A non-viral gene vector based on bioaffinity and its preparation method and application

Abstract

The present invention discloses a non-viral gene vector based on bioaffinity, and its preparation method and application. The present invention provides a non-viral gene vector based on bioaffinity, characterized in that the non-viral gene vector comprises (i) a vector core, and (ii) a vector shell; wherein the vector core is composed of a cationic material modified by a lipid substance and a nucleic acid molecule, and the vector shell is composed of a protein having a bioaffinity effect on the lipid substance in the vector core. The non-viral gene vector provided by the present invention is stable, can efficiently deliver genes into the body and produce target products or interfere with the expression of target products, thereby treating diseases; it can also efficiently transfect target cells in vitro to prepare cell preparations required for cell therapy. In addition, the non-viral gene vector can reduce the toxicity caused by the use of cationic materials, thereby avoiding damage to normal cells in the body, and has good biocompatibility.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and relates to a non-viral gene carrier and a preparation method and application thereof, and more specifically, to a non-viral gene carrier based on bioaffinity and a preparation method and application thereof. Background Art

[0002] Gene therapy refers to the introduction of normal exogenous genes into target cells via gene vectors to correct or compensate for diseases caused by defective or abnormal genes, thereby achieving therapeutic goals. This also includes the application of technologies such as transgenics, where exogenous genes are inserted into appropriate recipient cells of a patient through gene transfer, causing them to produce or reduce the expression of their products, thereby treating a specific disease. Broadly speaking, gene therapy also encompasses measures and new technologies for treating certain diseases at the DNA level.

[0003] The gene vectors currently in use can be divided into two major categories, viral vectors and non-viral vectors, according to their sources. Viral vectors are viruses (such as retroviruses, slow viruses and adenoviruses) that are modified so that they can carry therapeutic genes. The modified viral vectors have the same ability to infect organisms as wild-type viruses and have higher gene transfection efficiency, but have defects such as small gene capacity, large toxic side effects, and high preparation costs. Non-viral vectors have good clinical application prospects because they have advantages such as low immunogenicity, low cost, and easy scalability. However, the transduction efficiency of non-viral vectors is low, and the size of the entire delivery system is large, which easily triggers an immune response and is cleared by the body.

[0004] In recent years, non-viral gene delivery vehicles using cationic polymer materials have emerged. These carriers, which compact genes through high-density positive charges, facilitate cellular uptake and thus have high transfection efficiency. However, the highly concentrated positive charges on the surface of cationic polymer carriers also result in high toxicity in vivo, limiting their application.

[0005] Coating positively charged carriers with negatively charged materials through charge adsorption can effectively shield the carrier's positive charge and is a proven method for reducing the toxicity of cationic carriers. However, nanocarriers with core-shell structures that rely solely on charge interactions are prone to shedding their outer shell during in vivo transport, exposing the positively charged inner core, which can cause toxic side effects. Furthermore, even if the nanocarriers reach the target site intact, the negative charge of the outer shell prevents them from being taken up by target cells, resulting in low transfection efficiency. Therefore, increasing the stability of these core-shell carriers and improving their efficiency in target cell uptake has become an urgent issue.

[0006] Bioaffinity refers to the phenomenon that a biological molecule can specifically recognize a substance and attract and bind to it under certain circumstances. Among them, proteins such as albumin (also known as albumin), apolipoprotein, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), and high-density lipoprotein (HDL) have good bioaffinity for lipids and have good targeting to lesion tissues such as tumors, articular cavities, and cardiovascular plaques. Therefore, they are expected to be used to improve the stability of gene delivery vectors with core-shell structures and achieve targeted treatment of lesions. Jianping Zhou et al. (Biomaterials vol.35, 25 (2014): 7214-27.) reported a nano-delivery system with a core-shell structure by cholesterol-forming siRNA to obtain lipophilic siRNA, which was then encapsulated with recombinant high-density lipoprotein. Although this nano-delivery system has high safety, the modification of the gene itself will affect its function, and the gene is present outside the system and is easily degraded by the complex environment in the body.

[0007] Therefore, it is necessary to develop a non-viral gene vector that is safe, stable, has good biocompatibility, and can improve the targeted therapeutic effect of lesions. Summary of the Invention

[0008] Problems to be solved by the invention

[0009] In response to the defects in the existing technology, namely the small gene capacity, large toxic side effects, and high preparation cost of traditional viral gene vectors, and the strong toxicity of traditional cationic non-viral gene vectors, the present invention provides a non-viral gene vector with a novel structure that has good biocompatibility and stability, as well as good biosafety, while being able to improve the targeted treatment effect of lesions.

[0010] Solutions for solving problems

[0011] In view of the problems existing in the above-mentioned prior art, the inventors, after in-depth research and repeated experiments, have proposed a non-viral gene vector with a novel core-shell structure constructed based on the bioaffinity between lipidated cationic materials and protein materials. This non-viral gene vector uses lipid-modified cationic materials to efficiently compress the gene to form the core of the vector, and selects proteins with high bioaffinity for lipids to form the outer shell of the vector. This outer shell can be firmly adsorbed on the surface of the lipidated cationic core, on the one hand, effectively shielding the positive charge of the core and improving in vivo safety, and on the other hand, achieving passive or active targeting of diseased cells, thereby completing the present invention. That is, the present invention is as follows:

[0012] The first aspect of the present invention provides a non-viral gene vector based on bioaffinity, characterized in that the non-viral gene vector comprises (i) a vector core, and (ii) a vector shell;

[0013] The carrier core is composed of cationic materials modified with lipid substances and nucleic acid molecules, and the carrier shell is composed of proteins having biological affinity for the lipid substances in the carrier core.

[0014] In some embodiments of the present invention, the lipid substance is selected from any one of the group consisting of: fatty acids (esters), glycerolipids, phospholipids, glycolipids, cholesterol esters, cholesterol, bile acids, vitamin D, and structural analogs and derivatives thereof;

[0015] In some preferred embodiments, the lipid substance is either cholesterol or stearic acid.

[0016] In some embodiments of the present invention, the cationic material includes organic cationic materials and inorganic cationic materials;

[0017] In some preferred embodiments, the organic cationic material is selected from any one of the group consisting of polyacrylamide, polyethyleneimine, polyglutamic acid, polylysine, polyarginine, ferrocene, DOTAP, chitosan and protamine; the inorganic cationic material is selected from any one of the group consisting of aluminum hydroxide, ferric hydroxide, hollow mesoporous silica and calcium ions;

[0018] In some more preferred embodiments, the cationic material is selected from any one of the group consisting of polyethyleneimine, polylysine and protamine.

[0019] In some embodiments of the present invention, the nucleic acid molecules include DNA, RNA, and hybrids thereof;

[0020] In some preferred embodiments, the nucleic acid molecule is selected from any one of the group consisting of complementary DNA (cDNA), plasmid DNA (pDNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, viral RNA (vRNA) and CRISPR RNA sequences;

[0021] In some more preferred embodiments, the nucleic acid molecule is any one of pDNA, siRNA or mRNA.

[0022] In some embodiments of the present invention, the protein in the vector shell has passive or active targeting to target cells, and the protein is selected from any one of the group consisting of albumin, apolipoprotein, very low density lipoprotein, low density lipoprotein, intermediate density lipoprotein, high density lipoprotein and their structural analogs and derivatives;

[0023] In some preferred embodiments, the protein is any one of low-density lipoprotein, serum albumin, or high-density lipoprotein.

[0024] In some embodiments of the present invention, the mass ratio of the carrier shell to the carrier core is 0.1:1 to 1000:1, the molar ratio of the lipid substance to the cationic material in the carrier core is 0.1:1 to 100000:1, and the mass ratio of the cationic material modified with the lipid substance to the nucleic acid molecule is 0.1:1 to 10000:1;

[0025] In some preferred embodiments, the molar ratio of the carrier shell to the carrier core is 1:1 to 50:1, the molar ratio of the lipid substance in the carrier core to the cationic material is 0.1:1 to 30000:1, and the mass ratio of the cationic material modified by the lipid substance to the nucleic acid molecule is 1:1 to 80:1.

[0026] In some embodiments of the present invention, the particle size of the non-viral gene vector is 10 to 1000 nm, and the Zeta potential of the non-viral gene vector is -25 to 25 mV;

[0027] In some preferred embodiments, the particle size of the non-viral gene vector is 30 to 200 nm, and the Zeta potential of the non-viral gene vector is -20 to -2 mV;

[0028] In some more preferred embodiments, the particle size of the non-viral gene vector is 35 to 130 nm, and the Zeta potential of the non-viral gene vector is -12 to -4 mV.

[0029] The second aspect of the present invention provides a method for preparing the non-viral gene vector as described in the first aspect of the present invention, characterized in that the preparation method comprises the following steps:

[0030] 1) chemically coupling the lipid substance and the cationic material to obtain a lipid substance-modified cationic material;

[0031] 2) mixing the nucleic acid molecule and the cationic material modified with the lipid substance prepared in step 1) to obtain a carrier core loaded with the nucleic acid molecule;

[0032] 3) mixing the carrier core loaded with the nucleic acid molecule prepared in step 2) with a protein, wherein the protein has an affinity for the lipid substance in the carrier core.

[0033] In some embodiments of the present invention, the molar ratio of the lipid substance to the cationic material in step (1) of the preparation method is 0.1:1 to 100,000:1; the mass ratio of the cationic material modified with the lipid substance to the nucleic acid molecule in step (2) of the preparation method is 0.1:1 to 10,000:1; and the mass ratio of the protein to the carrier core carrying the nucleic acid molecule in step (3) of the preparation method is 0.1:1 to 1,000:1.

[0034] In some embodiments of the present invention, the solvent used in steps (1) to (3) is selected from any one of the group consisting of water, ethanol, isopropanol, triethylamine, glycerol, petroleum ether, acetonitrile, acetone, n-hexane, cyclohexane, trifluoroacetic acid, 1,1,1-trichloroethane, N,N-dimethylformamide, carbon tetrachloride, anhydrous chloroform, dichloromethane, 1,4-dioxane, dimethyl sulfoxide, ethyl acetate, butyl acetate, tetrahydrofuran and diethyl ether.

[0035] The third aspect of the present invention provides a use of the non-viral gene vector as described in the first aspect of the present invention in the preparation of a drug for preventing or treating a disease;

[0036] In some preferred embodiments, the disease includes genetic disease, cancer, immune disease, inflammation, cardiovascular disease, infectious disease;

[0037] In some more preferred embodiments, the disease is cancer (eg, breast cancer, gastric cancer, liver cancer, endometrial cancer, prostate cancer, colon cancer, esophageal cancer, skin cancer, bladder cancer, nasopharyngeal cancer, brain tumor, etc.).

[0038] The third aspect of the present invention provides a use of the non-viral gene vector as described in the first aspect of the present invention in preparing a cell preparation, characterized in that the non-viral gene vector can efficiently transfect cells in vitro, and the obtained cell preparation can be used for cell therapy;

[0039] In some preferred embodiments, the cell preparation includes a stem cell preparation, a red blood cell preparation, a T cell preparation, a natural killer cell preparation, a macrophage preparation, a dendritic cell preparation, and combinations thereof.

[0040]

Term Definition

[0041] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0042] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0043] In this specification, the use of “substantially” or “essentially” means that the standard deviation from a theoretical model or theoretical data is within a range of 5%, preferably 3%, and more preferably 1%.

[0044] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0045] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0046] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0047] As used herein, the term "nucleic acid molecule" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA, RNA, and hybrids thereof. DNA can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA can be in the form of a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a messenger RNA (mRNA), an antisense RNA, miRNA, micRNA, a multivalent RNA, a dicer substrate RNA, or a viral RNA (vRNA), and combinations thereof. Nucleic acid molecules include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring, and non-naturally occurring and have binding properties similar to a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNA). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to a reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as sequences explicitly indicated. In particular, degenerate codon substitutions (Batzer et al., Nucleic Acid Res., 19:5081 (1991)) can be achieved by generating sequences in which three of one or more selected (or all) codons are replaced by mixed bases and / or deoxyinosine residues; Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). "Nucleotide" contains sugar (deoxyribose (DNA) or ribose (RNA)), bases, and phosphate groups. Nucleotides are linked together by phosphate groups. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications to provide new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0048] The term "lipid substances" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by poor solubility in water but soluble in a wide variety of organic solvents. They are generally divided into at least three categories: (1) "simple lipids," which include fats and oils, as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derivative lipids," such as steroids. In addition, as used herein, lipids also include lipid-like compounds. The term "lipid-like compound," also referred to as "lipid," refers to a lipid-like compound (e.g., an amphiphilic compound having lipid-like physical properties).

[0049] According to the present invention, the terms "polypeptide", "protein" and "peptide" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with similar peptide backbones.

[0050] According to the present invention, the terms "nucleic acid molecule," "polynucleotide," "polynucleic acid," and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0051] According to the present invention, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny that possess the same function or biological activity as that screened for in the originally transformed cell are included. Where a different designation is intended, this is clear from the context.

[0052] The term "treatment" refers to a clinical intervention intended to reverse, alleviate a disease or disorder or one or more symptoms thereof, delay the onset of a disease or disorder or one or more symptoms thereof, or suppress a disease or disorder or one or more symptom progress thereof as described herein. As used herein, the term "treatment" refers to a clinical intervention intended to reverse, alleviate a disease or disorder or one or more symptoms thereof, delay the onset of a disease or disorder or one or more symptoms thereof, or suppress a disease or disorder or one or more symptom progress thereof as described herein. In some embodiments, treatment can be administered after one or more symptoms have been formed and / or after the disease has been diagnosed. In other embodiments, treatment can be administered in the absence of symptoms, for example, for preventing or delaying the onset of symptoms or suppressing the onset or progress of a disease. For example, treatment can be administered in susceptible individuals before the onset of symptoms (for example, in view of the history of symptoms and / or in view of genetic or other susceptibility factors). Treatment can also be continued after symptom regression, for example, to prevent or delay its recurrence.

[0053] Effects of the Invention

[0054] It can be seen from the technical solution of the present invention that, compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0055] 1. The non-viral gene vector based on bioaffinity provided by the present invention has good biocompatibility and stability, can transfect genes into cells with high transfection efficiency, and can efficiently deliver genes into the body to produce target gene products, thereby treating diseases;

[0056] 2. The non-viral gene carrier based on bioaffinity provided by the present invention can reduce the toxicity caused by the use of cationic materials, thereby avoiding damage to normal cells in the body and having good biosafety;

[0057] 3. The non-viral gene vector based on bioaffinity provided by the present invention uses a portion of the protein shell that has the ability to passively or actively target specific sites (such as some organs) in the body, thereby enabling the accumulation of genes at the target site and improving the therapeutic effect.

[0058] 4. The bioaffinity-based non-viral gene vector provided by the present invention does not require any modification of the gene drug, which helps maintain the biological activity of the gene drug. It is a highly valuable gene delivery vector in the field of gene therapy. Furthermore, the preparation method of the bioaffinity-based non-viral gene vector provided by the present invention has a clear overall design concept, a simple preparation process, mild conditions, and is easily applicable to clinical translation, showing great application potential.

[0059] In order to make the above and other objects, features and advantages of the present invention more clearly understood, preferred embodiments are given below with reference to the accompanying drawings for detailed description as follows: BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Schematic diagram of the structure of the low-density lipoprotein / cholesterolized polyethyleneimine gene vector loaded with siRNA prepared in Example 1.

[0061] Figure 2 Schematic diagram of the principle of releasing nucleic acid molecules into cells from the low-density lipoprotein / cholesterolized polyethyleneimine gene vector loaded with siRNA prepared in Example 1.

[0062] Figure 3 This is a transmission electron micrograph of the low-density lipoprotein / cholesterolized polyethyleneimine gene vector loaded with siRNA prepared in Example 1.

[0063] Figure 4 Line graphs showing the particle size changes of the non-viral gene vectors prepared as described in Example 1, Example 1, Example 2, Example 3, Example 4, and Example 5 (named "CP@LDL", "SL@HSA", "SPr@HDL", "SP@HSA", and "CP@HSA", respectively) and the siRNA-loaded human serum albumin / polyethyleneimine vector gene vector (named P@HSA) after being diluted 5-fold and 20-fold, respectively.

[0064] Figure 5 The non-viral gene vectors prepared by the methods described in Examples 1, 1, 2, 3, 4 and 5 (named "CP@LDL", "SL@HSA", "SPr@HDL", "SP@HSA", "CP@HSA") were loaded 32 After the modified gene was injected into the tail vein of tumor-bearing mice, the tissues in the body 32 P counting result graph.

[0065] Figure 6 The fluorescence results of transfecting NIH-3T3-GFP cells under a laser confocal microscope are shown below:

[0066] Figure 7The fluorescence results of transfecting NIH-3T3-GFP cells under a laser confocal microscope are shown below:

[0067] Figure 8 The fluorescence results of transfecting 4T1 cells under a laser confocal microscope are shown, after the human serum albumin / cholesterolized polyethyleneimine gene vector (named CP@HSA) prepared by the method described in Example 5 and the non-viral gene vector containing only the vector core (named CP) were loaded with mRNA capable of expressing GFP.

[0068] Figure 9 Figure 2 shows the fluorescence results of transfection of human umbilical cord mesenchymal stem cells under an inverted fluorescence microscope after loading mRNA capable of expressing GFP with a human serum albumin / cholesterolized polyethyleneimine gene vector (named CP@HSA) and a non-viral gene vector containing only a vector core (named CP) prepared according to the method described in Example 5.

[0069] Figure 10 Figure 2 shows the qRT-PCR results of human serum albumin / stearoyl polylysine gene vector (named SL@HSA) and a non-viral gene vector containing only a vector core (named CP), and high-density lipoprotein / stearoyl protamine (named SPr@HDL) and a non-viral gene vector containing only a vector core (named SPr), prepared by the methods described in Examples 2 and 3, after being loaded with pDNA capable of expressing hyaluronidase.

[0070] Figure 11 The low-density lipoprotein / cholesterolized polyethyleneimine gene vector (named CP@LDL) and the non-viral gene vector containing only the vector core (named CP) and the human serum albumin / stearoyl polyethyleneimine gene vector (named SP@HSA) and the non-viral gene vector containing only the vector core (named SP), prepared by the methods described in Examples 1 and 4, were loaded with siRNA capable of silencing survivin and injected into tumor-bearing mice through the tail vein. A line graph shows the changes in tumor volume.

[0071] Figure 12The low-density lipoprotein / cholesterolized polyethyleneimine gene vector (named CP@LDL) and the non-viral gene vector containing only the vector core (named CP) and the human serum albumin / stearoyl polyethyleneimine gene vector (named SP@HSA) and the non-viral gene vector containing only the vector core (named SP), prepared by the methods described in Examples 1 and 4, were loaded with siRNA capable of silencing survivin and injected into tumor-bearing mice via the tail vein. Mouse survival curves are shown. DETAILED DESCRIPTION

[0072] The specific embodiments listed herein are merely exemplary of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions of the embodiments described below are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to be encompassed within the scope of the present invention.

[0073] The test materials, test reagents and instruments used in the examples of the present invention can all be purchased commercially.

[0074] Principle of the present invention:

[0075] The bioaffinity-based non-viral gene vector described in the present invention works as follows: a lipid-modified cationic material binds to nucleic acid molecules through electrostatic interactions to form the core of the non-viral gene vector, while a protein binds to the lipids modified on the cationic material through bioaffinity to form the outer shell of the non-viral gene vector. Once the non-viral gene vector enters an organism, the protein shell protects the nucleic acid molecules in the core from degradation and prevents the highly dense positive charges on the cationic material's surface from toxicating the organism's cells. The modified protein shell or the properties of the protein shell itself enable the vector to passively or actively target the nucleic acid molecules, thereby delivering them to the target site for gene therapy.

[0076] Example 1 Preparation of siRNA-Carrying Low-Density Lipoprotein / Cholesterolized Polyethyleneimine Gene Vector

[0077] This embodiment provides a non-viral gene vector comprising low-density lipoprotein / cholesterolized polyethyleneimine and capable of delivering siRNA. The specific preparation method is as follows:

[0078] (1) 0.2250 g of cholesterol chloroformate (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) and 0.2600 g of polyethyleneimine (PEI 1.3k, wherein 1.3k is the molecular weight of the PEI, purchased from Merck Group, Germany, and used after freeze-drying) were dissolved in 10 mL of anhydrous chloroform and cooled in an ice bath for 30 min to obtain a cholesterol chloroformate solution and a PEI solution, respectively; then, 10 μL of triethylamine (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to the PEI solution to obtain a PEI solution containing triethylamine;

[0079] (2) In an ice bath, slowly adding the cholesterol chloroformate solution prepared in step (1) to the PEI solution containing triethylamine prepared in step (1) while stirring, and then stirring the mixture solution in an ice bath for 30 minutes, and then stirring at room temperature for 6 hours;

[0080] (3) Rotary evaporation of the mixture solution prepared in step (2) at 60° C. for 30 min to remove anhydrous chloroform and triethylamine from the mixture solution; dialyzing with deionized water (MWCO: 1000 Da) for 48 h; and freeze-drying to obtain cholesterol-modified PEI. 1.3k ;

[0081] (4) Add 1 μL of diethyl pyrocarbonate (DEPC) to 1 mL of ultrapure water to obtain nuclease-free water;

[0082] (5) 13 μg of siRNA (purchased from Guangzhou Ruibo Biotechnology Co., Ltd., catalog number: siN0000001-1-5) was added to 500 μL of nuclease-free water described in step (4), and then 0.304 mg of cholesterol-modified PEI prepared in step (3) was added. 1.3k , vortexed for 30 seconds, and incubated at room temperature for 30 minutes to obtain a vector core solution containing the gene;

[0083] (6) Add 4.560 mg of low-density lipoprotein (LDL, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) to the gene-carrying vector core solution prepared in step (5), vortex for 30 seconds, and incubate at room temperature for 1 hour to obtain a low-density lipoprotein / cholesterolized polyethyleneimine gene vector carrying siRNA.

[0084] The non-viral gene vector prepared in this example, namely the low-density lipoprotein / cholesterolized polyethyleneimine gene vector carrying siRNA, is shown in FIG. Figure 1 ; The schematic diagram of the principle of the non-viral gene vector releasing nucleic acid molecules in cells is shown in Figure 2Specifically, after the LDL receptor on the cell surface recognizes the LDL on the surface of the carrier, it forms clathrin pits on the cell membrane, which then coat the carrier to form coated vesicles. After being delivered to the lysosomes in the cell, the coated vesicles are converted into circulating vesicles and return to the cell membrane. The carrier is degraded in the lysosomes to form amino acids, cholesterol-containing polyethyleneimine and siRNA, and then the siRNA plays a further role in the cell.

[0085] 5 μL of the non-viral gene vector was dropped onto the surface of a copper mesh and negatively stained with 2% phosphotungstic acid for 5 min. After drying at 25°C under nitrogen for 20 min, the mesh was observed using a transmission electron microscope (LIBRA 120, Carl Zeiss Microscopy GmbH) at a voltage of 120 kV and a magnification of 100,000 times. The photographs are shown in Figure 3 .

[0086] Example 2 Preparation of pDNA-carrying human serum albumin-encapsulated stearoyl-modified polylysine gene vector

[0087] This embodiment provides a non-viral gene vector comprising human serum albumin / stearoyl polylysine and capable of delivering pDNA. The specific preparation method is as follows:

[0088] (1) 0.3339 g of stearyl bromide (purchased from Shanghai Myrel Chemical Technology Co., Ltd.) was dissolved in 20 mL of a mixed solution of dioxane and 200 μL of 1 M NaOH to obtain solution A; 0.1000 g of poly-L-lysine hydrobromide (PLL-HBr, molecular weight 50 kDa, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) was dissolved in 20 mL of a dimethyl sulfoxide solution to obtain solution B; the solutions A and B were mixed and stirred at room temperature for 24 h to obtain a mixture solution;

[0089] (2) pouring the mixture solution prepared in step (1) into excess ether, and the resulting product was purified by dialysis against deionized water (MWCO: 1000 Da) for 48 h, and freeze-dried to obtain stearoyl-modified poly-L-lysine;

[0090] (3) Add 1 μL of diethyl pyrocarbonate (DEPC) to 1 mL of ultrapure water to obtain nuclease-free water;

[0091] (4) 10 μg of pDNA (purchased from Thermo Fisher Scientific, catalog number: SD0061) was added to 500 μL of the nuclease-free water described in step (3), and then 0.100 mg of the stearoyl-modified poly-L-lysine prepared in step (2) was added, vortexed for 30 seconds, and incubated at room temperature for 30 minutes to obtain a vector core solution containing the gene;

[0092] (5) Add 0.1500 g of human serum albumin (HSA, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) to the vector core solution prepared in step (4), vortex for 30 seconds, and incubate at room temperature for 1 hour to obtain a stearoyl-modified polylysine gene vector coated with human serum albumin carrying pDNA.

[0093] Example 3 Preparation of pDNA-carrying high-density lipoprotein-encapsulated stearoyl-modified protamine gene vector

[0094] This embodiment provides a non-viral gene vector comprising high-density lipoprotein / stearoyl protamine and capable of delivering pDNA. The specific preparation method is as follows:

[0095] (1) Under nitrogen protection, 0.1151 g of N-hydroxysuccinimide (NHS, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and 0.1422 g of stearic acid (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were dissolved in 15.3 mL of tetrahydrofuran (THF), and 0.2063 g of N,N'-dicyclohexylcarbodiimide (DCC, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added. The mixture was stirred at 0°C for 2 h and then allowed to stand at room temperature overnight.

[0096] (2) adding a small amount of water to the solution prepared in step (1) to convert the excess DCC in the solution into dicyclohexylurea (DCU), and removing the dicyclohexylurea by suction filtration using a G6 sand core filter;

[0097] (3) The solution obtained in step (2) was rotary evaporated at 60° C. for 30 min to remove the solvent; the solution was redissolved in ethyl acetate and washed with water three times to remove excess NHS; 10 g of anhydrous sodium sulfate was added and dried overnight; the anhydrous sodium sulfate was removed by suction filtration using a G6 sand core filter; the filtrate was subjected to reduced pressure distillation to remove the ethyl acetate; and the solution was dried in a vacuum drying oven overnight to obtain stearic acid-NHS;

[0098] (4) 0.5100 g of protamine sulfate (molecular weight 5100 kDa, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was dissolved in 35.7 mL of boric acid buffer solution (pH = 9.0) containing 8 M urea, and stirred at room temperature until completely dissolved to obtain a protamine sulfate solution; 0.2573 g of stearic acid-NHS prepared in step (3) was dissolved in 15.3 mL of THF, and the solution was added to the above protamine sulfate solution; and stirred at room temperature for 1.5 h to obtain a mixture solution;

[0099] (5) The mixture solution prepared in step (4) was dialyzed (MWCO: 1000 Da) against phosphate buffer (pH = 7.4) containing 150 nM NaCl at 4°C overnight; the pH of the solution was adjusted to 5.0 with 1 M HCl; the solution was washed with ether five times; the solution was dialyzed against ultrapure water (MWCO: 1000 Da) for 72 h; and the solution was freeze-dried to obtain stearoyl protamine;

[0100] (6) Add 1 μL of diethyl pyrocarbonate (DEPC) to 1 mL of ultrapure water to obtain nuclease-free water;

[0101] (7) 13 μg of pDNA (purchased from Thermo Fisher Scientific, catalog number: SD0061) was added to 500 μL of the nuclease-free water described in step (6), and then 0.015 mg of the stearoyl protamine described in step (5) was added, vortexed for 30 seconds, and incubated at room temperature for 30 minutes to obtain a vector core solution containing the gene;

[0102] (8) 0.2250 g of high-density lipoprotein (HDL, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was added to the vector core solution described in step (7), vortexed for 30 seconds, and incubated at room temperature for 1 hour to obtain a stearoyl-modified protamine gene vector encapsulated by high-density lipoprotein carrying pDNA.

[0103] Example 4 Preparation of siRNA-loaded human serum albumin-encapsulated stearoyl-modified polyethyleneimine gene vector

[0104] This embodiment provides a non-viral gene vector comprising human serum albumin / stearoyl polyethyleneimine and capable of delivering siRNA. The specific preparation method is as follows:

[0105] (1) Under nitrogen protection, 0.1151 g of N-hydroxysuccinimide (NHS, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and 0.1422 g of stearic acid (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were dissolved in 20 mL of tetrahydrofuran (THF), and 0.2063 g of N,N'-dicyclohexylcarbodiimide (DCC, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added. The mixture was stirred at 0°C for 6 h and then allowed to stand at room temperature overnight.

[0106] (2) Using a G6 sand core funnel, remove impurities from the solution in step (1); centrifuge at 4000 rpm for 10 min, and take the supernatant for later use;

[0107] (3) Rotary evaporate the supernatant of step (2) at 60° C. for 30 min to remove the solvent; redissolve in ethyl acetate and wash with water three times to remove excess NHS; add 10 g of anhydrous sodium sulfate and dry overnight; use a G6 sand core funnel to filter and remove the anhydrous sodium sulfate; distill the filtrate under reduced pressure to remove the ethyl acetate contained therein; and dry in a vacuum drying oven overnight to obtain stearic acid-NHS;

[0108] (4) 0.6500g polyethyleneimine (PEI 1.3k , purchased from Merck Group, Germany, and used after freeze-drying) was dissolved in 20 mL of boric acid buffer solution (pH = 9.0) containing 8 M urea, and stirred at room temperature until completely dissolved to obtain a PEI solution; 0.2573 g of stearic acid-NHS prepared in step (3) was dissolved in 20 mL of THF, and the solution was added to the PEI solution described above; stirred at room temperature for 4 h to obtain a mixture solution;

[0109] (5) The mixture solution was dialyzed against phosphate buffer (pH = 7.4) containing 150 nM NaCl at 4°C (MWCO: 1000 Da) overnight; the pH of the solution was adjusted to 5.0 with 1 M HCl; the solution was washed with ether five times; the solution was dialyzed against ultrapure water (MWCO: 1000 Da) for 72 h; and the solution was freeze-dried to obtain stearoyl polyethyleneimine (stearoyl PEI);

[0110] (6) Add 1 μL of diethyl pyrocarbonate (DEPC) to 1 mL of ultrapure water to obtain nuclease-free water;

[0111] (7) 20 μg of siRNA (purchased from Guangzhou Ruibo Biotechnology Co., Ltd., catalog number: siN0000001-1-5) was added to 500 μL of nuclease-free water prepared in step (6), and then 0.033 mg of stearoyl polyethyleneimine prepared in step (5) was added, vortexed for 30 seconds, and incubated at room temperature for 30 minutes to obtain a vector core solution containing the gene;

[0112] (8) Add 0.2250 g of human serum albumin (HSA, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) to the vector core solution described in step (7), vortex for 30 seconds, and incubate at room temperature for 1 hour to obtain a stearoyl-modified polyethyleneimine gene vector coated with human serum albumin carrying siRNA.

[0113] Example 5 Preparation of Human Serum Albumin / Cholesterolized Polyethyleneimine Gene Vector Carrying mRNA

[0114] This embodiment provides a non-viral gene vector comprising human serum albumin / cholesterolized polyethyleneimine and capable of delivering mRNA. The specific preparation method is as follows:

[0115] (1) 0.2250 g of cholesterol chloroformate (purchased from Shanghai Yuanye Biotechnology Co., Ltd.), 0.2600 g of polyethyleneimine (PEI 1.3k , purchased from Merck Group, Germany, and used after freeze-drying) were dissolved in 10 mL of anhydrous chloroform and cooled in an ice bath for 30 min to obtain a cholesterol chloroformate solution and a PEI solution, respectively; then, 10 μL of triethylamine (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to the PEI solution to obtain a PEI solution containing triethylamine;

[0116] (2) slowly dripping the cholesterol chloroformate solution in step (1) into the PEI solution containing triethylamine in an ice bath while stirring, and then stirring the mixture solution in an ice bath for 30 minutes, and then stirring at room temperature for 6 hours;

[0117] (3) The mixture solution prepared in step (2) was rotary evaporated at 60°C for 30 min to remove anhydrous chloroform and triethylamine; dialyzed with deionized water (MWCO: 1000Da) for 48 h; and freeze-dried to obtain cholesterol-modified PEI. 1.3k ;

[0118] (4) Add 1 μL of diethyl pyrocarbonate (DEPC) to 1 mL of ultrapure water to obtain nuclease-free water;

[0119] (5) Add 13 μg of mRNA (GenBank Accession No: X83959.1) to 500 μL of the nuclease-free water in step (4), and then add 0.650 mg of the cholesterol-modified PEI prepared in step (3). 1.3k , vortexed for 30 seconds, and incubated at room temperature for 30 minutes to obtain a vector core solution containing the gene;

[0120] (6) Add 9.750 mg of human serum albumin (HSA, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) to the vector core solution described in step (5), vortex for 30 seconds, and incubate at room temperature for 1 hour to obtain the human serum albumin / cholesterolized polyethyleneimine gene vector carrying mRNA.

[0121] Example 6 Characterization of non-viral gene vectors based on bioaffinity

[0122] (1) Preparation of non-viral gene vectors based on bioaffinity: The specific preparation method is as described in Examples 1, 2, 3, 4 and 5; at the same time, referring to the preparation method described in Example 4, a human serum albumin / polyethyleneimine non-affinity vector loaded with siRNA is prepared as a control example (named "non-affinity control example" in this example), the difference being that the same molar amount of polyethyleneimine is used instead of stearyl polyethyleneimine.

[0123] (2) Particle size and zeta potential determination: The particle size and zeta potential of the samples were measured at 25°C using a Malvern Zetasizer NanoZS. The measurement results are shown in Table 1.

[0124] Table 1 Characterization of non-viral gene vectors using bioaffinity (n=3)

[0125]

[0126] As can be seen in Table 1 above, the particle size of the non-viral gene carrier prepared by the present invention, which utilizes bioaffinity, is approximately 60 nm, while the particle size of the control is close to the micron level. This demonstrates that the particle size of the non-viral gene carrier prepared by the present invention is significantly reduced. Small-sized nanoparticles are less susceptible to degradation in vivo, more easily enter cells, and can accumulate at the site of solid tumors through the EPR effect. Furthermore, the protein shell reduces the dense positive charge on the surface of the bare core, significantly reducing the cytotoxicity caused by dense surface positive charge compared to traditional cationic gene carriers. Furthermore, the particle size of the non-affinity control group was as high as nearly 1 μm, confirming that affinity is essential for the formation of this gene delivery system.

[0127] Example 7 Evaluation of dilution stability of non-viral gene vectors based on bioaffinity

[0128] The dilution stability of non-viral gene vectors with bioaffinity was evaluated using a Malvern nanoparticle size potential analyzer. The specific evaluation method is as follows:

[0129] (1) Non-viral gene vectors (named "CP@LDL", "SL@HSA", "SPr@HDL", "SP@HSA", and "CP@HSA" in this example) were prepared according to the preparation methods described in Examples 1, 2, 3, 4, and 5. SiRNA-loaded human serum albumin / polyethyleneimine vectors were prepared as a control example (named "P@HSA" in this example) with reference to the preparation method described in Example 4. The difference was that the same molar amount of polyethyleneimine was used instead of stearyl polyethyleneimine.

[0130] (2) The prepared complex solution was diluted 5 times and 20 times, respectively, and the particle size was measured at 25°C using a Malvern Zetasizer Nano ZS.

[0131] The line graph of particle size change after dilution of the two complex solutions is shown in Figure 2. Figure 4 shown.

[0132] Result analysis: From Figure 4 As can be seen from the results, the non-viral gene vector prepared by the present invention using bioaffinity exhibits no significant change in particle size before and after dilution, indicating excellent dilution stability. In contrast, the control (i.e., "P@HSA") exhibits a significant increase in particle size to over 1000 nm after a 5-fold dilution, and further increases after a 20-fold dilution, indicating extremely poor dilution stability. This demonstrates that non-viral gene vectors constructed using bioaffinity exhibit superior stability.

[0133] Example 8 Evaluation of in vivo targeting ability of non-viral gene vectors based on bioaffinity

[0134] (1) According to the preparation methods described in Examples 1, 2, 3, 4 and 5, non-viral gene vectors (named in this example as "CP@LDL", "SL@HSA", "SPr@HDL", "SP@HSA", and "CP@HSA") were prepared; wherein the siRNA, pDNA, and mRNA contained in the non-viral gene vectors were all 32 P (purchased from China Isotope Company) modified.

[0135] (2) Thirty mice (BALB / c Nude Mice, purchased from Qinglongshan Animal Breeding Farm) were subcutaneously injected with 100 μL MDA-MB-231 cells (containing approximately 2×10 6 cells, purchased from Shanghai Meixuan Biotechnology Co., Ltd.). When the tumor volume reached 50 mm 3 Six mice in each group were injected via the tail vein with 200 μL of CP@LDL, SL@HSA, SPr@HDL, SP@HSA, and CP@HSA. Twelve hours later, the hearts, livers, spleens, lungs, kidneys, brains, and tumors of the mice were harvested and weighed. A cryogenic grinding apparatus (JXFSTPRP-I, Tuohe Electromechanical Technology Co., Ltd.) was used to prepare a grinding solution (5 mg / mL) for each tissue. The samples were centrifuged at 12,000 rpm for 5 minutes, and 500 μL of the supernatant was collected from each sample.

[0136] (3) Prepare scintillation fluid by dissolving 5 g of 2,5-diphenyloxazole and 0.2 g of 1,4-bis(5-phenyl-2-oxazole)benzene in 1000 mL of xylene. Add 500 μL of the supernatant from each tissue obtained in step (2) to a glass scintillation vial (M1152, purchased from Merck, Germany) containing 5 mL of scintillation fluid. Count the scintillation fluid using a liquid scintillation counter (LS 6500, Beckman Coulter).

[0137] In each group 32 P count / mg tissue results are as follows Figure 5 shown.

[0138] Results: Due to the overexpression of corresponding receptors (such as albumin receptors, LDL receptors, and HDL receptors) on the surface of breast cancer cells and the nanometer size of the prepared non-viral gene vector, the non-viral gene vector prepared by the present invention, which utilizes bioaffinity, has excellent targeting ability to tumor tissue in vivo. Therefore, it can be demonstrated that the non-viral gene vector constructed through bioaffinity has excellent targeting ability to lesion cells in vivo.

[0139] Example 9 Evaluation of in vitro transfection ability of non-viral gene vectors based on bioaffinity

[0140] The in vitro transfection ability of non-viral gene vectors with bioaffinity was evaluated by laser confocal microscopy. The specific operation method is as follows:

[0141] (1) According to the preparation method described in Example 4, a non-viral gene vector comprising human serum albumin / stearoyl polyethyleneimine and siRNA was prepared, wherein the siRNA was capable of silencing green fluorescent protein (GFP) expression (purchased from Guangzhou Ruibo Biotechnology Co., Ltd., catalog number: siP0000005-1-5);

[0142] (2) NIH-3T3-GFP cells (purchased from Shanghai Meixuan Biotechnology Co., Ltd.) grown to 60% to 80% in the culture flask were washed with PBS buffer at pH 7.0 to 7.4, dispersed with 1 mL of 0.25% trypsin, and plated at 1×10 5 pieces / cm 2The cells were seeded at a density of 100 μg / mL on the bottom of a confocal microplate, and 2 mL of DMEM medium containing 10% inactivated FBS (purchased from Thermo Fisher Scientific) was added to each microplate. The vector core containing the gene (named "SP" in this example) and the core coated with the shell (named "SP@HSA" in this example) prepared according to the above method were added to four microplates as experimental groups. Lipofectamine 3000 reagent (purchased from Thermo Fisher Scientific) and DMEM medium were added to two other microplates as positive control (named Lipo3000 in this example) and negative control (named Control in this example), respectively. The cells were incubated at 37°C and 5% CO2 for 24 h.

[0143] (3) The green fluorescence intensity of NIH-3T3-GFP cells was detected using a laser confocal microscope to compare and evaluate the in vitro gene transfection ability of the gene vector.

[0144] Test results: The fluorescence results obtained by laser confocal microscopy after transfection of the gene vector are shown in the figure Figure 6 As shown. Figure 6 It can be seen that the green fluorescence of the SP group and SP@HSA group was significantly weakened compared with the Control group, and was weaker than that of the Lipo3000 group. That is, the SP group and SP@HSA group had stronger gene silencing effects than the Lipo3000 group, indicating that SP and SP@HSA have good in vitro gene transfection ability.

[0145] Similarly, the in vitro transfection ability of the non-viral gene vector containing low-density lipoprotein / cholesterolized polyethyleneimine and siRNA (named "CP@LDL") and the non-viral gene vector containing only the vector core (named "CP") and the complex with siRNA prepared in Example 1 was evaluated according to the above method. The fluorescence results obtained by laser confocal microscopy after transfection of the gene vector are shown in Figure 2. Figure 7 As shown. Figure 7 It can be seen that the green fluorescence of the CP group and CP@LDL group was significantly weakened compared with the Control group, and was weaker than that of the Lipo3000 group. That is, the CP group and CP@LDL group had stronger gene silencing effects than the Lipo3000 group, indicating that CP and CP@LDL have good in vitro gene transfection ability.

[0146] Similarly, the in vitro transfection ability of the non-viral gene vector (named "CP@HSA") containing human serum albumin / cholesterolized polyethyleneimine and mRNA prepared in Example 5 and the non-viral gene vector containing only the vector core (which contains mRNA and is named "CP") was evaluated according to the above method. The difference from the above process is that the gene used is mRNA that allows cells to express green fluorescent protein (GenBank Accession: X83959.1), and the cells used are 4T1 cells (purchased from Shanghai Meixuan Biotechnology Co., Ltd.) and human umbilical cord mesenchymal stem cells (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.). The fluorescence results obtained by laser confocal microscopy after the gene vector was transfected into 4T1 cells are shown in the figure below. Figure 8 As shown in the figure, the fluorescence results obtained by inverted fluorescence microscope after transfection of human umbilical cord mesenchymal stem cells are shown in the figure Figure 9 As shown. Figure 8 and Figure 9 It can be seen that the CP group and CP@HSA group have obvious green fluorescence expression compared with the Control group, and are stronger than the Lipo3000 group, that is, the CP group and CP@HSA group have stronger gene expression effects than the Lipo3000 group, indicating that CP and CP@HSA have good in vitro gene transfection ability, and the vector can well transfect genes into stem cells.

[0147] The in vitro transfection ability of non-viral gene vectors with bioaffinity was evaluated by qRT-PCR. The specific operation method is as follows:

[0148] (1) According to the preparation method described in Example 2, a non-viral gene vector comprising human serum albumin / stearoyl polylysine and a complex thereof with pDNA was prepared, wherein the pDNA was capable of highly expressing hyaluronidase (GenBank Accession: AF069741.1);

[0149] (2) M14 cells (purchased from Shanghai Meixuan Biotechnology Co., Ltd.) grown to 60% to 80% in the culture flask were washed with PBS buffer at pH 7.0 to 7.4, dispersed with 1 mL of 0.25% trypsin, and plated at 2 × 10 5The cells were seeded at a density of 100 μg / well in a 6-well plate, 2 mL of DMEM medium containing 10% inactivated FBS (purchased from Thermo Fisher Scientific) was added to each well, and then the gene-carrying vector core (named "SL" in this example) and the shell-encapsulated core (named "SL@HSA" in this example) prepared according to the above method were added to two of the wells as experimental groups, and Lipofectamine 3000 reagent (purchased from Thermo Fisher Scientific) and DMEM medium were added to the other two wells as positive control (named "Lipo3000" in this example) and negative control (named "Control" in this example), respectively. The plates were incubated at 37°C and 5% CO2 for 24 h.

[0150] (3) Total RNA was extracted from M14 cells using the RNAqueous Micro RNA Isolation Kit (purchased from Thermo Fisher Scientific). After reverse transcription, the cells were stained using the PowerTrack SYBR Green Master Mix Kit (purchased from Thermo Fisher Scientific), and the expression of hyaluronidase was detected using a real-time fluorescence quantitative PCR system (7300Plus, Thermo Fisher Scientific). The qRT-PCR amplification conditions were: 95°C for 15 min, 1 cycle; 95°C for 10 s, 65°C for 30 s, 40 cycles. The amplification primer sequences for hyaluronidase were: SEQ ID NO: 1: 5'-CGA TAT GGC CCA AGGCTT TAG-3' (sense chain), and SEQ ID NO: 2: 5'-ACC ACA TCG AAG ACA CTG ACA T-3' (antisense chain).

[0151] Similarly, the in vitro transfection ability of the non-viral gene vector prepared in Example 3 comprising high-density lipoprotein / stearoyl protamine and pDNA (named "SPr@HDL") and the non-viral gene vector comprising only the vector core (named "SPr") and its complex with pDNA were evaluated according to the above method.

[0152] Test results: The qRT-PCR results obtained after transfection of the above two gene vectors are shown in the figure Figure 10 As shown. Figure 10 It can be seen that the expression of hyaluronidase mRNA in the SL group, SL@HSA group, SPr group and SPr@HDL group was significantly increased compared with the Control group, and the effects of the SL@HSA group and SPr@HDL group were close to those of the Lipo3000 group, indicating that SL@HSA and SPr@HDL have good in vitro gene transfection ability.

[0153] Example 10 Evaluation of in vivo transfection ability of non-viral gene vectors based on bioaffinity

[0154] The in vivo transfection ability of biocompatible non-viral gene vectors was evaluated using a mouse breast cancer model. The specific procedures are as follows:

[0155] (1) According to the preparation methods described in Examples 1 and 4, non-viral gene vectors (named "CP@LDL" and "SP@HSA" in this example) were prepared. Similarly, gene-carrying vector cores (named "CP" and "SP" in this example) were prepared by referring to the above method. Lipofectamine 3000 reagent (purchased from Thermo Fisher Scientific Inc.) was used to co-incubate with siRNA according to the corresponding instructions as a positive control (named "Lipo3000" in this example). 0.9% sterile saline (purchased from Beijing Myrida Technology Co., Ltd.) was used as a negative control (named "Control" in this example). The siRNA used above was capable of silencing survivin expression (purchased from Guangzhou Ruibo Biotechnology Co., Ltd., product number: siB150723094012-1-5).

[0156] (2) Thirty-six mice (BALB / c Nude Mice, purchased from Qinglongshan Animal Breeding Farm) were subcutaneously injected with 100 μL MDA-MB-231 cells (containing approximately 2×10 6 cells, purchased from Shanghai Meixuan Biotechnology Co., Ltd.). When the tumor volume reached 50 mm 3 Six mice in each group were injected with 200 μL of CP@LDL, SP@HSA, CP, SP, Lipo3000, and 0.9% saline via the tail vein, once every three days for a total of four injections. The tumor volume and mouse body weight were monitored during the period. Animal welfare requirements were followed. When the tumor volume exceeded 1200 mm 3 The mice were considered dead and sacrificed.

[0157] (3) The tumor volume and body weight of each group of mice were measured daily, and a tumor volume line graph and a mouse survival curve were drawn to compare and evaluate the in vivo gene transfection ability of the gene vector.

[0158] Experimental results: The line graph of tumor volume changes in each group of mice is as follows Figure 11 The survival curves of mice in each group are shown in Figure 12 As shown. Figure 11It can be seen that the non-viral gene vector prepared by the present invention has a more significant inhibitory effect on tumor volume growth than the positive control Lipo3000. After being encapsulated with a protein shell, tumor growth is further slowed down. Moreover, thanks to the protein shell, the vector will not be easily degraded in the body, and its anti-tumor effect will last longer. Figure 12 As can be seen, the non-viral gene vector prepared by the present invention is less toxic than the positive control Lipo3000 and has a smaller impact on mouse survival. This impact is further reduced after encapsulation with a protein coat. These results demonstrate that the non-viral gene vector prepared by the present invention has excellent in vivo transfection ability and biosafety.

Claims

1. A non-viral gene vector based on bioaffinity, characterized in that: The non-viral gene vector comprises (i) a vector core, and (ii) a vector shell; The carrier core is composed of cationic materials modified with lipid substances and nucleic acid molecules, and the carrier shell is composed of proteins having biological affinity for the lipid substances in the carrier core; Wherein, the lipid substance is any one of cholesterol or stearic acid; The cationic material is selected from any one of the group consisting of polyethyleneimine, polylysine and protamine; The nucleic acid molecule is any one of pDNA, siRNA or mRNA; The protein is any one of low-density lipoprotein, serum albumin or high-density lipoprotein; The Zeta potential of the non-viral gene vector is -20 to -2 mV.

2. The non-viral gene vector according to claim 1, wherein The mass ratio of the carrier shell to the carrier core is 0.1:1 to 1000:1, the molar ratio of the lipid substance in the carrier core to the cationic material is 0.1:1 to 100000:1, and the mass ratio of the cationic material modified by the lipid substance to the nucleic acid molecule is 0.1:1 to 10000:

1.

3. The non-viral gene vector according to claim 2, wherein the molar ratio of the vector shell to the vector core is 1:1 to 50:1, the molar ratio of the lipid substance in the vector core to the cationic material is 0.1:1 to 30000:1, and the mass ratio of the lipid-modified cationic material to the nucleic acid molecule is 1:1 to 80:

1.

4. The non-viral gene vector according to any one of claims 1 to 3, wherein The particle size of the non-viral gene carrier is 35-130 nm. The non-viral gene vector according to claim 4 , wherein the zeta potential of the non-viral gene vector is -12 to -4 mV.

6. The method for preparing the non-viral gene vector according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: 1) chemically coupling the lipid substance and the cationic material to obtain a lipid substance-modified cationic material; 2) mixing the nucleic acid molecule and the cationic material modified with the lipid substance prepared in step 1) to obtain a carrier core loaded with the nucleic acid molecule; 3) mixing the carrier core loaded with the nucleic acid molecule prepared in step 2) with a protein, wherein the protein has an affinity for the lipid substance in the carrier core; Wherein, the lipid substance is any one of cholesterol or stearic acid; The cationic material is selected from any one of the group consisting of polyethyleneimine, polylysine and protamine; The nucleic acid molecule is any one of pDNA, siRNA or mRNA; The protein is any one of low-density lipoprotein, serum albumin or high-density lipoprotein; The Zeta potential of the non-viral gene vector is -20 to -2 mV.

7. The preparation method according to claim 6, wherein the molar ratio of the lipid substance to the cationic material in step (1) is 0.1:1 to 100000:1; the mass ratio of the cationic material modified with the lipid substance to the nucleic acid molecule in step (2) is 0.1:1 to 10000:1; and the mass ratio of the protein to the carrier core carrying the nucleic acid molecule in step (3) is 0.1:1 to 1000:

1.

8. The preparation method according to claim 6 or 7, wherein the solvent used in steps (1) to (3) is any one selected from the group consisting of water, ethanol, isopropanol, triethylamine, glycerol, petroleum ether, acetonitrile, acetone, n-hexane, cyclohexane, trifluoroacetic acid, 1,1,1-trichloroethane, N,N-dimethylformamide, carbon tetrachloride, anhydrous chloroform, dichloromethane, 1,4-dioxane, dimethyl sulfoxide, ethyl acetate, butyl acetate, tetrahydrofuran and diethyl ether.

9. Use of the non-viral gene vector according to any one of claims 1 to 5 in the preparation of cell preparations, characterized in that: The non-viral gene vector can efficiently transfect cells in vitro, and the obtained cell preparation can be used for cell therapy.

10. The use according to claim 9, wherein the cell preparation comprises a stem cell preparation, a red blood cell preparation, a T cell preparation, a natural killer cell preparation, a macrophage preparation, a dendritic cell preparation, and a combination thereof.

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