KR20140137739A - Gene Delivery System Comprising a New Cationic Lipid - Google Patents
Gene Delivery System Comprising a New Cationic Lipid Download PDFInfo
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Abstract
The present invention relates to a gene transfer complex comprising a cationic lipid having excellent gene transfer efficiency or a liposome prepared using the cationic lipid and a method for producing the same. The cationic lipids or liposomes of the present invention have significantly improved gene transfer efficiency as compared to conventional gene carriers. In addition, the cationic lipids or liposomes of the present invention are extremely cytotoxic and can be developed as effective gene carriers.
Description
The present invention relates to a gene transfer complex comprising a cationic lipid and a method for producing the same.
Research on human genome has been actively carried out, and studies on the cause and mechanism of disease have been actively conducted. However, due to the difficulty in efficiently transferring genes to cells or tissues, there is a great demand for development of efficient gene carriers.
Generally, gene carriers are divided into viral vectors and non-viral vectors. Examples of viral vectors include retrovirus, adenovirus, adeno-associated virus (AAV), lentivirus, (vaccine virus). They have high gene transfer efficiency, but they are difficult to deliver DNA having a high molecular weight, and have serious problems such as reinfection of virus, induction of immune response or activation of oncogenes gene. 5 (1), 2000. Kremer et al., 2000, Adenovirus technology for gene manipulation, J. Immunol. Br Med Bull 51, 31-44, 1995).
The gene vectors that do not use these viral vectors include cationic liposomes (polyethyleneimine (PEI), dendrimer), which are composed of cationic polymers that can be chemically synthesized, and cationic liposomes Vector is the most widely used. The non-viral vector and DNA molecule complex still have a total cation, approaching the anionic cell membrane and fusing the structure of the lipid membrane unstably and entering the cell, resulting in gene transfer. Non-viral vectors have the advantage that they have no specific immune response, are not limited by DNA size, and are capable of mass production (Eur. J. Pharm. Biopharm. 50, 101-119, 2000; Ch.Garcia-Chaumont et al Colin W Pouton et al., Adv. Drug Deliv. Rev. 46, 187-203, 2001), the delivery efficiency was lower than that of the viral vector, And thus there is a problem that the intracellular delivery efficiency is not constant and the cell membrane is damaged and toxicity is exhibited (Amarnath Sharma et al., International Journal of pharmaceutics, 154, 123-140, 1997; Saghir Akhtar et al., Adv.Drug Deliv. Rev. 44, 3-21, 2000). Therefore, there is a demand for development of a nonviral vector that enhances gene transfer efficiency by complementing and improving such problems of nonviral vectors.
Recently, studies have been conducted to apply a cholesterol component constituting a bilayer structure of a eukaryotic cell membrane as a nonviral vector. The eukaryotic membrane has a bilayer structure and consists mainly of phospholipids, sphingolipids and cholesterol, and cholesterol or cholesterol-like sterols are the most abundant species in the progressive membrane. Free cholesterol has been used as a component of liposome compositions with phospholipids. However, when liposomes composed of free cholesterol and phospholipids are present in biological fluids containing other biological lipids and serum, free cholesterol is rapidly transferred from liposomes to biological lipids. Loss of free cholesterol from the liposomes generally results in reduced stability of the lipid bilayer and subsequent loss of content from the liposomes. In addition, in the presence of serum, the absorption of free cholesterol by serum lipoprotein will result in the transfer of free cholesterol from the liposome to the protein, leading to a significant increase in liposome leakage. Therefore, studies on cationic cholesterol derivatives have been made by modifying cholesterol so as to enable more stable structure formation with amphiphilic lipids (Biochem. Biophys. Res. Commun. Vol. 337, pp387-388, 1991; Biochem. Biophys 221, p82-88, 1996; International journal of Pharmaceutics. 353, p260-269, 2008); WO 2009/064696). However, since vectors expressing wide range of transfer efficiencies have not yet been developed in various cell lines, researches on methods for enhancing transfer efficiency are required.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
The present inventors have endeavored to develop a carrier which is low in toxicity in vivo and can increase gene transfer efficiency. As a result, it has been found that the cationic lipid derived from cholesterol is less cytotoxic and the gene transfer efficiency is remarkably improved as compared with the conventional gene carrier, thereby completing the present invention.
Accordingly, it is an object of the present invention to provide a gene transfer complex comprising a cationic lipid.
It is another object of the present invention to provide a method for producing a cationic lipid for gene transfer.
It is another object of the present invention to provide a method for producing a gene transfer complex comprising the step of forming a liposome using the cationic lipid.
It is still another object of the present invention to provide a gene delivery method comprising the step of administering a gene transfer complex comprising the cationic lipid.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
According to one aspect of the present invention, there is provided a gene transfer complex comprising a cationic lipid of the following general formula (1).
In the above formulas,
X is an organic acid salt selected from the group consisting of trifluoroacetic acid salts, acetic acid salts and methanesulfonic acid salts or an inorganic acid salt selected from the group consisting of a carbonate, a hydrochloride, a sulfate, a acetate, a phosphate, a phosphate, a perchlorate and a hypochlorite ;
m is an integer of 1 to 2;
n is an integer of 1-5.
The present inventors have endeavored to develop a carrier which is low in toxicity in vivo and can increase gene transfer efficiency. As a result, it has been found that cationic lipids derived from cholesterol exhibit excellent gene transfer efficiency, thereby completing the present invention.
The cationic lipid of the present invention has a characteristic of being in the form of a salt with an organic acid or inorganic acid in the form of a cation of -NH 3 + . The organic acid or inorganic acid constituting the salt may include various acids having an ability to form the cationic lipid salt, and preferably an organic acid or carbonate such as trifluoroacetic acid salt, acetic acid salt, methanesulfonic acid salt, etc., Inorganic acids such as hydrochloride, sulfate, acetate, phosphate, fluoride, perchlorate and hypochlorite may be used, and trifluoroacetate or acetate may be used more preferably.
The cationic lipid of the present invention is preferably an amphipathic compound composed of a hydrophobic lipid moiety and a hydrophilic cationic amine moiety, wherein the hydroxy group at the 3-position of cholesterol is substituted with an amine and synthesized through an aminoalkylation reaction. Alkyl of various carbon numbers may be bonded through the aminoalkylation reaction. Preferably, n is an integer of 1-5, considering the transfection efficiency into cells and economical efficiency. More preferably, 1-2 It is an integer.
The cationic lipid of the present invention has an ability to form a complex with a gene having a negative charge.
In the present invention, the gene that forms a complex with the cationic lipid may be a single-stranded or double-stranded DNA, plasmid or RNA, and the RNA may be an antisense complementary to the target DNA or RNA sequence of any cell as well as mRNA, tRNA, RNA, siRNA, miRNA, and ribozymes. A gene encoding a polypeptide associated with the treatment or diagnosis of an arbitrary protein or disease can be inserted into the plasmid. Examples of such a polypeptide include various hormones, histocompatibility antigens, cell adhesion proteins, cytokines, various antibodies, , Intracellular or extracellular enzymes and fragments thereof, and the like.
In addition, the gene preferably includes a gene expression regulator, for example, a transcriptional promoter, an enhancer, a silencer, an operator, a terminator, an attenuator, and other expression control factors.
The proper charge ratio of the DNA: cationic lipid in the complex is 1: 1-1: 20, preferably 1: 1 to 1:10, and when the charge ratio exceeds the range, the gene transfer efficiency decreases There is a problem.
According to a preferred embodiment of the present invention, the cationic lipids of the present invention are in the form of liposomes.
The term " liposome " as used herein refers to a lipid carrier made by forming a closed lipid bilayer. Liposomes are lipid bilayer membranes that are biocompatible and biocompatible, allowing them to pass through hydrophobic membranes with internal hydrophilic materials. Liposomes can perform the function of transporting various pharmaceutically useful substances to the target cells inside. When the liposome is used for pharmaceutical use, the lipid membrane is slowly dissolved, so that the sustained-release pharmaceutical preparation can promote the absorption in the body by oral administration or injection, and the concentration of the drug only in the vicinity of the target organs or tissues There is an advantage that it can be increased.
The cationic liposome of the present invention may be constituted by using only the cationic lipid of the present invention, or may further comprise other kinds of lipids.
According to a preferred embodiment of the present invention, the other kind of lipid is a neutral lipid.
When the neutral lipid is additionally included, the mixing ratio of the cationic lipid compound of formula (1) and the auxiliary neutral lipid used in the cationic liposome is preferably 1: 1-10: 1 (wt: wt) Preferably 1: 1-5: 1 (wt: wt).
The cationic liposome can form a spherical vesicle of a very homogeneous size by treating the lipid molecules in an aqueous solution and treating the liposomes with ultrasonic waves, and the lipid solution in ethanol is formed by rapid mixing with water, . ≪ / RTI >
The other kind of lipid may be various lipids known to those skilled in the art. Preferably, a neutral lipid such as cholesterol, dioleoylphosphatidylethanolamine (DOPE), or dioleoylphosphatidylcholine (DOPC) Liposomes can be constructed.
According to another aspect of the present invention, the present invention provides a method for producing a cationic lipid for gene delivery comprising the steps of:
(a) introducing a tosyl group to the 3-hydroxy group of cholesterol;
(b) replacing the tosyl group of the compound produced in step (a) with an azide group;
(c) reducing the azide group of the compound produced in step (b) to an amine group;
(d) introducing an alkylamine in which an amine protecting group is bonded to an amine group of the compound produced in the step (c); And
(e) treating the compound formed in step (d) with an organic or inorganic acid to remove the amine protecting group and obtain in the form of a lipid salt.
Since the method of the present invention is a process for producing the cationic lipid for gene delivery of the present invention described above, the description common to both of them is omitted in order to avoid excessive complexity of the specification according to the repetitive description.
The cationic lipid of the present invention is obtained by replacing the 3-hydroxy group of the cholesterol with an amine group and then combining with an aminoalkyl group, as shown in Fig. However, the cationic lipid of the present invention should not be construed as being limited to being produced by the above method, but it is possible for a person skilled in the art to prepare the cationic lipid of the present invention by a procedure other than the method of the present invention.
The process of the present invention for preparing the cationic lipids of the present invention will be described step by step in detail as follows:
(a) to the 3-hydroxy group of cholesterol Toys Introduction
First, a tosyl group is introduced into cholesterol.
The introduction of the tosyl group can be carried out by various methods, preferably by dissolving cholesterol in an organic solvent, adding pyridine, and then adding toluenesulfonyl chloride to introduce a toluenesulfonyl group into the 3-hydroxy group of the cholesterol.
Through this method, 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-10,13-dimethyl-17- (6-methylheptane- 2-yl) -1H-cyclopenta [a] phenanthrene-3-yl-4-methylbenzenesulfonate.
(b) Toys Azaide substitution
Next, the tosyl group of the compound produced in the above step (a) is substituted with an azide group.
Substitution with the azide group is possible by various methods, and the compound produced in step (a) is 2,3,4,7,8,9,10,11,12,13,14,15,16, In the case of 17-tetradecahydro-10,13-dimethyl-17- (6-methylheptan-2-yl) -1H-cyclopenta [a] phenanthrene- It can be substituted by adding fluoride dimethyl ether and trimethylsilane azide.
Through the above process, 3-azido-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-10,13-dimethyl-17- ( 6-methylheptan-2-yl) -1H-cyclopenta [a] phenanthrene.
(c) Azaid Amine group restoration
Next, the azide group of the compound produced in step (b) is reduced to an amine group.
The reduction to the amine group is possible by various methods, and the compound produced in step (b) is selected from the group consisting of 3-azido-2,3,4,7,8,9,10,11,12,13,14, In the case of 15,16,17-tetradecahydro-10,13-dimethyl-17- (6-methylheptan-2-yl) -1H-cyclopenta [a] phenanthrene, it is dissolved in an appropriate organic solvent, The azide group can be reduced to an amine group. Preferred reducing agents include lithium aluminum borohydride and the like.
Through this method, 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-10,13-dimethyl-17- (6-methylheptane- Yl) -1H-cyclopenta [a] phenanthrene-3-amine can be obtained.
(d) an amine protecting group Combined Alkylamine introduction
Next, an alkylamine to which an amine protecting group is bonded is introduced into the amine group of the compound produced in the step (c).
The amine protecting group may be selected from various kinds of protecting groups known in the art, preferably t-butoxycarbonyl (Boc), 9H-fluoren-9-ylmethoxycarbonyl (Fmoc), trityl , Benzyl, chloroacetyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, formyl, trifluoroacetyl, p-toluenesulfonyl, benzenesulfonyl, methanesulfonyl, p-nitrobenzyloxycarbonyl, 2 , 2,2-trichloroethoxycarbonyl, and the like can be introduced.
When butoxycarbonyl is used as the amine protecting group, the 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-10,13-dimethyl- Butyl (2-bromoethyl) carbamate or tert-butyl (2-bromopropyl) carbamate was added to 17- (6-methylheptan- Carbamate may be reacted to introduce an aminoalkyl group.
Through the above process, tert-butyl 2- (2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-10,13-dimethyl-17- (6-methylheptan-2-yl) -1H-cyclopenta [a] phenanthrene-3- ylamino) ethylcarbamate or tert- butyl- 3- (2,3,4,7,8,9,10 , 11,12,13,14,15,16,17-? -Datahydro-10,13-dimethyl-17- (6-methylheptan-2-yl) -1H- cyclopenta [a] phenanthrene- - < / RTI > amino) propyl carbamate.
(e) removal of the amine protecting group and Lipid Salt yield
Finally, the amine protecting group of the compound produced in step (d) is removed to yield the cationic lipid of the present invention in the form of a lipid salt.
The removal of the amine protecting group can be carried out using various organic acids or inorganic acids known in the art. When an organic acid is used, trifluoroacetic acid, acetic acid, methanesulfonic acid, or the like can be used. When an inorganic acid is used, carbonic acid, hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, hydrofluoric acid, perchloric acid, hypochlorous acid, no.
When trifluoroacetic acid is used as the organic acid, the compound produced in step (d) above, preferably tert-butyl 2- (2,3,4,7,8,9,10,11,12,13,14 A] phenanthren-3-ylamino) ethylcarbamate or tert (tert-butoxycarbonylamino) ethyl] -Butyl-3- (2,3,4,7,8,9,10,11,12,13,14,15,16,17-? -Datahydro-10,13-dimethyl-17- (6- Yl) -lH-cyclopenta [a] phenanthren-3-ylamino) propyl carbamate was prepared from N- (2-aminoethyl) - 2,3,4,7,8,9,10,11 , 12,13,14,15,16,17-tetradecahydro-10,13-dimethylamine-17- (6-methylheptan-2-yl) -1H- cyclopenta [a] phenanthrene- Trifluoroacetic acid salt or N- (2-aminopropyl) -2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-10,13 -Dimethylamine-17- (6-methylheptan-2-yl) -1H-cyclopenta [a] phenanthrene-3-amine trifluoroacetate.
According to another aspect of the present invention, the present invention provides a method for producing a gene transfer complex comprising adding a neutral lipid to a cationic lipid or a cationic lipid prepared according to the above method to form a liposome.
Transfection of a gene delivery complex comprising a cationic lipid of the present invention or a liposome comprising the same into a target cell is accomplished by fusion of endocytosis or cell membrane with liposomes or complexes. In the case of the entocytosis, gene transfer takes place through the formation of endosomes in the cell. In the case of fusion, the membrane of the liposome is integrated with the target cell membrane and the content in the liposome is transferred into the cytoplasm. .
In addition, the gene to be delivered in the present invention can be used regardless of its size and shape, and can be mixed with the cationic lipid or a liposome containing the cationic lipid to include the gene. The preferred gene: liposome charge ratio is 1: 1 to 1:10.
When the gene is oligonucleotide, it is preferable to use a nucleic acid having a size of 15-120 bases. When the DNA is a plasmid vector, it is preferably 3-8 kb.
According to another aspect of the present invention, there is provided a gene delivery method comprising the step of administering a gene transfer complex comprising the cationic lipid.
The gene transfer complex of the present invention can be administered for the purpose of enhancing gene transfer ability and can be administered to a mammal including a human. Preferred mammals exclude humans.
The gene transfer complex of the present invention can be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, it can be administered by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, have.
The gene transfer complex of the present invention has a gene transfer efficiency of 1.2 times or more, preferably 2 times or more as compared to conventional lipofectamine.
According to one embodiment of the present invention, the expression level of a gene in MCF-7 cells or HEK293 cells is measured by using a plasmid vector containing Luciferase or GFP (green fluorecence protein) The efficiency was remarkably improved as compared with the lipofectamine.
As shown in FIGS. 2 and 3, the gene carrier using the cationic lipid or liposome of the present invention is superior as a gene carrier because it is an excellent gene carrier and has a remarkably low toxicity as compared with the conventional gene carriers.
The features and advantages of the present invention are summarized as follows:
(i) The present invention provides a gene transfer complex comprising a cationic lipid having excellent gene transfer efficiency or a liposome prepared using the cationic lipid and a method for producing the same.
(Ii) The cationic lipids or liposomes of the present invention have significantly improved gene transfer efficiency as compared with the conventional gene carriers.
(Iii) Furthermore, the cationic lipids or liposomes of the present invention are extremely cytotoxic and can be developed as effective gene carriers.
Figure 1 is an illustration of an example of a synthetic step of synthesizing an aminosterol derivative from cholesterol.
FIG. 2 is a graph comparing the activity of luciferase in MCF-7 cells transfected with the gene transfer complex prepared by mixing the charge ratio of DNA: liposome at 1: 3 or 1: 5.
FIG. 3 is a graph comparing the activity of luciferase in HEK293 cells transfected with the gene transfer complex prepared by mixing the charge ratio of DNA: liposome at 1: 3 or 1: 5.
FIG. 4 is a graph showing the results of toxicity tests on cells in HEK293 cells transfected with a gene transfer complex prepared by mixing the charge ratio of DNA and liposome at 1: 3 or 1: 5.
FIG. 5 is a photograph showing the degree of GFP (Green Fluorescent Protein) fluorescence expression in MCF-7 cells transfected with a gene transfer complex prepared by mixing a charge ratio of DNA: liposome of 1: 3 or 1: 5.
FIG. 6 is a photograph showing the degree of GFP (Green Fluorescent Protein) fluorescence expression in HEK293 cells transfected with a gene transfer complex prepared by mixing DNA: liposome at a charge ratio of 1: 3 or 1: 5.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
Example
Example 1: Synthesis of novel lipids
1-1: 2,3,4,7,8,9,10,11,12,13,14,15,16,17- Tetradecahydro -10,13-dimethyl-17- (6-methylheptan-2-yl) -1H- Cyclopenta [a] phenanthrene 3-yl-4- Methylbenzenesulfonate (Compound 2) Synthesis
Cholesterol (1 g, 2.59 mmol) was dissolved in pyridine (8 mL), and then toluenefonyl chloride (1.48 g, 7.76 mmol) was slowly added at room temperature. The reaction solution was stirred at room temperature for 24 hours. After completion of the reaction, the temperature of the reaction solution was cooled to 0 ° C, and 10% NaHCO 3 aqueous solution (10 ml) was slowly added thereto. The resulting white solid was filtered under reduced pressure, washed with distilled water (50 ml), and dried under vacuum to obtain 2,3,4,7,8,9,10,11,12,13,14,15,16, Cyclopenta [a] phenanthrene-3-yl-4-methylbenzenesulfonate (1.19 g) was added to a solution of 17-tetradecahydro-10,13-dimethyl- (Yield: 85%).
1 H-NMR (400 MHz, CDCl 3 )? (Ppm): 0.65 (s, 3H), 0.92 (d, 3H), 0.97 2H), 7.80 (d, 1H), 1.79-2.01 (m, 7H), 2.08 (d, 2H).
1-2: 3-azido-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-10,13-dimethyl-17- ( 6-methylheptan-2-yl) -1H-cyclopenta [a] phenanthrene (Compound 3)
10,13-dimethyl-17- (6-methylheptan-2-yl) -2,3-dihydro- -1H-cyclopenta [a] phenanthrene-3-yl-4-methylbenzenesulfonate (1 g, 2.15 mmol) was dissolved in methylene chloride (20 ml) and then cooled to 0 ° C and boron trifluoride dimethyl ether (0.578 mg, 4.3 mmol) was slowly added dropwise. Trimethylsilan azide (0.372 mg, 3.22 mmol) was slowly added to the reaction solution and stirred at room temperature for 3 hours. When the reaction was completed, distilled water (10 ml) was added to terminate the reaction. The methylene chloride layer was separated, concentrated under reduced pressure, and then purified by column chromatography (hexane: ethyl acetate, 10: 1, v: v) to obtain 3-azido-2,3,4,7,8,9 , 10,11,12,13,14,15,16,17-tetradecahydro-10,13-dimethyl-17- (6-methylheptan-2-yl) -1H-cyclopenta [a] phenanthrene 0.79 g) (yield 89%).
1 H-NMR (400MHz, CDCl 3) δ (ppm): 5.38 (s, 1H), 3.26-3.15 (m, 1H), 2.28 (d, 2H), 2.05-0.85 (m, 38H), 0.68 (s , 3H).
1-3: 2,3,4,7,8,9,10,11,12,13,14,15,16,17-Tetradecahydro-10,13-dimethyl-17- (6-methylheptane- 2-yl) -1H-cyclopenta [a] phenanthrene-3-amine (Compound 4)
3-azido-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-10,13-dimethyl-17- (6-methylheptane Cyclopenta [a] phenanthrene (1.5 g, 3.64 mmol) was dissolved in tetrahydrofuran (20 ml), cooled to 0 ° C, and lithium aluminum borohydride (64 mg, 5.46 mmol) was added slowly to the solution. The reaction solution was stirred at room temperature for 24 hours. When the reaction was completed, the reaction was terminated with a 1N-NaOH aqueous solution. Methylene chloride (30 ml) was added to the reaction solution and the mixture was extracted twice, and then the organic solvent was concentrated under reduced pressure. The obtained residue was purified by column chromatography (methylene chloride: methanol, 8: 1, v: v) to obtain 2,3,4,7,8,9,10,11,12,13,14,15, Cyclopenta [a] phenanthrene-3-amine (1.11 g) was obtained in a yield of 80% (yield 80%). ).
1 H-NMR (400MHz, CDCl 3) δ (ppm): 5.38 (s, 1H), 2.82-2.96 (m, 1H), 2.28 (d, 2H), 2.05-0.85 (m, 38H), 0.68 (s , 3H).
1-4: tert-Butyl 2- (2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-10,13-dimethyl-17- (6-methylheptan-2-yl) -1H-cyclopenta [a] phenanthrene-3-ylamino) ethylcarbamate (Compound 5-1)
10,13-dimethyl-17- (6-methylheptan-2-yl) -2,3-dihydro- Cyclopenta [a] phenanthrene-3-amine (400 mg, 0.934 mmol) was dissolved in DMF (5 ml), and potassium carbonate (322 mg, 2.33 mmol) was added and stirred. After the temperature of the reaction solution was raised to 90 캜, tert-butyl (2-bromoethyl) carbamate (174 mg, 0.78 mmol) was slowly added thereto. The temperature of the reaction solution was gradually lowered to 60 캜 and then stirred for 5 hours. The temperature of the reaction solution was cooled to room temperature, filtered under reduced pressure, and then the organic solvent was concentrated under reduced pressure. The concentrated residue was purified by column chromatography (methylene chloride: methanol, 5: 1, v: v) to give tert-butyl 2- (2,3,4,7,8,9,10,11,12 , 13,14,15,16,17-tetradecahydro-10,13-dimethyl-17- (6-methylheptan-2-yl) -1H- cyclopenta [a] phenanthrene- Carbamate (310 mg) (yield: 64%).
1 H-NMR (400MHz, CDCl 3) δ (ppm): 5.26 (d, 1H), 5.22 (br, 1H), 4.41 (t, 1H), 3.60 (t, 1H), 3.23 (t, 2H), 2H), 1.85 (m, 2H), 1.82 (m, 2H), 2.81 (m, 2H), 2.45 m, 25H), 0.65 (s, 3H); MS (M + H) m / z 530.
1-5: tert -Butyl-3- (2,3,4,7,8,9,10,11,12,13,14,15,16,17- Teradeta Heido -10,13-dimethyl-17- (6- Methyl heptane Yl) -1H- Cyclopenta [a] phenanthrene -3- Amino ) Propyl carbamate (Compound 5-2) Synthesis
10,13-dimethyl-17- (6-methylheptan-2-yl) -2,3-dihydro- (4-fluorophenyl) -1H-cyclopenta [a] phenanthrene-3-amine (400 mg, 0.934 mmol) and tert- butyl (2-bromopropyl) carbamate (184 mg, 0.78 mmol) 4, to give tert-butyl-3- (2,3,4,7,8,9,10,11,12,13,14,15,16,17-? -Radatahydro Cyclopenta [a] phenanthren-3-ylamino) propyl carbamate (275 mg) was obtained (yield 55%).
1 H-NMR (500MHz, CDCl 3) δ (ppm): 5.36 (d, 1H), 5.10 (br, 1H), 3.21 (m, 2H), 2.65 (t, 2H), 2.43 (m, 1H), 2.31 (m, 1H), 2.05 (m, 4H), 1.65-1.85 (m, 4H), 1.25-1.65 (m, 29H), 0.85-1.25 (m, 30H), 0.65 (s, 3H); MS (M + H) m / z 544.
1-6: N- (2- Aminoethyl ) -2,3,4,7,8,9,10,11,12,13,14,15,16,17- Tetradecahydro -10,13-dimethylamine-17- (6-methylheptan-2-yl) -1H- Cyclopenta [a] phenanthrene -3-amine Trifluoroacetic acid Salt (Compound 6-1, Chol - NEN ) synthesis
tert-butyl 2- (2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-10,13-dimethyl-17- (6-methyl (100 mg, 0.189 mmol) was dissolved in methylene chloride (2 mL), and then trifluoroacetic acid (2 mL) was added to the solution, ) Was added slowly and the mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure and then purified by column chromatography (methanol: chloroform, 10: 1, v: v) to obtain N- (2-aminoethyl) (6-methylheptan-2-yl) -1H-cyclopenta [a] phenanthrene-9,10,11,12,13,14,15,16,17-tetradecahydro- Thiophene-3-amine trifluoroacetate (92 mg) (yield 80%).
1 H-NMR (400MHz, CDCl 3) δ (ppm): 5.26 (d, 1H), 3.05 (s, 4H), 2.23 (t, 2H), 1.60-1.81 (m, 5H), 1.35-1.51 (m , 7H), 0.75-1.25 (m, 40H), 0.65 (s, 3H); MS (M + H) m / z 430.
1-7: N- (2-aminopropyl) -2,3,4,7,8,9,10,11,12,13,14,15,16,17- Tetradecahydro -10,13-dimethylamine-17- (6- Tiled Yl) -1H- Cyclopenta [a] phenanthrene -3-amine Trifluoroacetic acid Salt (compounds 6-2, Chol - NPN ) synthesis
tert-butyl-3- (2,3,4,7,8,9,10,11,12,13,14,15,16,17-? rdatahydro-10,13-dimethyl-17- (6 Cyclopenta [a] phenanthrene-3-ylamino) propyl carbamate (100 mg, 0.184 mmol) was reacted in the same manner as in Example 1-6 to give N- (2-aminopropyl) -2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-10,13-dimethylamine-17- Cyclopenta [a] phenanthrene-3-amine trifluoroacetate (82 mg) was obtained (yield: 72%).
1 H-NMR (400 MHz, CDCl 3 )? (Ppm): 5.46 (d, 1H), 3.05 (s, 4H), 2.23 (m, 2H), 1.80-2.11 , 10H), 0.85-1.40 (m, 44H), 0.65 (s, 3H); MS (M + 1) m / z 443.
Example 2 : Liposomal Produce
[0060] Compound 6-2 and dioleoylphosphatidylethanolamine (DOPE) (wt: wt, 1: 1 to 1: 3) were dissolved in chloroform, blended with nitrogen gas to remove chloroform, and dried under vacuum (Table 1). An appropriate amount of distilled water was added to the dried mixture, and the mixture was shaken and suspended. The liposomes were then treated at room temperature for several minutes using a sonicator to form liposomes. The liposome solution was homogenized by passing through a 200 nm carbon membrane several times.
Example 3: Production of gene transfer complex DNA Transfer efficiency
3-1: Cell and cell culture
In this embodiment, a breast cancer cell line (MCF-7) was used. The culture medium of the cell line is RPMI 164 (Welgene, Korea), and 10% heat-inactivated FBS (Welgene) and 2 mM L-glutamine e, 100 units / ml penicillin and 100 μg / ml streptomycin streptomycin (Welgene) And cultured in a 5
3-2: Reporter plasmid vector ( Reporter plasmid vector ) Production
The recombinant plasmids (pcDNA-Luc, pCMVTnT-CFP) containing Luciferase or GFP (green fluorecence protein) genes were amplified using XL1-Blue E. coli and amplified with maxi-kit (Qiagen Inc., USA (Chol-GS) as a New Gene Delivery Vector) was used as a template (see J. Microbiol. Biotechnol. (2011), 21 (1), 93-99 Synthesis and Optimization of Cholesterol-Based Diquaternary Ammonium Gemini Surfactant .
3-3: Nucleic acid body And cations Liposome Formation of complex
A gene transfer complex was prepared using the reporter plasmid pcDNA3 vector and the cationic liposome prepared in Example 2 as a nucleic acid construct. The gene transfer complex was prepared in TOM medium (Welgene) with serum.
At the time of mixing, the reporter plasmid pcDNA3 vector and cation liposome were mixed and reacted at room temperature for 15 minutes to obtain a gene transfer complex. The charge ratio of DNA: liposome was adjusted to 1: 3 and 1: 5.
As a control, a comparative experiment was carried out using lipofectamine.
3-4: Transfection of Gene Delivery Complex ( Transfection )
The MCF-7 or HEK293 cell line cultured the day before the experiment was divided into 48-well plates and the gene transfer complex was treated to transfection the cells and cultured at 37 DEG C for 24 hours.
3-5: Transfer efficiency of gene transfer complex
The luciferase activity was measured as follows. The transfected cells were washed twice with PBS buffer and washed with 100 μl of 1 × CCLR (cell culture lysis reagent, 25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM diaminocyclohexane-N 'N'N'-tetraacetic acid, 10% glycerol, 1% Triton X-100). The resulting cell lysate was centrifuged at 12,000 g for 1 minute, and the protein was quantified with BCATM Protein Assay (PIERCE, USA) to measure the activity of luciferase. 100 μl of a luciferase assay buffer (25 mM Glycylglycine pH 7.8, 15 mM Potassium Phosphate pH 7.8, 15 mM MgSO 4 , 4 mM EGTA, 2 mM ATP, 1 mM DTT) was added to the cell lysate, Luciferin was added. Luciferase activity was measured with a luminometer (Berthold Detection Systems, Germany) for 10 seconds, and the results are shown in FIGS. 2 and 3.
On the other hand, detection of GFP was performed by washing the cell line twice with PBS buffer, and then confirming the expression pattern of intracellular GFP gene using a fluorescence inversion microscope. The gene expression pattern through the fluorescence-based microscope is shown in FIGS.
As shown in FIGS. 2 to 5, the activity was remarkably superior to that of the control group. As a result, it was confirmed that the gene transfer complex had an ability to transfer DNA into a superior cell.
Example 4 : HEK293 Toxicity experiments on cells
HEK293 cells were seeded at a density of 6 × 10 4 cells / well in a 48-well plate and cultured in MEM (minimal essential medium) containing 10% fetal bovine serum (FBS) at 37 ° C. under 5% carbon dioxide. Liposome complex (DNA: liposome, 1: 3 or 1: 5) was added. After incubating the cells for 24 hours, 10 μl of MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazonium bromide) at a concentration of 5 mg / ml was added to each well. After further incubation for a time, the medium is removed. DMSO (dimethyl sulfoxide) (250 μl) was added to each well, and the reactants were dissolved. 200 μl of the reaction solution was dispensed into a 98-well plate and absorbance was measured at 550 nm.
As shown in FIG. 6,
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (17)
Formula 1
In the above formulas,
X is an organic acid salt selected from the group consisting of trifluoroacetic acid salts, acetic acid salts and methanesulfonic acid salts or an inorganic acid salt selected from the group consisting of a carbonate, a hydrochloride, a sulfate, a acetate, a phosphate, a phosphate, a perchlorate and a hypochlorite ;
m is an integer of 1 to 2;
n is an integer of 1-5.
(a) introducing a tosyl group to the 3-hydroxy group of cholesterol;
(b) replacing the tosyl group of the compound produced in step (a) with an azide group;
(c) reducing the azide group of the compound produced in step (b) to an amine group;
(d) introducing an alkylamine in which an amine protecting group is bonded to an amine group of the compound produced in the step (c); And
(e) treating the compound formed in step (d) with an organic or inorganic acid to remove the amine protecting group and obtain in the form of a lipid salt.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20180024231A (en) * | 2016-08-29 | 2018-03-08 | 영남대학교 산학협력단 | Composition for delivering bioactive substance comprising imipramine |
| WO2022191555A1 (en) * | 2021-03-08 | 2022-09-15 | 아이진 주식회사 | Composition for in vivo delivery of rna and preparation method therefor |
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| US5948767A (en) * | 1994-12-09 | 1999-09-07 | Genzyme Corporation | Cationic amphiphile/DNA complexes |
| US6331524B1 (en) * | 1994-12-09 | 2001-12-18 | Genzyme Corporation | Organ-specific targeting of cationic amphiphile / DNA complexes for gene therapy |
| KR20020013528A (en) * | 1999-05-28 | 2002-02-20 | 박종상 | Cationic lipids for gene transfer and preparation method thereof |
| KR20020022867A (en) * | 2000-09-21 | 2002-03-28 | 문우철 | Cationic lipids and method for preparing the same |
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| US5948767A (en) * | 1994-12-09 | 1999-09-07 | Genzyme Corporation | Cationic amphiphile/DNA complexes |
| US6331524B1 (en) * | 1994-12-09 | 2001-12-18 | Genzyme Corporation | Organ-specific targeting of cationic amphiphile / DNA complexes for gene therapy |
| KR20020013528A (en) * | 1999-05-28 | 2002-02-20 | 박종상 | Cationic lipids for gene transfer and preparation method thereof |
| KR20020022867A (en) * | 2000-09-21 | 2002-03-28 | 문우철 | Cationic lipids and method for preparing the same |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20180024231A (en) * | 2016-08-29 | 2018-03-08 | 영남대학교 산학협력단 | Composition for delivering bioactive substance comprising imipramine |
| WO2022191555A1 (en) * | 2021-03-08 | 2022-09-15 | 아이진 주식회사 | Composition for in vivo delivery of rna and preparation method therefor |
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