A composition for gene therapy or transfection, and a method for preparing the same and use thereof

Abstract

The present application provides a kind of gene therapy or transfection composition, including nucleic acid, natural polyphenol and cationic molecule. Wherein, natural polyphenol contains multiple phenolic hydroxyl group, has the effect of scavenging free radical, anti-tumor, anti-inflammatory, antioxidant and the like. Compared with the nucleic acid delivery using cationic molecule alone, the transfection efficiency of the composition can be significantly improved by about 60-70%, the effective transfection dose of cationic molecule is reduced by about 20 times, thereby reducing the toxicity of transfection, showing better biocompatibility. The composition described in the present application does not introduce other chemical reagents, does not need to be synthesized, has high biological safety, can efficiently and safely deliver a variety of nucleic acid molecules into cells, can be applied to the treatment of gene-related diseases as high-efficiency and low-toxicity nucleic acid drugs, and has good medicinal value.

Description

Technical Field

[0001] This invention relates to a composition for gene therapy or transfection, belonging to the fields of biochemistry and biomaterials technology. Background Technology

[0002] Gene therapy, as a novel treatment approach, is widely used to treat various diseases, such as tumors, genetic diseases, inflammation, immune disorders, endocrine imbalances, mental disorders, and cardiovascular diseases. It primarily works by introducing exogenous genes into target cells to alter a series of vital processes, including transcription and translation of existing genes, protein expression, and enzyme synthesis, thereby regulating these processes and achieving therapeutic effects. The success of gene therapy lies in its ability to efficiently and safely deliver exogenous genes into target cells. Therefore, highly efficient and safe gene vectors are crucial.

[0003] Cationic molecules, a major class of non-viral vectors, primarily enter cells via endocytosis after forming positively charged complexes with nucleic acid molecules through electrostatic interactions. However, a vicious cycle exists between the transfection efficiency and cytotoxicity of cationic molecules. The greater the positive charge and molecular weight of a cationic molecule, the better the transfection efficiency, but also the higher the cytotoxicity. Conversely, low-molecular-weight cationic molecules, such as peptides or polymers, are more biocompatible, but due to their lower surface positive charge, they bind weakly to nucleic acids. These weak complexes become unstable when interfered with by proteins, phospholipids, and other molecules, leading to premature release of nucleic acids and low transfection efficiency. This vicious cycle between transfection efficiency and cytotoxicity limits the development of cationic molecules in biomedicine and clinical gene therapy.

[0004] Many existing gene transfection methods are complex, the material synthesis process is complicated, the composition is uncontrollable, the gene transfection effect is poor, and the toxicity is high. Summary of the Invention

[0005] This invention addresses the problem of weak binding of cationic molecules to nucleic acids by introducing natural polyphenols. Natural polyphenols bind to nucleic acids through hydrogen bonds, forming negatively charged nanoparticles, which then bind to cationic molecules, helping to stabilize the nucleic acids and form more uniform nanoparticles (such as…). Figure 1 and 2This composition improves the transfection efficiency of cationic molecules, reduces the amount of cationic molecules needed, and exhibits good biocompatibility. Compared with the use of cationic molecules alone for delivery, this composition can significantly improve transfection efficiency by approximately 60-70%, reduce the effective transfection dose of cationic molecules by approximately 20 times, thereby reducing transfection toxicity and demonstrating better biocompatibility. The composition described in this invention does not introduce other chemical reagents, requires no synthesis, has high biocompatibility, and can efficiently and safely deliver various nucleic acid molecules into cells. Simultaneously, it utilizes natural polyphenols to scavenge reactive oxygen species generated during transfection, and leverages the antitumor, anti-inflammatory, and antioxidant effects of natural polyphenols to fully realize the therapeutic potential of this composition in the treatment of tumors and related inflammatory diseases.

[0006] This invention is achieved through the following technical solution:

[0007] A composition for gene therapy or transfection comprising nucleic acids, natural polyphenols, and cationic polymers.

[0008] As a preferred embodiment, the nucleic acid includes one of siRNA, miRNA, lncRNA, mRNA, or modified RNA.

[0009] As a preferred embodiment, the nucleic acid includes one of siRNA, miRNA, oligonucleotide, peptide nucleic acid, and modified siRNA.

[0010] As a preferred embodiment, the nucleic acid is siRNA or modified siRNA.

[0011] As a preferred embodiment, the natural polyphenols are selected from at least one of flavanols, anthocyanins, flavonoids, flavonols, and phenolic acid plant extracts.

[0012] As a preferred embodiment, the natural polyphenols include at least one selected from the following: epigallocatechin gallate, epicatechin, epigallocatechin (EGCG), epicatechin gallate, tannin (TA), 1,2,3,4,6-pentagalloglucoside, 1,2,3,6-tetragalloglucoside, 1,3,6-trigalloglucoside, proanthocyanidins, anthocyanins, ellagic acid, and caffeic acid.

[0013] As a preferred embodiment, the cationic molecule includes at least one of cationic polymers, cationic natural small molecule compounds, cationic polypeptides, and positively charged proteins.

[0014] As a preferred embodiment, the cationic molecule includes at least one of polyamide-amine dendritic polymer, polypropylene imine dendritic polymer, polylysine cationic polymer, branched polyethyleneimine cationic polymer, and linear polyethyleneimine cationic polymer.

[0015] As a preferred embodiment, the cationic molecule includes a compound of formula I, II, III, IV, and V:

[0016]

[0017] Wherein, M includes one of ammonia, ethylenediamine, butanediamine, hexamethylenediamine, octanediamine, decanediamine, and 1,12-dodecanediamine, n is an integer from 1 to 10, m is an integer from 2 to 4, a is an integer from 1 to 100,000, the mass-average molecular weight of compound III is 100 to 100,000, the mass-average molecular weight of compound IV is 100 to 100,000, and the mass-average molecular weight of compound V is 100 to 100,000; the cationic polypeptide includes cell-penetrating peptides.

[0018] A method for preparing the composition as described above includes the following steps: mixing nucleic acids, natural polyphenols and cationic molecules in a mass ratio of 1:0.01 to 100000:0.01 to 100000.

[0019] Use of a composition as described above in gene transfection or gene therapy drugs.

[0020] As a preferred embodiment, the drug includes one of the following: oncology drugs, anti-inflammatory drugs, immune disorder drugs, endocrine disorder drugs, mental disorder drugs, cardiovascular drugs, and dermatology drugs.

[0021] Natural polyphenols are a class of polyphenolic compounds widely found in plants, including phenolic acids and flavonoids. These polyphenolic compounds have several phenolic hydroxyl groups in their molecular structure. Numerous in vitro and in vivo experiments have demonstrated that these polyphenolic compounds have significant effects in scavenging free radicals, anti-oxidation, UV protection, anti-obesity, preventing cardiovascular disease, anti-inflammation, and preventing and treating cancer. They are commonly used as additives in health products and cosmetics. Simultaneously, these molecules are also excellent hydrogen bond donors, capable of forming complexes with nucleic acids through hydrogen bonds, protecting nucleic acids from degradation by nucleases.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The method described in this invention does not introduce other chemical reagents, requires no synthesis, uses natural materials, and has high biosafety. It can effectively and safely deliver nucleic acids into cells and can serve as a gene transfection method with the advantages of high efficiency, low toxicity, and low cost. It has good application prospects in the treatment of gene-related diseases such as tumors, chronic enteritis, skin wound healing, and immune disorders. Attached Figure Description

[0024] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0025] Figure 1 The sizes of the two complexes, siRNA / PLL and siRNA / EGCG / PLL, in Example 1;

[0026] Figure 2 The morphology of the two complexes, siRNA / PLL and siRNA / EGCG / PLL, in Example 1 is shown.

[0027] Figure 3 This illustrates the endocytosis of different complexes in Example 2.

[0028] Figure 4 This refers to the efficiency of knocking out the luciferase gene in Hela-luci cells using different combinations of the three components of this composition (EGCG) in Example 3.

[0029] Figure 5 The efficiency of this composition (EGCG) in knocking out the luciferase gene in Hela-luci cells using different cationic polymers in Example 4;

[0030] Figure 6 This demonstrates the specificity of this composition (EGCG) in knocking out the luciferase gene in Hela-luci cells, as shown in Example 5.

[0031] Figure 7 The efficiency and specificity of this composition (EGCG) in knocking out the luciferase gene in human breast cancer cells (MDA-MB-231) in Example 6;

[0032] Figure 8 The efficiency of this composition (EGCG) in knocking out matrix metalloproteinase MMP-9 in brain cancer cells U87 in Example 7;

[0033] Figure 9 The efficiency of this composition (EGCG) in knocking out glyceraldehyde-3-phosphate dehydrogenase GAPDH in human lung adenocarcinoma cells PC-9 in Example 8;

[0034] Figure 10 The efficiency of this composition (EGCG) in knocking out proline hydroxylase PHD2 in mouse fibroblasts NIH-3T3 is shown in Example 9.

[0035] Figure 11 This is the efficiency of the composition (EGCG) in Example 10 in knocking out proline hydroxylase PHD2 in mouse intestinal epithelial cells IEC;

[0036] Figure 12The efficiency of this composition (EGCG) in knocking out proline hydroxylase PHD2 in mouse macrophages RAW264.7 in Example 11;

[0037] Figure 13 The effect of this composition (EGCG) in knocking out proline hydroxylase PHD2 in mice with enteritis in Example 12 and its therapeutic effect on enteritis;

[0038] Figure 14 The efficiency of this composition (TA) in knocking out the luciferase gene in Hela-luci cells using different cationic polymers in Example 13.

[0039] Figure 15 This refers to the efficiency of this composition (catechin) in knocking out the luciferase gene in Hela-luci cells in Example 14.

[0040] Figure 16 The cytotoxicity of EGCG in Hela-luci cells in Example 15;

[0041] Figure 17 The cytotoxicity of EGCG and LPEI in Hela-luci cells as shown in Example 16;

[0042] Figure 18 The cytotoxicity of EGCG and BPEI in Hela-luci cells as shown in Example 17;

[0043] Figure 19 The cytotoxicity of EGCG and PLL in Hela-luci cells as shown in Example 18;

[0044] Figure 20 The cytotoxicity of EGCG and PAMAM G1 in Hela-luci cells as shown in Example 19;

[0045] Figure 21 The cytotoxicity of EGCG and PAMAM G2 in Hela-luci cells in Example 20. Detailed Implementation

[0046] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0047] Example 1 This example involves a comparison of the size and morphology of two complexes: siRNA / PLL and siRNA / EGCG / PLL.

[0048] A complex was formed at room temperature by combining natural polyphenol EGCG and luciferase siRNA (mass ratio 5:1), and then polylysine cationic polymer PLL was added. The size and morphology of the complex were observed by dynamic light scattering and transmission electron microscopy.

[0049] Experimental results:

[0050] like Figure 1 The figures show the sizes of two complexes: siRNA / PLL and siRNA / EGCG / PLL. Figure 2 The electron microscopy morphology of the two complexes, siRNA / PLL and siRNA / EGCG / PLL, is shown. The results indicate that, compared with PLL alone, the addition of EGCG enables the complex to form nanoparticles with more uniform size and better morphology.

[0051] Example 2 This example involves the cellular uptake of different complexes.

[0052] Natural polyphenol EGCG and fluorescently tagged luciferase siRNA (FAM-siRNA) were combined to form a complex at room temperature. Then, cationic polymers PLL and PAMAM (mass ratio of siRNA:EGCG:PLL = 1:5:5, siRNA:EGCG:PAMAM = 1:10:10) were added, and the mixture was incubated before being added to cells. The cellular uptake of siRNA was then observed.

[0053] Experimental results:

[0054] like Figure 3 The results show that siRNA uptake by different complexes was enhanced after the addition of EGCG compared to cationic molecules alone.

[0055] Example 3 This example involves the efficiency of knocking out the luciferase gene in Hela-luci cells using different combinations of nucleic acids, natural polyphenols, and cationic molecules.

[0056] The three components of the composition were incubated at room temperature in different combinations, and then transfected into Hela-luci cells. The gene transfection efficiency of the complex was evaluated by detecting the expression level of luciferase.

[0057] Experimental results:

[0058] like Figure 4The results show the efficiency of different component combinations in knocking out the luciferase gene in Hela-luci cells. The results indicate that if there are only one or two components, no matter how they are combined, the luciferase gene cannot be effectively knocked out. However, when all three are combined (mass ratio of siRNA:EGCG:PLL = 1:5:5), the gene can be effectively knocked out by 77.2%. All three components are indispensable.

[0059] Example 4 This example demonstrates the efficiency of knocking out the luciferase gene in Hela-luci cells using different cationic polymers.

[0060] Natural polyphenol EGCG and luciferase siRNA (siLuc) were combined to form a complex at room temperature. Then, different cationic molecules (mass ratios of siRNA:EGCG:PLL = 1:5:5, siRNA:EGCG:LPEI = 1:5:5, siRNA:EGCG:DGL = 1:10:10, siRNA:EGCG:BPEI = 1:10:10, siRNA:EGCG:PPI = 1:10:10, siRNA:EGCG:PAMAM = 1:10:10) were added and incubated. After incubation, the complex was transfected into Hela-luci cells. The gene transfection efficiency of the complex was evaluated by detecting the expression level of luciferase.

[0061] Experimental results:

[0062] like Figure 5 The results show the efficiency of different cationic polymers in knocking out the luciferase gene in this composition. The results indicate that different cationic molecules can effectively knock out the gene by 65-80%.

[0063] Example 5 This example relates to the specificity of a composition in knocking out the luciferase gene in Hela-luci cells.

[0064] Natural polyphenol EGCG, luciferase siRNA (siLuc), and meaningless sequence siRNA (siNC) were combined to form a complex at room temperature. Then, polylysine cationic polymer PLL (mass ratio of siRNA:EGCG:PLL = 1:5:5, siNC:EGCG:PLL = 1:5:5) was added. After incubation, the complex was transfected into Hela-luci cells. The gene transfection efficiency of the complex was evaluated by detecting the expression level of luciferase.

[0065] Experimental results:

[0066] like Figure 6As shown, only the siRNA group effectively knocked out genes, while the siNC group did not effectively silence genes. The results indicate that the gene silencing effect of this combination is specific, and no non-targeted knockout occurred.

[0067] Example 6 This example relates to the efficiency and specificity of a composition in knocking out the luciferase gene in human breast cancer cells (MDA-MB-231).

[0068] A complex was formed by combining natural polyphenol EGCG, luciferase siRNA (siLuc), and meaningless sequence siRNA (siNC) at room temperature. Then, polylysine cationic polymer PLL (mass ratio of siRNA:EGCG:PLL = 1:20:20, siNC:EGCG:PLL = 1:20:20) was added. After incubation, the complex was transfected into human breast cancer cells (MDA-MB-231). The gene transfection efficiency of the complex was evaluated by detecting the expression level of luciferase.

[0069] Experimental results:

[0070] like Figure 7 As shown, this composition can also achieve 78% gene silencing in human breast cancer cells (MDA-MB-231) without nonspecific knockout.

[0071] Example 7 This example relates to the efficiency of a composition in knocking out matrix metalloproteinase MMP-9 in U87 brain cancer cells.

[0072] Natural polyphenol EGCG, MMP-9 siRNA, and meaningless sequence siRNA (siNC) were combined to form a complex at room temperature. Then, polylysine cationic polymer PLL (mass ratio of siRNA:EGCG:PLL = 1:5:5, siNC:EGCG:PLL = 1:5:5) was added. After incubation, the complex was transfected into U87 cells. The gene transfection efficiency of the complex was evaluated by detecting the expression level of MMP-9 mRNA using quantitative real-time RT-PCR.

[0073] Experimental results:

[0074] like Figure 8 As shown, this composition can perform specific gene knockout in U87 cells with an efficiency of 79%.

[0075] Example 8 This example relates to the efficiency of a composition in knocking out glyceraldehyde-3-phosphate dehydrogenase GAPDH in human lung adenocarcinoma cells PC-9.

[0076] Natural polyphenol EGCG and GAPDH siRNA and meaningless sequence siRNA (siNC) were combined to form a complex at room temperature. Then, polylysine cationic polymer PLL (mass ratio of siRNA:EGCG:PLL = 1:5:5, siNC:EGCG:PLL = 1:5:5) was added. After incubation, the complex was transfected into PC-9 cells. The gene transfection efficiency of the complex was evaluated by detecting the expression level of GAPDH mRNA using quantitative real-time RT-PCR.

[0077] Experimental results:

[0078] like Figure 9 As shown, this composition can perform specific gene knockout in PC-9 cells with an efficiency of 89%.

[0079] Example 9 This example relates to the efficiency of a composition in knocking out proline hydroxylase PHD2 in mouse fibroblast NIH-3T3 cells.

[0080] Natural polyphenol EGCG, PHD2 siRNA, and meaningless sequence siRNA (siNC) were combined to form a complex at room temperature. Then, polylysine cationic polymer PLL (mass ratio of siRNA:EGCG:PLL = 1:5:5, siNC:EGCG:PLL = 1:5:5) was added. After incubation, the complex was transfected into NIH-3T3 cells. The gene transfection efficiency of the complex was evaluated by detecting the expression level of PHD2H mRNA using quantitative real-time RT-PCR.

[0081] Experimental results:

[0082] like Figure 10 As shown, this composition can perform specific gene knockout in NIH-3T3 cells with an efficiency of 70%.

[0083] Example 10 This example relates to the efficiency of a composition in knocking out proline hydroxylase PHD2 in mouse intestinal epithelial cells (IECs).

[0084] Natural polyphenol EGCG, PHD2 siRNA, and meaningless sequence siRNA (siNC) were combined to form a complex at room temperature. Then, polylysine cationic polymer PLL (mass ratio of siRNA:EGCG:PLL = 1:5:5, siNC:EGCG:PLL = 1:5:5) was added. After incubation, the complex was transfected into IEC cells. The gene transfection efficiency of the complex was evaluated by detecting the expression level of PHD2H mRNA using quantitative real-time RT-PCR.

[0085] Experimental results:

[0086] like Figure 11As shown, this composition can perform specific gene knockout in IEC cells with an efficiency of 84%.

[0087] Example 11 This example relates to the efficiency of a composition in knocking out proline hydroxylase PHD2 in mouse macrophages RAW264.7.

[0088] A complex was formed at room temperature by combining natural polyphenol EGCG, PHD2 siRNA, and meaningless sequence siRNA (siNC). Then, polylysine cationic polymer PLL (mass ratio of siRNA:EGCG:PLL = 1:5:5, siNC:EGCG:PLL = 1:5:5) was added. After incubation, the complex was transfected into RAW264.7 cells. The gene transfection efficiency of the complex was assessed by detecting the expression level of PHD2 mRNA using quantitative real-time RT-PCR.

[0089] Experimental results:

[0090] like Figure 12 As shown, this composition can perform specific gene knockout in RAW264.7 cells with an efficiency of 79%.

[0091] Example 12 This example relates to a composition for treating chronic enteritis by knocking out proline hydroxylase PHD2 in mice.

[0092] BALB / c mice were induced to develop enteritis using dextran sulfate. A combination of siRNA, EGCG, and PLL (mass ratio siRNA:EGCG:PLL = 1:5:5, siNC:EGCG:PLL = 1:5:5) was administered rectally to deliver PHD2 siRNA into the mouse intestines. The development of enteritis was observed. After 8 days, the inflammatory manifestations (weight change, fecal condition, intestinal bleeding) were assessed and scored. Tissue RNA was then extracted, and PHD2 and TNF-α mRNA levels were detected by RT-PCR to evaluate the therapeutic effect of this combination on mouse enteritis. Normal mice, phosphate-buffered saline (PBS), and siNC served as control groups.

[0093] Experimental results:

[0094] like Figure 13 As shown, in mice, this composition can effectively knock out the PHD2 gene in the mouse intestine and downregulate the expression of the inflammation-related factor TNF-α, thus inhibiting the development of inflammation. Experimental results indicate that this composition can effectively control disease progression by regulating the expression of inflammation-related genes through gene silencing.

[0095] Example 13 This example demonstrates the efficiency of knocking out the luciferase gene in Hela-luci cells using different cationic polymers.

[0096] Natural polyphenol TA and luciferase siRNA (siLuc) were combined to form a complex at room temperature. Then, different cationic molecules were added (mass ratios of siRNA:TA:PLL = 1:2:5, siRNA:TA:LPEI = 1:2:5, siRNA:TA:PAMAM = 1:1:10). After incubation, the complex was transfected into Hela-luci cells. The gene transfection efficiency of the complex was evaluated by detecting the expression level of luciferase.

[0097] Experimental results:

[0098] like Figure 14 The results show the efficiency of different cationic polymers in knocking out the luciferase gene in this composition. The results indicate that different cationic molecules can effectively knock out the gene by 40-55%.

[0099] Example 14 This example relates to the efficiency of a composition (catechin) in knocking out the luciferase gene in Hela-luci cells.

[0100] A complex was formed between natural polyphenol catechin and luciferase siRNA (siLuc) at room temperature. Then, different cationic molecules (mass ratio of siRNA:catechin:LPEI = 1:10:10) were added, and after incubation, the complex was transfected into Hela-luci cells. The gene transfection efficiency of the complex was evaluated by detecting the expression level of luciferase.

[0101] Experimental results:

[0102] like Figure 15 The figure shows the efficiency of knocking out the luciferase gene in this composition. The results show that different cationic molecules can effectively knock out the gene by up to 52%.

[0103] Example 15 Cytotoxicity of natural polyphenol EGCG in Hela-luci cells

[0104] The cytotoxicity of the natural polyphenol EGCG described in this invention was investigated by incubating Hela-luci cells in 96-well plates at a cell density of 102. 4 Cells / wells were cultured for 12 hours, then the culture medium was removed, and 100 μL of fresh culture medium containing the same concentration of siRNA as the transfection concentration and a certain amount of EGCG was added. The EGCG concentration was increased from low to high (including the transfection concentration), with the final concentration being much higher than the transfection concentration. Cells were cultured for 24 hours. Cytotoxicity was detected using the MTT assay.

[0105] Experimental results:

[0106] like Figure 16The natural polyphenol EGCG is shown to have toxic effects on Hela-luci cells. Figure 16 This indicates that the natural polyphenol EGCG still exhibits low cytotoxicity even at doses far exceeding the transfection dose, with a cell survival rate of over 90%, demonstrating low cytotoxicity.

[0107] Example 16 Cytotoxicity of natural polyphenols EGCG and LPEI in HeLa-luci cells

[0108] The cytotoxicity of the natural polyphenol EGCG and cationic polymer LPEI described in this invention was investigated by incubating Hela-luci cells in 96-well plates at a cell density of 102. 4 Cells / wells were cultured for 12 hours, then the culture medium was removed, and 100 μL of fresh culture medium containing the same concentration of siRNA, LPEI, and a certain amount of EGCG as the transfection concentration was added. The EGCG concentration was increased from low to high (including the transfection concentration), with the final concentration being much higher than the transfection concentration. Cells were cultured for 24 hours. Cytotoxicity was detected using the MTT assay.

[0109] Experimental results:

[0110] like Figure 17 The natural polyphenols EGCG and LPEI are shown to have toxic effects on Hela-luci cells. Figure 17 This indicates that, in the presence of cationic polymers, the natural polyphenol EGCG, at doses far exceeding the transfection concentration, still exhibits low cytotoxicity, with cell survival exceeding 90%, demonstrating low cytotoxicity.

[0111] Example 17 Cytotoxicity of natural polyphenols EGCG and BPEI in Hela-luci cells

[0112] The cytotoxicity of the natural polyphenol EGCG and cationic polymer BPEI described in this invention was investigated using the following experimental method: Hela-luci cells were incubated in 96-well plates at a cell density of 102. 4 Cells / wells were cultured for 12 hours, then the culture medium was removed, and 100 μL of fresh culture medium containing the same concentration of siRNA, BPEI, and a certain amount of EGCG as the transfection concentration was added. The EGCG concentration was increased from low to high (including the transfection concentration), with the final concentration being much higher than the transfection concentration. Cells were cultured for 24 hours. Cytotoxicity was detected using the MTT assay.

[0113] Experimental results:

[0114] like Figure 18 The toxicity of natural polyphenols EGCG and BPEI to Hela-luci cells is shown. Figure 18This indicates that, in the presence of cationic polymers, the natural polyphenol EGCG, at doses far exceeding the transfection concentration, still exhibits low cytotoxicity, with cell survival exceeding 90%, demonstrating low cytotoxicity.

[0115] Example 18 Cytotoxicity of natural polyphenols EGCG and PLL in Hela-luci cells

[0116] The cytotoxicity of the natural polyphenol EGCG and cationic polymer PLL described in this invention was investigated using the following experimental method: Hela-luci cells were incubated in 96-well plates at a cell density of 102. 4 Cells / wells were cultured for 12 hours, then the culture medium was removed, and 100 μL of fresh culture medium containing the same concentration of siRNA, PLL, and a certain amount of EGCG as the transfection concentration was added. The EGCG concentration was increased from low to high (including the transfection concentration), with the final concentration being much higher than the transfection concentration. Cells were cultured for 24 hours. Cytotoxicity was detected using the MTT assay.

[0117] Experimental results:

[0118] like Figure 19 The natural polyphenols EGCG and PLL are shown to have toxic effects on Hela-luci cells. Figure 19 This indicates that, in the presence of cationic polymers, the natural polyphenol EGCG, at doses far exceeding the transfection concentration, still exhibits low cytotoxicity, with cell survival exceeding 90%, demonstrating low cytotoxicity.

[0119] Example 19 Cytotoxicity of natural polyphenols EGCG and PAMAM G1 in Hela-luci cells

[0120] The cytotoxicity of the natural polyphenol EGCG and cationic polymer PAMAM G1 described in this invention was investigated using the following experimental method: Hela-luci cells were incubated in 96-well plates at a cell density of 102. 4 Cells / wells were cultured for 12 hours, then the medium was removed, and 100 μL of fresh medium containing the same concentration of siRNA, PAMAM G1, and a certain amount of EGCG was added. The EGCG concentration was increased from low to high (including the transfection concentration), with the final concentration being much higher than the transfection concentration. Cells were cultured for 24 hours. Cytotoxicity was detected using the MTT assay.

[0121] Experimental results:

[0122] like Figure 20 The natural polyphenols EGCG and PAMAM G1 are shown to have toxic effects on Hela-luci cells. Figure 20This indicates that, in the presence of cationic polymers, the natural polyphenol EGCG, at doses far exceeding the transfection concentration, still exhibits low cytotoxicity, with cell survival exceeding 90%, demonstrating low cytotoxicity.

[0123] Example 20 Cytotoxicity of natural polyphenols EGCG and PAMAM G2 in Hela-luci cells

[0124] The cytotoxicity of the natural polyphenol EGCG and cationic polymer PAMAM G2 described in this invention was investigated using the following experimental method: Hela-luci cells were incubated in 96-well plates at a cell density of 102. 4 Cells / wells were cultured for 12 hours, then the medium was removed, and 100 μL of fresh medium containing the same concentration of siRNA, PAMAM G2, and a certain amount of EGCG was added. The EGCG concentration was increased from low to high (including the transfection concentration), with the final concentration being much higher than the transfection concentration. Cells were cultured for 24 hours. Cytotoxicity was detected using the MTT assay.

[0125] Experimental results:

[0126] like Figure 21 The natural polyphenols EGCG and PAMAM G2 are shown to have toxic effects on Hela-luci cells. Figure 21 This indicates that, in the presence of cationic polymers, the natural polyphenol EGCG, at doses far exceeding the transfection concentration, still exhibits low cytotoxicity, with cell survival exceeding 90%, demonstrating low cytotoxicity.

[0127] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A composition for gene therapy or transfection, characterized in that, It includes nucleic acids, natural polyphenols, and cationic macromolecules, wherein the nucleic acids are selected from at least one of siRNA, miRNA, lncRNA, mRNA, or other modified RNA; The natural polyphenols are selected from at least one of epigallocatechin gallate, epicatechin, epigallocatechin, epicatechin gallate, tannins, 1,2,3,4,6-pentagalloglucoside, 1,2,3,6-tetragalloglucoside, and 1,3,6-trigalloglucoside. The cationic polymer is selected from at least one of polylysine (PLL), polyamide-amine dendritic polymer (PAMAM), linear polyethyleneimine (LPEI), dendritic polylysine (DGL), branched polyethyleneimine (BPEI), or polypropyleneimine dendritic polymer (PPI).

2. The composition according to claim 1, characterized in that: This includes siRNA or modified siRNA, epigallocatechin gallate, and polylysine cationic polymers.

3. A method for preparing the composition according to claim 1, characterized in that, The process includes the following steps: mixing nucleic acids, natural polyphenols, and cationic molecules in a certain mass ratio; wherein the mass ratio of the nucleic acids, natural polyphenols, and cationic polymers is 1:1 to 100:1 to 100.

4. Use of the composition of claim 1 in the preparation of gene therapy or transfection medicaments.

5. The use as described in claim 4, characterized in that, The drug is selected from one of the following: oncology drugs, anti-inflammatory drugs, immune disorder drugs, endocrine disorder drugs, mental disorder drugs, cardiovascular drugs, and dermatology drugs.

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