KR101239283B1 - Complex composition of nanofiber and target DNA for effective transfection into the cells - Google Patents

Abstract

The present invention relates to a gene carrier for delivering a target gene to a cell, wherein the gene carrier according to the present invention stably delivers a target gene to diseased cells or tissues, and exhibits sufficient selectivity for target cells to normal cells. Not only does it significantly reduce toxicity and allow continuous delivery of target genes, it also has higher selectivity for diseased tissues than normal low-molecular-weight gene carriers, leading to greater amounts of accumulation in target cells or tissues, resulting in significant therapeutic effects. There is an advantage.

Description

Complex fiber of nanofiber and target DNA for effective transfection into the cells

The present invention relates to a gene carrier for delivering a gene of interest to a cell, and more particularly, to a gene carrier for delivering a gene of interest, including a gene immobilized on a biodegradable polyesteramine nanofiber, to a cell. It is about.

Cardiac diseases such as coronary heart disease and heart failure are a large part of the medical burden in developed countries in the western world. In the United States, more than seven million people have had myocardial infarction and 4.9 million suffer from congestive heart failure. Various treatment methods and medical technologies have been rapidly developed for the treatment of heart disease, but the incidence of heart disease is still increasing, and more fundamental treatment methods are urgently needed.

Recently, studies showing that skeletal myoblasts have been implanted in the heart disease site have been shown to suppress the remodeling of myocardium and contribute to the improvement of contractility, suggesting hope for the treatment of heart disease in patients with heart disease. However, in performing such a method, the possibility of inducing ventricular arrhythmias or the differentiation of transplanted myoblasts into cardiomyocytes is difficult, and thus, it is difficult to view the functional recovery by fundamental myocardial regeneration.

Angiogenesis, on the other hand, is generally referred to the development and differentiation of blood vessels. Many proteins, typically referred to as “angiogenic proteins”, are known to promote angiogenesis. The angiogenic proteins include fibroblast growth factor (FGF) family, vascular endothelial growth factor (VEGF) family, platelet-induced growth factor (PDGF) family, insulin-type growth factor (IGF) family, and the like. FGF and VEGF Members of the family are recognized as regulators of angiogenesis during growth and development, and a role has been reported to promote angiogenesis in mature animals.

Injecting these angiogenic proteins into cells to promote angiogenesis of the heart is used as a therapeutic method for the treatment of heart disease. Plasmids, retroviruses, adenoviruses and other vectors for in vivo gene transfer to the myocardium. Barr, et al., Supplement II, Circulation, 84 (4): Abstract 1673, 1991: Barr, et al., Gene Therapy, 1: 51-58, 1994; French et al., Circulation, 90 (5): 2414-2424 (1994); French et al., Circulation, 90: 1517 Abstract No. 2785 (1994); Giordano et al., Clin. Res., 42: 123A (1994); Giordano et al. J. Investigative Med., Supplement 2, 43: 287A (1995); Guzman et al., Circulation Research, 73 (6): 1202-1207 (1993); Kass-Eisler et al., PNAS (USA) 90: 11498-11502 (1993); Muhlhauser et al., Human Gene Therapy, 6: 1457-1465 (1995); Muhlhauser et al., Circulation Research, 77 (6): 1077-1086 (1995); French et al., Circulation, 90 (5): 2402-2413 (1994); Rowland et al. , Am. Thorao.Surg., 60 (3): 721-728 (1995)).

In general, however, the reported methods had one or more of the following defects: inadequate transduction efficiency and transgene expression; Significant immune response to the vector used, including inflammation and tissue necrosis; And importantly, the relative inability to target transduction and transgene expression to the heart (ie, gene transfer also causes transgenes to be delivered to non-heart sites such as liver, kidney, lung, brain and testes of the test animal). Results).

In addition, there is a difficulty in requiring the proper form, concentration required for the effective absorption and promotion of proteins for the formation of new blood vessels, and the repeated infusion of proteins for angiogenesis.

The present inventors have continued to develop a system for efficiently in vivo delivery of a substance capable of effectively inducing angiogenesis, and as a result, the objective of including genes immobilized on biodegradable polyesteramine nanofibers To prepare a gene carrier for delivering the gene to the cell, confirming that the gene carrier enables efficient high-efficiency expression of the delivered gene and the continuous delivery of the gene into the cell efficiently, to complete the present invention.

An object of the present invention is to stably deliver a continuous gene to diseased cells or tissues, the target gene comprising a gene immobilized on a biodegradable polyesteramine nanofiber (fiber) to enable high efficiency expression of the delivered gene into a cell It is intended to provide a gene carrier for delivery and a method of manufacturing the same.

Another object of the present invention to provide a pharmaceutical composition for angiogenesis, comprising the gene carrier as an active ingredient.

In order to achieve the above object, the present invention provides a gene carrier for delivering a target gene including a gene immobilized on a biodegradable polyesteramine nanofiber (fiber) to a cell and a method for producing the same.

The present invention provides a pharmaceutical composition for angiogenesis, comprising the gene carrier as an active ingredient.

The gene carrier according to the present invention is immobilized a target gene on a biodegradable polyesteramine nanofiber prepared by electrospinning a polymer solution containing polyethylenimine (PEI) and polycaprolactone (PCL). It is characterized by.

The gene carrier according to the present invention is characterized in that the target gene is complexed with branched polyethylenimine cross-linked via disulfide bonds (ssPEI).

The gene carrier according to the present invention is for delivering a gene of interest to a cell, and more particularly, for delivering a gene of interest to a heart cell.

Gene carrier according to the present invention is to deliver angiogenic factor genes to cells, more specifically the angiogenesis factor is vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin- It is characterized by one or more selected from the group consisting of a similar growth factor, erythropoietin and nitric oxide synthase.

The pharmaceutical composition according to the present invention is an angiogenic pharmaceutical composition comprising the gene carrier as an active ingredient, characterized in that it is used for preventing or treating coronary artery disease.

Hereinafter, the present invention will be described in detail.

The present invention

Biodegradable polyesteramine nanofibers prepared by electrospinning a polymer solution including polyethylenimine (PEI) and polycaprolactone (PCL); And

Target gene immobilized on the nanofiber:

It provides a gene carrier for delivering a target gene, including a cell.

The gene carrier according to the present invention comprises the steps of electrospinning a polymer solution containing polyethylenimine and polycaprolactone (PCL) to produce biodegradable polyesteramine nanofibers (fiber); And

Immobilizing a gene of interest on the nanofibers:

Characterized in that it is prepared to include.

In the present invention, the polymer solution is characterized in that the molar ratio of polyethyleneimine and polycaprolactone is 1: 1 to 5.

The polymer may have a number average molecular weight such that electrospinning is possible, preferably 10,000 to 100,000, and may be used regardless of whether it is crystalline or amorphous. In addition, the solvent of the polymer solution for electrospinning dissolves the polymer. It can be used without limitation as long as it can be made and electrospinning possible.

The electrospinning method used in the present invention is a method for producing nanofibers having a diameter in a range of 100 nm to 10 μm by accelerating a jet of charged polymer solution in an electric field. Due to the surface tension at the beginning of spinning, the polymer solution forms a liquid droplet at the end of the micronozzle. When exposed to an electric field, an electric charge is induced on the surface of the polymer solution.

The electrospinning apparatus and conditions are not limited, and the diameter of the nanofibers obtained by electrospinning is preferably 1 μm or less.

In the present invention, the target gene is complexed with branched polyethylenimine (PEI) or cross-linked via disulfide bonds (ssPEI) crosslinked through disulfide bonds (ssPEI). It may be complexed), and more preferably complexed with branched polyethylenimine cross-linked via disulfide bonds (ssPEI) through disulfide bonds.

More specifically, a rerotor gene using a carrier of a target gene (ssPEI / DNA nanoparticles) complexed with crosslinked polyethyleneimine through a disulfide bond and a carrier of a target gene (bPEI / DNA nanoparticles) complexed with polyethyleneimine As a result of investigating the activity of luciferase and the expression level of vascular endothelial growth factor (VEGF) gene, it was confirmed that the gene transfer efficiency of the present invention is superior to the gene transfer efficiency using a general method (bolus). In addition, the delivery of the target gene (ssPEI / DNA nanoparticles) complexed with the crosslinked polyethyleneimine via disulfide bonds is higher than the transporter of the target gene (bPEI / DNA nanoparticles) complexed with the polyethyleneimine. It was confirmed that toxicity is minimized.

In the present invention, the target gene may be prepared by containing 1 to 95 parts by weight based on the total weight of the carrier.

In the present invention, the gene carrier is characterized in that the delivery of the gene of interest to the heart cells, more specifically, characterized in that the delivery of angiogenic factor (angiogenic factor) gene, the target gene to the heart cells.

In the present invention, the angiogenic factor is one selected from the group consisting of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor, erythropoietin and nitric oxide synthase It includes two or more.

The gene carrier according to the present invention has a higher selectivity for disease tissue than a general low molecular weight gene carrier, so that a larger amount accumulates in target cells or tissues, thereby having an advantageous therapeutic effect.

In addition, the gene carrier according to the present invention is intended to be complexed with branched polyethylenimine cross-linked via disulfide bonds (ssPEI) crosslinked through disulfide bonds on biodegradable polyesteramine nanofibers (fiber). Since the gene is immobilized and produced sufficiently to select a target cell, it has the advantage of significantly reducing toxicity to normal cells and enabling continuous delivery of the target gene.

The present invention provides a pharmaceutical composition for angiogenesis comprising the gene carrier prepared by the production method as an active ingredient.

In the present invention, the pharmaceutical composition is for preventing or treating coronary artery disease, and the coronary artery disease is characterized in that myocardial infarction.

The pharmaceutical composition of the present invention may further comprise one or more pharmaceutically acceptable carriers in addition to the gene carrier according to the present invention for administration.

Pharmaceutically acceptable carriers should be compatible with the active ingredients of the present invention and may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. Other conventional additives such as antioxidants, buffers, bacteriostatics, etc. may be added as necessary. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions and the like.

It may also be formulated in various forms, such as powders, tablets, capsules, solutions, injections, ointments, syrups, and the like, and may also be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles. .

The pharmaceutical composition of the present invention can be administered orally or parenterally. The route of administration of the pharmaceutical composition according to the invention is not limited thereto, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, Sublingual or topical administration is possible. For such clinical administration, the pharmaceutical compositions of the present invention can be formulated into suitable formulations using known techniques. For example, during oral administration, it may be mixed with an inert diluent or an edible carrier, sealed in hard or soft gelatin capsules, or pressed into tablets.

For oral administration, the active compound may be mixed with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. In addition, various formulations, such as for injection and parenteral administration, can be prepared according to techniques known in the art or commonly used techniques.

Dosage of the composition of the present invention varies in the range depending on the weight, age, sex, health status, diet, time of administration, administration method, excretion rate and severity of the disease, etc. of the patient, easy to those skilled in the art Can decide.

Gene delivery system according to the present invention has the advantage of stably delivering the target gene to the diseased cells or tissues, while exhibiting sufficient selectivity for the target cell, significantly reducing the toxicity to normal cells, and enable the continuous delivery of the target gene.

The gene carrier according to the present invention has a higher selectivity for disease tissue than a general low molecular weight gene carrier, so that a larger amount accumulates in target cells or tissues, thereby having an advantageous therapeutic effect.

1 shows expression of a retorter gene for a target gene (ssPEI / DNA nanoparticles) complexed with polyethyleneimine cross-linked through disulfide bonds and a target gene (bPEI / DNA nanoparticles) complexed with polyethyleneimine Is the result of a survey
Figure 2 is transformed into cells for the target genes (ssPEI / DNA nanoparticles) complexed with crosslinked polyethyleneimine and the target genes (bPEI / DNA nanoparticles) complexed with polyethyleneimine via disulfide bond according to the present invention Is the result of comparing the efficiency,
Figure 3 is a result of confirming the cytotoxicity of the target genes (ssPEI / DNA nanoparticles) complexed with the crosslinked polyethyleneimine through the disulfide bond and the target genes (bPEI / DNA nanoparticles) complexed with polyethyleneimine according to the present invention ego,
4 is a photograph confirming the gene carrier prepared by the production method of the present invention with a scanning electron microscope,
5 is a photograph confirming the absorbance of YOYO-1 of the gene carrier of the present invention, the YOYO-1 gene is immobilized by a fluorescence microscope,
6 is a photograph confirming the gene carrier of the present invention attached to myoblasts by scanning electron microscopy,
Figure 7 is the result of examining the expression level of VEGF mRNA (A) and protein (B) of the gene delivery vehicle of the present invention in which the vascular endothelial growth factor (VEGF) is immobilized.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples, and it is apparent to those skilled in the art that various changes or modifications can be made within the idea and scope of the present invention.

Here, unless otherwise defined in the technical terms and scientific terms used, those having ordinary skill in the art to which the present invention belongs have a generally understood meaning.

Repeated descriptions of the same technical constitution and operation as those of the conventional art will be omitted.

[Manufacturing Example]

(1) Preparation of biodegradable polyesteramine nanofiber

Biodegradable polyesteramine nanofibers (PCL / PEI nanofibers and PCL / ssPEI nanofibers) were prepared using an electrospinning system (CHUNGPAEMT Co., Ltd, Korea).

10 g of polycaprolactone (molecular weight: 10,000) and 10 g of polyethylenimine (PEI) (molecular weight: 25,000) or disulfide bond in 50 ml of Chloroform (C) / Dimethyl Formamide (DMF) solvent A polymer solution was prepared by dissolving 5 g of polyethyleneimine (Branched polyethylenimine cross-linked via disulfide bonds; ssPEI) (molecular weight: 25,000).

Electrospinning system consists of 1) DC High Voltage Generator (CPS-40K03VIT), 2) Universal Collecting Drum Unit (CPS-UD2S), 3) Universal Collecting Drum Controller (CPS-UC2000), 4) Syringe Pump (kd Scientific, USA) It consisted of. The prepared polymer solution was placed in a 10 mL syringe. After installing the positive pole of the high voltage generator in the syringe needle, the needle was mounted on the counter electrode at a speed of 20 μl / min, pushed and electrospun with a syringe pump to prepare a nanofiber. At this time, the voltage of the high voltage generator was used 14kV.

(2) Preparation of gene carrier complexed with the target gene

In order to prepare a gene carrier complexed with the target gene, the target gene was prepared by attaching the target gene to the biodegradable polyesteramine nanofiber prepared in (1) prepared at a ratio of 2 μg / cm 2. The target gene was PBS buffer.

In order to attach the gene of interest to the surface of the nanofiber, condensation of the gene with nanoparticles was used. For this purpose, biodegradable ssPEI was used. The pH of the branched polyethylene imine (average molecular weight approximately 1,200 or so) solution was adjusted to 7.2 and then lyophilized to obtain a powder. The powder was dissolved in methanol in a nitrogen environment and reacted by adding propylene sulfide. The reaction was evaporated under reduced pressure and then purified by precipitation in diethyl ether twice to give ssPEI.

That is, the gene carrier complexed with polyethylenimine (ssPEI or PEI) is dropped by dropping the target gene (DNA) solution into polyethyleneimine solution and mixed at room temperature (PEI / DNA nanoparticles). After treatment, DNA was attached to the surface of the biodegradable polyesteramine nanofiber prepared in (1) to prepare a gene carrier complexed with the target gene.

(3) Immobilization on the surface of nanofibers of gene carriers complexed with the gene of interest

The gene carrier complexed with the polyethylenimine crosslinked through disulfide bonds, the target gene (Ds) solution is dropped into the crosslinked polyethylenimine solution via disulfide bond (ssPEI / DNA) After incubation with nanoparticles), DNA was attached to the surface of the biodegradable polyesteramine nanofiber prepared in (1) to prepare a gene carrier complexed with the target gene.

[ Example And the target gene Complexed  Expression of the target gene of the gene carrier

(1) Expression investigation of target gene according to nanofiber

In the gene carrier complexed with the target gene, the expression level of the target gene according to the type of nanofibers prepared was investigated.

Luciferase, a rerotor gene, was used by using a transporter of ssPEI / DNA nanoparticles complexed with polyethyleneimine cross-linked through disulfide bonds and a transporter of bPEI / DNA nanoparticles complexed with polyethyleneimine. luciferase activity and the expression level of vascular endothelial growth factor (VEGF) gene were investigated.

Gene transfection was performed using NIH / 3T3 (ATCC, USA), fibroblasts, and H9C2 cells, muscle cells. Each cell was treated with the two kinds of gene carriers by 5 × 10 ⁴ cells cultured in DMEM medium containing 10% of FBS (bovine serum) at 37 ° C. and 5% carbon dioxide. For transformation, cells were attached to gene carriers for 14 hours, and then luciferase and VEGF expression levels were examined. Thereafter, after 24 to 48 hours, the expression level of the protein was quantified through the cell hemolysis process, and the expression level was monitored through the fluorescence microscope.

In addition, the two kinds of gene carriers were left at room temperature for 24 hours and then treated with fibroblasts (NIH / 3T3) and neurons (A10). After that, the survival rate of the cells was confirmed through time through LDH analysis.

As a result, as can be seen in Figures 1 and 2 it was confirmed that the time-dependent expression of luciferase, the gene delivery of the preparation example was confirmed that the gene delivery efficiency using a common method (bolus) is good. In addition, the delivery of the target genes (ssPEI / DNA nanoparticles) complexed with the crosslinked polyethyleneimine via disulfide bonds showed higher gene transfer efficiency than that of the target genes (bPEI / DNA nanoparticles) complexed with the polyethyleneimine. I could confirm it.

In addition, the cytotoxicity due to the gene delivered into the cell was confirmed that there is no cytotoxicity does not cause side effects on cell growth, as can be confirmed in the results of FIG.

(2) nano On fiber  Expression study of immobilized target gene

In the two types of gene carriers prepared above, YOYO-1 was labeled (green fluorescent marker) or vascular endothelial growth factor (VEGF) to examine the expression of the target gene immobilized on the prepared nanofibers. The plasmid was attached to the YOYO-1 gene and the VEGF gene on the surface of the nanofiber in the same manner as in (2) of Preparation Example. After 24 hours of further incubation, attachment images were confirmed by fluorescence microscopy and scanning electron microscopy.

As a result, as can be seen in Figures 4 to 6, the gene carrier of the preparation example has a cylindrical geometry, the yellow cross-section is circular, it was confirmed that the diameter is 100 nm to 10 ㎛. In addition, in the gene carrier, genes attached to the nanofibers are evenly distributed, and through the scanning electron microscopy of the gene carriers attached to myoblasts, the gene carriers of the present invention directly transfer the target gene directly into the cells. Not only could it be delivered to the environment, but it was also found to increase uptake and expression into cells.

(3) Expression of the gene of interest transferred from the gene carrier into the cell

Reverse transcriptome gene amplification analysis (RT-PCR) was performed to confirm the expression of VEGF. RT-PCR confirmed the amount of GAPDH and VEGF mRNA expression of housekeeping (genes in which a certain amount is present in living cells) genes, and the amount of protein expressed through immunoblot analysis (western blot).

As a result, as can be seen in Figure 7, it was confirmed that the mRNA expression amount and the protein expression amount of the VEGF gene delivered into the cell can be expressed with high efficiency in the cell.

From the above results, it was confirmed that the continuous gene delivery into cells and high efficiency expression of the delivered genes were possible without causing side effects on cell growth.

In addition, the above result is a result confirming that the gene carrier of the present invention can selectively express angiogenic factor (angiogenic factor) genes that lead to normal angiogenesis can greatly contribute to the treatment of heart disease.

Claims (12)

delete delete Biodegradable polyesteramine nanofibers prepared by electrospinning a polymer solution including polyethylenimine (PEI) and polycaprolactone (PCL); And
A target gene immobilized on the nanofiber; In a gene carrier comprising:
The gene of interest is a gene carrier for delivering a gene of interest to the cell, characterized in that complexed with (branched polyethylenimine cross-linked via disulfide bonds; ssPEI) cross-linked through disulfide bonds. The method of claim 3, wherein
The gene of interest for delivering the gene of interest to the cell, characterized in that contained in 1 to 95 parts by weight relative to the total weight of the carrier. The method of claim 3, wherein
The gene of interest for delivering the gene of interest to the cell, characterized in that the gene of interest (angiogenic factor) gene. 6. The method of claim 5,
The angiogenesis factor is one or more genes selected from the group consisting of vascular endothelial growth factor (VEGF), fibroblast growth factor, insulin-like growth factor, erythropoietin and nitric oxide synthase Gene carrier for delivering to cells. The method of claim 3, wherein
The gene carrier is a gene carrier for delivering the target gene to the cell, characterized in that for delivering the target gene to the heart cell. Claims 3 to 7, including the gene carrier according to any one of the active ingredient, angiogenesis pharmaceutical composition. The method of claim 8,
The pharmaceutical composition is an angiogenic pharmaceutical composition, characterized in that for preventing or treating coronary artery disease. The method of claim 9,
The coronary artery disease is myocardial infarction (Myocardial infarction) characterized in that the pharmaceutical composition for angiogenesis. Preparing a biodegradable polyesteramine nanofiber by electrospinning a polymer solution including polyethylenimine (PEI) and polycaprolactone (PCL); And
Immobilizing a gene of interest on the nanofiber;
A method for producing a gene delivery system according to any one of claims 3 to 7, including. 12. The method of claim 11,
The gene of interest is a method for producing a gene carrier, characterized in that complexed with (branched polyethylenimine cross-linked via disulfide bonds (ssPEI) cross-linked through disulfide bonds.

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