Method for enhancing nucleic acid delivery efficiency of polyethyleneimine by using free amino acid

Abstract

Provided is a method for enhancing the nucleic acid delivery efficiency of polyethyleneimine by using a free amino acid, which method comprises contacting cells with polyethyleneimine and a nucleic acid to be delivered in the presence of a free amino acid, so as to carry out transfection, thereby delivering the nucleic acid into the cells. By means of the method, the transfection efficiency of polyethyleneimine can be significantly improved without increasing cytotoxicity.

Description

A method for enhancing the efficiency of nucleic acid delivery by polyethyleneimine using free amino acids Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a method for enhancing the efficiency of polyethyleneimine in delivering nucleic acids by using free amino acids. Background Art

[0002] As a common technical means, nucleic acid delivery has been widely used in scenarios such as gene therapy, biological preparation production and research. Nucleic acid delivery vectors are mainly divided into viral vectors and non-viral vectors. Viral vectors perform well in gene delivery, but they also have some disadvantages, such as limited DNA packaging capacity, difficulty in vector production, and safety issues such as mutations caused by viral insertion, uncontrolled immunogenicity and inflammatory response. These shortcomings seriously limit their application. Non-viral vectors use liposomes or cationic polymer complexes to encapsulate nucleic acids (DNA and / or RNA molecules) to form nanocomplexes. They can spontaneously compress nucleic acid molecules and protect them from degradation by DNase / RNase, and the resulting nanocomplexes can enter cells through endocytosis / phagocytosis pathways or direct fusion / penetration of the plasma membrane. Non-viral vectors have lower immunogenicity and have the potential to overcome many limitations of viral vectors, especially safety issues.

[0003] Currently, the most common nucleic acid delivery carriers are mainly the following categories: liposomes (including ordinary double-layer cationic lipids, liposomes DOTAP, Polyplus, Lipofectamine, Lipofectine, etc.), cationic polymer complexes [such as polyethyleneimine (PEI), polyamide-amine (PAMAM)), etc.], dendrimers (DEN) and various polymer microparticles (such as polycyanoacrylate nanoparticles, polylactic acid nanoparticles, etc.), ligand-mediated nucleic acid molecule targeted delivery systems and polypeptide protein delivery carriers based on specific receptors, and nanoparticles with active targeting effects modified with targeting molecules such as sialic acid proteins, polysaccharides, epidermal growth factor, folic acid, transferrin, steroid hormones, monoclonal antibodies, etc.

[0004] Cationic liposomes have excellent transfection efficiency but are very expensive and highly cytotoxic. Compared to cationic liposomes, cationic polymers offer higher transfection efficiency and lower cost, but are also more cytotoxic. Since 1995, polyethylenimine (PEI) has become the most widely used cationic polymer for nucleic acid delivery in vitro and in vivo. Its molecular weight, degree of polymerization, molecular size, and formulation method can all affect transfection efficiency, with 25kDa PEI solution being the most widely used cationic polymer transfection reagent.

[0005] The advantage of PEI in delivering nucleic acids is that it can bind and protect nucleic acid molecules and provide proton buffering capacity for endosome escape. However, when the high molecular weight or high N / P ratio of PEI (N is the free amino group in PEI, P is the phosphate group in DNA / RNA, and N / P refers to the ratio of the molar number of the two) is used to deliver nucleic acids to mammalian cells, due to its cationic properties and proton sponge effect, it can avoid being degraded by the endosome, making PEI have higher cytotoxicity. In order to solve this problem, the methods generally used include: using relatively low molecular weight PEI, such as PEI with a molecular weight of about 25kDa, or modifying PEI (such as coupling PEG). The former still has certain toxicity, and the latter has increased difficulty in synthesis and higher cost.

[0006] Summary of the Invention

[0007] Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. Experimental methods not specifically described in the present invention are performed according to the specific methods in the book "Molecular Cloning Laboratory Manual" (4th edition) by J. Sambrook, or in accordance with the instructions of the relevant products. The biological reagents used in the present invention, unless otherwise specified, can be obtained from commercial channels. Those skilled in the art can make various changes, modifications and substitutions without departing from the spirit of the present invention.

[0008] The present invention provides a method for enhancing the efficiency of nucleic acid delivery via polyethyleneimine using free amino acids, significantly improving the transfection efficiency of polyethyleneimine without increasing cytotoxicity. The method is simple, easy to implement, highly safe, and low-cost.

[0009] In one aspect, the present invention provides a method for delivering a nucleic acid to a cell, comprising: contacting polyethyleneimine, a nucleic acid to be delivered, and the cell in the presence of free amino acids, wherein the amino acids are selected from one or more of histidine, lysine, glycine, arginine, alanine, proline, leucine, glutamine, isoleucine, phenylalanine, methionine, tyrosine, cysteine, serine, tryptophan, and threonine, preferably one or more of histidine, glycine, alanine, proline, and threonine.

[0010] As used herein, "free amino acid" refers to the amino acid present in a free state in the medium in which the PEI, the nucleic acid to be delivered, and the cells are in contact. When amino acids are mentioned herein, unless explicitly excluded or can be excluded based on the context, they include the acid and its soluble salt form in the medium, for example, including but not limited to sodium, potassium, and magnesium salts. In some embodiments, the medium described herein is an aqueous medium, such as a solution or culture medium for cell nucleic acid transfection.

[0011] In one embodiment, the amino acid is selected from one or more of histidine, glycine, alanine, proline, serine, threonine, arginine, lysine and water-soluble salts thereof, preferably one or more of histidine, glycine, alanine, proline, threonine and water-soluble salts thereof.

[0012] The concentration of the amino acid in the medium can be readily determined by one skilled in the art. In one embodiment, the concentration of the amino acid in the medium is about 1-25 mmol / L, for example, about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-25, 9-25, 10-25, -20, 10-20, 10-20, 1-15, 1.25-15, 2-15, 2.5-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, for example about 2.5-20 or 5-10 mmol / L, such as about 5, 10 or 20 mmol / L.

[0013] As used herein, "nucleic acid" refers to a nucleotide polymer connected by 3'-5'-phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The nucleic acid includes single-stranded or double-stranded, linear or circular ribonucleic acids (RNA) composed of ribonucleotides (adenine ribonucleotides (A), guanine ribonucleotides (G), cytosine ribonucleotides (C), uracil ribonucleotides (U)) and deoxyribonucleic acids (DNA) composed of deoxyribonucleotides (deoxyadenine ribonucleotides (A), deoxyguanine ribonucleotides (G), deoxycytosine ribonucleotides (C), deoxythymidine ribonucleotides (T)). The nucleic acid may include any desired modifications, such as methylation, thiolation, glycosylation, etc.

[0014] Herein, the nucleic acid to be delivered refers to a nucleic acid that needs to be transferred into target cells, such as a construct containing a transgene, such as an expression vector or a transfer vector for gene recombination.

[0015] In one embodiment, the nucleic acid comprises DNA and / or RNA. In one embodiment, when the nucleic acid is DNA, the amino acid is selected from one or more of histidine, glycine, alanine, proline, serine, threonine, arginine and lysine, preferably selected from one or more of histidine, glycine, alanine, proline and threonine. In one embodiment, when the nucleic acid is RNA, the amino acid is selected from one or more of histidine, glycine, alanine, proline and threonine. In one embodiment, the DNA comprises plasmid DNA. In one embodiment, the RNA comprises mRNA.

[0016] As used herein, "delivery" refers to the process of using PEI as a delivery vector to allow the desired nucleic acid to pass through the cell membrane into the cytoplasm or into the nucleus. The conditions, steps, and conventional reagents required for delivering nucleic acids to cells using PEI as a delivery vector are known in the art.

[0017] In one embodiment, the delivering is performed in an aqueous medium, such as a solution or culture medium.

[0018] In one embodiment, when delivery is in an aqueous medium and the nucleic acid is DNA, the amino acid is selected from one or more of histidine, glycine, alanine, proline, serine, threonine, arginine, lysine, and water-soluble salts thereof.

[0019] In one embodiment, when delivery is in an aqueous medium and the nucleic acid is RNA, the amino acid is selected from one or more of histidine, glycine, alanine, proline, serine, threonine, arginine, lysine, and water-soluble salts thereof.

[0020] As used herein, "cell" is any cell to which nucleic acid can be delivered, including primary cells or cell lines. Numerous cells known in the art to which nucleic acid can be delivered include, but are not limited to, eukaryotic cells, such as animals (such as non-human mammals), insect cells.

[0021] In one embodiment, the cell is a human cell or cell line, including, for example, the cell is selected from eukaryotic cells, such as animal (such as non-human mammals), insect cells, human cells or cell lines, such as HEK-293 cells (ATCC No.: CCL-1573) and their derivatives, such as HEK-293T cells (ATCC No.: CRL-3216), N2a cells (Neuro-2a, ATCC No.: CCL-131) and CHO cells (ATCC No.: CCL-61) and their derivatives.

[0022] In one embodiment, the cells are transfected with the PEI and the nucleic acid to be delivered in the presence of the free amino acid for no more than 48 hours, such as no more than 36 hours, no more than 24 hours, no more than 12 hours, preferably no more than 24 hours.

[0023] Before nucleic acid delivery, cells are cultured to an appropriate confluence density, for example, 70% to 80%. Culture conditions, culture media, and additives for specific cells are well known in the art.

[0024] As used herein, "polyethyleneimine" (PEI, CAS: 9002-98-6) is a cationic polymer known in the art for nucleic acid delivery. PEI contains primary, secondary, and tertiary amine groups in its molecular structure and has high chemical reactivity.

[0025] In one embodiment, the average molecular weight (weight average molecular weight) of PEI described herein is about 20-30kDa, for example, about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30kDa, preferably about 25kDa. In particular, the weight average molecular weight is, for example, measured by LS method.

[0026] In one embodiment, the average molecular weight (number average molecular weight) of PEI described herein is about 8-12 kDa, such as about 8, 9, 10, 11 or 12 kDa, preferably about 10 kDa. In particular, the number average molecular weight is measured, for example, by GPC method.

[0027] As used herein, "contacting" refers to physical contact of the referenced substances, for example, the free amino acids, PEI, nucleic acid to be delivered and cells are in the same medium such as aqueous solution or culture medium and thus can contact each other.

[0028] Herein, in the nucleic acid delivery system, the ratio of the nucleic acid to be delivered to PEI can be adjusted according to the technical knowledge in the art, for example, the weight ratio can be about 1:5 to about 5:1, such as about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1 or about 5:1, preferably about 1:1.

[0029] Herein, in the nucleic acid delivery system, the free amino acid is present at a concentration of about 1-25 mmol / L, for example, about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 1-15, 1.25-15, 2-15, 2.5-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, for example about 2.5-20 or 5-10 mmol / L, such as about 5, 10 or 20 mmol / L.

[0030] In one embodiment, the solution for delivering nucleic acid has a ratio of nucleic acid to be delivered: free amino acids present: PEI of 1 μg nucleic acid: 0.5-2×10 -2 mmol amino acid: 1-3 μg PEI.

[0031] In one embodiment, the present invention provides a method for delivering a nucleic acid to a cell, comprising: mixing PEI, a nucleic acid to be delivered, and the free amino acid of the present invention, and then adding the mixture to a cell to deliver the nucleic acid into the cell.

[0032] In one embodiment, the present invention provides a method for enhancing the delivery of nucleic acids by PEI using free amino acids, comprising adding a certain concentration of amino acids to a mixture of PEI and the nucleic acid to be delivered (e.g., when delivering DNA, the preferred amino acids are histidine, glycine, alanine, proline, and / or threonine; when delivering RNA, histidine, glycine, alanine, proline and / or threonine are preferred), preferably after premixing for 20-40 minutes, adding the cell culture medium for transfection to deliver the nucleic acid into the cells.

[0033] In a further embodiment, the present invention provides a method for improving the efficiency of nucleic acid delivery by PEI using free amino acids, comprising:

[0034] (1) Cultivating cells (e.g., HEK-293T cells, N2a cells, or CHO cells) to an appropriate confluence density, e.g., 70%-80% confluence density and / or performing the following operations within 24 hours after adherence;

[0035] (2) Mix the amino acid, PEI, and the nucleic acid to be delivered and let it stand at room temperature for a period of time, for example, 20-40 minutes;

[0036] (3) Remove the cell culture medium, then add an equal volume of fresh culture medium and the mixed solution obtained in step (2), and continue culturing for 24 hours or 48 hours.

[0037] In one aspect, the present invention provides a method for delivering DNA to a cell, comprising: contacting polyethyleneimine, the DNA to be delivered, and the cell in the presence of free amino acids, wherein the amino acids are selected from one or more of histidine, lysine, glycine, arginine, alanine, proline, leucine, glutamine, isoleucine, phenylalanine, methionine, tyrosine, cysteine, serine, tryptophan, and threonine, preferably selected from one or more of histidine, glycine, alanine, proline, and threonine, and more preferably selected from one or more of glycine, alanine, and threonine.

[0038] In one embodiment, the amino acid is present at a concentration of about 1-25 mmol / L, for example, about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-2 0, 10-20, 1-15, 1.25-15, 2-15, 2.5-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, for example about 2.5-20 or 5-10 mmol / L, such as about 5, 10 or 20 mmol / L.

[0039] In one embodiment, the delivery is carried out in an aqueous medium such as a solution or culture medium. In one embodiment, when the delivery is carried out in an aqueous medium and the nucleic acid is DNA, the amino acid is selected from one or more of histidine, glycine, alanine, proline, serine, threonine, arginine, lysine and water-soluble salts thereof.

[0040] In one embodiment, the histidine is present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 2.5-20 mmol / L, more preferably about 10 mmol / L.

[0041] In one embodiment, the glycine is present at a concentration of about 1-25 mmol / L, e.g., about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10, or 5-10 mmol / L, preferably about 2.5-20 mmol / L, more preferably about 10 or 20 mmol / L, and most preferably about 20 mmol / L.

[0042] In one embodiment, the alanine is present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 20 mmol / L.

[0043] In one embodiment, the proline is present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 20 mmol / L.

[0044] In one embodiment, the serine is present at a concentration of about 1-25 mmol / L, for example about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 10 or 20 mmol / L, and most preferably about 10 mmol / L.

[0045] In one embodiment, the threonine is present at a concentration of about 1-25 mmol / L, for example about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 10 or 20 mmol / L, and most preferably about 10 mmol / L.

[0046] In one embodiment, the arginine is present at a concentration of about 1-25 mmol / L, e.g., about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10, or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 10 or 20 mmol / L, and most preferably about 20 mmol / L.

[0047] In one embodiment, the lysine is present at a concentration of about 1-25 mmol / L, for example about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 5-25 mmol / L, more preferably about 20 mmol / L.

[0048] In one embodiment, the cell is a HEK-293T cell. In one embodiment, the cell is transfected with the PEI and the DNA to be delivered in the presence of one or more free amino acids selected from histidine, glycine, alanine, proline, serine, threonine, arginine and lysine.

[0049] In one embodiment, the cell is an N2a cell and its derivatives, and wherein the free amino acid is one or more of histidine, glycine, alanine, proline, serine, threonine, arginine and lysine, preferably threonine. Preferably, the threonine is present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 10 or 20 mmol / L, and most preferably about 10 mmol / L.

[0050] In one embodiment, the cell is a CHO cell and its derivatives, and wherein the free amino acid is one or more of histidine, glycine, alanine, proline, serine, threonine, arginine and lysine, preferably one or more of glycine, alanine and threonine. Preferably, the glycine, alanine and / or threonine are present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 10 or 20 mmol / L, most preferably about 10 mmol / L.

[0051] In one aspect, the present invention provides a method for delivering RNA to a cell, comprising: contacting polyethyleneimine, the RNA to be delivered, and the cell in the presence of free amino acids, wherein the amino acids are selected from one or more of histidine, glycine, alanine, proline, serine, threonine, arginine, and lysine, preferably selected from one or more of histidine, glycine, alanine, proline, and threonine, and more preferably selected from one or more of glycine, alanine, and proline.

[0052] In one embodiment, the amino acid is present at a concentration of about 1-25 mmol / L, e.g., about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, or 5-10 mmol / L, such as about 5, 10 or 20 mmol / L.

[0053] In one embodiment, the histidine is present at a concentration of about 1-25 mmol / L, for example about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 5-10 mmol / L, more preferably about 5 mmol / L.

[0054] In one embodiment, the glycine is present at a concentration of about 1-25 mmol / L, e.g., about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 1-10, 1.25-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 1.25 or 5 mmol / L.

[0055] In one embodiment, the alanine is present at a concentration of about 1-25 mmol / L, for example, about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 1-10, 1.25-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 5-20 mmol / L, more preferably about 5 or 20 mmol / L, and most preferably about 5 mmol / L.

[0056] In one embodiment, the proline is present at a concentration of about 1-25 mmol / L, for example, about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 1-10, 1.25-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 2.5-20 mmol / L, more preferably about 5 or 10 mmol / L, and most preferably about 10 mmol / L.

[0057] In one embodiment, the threonine is present at a concentration of about 1-25 mmol / L, for example, about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 1-10, 1.25-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 1-20 or 2.5-10 mmol / L, more preferably about 2.5 or 5 mmol / L.

[0058] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the delivery is carried out in an aqueous medium and the nucleic acid is RNA, preferably, the amino acid is selected from one or more of histidine, glycine, alanine, proline, threonine and water-soluble salts thereof.

[0059] In one embodiment, the cell is selected from eukaryotic cells, such as animal (such as non-human mammals), insect cells, human cells or cell lines, for example, HEK-293 cells (ATCC No.: CCL-1573) and its derivatives, such as HEK-293T cells (ATCC No.: CRL-3216), N2a cells (Neuro-2a, ATCC No.: CCL-131) and CHO cells (ATCC No.: CCL-61) and their derivatives.

[0060] In one embodiment, the cell is a HEK-293T cell. In one embodiment, the cell is transfected with the PEI and the RNA to be delivered in the presence of one or more free amino acids selected from histidine, glycine, alanine, proline, serine and threonine. Preferably, the concentration of the amino acid present is about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 1.25, 2.5, 5, 10 or 20 mmol / L, most preferably about 10 mmol / L.

[0061] In one embodiment, the cell is an N2a cell and its derivatives, and wherein the free amino acid is one or more of histidine, glycine, alanine, proline, serine, threonine, arginine and lysine, preferably histidine, glycine, alanine, proline and threonine. Preferably, the amino acid is present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 10 or 20 mmol / L, and most preferably about 10 mmol / L.

[0062] In one embodiment, the cell is a CHO cell and its derivatives, and wherein the free amino acid is one or more of histidine, glycine, alanine, proline and threonine, preferably one or more of glycine, alanine and proline. Preferably, the glycine, alanine and / or proline are present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 10 or 20 mmol / L, most preferably about 10 mmol / L.

[0063] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of free amino acid histidine.

[0064] In one embodiment, the histidine is present at a concentration of about 1-25 mmol / L, for example, about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-2 0, 10-20, 1-15, 1.25-15, 2-15, 2.5-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, for example about 2.5-20 or 5-10 mmol / L, such as about 5, 10 or 20 mmol / L.

[0065] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of histidine and a water-soluble salt thereof.

[0066] In one embodiment, when the nucleic acid is DNA, the histidine is present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 2.5-20 mmol / L, more preferably about 10 mmol / L.

[0067] In one embodiment, when the nucleic acid is RNA, the histidine is present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 5-10 mmol / L, more preferably about 5 mmol / L.

[0068] In one embodiment, the present invention provides a method for delivering nucleic acid DNA to a cell, comprising: contacting polyethyleneimine, the DNA to be delivered, and the cell in the presence of free amino acid lysine.

[0069] In one embodiment, the lysine is present at a concentration of about 1-25 mmol / L, for example about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 5-25 mmol / L, more preferably about 20 mmol / L.

[0070] In one embodiment, the delivery is carried out in an aqueous medium such as a solution or culture medium. In one embodiment, when the nucleic acid DNA is delivered in an aqueous medium, the amino acid is selected from one or more of lysine and its water-soluble salts.

[0071] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of the free amino acid glycine.

[0072] In one embodiment, the glycine is present at a concentration of about 1-25 mmol / L, e.g., about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-2 0, 10-20, 1-15, 1.25-15, 2-15, 2.5-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, for example about 2.5-20 or 5-10 mmol / L, preferably about 5, 10 or 20 mmol / L.

[0073] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of glycine and a water-soluble salt thereof.

[0074] In one embodiment, when the nucleic acid is DNA, the glycine is present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 2.5-20 mmol / L, more preferably about 10 or 20 mmol / L, and most preferably about 20 mmol / L.

[0075] In one embodiment, when the nucleic acid is RNA, the glycine is present at a concentration of about 1-25 mmol / L, e.g., about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 1-10, 1.25-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 5 mmol / L.

[0076] In one embodiment, the present invention provides a method for delivering nucleic acid DNA to a cell, comprising: contacting polyethyleneimine, the DNA to be delivered, and the cell in the presence of free amino acid arginine.

[0077] In one embodiment, the arginine is present at a concentration of about 1-25 mmol / L, e.g., about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10, or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 10 or 20 mmol / L, and most preferably about 20 mmol / L.

[0078] In one embodiment, the delivery is carried out in an aqueous medium such as a solution or culture medium. In one embodiment, when the nucleic acid DNA is delivered in an aqueous medium, the amino acid is selected from one or more of arginine and its water-soluble salts.

[0079] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of the free amino acid alanine.

[0080] In one embodiment, the alanine is present at a concentration of about 1-25 mmol / L, for example, about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-2 0, 10-20, 1-15, 1.25-15, 2-15, 2.5-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, for example about 2.5-20 or 5-10 mmol / L, preferably about 5, 10 or 20 mmol / L.

[0081] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of alanine and a water-soluble salt thereof.

[0082] In one embodiment, when the nucleic acid is DNA, the alanine is present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 20 mmol / L.

[0083] In one embodiment, when the nucleic acid is RNA, the alanine is present at a concentration of about 1-25 mmol / L, such as about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 1-10, 1.25-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 5-20 mmol / L, more preferably about 5 or 20 mmol / L, and most preferably about 5 mmol / L.

[0084] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of free amino acid proline.

[0085] In one embodiment, the proline is present at a concentration of about 1-25 mmol / L, e.g., 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20 , 10-20, 1-15, 1.25-15, 2-15, 2.5-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, for example, about 2.5-20 or 5-10 mmol / L, preferably about 5, 10 or 20 mmol / L.

[0086] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of proline and a water-soluble salt thereof.

[0087] In one embodiment, when the nucleic acid is DNA, the proline is present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 20 mmol / L.

[0088] In one embodiment, when the nucleic acid is RNA, the proline is present at a concentration of about 1-25 mmol / L, such as about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 1-10, 1.25-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 2.5-20 mmol / L, more preferably about 5 or 10 mmol / L, and most preferably about 10 mmol / L.

[0089] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of free amino acid leucine.

[0090] In one embodiment, the leucine is present at a concentration of about 1-25 mmol / L, e.g., about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-2 0, 10-20, 1-15, 1.25-15, 2-15, 2.5-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, for example about 2.5-20 or 5-10 mmol / L, such as about 5, 10 or 20 mmol / L.

[0091] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of leucine and a water-soluble salt thereof.

[0092] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of free amino acid glutamine.

[0093] In one embodiment, the glutamine is present at a concentration of about 1-25 mmol / L, for example, about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-25, 10, 15, 20, 100, 200, 150, 300, 400, 500, 600, 700, 800, 900, 1000, 200, 1000, 3000, 4000, 800, 900, 1000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 2000, 10000, 3000, 4000, 8000, 9000, 10000, 2000, 10000, 30000, 40000, 8000, 9000, 10000, 20000, 10000, 30000, 40000, 8000

[0094] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of glutamine and its water-soluble salts.

[0095] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of free amino acid isoleucine.

[0096] In one embodiment, the isoleucine is present at a concentration of about 1-25 mmol / L, e.g., about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-25, 10, 15, 20, 100, 200, 150, 300, 400, 500, 600, 700, 800, 900, 1000, 200, 1000, 3000, 4000, 800, 900, 1000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 2000, 10000, 3000, 4000, 8000, 9000, 10000, 2000, 10000, 30000, 40000, 8000, 9000, 10000, 20000, 10000, 30000, 40000, 8000

[0097] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of isoleucine and a water-soluble salt thereof.

[0098] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of free amino acid phenylalanine.

[0099] In one embodiment, the phenylalanine is present at a concentration of about 1-25 mmol / L, for example, about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-25, 10, 15, 20, 100, 200, 150, 300, 400, 500, 600, 700, 800, 900, 1000, 200, 1000, 3000, 4000, 800, 900, 1000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 2000, 10000, 3000, 4000, 8000, 9000, 10000, 2000, 10000, 30000, 40000, 8000, 9000, 10000, 20000, 10000, 30000, 40000, 8000

[0100] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of phenylalanine and water-soluble salts thereof.

[0101] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of free amino acid methionine.

[0102] In one embodiment, the methionine is present at a concentration of about 1-25 mmol / L, for example, about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-25, 10, 15, 20, 100, 200, 150, 300, 400, 500, 600, 700, 800, 900, 1000, 200, 1000, 3000, 4000, 800, 900, 1000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 2000, 10000, 3000, 4000, 8000, 9000, 10000, 2000, 10000, 30000, 40000, 8000, 9000, 10000, 20000, 10000, 30000, 40000, 8000

[0103] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of methionine and water-soluble salts thereof.

[0104] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of free amino acid tyrosine.

[0105] In one embodiment, the tyrosine is present at a concentration of about 1-25 mmol / L, e.g., about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, or 5-10 mmol / L, such as about 5, 10 or 20 mmol / L.

[0106] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of tyrosine and water-soluble salts thereof.

[0107] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of free amino acid cysteine.

[0108] In one embodiment, the cysteine ​​is present at a concentration of about 1-25 mmol / L, e.g., about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-25, 10, 15, 20, 100, 200, 150, 300, 400, 500, 600, 700, 800, 900, 1000, 200, 1000, 3000, 4000, 800, 900, 1000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 2000, 10000, 3000, 4000, 8000, 9000, 10000, 2000, 10000, 30000, 40000, 8000, 9000, 10000, 20000, 10000, 30000, 40000, 8000

[0109] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of cysteine ​​and a water-soluble salt thereof.

[0110] In one embodiment, the present invention provides a method for delivering nucleic acid DNA to a cell, comprising: contacting polyethyleneimine, the DNA to be delivered and the cell in the presence of free amino acid serine.

[0111] In one embodiment, the serine is present at a concentration of about 1-25 mmol / L, for example about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 10 or 20 mmol / L, and most preferably about 10 mmol / L.

[0112] In one embodiment, the delivery is carried out in an aqueous medium such as a solution or culture medium. In one embodiment, when the nucleic acid DNA is delivered in an aqueous medium, the amino acid is selected from one or more of serine and its water-soluble salts.

[0113] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of free amino acid tryptophan.

[0114] In one embodiment, the tryptophan is present at a concentration of about 1-25 mmol / L, for example, about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-2 0, 10-20, 1-15, 1.25-15, 2-15, 2.5-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, for example about 2.5-20 or 5-10 mmol / L, such as about 5, 10 or 20 mmol / L.

[0115] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of tryptophan and a water-soluble salt thereof.

[0116] In one embodiment, the present invention provides a method for delivering a nucleic acid (eg, DNA or RNA) to a cell, comprising contacting polyethyleneimine, the nucleic acid to be delivered, and the cell in the presence of free amino acid threonine.

[0117] In one embodiment, the threonine is present at a concentration of about 1-25 mmol / L, e.g., about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 1-15, 1.25-15, 2-15, 2.5-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, for example about 2.5-20 or 5-10 mmol / L, preferably about 5, 10 or 20 mmol / L.

[0118] In one embodiment, the delivery is carried out in an aqueous medium, such as a solution or culture medium. In one embodiment, when the nucleic acid is delivered in an aqueous medium, the amino acid is selected from one or more of threonine and a water-soluble salt thereof.

[0119] In one embodiment, when the nucleic acid is DNA, the threonine is present at a concentration of about 1-25 mmol / L, such as about 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 10-20, 1-10, 1.25-10, 2-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 10-25 mmol / L, more preferably about 10 or 20 mmol / L, and most preferably about 10 mmol / L.

[0120] In one embodiment, when the nucleic acid is RNA, the threonine is present at a concentration of about 1-25 mmol / L, such as about 1-25, 1.25-25, 2-25, 2.5-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 1-20, 1.25-20, 2-20, 2.5-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 1-10, 1.25-10, 2.5-10, 3-10, 4-10 or 5-10 mmol / L, preferably about 1-20 or 2.5-10 mmol / L, more preferably about 2.5 or 5 mmol / L.

[0121] The amino acid used in the method of the present invention is a naturally occurring substance with good biosafety and is charged. By participating in the binding of PEI to nucleic acids, the amino acid can reduce the amount of PEI used and the N / P ratio (N is the free amino group in PEI, P is the phosphate group in DNA / RNA, and N / P refers to the molar ratio of the two) during the transfection process, and significantly enhance the transfection effect of PEI.

[0122] The beneficial effect of the present invention is that the improved polyethyleneimine-based nucleic acid delivery method of the present invention can significantly improve the transfection efficiency of polyethyleneimine without increasing cytotoxicity. The method is simple, easy to implement, highly safe, and low-cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] FIG1 is the observation result under a fluorescence microscope of Example 1.

[0124] FIG2 is the statistical result of the mean fluorescence intensity of Example 1.

[0125] FIG3 and FIG4 are the observation results of Example 2 under bright field and fluorescence microscope.

[0126] FIG5 is the statistical result of the mean fluorescence intensity of Example 2.

[0127] FIG6 shows the effect of the transfection reagent on cell proliferation and the cytotoxicity (CCK-8) test results during DNA transfection in Example 3.

[0128] FIG7 is the statistical result of the mean fluorescence intensity of Example 4.

[0129] FIG8 shows the effect of the transfection reagent on cell proliferation and the cytotoxicity (CCK-8) test results when the RNA was transfected in Example 4.

[0130] FIG9 is the statistical result of the mean fluorescence intensity of Example 5.

[0131] FIG10 is the statistical result of the average fluorescence intensity of Example 6.

[0132] FIG11 is the statistical result of the average fluorescence intensity of Example 7.

[0133] FIG12 is the statistical results of the average fluorescence intensity of Example 8.

[0134] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, an optionally included step means that the step exists or does not exist.

[0135] As used herein, the term "about" refers to a range of values ​​that include the specific value and that one skilled in the art would reasonably consider to be similar to the specific value. In some embodiments, the term "about" refers to within the standard error of measurement using commonly accepted measurements in the art. In some embodiments, approximately refers to + / - 10% of the specific value.

[0136] Ranges disclosed herein should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, description of a range from 1 to 6 should be considered to have specifically disclosed subranges from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0137] The present invention will be further illustrated by the following non-limiting examples. It is well known to those skilled in the art that many modifications may be made to the present invention without departing from the spirit of the invention, and such modifications also fall within the scope of the invention. The scope of the present invention should be described by the appended claims, and any variations equivalent to the intent and scope of the claims should also be included within the scope of the present invention.

[0138] Unless otherwise specified, the following experimental methods are conventional methods, and the experimental materials used are easily obtained from commercial companies unless otherwise specified. Example

[0139] Example 1:

[0140] 12 hours in advance according to 1×10 4 HEK-293T cells (ATCC No.: CRL-3216) were seeded in a 96-well plate and cultured at 37° C. with 5% CO 2 .

[0141] Prepare 1 mg / mL of 25 kDa PEI (i.e., PEI stock solution) and 1 mg / mL of a plasmid DNA containing a eukaryotic GFP reporter gene sequence (SEQ ID NO: 1) stock solution in PBS. Add the PBS solution containing the indicated amino acids to sterile tubes. Add 0.1 μg of the plasmid DNA containing a eukaryotic GFP reporter gene sequence and 0.1 μg of 25 kDa PEI (CAS: 9002-98-6, average Mw ~25,000 by LS, average Mn ~10,000 by GPC) to each sterile tube. The total volume of each tube is 10 μL. The amino acid concentration is 50 mM. Vortex to mix thoroughly, let stand at room temperature for 30 minutes, and then add 90 μL of DMEM to prepare the transfection working solution. The final amino acid concentration in each tube is 5 mM. The old cell culture medium was discarded, and 100 μL of the transfection working solution supplemented with fresh culture medium was added to each well of the 96-well plate. The plate was returned to the incubator and cultured. After 48 h, the expression of green fluorescent protein was observed using an inverted fluorescence microscope using a 488 nm (excitation light) / 520 nm (emission light) filter. The results are shown in Figure 1.

[0142] The fluorescent images were processed using ImageJ software. Six random fields of view of the same position and size were taken for each sample. The fluorescence intensity values ​​of the cells in the field of view were calculated. The fluorescence intensity values ​​of each group were compared with those of the PEI control group to obtain the relative percentage of the control (% of Control). The results are shown in Figure 2.

[0143] As shown in Figure 1,2, compared with PBS, after adding amino acid, the mean fluorescence intensity of the cell increases, and the positive ratio on the photo also significantly improves. It can be seen that some specific amino acids that add 5mM have varying degrees of enhancement to the cell transfection effect. Wherein, histidine, lysine, glycine, arginine, alanine, proline, leucine, glutamine, isoleucine, phenylalanine, methionine, tyrosine, cysteine, serine, tryptophan, threonine all have the effect of certain enhancing transfection effect. Among the embodiment 2 of the follow-up concentration test, selected these 8 kinds of amino acids of histidine, glycine, alanine, proline, serine, threonine, arginine, lysine to do the test of further transfection enhancing ability.

[0144] Example 2:

[0145] 12 hours in advance according to 2×10 4 HEK-293T cells (ATCC No.: CRL-3216) were seeded in a 96-well plate and cultured at 37° C. with 5% CO 2 .

[0146] Prepare 1 mg / mL of 25 kDa PEI (i.e., PEI stock solution) and 1 mg / mL of a plasmid DNA stock solution containing a eukaryotic GFP reporter gene sequence (SEQ ID NO: 1) in PBS. Prepare 200 mM solutions of histidine, glycine, alanine, proline, serine, threonine, arginine, and lysine in PBS. Place 0.625 μL, 1.25 μL, 2.5 μL, 5 μL, and 10 μL of each amino acid-PBS solution into sterile tubes and bring the volume up to 10 μL with PBS. The amino acid concentrations in each tube are now 12.5 mM, 25 mM, 50 mM, 100 mM, and 200 mM. To each of the sterile tubes, add 0.1 μg of plasmid DNA containing a eukaryotic GFP reporter gene sequence and 0.1 μg of 25 kDa PEI (CAS: 9002-98-6, average Mw ~25,000 by LS, average Mn ~10,000 by GPC). Vortex to mix thoroughly. After standing at room temperature for 30 minutes, add 90 μL of DMEM to prepare the transfection working solution. The final amino acid concentrations in each tube were 1.25 mM, 2.5 mM, 5 mM, 10 mM, and 20 mM. Transfection controls were performed using the same ratios of PEI or Lipo2000 (Thermo Fisher Scientific) without amino acids.

[0147] Discard the old cell culture medium and add 100 μL of the transfection solution to each well of the 96-well plate. Return the cells to the incubator and continue culturing. After 48 hours, observe the expression of green fluorescent protein using an inverted fluorescence microscope with a 488 nm (excitation) / 520 nm (emission) filter. The results are shown in Figures 3 and 4.

[0148] Use ImageJ software to process the fluorescence photos taken, and take 6 random visual fields of the same position and size for each fluorescence photo. The fluorescence intensity values ​​of the cells in the visual field are counted, and the fluorescence intensity values ​​of each group and the PEI control group are compared respectively to obtain the relative percentage (% control) with the control, and the results are shown in Figure 5. Different amino acids have a significant promoting effect on PEI transfection as their concentrations increase, among which serine and threonine have the highest MFI (mean fluorescence intensity) improvement relative to other amino acids at this concentration at a working concentration of 10mM. Although other amino acids such as alanine, proline, arginine, and lysine also have a transfection enhancing effect at the 20mM group, the 20mM alanine, proline, arginine, and lysine test group have shown more obvious cytotoxicity at 48h, and it can be observed that the culture medium of the corresponding sample turns yellow significantly, and cell shrinkage causes phenomena such as plaques to appear on the culture surface.

[0149] Example 3:

[0150] 12 hours in advance according to 2×10 4 HEK-293T cells (ATCC No.: CRL-3216) were seeded in a 96-well plate and cultured at 37° C. with 5% CO 2 .

[0151] Prepare 1 mg / mL of 25kDa PEI (i.e., PEI stock solution) and 1 mg / mL of a plasmid DNA containing a eukaryotic GFP reporter gene sequence (SEQ ID NO: 1) stock solution in PBS. Add 10 μL of each of the prepared 100 mM PBS solutions of glycine, alanine, histidine, proline, serine, threonine, arginine, and lysine to the sterile tubes described above, adding 0.1 μg of the plasmid DNA containing a eukaryotic GFP reporter gene sequence and 0.1 μg of 25kDa PEI (CAS: 9002-98-6, average Mw ~25,000 by LS, average Mn ~10,000 by GPC), respectively. Vortex to mix, let stand at room temperature for 30 minutes, and then add 90 μL of fresh culture medium (e.g., DMEM) to prepare the transfection working solution. The final amino acid concentration in each tube is 10 mM. The blank control group only received 10 μL of PBS and 0.2 μg of DNA, and the transfection control group used PEI or Lipo2000 (Thermo Fisher Scientific) in the same ratio without amino acids.

[0152] Discard the old cell culture medium and add 100 μL of the above transfection solution to each well of the 96-well plate. Return the plate to the incubator and continue culturing.

[0153] At 24 and 48 hours, 10 μL of CCK8 solution was added to each well, and the culture plate was incubated in a 37°C incubator for 1-4 hours. The absorbance at 450 nm and 630 nm was measured using a microplate reader to detect cell viability and calculate the cell viability. The results are shown in Figure 6.

[0154] Compared with the PBS group, the PEI + alanine and PEI + histidine groups showed significant cytotoxicity at 24 and 48 hours. The PEI + glycine and PEI + lysine groups showed weak cytotoxicity to HEK-293T cells at 24 hours, and the PEI + arginine group showed weak cytotoxicity to HEK-293T cells at 48 hours. In addition, the PEI + glycine group had a significant growth-promoting effect on HEK-293T cells at 48 hours. The PEI + proline and PEI + serine groups had a weak growth-promoting effect on HEK-293T cells at 24 hours, but no significant effect after 48 hours. The PEI + threonine group showed no cytotoxic or growth-promoting effects at either 24 or 48 hours.

[0155] Example 4:

[0156] 12 hours in advance according to 2.5×10 4 HEK-293T cells (ATCC No.: CRL-3216) were seeded in a 96-well plate and cultured at 37° C. with 5% CO 2 .

[0157] Prepare 1 mg / mL of 25 kDa PEI (i.e., PEI stock solution) and 1 mg / mL of mRNA containing the eukaryotic expression NeonGreen reporter gene sequence (SEQ ID NO: 3) in PBS. Prepare 200 mM solutions of histidine, glycine, alanine, proline, serine, threonine, arginine, and lysine in PBS. Take 0.625 μL, 1.25 μL, 2.5 μL, 5 μL, and 10 μL of the amino acid-PBS solution and place them in sterile tubes. Fill the tubes with PBS to the same volume of 10 μL. At this point, the amino acid concentrations in each tube are 12.5 mM, 25 mM, 50 mM, 100 mM, and 200 mM. To each of the above sterile tubes, add 0.2 μg of mRNA containing the eukaryotic expression NeonGreen reporter gene sequence and 0.2 μg of 25 kDa PEI (CAS: 9002-98-6, average Mw ~25,000 by LS, average Mn ~10,000 by GPC). Vortex to mix thoroughly. After standing at room temperature for 20 minutes, add 90 μL of DMEM to prepare the transfection working solution. The final amino acid concentrations in each tube were 1.25 mM, 2.5 mM, 5 mM, 10 mM, and 20 mM. A transfection control group used PEI without amino acids, Lipo RNAiMAX (Thermo Fisher), and Zeta Life in the same ratio.

[0158] The old cell culture medium was discarded, and 100 μL of the above transfection working solution was added to each well of the 96-well plate. The cells were returned to the incubator and cultured. After 24 hours, the expression of green fluorescent protein was photographed using an inverted fluorescence microscope using a 488 nm (excitation light) / 520 nm (emission light) filter. The fluorescent images taken were processed using ImageJ software. Six random fields of view of the same position and size were taken for each fluorescent image. The fluorescence intensity values ​​of the cells in the field of view were counted, and the fluorescence intensity values ​​of each group were compared with those of the PEI control group to obtain the relative percentage of the control (% control). The results are shown in Figure 7.

[0159] Then, 10 μL of CCK8 solution was added to each well, and the culture plate was incubated in a 37°C incubator for 1-4 hours. The absorbance at 450 nm and 630 nm was measured using a microplate reader to detect cell activity and calculate the cell viability. The results are shown in Figure 8.

[0160] Judging by the fluorescence intensity after reporter gene expression, histidine, glycine, alanine, proline, and threonine all significantly promoted mRNA transfection. Histidine showed the best effect at a concentration of 5 mM, approximately double that of the control group. Variations in glycine concentration had little effect on transfection efficiency, with overall levels approximately 40% higher than the control group. Fluorescence values ​​for alanine and proline showed a positive correlation with concentration, reaching maximum values ​​at 20 mM and 10 mM, respectively. Arginine and lysine significantly inhibited mRNA transfection, with fluorescent protein expression virtually non-existent at high concentrations. Threonine exhibited a strong promoting effect at low concentrations, with some inhibitory effect observed at 20 mM. Serine had no significant enhancing effect and exhibited an inhibitory effect at 20 mM.

[0161] The results of CCK8 showed that there was no significant statistical difference between each test group and the PBS control group, indicating that each group of transfection reagents had no significant toxicity to cells when transfecting mRNA.

[0162] Example 5:

[0163] 12 hours in advance according to 1×10 5 Cells / well N2a cells (ATCC No.: CCL-131) were seeded in 24-well plates and cultured at 37°C and 5% CO2.

[0164] Prepare 1 mg / mL of 25 kDa PEI (i.e., PEI stock solution) and 1 mg / mL of a plasmid DNA stock solution containing the eukaryotic expression NeonGreen reporter gene sequence (SEQ ID NO: 2) in PBS. Prepare 200 mM solutions of histidine, glycine, alanine, proline, serine, threonine, arginine, and lysine in PBS. Place 25 μL of the amino acid-PBS solution in a sterile tube. Add 0.5 μg of the plasmid DNA containing the eukaryotic expression NeonGreen reporter gene sequence and 0.5 μg of 25 kDa PEI (CAS: 9002-98-6, average Mw ~25,000 by LS, average Mn ~10,000 by GPC) to each sterile tube. Vortex to mix thoroughly. Let stand at room temperature for 30 minutes, then add 475 μL of DMEM to prepare the transfection working solution. The final amino acid concentration in each tube is 10 mM. The transfection control group used the same ratio of PEI and Lipo 3000 (Thermo Fisher Scientific) without amino acids.

[0165] The old cell culture medium was discarded, and 500 μL of the above transfection working solution was added to each well of the 24-well plate. The cells were returned to the incubator and cultured. After 48 hours, the expression of green fluorescent protein was photographed using an inverted fluorescence microscope using a 488 nm (excitation light) / 520 nm (emission light) filter. The fluorescent images taken were processed using ImageJ software. Six random fields of view of the same position and size were taken for each fluorescent image. The fluorescence intensity values ​​of the cells in the field of view were counted, and the fluorescence intensity values ​​of each group were compared with those of the PEI control group to obtain the relative percentage of the control (% control). The results are shown in Figure 9.

[0166] The results showed that when N2a cells were transfected, unlike HEK-293T cells, threonine still had a significant promoting effect at a concentration of 10mM, suggesting that the PEI transfection effect of different amino acids in promoting plasmid DNA has certain cell specificity.

[0167] Example 6:

[0168] 12 hours in advance according to 1×10 5 Cells / well N2a cells (ATCC No.: CCL-131) were seeded in 24-well plates and cultured at 37°C and 5% CO2.

[0169] Prepare 1 mg / mL of 25 kDa PEI (i.e., PEI stock solution) and 1 mg / mL of mRNA containing the eukaryotic expression NeonGreen reporter gene sequence (SEQ ID NO: 3) in PBS. Prepare 200 mM solutions of histidine, glycine, alanine, proline, serine, threonine, arginine, and lysine in PBS. Place 25 μL of the amino acid-PBS solution in a sterile tube. Add 0.5 μg of mRNA containing the eukaryotic expression NeonGreen reporter gene sequence and 0.5 μg of 25 kDa PEI (CAS: 9002-98-6, average Mw ~25,000 by LS, average Mn ~10,000 by GPC) to each sterile tube. Vortex to mix thoroughly. Let stand at room temperature for 20 minutes, then add 475 μL of DMEM to prepare the transfection working solution. The final amino acid concentration in each tube is 10 mM. The transfection control group used the same ratio of PEI without amino acids, jetMESSENGER (Polyplus), Lipo RNAiMAX (Thermo Fisher Scientific), and Zeta Life.

[0170] The old cell culture medium was discarded, and 500 μL of the above transfection working solution was added to each well of the 24-well plate. The cells were returned to the incubator and cultured. After 24 hours, the expression of green fluorescent protein was photographed using a fluorescence inverted microscope through a 488 nm (excitation light) / 520 nm (emission light) filter. The fluorescent photos taken were processed using ImageJ software. Six random fields of view of the same position and size were taken for each fluorescent photo. The fluorescence intensity values ​​of the cells in the field of view were counted, and the fluorescence intensity values ​​of each group were compared with the PEI control group to obtain the relative percentage (% control) of the control. The results are shown in Figure 10.

[0171] During mRNA transfection, the situation was basically similar to that of HEK-293T cells. Histidine, glycine, alanine, proline, and threonine still had significant promoting effects, among which alanine had the most obvious effect. However, the promotion extent was also different from that in HEK-293T cells, suggesting that the PEI transfection effect of different amino acids promoting RNA has certain cell specificity.

[0172] Example 7:

[0173] 12 hours in advance according to 1×10 5 Cells / well CHO cells (ATCC No.: CCL-61) were seeded in 24-well plates and cultured at 37°C and 5% CO2.

[0174] Prepare 1 mg / mL of 25 kDa PEI (i.e., PEI stock solution) and 1 mg / mL of a plasmid DNA stock solution containing the eukaryotic expression NeonGreen reporter gene sequence (SEQ ID NO: 2) in PBS. Prepare 200 mM solutions of histidine, glycine, alanine, proline, serine, threonine, arginine, and lysine in PBS. Place 25 μL of the amino acid-PBS solution in a sterile tube. Add 0.5 μg of the plasmid DNA containing the eukaryotic expression NeonGreen reporter gene sequence and 0.5 μg of 25 kDa PEI (CAS: 9002-98-6, average Mw ~25,000 by LS, average Mn ~10,000 by GPC) to each sterile tube. Vortex to mix thoroughly. Let stand at room temperature for 30 minutes, then add 475 μL of DMEM to prepare the transfection working solution. The final amino acid concentration in each tube is 10 mM. The transfection control group used the same ratio of PEI and jetPRIME (Polyplus) without amino acids.

[0175] The old cell culture medium was discarded, and 500 μL of the above transfection working solution was added to each well of the 24-well plate. The cells were returned to the incubator and cultured. After 48 hours, the expression of green fluorescent protein was photographed using a fluorescence inverted microscope through a 488 nm (excitation light) / 520 nm (emission light) filter. The fluorescent photos taken were processed using ImageJ software. Six random fields of view of the same position and size were taken for each fluorescent photo. The fluorescence intensity values ​​of the cells in the field of view were counted, and the fluorescence intensity values ​​of each group were compared with the PEI control group to obtain the relative percentage (% control) of the control. The results are shown in Figure 11.

[0176] When plasmids were transfected into CHO cells, glycine, alanine, and threonine showed significant promoting effects, unlike those in HEK-293T and N2a cells. Alanine was the most significant, suggesting that the effects of different amino acids in promoting PEI transfection of plasmid DNA have certain cell specificity.

[0177] Example 8:

[0178] 12 hours in advance according to 1×10 5 Cells / well CHO cells (ATCC No.: CCL-61) were seeded in 24-well plates and cultured at 37°C and 5% CO2.

[0179] Prepare a 1 mg / mL 25 kDa PEI stock solution and a 1 mg / mL stock solution of mRNA containing the eukaryotic expression NeonGreen reporter gene sequence (SEQ ID NO: 3) in PBS. Prepare 200 mM solutions of histidine, glycine, alanine, proline, serine, threonine, arginine, and lysine in PBS. Place 25 μL of the amino acid-PBS solution in a sterile tube. Add 0.5 μg of mRNA containing the eukaryotic expression NeonGreen reporter gene sequence and 0.5 μg of 25 kDa PEI (CAS: 9002-98-6, average Mw ~25,000 by LS, average Mn ~10,000 by GPC) to each sterile tube. Vortex to mix thoroughly. Let stand at room temperature for 20 minutes, then add 475 μL of DMEM to prepare the transfection working solution. The final amino acid concentration in each tube is 10 mM. The transfection control group used the same ratio of PEI, jetMESSENGER (Polyplus), and Zeta Life without amino acids.

[0180] The old cell culture medium was discarded and 500 μL of the above transfection working solution was added to each well of the 24-well plate. The cells were returned to the incubator and cultured. After 24 hours, the expression of green fluorescent protein was photographed using a fluorescence inverted microscope through a 488 nm (excitation light) / 520 nm (emission light) filter. The fluorescent photos taken were processed using ImageJ software. Six random fields of view of the same position and size were taken for each fluorescent photo. The fluorescence intensity values ​​of the cells in the field of view were counted. The fluorescence intensity values ​​of each group were compared with those of the PEI control group to obtain the relative percentage (% control) of the control. The results are shown in Figure 12.

[0181] During mRNA transfection, unlike the performance in HEK-293T and N2a cells, alanine still showed a significant promoting effect, and glycine and proline also showed a certain promoting trend, suggesting that the PEI transfection effect of different amino acids in promoting RNA has certain cell specificity.

[0182] Sequence Listing

[0183] SEQ ID NO: 1: The complete sequence of the GFP reporter gene expressing plasmid used.

[0184] SEQ ID NO: 2: The complete sequence of the NeonGreen reporter gene expressing plasmid used.

[0185] SEQ ID NO: 3: The complete sequence of the mRNA used to express the NeonGreen reporter gene.

Claims

1. A method for delivering a nucleic acid to a cell, comprising: In the presence of free amino acids, polyethyleneimine, the nucleic acid to be delivered and the cells are contacted, wherein the amino acids are selected from one or more of histidine, lysine, glycine, arginine, alanine, proline, leucine, glutamine, isoleucine, phenylalanine, methionine, tyrosine, cysteine, serine, tryptophan and threonine, preferably selected from one or more of histidine, glycine, alanine, proline and threonine.

2. The method of claim 1, wherein the nucleic acid comprises DNA and / or RNA, Preferably, when the nucleic acid is DNA, the amino acid is selected from one or more of histidine, glycine, alanine, proline and threonine; or, when the nucleic acid is RNA, the amino acid is selected from one or more of histidine, glycine, alanine, proline and threonine.

3. The method of claim 1 or 2, wherein the method comprises: (i) mixing the amino acid, polyethyleneimine and the nucleic acid to be delivered to obtain a mixed solution; and (ii) contacting the mixture of (i) with cells.

4. The method of any one of claims 1 to 3, wherein: (a) the average molecular weight (weight average molecular weight) of polyethyleneimine is about 20-30 kDa, preferably about 25 kDa; and / or (b) the average molecular weight (number average molecular weight) of the polyethyleneimine is about 8-12 kDa, preferably about 10 kDa; and / or (c) the weight ratio of the nucleic acid to be delivered to the polyethyleneimine is about 1:5 to about 5:1, preferably about 1:1; and / or (d) the concentration of free amino acids is about 1-25 mmol / L, for example about 2.5-20, 5-10 or 10-25 mmol / L, such as about 5, 10 or 20 mmol / L; and / or (e) The cell is selected from eukaryotic cells, such as animal (such as non-human mammal) cells, insect cells, human cells or cell lines, for example HEK-293 cells (ATCC No.: CCL-1573) and their derivatives, such as HEK-293T cells (ATCC No.: CRL-3216), N2a cells (Neuro-2a, ATCC No.: CCL-131) and CHO cells (ATCC No.: CCL-61) and their derivatives.

5. The method of any one of claims 1 to 4, wherein the ratio of nucleic acid to be delivered: free amino acids present: PEI is 1 μg nucleic acid: 0.5-2×10 -2 mmol amino acid: 1-3μg polyethyleneimine.

6. The method of any one of claims 1 to 5, wherein When the nucleic acid is DNA, the amino acid is selected from one or more of histidine, glycine, alanine, proline and threonine, Preferably, the concentration of histidine is about 1-25 mmol / L, more preferably about 2.5-20 mmol / L, more preferably about 10 mmol / L; and / or Preferably, the concentration of glycine is about 1-25 mmol / L, more preferably about 2.5-20 mmol / L, more preferably about 10 or 20 mmol / L, most preferably about 20 mmol / L; and / or Preferably, the concentration of alanine is about 1-25 mmol / L, more preferably about 10-25 mmol / L, more preferably about 20 mmol / L; and / or Preferably, the concentration of proline is about 1-25 mmol / L, more preferably about 10-25 mmol / L, more preferably about 20 mmol / L; and / or Preferably, the concentration of threonine is about 1-25 mmol / L, more preferably about 10-25 mmol / L, more preferably about 10 or 20 mmol / L, most preferably about 10 mmol / L.

7. The method of any one of claims 1 to 5, wherein When the nucleic acid is RNA, the amino acid is selected from one or more of histidine, glycine, alanine, proline and threonine, Preferably, the concentration of histidine is about 1-25 mmol / L, more preferably about 5-10 mmol / L, more preferably about 5 mmol / L; and / or Preferably, the concentration of glycine is about 1-25 mmol / L, more preferably about 5 mmol / L; and / or Preferably, the concentration of alanine is about 1-25 mmol / L, more preferably about 5-20 mmol / L, more preferably about 5 or 20 mmol / L, most preferably about 5 mmol / L; and / or Preferably, the concentration of proline is about 1-25 mmol / L, more preferably about 2.5-20 mmol / L, more preferably about 5 or 10 mmol / L, most preferably about 10 mmol / L; and / or Preferably, the concentration of threonine is about 1-25 mmol / L, more preferably about 1-20 or 2.5-10 mmol / L, more preferably about 2.5 or 5 mmol / L.

8. The method of any one of claims 1 to 7, comprising: (1) culturing the cells to a suitable density, for example, a density of 70%-80%; (2) mixing the amino acid, polyethyleneimine, and nucleic acid to obtain a mixed solution, and standing the mixed solution at room temperature, for example, for 20 to 40 minutes; and (3) Collect the cells, add an equal volume of fresh culture medium and the mixed solution of step (2), and perform nucleic acid transfection, preferably for no more than 24 hours or 48 hours.

Images