is at least one of polyethylene glycol-modified poly(glycolic acid-co-lactide) and poly(glycolic acid-co-lactide); and / or, the LA / GA ratio in the polyethylene glycol-modified poly(glycolic acid-co-lactide) and the poly(glycolic acid-co-lactide) ranges from 95:5 to 50:50; and / or, the molecular weight of the polyethylene glycol ranges from 1000 to 10000 Daltons.

[0014] A second aspect of the present invention provides a method for preparing the gene nanomedicine for inducing tumor cell apoptosis as described in any of the first aspects, comprising the following steps:

[0015] The plasmid that induces tumor cell apoptosis is mixed with DEPC water to obtain an aqueous phase; ionizable lipids, cationic lipids, and ethanol are mixed to obtain a lipid ethanol solution; the polymer and organic solvent are mixed to obtain a mixture, and then the lipid ethanol solution and the mixture are mixed to obtain an oil phase; the oil phase and the aqueous phase are mixed to prepare an oil-in-water emulsion; after the emulsion is allowed to stand at room temperature for 15 minutes, gene nanomedicine particles for inducing tumor cell apoptosis are obtained.

[0016] The gene nanoparticles for inducing tumor cell apoptosis prepared in this invention are nanoscale spherical structures with a polyethylene glycol (PEG) shell, exhibiting uniform particle size distribution and positive charge. With an average particle size of approximately 140 nm, the nanoscale size can increase particle accumulation at the tumor site through the high permeability and retention effect of solid tumors, achieving efficient delivery of the target gene nanomedicine.

[0017] A third aspect of the present invention provides a gene nanomedicine for inducing tumor cell death as described in the first aspect, which converts a prodrug (such as ganciclovir, acyclovir, valacyclovir) into a toxic substance (such as ganciclovir triphosphate) to kill tumor cells.

[0018] Compared with the prior art, the advantages and beneficial effects of the present invention include at least the following:

[0019] The gene nanomedicine for inducing tumor cell apoptosis provided in this invention utilizes cationic lipids and ionizable lipid-assisted polymer nanoparticles to deliver plasmids. The plasmids are loaded via water-in-oil nanoparticles formed through hydrophilic-hydrophobic interactions, exhibiting excellent stability. The polyethylene glycol shell allows for long-term circulation in vivo. The nanoscale (approximately 100 nm) size can increase particle accumulation in tumors due to the high permeability and retention effect (EPR effect) of solid tumors. Cationic lipids facilitate cellular uptake of the nanoparticles, while ionizable lipids facilitate lysosomal escape. Examples of this invention include a molar ratio of cationic lipids to ionizable lipids of 1:2; and / or a molar ratio of cationic lipids to plasmid nucleic acids of 1:3. The prepared nanoparticles, combined with the prodrug ganciclovir, effectively induce tumor cell apoptosis in vivo. Attached Figure Description

[0020] Figure 1 Maps of the tumor-specific gene expression plasmids pTyr-HSV-SR39TK (pTyr-SR39TK) and pSur-HSV-SR39TK (pSur-SR39TK) prepared in Example 1 of this invention.

[0021] Figure 2 This is a schematic diagram illustrating the preparation process of the gene nanomedicine for inducing tumor cell apoptosis provided by the present invention.

[0022] Figure 3 iCLAN prepared in Example 2 of the present invention pTyr-SR39TK Figure showing the particle size, PDI potential, and morphology characterization results.

[0023] Figure 4 iCLAN prepared in Example 2 of the present invention pTyr-SR39TK iCLAN pSur-SR39TK Fluorescence flow cytometry results of tumor-specific expression of EGFP mediated by this method.

[0024] Figure 5 iCLAN prepared in Example 2 of the present invention pTyr-SR39TK iCLAN pSur-SR39TK The image shows the results of Western blotting analysis of tumor-specific expression of SR39TK protein.

[0025] Figure 6 iCLAN prepared in Example 2 of the present invention pTyr-SR39TK iCLAN pSur-SR39TK The image shows the results of in vitro detection of tumor cell killing by the prodrug ganciclovir combined with ganciclovir.

[0026] Figure 7 The iCLAN prepared in Example 2 of this invention pTyr-SR39TKThe results of in vivo testing of the combined effects of the prodrug ganciclovir and melanoma growth inhibition.

[0027] Figure 8 The iCLAN prepared in Example 2 of this invention pSur-SR39TK The results of in vivo testing of the combined effects of the prodrug ganciclovir and melanoma and colon cancer tumor growth. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be considered as limitations on the invention. All other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. In the following description, the term "some embodiments" refers to a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same or different subsets of all possible embodiments and can be combined with each other without conflict. Unless otherwise defined, all technical and scientific terms used in the embodiments of this invention have the same meaning as commonly understood by those skilled in the art to which the embodiments of this invention pertain. The terminology used in the embodiments of this invention is for the purpose of describing the embodiments of this invention only and is not intended to limit the invention. In the following description of these embodiments, the terms "comprising," "including," "having," and "containing," etc., are open-ended terms, meaning including but not limited to. It should be noted that all raw materials / reagents in the embodiments of the present invention can be purchased on the market or prepared according to conventional methods known to those skilled in the art; the term "and / or" in the embodiments of the present invention is only used to describe the relationship between related objects, indicating that there can be three relationships. For example, A and / or B means three cases: A exists alone, B exists alone, and A and B exist simultaneously. A and B can be singular or plural. The character " / " generally indicates that the related objects before and after are in an "or" relationship. In the following description of this embodiment, the term "at least one" means one or more, and "more" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b or c", or "at least one of a, b and c", can both mean: a, b, c, ab (a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0029] Those skilled in the art should understand that, in the following description of the embodiments of the present invention, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention. The terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments only and is not intended to limit the present invention. The singular forms "a" and "the" as used in the embodiments of the present invention and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0030] Those skilled in the art will understand that the numerical ranges in the embodiments of this invention should be understood as each intermediate value between the upper and lower limits of the specifically disclosed range. Each smaller range between any stated value and an intermediate value within the stated range, as well as any other stated value or an intermediate value within the stated range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range. Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein. While only preferred methods and materials are described herein, any methods and materials similar to or equivalent to those described herein may be used in embodiments or test cases of this invention. All references to this specification are generally incorporated herein by reference to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the contents of this specification shall prevail.

[0031] It should be noted that all raw materials and / or reagents in the embodiments of the present invention were purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0032] In a first aspect, embodiments of the present invention provide a gene nanomedicine for inducing tumor cell apoptosis, comprising: a plasmid and polymer nanoparticles; wherein the plasmid is a plasmid that can induce the expression of genes related to tumor cell apoptosis; and the polymer nanoparticles are composed of ionizable lipids, cationic lipids and polymers, and can be used for in vivo and in vitro encapsulation and delivery of nucleic acid drugs.

[0033] In a specific embodiment, the plasmid used in this invention is preferably a plasmid expressing the herpes simplex virus thymidine kinase mutant HSV-SR39TK.

[0034] In a specific embodiment, the prodrug in this invention is preferably one of ganciclovir and acyclovir.

[0035] In specific embodiments, the plasmids used in the embodiments of the present invention are preferably at least one of human and mouse genes.

[0036] In specific embodiments, the promoter of the plasmid in this invention is preferably a tumor-specific promoter; the tumor-specific promoter is preferably at least one of the melanoma-specific tyrosinase Tyr promoter and the tumor-general survivin Sur promoter. It should be noted that tumor-specific promoters can selectively drive gene transcription and expression in tumor cells, while having no effect on other normal cells. Tyrosinase promoters can achieve melanoma-specific gene expression, and survivin promoters can achieve the expression of various tumor-specific genes.

[0037] In a specific embodiment, the terminator of the plasmid in this invention is preferably bGH polyA.

[0038] In specific embodiments, the ionizable lipids used in this invention are preferably at least one of Dlin-MC3-DMA, SM-102, ALC-0315, Dlin-KC2-DMA, BHEM-DBA, BHEM-APMP, BHEM-EAA, and BHEM-AEA. It should be noted that these ionizable lipids, when forming gene nanomedicines, are electrically neutral at physiological pH. After being taken up by endocytosis, they become positively charged in the acidic endosome environment, interacting with negatively charged phospholipids on the endosome membrane, thereby disrupting the endosome membrane and enabling endosome escape. This helps promote the release of delivered nucleic acid drugs and improve transfection efficiency.

[0039] In specific embodiments, the cationic lipids of the present invention are preferably one of trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA), trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), dimethyl-2-hydroxyethyl-2,3-dioleoyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-bisoctadecyloxypropylammonium bromide, N-(2-speryl)-N',N'-bisoctadecylglycineamide (DOGS), and 1,2-dioleoyl-3-succinyl-sn-glycerolcholine ester (DOSC); further, the cationic lipid is more preferably trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP). It should be noted that these cationic lipids have the following effects when forming gene nanomedicines: (1) They can form stable complexes with negatively charged nucleic acids (such as DNA or RNA), and these complexes can easily enter cells through the cell membrane, thereby improving the gene transfection of gene nanomedicines. These cationic liposomes bind to nucleic acids through charge interactions, which can greatly compress the volume of nucleic acid molecules and promote their transfection and expression in cells. (2) After gene transfer mediated by cationic liposomes, the liposomes are degraded by intracellular enzymes, with no toxic side effects on cells, and exhibit good biocompatibility. Compared with viral vectors, cationic liposomes as gene vectors have lower immunogenicity and toxicity, which can reduce immune response and cytotoxicity. (3) The complex formed by cationic liposomes and nucleic acids can promote endocytosis in cells and effectively deliver nucleic acids into cells. In cells, the complex can release nucleic acids, enabling them to be transcribed and expressed in cells, thereby achieving the purpose of gene therapy. (4) Cationic liposomes such as DOTAP and DOTMA have hydrophobic tails and hydrophilic positively charged heads, which can form a stable interface between the aqueous and organic phases, which is conducive to binding with nucleic acids and forming complexes. Cationic liposomes such as DOGS have specific chemical structures, such as hydrophobic tails and hydrophilic positively charged heads, which give them unique biological characteristics and application potential.

[0040] In a specific embodiment, the molar ratio of cationic lipid to ionizable lipid in the present invention is preferably 1:1-4.

[0041] In a specific embodiment, the molar ratio of cationic lipid to nucleic acid in this invention is preferably 1:1-4.

[0042] In a specific embodiment, the average particle size of the polymer nanoparticles in this invention is preferably 50nm-350nm.

[0043] In specific embodiments, the polymer in the polymer nanoparticles of the present invention is preferably one of polyethylene glycol-modified poly(glycolic acid-co-lactide) (PEG-b-PLGA) and poly(glycolic acid-co-lactide) (PLGA). It should be noted that the LA / GA ratio in the polyethylene glycol-modified poly(glycolic acid-co-lactide) and the poly(glycolic acid-co-lactide) is preferably in the range of 95:5 to 50:50.

[0044] In a specific embodiment, the molecular weight range of the polyethylene glycol in this invention is preferably 1000-10000 Daltons.

[0045] In a second aspect, embodiments of the present invention provide a method for preparing the gene nanomedicine for inducing tumor cell apoptosis as described in the first aspect, the method comprising:

[0046] S1: The plasmid that induces tumor cell death is mixed with DEPC water to obtain the aqueous phase;

[0047] S2: Ionizable lipids, cationic lipids and ethanol are mixed to obtain a lipid ethanol solution; a polymer and an organic solvent are mixed to obtain a mixture, and then the lipid ethanol solution and the mixture are mixed to obtain an oil phase;

[0048] S3: Mix the oil phase and the aqueous phase to prepare an oil-in-water emulsion;

[0049] S4: After the emulsion is left to stand at room temperature, gene nanomedicine particles for inducing tumor cell death are obtained.

[0050] In the preparation method provided by this invention, in step S1, the plasmid inducing tumor cell apoptosis is mixed with DEPC-containing water. There are no restrictions on the mixing method; for example, the DEPC-containing water can be prepared as a DEPC-containing solution, which can be an aqueous solution of DEPC and water. Then, the DEPC-containing water and the plasmid inducing tumor cell apoptosis are directly mixed, and stirring can be used to ensure a uniform reaction. Preferably, the plasmid inducing tumor cell apoptosis is slowly added to the DEPC-containing water under stirring to obtain an aqueous phase, making the reaction more uniform and resulting in a water phase with uniform particle size. There are no particular restrictions on stirring; for example, magnetic stirring can be used. Generally, to ensure a more complete reaction, stirring should continue for a period of time after the plasmid is added, such as 0.3-2 hours. The preferred slow addition rate is 6-50 mL / min. The preferred environment for this step is room temperature to 80°C, meaning the mixing and reaction temperature in step S1 can be room temperature to 80°C. Heating can be done using a water bath.

[0051] In the preparation method provided by this invention, in step S2, ionizable lipids, cationic lipids, and ethanol are mixed to obtain a lipid ethanol solution; the polymer and an organic solvent are mixed to obtain a mixture; then, the lipid ethanol solution and the mixture are mixed to obtain an oil phase. This invention does not impose specific limitations on the mixing process. For example, the ionizable lipids and cationic lipids can be directly dissolved in ethanol simultaneously to obtain a lipid ethanol solution; the ionizable lipids can be dissolved in ethanol first to obtain a mixture, and then the cationic lipids can be dissolved in the aforementioned ionizable lipid ethanol solution to obtain a lipid ethanol solution; or the cationic lipids can be dissolved in ethanol first, and then the ionizable lipids can be dissolved in the aforementioned cationic lipid ethanol solution to obtain a lipid ethanol solution. During this mixing process, direct stirring or heating and stirring can be performed to ensure a uniform reaction. Preferably, this invention first prepares separate ethanol solutions of the ionizable lipids and cationic lipids, and then mixes the two mixtures to obtain a lipid ethanol solution, resulting in a more uniform reaction and the formation of an oil phase. There are no particular limitations on stirring in this invention; for example, magnetic stirring can be used. Generally, to ensure a more complete reaction, stirring should continue for a period of time after the addition is complete, which can be 0.3-2 hours. The preferred rate of slow addition is 6-50 mL / min. The preferred environment for this step is room temperature to 80°C, meaning the mixing and reaction temperature in step S2 can be between room temperature and 80°C. Heating can be done using a water bath.

[0052] In the preparation method provided by this invention, in step S3, the oil phase and the aqueous phase are mixed to prepare an oil-in-water emulsion. This invention does not impose any particular limitation on the mixing method; for example, the oil phase and the aqueous phase can be mixed directly. Preferably, the method for preparing the oil-in-water emulsion from the oil phase and the aqueous phase can be any one of manual mixing, microfluidic, and microchannel reaction methods.

[0053] In the preparation method provided by this invention, after the emulsion is allowed to stand at room temperature in step S4, the gene nanomedicine that induces tumor cell apoptosis is obtained. This invention does not have specific limitations on the standing time; it can be left to stand for 10-60 minutes.

[0054] Thirdly, embodiments of the present invention provide a gene nanomedicine for inducing tumor cell apoptosis as described in the first aspect, or a gene nanomedicine for inducing tumor cell apoptosis prepared by the method described in the second aspect, which is converted into a toxic substance by binding a prodrug to kill tumor cells.

[0055] The gene nanomedicine for inducing tumor cell death provided in this invention specifically expresses thymidine kinase in tumor cells and, in combination with the prodrug ganciclovir, triphosphorylates it in tumor cells to form ganciclovir triphosphate compound, thereby competitively inhibiting DNA replication and ultimately inducing tumor cell death; in addition, the generated ganciclovir triphosphate diffuses to surrounding tumor cells, expanding the tumor killing effect.

[0056] The technical method of the present invention will be further described below with reference to specific embodiments.

[0057] Sources of raw materials used in the examples:

[0058] DOTAP and Dlin-MC3-DMA were purchased from Aivito Pharmaceutical Technology Co., Ltd.

[0059] The polyester material was purchased from Guangzhou Xinheng Biotechnology Co., Ltd.

[0060] Dimethyl sulfoxide was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0061] B16-F10, Hepa1-6, Panc02, 4T1, C2C12, NIH-3T3, CT26, and RAW264.7 cells were purchased from ATCC;

[0062] Primary mouse skin cells, primary colon cells, primary pancreatic cells, and primary mammary cells were extracted from C57 BL / 6 mice;

[0063] Anti-HHV1-TK antibody was purchased from Thermo Fisher Scientific.

[0064] The anti-GAPDH antibody was purchased from Proteintech.

[0065] CCK-8 reagent was purchased from Biosharp.

[0066] Instrument models and manufacturers used in the examples:

[0067] Nano particle size and Zeta potential meter: Model Nano ZSE, Malvern, UK;

[0068] Field emission transmission electron microscope: Model Talos F200x, Thermo Fisher Scientific, USA;

[0069] Analytical flow cytometer: Model FACSCelesta, BD Biosciences, USA;

[0070] Chemiluminescence imager: Model ChemiDoc MP, Bio-RAD Corporation, USA;

[0071] Benchtop micro-refrigerated centrifuge: Model Microfuge 20R, Beckman Coulter, USA;

[0072] Microplate reader: Model 800TS, BioTek, USA.

[0073] Example 1

[0074] Construction of a plasmid for a simple thymidine kinase mutant that induces tumor cell self-death

[0075] Using genetic engineering methods, the mutant sequence of herpes simplex virus thymidine kinase and the gene sequence of melanoma Tyr promoter or tumor-universal Sur promoter were constructed into a plasmid with pUC57 as the backbone, resulting in tumor-specific herpes simplex virus thymidine kinase (HSV-SR39TK) expression plasmids applicable to melanoma or different types of tumors: denoted as pTyr-SR39TK and pSur-SR39TK. Furthermore, a series of plasmids carrying T2A, EGFP reporter genes, etc., were constructed using similar methods: pTyr-SR39TK-EGFP, pSur-SR39TK-EGFP, etc.

[0076] like Figure 1 The images shown are the plasmid maps of pTyr-SR39TK and pSur-SR39TK, respectively. The main elements of this expression plasmid include the Ori replication start site, Tyr or Sur promoter, HSV-SR39TK functional fragment, T2A, reporter gene EGFP, ampicillin resistance gene, and bGH polyA terminator.

[0077] Example 2

[0078] Preparation method of gene nanomedicines for inducing tumor cell apoptosis and characterization of particle size, potential and morphology.

[0079] Ethanol solutions of Dlin-MC3-DMA and DOTAP were prepared separately, with a concentration of 20 mg / mL for both. Polyethylene glycol-modified poly(glycolic acid-co-lactide) (PEG) was also prepared. 2k -b-PLGA 2k Prepare a dimethyl sulfoxide solution to a concentration of 150 mg / mL. Add 10 μL of Dlin-MC3-DMA ethanol solution, 5 μL of DOTAP ethanol solution, and 8 μL of PEG to a 1.5 mL enzyme-free EP tube. 2k -b-PLGA 2k Dimethyl sulfoxide solution and 5 μL of anhydrous ethanol were mixed by pipetting to obtain the oil phase. 20 μg of the pTyr-SR39TK and pSur-SR39TK plasmids prepared in Example 1 were dissolved in enzyme-free DEPC water to a total volume of 60 μL. The mixture was then pipetted to obtain the aqueous phase. The aqueous phase was added to the oil phase, pipetted for 15 seconds, and allowed to stand at room temperature for 10 minutes to obtain the nanomedicine. 4 mL of enzyme-free DEPC water was added, mixed by pipetting, and then transferred to a 100 kDa ultrafiltration tube. The mixture was centrifuged at 3000g for 5 minutes, repeated three times to remove the organic solvent, yielding the gene nanomedicine for inducing tumor cell apoptosis. Figure 2 As shown, denoted as iCLANpTyr-SR39TK or iCLAN pSur-SR39TK .

[0080] To verify the efficacy of the iCLAN gene nanomedicine prepared in Example 2, which induces tumor cell apoptosis. pTyr-SR39TK The relevant performance of iCLAN pTyr-SR39TK The relevant biological properties were characterized, such as Figure 3 As shown, Figure 3 iCLAN as an example pTyr-SR39TK Figure showing the results of particle size, potential and morphology characterization.

[0081] Take the prepared iCLAN pTyr-SR39TK In the sample cell, the nanoparticles were characterized using a nanoparticle size analyzer and a zeta potential analyzer to determine their hydration diameter, corresponding particle size, and zeta potential distribution. Figure 3 As shown in Figure A, this gene nanomedicine for inducing tumor cell apoptosis is nanoscale in size, with a uniform particle size distribution and positive charge. The average particle size is 143.7 nm, the PDI is 0.235, and the Zeta potential is 42.8 mV. In this invention, the prepared iCLAN... pTyr-SR39TK The particles were diluted 50 times with ultrapure water and then ultrasonically dispersed. 10 μL of the particle solution was dropped onto a copper grid and allowed to stand at room temperature for 5-10 minutes. The liquid was then absorbed using filter paper. Next, under light-protected conditions, 10 μL of 2% phosphotungstic acid solution was dropped into a clean 60 mm petri dish. The copper grid was gently held at the edge with tweezers to bring it into contact with the droplet. After negative staining for 30 seconds, the dish was placed on filter paper and allowed to dry. The morphology of the nanomedicine was observed using a field emission transmission electron microscope at 200 kV. Figure 3 As shown in B, the gene nanomedicine used to induce tumor cell apoptosis is a nanoscale spherical structure with a polyethylene glycol (PEG) shell.

[0082] Example 3

[0083] In vitro EGFP fluorescence detection of gene nanomedicines for inducing tumor cell apoptosis

[0084] In this invention, 5 × 10⁵ seeds are inoculated into each well of a 24-well plate. 4 Primary cells from mouse tumors (B16-F10, Panc02, CT26, 4T1, Hepa1-6), normal mice (NIH-3T3, RAW264.7, C2C12), mouse epithelial cells, pancreatic cells, colon cells, and mammary cells were transfected when cell confluence reached 70%. The old culture medium in the 24-well plate was discarded and replaced with antibiotic-free basal medium. Following the preparation method in Example 2, 500 ng of iCLAN plasmid was added to each well. Control iCLAN pTyr-SR39TK-EGFP or iCLANpSur-SR39TK-EGFP After gently shaking, incubate in an incubator for 6 hours, then replace with fresh complete culture medium. After 48 hours, detect EGFP fluorescence by flow cytometry. The test results are as follows: Figure 4 As shown, Figure 4 As shown in A, iCLAN Control and iCLAN pTyr-SR39TK-EGFP B16-F10, Panc02, CT26, 4T1, and Hepa1-6 mouse tumor cells and NIH-3T3, RAW264.7, and C2C12 mouse normal cells were transfected, respectively. After 48 hours, flow cytometry detected that only B16-F10 cells expressed EGFP fluorescence, while the other cells did not. Figure 4 From B, we know that iCLAN can be used. pSur-SR39TK-EGFP and iCLAN Control B16-F10, Panc02, CT26, and 4T1 mouse tumor cells and mouse-derived primary epithelial, pancreatic, colonic, and mammary cells were transfected, respectively. After 48 hours, flow cytometry detected that B16-F10, Panc02, CT26, and 4T1 cells expressed EGFP fluorescence, while mouse-derived primary cells did not express it.

[0085] Example 4

[0086] In vitro expression of thymidine kinase protein in gene nanomedicines for inducing tumor cell apoptosis.

[0087] In this invention, 5 × 10⁵ seeds are inoculated into each well of a 24-well plate. 4 Primary cells from mouse tumors (B16-F10, Panc02, CT26, 4T1, Hepa1-6), normal mice (NIH-3T3, RAW264.7, C2C12), mouse epithelial cells, pancreatic cells, colon cells, and mammary cells were transfected when cell confluence reached 70%. The old culture medium in the 24-well plate was discarded and replaced with antibiotic-free basal medium. Following the preparation method in Example 2, 500 ng of iCLAN plasmid was added to each well. Control iCLAN pTyr-SR39TK or iCLAN pSur-SR39TK After gently shaking, the cells were incubated in an incubator for 6 hours. The medium was then replaced with fresh complete culture medium. After 48 hours, total cell protein was extracted, and the expression of thymidine kinase protein in the cells was detected by Western blotting. Results are as follows: Figure 5 As shown in A and B, iCLAN pTyr-SR39TK iCLAN can only induce efficient expression of SR39TK protein in B16-F10 cells. pSur-SR39TK It can induce efficient expression of SR39TK protein in four cell types: B16-F10, Panc02, CT26, and 4T1, while other cell types do not express it.

[0088] Example 5

[0089] Results of in vitro assays on tumor cell killing effects of gene nanomedicine combined with the prodrug ganciclovir for inducing tumor cell apoptosis.

[0090] In this invention, 5 × 10⁵ seeds are inoculated into each well of a 24-well plate. 4 B16-F10 cells were cultured until 70% confluence was achieved. The old culture medium in the 24-well plate was discarded and replaced with antibiotic-free basal medium. Following the preparation method in Example 2, 500 ng of iCLAN plasmid was added to each well. Control iCLAN pTyr-SR39TK After gently shaking and incubating in an incubator for 6 hours, the medium was replaced with fresh complete culture medium. After 24 hours, different concentrations of GCV dilution buffer (0, 1, 2.5, 5, 10, 50, 100 μg / mL) were added, and the mixture was incubated for a total of 36 hours. Then, CCK-8 reagent was added, and after 1.5 hours of incubation, the absorbance was measured using a microplate reader. Additionally, 5 × 10⁵ cells were seeded per well in a 24-well plate. 4 B16-F10 or CT26 cells were cultured until 70% confluence was achieved. The old culture medium in the 24-well plate was then discarded and replaced with antibiotic-free basal medium. Following the preparation method in Example 2, 500 ng of iCLAN plasmid was added to each well. Control iCLAN pSur-SR39TK After gently shaking and incubating in an incubator for 6 hours, the medium was replaced with fresh complete culture medium. After 24 hours, different concentrations of GCV dilution buffer (0, 1, 2.5, 5, 10, 50, 100 μg / mL) were added, and the mixture was incubated for a total of 36 hours. Then, CCK-8 reagent was added, and after 1.5 hours of incubation, the absorbance was measured using a microplate reader. The results are as follows: Figure 6 As shown in A and B, iCLAN pTyr-SR39TK The combination of GCV and the nanoparticles effectively killed B16-F10 cells in a dose-dependent manner. The killing effect of the nanoparticles combined with the prodrug increased with increasing prodrug concentration. For subsequent experiments, we selected a prodrug concentration exceeding 50% as the prodrug addition concentration. (iCLAN) pSur-SR39TK The combination of GCV and other drugs effectively killed B16-F10 and CT26 cells in a dose-dependent manner. The killing effect of the prepared nanoparticles combined with the prodrug increased with the increase of prodrug concentration.

[0091] Example 6

[0092] iCLAN pTyr-SR39TK Results of in vivo testing of the combined effects of the prodrug ganciclovir and melanoma growth inhibition.

[0093] To verify iCLAN pTyr-SR39TKIn combination with the antitumor effect of GCV, 35 C57 BL / 6 mice implanted with B16-F10 subcutaneous melanoma were randomly divided into 7 groups of 5 mice each. Each group was injected via tail vein with 100 μL of PBS or iCLAN. Control iCLAN pTyr-SR39TK (1 mg plasmid / kg body weight), administered every 2 days for a total of 4 doses. The day after tail vein injection, different concentrations of GCV (0, 25, 50, 100 mg / kg) were administered intraperitoneally. Throughout the treatment, tumor size was measured daily using calipers. The tumor volume was calculated using the following formula: Volume (mm²) 3 = 0.5 × length × width 2 After treatment, the mouse tumor tissue was isolated and weighed, such as... Figure 7 As shown, iCLAN pTyr-SR39TK The combination of GCV and GCV significantly inhibited the growth of melanoma in mice.

[0094] Example 7

[0095] iCLAN pSur-SR39TK Results of in vivo testing of the combined effects of the prodrug ganciclovir and melanoma on the growth of colon cancer tumors.

[0096] To verify iCLAN pSur-SR39TK To assess its versatility in treating various solid tumors, 25 C57 BL / 6 mice implanted with B16-F10 subcutaneous melanoma and 25 BalB / c mice implanted with CT26 colorectal cancer were randomly divided into 5 groups of 5 mice each. Each group received a tail vein injection of 100 μL of PBS or iCLAN. Control iCLAN pSur-SR39TK (1 mg plasmid / kg body weight), administered every 2 days for a total of 4 doses. The day after tail vein injection, different concentrations of GCV (0, 50, 100 mg / kg) were administered intraperitoneally. Throughout the treatment, tumor size was measured daily using calipers. The tumor volume was calculated using the following formula: Volume (mm²) 3 = 0.5 × length × width 2 After treatment, mouse tumor tissue was isolated and weighed. The results of melanoma and colon cancer tumor detection are as follows: Figure 8 As shown in A and B, iCLAN pSur-SR39TK The combination of GCV and other drugs significantly inhibited the growth of various types of tumors.

[0097] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A gene nanomedicine for inducing tumor cell apoptosis, characterized in that, include: Plasmids and polymer nanoparticles; the plasmid is a plasmid that can bind to a prodrug to induce the expression of genes related to tumor cell death; the polymer nanoparticles are composed of ionizable lipids, cationic lipids and polymers.

2. The gene nanomedicine for inducing tumor cell apoptosis according to claim 1, characterized in that, The plasmid is a plasmid expressing the herpes simplex virus thymidine kinase mutant HSV-SR39TK.

3. The gene nanomedicine for inducing tumor cell apoptosis according to claim 1, characterized in that, The plasmid is at least one of human and mouse genes; and / or, the promoter of the plasmid is a tumor-specific promoter; the tumor-specific promoter is at least one of the melanoma-specific tyrosinase Tyr promoter and the tumor-universal survivin Sur promoter; and / or, the terminator of the plasmid is bGH polyA.

4. The gene nanomedicine for inducing tumor cell apoptosis according to claim 1, characterized in that, The ionizable lipid is at least one of Dlin-MC3-DMA, SM-102, ALC-0315, Dlin-KC2-DMA, BHEM-DBA, BHEM-APMP, BHEM-EAA, and BHEM-AEA.

5. The gene nanomedicine for inducing tumor cell apoptosis according to claim 1, characterized in that, The cationic lipid is at least one of trimethyl-2,3-diolenooxypropylammonium chloride, trimethyl-2,3-dioleoyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleoyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-bisoctadecyloxypropylammonium bromide, N-(2-speramido)-N',N'-bisoctadecylglycineamide, and 1,2-dioleoyl-3-succinoyl-sn-glycerolcholine ester.

6. The gene nanomedicine for inducing tumor cell apoptosis according to claim 1, characterized in that, The molar ratio of the cationic lipid to the ionizable lipid is 1:1-4; and / or the molar ratio of the cationic lipid to the plasmid is 1:1-4.

7. The gene nanomedicine for inducing tumor cell apoptosis according to claim 1, characterized in that, The average particle size of the polymer nanoparticles is 50 nm to 350 nm; and / or, the polymer in the polymer nanoparticles is at least one of polyethylene glycol-modified poly(glycolic acid-co-lactide) and poly(glycolic acid-co-lactide); and / or, the LA / GA ratio in the polyethylene glycol-modified poly(glycolic acid-co-lactide) and the poly(glycolic acid-co-lactide) ranges from 95:5 to 50:50; and / or, the molecular weight of the polyethylene glycol ranges from 1000 to 10000 Daltons.

8. The method for preparing the gene nanomedicine for inducing tumor cell apoptosis according to any one of claims 1-7, characterized in that, Includes the following steps: A plasmid that induces tumor cell apoptosis is mixed with DEPC water to obtain an aqueous phase; an ionizable lipid, cationic lipid, and ethanol are mixed to obtain a lipid ethanol solution; a polymer and an organic solvent are mixed to obtain a mixture, and then the lipid ethanol solution and the mixture are mixed to obtain an oil phase; the oil phase and the aqueous phase are mixed to prepare an oil-in-water emulsion; after the emulsion is allowed to stand at room temperature, a gene nanomedicine for inducing tumor cell apoptosis is obtained.

9. The gene nanomedicine for inducing tumor cell apoptosis according to any one of claims 1-7, or the gene nanomedicine for inducing tumor cell apoptosis prepared by the method of claim 8, is converted into a toxic substance by binding a prodrug to kill tumor cells.

10. The application according to claim 9, wherein the prodrug comprises ganciclovir, acyclovir, or valacyclovir.

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