CN115814109A - Non-viral gene vector based on bioaffinity and preparation method and application thereof - Google Patents

Abstract

The invention discloses a non-viral gene vector based on bioaffinity, a preparation method and application thereof. The invention provides a non-viral gene vector based on bioaffinity, which is characterized in that the non-viral gene vector comprises (i) a vector inner core and (ii) a vector outer shell; wherein the carrier inner core is composed of cationic materials modified by lipid substances and nucleic acid molecules, and the carrier outer shell is composed of proteins with bioaffinity effect on the lipid substances in the carrier inner core. The non-viral gene vector provided by the invention has stability, can efficiently deliver genes into a body and generate a target product or interfere the expression of the target product, thereby treating diseases; target cells can also be transfected efficiently in vitro to prepare cell preparations required by cell therapy. In addition, the non-viral gene vector can reduce toxicity caused by using a cationic material, so that damage to normal cells in vivo is avoided, and the non-viral gene vector has good biocompatibility.

Description

Non-viral gene vector based on bioaffinity and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, relates to a non-viral gene vector, a preparation method and application thereof, and more particularly relates to a non-viral gene vector based on bioaffinity, a preparation method and application thereof.

Background

Gene therapy (gene therapy) refers to a technique of introducing exogenous normal genes into target cells through a gene vector to correct or compensate diseases caused by defective and abnormal genes, so as to achieve the purpose of treatment. Also included are the use of transgenes, which are produced by inserting an exogenous gene into the appropriate recipient cell of a patient by gene transfer techniques, which can treat a disease by producing a product or reducing the expression of the product. In a broad sense, gene therapy may also include measures and new technologies taken from the DNA level to treat certain diseases.

The gene vectors currently used can be classified into viral vectors (viral vectors) and non-viral vectors (non-viral vectors) according to their origin. Viral vectors are viruses (e.g., retroviruses, lentiviruses, and adenoviruses) engineered to carry therapeutic genes. The modified virus vector has the same organism infection capacity as wild virus, has higher gene transfection efficiency, but has the defects of small gene capacity, large toxic and side effects, high preparation cost and the like. The non-viral vector has good clinical application prospect due to the advantages of low immunogenicity, low cost, easy scale production and the like. However, the transduction efficiency of non-viral vectors is low, the size of the entire delivery system is large, and immune responses are easily induced and eliminated by the body.

In recent years, non-viral gene vectors adopting cationic polymer materials to deliver genes are emerging, and the cationic polymer gene vectors compress genes through high-density positive charges, are beneficial to the uptake of the genes by cells, and therefore have higher transfection efficiency. However, the highly dense positive charges on the surface of the cationic polymer gene vector also have strong toxicity in vivo, which limits the application thereof.

The method adopts electronegative materials to coat positive charge carriers through charge adsorption, can effectively shield positive charges of the carriers, and is an effective method for reducing toxicity of cation carriers. However, the nanocarrier having a core-shell structure formed merely by relying on charge interaction has the disadvantages that, on the one hand, the outer shell is easily peeled off during in vivo transportation, and the exposed positively charged inner core may cause toxic and side effects, and, on the other hand, the nanocarrier, even if it completely reaches the target site, is not easily taken up by the target cell due to the electronegativity of the outer shell, and thus the transfection efficiency is low. Therefore, how to increase the stability of such a vector having a core-shell structure and improve the target cell uptake efficiency of the vector becomes an urgent problem to be solved.

Bioaffinity refers to the phenomenon in which a biomolecule is capable of specifically recognizing and attractively binding to a substance under certain conditions. Among them, albumin (albumin, also called albumin), apolipoprotein (apolipoprotein), very Low Density Lipoprotein (VLDL), low Density Lipoprotein (LDL), intermediate Density Lipoprotein (IDL), high Density Lipoprotein (HDL), and other proteins have good bioaffinity to lipid, and have good targeting to focal tissues such as tumor, articular cavity (cardiovascular plaque), and the like, and thus, the albumin (albumin, also called albumin), apolipoprotein (apolipoprotein), and other proteins are expected to be used for improving the stability of a gene delivery vector having a core-shell structure and realizing targeted therapy to a focal. Jianping Zhou et al (Biomaterials vol.35,25 (2014): 7214-27.) reported that lipophilic siRNA is obtained by cholesterizing siRNA, and then a nano delivery system with a core-shell structure is prepared by externally coating recombinant high-density lipoprotein, wherein the nano delivery system has high safety, but the modification on the gene per se can influence the function of the gene, and the gene exists at the periphery of the system and is easily degraded by a complex environment in vivo.

Therefore, there is a need to develop a safe, stable, biocompatible non-viral gene vector that can improve the therapeutic effect of the target lesion.

Disclosure of Invention

Problems to be solved by the invention

Aiming at the defects in the prior art, namely the problems of small gene capacity, large toxic and side effects and high preparation cost of the traditional viral gene vector and the problem of strong toxicity of the traditional cationic non-viral gene vector, the invention provides the non-viral gene vector with a novel structure, which has good biocompatibility and stability, better biological safety and can improve the targeted treatment effect of a focus.

Means for solving the problems

In view of the problems of the prior art, the present inventors have made extensive studies and experiments to provide a non-viral gene vector having a novel core-shell structure, which is constructed based on the bioaffinity between a lipidated cationic material and a proteinaceous material. The non-viral gene vector adopts lipid modified cationic materials to efficiently compress genes to form the inner core of the vector, and selects proteins with high biological affinity to lipids to form the outer shell of the vector. The shell can be firmly adsorbed on the surface of the lipidated cation inner core, on one hand, the positive charge of the inner core can be effectively shielded, the in vivo safety is improved, on the other hand, the passive or active targeting to focus cells is realized, and thus the invention is completed. Namely, the present invention is as follows:

in a first aspect, the present invention provides a non-viral gene vector based on bioaffinity, wherein the non-viral gene vector comprises (i) a vector core, and (ii) a vector shell;

wherein the carrier inner core is composed of cationic materials modified by lipid substances and nucleic acid molecules, and the carrier outer shell is composed of proteins with bioaffinity effect on the lipid substances in the carrier inner core.

In some embodiments of the 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;

in some preferred embodiments, the lipid substance is any one of cholesterol or stearic acid.

In some embodiments of the invention, the cationic material comprises an organic based cationic material and an inorganic based cationic material;

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

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

In some embodiments of the invention, the nucleic acid molecule comprises DNA, RNA, and hybrids thereof;

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, microrna, multivalent RNA, viral RNA (vRNA), and CRISPR RNA sequences;

in some more preferred embodiments, the nucleic acid molecule is any one of pDNA, siRNA or mRNA.

In some embodiments of the invention, the protein in the carrier coat has passive or active targeting to the cell of interest, and the protein is selected from any one of the group consisting of albumin, apolipoprotein, very low density lipoprotein, medium density lipoprotein, high density lipoprotein, and structural analogs and derivatives thereof;

in some preferred embodiments, the protein is any one of low density lipoprotein, serum albumin, or high density lipoprotein.

In some embodiments of the invention, the mass ratio of the carrier shell to the carrier core is from 0.1 to 1000, the molar ratio of lipid substance to the cationic material in the carrier core is from 0.1 to 100000, the mass ratio of the lipid substance-modified cationic material to the nucleic acid molecule is from 0.1;

in some preferred embodiments, the molar ratio of the carrier shell to the carrier core is 1:1 to 50, the molar ratio of lipid species to the cationic material in the carrier core is 0.1 to 30000, the mass ratio of the lipid species-modified cationic material to the nucleic acid molecule is 1:1 to 80.

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

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

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

In a second aspect of the present invention, there is provided a method for producing the non-viral gene vector according to the first aspect of the present invention, comprising the steps of:

1) Chemically coupling the lipid substance with a cationic material to obtain a lipid substance modified cationic material;

2) Mixing nucleic acid molecules with the cationic material modified by the lipid substance prepared in the step 1) to obtain carrier cores carrying the nucleic acid molecules;

3) Mixing the nucleic acid molecule-loaded carrier core prepared in step 2) with a protein, and wherein the protein has an affinity for the lipid material in the carrier core.

In some embodiments of the invention, wherein the molar ratio of the lipid substance to the cationic material in step (1) of the preparation method is 0.1; the mass ratio of the cationic material modified by the lipid substance to the nucleic acid molecule in the step (2) of the preparation method is 0.1; the mass ratio of the nucleic acid molecule-carrying carrier core to the protein in step (3) of the preparation method is 0.1 to 1.

In some embodiments of the present invention, wherein the solvent used in the steps (1) - (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-dimethylformamide, carbon tetrachloride, anhydrous chloroform, dichloromethane, 1,4-dioxane, dimethyl sulfoxide, ethyl acetate, butyl acetate, tetrahydrofuran, and diethyl ether.

A 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 for the preparation of a medicament for preventing or treating a disease;

in some preferred embodiments, the disease includes a genetic disorder, cancer, an immunological disorder, inflammation, cardiovascular disease, infectious disease;

in some more preferred embodiments, the disease is cancer (e.g., breast cancer, gastric cancer, liver cancer, endometrial cancer, prostate cancer, colon cancer, esophageal cancer, skin cancer, bladder cancer, nasopharyngeal cancer, brain tumors, etc.).

In a third aspect, 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 cell preparation, wherein the non-viral gene vector is capable of transfecting cells in vitro with high efficiency, and the obtained cell preparation is useful for cell therapy;

in some preferred embodiments, the cell preparation comprises a stem cell preparation, an erythrocyte preparation, a T cell preparation, a natural killer cell preparation, a macrophage preparation, a dendritic cell preparation, and combinations thereof.

[ definitions of terms ]

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present specification, the numerical range represented by "a value a to B value" means a range including the endpoint value A, B.

In the present specification, the term "substantially" or "essentially" means that the standard deviation from the theoretical model or theoretical data is within a range of 5%, preferably 3%, more preferably 1%.

In the present specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In this specification, "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.

Reference in the specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "embodiments," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

As used herein, the term "nucleic acid molecule" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-or double-stranded form, and includes DNA, RNA, and hybrids thereof. The DNA may be in the form of: antisense molecules, plasmid DNA, cDNA, PCR products or vectors. The RNA may be in the form of: a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a messenger RNA (mRNA), an antisense RNA, a miRNA, a microRNA, 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 similar binding properties as reference nucleic acids (reference nucleic acids). Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2' -O-methyl ribonucleotide, and peptide-nucleic acid (PNA). Unless specifically limited, the term encompasses nucleic acids comprising known analogs of natural nucleotides that have similar binding properties as the 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 the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which three positions of one or more selected (or all) codons are substituted with mixed-base and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.,19, 5081 (1991); ohtsuka et al, J.biol.chem., 260. "nucleotides" contain a sugar (deoxyribose (DNA) or Ribose (RNA)), a base, and a phosphate group. 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 that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

The term "lipid material" refers to a group of organic compounds, which include, but are not limited to, esters of fatty acids, and are generally characterized as having poor solubility in water, but being 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, and waxes; (2) "compound lipids" including phospholipids and glycolipids; and (3) "derivatized lipids", such as steroids. Furthermore, as used herein, lipids also include lipidoid compounds. The term "lipid-like compound" is also referred to simply as "lipid-like"; refers to lipid-like compounds (e.g., amphiphilic compounds having lipid-like physical properties).

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

According to the present invention, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogues thereof. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. 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. The nucleic acid molecule may be linear or circular.

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 "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of metastases. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clearly visible from the context.

The term "treatment" refers to a clinical intervention as described herein that is intended to reverse, alleviate, delay the onset of, or inhibit the progression of a disease or disorder, or one or more symptoms thereof. As used herein, the term "treatment" refers to a clinical intervention as described herein that is intended to reverse, alleviate, delay the onset of, or inhibit the progression of a disease or disorder, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after the disease has been diagnosed. In other embodiments, treatment may be administered without symptoms, e.g., for preventing or delaying the onset of symptoms or inhibiting the onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in view of the history of symptoms and/or in view of genetic or other susceptibility factors). Treatment may also be continued after the symptoms have subsided, for example to prevent or delay their recurrence.

ADVANTAGEOUS EFFECTS OF INVENTION

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

1. the non-viral gene vector based on the bioaffinity effect has good biocompatibility and stability, can transfect the gene into cells with higher transfection efficiency, and can efficiently deliver the gene into the body to generate a target gene product so as to treat diseases;

2. the non-viral gene vector based on the bioaffinity can reduce the toxicity caused by using a cationic material, thereby avoiding the damage to normal cells in vivo and having better biological safety;

3. the partial protein outer shell used by the non-viral gene vector based on the bioaffinity provided by the invention has passive or active targeting capability on a specific part (such as a part of organ) in vivo, thereby realizing the aggregation of genes on a target part and improving the treatment effect.

4. The non-viral gene vector based on the bioaffinity provided by the invention does not need to modify gene drugs, is favorable for maintaining the biological activity of the gene drugs, and is a valuable gene delivery vector in the field of gene therapy. In addition, the preparation method of the non-viral gene vector based on the bioaffinity has clear overall design thought, simple preparation process, mild conditions, easy realization of clinical transformation and good application potential.

In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below:

drawings

FIG. 1 is a schematic structural view of the siRNA-loaded low density lipoprotein/cholesterylated polyethyleneimine gene vector prepared in example 1.

FIG. 2 is a schematic diagram showing the release of nucleic acid molecules in cells from the siRNA-loaded LDH/PEPTIDE gene vector prepared in example 1.

FIG. 3 is a TEM image of the siRNA-loaded LDL/cholesteryl polyethyleneimine gene vector prepared in example 1.

FIG. 4 is a line graph showing the particle size change of the nonviral gene vectors (designated "CP @ LDL", "SL @ HSA", "SPr @ HDL", "SP @ HSA", "CP @ HSA") and the siRNA-loaded human serum albumin/polyethyleneimine vector gene vector (designated as P @ HSA) prepared in examples 1, 2, 3, 4 and 5, respectively, after dilution 5-fold and 20-fold.

FIG. 5 shows examples 1, 2 and 3,The non-viral gene vectors (designated "CP @ LDL", "SL @ HSA", "SPr @ HDL", "SP @ HSA", "CP @ HSA", in that order) prepared in examples 4 and 5 were loaded ³² P modified gene, tail vein injection into tumor-bearing mouse, and in vivo tissue ³² P count result chart.

FIG. 6 is a graph showing the fluorescence results of transfected NIH-3T3-GFP cells under a confocal laser confocal microscope after loading siRNA capable of silencing Green Fluorescent Protein (GFP) into a human serum albumin/stearoyl polyethyleneimine gene vector (designated SP @ HSA) and a non-viral gene vector (designated SP) containing only a vector core, which were prepared by the method described in reference example 4.

FIG. 7 is a graph showing the fluorescence results of NIH-3T3-GFP transfected cells under a confocal laser confocal microscope after loading siRNA capable of expressing silent GFP with the LDAP gene vector (designated as CP @ LDL) and a non-viral gene vector (designated as CP) containing only the vector core prepared by the method described in example 1.

FIG. 8 is a graph showing the fluorescence results of transfected 4T1 cells under a confocal laser scanning microscope after loading mRNA capable of expressing GFP onto the human serum albumin/cholesterylated polyethyleneimine gene vector (designated as CP @ HSA) prepared by the method described in example 5 and the non-viral gene vector (designated as CP) containing only the vector core.

FIG. 9 is a graph showing the fluorescence results of transfected human umbilical cord mesenchymal stem cells under an inverted fluorescence microscope after loading mRNA capable of expressing GFP with the human serum albumin/cholesterylated polyethyleneimine gene vector (designated as CP @ HSA) prepared by the method described in example 5 and the non-viral gene vector (designated as CP) containing only the vector core.

FIG. 10 is a graph showing the results of qRT-PCR in which pDNA capable of expressing hyaluronidase was loaded on the human serum albumin/stearylated polylysine gene vector (designated as SL @ HSA) prepared as described in reference examples 2 and 3, the non-viral gene vector (designated as CP) containing only the vector core, the high-density lipoprotein/stearylated protamine (designated as SPr @ HDL), and the non-viral gene vector (designated as SPr) containing only the vector core, respectively.

FIG. 11 is a line graph showing the change in tumor volume after tail vein injection of a low-density lipoprotein/cholesterylated polyethyleneimine gene vector (designated as CP @ LDL) prepared by the methods described in reference to examples 1 and 4, and a non-viral gene vector (designated as CP) containing only a vector core, a human serum albumin/stearoyl polyethyleneimine gene vector (designated as SP @ HSA), and a non-viral gene vector (designated as SP) containing only a vector core, each loaded with siRNA capable of silencing survivin, into tumor-bearing mice.

FIG. 12 is a graph showing the survival rate of mice after the low-density lipoprotein/cholesterylated polyethyleneimine gene vector (designated as CP @ LDL) prepared by the methods described in reference to examples 1 and 4, and a non-viral gene vector (designated as CP) containing only a vector core, a human serum albumin/stearoyl polyethyleneimine gene vector (designated as SP @ HSA), and a non-viral gene vector (designated as SP) containing only a vector core, each loaded with siRNA capable of silencing survivin (survivin), were injected into tumor-bearing mice via caudal veins.

Detailed Description

The embodiments of the present invention are described as examples of the present invention, and the present invention is not limited to the embodiments described below. Any equivalent modifications and substitutions to the embodiments described below are within the scope of the present invention for those skilled in the art. Accordingly, equivalent alterations and modifications are intended to be included within the scope of the present invention, without departing from the spirit and scope of the invention.

The test materials, test reagents and instruments used in the examples of the present invention are commercially available.

The principle of the invention is as follows:

the non-viral gene vector based on the bioaffinity effect has the following action principle: the lipid modified cationic material can be combined with nucleic acid molecules through electrostatic interaction to form an inner core of the non-viral gene vector, and the protein is combined with the modified lipid on the cationic material through bioaffinity to form an outer shell of the non-viral gene vector. When the non-viral gene vector enters an organism, the protein shell can protect nucleic acid molecules in the inner core from being degraded, and avoid the toxicity of highly dense positive charges on the surface of the cationic material to cells of the organism, and the vector has passive or active targeting property by means of modification or the characteristics of the protein shell, so that the nucleic acid molecules are delivered to a target position for gene therapy.

Example 1 preparation of siRNA-loaded low-density lipoprotein/cholesterylated polyethyleneimine gene vector

This example provides a non-viral gene vector containing low density lipoprotein/cholesterylated polyethyleneimine and capable of delivering siRNA, which is specifically prepared as follows:

(1) 0.2250g cholesteryl chloroformate (available from Shanghai-source leaf Biotech, inc.) and 0.2600g Polyethyleneimine (PEI) _1.3k Wherein 1.3k is the molecular weight of the PEI, purchased from Merck, germany, and used after freeze-drying treatment) are respectively dissolved in 10mL of anhydrous chloroform and cooled in an ice bath for 30min to respectively obtain a cholesteryl chloroformate solution and a PEI solution; then adding 10 μ L of triethylamine (purchased from Shanghai Arlatin Biotechnology Co., ltd.) to the PEI solution to obtain a PEI solution containing triethylamine;

(2) Slowly dripping the cholesteryl chloroformate solution prepared in the step (1) into the PEI solution containing triethylamine prepared in the step (1) in an ice bath while stirring, then continuously stirring the mixture solution in the ice bath for 30min, and then stirring at room temperature for 6h;

(3) Performing rotary evaporation on the mixture solution prepared in the step (2) at 60 ℃ for 30min, and removing anhydrous chloroform and triethylamine in the mixture solution; dialyzing with deionized water (MWCO: 1000 Da) for 48h; freeze drying to obtain cholesterol modified PEI _1.3k ；

(4) 1 mu L of diethyl pyrocarbonate (DEPC) is added into 1mL of ultrapure water to obtain nuclease-free water;

(5) Mu.g of siRNA (purchased from Ribobo Biotech, guangzhou, cat # siN 0000001-1-5) was added to 500. Mu.L of the nuclease-free water described in step (4), and 0.304mg of the cholesterol-modifying agent prepared in step (3) was addedDecorated PEI _1.3k Swirling for 30s, and incubating for 30min at room temperature to obtain a carrier inner core solution carrying the gene;

(6) And (3) adding 4.560mg of low-density lipoprotein (LDL, purchased from Shanghai Yuanye Biotechnology Co., ltd.) into the carrier core solution carrying the genes prepared in the step (5), swirling for 30s, and incubating for 1h at room temperature to obtain the low-density lipoprotein/cholesteryl polyethyleneimine gene carrier carrying the siRNA.

The structural schematic diagram of the non-viral gene vector prepared in this example, i.e., the siRNA-loaded low-density lipoprotein/cholesterylated polyethyleneimine gene vector, is specifically shown in fig. 1; the schematic diagram of the principle of the non-viral gene vector releasing nucleic acid molecules in cells is shown in FIG. 2. Specifically, after recognizing LDL on the surface of a carrier, an LDL receptor on the surface of the carrier forms a clathrin pit on a cell membrane, then the carrier is coated to form a coating vesicle, the coating vesicle is converted into a circulating vesicle to return to the cell membrane after being delivered to a lysosome in a cell, the carrier is degraded under the action of the lysosome to form amino acid, cholesteryl polyethyleneimine and siRNA, and then the siRNA plays a further role in the cell.

5 μ L of the non-viral gene vector was dropped onto the surface of a copper mesh, and the non-viral gene vector was stained with 2% phosphotungstic acid for 5min, dried at 25 ℃ under nitrogen for 20min, and observed with a transmission electron microscope (LIBRA 120, carl Zeiss Microcopy GmbH) under a voltage of 120kV and a magnification of 100000 times, and the photograph taken is shown in FIG. 3.

Example 2 preparation of pDNA-loaded human serum Albumin-coated stearoyl-modified polylysine Gene vectors

This example provides a non-viral gene vector containing human serum albumin/stearylated polylysine and capable of delivering pDNA, specifically prepared as follows:

(1) 0.3339g stearyl bromide (available from Shanghai Mirrel chemical technology, inc.) was dissolved in 20mL dioxane and 200. Mu.L 1M NaOH mixed solution to give solution A; 0.1000g of poly-L-lysine hydrobromide (PLL-HBr, molecular weight 50kDa, available from Mecanum Biotech, inc. of Shanghai) was dissolved in 20mL of dimethyl sulfoxide solution to obtain solution B; mixing the solution A and the solution B, and stirring at room temperature for 24 hours to obtain a mixture solution;

(2) Pouring the mixture solution prepared in the step (1) into excessive ether, dialyzing and purifying the obtained product with deionized water (MWCO: 1000 Da) for 48h, and freeze-drying to obtain stearoyl-modified poly-L-lysine;

(3) Adding 1 mu L of diethyl pyrocarbonate (DEPC) into 1mL of ultrapure water to obtain nuclease-free water;

(4) Adding 10 mu g of pDNA (purchased from Saimer Feishell science and technology Co., ltd., product number: SD 0061) into 500 mu L of the nuclease-free water obtained in the step (3), adding 0.100mg of the stearoyl-modified poly-L-lysine prepared in the step (2), vortexing for 30s, and incubating at room temperature for 30min to obtain a gene-loaded carrier core solution;

(5) And (3) adding 0.1500g of human serum albumin (HSA, purchased from Shanghai source leaf Biotechnology Co., ltd.) into the carrier core solution prepared in the step (4), swirling for 30s, and incubating for 1h at room temperature to obtain the human serum albumin-coated stearoyl-modified polylysine gene carrier loaded with the pDNA.

Example 3 preparation of high Density lipoprotein-Encapsulated stearoyl-modified protamine Gene vector carrying pDNA

This example provides a non-viral gene vector comprising high density lipoprotein/stearylated protamine and capable of delivering pDNA, which was specifically prepared as follows:

(1) 0.1151g N-hydroxysuccinimide (NHS, available from Shanghai Aladdin Biotechnology Co., ltd.) and 0.1422g stearic acid (available from Shanghai Aladdin Biotechnology Co., ltd.) were dissolved in 15.3mL Tetrahydrofuran (THF) under nitrogen protection, 0.2063g N, N' -dicyclohexylcarbodiimide (DCC, available from Shanghai Aladdin Biotechnology Co., ltd.) was added, stirred at 0 ℃ for 2 hours and then allowed to stand overnight at room temperature;

(2) Adding a little water into the solution prepared in the step (1) to convert excessive DCC in the solution into Dicyclohexylurea (DCU), and performing suction filtration by using a G6 sand core filter funnel to remove the dicyclohexylurea;

(3) Carrying out rotary evaporation on the solution obtained in the step (2) at 60 ℃ for 30min to remove the solvent; dissolving in ethyl acetate, washing with water for three times, and removing excessive NHS; adding 10g of anhydrous sodium sulfate and drying overnight; removing the anhydrous sodium sulfate by suction filtration by using a G6 sand core filter funnel; distilling the filtrate under reduced pressure to remove ethyl acetate; drying in a vacuum drying oven overnight to obtain stearic acid-NHS;

(4) 0.5100g of protamine sulfate (5100 kDa molecular weight, available from shanghai source, leaf biotechnology limited) was dissolved in 35.7mL of a boric acid buffer solution (pH = 9.0) containing 8M urea, and stirred at room temperature until completely dissolved, to obtain a protamine sulfate solution; 0.2573g of stearic acid-NHS prepared in step (3) was dissolved in 15.3mL of THF, and this solution was added to the protamine sulfate solution described above; stirring for 1.5h at room temperature to obtain a mixture solution;

(5) Dialyzing (MWCO: 1000 Da) the mixture solution prepared in step (4) with a phosphate buffer (pH = 7.4) containing 150nM NaCl at 4 ℃ overnight; adjusting the pH of the solution to 5.0 with 1M HCl; washing with diethyl ether for 5 times; dialyzing with ultrapure water (MWCO: 1000 Da) for 72h; freeze drying to obtain stearoyl protamine;

(6) 1 mu L of diethyl pyrocarbonate (DEPC) is added into 1mL of ultrapure water to obtain nuclease-free water;

(7) Adding 13 μ g of pDNA (purchased from Saimer Feishell science and technology Co., ltd., product number: SD 0061) into 500 μ L of the nuclease-free water obtained in the step (6), adding 0.015mg of the stearoyl protamine obtained in the step (5), vortexing for 30s, and incubating at room temperature for 30min to obtain a carrier core solution carrying the gene;

(8) Adding 0.2250g of high density lipoprotein (HDL, purchased from Shanghai Yuanye Biotechnology Co., ltd.) into the carrier core solution in the step (7), vortexing for 30s, and incubating for 1h at room temperature to obtain the high density lipoprotein-coated stearoyl modified protamine gene carrier carrying pDNA.

Example 4 preparation of siRNA-loaded human serum Albumin-coated stearoyl-modified polyethyleneimine Gene vectors

This example provides a non-viral gene vector containing human serum albumin/stearylated polyethyleneimine and capable of delivering siRNA, which is specifically prepared as follows:

(1) Under the protection of nitrogen, 0.1151g N-hydroxysuccinimide (NHS, available from Shanghai Aladdin Biotechnology Co., ltd.) and 0.1422g stearic acid (available from Shanghai Aladdin Biotechnology Co., ltd.) were dissolved in 20mL Tetrahydrofuran (THF), 0.2063g N, N' -dicyclohexylcarbodiimide (DCC, available from Shanghai Aladdin Biotechnology Co., ltd.) was added, stirred at 0 ℃ for 6 hours and then allowed to stand at room temperature overnight;

(2) Filtering and removing impurities in the solution in the step (1) by using a G6 sand core funnel; centrifuging at 4000rpm for 10min, and collecting supernatant;

(3) Performing rotary evaporation on the supernatant obtained in the step (2) at 60 ℃ for 30min to remove the solvent; dissolving in ethyl acetate, washing with water for three times, and removing excessive NHS contained in the solution; adding 10g of anhydrous sodium sulfate and drying overnight; removing anhydrous sodium sulfate by suction filtration by using a G6 sand core funnel; distilling the filtrate under reduced pressure to remove ethyl acetate contained therein; drying in a vacuum drying oven overnight to obtain stearic acid-NHS;

(4) 0.6500g of Polyethyleneimine (PEI) _1.3k Purchased from merck group, germany, used after freeze-drying) was dissolved in 20mL of boric acid buffer solution containing 8M urea (pH = 9.0) and stirred at room temperature until completely dissolved to obtain a PEI solution; 0.2573g of stearic acid-NHS prepared in step (3) was dissolved in 20mL THF and this solution was added to the PEI solution described above; stirring for 4h at room temperature to obtain a mixture solution;

(5) The mixture solution was dialyzed (MWCO: 1000 Da) overnight at 4 ℃ with a phosphate buffer (pH = 7.4) containing 150nM NaCl; adjusting the pH of the solution to 5.0 with 1M HCl; washing with diethyl ether for 5 times; dialyzing with ultrapure water (MWCO: 1000 Da) for 72h; freeze drying to obtain stearoyl polyethyleneimine (namely stearoyl PEI);

(6) 1 mu L of diethyl pyrocarbonate (DEPC) is added into 1mL of ultrapure water to obtain nuclease-free water;

(7) Adding 20 μ g siRNA (purchased from Ribo Biotechnology Ltd, guangzhou, cat: siN 0000001-1-5) into 500 μ L nuclease-free water prepared in step (6), adding 0.033mg stearoyl polyethyleneimine prepared in step (5), vortexing for 30s, and incubating at room temperature for 30min to obtain a gene-loaded vector core solution;

(8) Adding 0.2250g human serum albumin (HSA, purchased from Shanghai source leaf Biotechnology Co., ltd.) into the carrier core solution in the step (7), vortexing for 30s, and incubating at room temperature for 1h to obtain the human serum albumin-coated stearyl group-modified polyethyleneimine gene carrier carrying siRNA.

Example 5 preparation of mRNA-carrying human serum Albumin/Cholesterol polyethyleneimine Gene vectors

This example provides a non-viral gene vector containing human serum albumin/cholesterylated polyethyleneimine and capable of delivering mRNA, which is specifically prepared by the following method:

(1) 0.2250g cholesteryl chloroformate (available from Shanghai-source leaf Biotech, inc.), 0.2600g Polyethyleneimine (PEI) _1.3k Purchased from merck group of germany, used after freeze-drying treatment) were dissolved in 10mL of anhydrous chloroform, and cooled in an ice bath for 30min to obtain a cholesteryl chloroformate solution and a PEI solution, respectively; then adding 10 μ L of triethylamine (purchased from Shanghai Arlatin Biotechnology Co., ltd.) to the PEI solution to obtain a PEI solution containing triethylamine;

(2) Slowly dropping the cholesteryl chloroformate solution in the step (1) into the PEI solution containing triethylamine in an ice bath while stirring, and then continuously stirring the mixture solution in the ice bath for 30min and then stirring at room temperature for 6h;

(3) Carrying out rotary evaporation on the mixture solution prepared in the step (2) at 60 ℃ for 30min to remove anhydrous chloroform and triethylamine in the mixture solution; dialyzing with deionized water (MWCO: 1000 Da) for 48h; freeze drying to obtain cholesterol modified PEI _1.3k ；

(4) 1 mu L of diethyl pyrocarbonate (DEPC) is added into 1mL of ultrapure water to obtain nuclease-free water;

(5) Mu.g of mRNA (GenBank Accession No: X83959.1) was added to 500. Mu.L of the nuclease-free water of step (4), andadding 0.650mg of the cholesterol-modified PEI prepared in step (3) _1.3k Swirling for 30s, and incubating for 30min at room temperature to obtain a carrier inner core solution carrying the gene;

(6) Adding 9.750mg of human serum albumin (HSA, purchased from Shanghai leaf Biotechnology Co., ltd.) into the carrier inner core solution in the step (5), swirling for 30s, and incubating for 1h at room temperature to obtain the mRNA-loaded human serum albumin/cholesteryl polyethyleneimine gene carrier.

Example 6 characterization of non-viral Gene vectors based on bioaffinity

(1) Preparation of a non-viral gene vector based on bioaffinity: the specific preparation method is as described in example 1, example 2, example 3, example 4 and example 5; also referring to the preparation method described in example 4, a human serum albumin/polyethyleneimine non-affinity carrier loaded with siRNA was prepared as a control (designated as "non-affinity control" in this example) except that polyethyleneimine was used in the same molar amount as that of stearoyl polyethyleneimine.

(2) Particle size and Zeta potential measurement: the particle size and Zeta potential of the samples were measured using a Malvern Nano-particle size potentiometer (Malvern Zetasizer Nano ZS) at 25 ℃ and the results are given in table 1.

TABLE 1 characterization of non-viral gene vectors using bioaffinity (n = 3)

As can be seen from the above table 1, the particle size of the non-viral gene vector prepared by the invention and utilizing the bioaffinity effect is about 60nm, while the particle size of the comparative example is close to micron, so that the particle size of the non-viral gene vector prepared by the invention is obviously reduced, and the nano particles with small particle size are difficult to degrade in vivo and can enter the inside of cells more easily, and can realize the aggregation at the solid tumor part through the EPR effect; meanwhile, the coating of the protein shell reduces the dense positive charge on the surface of the naked core, and compared with the traditional cationic gene vector, the cytotoxicity caused by the dense positive charge on the surface is greatly reduced. In addition, the particle size of the non-affinity control group was as high as approximately 1 μm, confirming that affinity force is indispensable for the formation of the gene delivery system.

Example 7 evaluation of dilution stability of non-viral Gene vectors based on bioaffinity

The dilution stability of the non-viral gene vector with the bioaffinity effect is evaluated by a Malvern nano-particle size potentiometer, and the specific evaluation method is as follows:

(1) A human serum albumin/polyethyleneimine vector carrying siRNA was prepared as a control example (in this example, designated as "P @ HSA") according to the preparation method described in example 1, example 2, example 3, example 4 and example 5, except that a same molar amount of polyethyleneimine was used instead of stearoyl polyethyleneimine (non-viral gene vector (in this example, designated as "CP @ LDL", "SL @ HSA", "SPr @ HDL", "SP @ HSA", "CP @ HSA", "SP @ HSA") was prepared according to the preparation method described in example 4.

(2) The prepared complex solutions were diluted 5 and 20 times, respectively, and the particle size was measured at 25 ℃ using a Malvern Zetasizer Nano ZS.

The line of the particle size change of the two complex solutions after dilution is shown in FIG. 4.

And (4) analyzing results: as can be seen from FIG. 4, the particle size of the non-viral gene vector using bioaffinity prepared by the present invention does not significantly change before and after dilution, i.e., the dilution stability is good; whereas the control example (i.e. "p @ hsa") had a significant increase in particle size above 1000nm after 5-fold dilution and a further increase in particle size after 20-fold dilution, i.e. very poor dilution stability. Therefore, it can be proved that the non-viral gene vector constructed by the bioaffinity has more excellent stability.

Example 8 evaluation of in vivo targeting ability of non-viral Gene vectors based on bioaffinity

(1) Non-viral gene vectors (in this example) were prepared according to the preparation methods described in example 1, example 2, example 3, example 4 and example 5In turn named "CP @ LDL", "SL @ HSA", "SPr @ HDL", "SP @ HSA", "CP @ HSA"); wherein siRNA, pDNA and mRNA contained in the above non-viral gene vector are used ³² P (purchased from china isotope).

(2) 30 Mice (BALB/c Nude Mice, purchased from the Qinglongshan animal breeding farm) were each subcutaneously injected with 100. Mu.L of MDA-MB-231 cells (approximately 2X 10) ⁶ Individual cells, purchased from shanghai mexuan biotechnology limited). When the tumor volume reaches 50mm ³ In this case, 6 of each group were injected with 200. Mu.L of CP @ LDL, SL @ HSA, SPr @ HDL, SP @ HSA, and CP @ HSA via the tail vein. After 12h, the mouse heart, liver, spleen, lung, kidney, brain and tumor were taken, weighed, each tissue slurry (5 mg/mL) was obtained using a cryo-grinder (JXFSTPRP-I, touch electro-mechanical technologies, inc.), and subjected to cryocentrifugation at 12000rpm for 5min to obtain 500. Mu.L of each supernatant.

(3) 5g of 2, 5-diphenyloxazole and 0.2g of 1, 4-bis (5-phenyl-2-oxazole) benzene were dissolved in 1000mL of xylene to prepare a scintillation liquid. mu.L of each tissue supernatant obtained in step (2) was taken and added to a glass scintillation vial (M1152, available from Merck, germany) containing 5mL of scintillation fluid. The counting was performed using a liquid scintillation counter (LS 6500, beckmann coulter commercial limited).

In each group ³² The results of P counts/mg tissue are shown in FIG. 5.

And (4) analyzing results: due to the corresponding receptors (albumin receptor, LDL receptor, HDL receptor and the like) over-expressed on the surface of the breast cancer cells and the nano size of the prepared non-viral gene vector, the non-viral gene vector utilizing the bioaffinity effect prepared by the invention has good targeting capability on tumor tissues in vivo. Therefore, the non-viral gene vector constructed through the biological affinity effect has good targeting capacity on focus cells in vivo.

Example 9 evaluation of transfection Capacity of non-viral Gene vectors based on Bioaffinity Effect

The in vitro transfection capacity of the non-viral gene vector gene with the biological affinity effect is evaluated by a laser confocal microscope, and the specific operation method comprises the following steps:

(1) A non-viral gene vector comprising human serum albumin/stearylated polyethyleneimine and siRNA capable of silencing Green Fluorescent Protein (GFP) expression (available from Sharp Biotechnology Ltd, guangzhou, cathaki No. siP 0000005-1-5) was prepared according to the preparation method described in example 4;

(2) NIH-3T3-GFP cells (purchased from Shanghai Meixuan Biotech Co., ltd.) grown to 60% -80% in a culture flask were washed with PBS buffer at pH 7.0-7.4, dispersed with 1mL of 0.25% trypsin, and cultured at 1X 10 ⁵ Per cm ² The density of (2) was inoculated on the bottom of a confocal dish, 2mL of DMEM medium (purchased from Saimer Feishell Co., ltd.) containing 10% of inactivated FBS was added to each dish, 4 of the dishes were added with the gene-loaded vector core (named "SP" in this example) and the coated core (named "SP @ HSA" in this example) prepared as described above as experimental groups, and 2 of the dishes were added with Lipofectamine3000 reagent (purchased from Saimer Feishell Co., ltd.) and DMEM medium as a positive Control (named Lipo3000 in this example) and a negative Control (named Control in this example) at 37 ℃ and 5 CO, respectively ₂ Keeping the temperature for 24 hours under the condition;

(3) The green fluorescence intensity expressed by NIH-3T3-GFP cells is detected by a laser confocal microscope, and the in-vitro gene transfection capacity of the gene vector is evaluated in a comparative way.

And (3) test results: FIG. 6 shows fluorescence results obtained by laser confocal microscopy after transfection of the gene vector. As can be seen from FIG. 6, the SP group and the SP @ HSA group have significantly reduced green fluorescence compared to the Control group, and are weaker than the Lipo3000 group, i.e., the SP group and the SP @ HSA group have stronger gene silencing effect than the Lipo3000 group, indicating that the SP and the SP @ HSA have excellent in vitro gene transfection ability.

Similarly, the non-viral gene vector containing low-density lipoprotein/cholesterylated polyethyleneimine and siRNA (designated as "CP @ LDL") prepared in example 1 and the non-viral gene vector containing only the vector core (designated as "CP") and its complex with siRNA were evaluated for their in vitro transfection ability in accordance with the above-described method. FIG. 7 shows fluorescence results obtained by confocal laser scanning microscopy after transfection of the gene vector. As can be seen from FIG. 7, the CP group and CP @ LDL group had significantly reduced green fluorescence compared to Control group and was weaker than Lipo3000 group, i.e., the CP group and CP @ LDL group had stronger gene silencing effect than Lipo3000 group, indicating that CP and CP @ LDL had good in vitro gene transfection ability.

Similarly, the in vitro transfection ability of the non-viral gene vector containing human serum albumin/cholesterylated polyethyleneimine and mRNA (designated "CP @ HSA") prepared in example 5 and the non-viral gene vector containing only the vector core (containing mRNA therein, the vector designated "CP") was evaluated according to the above method. The difference from the above process is that the gene used was mRNA allowing the cells to express green fluorescent protein (GenBank Accession: X83959.1), and the cells used were 4T1 cells (purchased from Shanghai Meixuan Biotech Co., ltd.) and human umbilical cord mesenchymal stem cells (purchased from Qiao Xin boat Biotech Co., ltd. In Shanghai). The fluorescence result obtained by the gene vector through a laser confocal microscope after transfecting 4T1 cells is shown in figure 8, and the fluorescence result obtained by an inverted fluorescence microscope after transfecting human umbilical cord mesenchymal stem cells is shown in figure 9. As can be seen from FIGS. 8 and 9, the CP group and CP @ HSA group had significant green fluorescence expression compared to the Control group and were stronger than the Lipo3000 group, i.e., the CP group and CP @ HSA group had stronger gene expression than the Lipo3000 group, indicating that CP and CP @ HSA had good in vitro gene transfection ability and that the vector was able to transfect genes into stem cells well.

The non-viral gene vector gene in-vitro transfection capacity with the biological affinity effect is evaluated by qRT-PCR, and the specific operation method is as follows:

(1) According to the preparation method described in example 2, a non-viral gene vector comprising human serum albumin/stearylated polylysine and a complex thereof with pDNA capable of highly expressing hyaluronidase (GenBank Accession: AF 069741.1) was prepared;

(2) M14 cells (purchased from Shanghai Meixuan Biotech Co., ltd.) grown to 60% -80% in the culture flask were washed with PBS buffer solution having pH of 7.0 to 7.4,dispersed with 1mL of 0.25% trypsin at 2X 10 ⁵ Each well was inoculated with 6-well plates at a density of 2mL of DMEM medium (purchased from Saimer Feishell technology Co., ltd.) containing 10% inactivated FBS, 2 wells were each charged with the gene-loaded vector core (named "SL" in this example) and the coated core (named "SL @ HSA" in this example) prepared as described above as an experimental group, and the other 2 wells were each charged with Lipofectamine3000 reagent (purchased from Saimer Feishell technology Co., ltd.) and DMEM medium as a positive Control (named "Lipo3000" in this example) and a negative Control (named "Control" in this example) at 37 ℃ and 5 CO at 5 ℃ to prepare a drug ₂ Keeping the temperature for 24 hours under the condition;

(3) Total RNA was extracted from M14 cells using RNAqueous micro-extraction Total RNA isolation kit (purchased from Seimer Feishell technologies, ltd.). After reverse transcription, the expression of hyaluronidase was detected with a real-time fluorescent quantitative PCR system (7300 Plus, siemer feishel technologies ltd) using a PowerTrack SYBR Green Master Mix kit (purchased from siemer feishel technologies ltd). Wherein the qRT-PCR amplification conditions are as follows: circulating for 1 time at 95 ℃ for 15 min; circulating for 40 times at 95 deg.C 10s and 65 deg.C 30 s. The sequence of the amplification primer of the hyaluronidase is as follows: SEQ ID NO:1:5'-CGA TAT GGC CCA AGG CTT TAG-3' (sense strand), and SEQ ID NO:2:5'-ACC ACA TCG AAG ACA CTG ACA T-3' (antisense strand).

Similarly, the non-viral gene vector (named "SPr hdl") comprising high-density lipoprotein/stearylated protamine and pDNA prepared in example 3 and the non-viral gene vector (named "SPr") comprising only the vector core and its complex with pDNA were evaluated for their transfection ability in vitro according to the above-described methods.

And (3) test results: the qRT-PCR results obtained after transfection of the above two gene vectors are shown in FIG. 10. As can be seen from FIG. 10, compared with the Control group, the SL @ HSA group, the SPr group, and the SPr @ HDL group significantly increased the hyaluronidase mRNA expression, and the SL @ HSA group and the SPr @ HDL group were close in effect to the Lipo3000 group, indicating that SL @ HSA and SPr @ HDL had excellent in vitro gene transfection ability.

Example 10 evaluation of transfection Capacity of non-viral Gene vectors based on Bioaffinity

The in vivo transfection capability of the non-viral gene vector gene with the bioaffinity effect is evaluated by a mouse breast cancer model, and the specific operation method comprises the following steps:

(1) Non-viral gene vectors (designated "CP @ LDL", "SP @ HSA" in this example) were prepared according to the production methods described in examples 1 and 4, and gene-loaded vector cores (designated "CP", "SP" in this example) were prepared similarly with reference to the above-described methods; as a positive control, a Lipofectamine3000 reagent (purchased from seimer feishell science co., ltd) was used to co-incubate with siRNA according to the corresponding instructions (designated as "Lipo3000" in this example); as a negative Control (designated as "Control" in this example), 0.9% sterile physiological saline (purchased from beijing maireida technologies ltd.) was used; wherein, the siRNA used in the method can silence survivin (survivin) expression (purchased from Ribo Biotechnology Ltd, guangzhou, cat # siB 150723094012-1-5).

(2) 36 Mice (BALB/c Nude Mice, purchased from the Qinglongshan animal breeding farm) were each subcutaneously injected with 100. Mu.L of MDA-MB-231 cells (approximately 2X 10) ⁶ Individual cells, purchased from shanghai mexuan biotechnology limited). When the tumor volume reaches 50mm ³ When the composition is used, 6 per group are respectively injected with 200 μ L of CP @ LDL, SP @ HSA, CP, SP, lipo3000 and 0.9% physiological saline through tail vein, and injected once every three days for four times, during which, the tumor volume and the mouse weight are monitored, and the animal welfare requirements are followed, when the tumor volume exceeds 1200mm ³ Mice were considered dead and sacrificed.

(3) The in vivo gene transfection capability of the gene vector is evaluated by comparing a tumor volume line graph and a mouse survival curve graph which are drawn by measuring the tumor volume and the body weight of each group of mice every day.

And (3) test results: the line graphs of tumor volume changes of the mice in each group are shown in FIG. 11; the survival graphs of the mice in each group are shown in FIG. 12. As can be seen from FIG. 11, the non-viral gene vector prepared by the present invention has a more significant effect of inhibiting tumor volume growth than positive control Lipo3000, further slows down tumor growth after coating with protein coat, and does not easily degrade in vivo due to the protein coat, and has a longer time to exert anti-tumor effect. In addition, as can be seen from fig. 12, the non-viral gene vector prepared by the present invention was less toxic, had less effect on the survival of mice than the positive control Lipo3000, and had further reduced effect on the survival of mice after the outer coat of protein was coated. The above results indicate that the non-viral gene vector prepared by the present invention has good in vivo transfection ability and biosafety.

Claims (12)

1. A non-viral gene vector based on bioaffinity, wherein said non-viral gene vector comprises (i) a vector core, and (ii) a vector sheath;

wherein the carrier inner core is composed of cationic materials modified by lipid substances and nucleic acid molecules, and the carrier outer shell is composed of proteins with bioaffinity effect on the lipid substances in the carrier inner core.

2. The non-viral gene vector according to claim 1, wherein the lipid substance is any one selected from the group consisting of: fatty acids (esters), glycerolipids, phospholipids, glycolipids, cholesterol esters, cholesterol, bile acids, vitamin D, and structural analogs and derivatives thereof;

preferably, the lipid substance is any one of cholesterol or stearic acid.

3. The non-viral gene vector according to claim 1 or 2, wherein the cationic material comprises an organic cation-like material and an inorganic cation-like material;

preferably, 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 cation-like material is selected from any one of the group consisting of aluminum hydroxide, ferric hydroxide, hollow mesoporous silica and calcium ions;

more preferably, the cationic material is any one selected from the group consisting of polyethyleneimine, polylysine and protamine.

4. The non-viral gene vector according to any one of claims 1 to 3, wherein the nucleic acid molecule comprises DNA, RNA, and hybrids thereof;

preferably, 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, microrna, multivalent RNA, viral RNA (vRNA), and CRISPR RNA sequences;

more preferably, the nucleic acid molecule is any one of pDNA, siRNA or mRNA.

5. The non-viral gene vector according to any one of claims 1 to 4, wherein the protein in the vector coat has passive or active targeting to the cell of interest and is selected from any one of the group consisting of albumin, apolipoprotein, very low density lipoprotein, medium density lipoprotein, high density lipoprotein and their structural analogues and derivatives;

preferably, the protein is any one of low density lipoprotein, serum albumin, or high density lipoprotein.

6. The non-viral gene vector according to any one of claims 1 to 5, wherein the mass ratio of the vector shell to the vector core is 0.1 to 1000, the molar ratio of lipid substance to the cationic material in the vector core is 0.1 to 100000, and the mass ratio of the lipid substance-modified cationic material to the nucleic acid molecule is 0.1 to 10000;

preferably, the molar ratio of the carrier shell to the carrier inner core is 1:1-50, the molar ratio of the lipid substance to the cationic material in the carrier inner core is 0.1-30000.

7. The non-viral gene vector according to claim 1 to 6, wherein the particle diameter of the non-viral gene vector is 10 to 1000nm, and the Zeta potential of the non-viral gene vector is-25 to 25mV;

preferably, the particle size of the non-viral gene vector is 30-200 nm, and the Zeta potential of the non-viral gene vector is-20 to-2 mV;

more preferably, the particle size of the non-viral gene vector is 35 to 130nm, and the Zeta potential of the non-viral gene vector is-12 to-4 mV.

8. The method for producing a non-viral gene vector according to any one of claims 1 to 7, comprising the steps of:

1) Chemically coupling the lipid substance and the cationic material to obtain the cationic material modified by the lipid substance;

2) Mixing nucleic acid molecules with the cationic material modified by the lipid substance prepared in the step 1) to obtain a carrier core carrying the nucleic acid molecules;

3) Mixing the nucleic acid molecule-loaded carrier core prepared in step 2) with a protein, and wherein the protein has an affinity for the lipid material in the carrier core.

9. The production method according to claim 8, wherein the molar ratio of the lipid substance to the cationic material in step (1) is from 0.1; the mass ratio of the cationic material modified by the lipid substance to the nucleic acid molecule in the step (2) is 0.1; the mass ratio of the nucleic acid molecule-carrying vector kernel to the protein in the step (3) is 0.1.

10. The production method according to claim 8 or 9, wherein the solvent used in the 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-dimethylformamide, carbon tetrachloride, anhydrous chloroform, dichloromethane, 1,4-dioxane, dimethyl sulfoxide, ethyl acetate, butyl acetate, tetrahydrofuran and diethyl ether.

11. Use of the non-viral gene vector according to any one of claims 1 to 7 for the preparation of a medicament for the prevention or treatment of a disease; preferably, the disease includes genetic disease, cancer, immune disease, inflammation, cardiovascular disease, infectious disease; more preferably, the disease is cancer.

12. Use of the non-viral gene vector according to any one of claims 1 to 7 for the preparation of a cell preparation, wherein the non-viral gene vector is capable of transfecting cells efficiently in vitro and the obtained cell preparation is useful for cell therapy; preferably, the cell preparation comprises a stem cell preparation, an erythrocyte 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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