Abstract:
Provided is an electro-microneedle integrated body in which a dissolving microneedle and an electrode for electroporation are integrated into one, which enables a focused and efficient intracutaneous gene release for percutaneous gene delivery and intracellular gene delivery occurring in one in-situ treatment site The electro-microneedle integrated body according to the present invention includes an electrode for electroporation which is contacted with skin of a human body to apply an electric field pulse, including a base part and a plurality of electrode parts protruding from the base part, and a microneedle adhered to each electrode part and inserted into the skin of a human body, including a biocompatible and biodegradable viscous material and a genetic material, wherein the microneedle degrades within the skin, and the electric field pulse is applied through the electrode for electroporation in a site in which the microneedle is inserted.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This applications is a continuation of International Application No. PCT/KR2012/005777 filed on Jul. 19, 2012, which claims priority to Korean Application No. 10-2011-0074250 filed Jul. 26, 2011, which applications are incorporated herein by reference. 
     
    
     TECHNICAL FIELD 
       [0002]    The present invention relates to an electro-microneedle integrated body in which a dissolving microstructure and an electrode for electroporation are integrated into one structure for a percutaneous gene delivery in a treatment site, and to a method of preparing the same. 
       BACKGROUND ART 
       [0003]    Purposes of gene delivery (gene transfer, delivery) and gene therapy are to treat diseases or to develop treatment models by introducing foreign genes to inside of a body to replace a gene having defective cells or by introducing a required gene to provide a particular function. In particular, attentions were made to cutaneous gene transfer and gene therapy thanks to its easy accessibility and monitoring in skin and to existence of various intracutaneous immunocytes therein, yet during the development of delivery technology (gene delivery system) for effective delivery of the foreign gene into a target skin cell and stable expression thereof, penetration and introduction of a gene into biological walls, that are stratum corneum, the outer-most layer in skin, and cell membranes are the issues to be addressed. 
         [0004]    More and more attentions were made to non-viral chemico-physical methods which have a high stability as an adequate delivery system depending on genes and treatment sites, and among them, suggested by Genetronics Inc. is an electroporation method in which intracellular transfection of a gene is conducted with a temporal increase of penetration ratio in cell membrane in vivo by an electric field pulse (PCT application, WO99/062592 “Flow-through electroporation system for ex vivo gene therapy”). However, since the electroporation intended for intracellular transfection of a gene needed a sufficient gene concentration in an intracellular electric field pulse site, for this purpose, an additional transdermal delivery means which conventionally uses a disposal injection was required, and as such, an efficient intracutaneous gene delivery to a in-situ treatment site was not available with the said non-integrated system. Accordingly, an integrated system in which transdermal delivery and intracellular gene introduction occur in the in-situ treatment site was proposed, and a focus thereof has been made on an efficient integration between an apparatus for transdermal delivery and electroporation for intracellular gene delivery. 
         [0005]    BTX Inc. in the United States suggested a system in which transdermal delivery which uses a disposal injection and intracellular gene delivery method using electroporation were integrated into one apparatus (US granted U.S. Pat. No. 5,273,525 entitled “Injection and electroporation apparatus for drug and gene delivery”), enabling percutaneous gene delivery in a treatment site. However, the transdermal delivery using a disposal injection caused an intracutaneous stimulus and pain, and further, accurate release of a gene in fine skin membranes was limited. 
         [0006]    Johns Hopkins University introduced an electroporation gene gun in which a gene gun and electroporation were integrated (US patent publication No. 20090030364 entitled “Electroporation gene therapy gun system”), yet the accurate transdermal delivery in the treatment site was also limited with the gene gun. Accordingly, a novel transdermal delivery device integrated with electroporation is required in the art for percutaneous gene delivery in a treatment site, and as an accurate, efficient, and pain-free transdermal delivery device, attentions are paid to a microneedle. 
         [0007]    Generally, a microneedle has a diameter and length of micron size, penetrates skin without causing a pain, and a drug delivery with the microneedle is intended for topical intracutaneous release, not the release in circulatory systems, such as blood vessels or lymph ducts. Talon Biotech suggested a system in which a gene-coated microneedle and an electrode for electroporation were integrated (“Smallpox DNA vaccine delivered by novel skin electroporation device protects mice against intranasal poxvirus challenge,”  Vaccine  25, 1814-1823, 2007), though the coated microneedle had a limit on an amount of intracutaneous gene release because of a limited amount of gene load, and therefore, efficiency of percutaneous gene delivery was ultimately low. 
         [0008]    Accordingly, required for the efficient percutaneous gene delivery in a treatment site is a system in which a dissolving microneedle which enables more effective gene loading than the coated microneedle and an electrode for electroporation are integrated. However, it was difficult to prepare a monolithic integrated body in which the dissolving microneedle and the electrode for electroporation are integrated into one structure with a conventional molding method of preparing the dissolving microneedle, that is a micro-casting method. 
       SUMMARY 
       [0009]    The purpose of the present invention is therefore to provide an electro-microneedle integrated body in which a dissolving microneedle and an electrode for electroporation are integrated into one, which enables a focused and efficient intracutaneous gene release for percutaneous gene delivery and intracellular gene delivery occurring in an in-situ treatment site, and a method of preparing the same. 
         [0010]    In order to accomplish the said purpose, the electro-microneedle integrated body according to the present invention includes an electrode for electroporation which is contacted with skin of a human body to apply an electric field pulse, including a base part and a plurality of electrode parts protruding from the base part, and a microneedle adhered to each electrode part and inserted into the skin of a human body, including a biocompatible and biodegradable viscous material and a genetic material, wherein the microneedle degrades within the skin, and the electric field pulse is applied through the electrode for electroporation in a site in which the microneedle is inserted. 
         [0011]    A method of preparing an electro-microneedle integrated body according to the present invention includes preparing an electrode for electroporation including a base part and a plurality of electrode parts protruding from the base part, preparing a mixed composition by mixing a biocompatible and biodegradable viscous material and a genetic material, and forming a microneedle by supplying the mixed composition in a liquid state on the electrode parts and coagulate it. 
         [0012]    According to an exemplary embodiment of the present invention, the forming of the microneedle may include applying the mixed composition in a liquid state on a plate-like substrate, contacting the electrode parts with the applied mixed composition, drawing the mixed composition by making a relative movement of the substrate over the electrode parts, and coagulating the drawn mixed composition. 
         [0013]    According to another exemplary embodiment of the present invention, the method may further include, after coagulating the drawn mixed composition, liquefying the mixed composition on a side of the substrate by heating the substrate. 
         [0014]    According to the present invention, the genetic materials can be delivered intensively in the treatment site, and efficiency of intracellular gene delivery can be increased by an application of an electric field pulse in the same site. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0015]      FIG. 1  illustrates a flowchart showing a method of preparing an electro-microneedle integrated body according to one embodiment of the present invention. 
           [0016]      FIG. 2  and  FIG. 3  illustrate rough perspective views of an electrode for electroporation. 
           [0017]      FIG. 4  and  FIG. 5  illustrate rough flowcharts showing a method of forming a microneedle. 
       
    
    
     DETAILED DESCRIPTION 
       [0018]    An electro-microneedle integrated body and a method of preparing the same according to the exemplary embodiments of the present invention will be described in detail below with reference to the accompanying drawings. 
         [0019]      FIG. 1  illustrates a flowchart showing a method of preparing an electro-microneedle integrated body according to one embodiment of the present invention,  FIG. 2  and  FIG. 3  illustrate rough perspective views of an electrode for electroporation, and  FIG. 4  and  FIG. 5  illustrate rough flowcharts showing a method of forming a microneedle. 
         [0020]    Referring to  FIG. 1  to  FIG. 5 , the method of preparing an electro-microneedle integrated body according to the present embodiment (M 100 ) includes preparing an electrode for electroporation (S  10 ), preparing a mixed composition (S 20 ), and forming a microneedle (S 30 ). 
         [0021]    The preparing of the electrode for electroporation (S 10 ) is a step to prepare an electrode for electroporation. The electrode for electroporation ( 10 A to  10 D) is intended for electroporation, i.e. for an application of an electric field pulse so that a genetic material (gene) delivered within skin by a microneedle penetrate to cells smoothly. As illustrated in  FIG. 2  and  FIG. 3 , the electrode for electroporation ( 10 A to  10 D) possesses a plate-like base part  11 , and a plurality of electrode parts  12  protruding from the base part  11 . Herein, a shape of the electrode parts  12  can be various as illustrated in  FIG. 2  and  FIG. 3 . For instance, the electrode parts can be micro-blades, micro-knives, micro-fibers, micro-spikes, micro-probes, or micro-barbs, more preferably, micro-spikes, micro-probes, or micro-barbs, and most preferably micro-spikes or micro-probes. 
         [0022]    The electrode for electroporation can be prepared using various micro-fabrication technologies and metal deposition technologies either alone or in combination thereof. Herein, the micro-fabrication technologies denote technologies for cutting and abrasion of a metal and polymer, and the metal deposition technologies denote technologies for selective metal deposition and metal plating on a fabricated polymer. 
         [0023]    As illustrated in  FIG. 2 , the electrode for electroporation ( 10 A and  10 B) can be prepared in a form of “metal-integrated microstructure” in which the base part and the electrode parts are integrated into one body using a metal. Herein, the metal is preferably a metal which can be applied in a living body, and can be formed with ceramic or semiconductor. In particular, materials for the electrode for electroporation are cobalt, titanium, stainless steel, chromium, nickel, copper, silver, gold, aluminum, or alloys thereof, more preferably cobalt, titanium, stainless steel, chromium, nickel, copper, aluminum, or alloys thereof, and most preferably titanium, chromium, nickel, aluminum, or alloys thereof. 
         [0024]    The metal-integrated electrode for electroporation can be prepared with various metal micro-fabrication technologies known in the art. For instance, the metal micro-fabrication can be conducted using rolling, forging, milling, cutting, or turning processing. Further, an effective bottom diameter of the electrode parts of the electrode for electroporation is 10 to 1000 μm, and more preferably 50 to 500 μm. An effective aspect ratio (length:bottom diameter) is 0.1 to 10, and more preferably 0.5 to 5. 
         [0025]    Meanwhile, as illustrated in  FIG. 3 , the electrode for electroporation ( 10 C and  10 D) can be prepared in a “metal-patterned polymer form.” That is, the patterned form can be prepared by first preparing a basic structure having the base part and electrode parts using a polymer, and by a subsequent metal deposition or metal plating on an entire structure, or selective metal deposition or metal plating on the electrode parts. 
         [0026]    Herein, the polymer is preferably biocompatible, and can be, in particular, polymers such as acrylic acid, acrylic acid ester, acryl amide, acrylonitrile, methacrylic acid, methacrylic acid ester, and copolymers thereof, and more preferably methacrylic resin or polyacrylic acid resin. The metal deposition can be performed by various methods known in the art, physical metal deposition or chemical metal deposition can be preferably used, and most preferably, the metal can be deposited using physical metal deposition of sputtering or evaporation, or chemical metal deposition of Tollens&#39; reaction. In addition, metal plating can be further included after the metal deposition. The metal used in deposition or plating is the one can be applicable in a living body, preferably cobalt, titanium, stainless steel, chromium, nickel, copper, silver, gold, aluminum, or alloys thereof, more preferably cobalt, titanium, stainless steel, chromium, nickel, copper, aluminum, or alloys thereof, and most preferably titanium, chromium, nickel, aluminum, or alloys thereof. 
         [0027]    Meanwhile, the metal-integrated electrode for electroporation illustrated in  FIG. 2  can be only used as one electrode, and the metal-patterned electrode for electroporation illustrated in  FIG. 3  can be used as both electrodes (cathode and anode). 
         [0028]    In the preparing of the mixed composition (S 20 ), the mixed composition is prepared by mixing a viscous material and a genetic material. Herein, the viscous material is biocompatible, biodegradable, and a material dissolvable in vivo. The biocompatible material denotes a material which does not have any substantial toxicity in a human body, which is chemically inert, and which does not have immunogenicity, the biodegradable material denotes a material which can be decomposed by body fluids or microorganisms in a living body, and the material dissolvable in vivo denotes a material which can be dissolved by temperature or body fluids in a living body. 
         [0029]    The viscous material used in the present invention is carbohydrate, more preferably monosaccharide, disaccharide, trisaccharide, oligosaccharide, polysaccharide, or alcohol derivatives thereof. More preferably, the viscous material includes glucose, lactose, fructose, galactose, mannose, malturose, lacturose, maltose, sucrose, trehalose, raffinose, melezitose, melibiose, xylobiose, cellobiose, stachyose, sorbitol, mannitol, erythritol, xylitol, lacitol, maltitol; aldonic acids and lactone derivatives thereof, such as gluconic acid and gluconic acid γ-lactone, aldaric acids and lactone derivatives thereof, such as ribaraic acid, arabinaric acid, and galactaric acid; uronic acids, such as glucuronic acid, galaccuronic, acid and mannuronic acid; starch, vegetable gums, substituted cellulose (e.g., carboxymethylcellulose, hydroxyethylcellulose, and alkylcellulose), crystalline cellulose, heparin, hyaluronic acid, chitosan, dextran, alginate, tragacanth, agar, and carrageenan. 
         [0030]    Furthermore, the viscous material used in the present invention has a viscosity in a liquid state. Such a viscosity can be varied depending on type, concentration, air condition, and temperature, and can be adjusted in accordance with the purpose of the present invention. Preferably, the viscous material used in the present invention shows a viscosity of no more than 200000 cSt. 
         [0031]    Meanwhile, liquefying of the viscous material can be conducted by various methods known in the art. According to an exemplary embodiment of the present invention, the liquefying can be performed by heating at a temperature of at least a melting point of the biocompatible viscous material, or by dissolving the biocompatible material in an appropriate solvent (for example, water, water-free or water-containing lower alcohol with 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform, 1,3-butylene glycol, hexane, diethyl ether, butylacetate, and so on). 
         [0032]    According to the present embodiment, the viscous material has a melting point of at least 50° C., more preferably at least 60° C., still more preferably at least 70° C., and most preferably at least 80° C. When describing the melting point, an upper limit is not particularly limited, and can be preferably 500° C., more preferably 400° C., still more preferably 300° C., and most preferably 200° C. 
         [0033]    The genetic material (gene) is intended for gene therapy, and is a nucleic acid molecule, such as DNA, RNA, siRNA, and microRNA for antiinflammatory drug, antiarthritic drug, anticonvulsive drug, antidepressant drug, antipsychotic drug, tranquilizer, antianxiety drug, narcotic antagonist, antiparkinsonism drug, cholinergic agonist, anticancer drug, antiangiogenic drug, immunosuppressive drug, antiviral drug, antibiotic, orexigenic drug, anticholinergic drug, antihistamine drug, antimigraine drug, hormone drug, vasodilator for coronary artery, cerebrovascular, or peripheral blood vessel, contraceptive, antithrombotic drug, diuretic drug, antihypertensive drug, and drug for cardiovascular disease. Preferably, the genetic material includes hormone, hormone agonist, enzyme, enzyme inhibitor, signal delivery protein or portion thereof, antibody or portion thereof, single chain antibody, binding protein or binding domain thereof, antigen, adhesion protein, structural protein, regulatory protein, toxoprotein, cytokine, transcriptional regulatory factor, blood coagulation factor, and vaccine, but the present invention is not limited thereto. 
         [0034]    More particularly, the genetic material related to protein/peptide/vaccine includes insulin, insulin-like growth factor 1 (IGF-1), growth hormone, erythropoietin, granulocyte-colony stimulating factors (G-CSFs), granulocyte/macrophage-colony stimulating factors (GM-CSFs), interferon alpha, interferon beta, interferon gamma, interleukin-1 alpha and beta, interleukin-3, interleukin-4, interleukin-6, interleukin-2, epidermal growth factors (EGFs), calcitonin, adrenocorticotropic hormone (ACTH), tumor necrosis factor (TNF), atobisban, buserelin, cetrorelix, deslorelin, desmopressin, dynorphin A (1-13), elcatonin, eleidosin, eptifibatide, growth hormone releasing hormone-II (GHRH-II), gonadorelin, goserelin, histrelin, leuprorelin, lypressin, octreotide, oxytocin, pitressin, secretin, sincalide, terlipressin, thymopentin, thymosin α1, triptorelin, bivalirudin, carbetocin, cyclosporine, exedine, lanreotide, luteinizing hormone-releasing hormone (LHRH), nafarelin, parathyroid gland hormone, pramlintide, enfuvirtide (T-20), thymalfasin, ziconotide, antivirus, and vaccine protein. 
         [0035]    A mixing of the genetic material and the viscous material can be performed using various methods known in the art. A basic mixing may be available through a stirrer, and the preparation can be conducted in oil-in-water or water-in-oil emulsion type and multiple-emulsion type. A method for preparing an emulsion can be performed by dispersing the genetic material directly in the viscous material to prepare the emulsion in which the genetic material is included therein, preferably in the absence of a emulsifier, or using various natural or synthetic emulsifiers. 
         [0036]    When the emulsifier is used, the emulsion in which the gene is included therein is more preferably stabilized using natural emulsifiers, such as lecithin, borax, stearic acid, amizole soft, helio gel, beeswax, xanthan gum, emulsifying wax, and solubilizer, or is prepared using at least one selected from group consisting of synthetic emulsifiers, including emulsifiers for oil-in-water, such as PEG-8 dilaurate, PEG-150 distearate, PEG-8 stearate, PEG-40 distearate, PEG-100 distearate, or emulsifiers for water-in-oil emulsion, such as sorbitan stearate, sorbitan oleate, sorbitan sesquioleate, sorbitan trioleate, and combinations thereof. Most preferably, the emulsion is prepared without emulsifier. For instance, the mixed composition can be prepared by emulsifying a lipid-soluble liquid containing the genetic material in an aqueous viscous material in an oil-in-water (W/O) type with a homogenizer, or by emulsifying an aqueous liquid containing the genetic material in a lipid-soluble viscous material in a water-in-oil (O/W) type with a homogenizer. 
         [0037]    The forming of the microneedle (S 30 ) is forming a microneedle on the electrode parts  12  of the electrode for electroporation using the mixed composition, and in particular, is proceeded as follows. 
         [0038]    As illustrated in  FIG. 4 , the mixed composition is applied on a plate-like substrate g, and then, the electrode parts of the electrode for electroporation are contacted with the applied mixed composition. Thereafter, a relative movement of an electrode for electroporation  10  is performed over the substrate g to draw the mixed composition, and when time passes, both the drawn mixed composition and remaining mixed composition on the substrate coagulate. When the substrate is heated in this state, the viscous composition on the substrate is liquefied and thus the drawn viscous composition and the viscous composition on the substrate are separated, thereby forming the microneedle including the mixed composition on each electrode part. 
         [0039]    Meanwhile, the microneedle may be formed by spotting the mixed composition in each electrode part  12 , and then, coagulating the same, as illustrated in  FIG. 5 . 
         [0040]    Furthermore, the electrode parts of the electrode for electroporation are contacted with the mixed composition in a state in which the mixed composition is applied on a plate-like substrate. Thereafter, when the mixed composition is drawn in a state maintaining at least a certain temperature of the substrate so that the mixed composition on the substrate does not coagulate, the viscous composition on a side of the electrode for electroporation coagulates quickly while the viscous composition on a side of the substrate coagulates relatively slowly, and thus, it is drawn in a structure of which diameter decreases as being closer to the substrate. When the drawing is continuously performed in this state, the coagulated viscous composition is separated from the substrate, thereby forming the microneedle in each electrode part. 
         [0041]    Meanwhile, the thusly-formed microneedle has a top diameter of 1 to 100 μm, more preferably 2 to 50 μm, and most preferably 5 to 20 μm. 
         [0042]    In addition, an effective length of the microneedle is not particularly limited, and the length can be varied depending on the type of genes to be delivered, a gene delivery site, and a position for conducting electroporation, and so on. According to the present embodiment, the effective length of the microneedle is preferably 100 to 10,000 μm, more preferably 200 to 10,000 μm, still more preferably 300 to 8,000 μm, and most preferably 500 to 2,000 μm. 
         [0043]    Herein, “top” of the microneedle denotes an end part of the microneedle having a minimum diameter. Further, “effective length of microneedle” denotes a vertical length from the top of the microneedle to the upper surface of the electrode parts. 
         [0044]    In the thusly-structured electro-microneedle integrated body, the microneedle and the electrode parts are inserted into skin, the microneedle degrades within the skin, thereby releasing the genetic material (gene) contained therein. When power supply is applied in the electrode for electroporation in this state, an electric field pulse is applied in a region in which the genetic material is released, thereby enabling a smooth delivery of the genetic material into cells. 
         [0045]    As explained above, according to the present embodiment, a genetic material can be intensively delivered in a treatment site, an electric field pulse is applied in the same site, and as such, efficiency of intracellular gene delivery can be increased. 
         [0046]    It will be apparent to those skilled in the art that various modifications can be made to the above-described exemplary embodiments of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention covers all such modifications provided they come within the scope of the appended claims and their equivalents.