KR100999116B1 - Biodegradable coaxial fiber complex - Google Patents

Abstract

The present invention relates to a biodegradable coaxial fiber composite containing a bioactive material, a method for producing the same, and a use thereof. The biodegradable coaxial fiber composite of the present invention is a poly (ε-caprolactone) and polycaprolactone-polyethylene glycol (Poly (ε-caprolactone) -Poly (ethylene glycol) -NH ₂ ) block copolymer The liquid mixture is electrospun through the outer nozzle of the hollow fiber nozzle, and the solution containing the first bioactive material is formed through the inner nozzle of the hollow fiber nozzle to form coaxial fibers, and then the second bioactive material is formed on the surface of the fiber. It is prepared by chemically bonding. Therefore, the biodegradable coaxial fiber composite of the present invention is characterized in that the first bioactive material is enclosed within the coaxial fiber and the second bioactive material is chemically bonded to the fiber surface, and the bioactive materials are released differently. This can be useful where you need to provide a pattern.

Coaxial Fiber, Bioactive Material, Biodegradable

Description

Biodegradable coaxial fiber complex

This study was conducted as part of the next-generation new technology development project of Kangwon National University, Ministry of Commerce, Industry and Energy [00014827-2007-31, Development of intelligent nanofibers for tissue regeneration to promote cell differentiation].

The present invention relates to a biodegradable coaxial fiber composite containing a bioactive material, a method for producing the same and a use thereof.

Fibers have a large surface area and high porosity, and have the ability to provide sufficient nutrients and oxygen, which are essential conditions for cell culture. Therefore, recently, a fiber complex containing a bioactive material is used for tissue regeneration, stem cell differentiation, wound healing or dressing, wound healing products for cell growth, tissue engineering support, tissue regeneration support, and the like. Doing. In addition, when the drug is included in the fiber complex, it can be utilized in drug delivery systems [ J. Nanosci . Nanotechnol . 2004, 4 (1-2), pp 52-65]. Differentiation of stem cells including stem cells requires cytokines such as growth factors.In order to differentiate stem cells into specific cells, the growth factors required for differentiation vary depending on the time of differentiation. Growth factors are required. As such, two or more bioactive substances are required for the full growth and differentiation of cells, and it is necessary to continuously provide an appropriate amount of the appropriate bioactive substances at each time. However, the fiber composites known to date have not met this demand.

In one example, US Patent Publication 2004 / 0241436A1 discloses a protein film forming composition formed by electrospinning a composition comprising a protein and a polymer, in which case all of the protein is initially released to provide the appropriate protein at the right time. Can't. When the bioactive material contained in the fiber is initially released in a large amount, the differentiation and growth of cells cannot be continuously stimulated, so the efficacy of the bioactive material cannot be maximized, and thus, a large amount of bioactive material is required. It is uneconomical.

In addition, Korean Patent Application Publication No. 2008-13224, filed by the present inventors, describes a fiber composite in which a bioactive material is chemically bonded to the surface of the fiber, but may be provided by continuously releasing the bioactive material to some extent. Two or more bioactive substances cannot be acted upon at a time when they are needed.

In addition, according to Korean Patent Publication No. 2008-21306, which is an example of using biodegradable fibers as a drug release system, starch and polymer may be added to the drug release system by further including and spinning the drug of the active ingredient in a spinning solution in which starch and polymer are dissolved. Examples of use as a support for the same are disclosed. However, even in such a spinning method, it is necessary to mix and use two or more drugs that are necessary, so that the drugs can not be properly operated at the required time.

Therefore, according to the growth and differentiation time of the cell or other needs, it is necessary to develop a multifunctional fiber composite which allows the appropriate bioactive material to act sequentially at the appropriate time.

The problem to be solved by the present invention is to develop a multifunctional fiber composite that allows the appropriate bioactive material to act sequentially at the appropriate time according to the growth and differentiation time of the cell or other needs.

It is therefore an object of the present invention to provide a fiber composite in which two or more bioactive materials can act sequentially, and the use thereof.

It is also an object of the present invention to provide a fiber composite in which two or more bioactive materials can be released slowly, and the use thereof.

Another object of the present invention is to provide a method for producing the fiber composite of the present invention.

In order to solve the above problems, the present inventors, by enclosing the bioactive material in the interior of the fiber and bonding another bioactive material to the surface of the fiber, the two or more bioactive materials are released and act in different orders, and By devising a biodegradable coaxial fiber composite that can be released slowly, the present invention has been completed.

Hereinafter, the biodegradable coaxial fiber composite of the present invention and its use will be described.

The present invention is poly (ε-caprolactone) and polycaprolactone-polyethylene glycol (Poly (ε-caprolactone) -Poly (ethylene glycol) -NH ₂ ) block copolymer (amine and 3) A biodegradable coaxial fiber composite composed of fibers including all of the circles), wherein the first bioactive material is encapsulated inside the fiber and the second bioactive material is conjugated to the surface of the fiber. It provides a biodegradable coaxial fiber composite characterized in that.

The biodegradable fiber composite of the present invention, as shown in Fig. 1 or 2, poly (pro-caprolactone) -Poly (ethyleneglycol) containing a poly (ε- caprolactone) and an amine group at the end -NH ₂ ) by electrospinning the mixed solution containing the block copolymer through the outer nozzle of the hollow fiber nozzle, the solution containing the first bioactive material through the inner nozzle of the hollow fiber nozzle to form coaxial fibers, It is prepared by chemically bonding a second bioactive material to the surface of the fiber. Therefore, the first bioactive material is enclosed in the fabric, and the second bioactive material is bound to the surface of the fiber through modification.

In the biodegradable coaxial fiber composite of the present invention, the poly (ε-caprolactone) and polycaprolactone-polyethylene glycol block copolymers are mixed in a weight ratio of 99.9: 0.1 to 0.1: 99.9, preferably 95: 5 to 80:20 do. At this time, if the content of polycaprolactone is too low, the content of polyethyleneglycol having a relatively low glass transition temperature is high, so when the fibers are spun out by the low glass transition temperature, the fibers tend to be associated with each other and the thickness of the fiber is thick. If the content of polycaprolactone is too high, the thickness of the fiber tends to be thicker than that of the copolymer when mixed, so it is important to properly control the mixing ratio thereof.

The first bioactive material is included in the fiber and may be a predetermined material that is lipophilic or hydrophilic. Preferably, the first bioactive material may be a hydrophilic material. When the first bioactive material is a hydrophilic material, the solution including the first bioactive material may further include a material for facilitating inclusion of the first bioactive material into the fiber. The material for facilitating the inclusion may include a solution comprising a lipophilic poly (ε-caprolactone) and a polycaprolactone-polyethylene glycol block copolymer and a solution containing a hydrophilic first bioactive material and a hollow fiber nozzle. Through the role of electrospinning it does not separate from each other. Such materials include polyethylene glycol, polyoxyethylene alkyl ether, alkyl dimethyl amine oxide, fatty acid alkanolamide, alkyl polyglucoside, hydroxy ethylene alkyl polyphenyl ether, polyethylene oxide-polypropylene oxide-polyethylene oxide (Pluronic), poly ( It may be a nonionic surfactant such as vinyl alcohol) (PVA), and preferably poly (vinyl alcohol) (PVA). Thus, in the biodegradable fiber composite of the present invention, the first bioactive material can be encapsulated together with the nonionic surfactant when encapsulated inside the fiber. The nonionic surfactant prevents the polycaprolactone-polyethylene glycol block copolymer and the first bioactive material from being separated from each other.

The second bioactive material is combined with the surface of the fiber after the electrospinning through the hollow fiber nozzle, and is not limited to the type of material that is lipophilic or hydrophilic. The second bioactive material can be bound to the surface of the fiber by chemical or physical reaction. Preferably, the second bioactive material may be a bioactive material having a carboxyl group. In this case, the second bioactive material is chemically bonded to the amine group produced as the polyethylene glycol of the polycaprolactone-polyethylene glycol block copolymer extends into the hydrophilic solution.

The first bioactive substance and the second bioactive substance include all substances in an organism that are extracted from an organism, produced or synthesized by a recombinant method such as proteins, enzymes, antibodies, growth factors, cytokines, toxins, nucleic acids, and carbohydrates. In addition, the bioactive substance includes all kinds of drugs such as antibiotics, antifungal agents, anticancer agents. In addition, the bioactive material may be a growth factor or drug necessary for tissue regeneration, stem cell differentiation, wound healing or dressing, or cell growth. Depending on the application of the biodegradable fiber composite, the first bioactive material and the second bioactive material may be the same or different, and each of the first bioactive material and the second bioactive material may include a plurality of bioactive materials. Can be. For example, the first bioactive material and the second bioactive material are fibroblast growth factor (FGF), epidermal growth factor (EGF), and tumor cell growth factor-beta (transforming growth factor). -b, TGF-b), platelet-derived growth factor (PDGF) or mixtures thereof.

The fibers of the present invention have a diameter of 50-5000 nm, preferably 500-2000 nm. The first bioactive material entrapped in the fiber diffuses. Therefore, if the diameter of the fiber is too thick, the first bioactive material can not act at the appropriate time, if too thin, the first bioactive material can be released in a large amount initially, the half-life is short due to the influence of many enzymes around Can lose. Therefore, the fibers preferably have a diameter in the above range. In addition, it is preferable to have a diameter in the above range in order to form a structure for effectively growing cells by supplying nutrients which are one of the essential conditions of cell culture in the fiber complex.

The pores produced by the entanglement of the fiber is adjustable from several nm to several μm, preferably 700-8000 nm, and the pore size is known to be effective in blocking the penetration of bacteria having a size of μm [WJ Li et al . , Biomaterials 26 (2005) 599-609. In the fiber complex, it is preferable to have a size in the above range in order to sufficiently supply oxygen, which is one of the essential conditions for cell culture, and to have a shape of a surface area suitable for the cells to grow.

When differentiating stem cells into specific cells, the type and amount of bioactive substances such as growth factors required change depending on the differentiation time of stem cells. In addition, during tissue regeneration or wound treatment, different growth factors are required depending on the growth time of the cells. When two or more bioactive materials required for differentiation or growth of cells are simply bound to the surface of the fiber, the two or more bioactive materials work at the same time, making it difficult to provide appropriate growth factors in a timely manner. There is a limit to achieving differentiation effects.

The biodegradable fiber composite of the present invention is in the form of coaxial fiber in which the first bioactive material is enclosed in the fiber and the second bioactive material is bound to the surface of the fiber. Typically, the fibers used in the fiber composites are typically degraded and thus release and act on the bioactive materials attached to the fibers. However, the fiber produced by electrospinning the poly (ε-caprolactone) and polycaprolactone-polyethylene glycol block copolymers used in the present invention was very slow in decomposition and hardly decomposed. In the present invention, the first bioactive material is first released from the fiber by diffusion, and the released first bioactive material acts initially and loses activity by surrounding enzymes. Since the second bioactive material is chemically bonded to the surface, it may be activated at the beginning, but the amount released is very small and bound to the fiber even after 7 days, and thus may be active while maintaining activity for a long time. Thus, each bioactive material entrapped on the surface and inside of the fiber can act sequentially at the appropriate time, thereby continually stimulating cell growth.

In addition, the fiber of the present invention is versatile by allowing two or more bioactive materials to be released and function in one fiber. Therefore, the biodegradable fiber complex of the present invention has a suitable use as a wound healing product, wound dressing agent, tissue engineering support, tissue regeneration support in tissue regeneration, stem cell differentiation, wound treatment or dressing, or cell growth.

In addition, the biodegradable fiber composites of the present invention can be used for applications such as supports for drug delivery systems. If all of the bioactive substances trapped inside the fiber and bound to the surface are drugs, the drug trapped inside the fiber and the drug bound to the surface can be released in a timely manner. Therefore, as the differentiation of the cell progresses, the required drug can be continuously acted on the cell.

Hereinafter, the manufacturing method of the biodegradable coaxial fiber composite of this invention is demonstrated.

The manufacturing method of the present invention

(1) Poly (ε-caprolactone) (PCL) and polycaprolactone-polyethylene glycol (Poly (ε-caprolactone) -Poly (ethylene glycol) -NH ₂ ; PCL-PEG) block copolymer having an amine bonded to the terminal Preparing a mixed solution of;

(2) preparing a solution of the first bioactive material;

(3) The mixed liquid of the block copolymer of (1) is coaxially electrospun through the internal nozzle of the hollow fiber nozzle through the external nozzle of the hollow fiber nozzle, and the solution containing the first bioactive material of (2) Preparing a fiber; And

(4) binding the second bioactive material to the surface of the coaxial fiber prepared above.

In the preparation of the mixed liquid, a polycaprolactone-polyethylene glycol (Poly (ε-caprolactone) -Poly (ethylene glycol) -NH ₂ ) block copolymer having an amine bound to the terminal may be used commercially, and Example 1 It can be prepared and used according to the method given in the following. PCL and PCL-PEG block copolymers are mixed in a weight ratio of 99.9: 0.1 to 0.1: 99.9, preferably in a 95: 5 to 80:20 weight ratio. Since the content of the PCL and PCL-PEG block copolymer affects the release rate of the first bioactive material, the content of the PCL and the PCL-PEG block copolymer may be modified so as to deviate from the above range depending on the type and the required content of the first bioactive material.

In the preparation of mixed liquors, PCL and PCL-PEG block copolymers are dissolved in organic solvents at a concentration of 15-20%. If the concentration is too low, even if the fibers do not form or become non-uniform fibers with beads, or spun, The fibers may not be formed, and if the concentration is too high, the viscosity of the solution may be large and the fibers may become too thick or not be spun.

In the preparation of the mixed liquid, the organic solvent used may be a hydrocarbon-based or alcohol-based organic solvent alone or a mixture thereof in the range of 1-5 carbon atoms. Preferably, it is preferable to use a mixture of chloroform and methanol.

In the preparation of the solution of the first bioactive material, the solution of the first bioactive material can be hydrophilic or lipophilic, preferably hydrophilic. It is prepared by dissolving a hydrophilic first bioactive material in a hydrophilic solvent, preferably water. At this time, it may further include a material for preventing separation from each other during the electrospinning through the mixed solution and the hollow fiber nozzle so that the first bioactive material can be properly contained in the interior of the fiber. If such a substance is not added, when the solution of the hydrophilic first bioactive material and the lipophilic mixed solution are electrospun at the same time, they may be separated from each other and may be formed without forming fibers, thereby forming beads. It may not be properly captured inside this fiber. Such materials include polyethylene glycol, polyoxyethylene alkyl ethers, alkyldimethylamine oxides, fatty acid alkanolamides, alkyl polyglucosides, lyoxyethylene alkyl polyphenyl ethers, polyethylene oxide-polypropylene oxide-polyethylene oxides, and poly Nonionic surfactants, such as (vinyl alcohol) (PVA), can be used, Preferably poly (vinyl alcohol) (PVA) can be used. The concentration of poly (vinyl alcohol) can be appropriately adjusted according to the type of the first bioactive material used, the capacity of the mixed solution and the solution of the first bioactive material. Preferably it can be used at a concentration of 0-10% by weight, more preferably 5% by weight.

In the manufacture of fibers, the conditions of electrospinning through the hollow fiber nozzles affect the thickness and shape of the fibers, so that the concentration of the solution, the type of organic solvent, the voltage, the thickness of the needle, the ground from the end of the needle or Variables such as distance to the collector, flow rate, temperature, and humidity can be adjusted accordingly. By controlling these parameters it is possible to form pores due to the desired fiber thickness and fiber entanglement. In the example of the present invention, the spinning condition was a voltage of 15 kV, the height of the end of the hollow fiber nozzle and the ground (using aluminum foil) was 11 cm, the temperature and humidity during spinning was 25 ℃ and 30-50% Maintained.

The second bioactive material bound to the surface of the coaxial fiber is bound by chemical or physical reaction with the surface of the fiber. In particular, when the second bioactive material is a bioactive material having a carboxyl group, polyethylene glycol of the PCL-PEG block copolymer is chemically bonded to the generated amine group as it extends into the hydrophilic solution. At this time, the second bioactive material can be bound by simply adding to the fiber, preferably EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide), DCC (1,3-dicyclohexyl) Carbodiimide), BOP (benzotriazole-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate), HOBT (1-hydroxybenzotriazole), or HBTU (2- (1H-benzo) It can be combined by further adding a coupling agent such as triazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate). In one example of the present invention, a mixture of HOBT and EDC was used as a coupling agent when binding the second bioactive material.

The biodegradable coaxial fiber composite of the present invention contains two or more different bioactive materials which are multifunctional and economical. In addition, the first bioactive material is enclosed in the fiber and the second bioactive material is bound to the fiber surface, so that the bioactive material may act at the appropriate time in accordance with the growth rate of the cells according to the decomposition rate of the polymer constituting the fiber. To help. In addition, it is possible to act at the appropriate time in accordance with the growth of the cell, thereby continually stimulate cell growth. In addition, when the bioactive material is a drug, it is possible to release the initial release followed by a sustained release sequentially.

Hereinafter, the present invention will be described in detail by the following examples. However, the present invention is not limited by the following examples.

Components used in the embodiment of the present invention is caprolactone (ε-caprolactone, Mw 114.14), polycaprolactone [poly (ε-caprolactone), PCL, Mw 65000] is Aldrich (Aldrich, [St. Louis, Mo]) Purchased). PEG- (amine) 2 (Mw 3400) was purchased from SunBio (Korea). Poly-vinyl alcohol (Mw ~ 27,000) was purchased from Fluka. Fluorescein 5 (6) -isothiocyanate (FITC) and calf serum albumin (Albumin bovine serum, BSA) were purchased from Sigma (St. Louis, Mo). Texas-red was purchased from Invitrogen, USA.

Example  1: biodegradable Coaxial  Preparation of Fiber Composites

(One) PCL  - PEG  Preparation of Block Copolymers

3 g of PCL was completely dissolved in 30 ml of dichloromethane, and 19 mg of p-nitrophenyl chloroformate and 15 mg of pyridine were slowly added while stirring was continued (PCL: p-nitrophenyl chloroformate: pyridine = 1). : 2: 4, molar ratio). The reaction was further stirred at room temperature for 3 hours. About two-thirds of the dichloromethane used from the resulting mixture was removed by solvent evaporation, and the PCL obtained by filtration by precipitation of activated PCL by the addition of cold diethylene ether was dried in vacuo for one day. The precipitation reaction was repeated one more time.

2.29 g of the activated PCL and 0.595 g of polyethylene glycol (PEG) were dissolved in 30 ml and 3 ml of dichloromethane, respectively, to prepare a PCL solution and a PEG solution. The PCL solution was slowly added dropwise to the obtained PEG solution, whereby the stirrer was quickly turned to allow the mixture to be mixed uniformly throughout. After further reacting at room temperature for 24 hours, the produced PCL-PEG copolymer was precipitated by adding 3,000 ml of cold ethanol. The precipitated PCL-PEG copolymer obtained by filtration was dried at 40 ° C. under vacuum. Thus synthesized PCL-PEG copolymer (binary and ternary copolymer coexist) was sealed and refrigerated. The prepared PCL-PEG block copolymer is used as one component of the polymer solution.

(2) Hollow fiber  Electrospinning furnace first using the nozzle Bioactive substances (BSA) Included  Manufacture of fibers

The hollow fiber spinning nozzle has a dual-nozzle structure comprising a nozzle for the mixed liquid (outer nozzle) and a nozzle for the solution of the first bioactive material (inner nozzle). The diameter of the outer nozzle was 18G and the diameter of the inner nozzle was 25G.

A mixture of PCL-PEG block copolymer and PCL (including 10% by weight of PCL-PEG block copolymer) was dissolved in 10 ml (volume ratio, 3: 1) of a mixed solvent of chloroform and methanol to prepare a mixed solution having a concentration of 15% by weight. It was. 1.5 g of PVA was dissolved in 30 ml of distilled water to dilute the 5 wt% PVA solution to make 1 wt% PVA. A solution containing BSA was prepared by adding 50 mg (10 mg / ml) of BSA, which is a first bioactive substance, to 5 ml of prepared PVA solution PVA.

10 mL and 5 mL of the prepared solution were put into the outer nozzle and the inner nozzle of the hollow fiber nozzle, respectively, and the discharge rate of the mixed solution and the discharge rate of the solution containing BSA were 0.06-0.6 (in-out), respectively. Electrospinning was performed at 0.1-1.0, 0.2-0.6, and 0.3-1.0 ml / h. The overall model for the production of fibers by electrospinning using hollow fiber nozzles is described in Advanced functional materials 2006, 16, pp. 2110-2116 was followed. The spinning condition was a voltage of 15 kV, the height of the end of the hollow fiber nozzle and the ground (using aluminum foil) was 11 cm, and the temperature and humidity during spinning were maintained at 25 ℃ and 30-50%.

(3) second to the fabric produced Bioactive substance (BSA)  Preparation of Modified Fiber

0.5 mg of the prepared fiber was soaked in 100 ml of 70% ethanol in a full volume 10 ml vial, and 1 ml distilled water was added and washed by stirring slowly for 3 minutes. After dispensing 152 ml of BSA dissolved at a concentration of 1.18 mg / ml in PBS at pH 8 (two times the theoretical value of the amine group exposed on the fiber surface, mol ratio), 500 ml of PBS at pH 8 was further added. 1.97 mg HOBT dissolved in 100 ml of acetone and 2.8 mg EDC dissolved in PBS at pH 8 were added (10 times the theoretical value of the amine group exposed on the surface of the fiber, mol ratio) and stirred with the inlet open at room temperature for 30 minutes. As a result, acetone was removed. Thereafter, the inlet was sealed for reaction of the amine group and BSA on the surface of the fiber and stirred at room temperature for 4 hours 30 minutes. After the reaction, the mixture was stirred for 5 minutes using 1 ml distilled water and then removed. This washing procedure was repeated five times.

Experimental Example  1: inside the fiber Included  First With bioactive substances  On fiber surface Combined  Identify the Second Bioactive Material

Instead of BSA used in the solution containing the first bioactive material in Example 1, FITC (Fluorescein 5 (6) -isothiocyanate) -BSA was used in the same amount and electrospun onto the cover glass. The release rate of the solution containing the polymer mixture and the first bioactive material during the electrospinning was controlled as shown in Table 1 below, 0, 1 or 5% by weight PVA solution was used for each.

TABLE 1 Release rate during electrospinning (ml / h)

Release rate (ml / h) Solution containing the first bioactive material 0.06 0.1 0.2 0.3 Polymer mixture 0.6 1.0 0.6 1.0

The prepared fibers were transferred to a 24-well plate coated with silicone, and 100 µl of 70% ethanol was added to wet the fibers. 3 ml of distilled water was added to the soaked fiber and shaken in a 2-D shaker for 10 minutes, and then distilled water was removed. This procedure was repeated three times to completely remove the FITC-BSA not entrapped into the fiber. Then, a solution obtained by dissolving 39.46 µg Texas red-BSA, 0.39 µg HOBT, and 0.56 µg EDC in 1 ml of PBS (pH 8) was added to the prepared fiber. Stir slowly for 12 hours, add 3 ml of distilled water and wash for 10 minutes. The washing process was repeated three times to completely remove Texas red-BSA that did not bind to the fiber surface.

The prepared fiber was observed using a Confocal Laser Scanning Microscpoe (CLSM), and the results are shown in FIG. 3. If Texas red-BSA is bound on the fiber surface, it appears red when observed with CLSM. If FITC-BSA is included inside the fiber, it appears green when observed with CLSM. As shown in FIG. 3, it can be confirmed that BSA (FITC-BSA) is included in the fiber through CLSM and BSA (Texas red-BSA) is bonded to the fiber surface. In addition, it can be seen that when the ratio of the release rate of the solution containing the first bioactive material and the polymer solution is 1:10, a larger amount of BSA is contained than when the ratio is 1: 3.

In addition, it was confirmed whether the second bioactive material was bound even when only PCL was used as the polymer mixture. PCL was used in the same amount in place of the mixture of PCL-PEG block copolymer and PCL in the polymer mixture of Example 1, and the release rate and subsequent experimental procedures were performed in the same manner as described above. The results are shown in Fig. As shown in Figure 3, when using only PCL (A-C) it can be seen that the BSA is not bonded because the amine group is not exposed on the surface of the fiber.

Experimental Example  2: PVA  Observation of the fiber surface according to the invention according to the ratio of concentration and release rate

In Example 1, the release rate of the polymer mixed solution and the release rate of the solution including the first bioactive material were adjusted and controlled as shown in Table 1 above. It was also spun using 0, 1 and 5 wt% PVA solutions for each. The prepared fibers were observed using a Field Emission-Scanning Electron Microscope (FE-SEM). The results are shown in FIG. As shown in Figure 4, when the electrospinning using a hollow fiber nozzle, it can be seen that the shape of the fiber is in the form of fibers without beads. When the degree of bead formation according to the presence or absence of PVA was observed, PVA was used (corresponding to B, C, E and F in FIG. 4) compared to the case where PVA was not used (corresponding to A and D in FIG. 4). It can be confirmed that the degree of bead formation is small. In addition, it can be seen that using 5% by weight of PVA showed much less beads than when using 0 and 1% by weight of PVA. In addition, the diameter of the prepared fiber is shown in FIG. The diameter of the fiber thus prepared was about 500 nm to 1800 nm.

Experimental Example  3: inside fiber Included  First Of bioactive substances (BSA) Inclusion amount  Measure

In Example 1, the release rate of the solution containing the polymer mixed solution and the first bioactive material during electrospinning was adjusted as shown in Table 1 and above, and 0, 1 or 5 wt% PVA solution was used for each. About 1 mg of the prepared fibers were cut out and placed in a microtube subjected to silicone coating. 20 μl of 70% ethanol was added to the microtube, the fibers were wetted, then 1 ml of distilled water was added and shaken vigorously for 10 minutes and then distilled water was removed. This procedure was repeated three times to remove all non-inclusion BSA inside the fiber. After distilled water was completely removed, 200 µl of dichloromethane was added to dissolve the fibers. 800 µl of distilled water was further added to precipitate the fiber portion and BSA that were not dissolved in dichloromethane. Then, it was stirred while shaking vigorously for 1 hour. Centrifugation gave 100 μl of the solution in the upper layer and transferred to a 96-well plate. Then 100 μl of BCA (Bicinchoninc Acid) was added and stirred for 2 hours at 37 ° C. The content of the inclusion BSA was measured by observing the absorbance at 570 nm. The results are shown in FIG. As shown in Figure 6, as the release rate of the solution containing the BSA increases from 0.06 to 0.3 ml / h it can be seen that the amount of BSA contained in the fiber increases. In addition, it can be seen that as the concentration of PVA increases, the content of BSA contained in the fiber increases. In particular, when the ratio of the release rate of the solution containing the BSA and the polymer mixture is 1:10, it can be seen that the amount of BSA contained more than doubled when using 5% by weight PVA.

Experimental Example  4: first from fiber Bioactive substances (BSA)  2nd Of bioactive substances (BSA)  Release aspect measurement

(1) first Of bioactive substances  Release aspect

10 mg of the fiber prepared by the method of Example 1 (2) was placed in a 10 ml conical tube with a silicone coating. 200 μl of 70% ethanol was added to wet the fibers. Then, 5 ml of PBS (pH 7.4) was added and the tube was kept at room temperature for 1 hour with the lid open at room temperature to remove 70% ethanol. The release pattern was then monitored with stirring at 37 ° C. and 100 rpm conditions for 7 days. 100 μl of the release solution obtained on days 1, 3, 5 and 7 respectively were transferred to 96-well plates. 100 μl of BCA was added and stirred at 37 ° C. for 2 hours. The content of BSA released from the inside of the fiber was measured by observing absorbance at 570 nm. The results are shown in FIG. As shown in FIG. 7, when fibers were prepared using 0 and 5 wt% PVA (see A and B), it was confirmed that a large amount of the BSA contained in 1 day after the release experiment was released. being). On the other hand, when the fiber was prepared using 10% by weight PVA (see C), it can be seen that about 4 times less amount of BSA was released than when 0 and 5% by weight PVA was used (about 200 μg of BSA released). ). It can also be seen that the inclusion BSA is released slowly. Even when 10 wt% PVA was used, the ratio of the release rate of the solution containing BSA and the release rate of the polymer mixture was 1:10 (see a and b), and 1: 3 (see c and d). It can be seen that BSA is released slowly in about 2 times less amount.

(2) second Of bioactive substances  Release aspect

The release pattern according to the binding method of the second bioactive material to the fiber was investigated.

FITC-BSA was used to determine the degree of release of the protein bound to the amine group. First, FITC was bound to BSA. 2 mg / ml BSA and 52.9 mg of 0.1 M sodium carbonate were placed in a round bottom flask and the pH was adjusted to 9. 500 μl of FITC stock solution dissolved in 1 ml FITC in 1 ml DMSO was added to BSA and reacted for 12 hours while stirring in a dark place at 4 ° C. After the reaction, dialysis was performed three times in PBS to remove unbound FITC. FITC-BSA solution was lyophilized.

In order to conjugate FITC-BSA to the fiber, FITC-BSA was first activated. Different amounts of FITC-BSA and an activator were added depending on the mixing ratio (5, 10, 20 wt%) of PCL-PEG. 0.96 mg, 1.5 mg and 2.88 mg of FITC-BSA were weighed and dissolved in 5 ml of PBS. EDAC and HOBt were added to each FITC-BSA solution to double the mol of FITC-BSA. EDAC was added in 5.2mg, 8.4mg and 16mg, respectively, and HOBt in 3.8mg, 5.9mg and 11mg, respectively. After dissolving gently at RT, 1ml was dispensed into 2mg of fiber which was previously wetted with ethanol. At this time, the FITC-BSA put into the fiber was dispensed to the fiber obtained by spinning at the respective mixing ratio of what was prepared according to the mixing ratio of PCL-PEG. The mixture was slowly stirred at RT for 6 h to allow sufficient reaction between the amine groups of the fiber and FITC-BSA.

After the reaction, wash 3 times with PBS to remove unbound FITC-BSA. The combined fibers were placed in a silicon-coated 12-well culture plate with 1 ml PBS and the amount of FITC-BSA released for 3, 5 and 7 days in a shaking incubator at 37 ° C. Quantitation was performed using a meter (Fluorescence Spectrophotometer, F-2500, HITACHI, Japan).

Unlike the fiber composite where the fiber and FITC-BSA are chemically bonded, FITC-BSA was mixed and spun in PCL to observe the degree of release when the fiber and FITC-BSA were physically mixed. At this time, the concentration of the total solution was 15% by weight.

In order to make the mixing ratio of PCL-PEG and FITC-BSA 5, 10 and 20% by weight, PCL-PEG was added 48.8mg, 48.125mg and 46.4mg, respectively, and FITC-BSA was 1.2mg, 1.875mg and 3.6mg. Put it. FITC from PCL-PEG / FITC-BSA fibers cut into 1x1 cm ² and released in a 12-wells culture plate with 1 ml PBS for 1, 3, 5 or 7 days at 37 ° C, in a shaker incubator. The amount of -BSA was quantified by Fluorescence Spectrophotometer (F-2500, HITACHI, Japan). The results are shown in FIG. As shown in FIG. 8, when BSA was bound at the surface of the fiber (b), less than about 10% of BSA was released even after 7 days of the release experiment. On the other hand, when the fiber was prepared by mixing the BSA in the external polymer solution and then electrospinning (a), about 90% of the BSA was released within one day of the release experiment.

Figure 1 schematically shows the production process and structure of the biodegradable coaxial fiber comprising the first bioactive material and the second bioactive material of the present invention.

Figure 2 schematically shows the process and results of confirming that the first bioactive material is included in the fiber and the second bioactive material is bonded to the surface of the fiber in the biodegradable coaxial fiber of the present invention.

Figure 3 is a first bioactive material (FITC-BSA, green) is included in the fiber formed according to the composition, release rate (ml / h) and PVA concentration of the polymer mixture in the manufacturing method of the present invention and the second bioactive material It is confirmed that (Texas red-BSA, red) is combined.

i) A-C: polymer mixture containing only PCL, D-F: mixture of PCL and PCL-PEG block copolymer

ii) The release rate of the solution containing the first bioactive material and the polymer mixed solution (or the block copolymer mixed solution) was (a) 0.06-0.6 (in-out), (b) 0.1-1.0, (c) 0.2- 0.6, and (d) 0.3-1.0 ml / h.

iii) The concentrations of PVA were 0 (A and D), 1 (B and E), and 5 (C and F)%.

Figure 4 shows the field emission-scanning electron microscope (FE-SEM) image of the fiber formed according to the composition, the release rate (ml / h) and the concentration of PVA in the polymer mixture in the production method of the present invention.

i) A-C: polymer mixture containing only PCL, D-F: mixture of PCL and PCL-PEG block copolymer

ii) The release rate of the solution containing the first bioactive material and the polymer mixed solution (or the block copolymer mixed solution) was (a) 0.06-0.6 (in-out), (b) 0.1-1.0, (c) 0.2- 0.6, and (d) 0.3-1.0 ml / h.

iii) The concentrations of PVA were 0 (A and D), 1 (B and E), and 5 (C and F)%.

Figure 5 shows the diameter of the fiber formed according to the composition of the polymer solution, the release rate (ml / h) and the concentration of PVA in the production method of the present invention.

i) A: polymer mixed liquid containing only PCL, B: mixed liquid of PCL and PCL-PEG block copolymer

ii) The release rate (ml / h) of the solution containing the first bioactive material and the polymer mixed solution (or the block copolymer mixed solution) was (a) 0.06-0.6 (in-out), (b) 0.1-1.0, (c) 0.2-0.6, and (d) 0.3-1.0.

Figure 6 shows the inclusion efficiency of the first bioactive material of the fiber formed according to the composition of the polymer solution, the release rate (ml / h) and the concentration of PVA in the present invention.

i) A: polymer mixed liquid containing only PCL, B: mixed liquid of PCL and PCL-PEG block copolymer

ii) The release rate of the solution containing the first bioactive material and the polymer mixed solution (or the mixed solution of the block copolymer) is (a) 0.06-0.6 (in-out), (b) 0.1-1.0, (c) 0.2, respectively. -0.6, and (d) 0.3-1.0.

Figure 7 shows the release pattern of the first bioactive material of the fiber formed according to the release rate (ml / h) and the concentration of PVA in the present invention over time.

i) The concentrations of PVA were 0 (A), 5 (B) and 10 (C)%.

ii) The release rates of the solution containing the first bioactive material and the polymer mixture were (a) 0.06-0.6 (in-out), (b) 0.1-1.0, (c) 0.2-0.6, and (d) 0.3, respectively. -1.0.

Figure 8 shows the release aspect of the second bioactive material according to the binding method of the second bioactive material in the present invention.

(a) When BSA is mixed with polymer mixture and then electrospun

(b) BSA is bonded on the surface of the fiber

Claims (16)

Biodegradable coaxially composed of fibers comprising poly (ε-caprolactone) and polycaprolactone-polyethylene glycol (Poly (ε-caprolactone) -Poly (ethylene glycol) -NH ₂ ) block copolymers with amines bonded at the ends A fiber composite, wherein a biodegradable coaxial fiber is characterized in that a first bioactive material is encapsulated with a nonionic surfactant and a second bioactive material is conjugated to a surface of the fiber. (coaxial fiber) composite. The biodegradable coaxial fiber composite of claim 1, wherein the poly (ε-caprolactone) and the polycaprolactone-polyethylene glycol block copolymer are included in a weight ratio of 99.9: 0.1 to 0.1: 99.9. According to claim 1, wherein the nonionic surfactant encapsulated with the first bioactive material is polyethylene glycol, polyoxyethylene alkyl ether, alkyl dimethyl amine oxide, fatty acid alkanolamide, alkyl polyglucoside, hydroxy ethylene alkyl polyphenyl Biodegradable coaxial fiber composite, characterized in that selected from the group consisting of ether, polyethylene oxide-polypropylene oxide-polyethylene oxide (Pluronic), poly (vinyl alcohol). The biodegradable material according to claim 1, wherein the second bioactive material is a bioactive material having a carboxyl group, and the polyethylene glycol of the polycaprolactone-polyethylene glycol block copolymer is bonded to an amine group formed while extending into a hydrophilic solution. Coaxial fiber composite. According to claim 1, wherein the first bioactive material and the second bioactive material may be the same or different, fibroblast growth factor (FGF), epidermal growth factor (EGF), Biodegradable coaxial fiber composites selected from the group consisting of tumor cell growth factor-beta (TGF-b), platelet-derived growth factor (PDGF), and mixtures thereof . The biodegradable coaxial fiber composite of claim 1, wherein the fiber has a diameter of 500-2000 nm. (1) Poly (ε-caprolactone) (PCL) and polycaprolactone-polyethylene glycol (Poly (ε-caprolactone) -Poly (ethylene glycol) -NH ₂ ; PCL-PEG) block copolymer having an amine bonded to the terminal Preparing a mixed solution of; (2) preparing a solution of the first bioactive material comprising the first bioactive material and a nonionic surfactant; (3) The mixed liquid of the block copolymer of the above (1) is coaxial fibers by electrospinning through the inner nozzle of the hollow fiber nozzle, the solution containing the (2) the first bioactive material through the outer nozzle of the hollow fiber nozzle Preparing a; And (4) A method of producing a biodegradable coaxial fiber composite comprising the step of binding a second bioactive material to the surface of the coaxial fiber prepared above. The method of claim 7, wherein the PCL and PCL-PEG block copolymers are mixed in a weight ratio of 99.9: 0.1 to 0.1: 99.9. The method of claim 7, wherein the solution containing the first bioactive material is hydrophilic or lipophilic. The method of claim 7, wherein the nonionic surfactant is polyethylene glycol, polyoxyethylene alkyl ether, alkyl dimethyl amine oxide, fatty acid alkanolamide, alkyl polyglucoside, rioxy ethylene alkyl polyphenyl ether, polyethylene oxide-polypropylene oxide- Polyethylene oxide (Pluronic), poly (vinyl alcohol) The production method characterized in that it is selected from the group consisting of. The method of claim 10, wherein the nonionic surfactant is included at a concentration of 0.01-10% by weight. delete A biodegradable coaxial fiber composite prepared by the method of claim 7. delete Article for tissue regeneration comprising the biodegradable coaxial fiber complex of any one of claims 1 to 6. A drug delivery system comprising the biodegradable coaxial fiber complex of any one of claims 1 to 6.

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