KR20110012817A - Nano scaffold for gene delivery and a method for preparing the same - Google Patents

Abstract

PURPOSE: A nanostructure for gene delivery and a kit or pharmaceutical composition containing the same are provided to prevent random gene release and to gene deliver to a specific region. CONSTITUTION: A nanostructure for gene delivery contains a nanofiber, an enzyme substrate peptide and a complex of a cationic polymer and gene. The nanofiber and complex are linked by enzyme substrate peptide. The nanofiber contains biodegradable polymer. A method for preparing the nanostructure for gene delivery comprises: a step of preparing the nanofiber; a step of binding the enzyme substrate peptide to the nanofiber; a step of binding the cationic polymer to the enzyme substrate peptide; and a step of binding a gene to the cationic polymer.

Description

Nano scaffold for gene delivery and a method for preparing the same

The present invention relates to a nanostructure for gene delivery, a method for preparing the same, a kit comprising the same, a gene delivery method using the same and a wound treatment method. Specifically, the present invention provides a nanostructure for gene delivery comprising (i) a nanofiber, (ii) an enzyme substrate peptide, and (iii) a complex of a cationic polymer and a gene, wherein the nanofiber and the complex are the enzyme The present invention relates to a nanostructure connected by a substrate peptide, a method for preparing the same, a kit including the same, a gene transfer method using the same, and a wound treatment method.

Gene therapy is a new therapeutic method that delivers genes to specific cells to treat or prevent disease. Specifically, gene therapy is a method of treating a gene that is missing in a cell with a normal gene, inserting a new gene to give a new function to the body, or regulating the function of a gene that is over or underacting. In gene therapy, a vehicle required for introducing a therapeutic gene into a target cell is a gene carrier.

Such gene carriers are viral and non-viral carriers. However, due to the disadvantage that the virus is not only toxic due to a strong immune response, but also difficult to administer again, a lot of research has been conducted on the use of the virus.

On the other hand, recent studies to use nanofibers as gene carriers have been actively conducted (M Hadjiargyrou et al., Journal of Controlled Release, 89, "Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers). ", 2003, 341-353). Nanofibers can be obtained using an electrospinning method, which is economical and has the advantage of being easily obtained using desired raw materials. In particular, in terms of wound healing, nanostructures obtained through electrospinning, that is, nanofibers, have a large surface area, which can effectively modify a desired drug or gene, and are similar in shape to an extracellular matrix (ECM). Thus, it serves to promote differentiation or growth of cells. In addition, the randomly arranged nature of the nanofibers can prevent the invasion of bacteria from the outside. Due to these characteristics, nanofibers are widely applied in the field of tissue engineering. It is also possible to modify drugs (or growth factors) that promote wound healing on nanofiber surfaces (Hyuk Sang Yoo et al., Biomaterials, "In vivo wound healing of diabetic ulcers using electrospun nanofibers immobilized with human epidermal growth factor", 29 , 2008, 587-596).

Korean Patent Laid-Open Publication No. 10-2006-0097806 discloses a spinning solution in which a polymer resin is dissolved in a volatile solvent by electrospinning a spinning solution on a collector under a high voltage to a nanofiber having a thickness of 60-4000 nm. Disclosed is a method for producing porous nanofibers, characterized in that the collector is heated to a temperature range that is equal to or higher than the boiling point of the volatile solvent constituting and is equal to or less than the secondary glass transition temperature (Tg) of the polymer resin. The nanofibers prepared in this way have been described as having a large number of fine pores in the form of slits that do not penetrate the nanofibers, and thus can be used as gene carriers.

US 2008/0149561 discloses a fibrous support comprising nanofibers; And an interfacial polymerized polymer layer disposed on the fibrous support surface. The interfacially polymerized polymer layer may comprise DNA, RNA, etc. as functional molecules, such that biological substrates (e.g., scaffolds for tissue regeneration, immobilized enzyme and catalyst systems, wound dressings, artificial blood vessels, post-surgery) It can be used as a material for the prevention of induced stenosis).

However, the method of delivering genes using nanofibers has various problems. As a problem with most gene delivery systems, there is a problem that the time taken for the delivered gene to be actually expressed as a protein is too long.

On the other hand, when a wound is formed, bioactive substances such as various growth factors or enzymes for wound healing are generated at the wound site. One of them is matrix metalloproteinase (MMP). MMP is an enzyme that dissolves the extracellular matrix and facilitates cell migration to the wound site, thereby promoting wound healing. The enzyme, which is known to be overexpressed in cancer cells, is usually expressed in cells, but it is also known to overexpress at the wound site. Therefore, researches have been conducted using the enzyme substrate as a linker to make a gene carrier so that the gene is specifically released only at cancer cells or wound sites, thereby reducing the loss of the gene and accurately delivering the desired gene without side effects. Youngro Byun et al., European Journal of Pharmaceutics and Biopharmaceutics, 67, 2007, 646-654.

The present invention provides a nanostructure for gene delivery, a method for preparing the same, a kit or pharmaceutical composition comprising the same, a gene delivery method and a wound treatment method using the same.

The present invention provides a nanostructure for gene delivery, a method for preparing the same, a kit including the same, a gene delivery method using the same and a wound treatment method.

Hereinafter, the present invention will be described in detail.

Nanostructure for gene delivery of the present invention is a nanostructure for gene delivery comprising (i) a nanofiber, (ii) an enzyme substrate peptide, and (iii) a complex of a cationic polymer and a gene, wherein the nanofiber and the The complex may be linked by the enzyme substrate peptide.

One end of the enzyme substrate peptide in the nanostructure is nanofibers are bonded, and the other end may be a complex of a cationic polymer and a gene.

The nanofiber may be polycaprolactone or polycaprolactone-polyethylene glycol block copolymer, polylactic glycolic acid (PLGA), polylactic acid (PLA), polyglycolic acid (PGA) or polylactic glycolic acid-polyethylene glycol copolymer (PLGA It may include, but is not limited to, polyester-based polymers including -PEG) or biodegradable polymers including collagen. For example, the nanofibers may be an amine group exposed on the surface of the nanofibers. The diameter of the nanofibers may be 560-860 nm, but is not limited thereto. The method for producing nanofibers may use a conventional method known in the art for producing nanofibers from a polymer using electrospinning.

The enzyme substrate peptide may include a hydrolase-specific peptide including a matrix metalloproteinase (MMP) family. Preferably, the enzyme substrate peptide may comprise MMP-2 or MMP-9. More preferably the enzyme substrate peptide may comprise the amino acid sequence of SEQ ID NO: 1 (DGPLGVC). The enzyme substrate peptide may include functional groups available for binding to nanofibers and cationic polymers. For example, enzyme substrate peptides may include carboxyl groups, thiol groups, and the like.

The nanofibers and enzyme substrate peptides can be combined in a variety of ways. For example, the nanofibers and the enzyme substrate peptides can be bound by reacting the functional groups of the nanofibers with the functional groups of the enzyme substrate peptides. The functional group may include an amino group, a carboxyl group, a hydroxyl group, a thiol group, and the like. For example, the nanofibers and the enzyme substrate peptides can be bound by reacting the amine groups exposed on the surface of the nanofibers with the carboxyl groups of the enzyme substrate peptides.

The cationic polymer may include polyethyleneimine, chitosan, protamine and derivatives thereof. For example, the cationic polymer may comprise polyethyleneimine. Also, for example, the cationic polymer may be an activated cationic polymer for binding to an enzyme substrate peptide. The cationic polymer may be in linear or branched form having a molecular weight of 25000 Daltons, but is not limited thereto.

The cationic polymer may be bound to the other end of the enzyme substrate peptide to which the nanofibers are bound. For example, cationic polymers can be bound to enzyme substrate peptides by disulfide bonds. For example, cationic polymers can be activated and bound to enzyme substrate peptides.

The gene may be selected from the group consisting of gDNA, cDNA, plasmid DNA, mRNA, tRNA, rRNA, antisense nucleotides, missense nucleotides and protein producing nucleotides. In addition, the gene may be a gene expressing a growth factor that promotes wound healing. For example, the gene may be an epidermal growth factor (EGF), a fibroblast growth factor (FGF), a platelet-derived growth factor (PDGF), a tumor growth factor-b (transforming growth factor-b), TGF-b), vascular endothelial growth factor (vEGF), or a gene expressing insulin. For example, the gene may be an EGF having a DNA sequence of SEQ ID NO: 2 and an FGF having a DNA sequence of SEQ ID NO: 3. Or the gene may be an anti-tumor gene. For example, the anti-tumor gene may be, but is not limited to, a tumor suppressor gene, an apoptosis inducer gene, a cell cycle regulator gene or an angiogenesis suppressor gene.

The gene may be bound to the cationic polymer to form a complex of the cationic polymer and the gene. For example, the negative charge of a gene and the positive charge of a cationic polymer can form a complex by ionic bonding.

In the nanostructure according to the present invention, the N / P ratio (ratio of the number of nitrogen atoms / nucleic acid phosphate atoms of the nanostructure) may be in the range of 0.1-100.

Figure 1 shows an embodiment of the nanostructures according to the present invention and the release of growth factors in epidermal cells at the wound site after these nanostructures are enzyme cleaved by MMP.

As shown in FIG. 1, the nanostructure according to the present invention is, in one embodiment, an MMP-specific peptide bound to an amino group of a nanofiber, and further, a complex of polyethyleneimine and a gene forms a disulfide bond with an MMP-specific peptide. It has a structure that is bonded while forming. When the nanostructure according to the present invention comes into contact with the cells at the wound site, the MMP specific peptide is degraded by MMPs formed in excess at the wound site, thereby releasing polyethyleneimine and gene particles. The released polyethyleneimine and gene particles are injected into epidermal cells with wound sites to help heal wounds by expressing specific genes. Through this, it may be possible to promote wound healing by specifically releasing the gene only at the wound site, using MMPs that are overexpressed at the wound site, rather than randomly releasing the gene. In addition, the nanostructures of the present invention can control the rate of gene release.

The present invention also provides a method for producing a nanostructure according to the present invention.

Method for producing a nanostructure according to the present invention comprises the steps of (i) preparing a nanofiber; (ii) binding an enzyme substrate peptide to the nanofibers to prepare an enzyme substrate peptide to which nanofibers are bound; (iii) binding a cationic polymer to the enzyme matrix peptide to which the nanofibers are bound; And (iv) binding a gene to the cationic polymer.

Details of nanofibers, enzyme substrate peptides, cationic polymers and genes apply in the same manner as described in the nanostructures above.

Nanofibers can be prepared using conventional methods known in the art of making nanofibers from polymers using electrospinning. In addition to the organic solvent for the nanofiber manufacturing polymer, the emission conditions including the diameter of the nozzle, the release rate, the voltage, the distance between the nozzle and the ground, the temperature and humidity during the electrospinning, etc. can be appropriately modified. The organic solvent may include an organic solvent which is highly volatile and capable of dissolving the polymer. For example, dichloromethane, chloroform or dioxane, dimethyl sulfoxide may be used, the nozzle diameter is 27 G, and the discharge rate is 1 ml. / h, the voltage is 15kV, the distance between the nozzle and the ground is 15cm, the temperature and humidity during the electrospinning can be adjusted to 25 ℃ and 30-50%, but is not limited to these.

By binding the enzyme substrate peptide to the nanofibers, the enzyme substrate peptides to which the nanofibers are bound may be prepared. Nanofibers and enzyme substrate peptides can be bound using a variety of methods. For example, the nanofibers and the enzyme substrate peptides can be bound by reacting the functional groups of the nanofibers with the functional groups of the enzyme substrate peptides. The functional group may include an amino group, a carboxyl group, a hydroxyl group, a thiol group, and the like. In this linkage, a coupling agent comprising EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide), NHS (N-hydroxysuccinimide) or HOBt (hydroxybenzotriazole) is added. Can be used as Figure 2 shows that the PCL-PEG block copolymer and MMP-specific peptide is bonded to one embodiment of the present invention.

Cationic polymer may be bound to the enzyme substrate peptide to which the nanofibers are bound. The cationic polymer may be bound to the other end of the enzyme substrate peptide to which the nanofibers are bound. The linkage can be carried out using a variety of methods including chemical linkages. For example, the thiol group of the enzyme substrate peptide and the cationic polymer may be bonded. In addition, the cationic polymer may be activated for binding to the enzyme substrate peptide. Cationic polymers are either SPDP (N-succinimidyl 3- (2- (pyridylthio) -propionate) or LC-SPDP (succinimidyl 6- (3- [2-pyridylthio] -propionamido Hexonate), which activates the thiol group of the cationic polymer and binds the activated cationic polymer to the enzyme substrate peptide, Figure 2 shows a thiol of an MMP-specific peptide that is an embodiment of the invention. Group and SPDP-activated polyethyleneimine are shown to be bonded through disulfide bonds.

A gene may be coupled to the cationic polymer. The binding may be performed using various methods including ionic bonds, hydrophobic bonds, and coordinative bonds. For example, cationic polymers and genes can be combined to form a complex by ionic bonding of positive charges of cationic polymers and negative charges of genes. For example, the cationic polymer in which the nanofibers and the enzyme substrate peptide are bound can be bound by adding and reacting a solution containing a gene.

The present invention also provides a kit comprising the nanostructure of the present invention.

The kit of the present invention may comprise a nanostructure according to the present invention, or a nanostructure produced by the manufacturing method of the present invention. The present invention also provides a pharmaceutical composition or gene therapy agent comprising the nanostructure of the present invention. The kit, pharmaceutical composition or gene therapeutic agent according to the present invention may be used for wound treatment or tumor treatment in one embodiment, wherein the substrate peptide of MMP overexpressed at the wound site or tumor site may be used as the enzyme substrate peptide.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive in addition to the active ingredient, and may be formulated into a unit dosage form suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field. . Formulations suitable for this purpose are preferably oral or parenteral preparations, such as injection or topical preparations, wound dressings and the like. For example, it can be used parenterally in the form of injections of sterile solutions or suspensions using water or other pharmaceutically acceptable solvents as necessary. For example, in combination with a pharmaceutically acceptable carrier or medium such as sterile water, saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, excipients, vehicles, preservatives, binders and the like, It can be formulated by blending in a generally accepted unit dosage form. Dosage and timing of the pharmaceutical composition of the present invention will vary depending on the age, sex, condition, weight, route of administration, frequency of administration, and form of the subject. The daily dosage is about 0.01 ug / kg to 10 g / kg, preferably 0.01 mg / kg to 100 mg / kg.

In addition, the present invention in vitro (in vitro) or in vivo (in It provides a method for delivering genes to cells in vivo . The method comprises contacting the cells with the nanostructures of the invention. The contacting method may use, for example, but is not limited to, topical administration, systemic administration, intravenous administration, parenteral administration of the nanostructures.

The present invention also provides a method of treating wounds in cells in vitro or in vivo. The method comprises contacting the nanostructures of the present invention in vitro or with cells of in vivo. The contacting method may use, for example, but is not limited to, topical administration, systemic administration, intravenous administration, parenteral administration of the nanostructures.

The present invention does not randomly release a gene, but can use an enzyme expressed in excess at a specific site, so that the gene can be specifically delivered only to that site.

Hereinafter, an Example demonstrates this invention in more detail. However, the present invention is not limited by the following examples.

<Reagents and Samples>

Polycaproloktan [poly (ε-caprolactone), Mw 70,000-100,000] was purchased from Wako. PEG (polyethyleneglycol, Mw 2,000) with an amine group was purchased from SunBio, Korea. Linear PEI [polyethyleneimine, Mw 25,000] was purchased from Polyscience, Inc. MMP substrate peptides cleaved by MMP were made in Anygen (Korea). Fluorescamine was purchased from Sigma. Advanced Protein Assay (APA) reagents for PEI assay were purchased from Cytoskeleton. Quantum dot (QD-NH ₂ ) having an amine group at the end was purchased from ebioscience. NIH 3T3 cells were distributed by the Korea Cell Line Bank.

Example  1: Fabrication of Nanostructures

(1) Preparation of Polycaprolactone-Polyethylene Glycol Block Copolymer

3 g of polycaprolactone (PCL) (Mw 70,000-10,000) was dissolved in 30 ml of dichloromethane and stirred at 0 ° C. To the obtained solution was added 9.7 μl of pyridine and 12 mg of p-nitro phenyl chloroformate (molar ratio of PCL, p-nitro phenyl chloroformate, and pyridine is 1: 2: 4). The resulting mixture was further reacted for 3 hours at room temperature, then precipitated in ice-cold diethyl ether and dried overnight in vacuo. The precipitation reaction was repeated twice and dried to obtain 2 g of activated polycaprolactone. 2 g of the obtained polycaprolactone and 0.2 g of polyethylene glycol were dissolved in 20 ml of dichloromethane, respectively (molar ratio of polycaprolactone and polyethylene glycol was 5: 1). The obtained polycaprolactone dichloromethane solution was slowly added dropwise to the polyethylene glycol dichloromethane solution, at which time the stirring was accelerated to give a uniform reaction. After dropwise addition, the mixture was allowed to react at room temperature for 24 hours, and then poured into ice-cold ethanol to precipitate. The precipitated polycaprolactone-polyethyleneglycol block copolymers were dried in vacuo overnight. The synthetic yield of the obtained block copolymer was confirmed by 400 Hz ¹ H NMR. The results are shown in Fig.

As shown in FIG. 3, it was confirmed that polycaprolactone and polyethylene glycol were synthesized in a ratio of 1: 1 to form a diblock copolymer. The prepared copolymer was sealed and frozen.

(2) nanofiber production

The synthesized polycaprolactone-polyethylene block copolymer was dissolved in an organic solvent in which chloroform and methanol were mixed 1: 3 at a concentration of 10% by weight. The resultant solution was flowed through an electrospinning machine through a nozzle having a discharge rate of 1 m / h, a voltage of 15 kV, a nozzle distance of 15 cm and a diameter of 27 G. Temperature and humidity were 25 ° C. and 30-50%, respectively, during spinning. The shape of the fiber obtained after electrospinning was observed through FE-SEM (Field Emission Scanning Electron Microscope), and the diameter was measured through image analysis. The results are shown in FIG.

As shown in FIG. 4, it was confirmed that nanofibers without beads were produced. In addition, the thickness of the prepared nanofibers was about 500 nm to 1 μm.

(3) binding MMP substrate peptide and polyethyleneimine to nanofibers

(i) Determination of amines exposed on the surface

The prepared nanofibers were wetted with 30% ethanol and then hydrated with PBS (pH 7.5). Fluorescamin (0.3 mg / ml) was dissolved in acetone and dispensed into nanofibers, followed by reaction at room temperature for 30 minutes. PBS was removed and washed with methanol for 30 minutes and the washing process was repeated three times. After the obtained nanofibers were dried, they were completely dissolved in 1,4-dioxane to measure fluorescence. As a result of quantifying the exposed amine on the surface of the nanofibers, it was confirmed that the amine of 0.387 nmol / mg was exposed. This is about 3-4% of the total amines of the nanofibers, which is a large amount of amines piled up in the electrospinning process of receiving the nanofibers in a finite area, the amines enter into the interior of the nanofibers, which is substantially exposed to the surface of the nanofibers. The content of is considered to be small.

(ii) binding MMP substrate peptide and polyethyleneimine to nanofibers

Polycaprolactone-polyethyleneglycol nanofibers were wetted with 30% ethanol and then hydrated with PBS-EDTA (pH 7.5) buffer. To the obtained solution was added MMP substrate peptide dissolved in the same buffer (wherein the molar ratio of amine and peptide exposed on the surface of the nanofibers was 1: 1). EDC and NHS were added to the obtained mixed solution and reacted at room temperature for 2 hours (moles of EDC and NHS were added at about 10 times the number of moles of peptide). Polyethyleneimine was dissolved in PBS-EDTA, mixed with 20mM SPDP and reacted at room temperature for 30 minutes to activate polyethyleneimine. Activated polyethyleneimine and peptide-bound nanofibers were mixed, allowed to react at room temperature overnight, and then dried.

The extent to which polyethyleneimine is bound to the surface of polycaprolactone-polyethylene glycol nanofibers was measured using X-ray photoelectron spectroscopy (XPS, ESCA-2000 (Thermo Scientific)). XPS used non-monochromated Al irradiation as the x-ray source. As a result of XPS, a spectrum including C1s, O1s, and N1s is obtained, and the degree of binding of polyethyleneimine is measured by comparing the intensity of the C1s peak and the O1s peak with the intensity of the peak of N1s. For comparison, XPS was also performed on polycaprolactone-polyethylenimine nanofibers not bound with polyethyleneimine. The results are shown in Fig.

As shown in Figure 5, the polyethyleneimine-bonded nanofibers confirmed that there is 0.384 nmol / mg of nitrogen. This indicates that 99.2% (0.384 / 0.387 = 0.99) of ethylene glycol exposed to the surface of the nanofibers is bound to polyethyleneimine.

(4) binding genes to polyethylenimine

The prepared nanofibers were added to the silicon-coated microtubes, soaked with 30% ethanol, and hydrated with 1 ml PBS. Here, plasmid DNA expressing GFP (GenBank Accession # U55762) (SEQ ID NO: 4) was added at an N / P ratio according to Table 1 below. Since the nanofibers to which the gene binds are fixed and thus the polyethyleneimine content is fixed, the N / P ratio was controlled by adding different amounts of DNA. As a result, the lower the N / P ratio, the greater the amount of DNA added. After the addition of the DNA was reacted overnight with stirring at 37 ℃. The amount of DNA not bound to the nanofibers was measured by taking out the nanofibers and measuring the absorbance of the remaining supernatant. In this way it was confirmed whether the gene is bound to the nanofibers and the extent of gene binding according to the N / P ratio. The results are shown in Table 1 below.

TABLE 1

Nanofiber NF according to N / P ratio Amount of DNA added
(Μg / device) Amount of DNA bound to nanofibers (µg / device) Nanofiber-DNA Bonding Ratio (%) NF 2 36.7 6.4 ± 0.5 17.4 ± 1.4 NF 4 18.3 5.7 ± 0.8 30.9 ± 4.5 NF 8 9.2 4.4 ± 0.7 47.8 ± 7.7 NF 16 4.6 3.9 ± 0.2 79.5 ± 3.3

As shown in Table 1, it can be seen that the gene is bound to the nanofibers, and when the N / P ratio is 16, it can be seen that the gene is effectively bound to the nanofibers.

Example  2: Polyethylenimine-Plasmids from Nanostructures DNA Release of (1)

Nanostructures having N / P ratios (2, 4, 8, 16) prepared according to Example 1 were placed in a 50 ml conical tube coated with silicone and wetted with 30% ethanol, and then 5 ml of PBS (pH 7.5). ) Was added to hydrate the nanostructures. MMP-2 was added thereto in the same mole number as the MMP substrate peptide, followed by stirring at 37 ° C. at 250 rpm. The stirring time was 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours and 72 hours. The solution was aliquoted for each stirring time, and polyethyleneimine and DNA were quantified from the aliquoted solution.

50 μl of the solution was aliquoted at each stirring time, and 1.5 ml of 1 × APA reagent was added to the aliquot of the solution, followed by reaction at room temperature for 30 minutes, and the absorbance value was measured at 590 nm. Quantification In the same way, absorbance was measured at 260 nm to quantify the released DNA. The results are shown in Figure 6 (a and c).

In addition, the control group not treated with MMP quantified the amount of polyethyleneimine and DNA released in the same manner as described above, the results are shown in Figure 6 (b and d).

As salpin in Figure 6, the DNA release of NF 2 was the greatest increase over time. This is because the lower the N / P ratio, the lower the binding force between PEI and DNA. On the other hand, NF 16 was the highest in PEI. Referring to Table 1, as the N / P ratio decreased, the amount of bound DNA increased. Because of this, MMP interferes with the action of breaking the linker (MMP specific peptide), which seems to result in the least release of PEI of NF 2. By confirming the low degree of release of the control, it was confirmed that the MMP specific peptide effectively regulates the release of the PEI / DNA complex.

Example  3: Polyethylenimine-Plasmids from Nanostructures DNA Release of (2)

0.5M NaHCO ₃ at 0.5mg / ml DNA (pH 9.6) After adding 3 ml, NBS was added 10 times (molar ratio) of DNA and reacted for 10 minutes on ice. Then, the Quantum dot having an amine functional group at the end was added in 1/10 (molar ratio) and reacted at 50 ° C. for 1 hour and at room temperature overnight. The reaction finished QD-DNA was dialyzed using a dialysis membrane (cut off = 10 kDa) to obtain a QD-DNA.

The obtained QD-DNA was bonded to the nanofibers by varying the N / P ratio according to Example 1. Then, according to Example 2, the emission difference was confirmed using a confocal laser scanning microscopy (CLSM) of nanofibers before treatment (0 hours) and 72 hours after MMP treatment. The results are shown in FIG.

As shown in FIG. 7, before and after the MMP treatment, it can be visually confirmed that the PEI / DNA complex was released from the nanofibers.

In addition, the size of the released PEI / DNA complex was confirmed by using dynamic laser scattering (DLS). The results are shown in Table 2.

TABLE 2

Average particle size (nm) NF 2 505.1 ± 19.3 NF 4 471.9 ± 9.5 NF 8 379.9 ± 15.7 NF 16 248.5 ± 21.1

As shown in Table 2, as the N / P ratio was increased, the particle size tended to decrease. This is because the binding force between PEI and DNA increases as the N / P ratio increases.

In addition, a gel retardation assay was performed to confirm that complexes measured by DLS were properly formed. 0.32 g agarose was dissolved in 39.68 ml 1 × TBE buffer, 2 μl of EtBr (Ethidium bromide) was added to the mixture, poured into a mold, and placed in a comb (dmf) for 30 minutes. After the release was completed by 72 hours, the remaining solution was freeze-dried and dissolved in 100ul 3 distilled water to prepare a sample. 2.5 μl 5 × loading buffer and 10 μl sample were mixed and dispensed into each well, followed by electrophoresis at 100 V for 20 minutes. After 20 minutes, the movement of the particles was confirmed by a UV transilluminator. The results are shown in FIG. As shown in FIG. 8, gel delay analysis showed that particles remained in the wells of all groups. This confirmed that the PEI / DNA complex was formed surely.

The released complex was used to perform transfection (an experiment for measuring gene transfer efficiency). The powder obtained by lyophilizing the solution after 72 hours of dissolution was prepared by dissolving in 100 µl tertiary distilled water. In 12 wells, NIH 3T3 was passaged at ⁵ × 10 ⁵ cells / well and stabilized for 24 hours, followed by transfection for 5 hours by treatment with 900 μl serum-free DMEM medium and 100 μl samples. NF proceeded to 2, 4, 8, 16. At this time, to determine the difference in transfection results according to the amount of DNA, the experiment was performed in the same amount (100 µl) of the dissolved group and the same amount of DNA (5 µg) in order to know the effect of PEI. (I.e. the highest release of PEI / DNA complex in NF 2, so all groups were dissolved in the same volume and the highest amount of PEI / DNA complex entered in the wells treated with the released fraction of NF 2, If the amount of DNA in the group is calculated to be the same and treated with the released fraction of NF 16, the most PEI is contained, so each experiment was performed to compare the differences.) After 5 hours, The serum was changed to DMEM medium. After 43 hours, the cells were lysed by treating with 2 ml lysis buffer to measure the total amount of protein expressed by BCA (Bicinchoninic acid assay), and the fluorescence of the expressed GFP was measured using a spectrofluorometer. Measured. This is shown in FIG. In FIG. 9, the volume refers to a powder obtained by lyophilizing the released fraction and dissolving it in 100 µl of distilled water. As shown in FIG. 9, as the N / P ratio was increased, the transfection efficiency increased.

Figure 1 shows the nanostructures according to the invention, and the nanostructures release the growth factor in epidermal cells at the wound site after enzyme cleavage by MMP.

Figure 2 shows an embodiment of the process of binding the MMP specific peptide, and polyethyleneimine to the polycaprolactone-polyethylene glycol block copolymer.

Figure 3 shows the ¹ H-NMR results of the polycaprolactone-polyethylene block copolymer.

4 is an enlarged magnification of 2000 times (a) and 5000 times (b) the surface of a nanofiber prepared by electrospinning a polycaprolactone-polyethylene glycol block copolymer.

FIG. 5 shows the XPS of the case in which MMP substrate peptide is bonded to the nanofibers prepared by electrospinning polycaprolactone-polyethylene glycol block copolymer and then polyethyleneimine is not bound (a) and polyethyleneimine is bound (b). The results are shown.

FIG. 6 shows the results of quantifying DNA and PEI, respectively, in a release experiment of PEI / DNA complexes from nanofibers. It shows the group (A, C) which processed MMP and the group (B, D) which did not process MMP.

Figure 7 is observed with a confocal laser microscope to compare the before and after the treatment of MMP by quantum dot stained genes to the nanofibers.

Figure 8 shows the results of electrophoresis to confirm the PEI / DNA complex.

9 shows the results of transfection of the released PEI / DNA complex with NIH 3T3.

<110> KNU-Industry Cooperation Foundation <120> NANO SCAFFOLD FOR GENE DELIVERY AND A METHOD FOR PREPARING THE          SAME <130> PN084215 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 7 <212> PRT <213> MMP Peptide <400> 1 Asp Gly Pro Leu Gly Val Cys   1 5 <210> 2 <211> 165 <212> DNA <213> EGF <400> 2 atgaactcag atagtgaatg ccctttgagc cacgatggat attgcctaca tgacggtgtt 60 tgtatgtata tcgaggcttt agacaaatac gcatgcaact gcgttgttgg ttacatcgga 120 gaaagatgtc aataccgtga cttaaaatgg tgggaattac gttaa 165 <210> 3 <211> 570 <212> DNA <213> FGF <400> 3 atggcgagca aagaacccca gctcaaaggg attgtgacaa ggttattcag ccagcaggga 60 tacttcctgc agatgcaccc agatggtacc attgatggga ccaaggacga aaacagcgac 120 tacactctct tcaatctaat tcccgtgggc ctgcgtgtag tggccatcca aggagtgaag 180 gctagcctct atgtggccat gaatggtgaa ggctatctct acagttcaga tgttttcact 240 ccagaatgca aattcaagga atctgtgttt gaaaactact atgtgatcta ttcttccaca 300 ctgtaccgcc agcaagaatc aggccgagct tggtttctgg gactcaataa agaaggtcaa 360 attatgaagg ggaacagagt agagaaaacc aagccctcat cacattttgt accgaaacct 420 attgaagtgt gtatgtacag agaaccatcg ctacatgaaa ttggagaaaa acaagggcgt 480 tcaaggaaaa gttctggaca ccaccatgat ggaggcaagt tgtgatcagt tcacatgctg 540 agactcccct tcttccctct ctcatccctt 570 <210> 4 <211> 4733 <212> DNA <213> plasmid DNA <400> 4 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg 660 gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 720 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 780 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 840 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 900 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 960 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 1020 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 1080 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 1140 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac 1200 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 1260 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 1320 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 1380 gacgagctgt acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg 1440 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa 1500 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca 1560 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 1620 ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc 1680 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc 1740 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag 1800 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc 1860 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa 1920 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg 1980 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt 2040 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc 2100 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 2160 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 2220 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt 2280 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt 2340 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc 2400 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc 2460 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg 2520 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc 2580 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa 2640 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg 2700 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc 2760 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca 2820 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc 2880 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca 2940 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat 3000 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca 3060 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg 3120 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc 3180 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct 3240 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct 3300 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac 3360 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc 3420 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag 3480 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg 3540 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg 3600 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa 3660 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt 3720 cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg 3780 tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca 3840 acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt tagattgatt 3900 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 3960 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 4020 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 4080 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 4140 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 4200 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 4260 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 4320 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 4380 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 4440 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 4500 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 4560 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 4620 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 4680 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cat 4733  

Claims (19)

A nanostructure for gene delivery comprising (i) a nanofiber, (ii) an enzyme substrate peptide, and (iii) a complex of a cationic polymer and a gene, wherein the nanofiber and the complex are linked by the enzyme substrate peptide Nanostructure that is. The nanostructure of claim 1, wherein an amino group of the nanofibers is bonded to a carboxy group of the enzyme substrate peptide, and a thiol group of the enzyme substrate peptide is bonded to a cationic polymer constituting the complex. The method of claim 1, wherein the nanofibers are polycaprolactone or polycaprolactone-polyethylene glycol block copolymer, polylactic glycolic acid (PLGA), polylactic acid (PLA), polyglycolic acid (PGA) or polylactic glycolic acid- Nanostructure comprising a polyester-based polymer comprising a polyethylene glycol copolymer (PLGA-PEG) or a biodegradable polymer comprising collagen. The nanostructure of claim 1, wherein the enzyme substrate peptide comprises a hydrolase specific peptide comprising a matrix metalloproteinase (MMP). The nanostructure of claim 4, wherein the enzyme substrate peptide comprises the sequence of SEQ ID NO: 1. The nanostructure of claim 1, wherein the cationic polymer comprises polyethyleneimine, chitosan, protamine or derivatives thereof. The nanostructure of claim 1, wherein the gene is selected from the group consisting of gDNA, cDNA, plasmid DNA, mRNA, tRNA, rRNA, antisense nucleotides, missense nucleotides, and protein producing nucleotides. According to claim 1, wherein the gene is epidermal growth factor EGF (epidermal growth factor), fibroblast growth factor FGF (fibroblast growth factor), platelet-derived growth factor (PDGF), tumor growth factor-b (transforming growth factor-b, TGF-b), vascular endothelial growth factor (vEGF) or a nanostructure comprising a gene expressing insulin (insulin). The nanostructure of claim 1, wherein the gene comprises a gene expressing an anti-tumor gene. The nanostructure of claim 1, wherein the N / P (ratio of nitrogen atoms / nucleic acid phosphate atoms of the nanostructure) of the nanostructure is in the range of 0.1-100. (i) preparing nanofibers; (ii) binding an enzyme substrate peptide to the nanofibers to prepare an enzyme substrate peptide to which nanofibers are bound; (iii) binding a cationic polymer to the enzyme matrix peptide to which the nanofibers are bound; And (iv) binding a gene to the cationic polymer. The method of claim 11, wherein the functional group of the nanofibers and the functional group of the enzyme substrate peptide are bonded. The method of claim 11, wherein the enzyme substrate peptide and the cationic polymer are bound by chemical bonds. The method of claim 11, wherein the cationic polymer and the gene are bound by ionic bonds, hydrophobic bonds, or coordinate bonds. The nanostructure for gene delivery produced by any one of claims 11-14. Kit comprising a nanostructure of any one of claims 1 to 10. A method of delivering a gene comprising contacting a nanostructure of any one of claims 1 to 10 in vitro or in vivo with a cell. A method of treating wounds, comprising contacting the nanostructures of any one of claims 1 to 10 with cells in vitro or in vivo. A pharmaceutical composition for treating wounds or tumors, comprising the nanostructures of any one of claims 1 to 10.

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