KR101741998B1 - Use of aminopropyl magnesium phyllosilicate or aminopropyl calcium phyllosilicate for promoting gene delivery of virus vector - Google Patents

Use of aminopropyl magnesium phyllosilicate or aminopropyl calcium phyllosilicate for promoting gene delivery of virus vector Download PDF

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KR101741998B1
KR101741998B1 KR1020150178855A KR20150178855A KR101741998B1 KR 101741998 B1 KR101741998 B1 KR 101741998B1 KR 1020150178855 A KR1020150178855 A KR 1020150178855A KR 20150178855 A KR20150178855 A KR 20150178855A KR 101741998 B1 KR101741998 B1 KR 101741998B1
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임수정
한효경
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세종대학교 산학협력단
동국대학교 산학협력단
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Abstract

The present invention relates to the use of aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate for promoting gene transfer of viral vectors. According to the present invention, the use of aminopropyl magnesium phyllosilicate or aminopropyl calcium phyllosilicate together with a viral vector makes it possible to achieve high gene expression even with a small amount of viral vector, and therefore, cytotoxicity , Immunogenicity and the risk of hepatotoxicity can be reduced. Therefore, the aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate according to the present invention can be usefully used as an adjuvant for improving the gene transfer efficiency of a viral vector.

Description

Use of aminopropyl magnesium phyllosilicate or aminopropyl calcium phyllosilicate for promoting gene transfer of viral vectors. ≪ Desc / Clms Page number 2 > Use of aminopropyl magnesium phyllosilicate or aminopropyl calcium phyllosilicate for promoting gene delivery of virus vector.

The present invention relates to a composition for promoting gene transfer of a viral vector comprising aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate, that is, a composition for promoting gene transfer of a viral vector of aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate .

Vectors for transferring genes into cells are largely divided into two regions, non-viral vectors and viral vectors. Although safety concerns remain, the advances in gene recombination technology are leading to the solution, and due to their ability to transfer genes far superior to non-viral vectors, viral vectors have become important gene carriers in biomedical research and industry .

The entry of viral vectors into eukaryotic cells begins with the specific binding of the virus to the receptors present in the cell membrane. Therefore, when a gene is introduced into a specific cell using a recombinant virus, the absence of such a receptor in the cell membrane makes it difficult to infect the virus into the cell, thereby causing a significant reduction in gene introduction efficiency.

Adenovirus can be infected with various types of cells in each cell division stage, and is particularly popular as a viral vector for gene delivery. In this case, the gene transfer rate by the recombinant adenovirus is determined by the Coxakievirus-adenovirus receptor (Coxakievirus and adenovirus receptor (CAR). This is because the adenovirus fiber knob binds to the CAR to activate the adjacent integrin receptor, resulting in endocytosis of the viral particle, and the endocytosis virus particles are released from the endosome due to the unique nature of the viral protein itself I go through the cytoplasm and into the nucleus to express the gene. In addition, after administration in vivo, there is a possibility that the presence of antiviral antibodies in advance, the degree of generation of antiviral antibodies induced by repeated administration of the viral vectors, and the hepatic accumulation of viral particles due to the hepatic accumulation of viral particles Toxicity is also a limiting factor for adenovirus mediated gene transfer.

Adhesion of adenovirus surface protein with a polymer such as polyethylene glycol is effective in inhibiting the accumulation of adenovirus in the liver and neutralization (neutralization) by binding to the antiviral antibody, thereby overcoming the limit of gene therapy using adenoviral vectors . However, chemical modification of the viral proteins may reduce the production efficiency of viruses and make it difficult to purify or interfere with intracellular migration after cell entry. On the other hand, a method of forming a complex by electrostatic bonding by simply mixing a cationic lipid or polymer with a negative charge on the surface of adenovirus particles reduces the electrostatic repulsion between the cell membrane and the virus, The effect of adenovirus on the expression of genes was enhanced by the addition of the CAR receptor. Thus, such a complex system is useful in that it facilitates the gene transfer efficiency of the viral vector in a simple manufacturing process without chemical covalent bonds, thus allowing gene delivery to a similar extent with smaller amounts of viral vectors and reducing hepatotoxicity.

However, the enhancement of gene transfer of viral vectors by complex formation with cationic lipids or polymers causes cytotoxicity, and the degree of cytotoxicity depends on the concentration of the cationic lipid or polymer in the carrier complexed with the virus Respectively.

On the other hand, the natural clay is not well dispersed in water in spite of excellent biocompatibility, and thus it has a problem that its use in the field of biomedical medicine is limited. In order to solve these problems, studies have been made to synthesize a clay material which can be dispersed in water. In this process, an aminopropyl group is covalently bonded to the surface of magnesium phosphate silicate to form aminopropyl A method of synthesizing magnesium phyllosilicate is known (non-patent document 1). Such aminopropyl magnesium phyllosilicate can be easily dispersed in water and has no bioaccumulation property (Non-Patent Document 2). Recently, it has been applied as a drug delivery material for enhancing absorption of poorly soluble drugs (Patent Document 2).

Accordingly, the present inventors have studied a gene delivery carrier having excellent gene expression assisting ability, low cytotoxicity, and biocompatibility using the natural clay material.

Korea Patent Publication No. 2015-0004859 Korean Patent Publication No. 2014-0043587

 Mann et al., Journal of materials Chemistry, 1998, 8 (8), 1927-32  Yang et al., J Materials Chemistry B., 2014, 2, 7567-7574

Thus, the present inventors have found that by using a viral vector and aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate together as a gene delivery carrier, high gene expression can be achieved with a small amount of viral vector, and cytotoxicity, immunogenicity and hepatotoxicity The present invention has been completed.

Accordingly, an object of the present invention is to provide a composition for promoting gene transfer of a viral vector comprising aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate excellent in gene expression assisting ability, low cytotoxicity and excellent biocompatibility, that is, And to provide its use for promoting gene transfer.

In order to achieve the above object, the present invention provides a composition for promoting gene transfer of a viral vector comprising aminopropyl magnesium phosphate or aminopropyl calcium phosphate silicate.

The present invention also provides a method for enhancing the gene transfer capability of a viral vector into a cell, comprising contacting the composition with a cell together with the viral vector in vitro.

The present invention also provides a phyllosilicate-viral complex in which a viral vector is introduced into aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate.

The invention also relates to aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate; And a viral vector containing a gene to be introduced into the target cell.

The invention also relates to aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate; A viral vector containing the gene to be introduced into the target cell; And a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition for gene therapy comprising a pharmaceutically acceptable carrier.

According to the present invention, by using aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate together with a viral vector, higher gene expression can be achieved than when the same amount of viral vector is used alone, The target gene expression in vivo is enabled to reduce the risk of hepatotoxicity occurring when a high dose of virus is administered to a living body and the side effect of reducing gene expression efficiency due to immunogenicity upon re-administration. Compared with the case of using aminopropyl magnesium phosphate or aminopropyl calcium phyllosilicate as an adjuvant and the case of using cationic lipid or polymer as an adjuvant, the use of the phyllosilicate as an adjuvant provides better gene expression enhancement, I confirmed that there is almost no. Therefore, the aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate according to the present invention can be usefully used as an adjuvant for enhancing the gene transfer efficiency in cultured cells of a viral vector and in vivo.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention provides a composition for promoting gene transfer of a viral vector comprising aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate.

Specifically, aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate in the composition may be contained in the total composition at a concentration of 0.1 mg / mL to 10 mg / mL, but may be aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate The upper limit of the concentration range is not limited as long as the cytotoxicity caused by the cytotoxicity is not observed.

In the present invention, the composition may further comprise a physiologically acceptable carrier. The physiologically acceptable carrier may be applied in the same manner as the pharmaceutically acceptable carrier described below.

In the present invention, a viral vector is a virus that delivers a gene for synthesizing a function-regulating substance having biological activity that regulates all physiological phenomena in a living body, and is not particularly limited as long as it can carry a gene to be transduced. For example, the viral vector may be an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, a baculovirus vector, a parvovirus a parvovirus vector, a semiliniforest virus vector, a canarypoxvirus vector, a vaccinia virus vector, a fowl pox virus vector, a sindbis virus vector, a picornavirus vector, A picornavirus vector, and an alphavirus vector. In one embodiment, the preferred viral vector that can be used for gene transfer may be an adenovirus or an adeno-associated virus.

The kind of gene that can be contained in the viral vector according to the present invention is not particularly limited. The gene may be any gene that is desired to be introduced into the target cell. For example, cytokines, chemokines, antigens, tumor suppressor genes, suicide genes, anti-angiogenic factors (eg, ), A prodrug activating gene, an immunostimulatory gene, and a cellular differentiation regulatory gene may be contained in the viral vector.

The present invention also provides a method of enhancing the gene transfer capability of a viral vector comprising contacting said composition with a viral vector in vitro in vitro.

In one embodiment, all of the above-described contents of a viral vector and a gene can be applied or applied as they are.

The present invention also provides a phyllosilicate-viral complex in which a viral vector is introduced into aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate.

The aminopropyl magnesium phosphate or aminopropyl calcium phyllosilicate according to the present invention can exhibit a remarkably high gene expression enhancing effect as compared with the case of gene transfer in the form of virus alone or virus-liposome complex, It is possible to reduce the amount of virus to be treated for the cell itself.

In the present invention, binding of aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate to a viral vector can provide a carrier with low cytotoxicity and biocompatibility, showing excellent gene expression ability.

The aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate according to the present invention may be provided as a supplement for promoting gene transfer of a viral vector and may be provided in a form in which the viral vector can be directly mixed by a user, And may be provided in the form of a phyllosilicate-viral complex in which a viral vector containing a gene in aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate according to the present invention is already introduced, if necessary. The phyllosilicate-viral complexes according to the present invention can be formed by simple mixing of aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate with virus.

The complex may contain a viral vector between or between the layers of aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate to form aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate having a cationic surface upon dispersion in water phase, Virus particles can interact with each other to form a complex by electrostatic attraction.

In one embodiment, all of the above-described contents of a viral vector and a gene can be applied or applied as they are.

A composition comprising such a complex of aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate and a viral vector, for example, aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate, and a viral vector comprising the gene to be introduced into the target cell Etc. may be utilized for research or medical use.

The present invention also relates to aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate; A viral vector containing the gene to be introduced into the target cell; And a pharmaceutically acceptable carrier. In this case, the target cell to which the gene is to be delivered via the viral vector may be, but not limited to, cancer cells, stem cells, neurons, and the like.

The administration route of the pharmaceutical composition of the present invention may be any suitable route of administration. Since the filosylate-virus complex according to the present invention is positively charged, negative charge and attraction of the cell or tissue membrane may act and adhere to or contact the cell or tissue membrane, and may enter the cell by endocytosis.

The medicinal composition according to the present invention is treated so that the phyllosilicate-virus complex can be contacted with the target cell in In vivo or xvivo.

In the present invention, contact between the phyllosilicate-viral complex and the target cell may be in vivo, ex vivo or intramuscularly, intraperitoneally, intravenously, orally, intranasally, subcutaneously, intradermally, intrathecal or by inhalation Means that the phyllosilicate-virus complex contacts the target cell, whereby the viral vector is transferred into the cytoplasm or nucleus of the target cell.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention include, for example, one or more of water, saline, phosphate buffered saline, dextrin, glycerol, ethanol as well as combinations thereof. Such compositions may be formulated to provide a rapid release, sustained or delayed release of the active ingredient after administration.

Pharmaceutically acceptable carriers can be prepared according to a variety of factors well known to those skilled in the art, including, for example, the particular physiologically active material employed, its concentration, stability and intended bioavailability; Diseases and conditions or conditions to be treated; The subject, age, size and general condition to be treated; But are not limited to, the route used to administer the composition, for example, nasal, oral, ocular, topical, transdermal, and muscular factors. In general, pharmaceutically acceptable carriers for administration of physiologically active substances other than the oral administration route include aqueous solutions containing D5W, dextrose and physiological salts within 5% of the volume. Pharmaceutically acceptable carriers may also include additional ingredients which may enhance the stability of the active ingredients such as preservatives and antioxidants.

In addition, the dosage of the pharmaceutical composition of the present invention is administered in a dose effective for the intended treatment. The therapeutically effective amount required to treat or inhibit the progression of a particular medical disorder can be readily determined by those skilled in the art using preliminary clinical and clinical studies known in the medical arts. The term "therapeutically effective amount " as used herein in the present invention refers to the amount of active ingredient that causes the biological or medical response of a particular tissue, system, and animal or human to be desired by the clinician or researcher.

Hereinafter, the present application will be described in detail by way of examples. The following examples are illustrative of the present application and the scope of the present application is not limited to the following examples.

[ Example  1 to 5] Aminopropylmagnesium phyllosilicate and  Production of complexes of viral vectors

Aminopropylmagnesium phyllosilicate was prepared by dissolving magnesium chloride (0.84 g, 8.82 mmol) in ethanol (20 g) and adding aminopropyltriethoxysilane (1.3 mL, 5.63 mmol) dropwise to this solution at room temperature. After 5 minutes, the obtained white slurry was stirred overnight, and the precipitate was collected by centrifugation, washed with ethanol (50 ml) and dried at 40 ° C to synthesize aminopropylmagnesium phyllosilicate. 200 mg of the synthesized aminopropylmagnesium phyllosilicate was dissolved in 1 ml of 5% D-glucose. After several times of voltexing, the solution was sonicated at 100 to 280 watts for 30 minutes to prepare an aqueous solution. Adenovirus (replication-inactivated adenovirus type 5, Ad-GFP) containing the GFP gene as a target gene to be transferred into the prepared aqueous solution and the cells were mixed at the ratios shown in Tables 1 and 2 and incubated at room temperature for 20 to 30 minutes And allowed to stand to form an aminopropylmagnesium phosphate silicate-virus complex. The contents of viruses and aminopropylmagnesium phosphate silicate in each example are shown in Tables 1 and 2 below.

Example  1-1 Example  1-2 Example  1-3 Example  1-4 Example  1-5 Example  1-6 Example  1-7 Adenovirus ( pfu ) 8X10 7 8X10 7 8X10 7 8X10 7 8X10 7 8X10 7 8X10 7 Aminopropylmagnesium phyllosilicate  ( ug ) 50 100 250 500 1,000 2,000 3,000

Example  2 Example  3 Example  4-1 Example  4-2 Example  5 Adenovirus ( pfu ) 1X10 7 3.2X10 6 1.6X10 6 1.6X10 6 1X10 6 Aminopropylmagnesium phyllosilicate  (ug) 500 500 200 500 500

[ Comparative Example  1 to 6]

In the same manner as in Example 1 except that plasmid vector pEGFP-C1 (Clontech) was used instead of adenovirus vector Ad-GFP or lipofectamine 2000 (Thermofischer Scientific) was used instead of aminopropylmagnesium phyllosilicate, . The contents of the virus vectors, plasmids, aminopropylmagnesium phyllosilicate and lipofectamine 2000 in the respective comparative examples are shown in Tables 3 and 4.

Comparative Example  One Comparative Example  2-1 Comparative Example  2-2 Comparative Example  2-3 Comparative Example  2-4 Adenovirus ( pfu ) - 8X10 7 1X10 7 8X10 6 1.6X10 6 pEGFP  Plasmid ( ug ) - - - - - Aminopropylmagnesium phyllosilicate  ( ug ) - - - - - Lipofectamine (ul) - - - - -

Comparative Example  3-1 Comparative Example  3-2 Comparative Example  4 Comparative Example  5 Comparative Example  6 Adenovirus ( pfu ) 8X10 7 8X10 7 - - - pEGFP  Plasmid ( ug ) - - 2 2 2 Aminopropylmagnesium phyllosilicate  ( ug ) - - - 500 - Lipofectamine (ul) 20 40 - - 10

 [ Experimental Example  1] Measurement of gene expression rate by gene introduction method

Mouse melanoma cell line B16-F10 was inoculated in a volume of 1 x 10 < 5 > to a 12-well plate. After overnight incubation, the cells were washed with phosphate buffered saline (PBS), 400 μl of DMEM medium without serum was added, and the virus complex prepared in the above Examples and Comparative Examples was added. After incubation for 4 hours in a 5% CO 2 cell incubator at 37 ° C, the cells were washed with PBS to remove the complex, and 1 ml of the medium containing 10% serum was added again for further 30 hours. After 30 hours, the medium was removed from the plate and the cells were washed with PBS. Cells were then scraped and scraped into 1.75 ml tubes. Cells were dispersed in PBS and washed twice. One ml of cells And redispersed in the culture. After centrifuging, the medium was removed, and the cells were fixed with 1 ml of 1% paraformaldehyde solution for 30 minutes. The supernatant was removed and the fixed cells were dispersed in PBS. GFP expression in dispersed cells was measured by flow cytometric method at 10,000 fluorescence per sample at 530 nm. The results are shown in Table 5 below.

Adeno
virus
( pfu )
Aminopropylmagnesium phyllosilicate  ( ug ) Lipo
Pectamin
(ul)
medium
( GFP  Expression cells,%)
Standard Deviation
Example  1-1 8X10 7 50 - 19.8 3.3 Example  1-2 8X10 7 100 - 21.1 1.0 Example  1-3 8X10 7 250 - 44.4 9.4 Example  1-4 8X10 7 500 - 91.3 4.6 Example  1-5 8X10 7 1,000 - 98.3 0.5 Example  1-6 8X10 7 2,000 - 96.6 3.5 Example  1-7 8X10 7 3,000 - 96.6 1.8 Comparative Example  One - - - 2.4 0.6 Comparative Example  2-1 8X10 7 - - 6.5 0.8 Comparative Example  3-1 8X10 7 - 20 32.0 3.5 Comparative Example  3-2 8X10 7 - 40 51.4 1.2

According to Table 5, it was confirmed that the expression rate of the GFP gene was significantly increased when the viral vector was conjugated with 500 ug of aminopropylmagnesium phyllosilicate to form a complex (Example 1-4). In other words, when Examples 1-4 and Comparative Example 2-1 were compared, it was confirmed that the expression rate of the GFP gene by the complex of the viral vector and the aminopropylmagnesium phyllosilicate was about 15 times higher than that when the viral vector alone was used .

[ Experimental Example  2] Confirmation of gene transfer enhancement effect by cell line

Vascular endothelial cell line EY.hy926, lung cancer cell line H460 and colon cancer cell line HCT116 were inoculated into 12 well plates at 1 × 10 5 . After overnight incubation, the cells were washed with phosphate buffered saline (PBS), 400 μl of DMEM medium without serum, 400 μl of RPMI 1640 medium without serum was added to HY460 and HCT116, And the virus complex prepared in Comparative Example were added. After incubation for 4 hours in a 5% CO 2 cell incubator at 37 ° C, the cells were washed with PBS to remove the complex, and 1 ml of the medium containing 10% serum was added again for further 30 hours. After 30 hours, the medium was removed from the plate and the cells were washed with PBS. Cells were then scraped and scraped into 1.75 ml tubes. Cells were dispersed in PBS and washed twice. One ml of cells And redispersed in the culture. After centrifuging, the medium was removed, and the cells were fixed with 1 ml of 1% paraformaldehyde solution for 30 minutes. The supernatant was removed and the fixed cells were dispersed in PBS. GFP expression in dispersed cells was measured by flow cytometric method at 10,000 fluorescence per sample at 530 nm. The results are shown in Tables 6 to 8 below.

Cell line Adeno
virus
( pfu )
Aminopropylmagnesium Phylosilicate ( ug ) Lipo
Pectamin
(ul)
medium
( GFP  Expressing cells, % )
Standard Deviation
EY . hy926 Example  2 1X10 7 500 - 89.8 2.8 Comparative Example  One - - - 1.8 0.4 Comparative Example  2-1 8X10 7 - - 65.2 2.4 Comparative Example  2-2 1X10 7 - - 21.2 0.4

Cell line Adeno
virus
( pfu )
Aminopropylmagnesium Phylosilicate ( ug ) Lipo
Pectamin
(ul)
medium
( GFP  Expressing cells, % )
Standard Deviation
H460 Example  3 3.2X10 6 500 - 95.2 0.2 Example  4-1 1.6X10 6 200 - 66.9 2.9 Example  4-2 1.6X10 6 500 - 66.3 1.6 Example  5 1X10 6 500 - 93.1 2.5 Comparative Example  One - - - 2.9 1.3 Comparative Example  2-1 8X10 7 - - 99.5 0.1 Comparative Example  2-4 1.6X10 6 - - 21.0 0.8

Cell line Adeno
virus
( pfu )
Aminopropylmagnesium Phyllosilicate
( ug )
Lipo
Pectamin
(ul)
medium
( GFP  Expressing cells, % )
Standard Deviation
HCT116 Example  4-2 1.6X10 6 500 - 74.5 3.5 Comparative Example  One - - - 1.8 0.5 Comparative Example  2-1 8X10 7 - - 96.6 0.7 Comparative Example  2-3 8X10 6 - - 71.3 0.6 Comparative Example  2-4 1.6X10 6 - - 32.6 0.6

According to the above Table 6, in the case of the EY.hy926 cell line, when the amount of the virus was reduced to 1/8 in the case of the comparison example 2-1 and the example 2, even when the aminopropyl magnesium phosphate silicate treatment was performed, Can be seen.

According to the above Table 7, in the case of the H460 cell line, comparing Example 3 and Comparative Example 2-1, it can be confirmed that similar gene transfer efficiency is shown even when 1/25 of the viral vector is used.

According to the above Table 8, in the case of the HCT116 cell line, when comparing the Comparative Example 2-3 and the Example 4-2 in which the gene expression rate satisfies the range of 71 to 75%, the virus amount of 1/5 of the Comparative Example 2-3 And a similar level of expression.

Therefore, the effect of aminopropylmagnesium phyllosilicate on the gene transfer ability is different according to the degree of virus infiltration due to differences in cell receptor-specific receptor expression, but the effect of aminopropylmagnesium phyllosilicate on the viral vector gene transfer ability is common As shown in Fig.

[ Experimental Example  3] Confirm cytotoxicity

Cationic lipids are known to cause cytotoxicity when introduced into cells. Therefore, in this Example, aminopropylmagnesium phyllosilicate showed excellent gene expression assisting ability and it was confirmed that cytotoxicity can be reduced compared to cationic lipid. For this purpose, mouse melanoma cell line B16-F10 was inoculated in a 12 well plate at 1 × 10 5 cells. After overnight incubation, the cells were washed with PBS, 400 μl of serum-free DMEM medium was added, and aminopropylmagnesium phyllosilicate or lipofectamine was added, respectively. After incubation for 4 hours in a 5% CO 2 cell incubator at 37 ° C, the cells were washed with PBS to remove the complex, and 1 ml of the medium containing 10% serum was added again for further 30 hours. After 30 hours, the medium was removed from the plate for cell viability assay (MTT assay), and the cells were washed with PBS, and then MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrasolium bromide ) (0.25 g / 50 ml in PBS) diluted to 10% with DMEM medium, add 1 ml each, and incubate for 4 hours. After incubation, cells were washed with PBS containing MTT, and 1 ml of DMSO was added. The DMSO solution was inhaled and ejected with a pipette to dissolve the formazan crystals. The dissolved DMSO solution was transferred into a 96-well plate in an amount of 100 μl, and the absorbance was measured at 540 nm by a spectroscopic method. Cell growth rate was analyzed and the results are shown in Tables 9 and 10 below.

Lipofectamine Well per ul Cell growth rate ( % ) Standard Deviation Comparative Example  One 0 100 1.6 Comparative Example  1-1 One 94.5 3 Comparative Example  1-2 5 91.9 2.5 Comparative Example  1-3 10 84.6 1.5 Comparative Example  1-4 20 83.6 0.7 Comparative Example  1-5 40 77.3 1.4 Comparative Example  1-6 80 67.3 2.6

Aminopropylmag Nesium phyllosilicate Well Party ug Cell growth rate ( % ) Standard Deviation Comparative Example  One 0 100 1.6 Example  6-1 50 96.8 0.3 Example  6-2 100 94.6 2.1 Example  6-3 300 97.5 0.8 Example  6-4 500 93.3 1.6 Example  6-5 800 88.8 0.3 Example  6-6 1200 81.7 2 Example  6-7 2000 84.3 2.8

According to the above Table 9, when 40 μl of lipofectamine was treated (Comparative Example 1-5), the cell growth rate was 77.3%, and the cell growth was inhibited by about 23%. On the other hand, when 500 μg of aminopropyl magnesium phyllosilicate was treated Example 6-4) The cell growth rate was 93.3%, confirming that cell growth was inhibited by 6.7%.

Thus, it can be seen that the aminopropyl magnesium phosphate silicate of the present invention is significantly less cytotoxic than the lipofectamine, which is a cationic liposome.

[ Experimental Example  4] Identification of gene transfer enhancement effect by vector type

In this experimental example, differences in the gene expression aiding ability of aminopropylmagnesium phyllosilicate according to the type of vector (virus or non-viral vector) were examined. For this, aminopropylmagnesium phyllosilicate-virus complex and aminopropylmagnesium phyllosilicate-plasmid complex were prepared by the contents of Examples and Comparative Examples, and then treated with mouse melanoma cell line B16-F10 to analyze GFP expression level, The effect of magnesium phosphate silicate on the gene transfer efficiency of the vector was confirmed. For comparison, GFP expression by the lipofectamine-plasmid complex was also analyzed. The results are shown in Table 11 below.

Adenovirus
( pfu )
pEGFP  Plasmid
( ug )
Aminopropylmagnesium Phylosilicate ( ug ) Lipofectamine
(ul)
medium(%) Standard Deviation
Example  1-4 8X10 7 - 500 - 91.3 4.6 Comparative Example  One - - - - 2.4 0.6 Comparative Example  2-1 8X10 7 - - - 6.5 0.8 Comparative Example  3-2 8X10 7 - - 40 51.4 1.2 Comparative Example  4 - 2 - - 5.5 0.1 Comparative Example  5 - 2 500 - 9.8 1.7 Comparative Example  6 - 2 - 10 59.6 0.9

According to the above Table 11, in Example 1-4 in which aminopropylmagnesium phyllosilicate was treated with the viral vector, the expression rate of GFP gene was 91.3%, and compared with the case where the viral vector was treated alone (Comparative Example 2-1) But the expression rate of the viral vector in Comparative Example 3-2 treated with lipofectamine was increased by about 7.9 times. On the other hand, in the case of the plasmid vector which is a non-viral vector, the gene expression ratio was increased about 11 times in the case of treatment with the lipofectamine mixed treatment (Comparative Example 6) as compared with the case where the plasmid vector alone was treated, In the case of treatment with phyllosilicate (Comparative Example 5), only about 1.8 times increased.

Therefore, the effect of promoting gene expression by lipofectamine appears in both plasmids (pEGFP) and adenovirus vectors, but the effect of promoting gene expression by aminopropylmagnesium phyllosilicate is specific for adenovirus vectors Can be seen.

Claims (9)

A composition for promoting gene transfer of a viral vector comprising aminopropyl magnesium phyllosilicate (AMP) or aminopropyl calcium phyllosilicate (ACP).
The method according to claim 1,
Wherein the aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate is contained in the whole composition including the viral vector at a concentration of 0.1 mg / mL to 10 mg / mL.
The method according to claim 1,
Wherein said composition further comprises a physiologically acceptable carrier.
The method according to claim 1,
The viral vector may be an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, a baculovirus vector, a parvovirus vector A seminiforest virus vector, a canarypoxvirus vector, a vaccinia virus vector, a fowl pox virus vector, a sindbis virus vector, a picornavirus ) Vector and an alphavirus vector, wherein the vector is selected from the group consisting of a virus vector and an alphavirus vector.
A method for promoting gene transfer into a cell of a viral vector comprising contacting the composition according to any one of claims 1 to 4 with a cell together with the viral vector in vitro.
Aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate, wherein the viral vector is introduced.
The method according to claim 6,
The viral vector may be an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, a baculovirus vector, a parvovirus vector A seminiforest virus vector, a canarypoxvirus vector, a vaccinia virus vector, a fowl pox virus vector, a sindbis virus vector, a picornavirus ) Vector and an alphavirus vector. ≪ RTI ID = 0.0 > 8. < / RTI >
Aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate; And
A composition for gene transfer comprising a viral vector comprising a gene to be introduced into a target cell.
Aminopropylmagnesium phyllosilicate or aminopropyl calcium phyllosilicate; A viral vector containing the gene to be introduced into the target cell; And a pharmaceutically acceptable carrier.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108714223A (en) * 2018-05-31 2018-10-30 吉林大学 A kind of contrast agent and preparation method thereof having both magnetic resonance and fluorescent dual imaging characteristic
CN111481740A (en) * 2020-04-17 2020-08-04 中山职业技术学院 High-dispersity amorphous calcium phosphate nano powder and preparation method and application thereof
CN111494706A (en) * 2020-04-17 2020-08-07 中山职业技术学院 Porous modified amorphous calcium phosphate nano powder and preparation method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Yang, L. et al. International Journal of Nanomedicine 2013:8 4147-4155

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108714223A (en) * 2018-05-31 2018-10-30 吉林大学 A kind of contrast agent and preparation method thereof having both magnetic resonance and fluorescent dual imaging characteristic
CN108714223B (en) * 2018-05-31 2021-04-13 吉林大学 Contrast agent with magnetic resonance and fluorescence dual imaging characteristics and preparation method thereof
CN111481740A (en) * 2020-04-17 2020-08-04 中山职业技术学院 High-dispersity amorphous calcium phosphate nano powder and preparation method and application thereof
CN111494706A (en) * 2020-04-17 2020-08-07 中山职业技术学院 Porous modified amorphous calcium phosphate nano powder and preparation method and application thereof

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