KR101616345B1 - Complex scaffold comprising nanofiber with nanoparticle to drug-delivery for artificial skin and filler, and method for preparing the same - Google Patents

Abstract

The present invention relates to a composite support for artificial skin and filler comprising biodegradable nanofibers and cationic nanoparticles for drug delivery adhered thereto, and a method for producing the composite support. The composite support of the present invention has high cell adhesion ability, low cytotoxicity, and gradually releases a drug to be delivered, and thus can be usefully used as artificial skin and filler.

Description

TECHNICAL FIELD [0001] The present invention relates to a composite support for artificial skin and filler containing nanofibers and nanoparticles, and a method for preparing the composite support,

The present invention relates to a composite support for artificial skin and filler containing biodegradable nanofibers and cationic nanoparticles for transferring a pharmacological substance attached thereto, and a method for producing the composite support.

In the case of damage to organs or tissues in the human body, it is necessary to regenerate tissues effectively by providing cells, drug scaffolds and the like. The scaffold for tissue regeneration must firstly be physically stable at the implant site, and secondly, And third, after formation of new tissue, it should be decomposed in vivo. Fourth, degradation products should not be toxic. Such a tissue regeneration support is conventionally made of a sponge-type, matrix-type nanofiber or gel-type cell culture support using a polymer having a certain strength and shape, and the tissue regeneration scaffold has a specific depth or height It plays an important role in creating a three-dimensional shaped structure. The technique of transplanting a scaffold that serves as a framework for tissue regeneration and regenerating tissue in vivo using self-healing power is called regenerative medicine or tissue engineering. As an example of tissue engineering, there is a method of regenerating articular cartilage. In the articular cartilage regeneration method, an artificial prosthesis made of cartilage cells as a support is formed, and the artificial prosthesis is implanted in a damaged area. Is reproduced.

Electrospinning is one of the simplest methods of manufacturing fibrous mates such as extracellular supports. Since electrospinning allowed a reduction in pore diameter from micrometers to nanometers, it presented a new direction for biopolymers, thereby increasing the surface area to volume or mass ratio and significantly increasing the mechanical properties of the polymer. The advantages of electrospinning are the ability to fabricate very thin fibers in the nanometer to micrometer range and are being attempted in a variety of tissue engineering applications due to their wide surface area, versatility, excellent mechanical properties and ease of manufacture. In the case of an electrospinning support, the cells are easy to grow if they have sufficient mechanical properties.

On the other hand, chitosan is a β-1,4-linked pyranose unit of glucosamine. It is composed of polysaccharide series with more than 5,000 glucosamine residues and a molecular weight of more than 1 million and a multivalent cation Is a biopolymer material that can be extracted from aquatic systems including crustaceans such as crab shells and squid and squid. In terms of its molecular structure, it is similar in structure to cellulose, which is a type of polysaccharide, and has excellent biocompatibility, Chitosan has been applied to the biotech industry and biomedical materials of the 21st century after being approved as a food in the US FDA.

In order to deliver the pharmacological substance, there is a method in which the drug is wetted to the feces or the pharmacological substance itself is administered to the wound site. However, such a method has the disadvantage that the drug can not be continuously delivered and the ease of storage due to the wet state is also low .

Accordingly, the present inventors prepared a composite support by supporting a pharmacological substance on cationic nanoparticles and binding the cationic nanoparticles to biodegradable nanofibers. The composite support thus prepared has high cell adhesion ability, low cytotoxicity, The inventors of the present invention have completed the present invention by confirming that they can be usefully used as artificial skin and filler.

Patent Application No. 10-2013-0056586 'A method for manufacturing a nanofiber support capable of controlling the thickness and the pore size, a nanofiber support fabricated thereby, and a device for manufacturing a nanofiber support used therein, Patent Application No. 10-2013-0015021 " Method for producing nanofiber-type biopolymer / bioactive glass composite support and nanofiber-type bio-polymer / bioactive glass composite support " Patent Application No. 10-2005-0087072 " Nanofiber-type cell culture support &

It is an object of the present invention to provide a composite support for artificial skin and filler containing biodegradable nanofibers and cationic nanoparticles for drug delivery adhered thereto.

It is still another object of the present invention to provide a method for producing the composite support.

In order to solve the above problems, the present invention provides a composite support for artificial skin and filler comprising biodegradable nanofibers and cationic nanoparticles for drug delivery adhered thereto.

The present invention also provides a method for producing the composite support.

The composite support comprising the biodegradable nanofibers of the present invention and the cationic nanoparticles for drug delivery adhered thereto has a high cell adhesion ability and low cytotoxicity and gradually releases the pharmacological substance to be delivered, Can be usefully used.

1 is a SEM image of chitosan (CS) nanoparticles.
2 is an SEM image of a nanofiber and a composite support.
FIG. 3 is a chart showing the cytotoxicity of chitosan nanoparticles as an MTT assay. FIG.
FIG. 4 is a diagram showing the change in cell activity by fucoidan-chitosan nanoparticles through an MTT assay. FIG.
5 is a fluorescence photograph showing that nanoparticles penetrate into cells.
FIG. 6 is a view showing the adhesion ability and cell activity of the nanoparticle-nanofiber composite support of the present invention through MTT assay.
7 is an SEM image of cells attached to the nanoparticle-nanofiber composite support of the present invention.

According to the present invention,

1) dissolving the cationic polymer in an aqueous solution of an organic acid to prepare a cationic polymer solution, adding a gelling agent to the cationic polymer solution to prepare a cationic nanoparticle;

2) dissolving the biodegradable polymer in an organic solvent to prepare a biodegradable nanofiber; And

3) adding the cationic nanoparticles prepared in step 1) to the biodegradable nanofibers prepared in step 2) and adsorbing the biodegradable nanoparticles to prepare nanoparticle-nanofiber composite support; A method for producing a support is provided.

Hereinafter, the present invention will be described in detail.

The composite support according to the present invention is characterized in that cationic nanoparticles are added to biodegradable nanofibers and adsorbed thereon.

The process for producing the composite support according to the present invention will be described in detail as follows.

The step 1) is a step of preparing cationic nanoparticles. The cationic polymer is put into an aqueous solution of an organic acid to prepare a cationic polymer solution. To this solution is added a gelling agent with ultrasonic treatment to produce cationic nanoparticles. In this case, the cationic polymer solution and the gelling agent are preferably mixed in a volume ratio of 1 to 10: 1.

The cationic polymer is preferably chitosan or poly-L-lysine, more preferably chitosan. Chitosan is not specifically limited and is a pyranose unit of glucosamine which is? -1,4-linked.

The aqueous organic acid solution is preferably lactic acid, acetic acid, propionic acid, formic acid, ascorbic acid and tartaric acid, more preferably acetic acid to be.

The concentration of the cationic polymer in the aqueous solution of the cationic polymer is not limited to a specific range, but is preferably 0.01% (W / V) or more, and the cation for each organic acid used in preparing the aqueous solution of the cationic polymer To dissolve to the solubility range of the polymer. The concentration of the cationic polymer aqueous solution depends on the concentration of the organic acid used in preparing the aqueous solution. For example, when the concentration of the aqueous acetic acid solution is increased, the solubility of the cationic polymer is also increased. Therefore, the maximum concentration of the cationic polymer in the aqueous solution of the cationic polymer is up to the solubility of the cationic polymer with respect to the organic acid used.

The gelling agent is preferably tripolyphosphate (TPP), but is not limited thereto. Tripolyphosphate is a compound present in the form of sodium salt and has been conventionally used for a variety of purposes such as sea food, meat food, poultry food, and food preservative for pet food, The United States Food and Drug Administration recognizes tripolyphosphate as a safe human compound. The cationic polymer aqueous solution to which the tripolyphosphate aqueous solution is added is stirred to be well mixed. Conditions for stirring are not particularly limited, but according to one specific embodiment of the present invention, the mixture is stirred at 37 DEG C and 300 rpm for 30 minutes. Centrifugation is carried out using a conventional centrifugal separator and centrifugation is carried out at 15,000 rpm for 30 minutes. After centrifugation, the supernatant is removed and lyophilized to prepare a powder of cationic nanoparticles.

The cationic nanoparticles exhibit an average particle size of less than 1 mu m, preferably from 0.1 to 800 nm, more preferably from 30 to 50 nm. The average size of the cationic nanoparticles is the average diameter of the group of cationic nanoparticles, which can be measured by standard procedures known to those skilled in the art. On the other hand, the charge size of cationic nanoparticles can range from 0 mV to + 60 mV.

Since the cationic nanoparticles can gradually release the drug, the drug can be expected to have a sustained drug delivery effect and can penetrate into the cell through the cytoplasm. Therefore, continuous drug delivery can be expected in the cell.

The step 2) is a step of preparing a biodegradable nanofiber. The biodegradable polymer is dissolved in an organic solvent and electrospun to produce a biodegradable nanofiber.

The biodegradable nanofiber is a porous support capable of three-dimensional cell culture, which means that the biodegradable nanofibers intersect to form a network structure. The biodegradable polymer is preferably selected from the group consisting of polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactic acid-co-glycolic acid) amide / polyester-urethane, poly (valerolactone), poly (hydroxybutyrate), poly (hydroxyvalerate), polyanhydride Polyorthoesters, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid and poly-N-isopropylacrylamide, and more preferably polycaprolactone (PCL).

The nanofiber of the present invention has a high specific surface area due to its high aspect ratio (ratio of length to diameter), that is, it has a large contact area with the cells in the nanofiber, , The nanofibers of the present invention have linear nanofibers crossed with each other to smoothly connect the pores in the nanofibers. Thus, nutrients and oxygen necessary for cell culture can be easily supplied to the upper, lower, left, and right sides. Considering the specific surface area of the support to be produced, the ease of contact with cells, the size of the pores to be produced, and the like, the diameters of the linear nanofibers constituting the crossed network structure in the cell support are preferably 50 to 2,000 nm desirable.

The organic solvent includes but is not limited to THF (tetrahydrofuran), DMF (dimethylformamide), DMSO (dimethylsulfoxide), chloroform, dichloromethane, hexane, diethyl ether, acetonitrile and the like. In the present invention, a mixed solvent of THF and DMF is preferable. In this case, the mixing ratio is preferably 5 to 10: 1 to 5, and more preferably 7: 3.

The nanofibers may be prepared by drawing, template synthesis, phaseseparation, self assembly, electrospinning, or the like. In the present invention, . Electrospinning is a process by which nanofibers can be produced continuously and in large quantities, producing liquid nonwoven fabrics made of nanofibers by ejecting millimeter-diameter liquid jets through needles (capillary tubes or nozzles). The electrospinning can produce nanofibers having various diameters ranging from several nanometers to several thousands nanometers by controlling process conditions, and has a high volume-to-surface area ratio as well as a very high porosity of the prepared membrane. The specific process conditions of such electrospinning are not particularly limited in the present invention, and can be appropriately adopted by a person having ordinary skill in the art. The inner diameter of the needle is 0.3 to 0.7 mm and the voltage between the needle and the collector is 10 to 25 kV to perform electrospinning. Preferably, a needle of 18 to 24 gauge is used.

The step 3) is a step of preparing the nanoparticle-nanofiber composite, and the cationic nanoparticles recovered in the step 1) are introduced into the biodegradable nanofibers prepared in the step 2) and adsorbed.

The composite support comprising the biodegradable nanofibers produced by the above method and the cationic nanoparticles for drug delivery attached thereto is excellent in adhesion ability and cell activity of the cells and thus can be used for the treatment of muscular, skin, tendon, cartilage, ligament, bone , Organs, epidermis, blood vessels, and the like. The cell scaffolds prepared in this manner can adhere to muscles or tendons to promote cell growth while enduring mechanical stresses associated with muscle or tendon contraction.

As described above, the composite support comprising the biodegradable nanofibers of the present invention and the cationic nanoparticles for drug delivery adhered thereto has a high cell adhesion ability, low cytotoxicity, and gradually releases a pharmacological substance to be delivered Artificial skin and fillers.

Hereinafter, the present invention will be described more specifically based on the embodiments of the present invention. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.

Example 1. Preparation of nanoparticle-nanofiber composite support

1. Formation of chitosan nanoparticles

Chitosan was dissolved in 1.75% acetic acid solution to prepare a chitosan solution of 2 to 5 mg / ml. The chitosan solution was sonicated at an output of 40 W. TPP (tripolyphosphate) solution dissolved in distilled water at a concentration of 1 mg / ml was added at a rate of 2 ml / hr (when Fucoidan was included, And TPP of 0.66 mg / ml, respectively). Volume of chitosan solution: added until the volume of the TPP solution became 5: 1, then reacted for 1 hour to 1 hour 30 minutes and recovered by centrifugation at 3,000 xg. The recovered nanoparticles were re-dissolved in ethanol and recovered by lyophilization, which was confirmed by scanning electron microscopy (SEM). The results are shown in Fig.

As shown in Fig. 1, an SEM image of uniformly spherical nanoparticles having an average size of about 40 nm was confirmed.

2. Fabrication of nanofibers

Nanofibers were prepared by electrospinning and THF (tetrahydrofuran) and DMF (dimethylformamide) were mixed at a volume ratio of 7: 3. 15% (w / v) polycaprolactone (PCL) was dissolved in the mixed solvent and injected at a rate of 1 ml / hr through an 18G needle. During the injection, 15 cm and a voltage of 15 kV was applied to fabricate nanofibers.

3. Fabrication of nanoparticle-nanofiber composite support (complex electrospun scaffold)

The nanoparticles produced from the 10-ml chitosan solution were collected and re-dissolved in 10 ml ethanol and added to the 24-well nanofiber samples in 500 ul increments. After the nanoparticles were added, the 24-well plate was stored at 4 ° C overnight to be adsorbed, which was confirmed by SEM. The results are shown in Fig.

As shown in FIG. 2, the morphology of the nanofibers and the composite support in which the nanoparticles were attached to the nanofibers was confirmed by SEM image.

Experimental Example 1. Cytotoxicity of chitosan nanoparticles

To investigate the effects of chitosan nanoparticles on cells, cytotoxicity experiments were performed. The nanoparticles from 10 ml chitosan solution were collected by centrifugation and lyophilization, dispersed in 10 ml PBS, and the solution was added to the medium at 0, 100, 200, 300, ug / ml , And then the cells were adhered to a 96-well plate. After 1, 3, and 5 days of treatment with nanoparticles, cytotoxicity was confirmed by MTT assay. The results are shown in Fig.

As shown in FIG. 3, when the chitosan nanoparticles were added, no significant change in the MTT value was observed compared with the control group. This indicates that chitosan nanoparticles do not have a significant effect on cell activity and growth, confirming the possibility that the chitosan nanoparticle is a beneficial substance carrier that affects cells only by the effect of pure drug.

Experimental Example 2. Drug Release of Nanoparticles

To investigate the release of the substance carried on chitosan nanoparticles, fucoidan was loaded on chitosan nanoparticles and treated with fibroblasts at an inoculation concentration of ¹ × 10 ³ cells / well, and fucoidan-free The control and fucoidan were dissolved in the medium and compared with those treated once. To confirm this, MTT assays were performed at 1, 3, and 5 days after treatment with nanoparticles. The results are shown in Fig.

As shown in FIG. 4, in the case of fucoidan-carrying chitosan nanoparticles, it was confirmed that the control and fucoidan were dissolved in a medium and had an intermediate form of cell activity when they were treated at once. In addition, when the cell concentration and the purple formazan transformed by the cell with MTT were compared, the use of nanoparticles was similar to that of the control group, but it was confirmed that there were fewer formazan groups.

Experimental Example 3. Confirmation of penetration of nanoparticles

To confirm the degree of penetration of nanoparticles into cells, FITC labeled chitosan was prepared. That is, a 0.25% chitosan (CS) solution prepared by dissolving chitosan in 0.1 M acetic acid and a methanol solution dissolving an excess amount of FITC (fluorescein isothiocyanate) so as to have a concentration of 1.5 times the chitosan solution concentration were mixed in the same volume And chitosan was labeled with FITC. Thereafter, the reaction was allowed to proceed at room temperature for 1 hour to neutralize, followed by dialyzing for 48 hours to remove residual pigment and solvent, and recovered FITC-labeled chitosan nanoparticles through lyophilization. FITC-labeled nanoparticles of chitosan-coated fibroblasts were treated and then on day 5, the fluorescence of FITC was confirmed by fluorescence microscopy to identify the nanoparticles. The results are shown in Fig.

As shown in FIG. 5, in the fluorescence photograph, it was confirmed that the nanoparticles remained in the cell portion even after a number of washing processes, and it was confirmed that the nanoparticles enter the cell through the cytoplasm.

Experimental Example 4. Intracellular role of nanoparticle-nanofiber composite support

MTT assays were performed on PCL (polycaprolactone), PCL + CS (chitosan), and TCPS (tissue culture polystyrene) to confirm cell adhesion and cell activity in the composite support. MTT assays were performed at 2 hours and 24 hours by inoculating the cells on a composite support at a concentration of 3x10 ³ cells / well in order to confirm the adhesion ability. Cells were cultured at a concentration of 1 × 10 ³ cells / well , And the changes were confirmed by MTT assay on days 1, 3 and 5. The results are shown in Fig.

As shown in FIG. 6, the degree of adhesion of cells with or without chitosan nanoparticles was not large in relation to the adhesion ability, and it was confirmed that the degree of adhesion was higher than that of a general cell culture plate (TCPS). And a lower value in PCL + CS for cell activity.

In addition, the cells attached to the nanoparticle-nanofiber composite support were observed by SEM, and the results are shown in FIG.

As shown in FIG. 7, it was found that there was no significant difference in the case where the cells were adhered in the case where the nanoparticles were attached (FIG. 7A) and the case where the nanoparticles were not adhered (FIG. 7C). In addition, it was confirmed that the nanoparticles attached to the nanofibers 5 days after the culture spread into the medium and the amount remaining in the nanofibers was reduced (FIG. 7B).

Claims (11)

1) preparing a cationic polymer solution by dissolving chitosan or poly-L-lysine in an aqueous organic acid solution, adding tripolyphophate (TPP) to the cationic polymer solution together with ultrasonic waves, and stirring the cationic polymer solution to prepare cationic nanoparticles ;
2) Poly (caprolactone) (PCL), polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactide-co- glycolide) ), Diol / isocyanate aliphatic polyesters, polyester-amide / polyester-urethanes, poly (valerolactone), poly (hydroxybutyrate), poly (hydroxyvalerate), polyanhydride, polyortho One or more biodegradable polymers selected from the group consisting of polyorthoesters, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid and poly-N-isopropylacrylamide are dissolved in an organic solvent and electrospun Preparing biodegradable nanofibers; And
3) adding the cationic nanoparticles prepared in step 1) to the biodegradable nanofibers prepared in step 2) and adsorbing the biodegradable nanoparticles to prepare nanoparticle-nanofiber composite support; / RTI > delete The method according to claim 1, wherein in step 1), the organic acid aqueous solution is at least one selected from the group consisting of lactic acid, acetic acid, propionic acid, formic acid, ascorbic acid, and tartaric acid. . delete The method of claim 1, wherein the cationic polymer solution and tripolyphosphate are mixed in a volume ratio of 1 to 10: 1 in the step 1). [2] The composite support for artificial skin and filler according to claim 1, wherein the cationic nanoparticles produced in step 1) have a particle size of 0.1 to 800 nm. delete [4] The method of claim 1, wherein the organic solvent in step 2) is one or more selected from the group consisting of THF (tetrahydrofuran), DMF (dimethylformamide), DMSO, chloroform, dichloromethane, hexane, diethyl ether, and acetonitrile By weight based on the total weight of the composite support for artificial skin and filler. [Claim 9] The method according to claim 8, wherein the organic solvent is a mixed solvent of THF and DMF at a volume ratio of 1: 10: 1. delete A composite support for artificial skin and filler, comprising the biodegradable nanofiber and the cationic nanoparticles for drug delivery attached thereto, prepared by the method of claim 1.

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