Composition for delivering nucleic acid into cells

Abstract

The present invention relates to a composition or transfection agent for delivering nucleic acids into cells containing a cationic polymer, in particular to a composition or transfection agent for delivering nucleic acids into cells containing a composition or transfection agent selected from the group consisting of polyethyleneimine and optionally protamine, optionally polyarginine and / or optionally polylysine. The composition or transfection reagent for delivering nucleic acid into cells has the advantages of low toxicity, high transfection efficiency and the like.

Description

Technical Field

[0001] The present invention relates to a composition or transfection reagent for delivering nucleic acid into cells containing a cationic polymer, in particular to a composition or transfection reagent for delivering nucleic acid into cells containing a cationic polymer selected from polyethyleneimine and optionally protamine, optionally polyarginine and / or optionally polylysine. The composition or transfection reagent for delivering nucleic acid into cells of the present invention has the advantages of low toxicity, high transfection efficiency and the like. Background Art

[0002] Gene therapy is the process of intervening in the expression of specific genes in target cells to treat pathological diseases by introducing exogenous RNA or DNA. Gene therapy based on nucleic acid drug delivery is believed to lead the third industrial revolution in biomedicine. At present, gene therapy can be used to treat a variety of diseases, including cancer, familial genetic diseases, and cardiovascular diseases. Gene therapy has shown excellent results in the treatment of rare diseases. At present, people have developed two major gene vector systems: viral vectors and non-viral vectors. However, the expensive production costs of transgenic cells or viral vectors and potential biosafety risks have greatly reduced their clinical applicability. Physical gene delivery methods include direct injection, electroporation, magnetic fields, photothermal and ultrasound-assisted, etc. The above methods are prone to tissue damage, which limits their clinical application. Non-viral gene vectors mainly include lipid delivery vectors, polymer delivery vectors, polypeptide delivery vectors, etc., which have unique advantages such as simple preparation, low immunogenicity, not easy to integrate with host genes, and unlimited cloning ability, and have become important candidates for nucleic acid drug delivery. The current non-viral gene vector delivery vector still has the disadvantages of high toxicity and low delivery efficiency, which restricts its wide and large-scale application. Therefore, providing a transfection reagent with advantages such as low toxicity and high transfection efficiency has important application value in the fields of gene editing, diagnosis, and treatment. Summary of the invention

[0003] The present invention relates to a composition or transfection reagent for delivering nucleic acid into cells containing a cationic polymer, in particular a composition or transfection reagent for delivering nucleic acid into cells containing a cationic polymer selected from polyethyleneimine and optionally protamine, optionally polyarginine and / or optionally polylysine. Further, the combination of the present invention and the transfection reagent further comprise a cationic lipid and a neutral lipid. The composition or transfection reagent for delivering nucleic acid into cells of the present invention has the advantages of low toxicity, high transfection efficiency, etc.

[0004] Aspect 1. A composition for delivering nucleic acids into cells, characterized in that it is prepared by mixing liquid A and liquid B, wherein liquid A is an aqueous liquid containing a cationic polymer and a water-soluble calcium salt, and liquid B is a solution containing cationic lipids, neutral lipids and optionally ethanol.

[0005] Aspect 2. The composition for delivering nucleic acids into cells as described in Aspect 1 is characterized in that the cationic polymer contained in liquid A is selected from protamine, polyarginine, polylysine or polyethyleneimine, and any combination thereof; preferably, the cationic polymer contained in liquid A is selected from polyethyleneimine and optional protamine, optional polyarginine and / or optional polylysine; more preferably, the cationic polymer contained in liquid A is selected from polyethyleneimine.

[0006] Aspect 3. The composition for delivering nucleic acids into cells as described in any one of Aspects 1 or 2 is characterized in that the concentration of the cationic polymer in liquid A is 0.001-1 mg / ml; preferably, the concentration of the cationic polymer in liquid A is 0.001-0.5 mg / ml; more preferably, the concentration of the cationic polymer in liquid A is 0.01-0.1 mg / ml; further preferably, the concentration of the cationic polymer in liquid A is 0.01-0.05 mg / ml.

[0007] Aspect 4. The composition for delivering nucleic acid into cells according to any one of Aspects 1 to 3, characterized in that the water-soluble calcium salt in liquid A is selected from calcium acetate, calcium chloride, calcium sodium EDTA, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium hydrogen sulfate, calcium bisulfite, calcium bromide, calcium iodide, calcium citrate, calcium lactate, and calcium gluconate, and is more preferably calcium acetate, calcium sodium EDTA, calcium gluconate, calcium citrate, calcium lactate, and calcium gluconate, and is more preferably calcium acetate;

[0008] Preferably, the calcium salt concentration is 50 mmol / L to 1000 mmol / L; preferably, the calcium salt concentration is 50 to 150 mmol / L, 150 to 200 mmol / L, 200 to 400 mmol / L, 400 to 600 mmol / L, or 600 to 800 mmol / L; more preferably, the calcium salt concentration is 100 ± 50 mmol / L, 200 ± 50 mmol / L, 300 ± 50 mmol / L, 400 ± 50 mmol / L, 500 ± 50 mmol / L, 600 ± 50 mmol / L, 700 ± 50 mmol / L, 800 ± 50 mmol / L or 900 ± 50 mmol / L;

[0009] More preferably, the calcium salt concentration is 200±20 mmol / L.

[0010] Aspect 5. The composition for delivering nucleic acids into cells as described in any one of Aspects 1-4, characterized in that the pH value of liquid A is weakly acidic or neutral; preferably, the pH value of liquid A is 4-7.5; more preferably, the pH value of liquid A is 5±0.5, 6±0.5, 7±0.5; more preferably, the pH value of liquid A is 5±0.5; more preferably, the pH value of liquid A is 5.

[0011] Aspect 6. The composition for delivering nucleic acids into cells as described in any one of Aspects 1-5, characterized in that the cationic lipid in liquid B is selected from one of DC-Chol, DOSPA, MVL5, DSDMA, DODMA, DOIC, DOTAP, DODAP, DOTMA or any combination of the foregoing cationic lipids; preferably, the cationic lipid is selected from DOSPA and / or MVL5; more preferably, the cationic lipid is selected from DOSPA or MVL5.

[0012] Aspect 7. A composition for delivering nucleic acid into cells as described in any one of Aspects 1-6, characterized in that the neutral lipid in liquid B is selected from egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, Preferably, the neutral phospholipid is selected from one or more of distearoylphosphatidylcholine, phosphatidylcholine, egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin and phosphatidylethanolamine; more preferably, the neutral phospholipid is selected from dioleoylphosphatidylethanolamine (DOPE).

[0013] Aspect 8. The composition for delivering nucleic acids into cells as described in any one of Aspects 1-7 is characterized in that the molar ratio of cationic lipids to neutral lipids in liquid B is 1:99 to 99:1; preferably, the molar ratio of cationic lipids to neutral lipids in liquid B is 20:80 to 80:20; more preferably, the molar ratio of cationic lipids to neutral lipids in liquid B is 40:60 to 60:40.

[0014] Aspect 9. The composition for delivering nucleic acids into cells according to any one of Aspects 1-8, characterized in that the total lipid concentration in liquid B is 1-100 mM; preferably, the total lipid concentration in liquid B is 3-80 mM; more preferably, the total lipid concentration in liquid B is 3-50 mM; more preferably, the total lipid concentration in liquid B is 3-5 mM.

[0015] Aspect 10. The composition for delivering nucleic acids into cells as described in any one of Aspects 1-9 is characterized in that the mixed volume ratio of liquid A and liquid B is 1:2 to 100:2; preferably, the mixed volume ratio of liquid A and liquid B is 2:2 to 80:2; more preferably, the mixed volume ratio of liquid A and liquid B is 3:2 to 50:2.

[0016] Aspect 11. A composition for delivering nucleic acids into cells according to any one of Aspects 1-10, characterized in that the ethanol solution is an ethanol aqueous solution with an ethanol concentration of less than 30% or an ethanol aqueous solution with an ethanol concentration of more than 60%; preferably, the ethanol solution is an ethanol aqueous solution with an ethanol concentration of less than 20% or an ethanol aqueous solution with an ethanol concentration of more than 80%. Aspect 12. A kit for delivering nucleic acids into cells, characterized in that it comprises independently packaged liquid A and liquid B, wherein liquid A is an aqueous liquid comprising a cationic polymer and a water-soluble calcium salt, and liquid B is a liquid comprising a cationic lipid, a neutral lipid and an ethanol solution, and liquid A and liquid B are mixed before use before delivering nucleic acids into cells.

[0017] Aspect 13. The kit for delivering nucleic acids into cells as described in Aspect 12 is characterized in that the cationic polymer contained in liquid A is selected from protamine, polyarginine, polylysine or polyethyleneimine, and any combination thereof; preferably, the cationic polymer contained in liquid A is selected from polyethyleneimine and optional protamine, optional polyarginine and / or optional polylysine; more preferably, the cationic polymer contained in liquid A is selected from polyethyleneimine.

[0018] Aspect 14. A kit for delivering nucleic acids into cells as described in any one of Aspects 12 or 13, characterized in that the concentration of the cationic polymer in liquid A is 0.001-1 mg / ml; preferably, the concentration of the cationic polymer in liquid A is 0.001-0.5 mg / ml; more preferably, the concentration of the cationic polymer in liquid A is 0.01-0.1 mg / ml; further preferably, the concentration of the cationic polymer in liquid A is 0.01-0.05 mg / ml. Aspect 15. A kit for delivering nucleic acids into cells as described in any one of Aspects 12-14, characterized in that the water-soluble calcium salt in liquid A is selected from calcium acetate, calcium chloride, calcium sodium EDTA, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium hydrogen sulfate, calcium bisulfite, calcium bromide, calcium iodide, calcium citrate, calcium lactate, calcium gluconate, further preferably calcium acetate, calcium sodium EDTA, calcium gluconate, calcium citrate, calcium lactate, calcium gluconate, further preferably calcium acetate;

[0019] Preferably, the calcium salt concentration is 50 mmol / L to 1000 mmol / L; preferably, the calcium salt concentration is 50 to 150 mmol / L, 150 to 200 mmol / L, 200 to 400 mmol / L, 400 to 600 mmol / L, or 600 to 800 mmol / L;

[0020] More preferably, the calcium salt concentration is 100±50mmol / L, 200±50mmol / L, 300±50mmol / L, 400±50mmol / L, 500±50mmol / L, 600±50mmol / L, 700±50mmol / L, 800±50mmol / L or 900±50mmol / L;

[0021] More preferably, the calcium salt concentration is 200±20 mmol / L.

[0022] Aspect 16. The kit for delivering nucleic acid into cells as described in any one of Aspects 12-15, characterized in that the pH value of liquid A is weakly acidic or neutral; preferably, the pH value of liquid A is 4-7.5; more preferably, the pH value of liquid A is 5±0.5, 6±0.5, 7±0.5; more preferably, the pH value of liquid A is 5±0.5; more preferably, the pH value of liquid A is 5.

[0023] Aspect 17. A kit for delivering nucleic acids into cells as described in any one of Aspects 12-16, characterized in that the cationic lipid is selected from one of DOSPA, MVL5, DSDMA, DODMA, DOIC, DOTAP, DODAP, DOTMA or any combination of the foregoing cationic lipids; preferably, the cationic lipid is selected from DOSPA and / or MVL5; more preferably, the cationic lipid is selected from DOSPA or MVL5.

[0024] Aspect 18. A kit for delivering nucleic acid into cells as described in any one of Aspects 12-17, characterized in that the neutral lipid in liquid B is selected from egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, One or more of ethanolamine, dioleoylphosphatidylethanolamine, distearoylphosphatidylinositol, dimyristoylphosphatidylinositol, dipalmitoylphosphatidylinositol, dioleoylphosphatidylinositol, 9A1P9, 10A1P10; preferably, the neutral phospholipid is selected from one or more of distearoylphosphatidylcholine, phosphatidylcholine, egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin and phosphatidylethanolamine; more preferably, the neutral phospholipid is selected from dioleoylphosphatidylethanolamine (DOPE).

[0025] Aspect 19. The kit for delivering nucleic acids into cells according to any one of Aspects 12-18, characterized in that the molar ratio of cationic lipids to neutral lipids in liquid B is 1:99 to 99:1; preferably, the molar ratio of cationic lipids to neutral lipids in liquid B is 20:80 to 80:20; more preferably, the molar ratio of cationic lipids to neutral lipids in liquid B is 40:60 to 60:40.

[0026] Aspect 20. The kit for delivering nucleic acid into cells according to any one of Aspects 12-19, characterized in that the total lipid concentration in liquid B is 1-100 mM; preferably, the total lipid concentration in liquid B is 3-80 mM; more preferably, the total lipid concentration in liquid B is 3-50 mM; more preferably, the total lipid concentration in liquid B is 3-10 mM.

[0027] Aspect 21. The kit for delivering nucleic acids into cells as described in any one of Aspects 12-20 is characterized in that A and B in the kit are mixed and used to deliver nucleic acids into cells, and the mixed volume ratio of A and B liquids is 1:2 to 100:2; preferably, the mixed volume ratio of A and B liquids is 2:2 to 80:2; more preferably, the mixed volume ratio of A and B liquids is 3:2 to 50:2.

[0028] Aspect 22. The composition for delivering a nucleic acid into a cell according to any one of aspects 1 to 11 or the kit for delivering a nucleic acid into a cell according to any one of aspects 12 to 21, characterized in that the nucleic acid is plasmid DNA, single-stranded DNA, double-stranded DNA, siRNA, shRNA, aiRNA, miRNA, mRNA, circular RNA, gRNA, aptamer, ribozyme, antisense oligonucleotide ASO or any combination thereof;

[0029] Aspect 23. A composition for delivering nucleic acid into cells, characterized in that the nucleic acid is siRNA or ASO, the cationic lipid in liquid B is one of DOSPA and MVL5, or a mixture thereof, the neutral lipid is DOPE, and the molar ratio of the cationic lipid to the neutral lipid is 4:6 to 6:4; liquid A is a solution containing 0.01 to 0.05 mg / ml polyethyleneimine and 200 to 400 mM calcium acetate.

[0030] Aspect 24. A kit for delivering nucleic acid into cells, characterized in that the nucleic acid is siRNA or ASO, the cationic lipid in liquid B is one of DOSPA and MVL5, or a mixture thereof, the neutral lipid is DOPE, and the molar ratio of the cationic lipid to the neutral lipid is 4:6 to 6:4; liquid A is a solution containing 0.01 to 0.05 mg / ml polyethyleneimine and 200 to 400 mM calcium acetate.

[0031] Aspect 25. A composition for delivering nucleic acid into cells, characterized in that the nucleic acid is DNA, the cationic lipid in liquid B is one of DOSPA and MVL5, or a mixture thereof, the neutral lipid is DOPE, and the molar ratio of the cationic lipid to the neutral lipid is 4:6 to 6:4; liquid A is a solution containing 0.01 to 0.05 mg / ml polyethyleneimine and 200 to 400 mM calcium acetate.

[0032] Aspect 26. A kit for delivering nucleic acid into cells, characterized in that the nucleic acid is DNA, the cationic lipid in liquid B is one of DOSPA and MVL5, or a mixture thereof, the neutral lipid is DOPE, and the molar ratio of the cationic lipid to the neutral lipid is 4:6 to 6:4; liquid A is a solution containing 0.01 to 0.05 mg / ml polyethyleneimine and 200 to 400 mM calcium acetate.

[0033] Aspect 27. A composition for delivering nucleic acid into cells, wherein the nucleic acid is mRNA, the cationic lipid in liquid B is one of DOSPA and MVL5, or a mixture thereof, the neutral lipid is DOPE, and the molar ratio of the cationic lipid to the neutral lipid is 4:6 to 6:4; liquid A is a solution containing 0.01 to 0.05 mg / ml polyethyleneimine and 200 to 400 mM calcium acetate.

[0034] Aspect 28. A kit for delivering nucleic acid into cells, wherein the nucleic acid is mRNA, the cationic lipid in liquid B is one of DOSPA and MVL5, or a mixture thereof, the neutral lipid is DOPE, and the molar ratio of the cationic lipid to the neutral lipid is 4:6 to 6:4; liquid A is a solution containing 0.01 to 0.05 mg / ml polyethyleneimine and 200 to 400 mM calcium acetate.

[0035] Unless otherwise indicated, the following terms have the meanings ascribed to them.

[0036] Unless the context requires otherwise, the word "comprise" and variations such as "include" and "comprising" in this specification and claims, and the open and inclusive meaning are to be interpreted as meaning "including, but not limited to".

[0037] The term "precipitated calcium ions" refers to calcium salts whose pure substances are in a poorly soluble state under normal conditions, such as calcium phosphate, calcium carbonate, calcium fluoride, etc. The term "non-precipitated calcium ions" refers to calcium ions whose pure substances are in a non-poorly soluble state under normal conditions, that is, calcium ions in a soluble, easily soluble, or slightly soluble state, including but not limited to calcium acetate, calcium chloride, calcium sodium EDTA, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium hydrogen sulfate, calcium hydrogen sulfite, calcium bromide, calcium iodide, calcium citrate, calcium lactate, calcium gluconate, etc.

[0038] The term "DNA" refers to a non-replicating gene or a replicating gene vector used as an expression vector.

[0039] The term "interfering RNA" or "RNAi" or "interfering RNA sequence" refers to a single-stranded RNA (e.g., a mature miRNA) or a double-stranded RNA (i.e., a duplex RNA such as siRNA, aiRNA, or pre-miRNA) that can reduce or inhibit the expression of a target gene or sequence when the interfering RNA and the target gene or sequence are in the same cell (e.g., by mediating degradation and inhibiting the translation of an mRNA complementary to the interfering RNA sequence). Interfering RNA therefore refers to a single-stranded RNA complementary to a target mRNA sequence or a double-stranded RNA formed by two complementary strands or by a single self-complementary strand. Interfering RNA may have substantial or complete identity to a target gene or sequence, or may include a mismatch region (i.e., a mismatch motif). The sequence of an interfering RNA may correspond to a full-length target gene or a subsequence thereof.

[0040] Interfering RNA includes "small interfering RNA" or "siRNA," for example, about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary strand of a double-stranded siRNA is about 100 nt long). In some embodiments, the siRNA duplex may include a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and a 5' phosphate terminus. Examples of siRNA include, but are not limited to, a double-stranded polynucleotide molecule assembled from two separate strand molecules, one of which is a sense strand and the other is a complementary antisense strand; a double-stranded polynucleotide molecule assembled from single-stranded molecules, wherein the sense region and the antisense region are connected by a nucleic acid-based or non-nucleic acid-based linker; a double-stranded polynucleotide molecule containing a hairpin secondary structure having self-complementary sense and antisense regions; and a circular single-stranded polynucleotide molecule containing a stem having two or more loop structures and self-complementary sense and antisense regions, wherein the circular polynucleotide can be processed in vivo or in vitro to generate an active double-stranded siRNA molecule.

[0041] The term "substantially identical" or "substantial identity" in the context of two or more nucleic acids refers to two or more sequences or subsequences that are identical or have a specific percentage of identical nucleotides (i.e., at least about 60%, preferably at least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity) when compared and aligned for maximum correspondence over a comparison window, or a specified region, by using one of the following sequence comparison algorithms or by manual alignment and visual measurement. This definition, when indicated in the context, also similarly refers to the complement of the sequence. Preferably, the substantial identity exists over a region of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

[0042] The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and includes DNA and RNA. DNA can take the following forms: for example, antisense molecules, plasmid DNA, pre-concentrated DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA can take the following forms: siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, and non-naturally occurring, and which have binding properties similar to reference nucleic acids. Examples of the analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides and peptide-nucleic acids (PNAs). Unless specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a specific nucleic acid sequence also inherently includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the explicitly indicated sequence. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed basic and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem., 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8: 91-98 (1994)). "Nucleotides" comprise the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked together by the 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.

[0043] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that contains a partial or full-length coding sequence necessary for producing a polypeptide or precursor polypeptide.

[0044] The term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into at least three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0045] When " lipid granule " is used herein, refer to can be used for delivering active agent or therapeutic agent, such as nucleic acid (for example, interfering RNA), to the lipid preparation of target target site.In the lipid granule formed by the combination lipid of cationic lipid, non-cationic lipid and prevention particle aggregation of the typical of the present invention, described active agent or therapeutic agent can be encapsulated in lipid, thus protecting reagent from enzymatic degradation.

[0046] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus, such as sphingolipids, sphingolipid family, diacylglycerol and β-acyloxy acid are also included in the group referred to as amphipathic lipids. In addition, the above-mentioned amphipathic lipids can be mixed with other lipids, and the other lipids include triglycerides and sterols.

[0047] The term "neutral lipid", when present in lipid particles, can be any of many lipid species present in the form of uncharged or neutral zwitterions at physiological pH. Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cholesterol, cephalin and cerebrosides. The selection of neutral lipids for particles described herein is usually guided by considerations, such as liposome size and the stability of liposomes in the bloodstream. Preferably, the neutral lipid component is a lipid with two acyl groups (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine). Liposomes with acyl chain groups of multiple different chain lengths and saturation are available or can be separated or synthesized by known technology. In one group of embodiments, lipids containing saturated fatty acids with carbon chain lengths within the range of C10 to C20 are preferred. In another group of embodiments, lipids with mono- or di-unsaturated fatty acids with carbon chain lengths within the range of C10 to C20 are used. In addition, lipids with mixtures of saturated and unsaturated fatty acid chains can be used. Preferably, neutral lipids used for the present invention are DOPE, DSPC, POPC, DPPC or any relevant phosphatidylcholine. Neutral lipids used for the present invention can also be made of sphingomyelin, dihydrosphingomyelin or have other head groups, for example the phospholipids of serine and inositol form.

[0048] The term "non-cationic lipid" refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.

[0049] The term "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as a physiological pH or a pH used during formulation preparation. It has been unexpectedly found that cationic lipids comprising alkyl chains with multiple unsaturated sites, e.g., at least 2 or 3 unsaturated sites, are particularly effective for forming lipid particles with increased membrane fluidity.

[0050] The term "analogs of cationic lipids" refers to cationic lipids with similar structures produced by changing the number of carbon atoms in any carbon chain of a cationic lipid.

[0051] The term "DSDMA" refers to the substance with CAS number 871258-14-9, which has the structure

[0052]

[0053] The term "DODMA" refers to the substance with CAS number 104162-47-2, which has the structure

[0054]

[0055] In addition to the cationic lipids explicitly described above, other cationic lipids that carry a net positive charge at approximately physiological pH may also be included in the lipid particles of the present invention. Such cationic lipids include, but are not limited to, N, N-dioleyl-N, N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl-N, N-triethylammonium chloride ("DOTMA"); N, N-distearoyl-N, N-diethylammonium bromide ("DDAB"); N-(2,3-dioleyloxy)propyl)-N, N, N-triethylammonium chloride ("DOTAP"); 1,2-dioleyloxy-3-trimethylaminopropane hydrochloride ("DOTAP.Cl"); 3-(N-(N', N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-C hol″), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (“DOSPA”), dioctadecylaminoglycylcarboxyspermine (“DOGS”), 1,2-dioleyl-sn-3-phosphoethanolamine (“DOPE”), 1,2-dioleoyl-3-dimethylpropylamine (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (“DMRIE”). In addition, a number of commercial preparations of cationic lipids may be used, such as, for example, LIPOFECTIN (including DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECTAMINE (including DOSPA and DOPE, available from GIBCO / BRL). In particular embodiments, the cationic lipid is an amino lipid.

[0056] The abbreviation "Chol" refers to cholesterol.

[0057] The ratio "N / P" refers to the number of moles of positive charge of all cationic lipids contained in the composition: the number of moles of phosphate.

[0058] The term "mammal" refers to any mammalian species such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, livestock, and the like.

[0059] The term "DOSPA" is a compound with CAS No. 282533-23-7. The name of DOSPA is 2,3-dioleyl-N-[2-(arginine carboxamido)ethyl]-N,N-dimethyl-1-propylamine hydrochloride. The structure of the compound DOSPA is shown below:

[0060]

[0061] The term "MVL5" is a compound with CAS No. 464926-03-2. The name of MVL5 is N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-amino-propyl)amino]butylamide)ethyl]-3,4-bis[oleyloxy]-benzamide. The structure of MVL5 is shown below:

[0062]

[0063] The term "DOPE" is a compound having the structural formula shown below:

[0064]

[0065] The term "DC-Chol" is a compound named 3β-[N-(N',N'-dimethylaminoethane)-formyl]cholesterol salt or its hydrochloride salt.

[0066] The term "DOTAP" is a compound with the structural formula shown below:

[0067]

[0068] Example 1. Preparation and characterization of transfection reagents with different formulations

[0069] Transfection reagent A solution: The following solutions were adjusted to pH 5 with acetic acid:

[0070] 1 25 mM sodium acetate solution 2 200 mM calcium acetate solution 3 200 mM calcium acetate solution containing 0.14 mg / ml poly-L-lysine 4 200 mM calcium acetate solution containing 0.14 mg / ml protamine 5 200 mM calcium acetate solution containing 0.14 mg / ml polyethyleneimine 6 200 mM calcium acetate solution containing 0.025 mg / ml polyethyleneimine 7 400 mM calcium acetate solution containing 0.025 mg / ml polyethyleneimine 8 25 mM sodium acetate solution containing 0.025 mg / ml polyethyleneimine 9 400 mM sodium acetate solution containing 0.025 mg / ml polyethyleneimine

[0071] Transfection reagent A solution: The following solutions were adjusted to pH 6 with acetic acid:

[0072] 10 200 mM calcium acetate solution containing 0.025 mg / ml polyethyleneimine 11 400 mM calcium acetate solution containing 0.025 mg / ml polyethyleneimine

[0073] Transfection reagent A solution: The following solutions were adjusted to pH 7.5 with acetic acid:

[0074] 12 200 mM calcium acetate solution containing 0.025 mg / ml polyethyleneimine 13 400 mM calcium acetate solution containing 0.025 mg / ml polyethyleneimine

[0075] Transfection reagent B:

[0076] 1. Mix X and DOPE in the following molar ratio, heat at 60°C and dissolve in ethanol. The total lipid concentration is 50 mM, which is the ethanol phase. Add the ethanol phase to water. The volume ratio of the ethanol phase to the water phase is 1:4. Stir for 20 min, extrude 5 times with a 100 nm polycarbonate membrane (1 ml manual extruder, Atson), dialyze overnight in a 1000-fold volume of physiological saline solution (dialysis bag 10 KDa) to remove ethanol, filter and sterilize to obtain transfection reagent B solution.

[0077] Take 0.1 ml of transfection reagent B solution, add 0.9 ml of water to dilute, and measure the Z-ave particle size and distribution by dynamic light scattering (Malvern NanoZS).

[0078] ①X is DC-Chol, X:DOPE (molar ratio) = 45:55, Z-ave particle size 68.90nm, PDI = 0.196②X is MVL5, X:DOPE (molar ratio) = 50:50, Z-ave particle size 93.76nm, PDI = 0.302③X is DOSPA, X:DOPE (molar ratio) = 40:60, Z-ave particle size 57.05nm, PDI = 0.046④X is DOTAP, X:DOPE (molar ratio) = 60:40, Z-ave particle size 100.27nm, PDI = 0.0872. X and DOPE are mixed in the following molar ratios, heated at 60°C and dissolved in 90% ethanol, the total lipid concentration obtained is 10mM, and transfection reagent B solution is obtained.

[0079] ⑤X is DOSPA, X:DOPE (molar ratio) = 40:60

[0080] ⑥X is MVL5, X:DOPE (molar ratio) = 50:50

[0081] Example 2. Study on transfection of siRNA with different transfection reagents

[0082] 1. Transfection complex preparation

[0083] siRNA-hGAPDH sequence

[0084]

[0085] siRNA-NC sequence

[0086]

[0087] Experimental Group:

[0088] siRNA-hGAPDH was dissolved in RNase-free water at a concentration of 10 μM.

[0089] Take 2 μL of siRNA-hGAPDH solution (10 μM), add 14 μL of transfection reagent A solution, mix well, add 4 μL of transfection reagent B solution, mix well, incubate at room temperature for 5 minutes, and add 180 μL of cell culture medium to obtain siRNA transfection complex.

[0090] Control group:

[0091] siRNA-NC was dissolved in RNase-free water at a concentration of 10 μM.

[0092] Take 2 μL of siRNA-NC solution (10 μM), add 14 μL of transfection reagent A solution, mix well, add 4 μL of transfection reagent B solution, mix well, incubate at room temperature for 5 minutes, and add 180 μL of cell culture medium to obtain siRNA transfection complex.

[0093] 2. Cell Transfection

[0094] HepG2 cells in the logarithmic growth phase were cultured at a rate of 1×10 5 / 0.5mL / well was inoculated in a 24-well plate and incubated at 37°C, 5% CO2 for 24 hours to allow the cells to adhere to the wall. After the incubation, 50μL or 10μL of siRNA transfection complex was added to each well, that is, the siRNA concentration in the well was 10nM and 2nM, respectively. After culturing in a cell culture incubator at 37°C, 5% CO2 for 24 hours, RNA was extracted, and RT-qPCR was used to quantify GAPDH mRNA using β-actin mRNA as an internal reference.

[0095] 3. RNA Extraction

[0096] a. Add 300 μL of lysis buffer to the 24-well plate and gently pipette 8-10 times.

[0097] b. Add an equal volume of binding buffer to the lysate and mix gently.

[0098] c. Transfer the mixture to a purification column, centrifuge at 12,000 g for 30 seconds, and discard the liquid in the collection tube.

[0099] d. Add 600 μL of washing solution I, centrifuge at 12000 g for 30 seconds, and discard the liquid in the collection tube.

[0100] e. Add 600 μL of washing solution II, centrifuge at 12000 g for 30 seconds, and discard the liquid in the collection tube.

[0101] f. Repeat step 5 once.

[0102] Centrifuge at 14000 g for 2 minutes and remove the remaining liquid.

[0103] h. Place the RNA purification column in the RNA elution tube provided by the kit, add 50 μL of elution buffer, leave at room temperature for 2-3 minutes, and centrifuge at 14000g for 30 seconds. The resulting solution is the total RNA sample.

[0104] i. Quantify RNA using Nanodrop 2000. Adjust RNA concentration to about 50 μg / mL for qRT-PCR.

[0105] 4.qRT-PCR detection

[0106] Primer information

[0107]

[0108] One-Step qRT-PCR reaction system setup

[0109]

[0110] qRT-PCR procedure

[0111] a. Reverse transcription: 50℃15min

[0112] b. Pre-denaturation: 95℃2min

[0113] c. Denaturation: 95℃15sec

[0114] d. Annealing / extension: 60℃15sec

[0115] e. Repeat steps c and d for a total of 40 cycles

[0116] f. Use 2 -△△CT Analytical methods for data analysis

[0117] 5. Knockdown efficiency of adding different transfection reagents A and B

[0118] According to the above method, different transfection reagent A liquid and transfection reagent B liquid were added, and the obtained transfection complex was transfected into cells. The GAPDH mRNA level of the experimental group was analyzed, and the relative value of the control corresponding concentration siRNA-NC group was shown in Table 1. It can be seen that for several transfection reagent B liquids, there is an enhancement effect after the introduction of calcium ions and cationic polymers, and the simultaneous introduction of calcium ions and cationic polymers can improve the knockdown efficiency of transfected siRNA more than the introduction of calcium ions alone. In addition, for the prescription of transfection reagent A liquid without cationic lipids and DOPE, the knockdown efficiency of transfected siRNA is very poor.

[0119] Table 1. GAPDH mRNA levels after cells were transfected with different transfection complexes

[0120]

[0121]

[0122] 6. Transfection complex preparation conditions

[0123] The transfection complex was prepared using the transfection reagent A solution 6 and the transfection reagent B solution ③, but the preparation method of the transfection complex was adjusted:

[0124] 6.1 Take 2 μL of siRNA-hGAPDH solution (10 μM), add 30 μL of transfection reagent A solution, mix well, add 4 μL of transfection reagent B solution, mix well, incubate at room temperature for 5 minutes, and add 164 μL of cell culture medium to obtain siRNA transfection complex.

[0125] 6.2 Take 2 μL of siRNA-hGAPDH solution (10 μM), add 7 μL of transfection reagent A solution, mix well, add 2 μL of transfection reagent B solution, mix well, incubate at room temperature for 5 minutes, and add 189 μL of cell culture medium to obtain siRNA transfection complex.

[0126] 6.3 Take 2 μL of siRNA-hGAPDH solution (10 μM), add 14 μL of transfection reagent A solution, mix well, add 4 μL of transfection reagent B solution, mix well, incubate at room temperature for 60 min, add 180 μL of cell culture medium to obtain siRNA transfection complex.

[0127] After transfection of cells with the transfection complex prepared by the above method, the GAPDH mRNA level of the experimental group was analyzed, and the relative value of the control siRNA-NC group with the corresponding concentration was shown in Table 2. It can be seen that the transfection complexes formed by several preparation ratios and time can achieve efficient GAPDH knockdown.

[0128] Table 2. GAPDH mRNA levels after transfection of cells with transfection complexes prepared under different conditions

[0129] Preparation method siRNA 10nM siRNA 2nM 6.1 4％ 10％ 6.2 5％ 15％ 6.3 3％ 14％

[0130] Example 3. Study on mRNA transfection using different transfection reagents

[0131] 1. Transfection complex preparation

[0132] Fluc mRNA was dissolved in RNase-free water at a concentration of 1 mg / mL

[0133] Take 2 μL of Fluc-mRNA solution (1 mg / mL), add 14 μL of transfection reagent A solution, mix well, add 4 μL of transfection reagent B solution, mix well, incubate at room temperature for 10 min, add 180 μL of cell culture medium to obtain mRNA transfection complex.

[0134] 2. Cell Transfection

[0135] HepG2 cells in the logarithmic growth phase were cultured at a rate of 1×10 4 / 0.1mL / well was inoculated in a 96-well white plate and incubated at 37°C, 5% CO2 for 24h to allow the cells to adhere to the wall. After the incubation, 10μL of mRNA transfection complex was added to each well, and 3 replicate wells were set up for each group. After culturing in a 37°C, 5% CO2 cell culture incubator for 24h, the luciferase expression was measured.

[0136] 3. Luciferase Quantification

[0137] Bright-Lite TM Preparation of detection reagents: Melt the Bright-Lite Luciferase Assay Buffer at room temperature, add the entire bottle of melted Bright-Lite Luciferase Assay Buffer to the Bright-Lite Luciferase Assay Substrate, and gently invert 3-5 times to mix to fully dissolve the substrate.

[0138] Take out the cell culture plate to be tested from the incubator and place it at room temperature for 30 minutes to allow the temperature of the culture plate to equilibrate to room temperature.

[0139] Add 100 μL of Bright-Lite to each well. TM Detection reagent. Leave at room temperature for at least 2 minutes to allow cells to fully lyse, and use a multifunctional microplate reader for bioluminescence detection.

[0140] 4. Protein expression efficiency after adding different transfection reagents A and B

[0141] According to the above method, different transfection reagent A solutions and transfection reagent B solutions were added, and the obtained transfection complexes were transfected into cells. The fluorescence values ​​in the fluorescein colorimetric experiment of the experimental group were analyzed, as shown in Table 3. It can be seen that the introduction of calcium ions and cationic polymers in transfection reagent A solution, combined with cationic lipids and DOPE in transfection reagent B solution, can greatly increase the protein expression of mRNA.

[0142] Table 3. Average fluorescence intensity of cells transfected with different mRNA transfection complexes

[0143]

[0144] Example 4. Study on DNA transfection using different transfection reagents

[0145] 1. Transfection complex preparation

[0146] pGL3 DNA was dissolved in enzyme-free water at a concentration of 1 mg / mL

[0147] Take 2 μL of pGL3 DNA solution (1 mg / mL), add 14 μL of transfection reagent A solution, mix well, add 4 μL of transfection reagent B solution, mix well, incubate at room temperature for 10 min, add 180 μL of cell culture medium to obtain DNA transfection complex.

[0148] 2. Cell Transfection

[0149] HEK293T cells in the logarithmic growth phase were cultured at a rate of 1×10 4 / 0.1mL / well was inoculated in a 96-well white plate and incubated at 37°C, 5% CO2 for 24h to allow the cells to adhere to the wall. After the incubation, 10μL of DNA transfection complex was added to each well, and 3 replicate wells were set up for each group. After culturing in a 37°C, 5% CO2 cell culture incubator for 24h, the luciferase expression was measured.

[0150] 3. Luciferase Quantification

[0151] Bright-Lite TM Preparation of detection reagents: Melt the Bright-Lite Luciferase Assay Buffer at room temperature, add the entire bottle of melted Bright-Lite Luciferase Assay Buffer to the Bright-Lite Luciferase Assay Substrate, and gently invert 3-5 times to mix to fully dissolve the substrate.

[0152] Take out the cell culture plate to be tested from the incubator and place it at room temperature for 30 minutes to allow the temperature of the culture plate to equilibrate to room temperature.

[0153] Add 100 μL of Bright-Lite to each well. TM Detection reagent. Leave at room temperature for at least 2 minutes to allow cells to fully lyse, and use a multifunctional microplate reader for bioluminescence detection.

[0154] 4. DNA expression efficiency after adding different transfection reagents A and B

[0155] According to the above method, different transfection reagent A solutions and transfection reagent B solutions were added, and the obtained transfection complexes were transfected into cells. The fluorescence values ​​in the fluorescein colorimetric experiment of the experimental group were analyzed, as shown in Table 4. It can be seen that the introduction of calcium ions and cationic polymers in transfection reagent A solution, combined with cationic lipids and DOPE in transfection reagent B solution, can greatly increase the protein expression of transfected DNA.

[0156] Table 4. Average fluorescence intensity of cells transfected with different DNA transfection complexes

[0157]

[0158]

Claims

1. A composition for delivering nucleic acid into a cell, characterized in that It is prepared by mixing liquid A and liquid B, wherein liquid A is an aqueous liquid containing a cationic polymer and a water-soluble calcium salt, and liquid B is a solution containing a cationic lipid, a neutral lipid and optionally an ethanol solution.

2. The composition for delivering nucleic acid into cells according to claim 1, characterized in that The cationic polymer contained in liquid A is selected from protamine, polyarginine, polylysine or polyethyleneimine, and any combination thereof; preferably, the cationic polymer contained in liquid A is selected from polyethyleneimine and optional protamine, optional polyarginine and / or optional polylysine; more preferably, the cationic polymer contained in liquid A is selected from polyethyleneimine.

3. The composition for delivering nucleic acid into cells according to any one of claims 1 or 2, characterized in that The concentration of the cationic polymer in liquid A is 0.001-1 mg / ml; preferably, the concentration of the cationic polymer in liquid A is 0.001-0.5 mg / ml; more preferably, the concentration of the cationic polymer in liquid A is 0.01-0.1 mg / ml; further preferably, the concentration of the cationic polymer in liquid A is 0.01-0.05 mg / ml.

4. The composition for delivering nucleic acid into cells according to any one of claims 1 to 3, characterized in that The water-soluble calcium salt in liquid A is selected from calcium acetate, calcium chloride, calcium sodium EDTA, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium hydrogen sulfate, calcium bisulfite, calcium bromide, calcium iodide, calcium citrate, calcium lactate, and calcium gluconate, and is further preferably calcium acetate, calcium sodium EDTA, calcium gluconate, calcium citrate, calcium lactate, and calcium gluconate, and is further preferably calcium acetate; Preferably, the calcium salt concentration is 50 mmol / L to 1000 mmol / L; preferably, the calcium salt concentration is 50 to 150 mmol / L, 150 to 200 mmol / L, 200 to 400 mmol / L, 400 to 600 mmol / L, or 600 to 800 mmol / L; More preferably, the calcium salt concentration is 100±50mmol / L, 200±50mmol / L, 300±50mmol / L, 400±50mmol / L, 500±50mmol / L, 600±50mmol / L, 700±50mmol / L, 800±50mmol / L or 900±50mmol / L; More preferably, the calcium salt concentration is 200±20 mmol / L.

5. The composition for delivering nucleic acid into cells according to any one of claims 1 to 4, characterized in that The pH value of liquid A is weakly acidic or neutral; preferably, the pH value of liquid A is 4-7.5; more preferably, the pH value of liquid A is 5±0.5, 6±0.5, 7±0.5; more preferably, the pH value of liquid A is 5±0.5; more preferably, the pH value of liquid A is 5.

6. The composition for delivering nucleic acid into cells according to any one of claims 1 to 5, characterized in that The cationic lipid in liquid B is selected from one of DC-Chol, DOSPA, MVL5, DSDMA, DODMA, DOIC, DOTAP, DODAP, DOTMA or any combination of the foregoing cationic lipids; preferably, the cationic lipid is selected from DOSPA and / or MVL5; more preferably, the cationic lipid is selected from DOSPA or MVL5.

7. The composition for delivering nucleic acid into cells according to any one of claims 1 to 6, characterized in that The neutral lipids in liquid B are selected from one or more of egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, distearoylphosphatidylinositol, dimyristoylphosphatidylinositol, dipalmitoylphosphatidylinositol, dioleoylphosphatidylinositol, 9A1P9, and 10A1P10; preferably, the neutral phospholipids are selected from one or more of distearoylphosphatidylcholine, phosphatidylcholine, egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, and phosphatidylethanolamine; more preferably, the neutral phospholipids are selected from dioleoylphosphatidylethanolamine (DOPE).

8. The composition for delivering nucleic acid into cells according to any one of claims 1 to 7, characterized in that The molar ratio of cationic lipids to neutral lipids in liquid B is 1:99 to 99:1; preferably, the molar ratio of cationic lipids to neutral lipids in liquid B is 20:80 to 80:20; more preferably, the molar ratio of cationic lipids to neutral lipids in liquid B is 40:60 to 60:

40.

9. The composition for delivering nucleic acid into cells according to any one of claims 1 to 8, characterized in that The total lipid concentration in liquid B is 1-100 mM; preferably, the total lipid concentration in liquid B is 3-80 mM; more preferably, the total lipid concentration in liquid B is 3-50 mM; more preferably, the total lipid concentration in liquid B is 3-5 mM.

10. The composition for delivering nucleic acid into cells according to any one of claims 1 to 9, characterized in that The volume ratio of liquid A to liquid B is 1:2 to 100:2; preferably, the volume ratio of liquid A to liquid B is 2:2 to 80:2; more preferably, the volume ratio of liquid A to liquid B is 3:2 to 50:2.

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