WO2022154239A1 - 유전자 전달능을 갖는 폴리에틸렌이민-콜산 이온결합 화합물 및 이의 용도 - Google Patents
유전자 전달능을 갖는 폴리에틸렌이민-콜산 이온결합 화합물 및 이의 용도 Download PDFInfo
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- WO2022154239A1 WO2022154239A1 PCT/KR2021/016986 KR2021016986W WO2022154239A1 WO 2022154239 A1 WO2022154239 A1 WO 2022154239A1 KR 2021016986 W KR2021016986 W KR 2021016986W WO 2022154239 A1 WO2022154239 A1 WO 2022154239A1
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- gene
- gene delivery
- delivery system
- polyethyleneimine
- cholic acid
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- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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- C—CHEMISTRY; METALLURGY
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Definitions
- the present invention relates to a polyethylenimine-cholic acid ionic compound having gene transfer ability and uses thereof, and more particularly, to a compound in which polyethyleneimine and cholic acid are ionically bound, a preparation method thereof, a gene transfer method thereof, and a use for gene transfer .
- Gene therapy is all genetic defects such as cancer, infectious disease, autoimmune disease, etc. caused by genetic modification of cells or by correcting a genetic defect by injecting genetic material such as pDNA or siRNA into the patient's cells. how to prevent or treat Such gene therapy is attracting attention as an innovative treatment method capable of treating cancer or diseases caused by genetic modification.
- biocompatible polymers are used in the development of effective systems for delivering various therapeutic agents, such as chemical drugs, contrast agents, peptides, proteins, and genetic materials, as constituents of drug delivery systems.
- polyethyleneimine polyethylenimine, PEI
- PEI polyethylenimine
- a cationic polymer consists of a high concentration of cationic amine groups, and can form colloidal particles by compressing negatively charged nucleic acid substances, and through endocytosis, intracellular Translocation is reported to be possible.
- Gene delivery is largely divided into three categories, such as cell membrane passage, endosome escape, and nuclear membrane passage. Unlike other gene delivery systems, the complex using polyethyleneimine effectively escapes endosomes through the proton sponge effect using pH buffering ability. process is possible.
- a relatively high molecular weight polyethyleneimine is capable of effective gene transfer but has strong cytotoxicity, and a low molecular weight polyethyleneimine has less cytotoxicity, but has a disadvantage in that the gene transfer efficiency is relatively low.
- cholic acid shows high hydrophilicity and biocompatibility compared to cholesterol, and can effectively destabilize cell membranes due to its amphiphilicity, so it is effective in constructing a gene delivery system.
- a ligand for a steroid receptor expressed on the nuclear membrane it helps in gene transfer efficiency. According to a previous study of synthesizing a polyethyleneimine derivative using cholic acid, the gene transfer efficiency was improved, but there is a difficulty in synthesizing the derivative through a complex chemical formula.
- the present invention easily forms derivatives of various types of cholic acid and polyethylenimine having various molecular weights, ionically bonding them, and reveals the effectiveness of their gene delivery system.
- An object of the present invention is to provide a gene delivery system having low toxicity and effective gene transfer efficiency, a method for preparing the same, and an intracellular gene delivery method using the same.
- the present invention provides a gene delivery system represented by the following formula (1) in which polyethyleneimine and cholic acid are ionically bonded.
- n is an integer of 58 to 930.
- the present invention is to provide a composition for gene delivery comprising the gene delivery system and the gene.
- the present invention comprises the steps of (a) dissolving polyethyleneimine in an alcohol solution and reacting by adding an acid solution; and (b) mixing cholic acid with the solution and sonicating after reaction to obtain the gene delivery system.
- the present invention provides a gene delivery method comprising the step of contacting the gene delivery system with a cell.
- the polyethylenimine-cholic acid derivative according to the present invention has low toxicity and excellent gene transfer efficiency, so it is useful for gene transfer and can be widely applied to gene therapy.
- 1 is a view showing a process for preparing a compound in which polyethyleneimine and cholic acid are ionically bonded using three kinds of polyethyleneimine and three kinds of cholic acid.
- Figure 2 shows the results of analysis of lithocholized linear polyethyleneimine (LPL) by Fourier transform infrared spectroscopy (FT-IR).
- LPL lithocholized linear polyethyleneimine
- Figure 3 shows the results of comparing the gene transfer efficiency and cytotoxicity of lithocylated linear polyethyleneimine (LPL) ionically bonded to PLC using covalent bonding in Chinese hamster ovary cells (CHO) and cervical cancer cells (HeLa).
- LPL lithocylated linear polyethyleneimine
- FIG. 4 shows the results of evaluating the transfection efficiency and cytotoxicity of the gene delivery system synthesized in Chinese hamster ovary cells (CHO).
- 5 shows the results of evaluating the transfection efficiency and cytotoxicity according to the amount of polyethylenimine and DNA used (weight ratio) of the gene delivery system synthesized in Chinese hamster ovary cells (CHO).
- the present invention confirmed that the compound in which polyethyleneimine and cholic acid are ionically bonded has low toxicity and has excellent gene transfer efficiency in several cell lines (Chinese hamster ovary cells (CHO) and cervical cancer cells (HeLa)). reached
- the present invention provides a gene delivery system represented by the following formula (1) in which polyethyleneimine and cholic acid are ionically bonded.
- m is an integer of 2 to 930
- n is an integer of 58 to 930.
- 1 is a schematic diagram showing the process of ion bonding between polyethyleneimine and cholic acid.
- the polyethyleneimine may be linear polyethyleneimine or branched polyethyleneimine. Preferably, it may be a linear polyethyleneimine.
- the molecular weight of the linear polyethyleneimine when the molecular weight of the linear polyethyleneimine is small, gene transfer efficiency may be low, and if the molecular weight is large, cytotoxicity may appear.
- one branched chain of branched polyethyleneimine exists per 3 to 3.5 of the main chain nitrogen atoms, and such polyethyleneimine is soluble in water, alcohol, glycol, dimethylformamide, tetrahydrofuran, esters, etc., It is known that it is insoluble in high molecular weight hydrocarbons, oleic acid, and diethyl ether.
- polyethyleneimine can be crosslinked with ketones by slowly reacting with most chlorinated solvents.
- the polyethyleneimine may have a weight average molecular weight of 2,500 to 40,000. If the weight average molecular weight is less than 2,500, there is a limit to transfection, and if it is more than 40,000, there is a limit to cytotoxicity, so it is better to use one within the above range.
- the cholic acid may be at least one selected from the group consisting of lithocholic acid, deoxycholic acid, and taurocholic acid, but is not limited thereto.
- the name of the compound was named according to the type of cholic acid and the type of polyethyleneimine.
- a compound using lithocholic acid and linear polyethyleneimine (PEI Linear) is called LPL ( Lithocholic acid PEI L inear ) .
- the gene may be selected from the group consisting of gDNA, cDNA, plasmid DNA, mRNA, tRNA, rRNA, antisense nucleotides, missense nucleotides and protein-producing nucleotides.
- genes include epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), tumor growth factor-b (transforming growth factor-b, TGF-b), vascular cell growth factor (vascular endothelial growth factor, vEGF) or may be a gene expressing insulin (insulin), but is not necessarily limited thereto.
- the present invention is a gene delivery system; And it provides a composition for gene delivery comprising a gene.
- the gene delivery system and the gene in a weight ratio of 4 to 6:1, because the gene transfer efficiency is the best while having low toxicity.
- lactose lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, which are commonly used as pharmaceutically acceptable carriers, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.
- composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.
- the present invention comprises the steps of (a) dissolving polyethyleneimine in an alcohol solution and reacting by adding an acid solution; and (b) mixing cholic acid with the solution and sonicating after the reaction to obtain the gene carrier represented by Formula 1 above.
- step (a) polyethyleneimine is dissolved in an alcohol solution and reacted by adding an acid solution.
- the alcohol solution is at least one selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, and hexalol, but is not necessarily limited thereto.
- step (b) cholic acid is mixed with the solution and sonicated after the reaction to obtain the gene carrier represented by Chemical Formula 1, and cholic acid is separately mixed with an alcohol solution and put into the solution obtained in step (a).
- step (b) The solution obtained in step (b) may be completely removed from the solvent through vacuum concentration under reduced pressure and then sonicated to obtain a compound in which polyethyleneimine and cholic acid are ionically bonded.
- step (b) it is preferable to adjust the pH of the solution containing polyethyleneimine and cholic acid obtained in step (b) to 6.9 to 7.1. This is because the polyethylenimine and cholic acid ionic compound can be prepared most efficiently.
- step (b) it is most preferable in terms of yield to react for 1 to 3 hours after mixing cholic acid in step (b), but this can be changed depending on the reaction conditions.
- the present invention provides a gene delivery method comprising the step of contacting the gene delivery system represented by Formula 1 with a cell in vitro or in vivo.
- TPL Taurocholated Linear Polyethylenimine
- Example 1-2 It was prepared in the same manner as in Example 1-2, except that linear polyethyleneimine having a weight average molecular weight of 4,500 was used instead of linear polyethyleneimine having a weight average molecular weight of 2,500, and deoxycholic acid was used instead of lithocholic acid (Fig. One).
- Example 1-2 It was prepared in the same manner as in Example 1-2, except that a linear polyethyleneimine having a weight average molecular weight of 40,000 was used instead of a linear polyethyleneimine having a weight average molecular weight of 2,500, and deoxycholic acid was used instead of lithocholic acid (Fig. One).
- the present inventors transfected the compound prepared in Example 1 into Chinese hamster ovary cells (CHO) and cervical cancer cells (HeLa) and evaluated the cytotoxicity.
- the CHO cell line (KCLB, Republic of Korea) contains F-12K (Hyclone, USA), 10% bovine serum (FBS, Hyclone), 1% penicillin/streptomycin (Hyclone), and 1% L-glutamine. cultured in the medium. Cells from passages 5-7 were used in the study. After culturing 8,000 CHO cells per well in a 96-well plate for one day, a transfection experiment was performed when the cells in each well grew to 70% or more.
- HeLa cell line (KCBL, Republic of Korea) was cultured with MEM (Hyclone, USA), 10% bovine serum (FBS, Hyclone), 1% penicillin/streptomycin (Hyclone), and 1% L-glutamine. Cultured in medium. Cells from passages 5-7 were used in the study. After culturing 10,000 HeLa cells per well of a 96-well plate for one day, a transfection experiment was performed when the cells in each well grew to 70% or more.
- a plasmid DNA-lipid (Example 1) mixture was prepared.
- green fluorescent (GFP) inserted plasmid DNA was used as plasmid DNA, and 1 ⁇ g of plasmid DNA was mixed with 10 ⁇ l of bovine serum-free medium.
- PLC synthesized using covalent bonding and 4 ⁇ g of the compound of Example 1-1 (LPL) synthesized using ionic bonding were mixed in 10 ⁇ l of each bovine serum-free medium and prepared.
- Figure 3-a shows the results of measuring the expression level of fluorescence in both cell lines through a fluorometer.
- the expression is similar to that of LFA2000, whereas in the case of LPL using an ionic bond, 20% or more is delivered. Efficiency increased. Therefore, it was found that the delivery system using ionic bonds better delivered the nucleic acid material into the cell than the delivery system using covalent bonds.
- Figure 3-b shows the results of cytotoxicity experiments in two cell lines, LFA2K showed a very large cytotoxicity compared to the untreated group, whereas the two synthesized gene carriers showed significantly reduced cytotoxicity.
- Figures 3-c and 3-d are the results of observing the cell viability and fluorescence expression under a microscope. In a bright field, it can be confirmed that the cell viability is significantly higher than that of the control group, and the green fluorescence In expression, a markedly increased green fluorescence can be observed. Through this, it was found that the gene delivery system (LPL) using ionic bonding reduced cytotoxicity compared to commercial products, and the delivery ability was increased compared to the gene delivery system (PLC) using covalent bonding.
- LPL gene delivery system
- PLC gene delivery system
- CHO cell line (KCLB, Republic of Korea) is culture medium containing F-12K (Hyclone, USA) + 10% bovine serum (FBS, Hyclone), 1% penicillin/streptomycin (Hyclone), and 1% L-glutamine was cultured, and passages 5-7 cells were used for the study. After culturing 8,000 CHO cells in a 96-well plate for one day, a transfection experiment was performed when the cells in each well grew to 70% or more.
- a plasmid DNA-lipid (Examples 1-1 to 1-9) mixture was prepared.
- green fluorescent (GFP) inserted plasmid DNA was used as plasmid DNA, and 1 ⁇ g of plasmid DNA was mixed with 10 ⁇ l of bovine serum-free medium.
- Examples 1-1 to 1-9 Compounds 4 ⁇ g were prepared by mixing 10 ⁇ l of each bovine serum-free medium. After mixing the two dilutions well, they were left at room temperature for 30 minutes, and the mixture thus prepared was added to the plate and incubated for 24 hours in a CO2 incubator at 37°C. The expressed green fluorescent protein was observed under a fluorescence microscope, and cytotoxicity was evaluated through WST assay (FIG. 4).
- Figure 4-a shows the result of measuring the expression level of fluorescence through a fluorometer.
- polyethyleneimine (2500, 40000) alone the expression was only half compared to LFA2000, whereas most of the synthesized gene delivery systems (implemented) Examples 1-1 to 1-9) increased significantly. Therefore, it was found that the synthesized gene delivery systems have the ability to deliver nucleic acid substances into cells with good efficiency.
- Figure 4-b shows the results of the cytotoxicity experiment, LFA2K showed a very large cytotoxicity compared to the untreated group, whereas the synthesized gene carriers showed reduced cytotoxicity than LFA2K. Therefore, it can be seen that the synthesized gene carriers are carriers with low cytotoxicity.
- Figures 4-c and 4-d are results of observing the cell viability and expression of fluorescence under a microscope. In a bright field, it can be confirmed that the cell viability is significantly higher than that of LFA2K, and the expression of green fluorescence markedly increased green fluorescence can be observed. It was found that the synthesized gene delivery system was an advanced gene delivery material with increased nucleic acid delivery ability while reducing cytotoxicity compared to commercial products.
- DNA and the compounds of Examples 1-1 to 1-9 were used in 1:4, 1:5, and 1:6 ratios to transform according to the DNA and compound ratio To check the efficiency (Fig. 5).
- the experimental method is the same as in Experimental Examples 1 and 2.
- Figure 5-a is the result of measuring the gene transfer efficiency according to the DNA:compound ratio by the expression amount of fluorescence, and showed better delivery ability than Lipofectamine 2000 (LFA2K) in most results, and at a specific ratio, the results of Experimental Example 2 It was confirmed that the nucleic acid delivery ability was further increased than in
- Figure 5-b shows the results of the cytotoxicity experiment, showing the result that the cytotoxicity increased as the ratio of the compound increased, and showed the best cell viability at a ratio of 1:4 to 1:5. The most optimal ratio is 1:4.
- Figures 5-c, 5-d, and 5-e are results obtained by observing the cell viability and fluorescence expression under a microscope, and show appearances corresponding to those of Figures 5-a and 5-b.
- FIG. 6-a shows the results of measuring the gene transfer efficiency according to pH as the expression level of fluorescence, confirming that the expression level of fluorescence is not significantly affected by pH.
- Figure 6-b shows the results of the cytotoxicity experiment, the compound adjusted to pH 6.9 to 7.1 showed a higher cell viability than that not.
- 6-c, 6-d, 6-e, 6-f, 6-g, and 6-h are the results of microscopic observation of cell viability and fluorescence expression, FIG. 6- a, and a figure corresponding to FIG. 6-b is shown.
- 7-c is the result of observing the cell viability and the expression of fluorescence under a microscope, and shows the appearance corresponding to FIGS. 7-a and 7-b.
- the present invention conveniently forms derivatives of various types of cholic acid and polyethylenimine having various molecular weights to form derivatives, and reveals the utility of the gene delivery system.
- the polyethylenimine-cholic acid ionic compound according to the present invention has low toxicity and excellent gene transfer efficiency, so it is useful for gene transfer and can be widely applied to gene therapy.
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Abstract
Description
Claims (10)
- 제 1 항에 있어서,상기 유전자는 gDNA, cDNA, 플라스미드 DNA, mRNA, tRNA, rRNA, 안티센스 뉴클레오티드, 미스센스 뉴클레오티드 및 단백질 생산 뉴클레오티드로 이루어진 군으로부터 선택되는 것을 특징으로 하는, 유전자 전달체.
- 제 1 항에 있어서,상기 폴리에틸렌이민은 중량평균분자량이 2,500 내지 40,000인 선형 또는 가지형 폴리에틸렌이민인 것을 특징으로 하는, 유전자 전달체.
- 제 1 항에 있어서,상기 콜산은 리토콜산(lithocholic acid), 데옥시콜산(deoxycholic acid) 및 타우로콜산(taurocholic acid)로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 유전자 전달체.
- 제 1 항 내지 제 4 항 중에서 선택된 어느 한 항의 유전자 전달체; 및 유전자;를 포함하는 유전자 전달용 조성물.
- 제 5 항에 있어서,상기 유전자는 gDNA, cDNA, 플라스미드 DNA, mRNA, tRNA, rRNA, 안티센스 뉴클레오티드, 미스센스 뉴클레오티드 및 단백질 생산 뉴클레오티드로 이루어진 군으로부터 선택되는 것을 특징으로 하는 유전자 전달용 조성물.
- 제 5 항에 있어서,상기 유전자 전달체 및 유전자는 4 내지 6 : 1 중량비로 포함하는 것을 특징으로 하는 유전자 전달용 조성물.
- (a) 알코올 용액에 폴리에틸렌이민을 용해하고 산 용액을 첨가하여 반응시키는 단계; 및(b) 상기 용액에 콜산을 혼합하고 반응 후 초음파 처리하여 제 1 항 내지 제 5 항 중에서 선택된 어느 한 항의 유전자 전달체를 수득하는 단계;를 포함하는 유전자 전달체의 제조방법.
- 제 8 항에 있어서,상기 (b) 단계는 폴리에틸렌이민 및 콜산이 포함된 용액의 pH를 6.9 내지 7.1로 조절하는 단계를 더 포함하는 것을 특징으로 하는 유전자 전달체의 제조방법.
- 제 1 항 내지 제 4 항 중에서 선택된 어느 한 항의 유전자 전달체를 세포와 접촉시키는 단계를 포함하는, 유전자의 전달방법.
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CN202180095399.4A CN116963783A (zh) | 2021-01-13 | 2021-11-18 | 具有基因转移活性的聚乙烯亚胺-胆酸的以离子键键合的化合物及其用途 |
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