KR101722857B1 - Spontaneous Organization of Numerous-layer Generation by Electrospray - Google Patents

Abstract

Advanced technologies that can mimic hierarchical architectures found in nature can provide pivotal clues for elucidating numerous biological mechanisms. Herein, a novel technology, spontaneous organization of numerous-layer generation by electrospray (SpONGE), was developed to create self-assembled and multilayered fibrous structures. The simple inclusion of salts in a polymer solution prior to electrospraying was key to mediating the structural versatilities of the fibrous structures. The SpONGE matrix demonstrated great potential as a crucial building block capable of inducing sequential, localized drug delivery or orchestrating cellular distribution in vivo, and thus the scope of the use thereof may be expanded to cover a variety of biomedical applications.

Description

[0002] Spontaneous Organization of Numerous-layer Generation by Electrospray by Electrospinning [

The present invention relates to a spontaneous multilayer structure formation (SpONGE) method by electrospinning.

Layered structures are common in nature. The macroscopic structure of the skin, consisting of the epidermis, dermis and subcutaneous layer, is a representative example of layered structure, and most organs such as the eye, heart and blood vessels of the human body contain various functional layered structures. As observed in the outer membrane of microorganisms, in the bark of insects, and in the coarse epidermis of plants, the lamellar structure generally exhibits versatility. Layered structures are found not only in biological structures but also in consumer goods. Examples of consumer goods include liquid crystal display panels and solar cells. The most distinct advantage offered by layered structures is that each layer has other functions that cooperate and interact with the function of adjacent layers.

To date, the range of techniques developed for the fabrication of multilayer structures is limited. Conventional methods can manually create each layer one by one. Assembly of a polymer electrolyte on a solid substrate, called layer-by-layer deposition, has been widely practiced. Multilayer microcapsules containing hydrophobic and hydrophilic drugs for drug delivery, and multi-layer structures in which the vaccine, enzyme and catalyst loaded multifunctional layers are prepared by LbL assembly. Spin coating is another method of producing a multi-layer structure. In particular, this technology has been used in semiconductor manufacturing. Recently, the fabrication of a hierarchical structure that mimics a biostructure has involved the use of multilayer spin-coating followed by lithography. Unlike non-spontaneous processes developed by researchers, in nature, cells with distinct functions are spatially distributed and secrete a variety of extracellular matrix (ECM), which are self-assembled to spontaneously form multilayer structures do. Unlike spontaneous formation of stratified structures found in nature, man-made methods require the fabrication of structures based on iterative processes for each layer.
[Prior Art Literature]
[Patent Literature]
1. Korean Patent Registration No. 10-1479755

It is an object of the present invention to provide a method for forming a spontaneous multilayer structure.

It is another object of the present invention to provide a multi-layer structure and a drug delivery system using the same, a skin patch, and a scaffold for tissue engineering.

In order to accomplish the above-mentioned object, the present invention provides a method for producing a polymer electrolyte membrane, comprising: mixing a solution of a polymer dissolved in a solvent and an ionic salt; And a step of spontaneously forming a multilayer structure composed of fibers and composed of a plurality of layers and including interspace between the layers by electrospinning a mixture of the polymer solution and the ionic salt so as to form a jet flow from the nozzle to the collector The method comprising the steps of:

In the present invention, the ionic salt induces high electron density and electrostatic repulsion of the solvent remaining in the fibers formed in the electrospinning and peripheral jets, and by this electrostatic repulsion, the multi-layer structure including interspace between the respective layers is spontaneously .

In the present invention, the polymer may be selected from the group consisting of polycaprolactone, poly (lactide-co-glycolide), poly (vinylpyrrolidone), polycarbonate, poly (ethylene glycol), poly (urethane), polyethyleneimine, , Gelatin, chitosan, collagen, elastin, hyaluronic acid, and fibrinogen.

In the present invention, the ionic salt is selected from the group consisting of FeCl ₃ , VCl ₅ , NiCl ₂ , CoCl ₂ , MnCl ₂ , CuCl ₂ , ZnCl ₂ , FeF ₃ , VF ₅ , NiF ₂ , CoF ₂ , MnF ₂ , CuF ₂ , ZnF ₂ , FeBr ₃ , VIr ₅ , NiBr ₂ , CoBr ₂ , MnBr ₂ , CuBr ₂ , ZnBr ₂ , FeI ₃ , VI ₅ , NiI ₂ , CoI ₂ , MnI ₂ , ZnI ₂ , NaIO ₄ , KIO ₄ , LiIO ₄ , HIO ₄ , At least one selected from the group consisting of LiClO ₄ , NaClO ₄ , KClO ₄ , HClO ₄ , LiBrO ₄ , NaBrO ₄ , KBrO ₄ , HBrO ₄ , ethylamine hydrochloride, dopamine, dopamine hydrochloride, norepinephrine, norepinephrine hydrochloride, have.

In the present invention, the electric conductivity measured after mixing the polymer solution and the ionic salt may be 30 to 1000 uS / cm.

In the present invention, electrospinning can be carried out continuously at a time.

The process according to the present invention may further comprise the step of removing the ionic salt.

In the present invention, the current measured between the collector and the nozzle may be 0.001 to 0.1 mA.

In the present invention, the diameter of the fiber may be 0.1 to 0.7 mu m.

In the present invention, the multilayer structure is separable into individual layers.

In the present invention, the volume ratio of the multi-layer structure may be 100 to 1000 cm < 3 > / g.

In the present invention, the contact angle of the multilayer structure may be 30 DEG or less.

In the present invention, the drug binding efficiency of the multi-layer structure can be 50% or more.

Further, the present invention provides a multi-layered structure made of the above-described method, which is made of fibers, and which has a multi-layer structure and includes interspace between the respective layers.

The present invention also relates to a multilayer structure as described above; And a drug delivery system.

In the drug delivery system according to the present invention, a plurality of drugs may be contained in different layers and sequentially discharged.

The present invention also relates to a multilayer structure as described above; And a skin patch comprising at least one selected from a drug, a protein, a gene, and a cell.

The present invention also relates to a multi-layered structure from which an ionic salt has been removed; And a scaffold for tissue engineering comprising at least one selected from drugs, proteins, genes, and cells.

The multi-layered nanofibers of the present invention have an increased volume compared to conventional nanofibers and can be used for the fabrication of a three-dimensional scaffold since cell penetration into a space between layers can be expected. In addition, it can be used in customized drug delivery systems using hydrophilic properties and layered properties. The present invention can easily form a multi-layered structure by a new method that has not been available in the past and can be applied to various kinds of polymers, so that it is possible to broaden the applicability of this structure in various fields where electrospinning is used.

1 shows a spontaneous multilayer structure formation (SpONGE) process by electrospinning according to the present invention.
Figure 2 shows the formation of a SpONGE matrix, wherein (A) is a digital and cross-sectional SEM image of sPCL and SpONGE, (B) is the electrical conductivity of the polymer solution, (C) is the work process proposed to form the SpONGE matrix, (D) shows the interspaces between the layers, (E) shows the ability of the SpONGE to form a number of uniform fiber mats by manual manipulation, and the scale bars are 10 μm (digital image) and 100 μm (SEM) .
Figure 3 shows the ability of the SpONGE matrix to be used as a drug carrier, wherein (A) is a rapid wetting of the SpONGE matrix with a water-soluble biomolecule, (B) is a visualization of the fast and complete absorption of the lgG-FITC molecule on the SpONGE matrix , (C) is the relative emission of lgG-FITC (red dot) or BSA-FITC (black square) emitted from sPCL (empty dot / quadrangle) and SpONGE system (filled dot / Shows local GFP expression at the transplantation site, and each matrix containing the AAV-GFP vector was subcutaneously transplanted, and the GFP-expressing cells were visualized using the Xenogen imaging system.
Figure 4 shows the sequential delivery of multiple drugs from the SpONGE matrix, where (A) is the way the dual drug can be loaded into the SpONGE matrix, (B) is the container capable of mediating unidirectional release, (C) (D) represents the drug accumulation amount released from the SpONGE matrix.
Figure 5 shows the in vivo cell distribution in the sPCL and SpONGE matrix subcutaneously implanted in rats. Cells permeated throughout the structure were stained with DAPI and H & E, S represents the matrix grafted into the mouse, 100 mu m.

Hereinafter, the present invention will be described in detail.

The present invention relates to a spontaneous multilayer structure (SpONGE) method by electrospinning, the method comprising: mixing a solution of a polymer dissolved in a solvent and an ionic salt; And a step of spontaneously forming a multilayer structure composed of fibers and composed of a plurality of layers and including interspace between the layers by electrospinning a mixture of the polymer solution and the ionic salt so as to form a jet flow from the nozzle to the collector .

First, a mixture of a polymer solution and an ionic salt is prepared.

The polymer is not particularly limited and any electrospun polymer may be used. For example, synthetic polymers such as polycaprolactone (PCL), poly (lactide-co-glycolide) (PLG, PLGA), poly (vinylpyrrolidone) (PVP), polycarbonate (Ethylene glycol) (PEG), poly (urethane) (PU), polyethyleneimine (PEI) and poly (vinyl alcohol) (PVA). As natural polymers, gelatin, chitosan, collagen, elastin, hyaluronic acid, fibrinogen and the like can be used. These polymers may be used alone or in combination of two or more. In particular, when applied to scaffolds for tissue engineering, it is preferable to use biodegradable polymers (PCL, PLGA, etc.).

According to the embodiment of the present invention, it was confirmed that the PLGA, PCL, and PVP, which are synthetic polymers, induce a multilayer structure by the presence of ionic salts. Therefore, it is expected that the same phenomenon will be induced in other synthetic polymers mainly used for electrospinning (PC, PEG, PU, PEI, PVA, etc.). In addition, it has been confirmed that the multi-layer structure is induced by the presence of ionic salts even in the case of natural polymers such as gelatin and chitosan. Therefore, the same phenomenon occurs in other natural polymers (collagen, elastin, hyaluronic acid, fibrinogen, etc.) It is expected that natural polymers will not be significantly affected by the type of polymer.

In the above description, polymers capable of forming a nanofiber scaffold are generally exemplified by electrospinning. However, since addition of an ionic salt is a major cause of formation of a multi-layer structure irrespective of the kind of a polymer in the present invention, It is not affected. However, depending on the kind of the polymer, a polymer having a molecular weight (Mw) of at least a certain level required for forming a fiber structure should be used. For example, the molecular weight of PCL is 30,000 to 100,000, the molecular weight of PVP is 5,000 to 400,000, the molecular weight of PVA is 20,000 to 30,000, and the molecular weight of PEO is 300,000 to 500,000. There is no significant influence on the molecular weight (Mw) of the polymer used, and solvent conditions capable of forming the fiber structure through electrospinning may be important.

The solvent for dissolving the polymer is not particularly limited and may vary widely depending on the kind of the polymer used. Examples thereof include acetic acid (acidic aqueous solution), chloroform, dimethylformamide, tetrahydrofuran, ethanol, methanol, deionized water, Fluorene, 2,2,2-trifluoroethanol, dichloromethane, etc. can be used.

The content of the polymer in the polymer solution is not particularly limited, and the concentration range for forming the nanofibers may be varied depending on the kind of the polymer, and may be, for example, 5 to 40% by weight, and preferably 15 to 30% by weight. If the concentration (concentration) of the polymer solution is too small, spherical particles of sub-micro size rather than nanofiber structure are formed and nanofiber structures can not be formed. In addition, if the content of the polymer solution is too large, the viscosity of the solution becomes large, which may interfere with the uniform mixing of the additional ions.

As the ionic salt, at least one selected from a transition metal salt, a peroxide salt, and an organic salt may be used. Specifically, the transition metal salt may be FeCl ₃ , VCl ₅ , NiCl ₂ , CoCl ₂ , MnCl ₂ , CuCl ₂ , ZnCl ₂ , FeF ₃ , VF ₅ , NiF ₂ , CoF ₂ , MnF ₂ , CuF ₂ , ZnF ₂ , FeBr ₃ , VBr ₅ , NiBr ₂ , CoBr ₂ , MnBr ₂ , CuBr ₂ , ZnBr ₂ , FeI ₃ , VI ₅ , NiI ₂ , CoI ₂ , MnI ₂ and ZnI ₂ . As the peroxide-based salt, NaIO ₄ , KIO ₄ , LiIO ₄ , HIO ₄ , LiClO ₄ , NaClO ₄ , KClO ₄ , HClO ₄ , LiBrO ₄ , NaBrO ₄ , KBrO ₄ and HBrO ₄ can be used. As organic salts, ethylamine hydrochloride, dopamine, dopamine hydrochloride, norepinephrine, norepinephrine hydrochloride, triamine hydrochloride and the like can be used.

According to an embodiment of the invention, it was to transition metal-based salt using FeCl _3, in this connection, because Fe is a typical transition metal and show similar properties to the transition metal region is chemically, Fe is other transition metals (V, Ni, Co, Mn, Cu, Zn, etc.). Likewise, since the same group 17 ions exhibit similar chemical properties, they can be replaced by F ^- , Br ^- , I ^- instead of Cl ^- , and thus various combinations of transition metals and anions described above are possible. Further, according to an embodiment of the present invention, because it shows similar chemical properties buffer were using NaIO ₄ in oxide-based salt, in this connection are the group of the elements such as Na ^+, K ^+, Li ⁺ instead of Na ^+, H ^{+ and} so on. Similarly, IO ₄ ^- is expected to show similar chemical properties as the replaced I to other group 17 element, and thus IO ₄ ^- is replaceable as such ^-, BrO ₄ ^- ClO ₄ instead. Also, according to the embodiment of the present invention, ethylamine hydrochloride is used as the organic salt, but other organic salts exemplified above can also be used.

With respect to the content of the ionic salt in the mixture of the polymer solution and the ionic salt, the measured value of electric conductivity after mixing the polymer solution and the ionic salt should be in the range of 30 to 1000 uS / cm. The system of the present invention is a phenomenon due to the conductivity of a salt of a polymer solution. When the conductivity is measured under ordinary conditions, a value of 45 uS / cm is obtained. When the conductivity in a polymer solution is 45 uS / cm or more, Structure, and it was confirmed that a structure other than a multilayer structure was formed under conditions of 1000 uS / cm. Therefore, a multilayer structure can be formed when the electric conductivity is 30 to 1000 uS / cm. It was also observed to be influenced by the solvent, type of salt, and humidity.

The ionic salts induce the high electron density and electrostatic repulsion of the solvent remaining in the fibers formed in the electrospinning process and the surrounding jets, and by this electrostatic repulsion, a multilayer structure including interspace is spontaneously formed between the respective layers .

Next, a mixture of the polymer solution and the ionic salt is electrospun.

The electrospinning may be performed using a conventional electrospinning apparatus including a spinneret and a collector. When a voltage is applied between the spinneret and the collector, fibers are formed as the jet flow forms from the nozzle toward the collector, the fibers are entangled to form a fiber layer, and the electrostatic repulsive force induced by the above- A sponge-like multi-layer structure including an interspace between the respective layers may be spontaneously formed by forming a next fibrous layer with a certain space therebetween.

The electrospinning may be carried out once or several times, preferably at one time. Further, preferably, in order to obtain a multi-layer structure in the form of a sponge, electrospinning can be performed continuously.

The process according to the present invention may further comprise the step of removing the ionic salt. When applied to scaffolds for tissue engineering, it is desirable to remove ionic salts as they may be harmful to organs and the like. The method for removing the ionic salt can be carried out by, for example, tapping with water. Specifically, for example, to remove the ionic salt, tapping with distilled water in a 1.5 ml microcentrifuge tube for 5 seconds can be repeated five times. IR shows that the salt is sufficiently removed.

The current measured between the collector and the nozzle may be 0.001 to 0.1 mA, preferably 0.01 to 0.05 mA. This current is associated with an increase in the electrical conductivity of the ionic salt-polymer mixture, and thus a significant volume expansion of the multilayer structure can be obtained. On the other hand, no current was typically observed during conventional electrospinning.

The diameter of the fibers may be 0.1 to 0.7 mu m, preferably 0.3 to 0.5 mu m. The multi-layered structure of the present invention can reduce the fiber diameter by about twice as compared with the conventional mat due to the improved tensile strength.

The multi-layer structure can be separated into individual layers, and the multi-layer structure can be divided and used. The number of layers of the multilayer structure is not particularly limited, and can be appropriately adjusted as necessary. Further, the thickness of each layer and the like can be appropriately adjusted as needed.

The specific volume of the multilayer structure may be 100 to 1000 cm3 / g, preferably 200 to 500 cm3 / g. The multi-layered structure of the present invention may have a lower specific volume or density than conventional mats due to significant volume expansion. The cell support of the nanofiber structure obtained by the conventional technique showed a volume specific value of about 50 cm 3 / g. It was confirmed that the thickness of the SpONGE cell support through the method according to the present invention was about 5.11 times higher than the conventional one , So the specific volume value of the SpONGE structure formed in accordance with the present invention is expected to have a value of about 250 cm3 / g.

The contact angle of the multilayer structure may be 30 DEG or less, preferably 10 DEG or less. That is, the multilayer structure of the present invention may have hydrophilicity. The hydrophilicity of the multi-layer structure can be attributed to the ionic salt and can be advantageous for drug delivery. The multi-layered nanofiber structure of the present invention can contain the drug within the structure in a shorter time than the conventional nanofiber structure. The lower limit of the contact angle may be 0 DEG, 0.1 DEG or 1 DEG.

The drug binding efficiency of the multi-layer structure may be 50% or more, preferably 70% or more, more preferably 90% or more. Considering that the drug binding efficiency of the conventional mat is 30% or less, the drug binding efficiency of the multi-layer structure according to the present invention is excellent. The upper limit of the drug binding efficiency may be 100%, 99% or 98%.

Further, the present invention provides a multi-layered structure made of the above-described method, which is made of fibers, and which has a multi-layer structure and includes interspace between the respective layers.

The present invention also relates to a multilayer structure as described above; And a drug delivery system.

Drugs applicable in the present invention may include chemical drugs, protein drugs, gene therapy drugs and the like. According to an embodiment of the present invention, one of the models of small molecule chemical drugs has observed the release kinetics of dopamine and thus can be utilized as a release carrier for functional chemical drugs. Available functional chemical agents include anti-inflammatory agents such as demamethasone, curcumin; Anticancer agents such as doxorubicin, peripherin, and fluorouracil (5-FU); Salvaric acid, and Proavine. In addition, according to the embodiment of the present invention, the release kinetics of BSA-FITC and BSA-rhodamine were utilized to confirm the controllability of the release rate of the protein. The types of functional protein drugs that can be used are antibodies, cytokines (IL-2, IL-10, etc.), growth factors (NGF, GDNF, FGF etc.) and functional enzymes. In addition, according to the embodiment of the present invention, local transmission of GFP reporter gene was confirmed through gene transfer using AAV, thereby confirming the possibility of efficient gene therapy through local gene transfer of viruses and non-viral gene carriers . The types of gene therapy drugs that can be utilized are viral vectors (adenovirus, adeno-associated virus, lentivirus, retrovirus, herpes virus, etc.), non-viral vectors (PEI conjugated plasmids, plasmid-encoded nanoparticles, etc.).

In the drug delivery system according to the present invention, a plurality of drugs may be contained in different layers and sequentially discharged. For example, the first drug may be included in the upper layer and the second drug may be included in the lower layer, wherein the first drug is released first and the second drug is released later.

The present invention also relates to a multilayer structure as described above; And a skin patch comprising at least one selected from a drug, a protein, a gene, and a cell. When applied as a skin patch, the ionic salt can be used as it is without being removed, and the absorption rate of drugs and the like can be increased by the ionic salt.

The present invention also relates to a multi-layered structure from which an ionic salt has been removed; And a scaffold for tissue engineering comprising at least one selected from drugs, proteins, genes, and cells. When applied to tissue engineering, it is preferable to use a biodegradable polymer, and it is preferable to remove the ionic salt in advance.

Electrospinning techniques that can easily make nanoseconds fibers have been widely used in energy conversion, sensors, catalysts, filtration, tissue engineering, and drug delivery. However, the structure of the electrospun fiber, which can be made by general electrospinning technology, is very limited and limits its applicability.

The present invention provides a method of easily forming an electrospun fiber structure having a multilayer structure by adding a small amount of an ionic salt to an electrospun polymer solution. The ionic salts added to the polymer solution can induce the high electron density of the solvent remaining in the fibers formed in the electrospinning process and the surrounding jets and induce electrostatic repulsion. By this electrostatic repulsion, an electrospun fiber having a multi-layered structure composed of several layers can be produced. The electrospun fiber of such a multi-layered structure exhibits an increased volume compared to a general electrospun fiber and can be easily separated into several sheets. In addition, even when a hydrophobic polymer is used by adding an ionic salt, a nanofiber having a hydrophilic structure can be produced. In the present invention, this technique is called "Spontaneous Organization of Numerous-layer Generation by Electrospray " (SpONGE) by Electrospinning.

There have been various attempts to fabricate electrospun fibers of a multi-layered structure. Electrospinning fibers of multilayer structures have been made using techniques such as sequential electrospinning, wet electrospinning or hydrospinning, and stacking. However, these prior art techniques have required the replacement of the polymer solution during the electrospinning process, additional operation is necessary, additional equipment is required, or the like has been carried out. In addition, due to the use of a limited number of polymers and solvents, it is not easy to form the electrospun fibers of a multilayer structure, and the interconnection between layers of the structure is remarkably deteriorated.

On the other hand, in the present invention, it is possible to easily produce a multi-layered electrospun fiber by a simple method of adding an ionic salt to a polymer solution. Above all, the multi-layer structure formed by spontaneous formation has a good interconnection between layers and can induce a multi-layer structure with various polymers.

[Example]

FIG. 1 shows a spontaneous multilayer structure (SpONGE) process according to the present invention in which a multilayer self-assembled and volume-expanded nanofiber structure can be formed by a new process called SpONGE. As a result of the introduction of organic or inorganic salts into the electrospinning process, a multi-fiber layer spontaneously formed. The SpONGE matrix has a unique ability to be partitioned into multiple individual fiber layers by manual separation and to position biomolecules spatially.

In the present invention, it has been demonstrated that electrospinning allows the spontaneous formation of a multilayer structure by a single continuous spinning of the polymer solution. The spontaneous formation of the multilayer structure by the electrospinning technique is an unusual result considering that conventional electrospinning forms a continuous fiber structure without being separated. Techniques such as physical stacking followed by sequential electrospinning or wet electrospinning have previously been conducted to fabricate structures that mimic blood vessels and other organs. However, the spontaneous formation of a multilayer structure by single continuous electrospinning of the polymer solution has not been reported. It was observed that the simple addition of organic / inorganic additives (sodium periodate, iron chloride, dopamine hydrochloride, ethylamine hydrochloride, etc.) formed a multilayer structure during the continuous electrospinning process. Due to the volumetric space between each layer, the thickness of the sponged structure (SpONGE) obtained according to the present invention increased sharply compared to that produced by conventional electrospinning (sPCL) (see FIG. 2A). In the present invention, this technique was named SpONGE because a multi-layer matrix such as a sponge was obtained by a single electrospinning process.

A significant volume expansion in the SpONGE technology as compared to conventional mats can be attributed to increased electrical conductivity of the salt-polymer mixture (see FIG. 2B). The inclusion of organic / inorganic salts could result in a net increase in electron density in the SpONGE matrix, which could be explained by the significant current (0.02 mA) measured between the ground collector and the nozzle (see FIG. 2C). No current was typically observed during conventional electrospinning, in which a random dispersion of fibers was observed. The residual solvent promotes electron transfer for net accumulation in the mat, resulting in electrostatic repulsion with the newly formed mat. Once the solvent has evaporated, the repulsion effect caused by the electron accumulation is extinguished, whereby a mat like sheet can be formed. Continuous electrospinning will then form a spontaneous layer and a thinner space by generating another set of net charge increments in the jet. Importantly, adjacent layers that internetwork through a fiber branch in the interspace (also the 2D arrow) are important features observed in the SpONGE matrix, while the conventional layered mat consists of physically stacked layers. These fibers may play an important role in maintaining the structural integrity of the SpONGE system. Repetition of this dual effect across several layers (i.e., electrostatic repulsion and continuous electrospinning) induces the SpONGE effect.

Regardless of the type of polymer (polycaprolactone (PCL), poly (lactide-co-glycolide) (PLG), poly (vinylpyrrolidone) (PVP) and gelatin) By adding organic or inorganic salts, it was consistently observed that a SpONGE matrix was formed. Thus, further studies were conducted to prepare multi-layered PCL mats using dopamine hydrochloride as SpONGE matrix. The thickness of the SpONGE matrix was 502 ± 87.8 μm to 1695.3 ± 7.5 μm. Thus, the matrix was 3 to 6 times thicker than a conventional sheet-type PCL mat (sPCL) and the other process parameters remained constant.

The SpONGE matrix could be divided into multiple discrete layers by hand removal like a paper sheet of a book (see Figure 2E). The procedure used to make the individual layers is very similar to stripping a strip of Scotch tape. SpONGE matrices collected over 2 hours formed 15 thin fiber mats by manual manipulation (see Figure 2E). The ability to remove the fiber layer by hand was observed regardless of the type of polymer used, in any case where organic or inorganic salts were introduced.

The detailed properties of the SpONGE matrix and of the five sub-layers were compared to sPCL mat. No substantial change in the shape of the external fibers was observed in either structure. The presence of dopamine in the SpONGE matrix was confirmed by FT-IR analysis with characteristic peaks (815 cm ^-1 , 1504 cm ^-1 ) corresponding to the phenyl ring at the catechol and amine groups (792 cm ^-1 ) . Importantly, the inclusion of ionic salts markedly altered both the surface and mechanical properties of the SpONGE matrix. As a result of the stationary contact angle measurement, the sPCL mat showed a typical hydrophobic property (125.6 ± 4 °), while the SpONGE matrix showed almost zero hydrophobicity, thereby promoting rapid absorption of water-soluble biomolecules. Both the SpONGE matrix and each individual layer had higher maximum tensile strength and lower elongation at fracture than sPCL mat. Interestingly, the SpONGE matrix exhibited a step-like fracture that could be attributed to the multilayer structure. Of all the conditions applied, no significant difference in Young's modulus was observed. Due to the improved tensile strength of the SpONGE matrix, the fiber diameter (0.43 mu m) was reduced by a factor of two compared to the fiber diameter (0.86 mu m) of the sPCL mat. This effect can be rationalized by the fact that a more ordered crystal structure capable of enhancing tensile strength is commonly observed in a fiber structure composed of thin fibers. In particular, the discrete discrete layers exhibited almost identical properties, indicating the homogeneity of each discrete layer.

The SpONGE matrix demonstrated a versatile ability to act as a controlled drug delivery vehicle. The hydrophilicity of the SpONGE matrix caused rapid absorption of immunoglobulin G and bovine serum albumin (IgG-FITC, BSA-FITC) bound to fluorescein isothiocyanate (FITC) through the entire multilayer structure (see Figure 3A) . However, the complete wetting of the sPCL mat by the drug-containing solution could not be achieved in a conventional sPCL mat due to its inherent hydrophobicity (see Fig. 3B). The drug-binding efficiency (97.59 ± 0.07%) of the SpONGE matrix was significantly higher than that of the sPCL mat (20.56 ± 8.39%). The lgG- / BSA-FITC molecules were continuously released from the SpONGE mat without initial overexpression (see Figure 3C). However, most of the lgG- or BSA-FITC molecules that were adsorbed predominantly to the outside of the sPCL mat were released within one hour, and no molecules were detected thereafter, presumably due to the lack of drug loaded on the sPCL mat. The sustained release of lgG- or BSA-FITC molecules from the SpONGE matrix can be attributed to the presence of a large number of interstitial spaces which form a reservoir that can extend the drug path length or delay the release of molecules from the matrix I could.

Immobilized AAV (adeno-associated viral) vector on SpONGE and subcutaneously transplanted into mice and the like resulted in highly localized green fluorescent protein (GFP) expressed at the graft site. However, on the outer surface of the sPCL mat, most of the AAV vector was not adsorbed, resulting in a strong GFP signal in the distal region of the graft site. Many interspaces within the SpONGE matrix functioned as a storage block that could store the gene vector stably and delay vector emission to the outer domain. This structural effect of the SpONGE matrix enabled local gene expression near the graft site as well as sustained release of the gene vector in vivo. Bolus AAV infusion was accompanied by rapid termination following extensive GFP expression, confirming the benefits of polymeric gene transfer for localization and extended gene expression.

The SpONGE matrix demonstrated the ability to deliver dual drugs sequentially. The SpONGE matrix was partially separated into two portions and two soluble model drugs, BSA-FITC and BSA-Rhodamine (BSA-Rho), were each added to the partially peeled layer (see FIG. To evaluate the release kinetics of the two molecules, a SpONGE matrix containing two molecules in different layers was placed in the piston container, which could lead to unidirectional drug release (see FIG. 4B). Variations in drug penetration rate as well as drug path length differences were the major factors controlling the sequential release of both drugs (see Figure 4C). As a result, the BSA-FITC molecules in the upper layer were released faster than those observed in the lower BSA-Rho as they were first exposed to aqueous solution, because the path length of BSA-FITC was shorter 4D). Due to the simplicity and ability to separate multiple drugs well, the SpONGE system will be easily applied as a platform carrier for the spatio-temporal delivery of multiple biomolecules.

Subcutaneous implantation of the SpONGE matrix resulted in cell sorting along the interstitial space between the fibrous layers, whereas infiltrating cells were distributed randomly across the sPCL mat (Fig. 5). The absence of toxicity of the SpONGE matrix to cells in vivo was confirmed prior to implantation. Organized cell populations are an important issue in regenerative medicine because accurate mimicry of cellular orientation and structural patterns can be crucial in restoring damaged organ function. Various approaches such as bioprinting, the use of patterned scaffolds and cell sheet technology, which generally require both complex equipment and specialized techniques, have been used to organize cell distribution. In particular, SpONGE systems can induce cell alignment in vivo without the need for additional complex steps, which represents a great potential as an advanced platform for manipulating patterns as well as cell orientation.

In conclusion, a new SpONGE technology has been developed to form a hierarchical multilayer fiber system by a single continuous electrospinning. The introduction of organic or inorganic salts into the electrospinning solution was suitable for spontaneously forming a multilayered fiber structure. This process caused volume expansion of the jetted material to form a sponge-like structure. The SpONGE matrix has demonstrated great potential as a system that can mediate sequential delivery of multiple drugs, local delivery, and organization of cell distribution. The application range of the SpONGE system can be extended to many areas that require control over biological events in a spatiotemporal manner, such as neovascularization, cytokine signaling and inflammation.

Claims (18)

Mixing a polymer solution in which a polymer is dissolved in a solvent and an ionic salt; And
Spontaneously forming a multilayer structure comprising fibers and consisting of a plurality of layers and including interspace between each layer by electrospinning a mixture of the polymer solution and the ionic salt so as to form a jet flow from the nozzle to the collector and,
The value obtained by measuring the electrical conductivity after mixing the polymer solution and the ionic salt is 30 to 1000 uS / cm,
The content of the polymer in the polymer solution is 5 to 40% by weight,
The polymer may be selected from the group consisting of polycaprolactone, poly (lactide-co-glycolide), poly (vinylpyrrolidone), polycarbonate, poly (ethylene glycol), poly (urethane), polyethyleneimine, poly (vinyl alcohol) Chitosan, collagen, elastin, hyaluronic acid, fibrinogen,
The ionic salts are selected from the group consisting of FeCl ₃ , VCl ₅ , NiCl ₂ , CoCl ₂ , MnCl ₂ , CuCl ₂ , ZnCl ₂ , FeF ₃ , VF ₅ , NiF ₂ , CoF ₂ , MnF ₂ , CuF ₂ , ZnF ₂ , FeBr ₃ , ₅ , NiBr ₂ , CoBr ₂ , MnBr ₂ , CuBr ₂ , ZnBr ₂ , FeI ₃ , VI ₅ , NiI ₂ , CoI ₂ , MnI ₂ , ZnI ₂ , NaIO ₄ , KIO ₄ , LiIO ₄ , HIO ₄ , LiClO ₄ , _{NaClO 4, KClO 4, HClO 4} , LiBrO 4, NaBrO 4, KBrO 4, HBrO 4, hydrochloride, dopamine, dopamine hydrochloride, norepinephrine, norepinephrine hydrochloride, tri-hydrochloride, characterized in that at least one member selected from A method for forming a spontaneous multilayer structure.
The method according to claim 1,
The ionic salts induce high electron density and electrostatic repulsion of the solvent remaining in the fibers formed in the electrospinning and peripheral jets, and by this electrostatic repulsion, spontaneous formation of a multilayer structure including interspace between each layer Layer structure.
delete delete delete The method according to claim 1,
Wherein the electrospinning is performed continuously at one time.
The method according to claim 1,
≪ / RTI > further comprising the step of removing the ionic salt.
The method according to claim 1,
Wherein the current measured between the collector and the nozzle is from 0.001 to 0.1 mA.
The method according to claim 1,
Wherein the diameter of the fibers is 0.1 to 0.7 mu m.
The method according to claim 1,
Wherein the multi-layer structure is separable into individual layers.
The method according to claim 1,
Wherein the volume ratio of the multi-layer structure is 100 to 1000 cm < 3 > / g.
The method according to claim 1,
Wherein the contact angle of the multilayer structure is 30 DEG or less.
The method according to claim 1,
Wherein the drug binding efficiency of the multi-layer structure is 50% or more.
delete The method according to claim 1,
Wherein the multi-layer structure formed according to the method is used in a drug delivery system comprising a drug.
16. The method of claim 15,
Wherein a plurality of drugs are contained in different layers and are sequentially discharged.
The method according to claim 1,
Wherein the multi-layer structure formed according to the method is used for a skin patch comprising at least one selected from drugs, proteins, genes, and cells.
8. The method of claim 7,
The method for forming a spontaneous multilayered structure according to claim 1, wherein the multi-layered structure from which the ionic salt is removed according to the above method is used in a tissue engineering scaffold comprising at least one selected from drugs, proteins, genes, and cells.

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