KR20200131054A - Injectable nanofiber-hydrogel composite particles and method for manufacturing the same - Google Patents

Abstract

An embodiment of the present invention provides: a complex particle comprising a photo-crosslinked nanofiber substrate; having a three-dimensional network structure, and a hydrogel coated and photo-crosslinked on at least a part of the surface of the substrate; and a manufacturing method thereof. The complex particle can be used for overall tissue engineering such as scaffolds and biosensors.

Description

Injectable nanofiber-hydrogel composite particle and its manufacturing method {INJECTABLE NANOFIBER-HYDROGEL COMPOSITE PARTICLES AND METHOD FOR MANUFACTURING THE SAME}

The present invention relates to composite particles, and more particularly, to composite particles comprising nanofibers and hydrogels and capable of being bioinjectable through injection.

Recently, in the field of tissue engineering, in order to regenerate damaged tissues of humans or animals, research has been actively conducted in which cells are cultured on a scaffold that mimics a living body and applied to the body. Polymers mainly used in these studies include polyethylene glycol (PEG), polycaprolactone (PCL), polyvinyl alcohol (PVA), and polyN-isopropylacrylamide, which are highly biocompatible. (N-isopropylacrylamide), PNIPAM) and the like. Typically, such a polymer was prepared in the form of a hydrogel or nanofiber substrate and used as a scaffold.

Hydrogel scaffolds can be manufactured simply and contain cells and growth factors in the gel, so that cell culture is easy, and they can be injected into the body through a syringe. However, although it is easy to manufacture a scaffold having a size of hundreds to thousands of micrometers, there is a limit to manufacturing a small scaffold of 500 μm or less due to entanglement between particles. In addition, there is a disadvantage that the surface area is small and it is difficult to control the cells cultured therein.

The nanofiber substrate scaffold has a large surface area and is composed of a porous structure, so it is easy to supply nutrients and oxygen during cell cultivation.However, since it is larger than a hydrogel scaffold, bioinjection through a syringe is impossible, so surgical therapy is required. As a result, there is a disadvantage in that side effects such as bacterial infection exist.

In order to overcome these limitations, research has been conducted to manufacture and apply a composite scaffold of nanofibers and hydrogels, but due to the problem of dissolving the nanofibers in the organic solvent used during manufacture, the hydrogel wraps the entire nanofibers. Only scaffolds of Such a scaffold is difficult to realize the advantage due to the surface area of the nanofibers.

Therefore, it can be inserted in a desired position in the body in a short period by a simple method such as injection through a syringe rather than surgery, and has a large surface area and a porous structure, so that nutrients and oxygen are supplied smoothly, and entanglement between particles can be suppressed. There is a need to develop a scaffold.

The present invention is to solve the problems of the prior art described above, and an object of the present invention is to provide a composite particle capable of simultaneously realizing the advantages of nanofibers and a hydrogel and capable of injecting and a method of manufacturing the same.

One aspect of the present invention, having a three-dimensional network structure, photo-crosslinked nanofiber substrate; And a hydrogel coated and photo-crosslinked on at least a part of the surface of the substrate.

In one embodiment, the nanofibers are chitosan, elastin, hyaluronic acid, alginate, gelatin, collagen, cellulose, polyethylene glycol, polyethylene oxide, polycaprolactone, polylactic acid, polyglycolic acid, poly[(lactic-co-( Glycolic acid))], poly[(L-lactide)-co-(caprolactone)], poly(ester urethane), poly[(L-lactide)-co-(D-lactide)], poly[ Ethylene-co-(vinyl alcohol)], polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, polystyrene, polyaniline, and a water-soluble polymer selected from the group consisting of two or more combinations thereof I can.

In one embodiment, the hydrogel may be formed in a ring shape on the outside of the composite particle, and the nanofiber substrate may be exposed on the inside of the ring shape.

In one embodiment, the hydrogel is polyethylene glycol, polyethylene oxide, polyhydroxyethyl methacrylate, poly(N-isopropylacrylamide), polycaprolactone, polylactic acid, polyglycolic acid, poly[(lactic- co-(glycolic acid))], polydioxanone, poly[(L-lactide)-co-(caprolactone)], poly(ester urethane), poly[(L-lactide)-co-(D- Lactide)], poly[ethylene-co-(vinyl alcohol)], polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, gelatin, alginate, carrageenan, chitosan, hydroxyalkyl cellulose, alkyl cellulose, silicone, rubber, It may be prepared from polyacrylic acetate and a photo-crosslinkable portion introduced into a hydrophilic polymer selected from the group consisting of a combination of two or more of them.

In one embodiment, the hydrogel may include a magnetic material, a light-emitting material, or a combination thereof.

In one embodiment, the composite particles have an average particle size of 500 μm or less, and the composite particles may be injectable.

In one embodiment, the composite particles may contain drugs, biomaterials, or cells.

Another aspect of the present invention, (a) preparing a nanofiber substrate by electrospinning a water-soluble polymer comprising a photocrosslinkable portion; (b) selectively photocrosslinking the substrate; (c) selectively photocrosslinking by applying a hydrogel precursor on the substrate; And (d) washing the substrate with distilled water to obtain the composite particles; containing, provides a method for producing composite particles.

In one embodiment, the selective light crosslinking may be performed by a photolithography method.

In one embodiment, the manufacturing method may not use an organic solvent.

In one embodiment, the hydrogel precursor may include a magnetic material, a light emitting material, or a combination thereof.

According to an aspect of the present invention, it is possible to simultaneously realize the advantages of nanofibers and hydrogels and to prepare injectable composite particles, and the composite particles can be used for overall tissue engineering such as scaffolds and biosensors.

According to another aspect of the present invention, it is possible to simply manufacture miniaturized composite particles of various shapes and sizes.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a diagram showing the properties of composite particles prepared according to an embodiment of the present invention and the composite particles capable of being injected into the body.
2 is a schematic diagram of a method of manufacturing a composite particle according to an embodiment of the present invention.
3 is an image photographed using an optical microscope and a scanning electron microscope of a composite particle manufactured according to an embodiment of the present invention.
4 is an image photographed using a fluorescence microscope after culturing cells in a composite particle prepared according to an embodiment of the present invention and then staining the cell nucleus.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be implemented in various different forms, and therefore is not limited to the embodiments described herein. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present invention, and similar reference numerals are assigned to similar parts throughout the specification.

Throughout the specification, when a part is said to be "connected" with another part, this includes not only "directly connected" but also "indirectly connected" with another member interposed therebetween. . In addition, when a part "includes" a certain component, it means that other components may be further provided, rather than excluding other components unless specifically stated to the contrary.

When a range of numerical values is described herein, the value has the precision of significant figures provided according to the standard rules in chemistry for significant figures, unless a specific range thereof is stated otherwise. For example, 10 includes a range of 5.0 to 14.9, and the number 10.0 includes a range of 9.50 to 10.49.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Composite particles

Composite particles according to an aspect of the present invention, having a three-dimensional network structure, photo-crosslinked nanofiber substrate; And a hydrogel coated and photo-crosslinked on at least a portion of the surface of the substrate. The composite particles can be used for tissue regeneration, wound treatment, or biosensor.

The nanofiber substrate may be a porous scaffold having a plurality of pores by forming a three-dimensional network structure of an aggregate of ultrafine fibers having a diameter of several tens of nanometers to several microns. According to the use of the composite particles, the pore structure of the scaffold may be controlled by adjusting the diameter and density of the fibers. For example, by controlling the pore size to be 20 to 100 μm and culturing cells with a size of 10 to 15 μm, it is possible to create an environment that facilitates the fixation, growth and differentiation of cells inside the scaffold.

Specifically, properties such as diameter and density of the nanofibers can be adjusted by changing conditions such as concentration of solution, flow rate, voltage, type and spinning distance of a solution during electrospinning. For example, high-density fibers can be manufactured using an integrated plate having high conductivity, and low-density fibers can be manufactured using an integrated plate having low conductivity. Conditions for such electrospinning can be appropriately adjusted by borrowing a known method according to the use of the composite particles.

The material constituting the nanofiber may be a water-soluble material soluble in water, for example, chitosan, elastin, hyaluronic acid, alginate, gelatin, collagen, cellulose, polyethylene glycol, polyethylene oxide, polycaprolactone, polylactic acid, Polyglycolic acid, poly[(lactic-co-(glycolic acid))], poly[(L-lactide)-co-(caprolactone)], poly(ester urethane), poly[(L-lactide)- water-soluble selected from the group consisting of co-(D-lactide)], poly[ethylene-co-(vinyl alcohol)], polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, polystyrene, polyaniline, and combinations of two or more thereof A photocrosslinkable portion such as a photocrosslinkable functional group may be introduced into the polymer. The water-soluble material into which the photocrosslinkable portion is introduced is easily soluble in water, but may not be dissolved in water when crosslinked by irradiation with ultraviolet (UV) light. As an example of the photocrosslinkable portion, a known photocrosslinkable functional group such as N-methyl-4(4-formylstyryl)pyridinium and diacrylate may be introduced.

In addition, the nanofiber substrate may be post-treated so that drugs, biomaterials, cells, etc. can be easily fixed. For example, oxygen plasma treatment may improve hydrophilicity, or a material capable of chemical bonding with a biomaterial may be bonded to the surface using a self-assembly monolayer (SAM) method.

The composite particle may be formed by photocrosslinking a hydrogel on at least a portion of the surface of the nanofiber substrate. The hydrogel may be formed by irradiating light to a polymer including a photo-crosslinkable portion, or by adding a photo initiator to a hydrogel precursor and then irradiating light.

For example, the hydrogel may be formed in a ring shape on the outside of the composite particle, and the nanofiber substrate may be exposed on the inside of the ring shape. Referring to FIG. 1, which is an image of an example of the composite particle, the composite particle may be manufactured in various shapes such as a circle, a triangle, and a square. Accordingly, the hydrogel is also It can have a shape. The composite particle has a larger surface area compared to the conventional hydrogel scaffold by exposing at least a portion of the nanofiber substrate to the surface, and at the same time, the outer hydrogel can improve the mechanical properties of the composite particle.

The scaffold formed of a nanofiber substrate has a porous structure having a large surface area compared to a hydrogel scaffold, so that nutrients and oxygen are easily supplied, and thus control is advantageous during cell culture. On the other hand, the hydrogel scaffold is simple to manufacture, can contain cells, growth factors, etc., and can be injected into the body through a syringe.

Conventional composite scaffolds of nanofibers and hydrogels for realizing these advantages at the same time have a problem in that the entire nanofibers are formed inside the hydrogel or it is difficult to control the size of the scaffold, so that bioinjection through injection is impossible. On the other hand, the composite particle according to an aspect of the present invention can be miniaturized through selective photocrosslinking of a water-soluble polymer, which will be described later, thereby implementing the advantages of each of the nanofiber scaffold and the hydrogel scaffold.

The hydrogel is polyethylene glycol, polyethylene oxide, polyhydroxyethyl methacrylate, poly(N-isopropylacrylamide), polycaprolactone, polylactic acid, polyglycolic acid, poly[(lactic-co-(glycolic acid) )], polydioxanone, poly[(L-lactide)-co-(caprolactone)], poly(ester urethane), poly[(L-lactide)-co-(D-lactide)], poly [Ethylene-co-(vinyl alcohol)], polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, gelatin, alginate, carrageenan, chitosan, hydroxyalkylcellulose, alkyl cellulose, silicone, rubber, polyacrylic acetate, and among them It may be prepared from the introduction of a photocrosslinkable portion to a hydrophilic polymer selected from the group consisting of two or more combinations, but is not limited thereto.

The hydrogel may include a magnetic material, a light emitting material, or a combination thereof. The magnetic material is, for example, Pt, Pd, Ag, Cu, Au, Co, Mn, Fe, Ni, Gd, Mo, CoCu, CoPt, FePt, CoSm, NiFe, NiFeCo, MM' ₂ O ₄ , M _x O _y and may be one selected from the group consisting of a combination of two or more of them, M and M'each independently represent Co, Fe, Ni, Mn, Zn, Gd or Cr, 0<x≤3, 0 <y≤5. If the hydrogel contains a magnetic material, entanglement between the composite particles can be prevented, so that it can be easily injected into the body.

The light-emitting material may be, for example, one selected from the group consisting of quantum dots, inorganic phosphors, organic phosphors, organic pigments, and combinations of two or more of them. The light-emitting material may impart color to the composite particles or impart a function of generating a fluorescent signal.

The light-emitting material may function as a sensor for detecting a target material. For example, if the composite particle contains an enzyme that reacts with glucose at the same time as the light-emitting material, glucose in saliva and the enzyme react to produce hydrogen peroxide, and since hydrogen peroxide decreases the fluorescence intensity of the light-emitting material, the light-emitting material Glucose concentration can be checked by measuring the degree of quenching.

When the hydrogel includes the magnetic material and the light-emitting material at the same time, a fluorescence signal is amplified, thereby improving the accuracy of the sensor when the above-described target material is detected.

The composite particles may have an average particle size of 1 μm or more, 10 μm or more, 50 μm or more, 100 μm or more, 150 μm or more, 200 μm or more, or 250 μm or more, and 500 μm or less, 450 μm or less, 400 μm or less, 350 It may be less than or equal to μm or less than or equal to 300 μm, but is not limited thereto.

The composite particles may contain drugs, biomaterials, or cells depending on the use. For example, the composite particles are drugs, biomarkers, growth factors, amino acids, proteins, lipids, carbohydrates, sugars, nucleic acids, enzymes, inorganic substances, hormones, biological substances such as antigens, fibroblasts, vascular endothelial cells, smooth muscle cells, It can be used as a biomaterial carrier, cell culture scaffold, and biosensor, including various cells such as nerve cells, cartilage cells, bone cells, skin cells, Schwann cells, and stem cells.

Method for producing composite particles

A method for producing a composite particle according to another aspect of the present invention includes the steps of: (a) preparing a nanofiber substrate by electrospinning a water-soluble polymer including a photocrosslinkable portion; (b) selectively photocrosslinking the substrate; (c) selectively photocrosslinking by applying a hydrogel precursor on the substrate; And (d) washing the substrate with distilled water to obtain composite particles.

The water-soluble polymer, nanofiber substrate, hydrogel, or composite particles are the same as described above.

Nanofiber substrates made from water-soluble polymers can be easily removed by dissolving in water or ethanol, but since the selective photocrosslinked portion is not dissolved in such a solvent, miniaturized particles having a desired shape can be easily prepared through the above manufacturing method. have.

The selective photocrosslinking may be performed by photolithography. More specifically, in the selective photocrosslinking of step (b), a photomask manufactured in a desired shape and size using an Auto CAD or the like is placed on the nanofiber substrate, and ultraviolet light irradiation for 1 to 100 seconds Thus, the nanofibers can be selectively photocrosslinked in the form of particles. The particles may have various shapes such as round, oval, triangle, and square, and the size of the particles is an injectable size, for example, an average particle size of 1 μm or more, 10 μm or more, 50 μm or more, 100 μm or more, It may be 150 μm or more, 200 μm or more, or 250 μm or more, and may be 500 μm or less, 450 μm or less, 400 μm or less, 350 μm or less, or 300 μm or less, but is not limited thereto. The selective photocrosslinking of step (c) may be performed so that a hydrogel can be formed on the outside of the nanofiber substrate, such as a circular ring, an elliptical ring, a triangular ring, and a square ring using an autocad or the like.

The manufacturing method may not use an organic solvent. More specifically, in the manufacturing method, only distilled water may be used for the electrospinning solution used in the electrospinning of step (a), the solvent of the hydrogel precursor of step (c) and the washing of step (d). When an organic solvent harmful to a living body is used in the electrospinning solution or the hydrogel precursor, the composite particles cannot be introduced into the body, and when an organic solvent is used for washing in step (d), the nanofibers exposed to the outside of the composite particles The substrate can be dissolved and removed.

Conventional hydrogel and nanofiber composite scaffold is a problem in that the entire nanofiber is located inside the hydrogel due to the problem that the nanofiber is dissolved in the organic solvent, making it difficult to control the loading of drugs, biomaterials or cells, and the supply of nutrients and oxygen. However, according to the above manufacturing method, only distilled water is used to produce a composite particle having less harmfulness and a part of the nanofibers exposed to the outside.

The hydrogel precursor may include a magnetic material, a light emitting material, or a combination thereof. The magnetic material or the light emitting material is the same as described above.

Hereinafter, embodiments of the present invention will be described in more detail. However, the following experimental results are only representative of the above examples, and cannot be interpreted as the scope and content of the present invention are reduced or limited by examples. Effects of each of the various embodiments of the present invention not explicitly presented below will be specifically described in the corresponding section.

2 is a schematic view showing a method of manufacturing a nanofiber scaffold according to an embodiment of the present invention.

Example

Poly(vinyl alcohol) N-methyl-4 (4-formyl styryl) pyridinium (poly(vinylalcohol) N-), a polyvinyl alcohol-based polymer having a formula weight value of about 45,000 and a photocrosslinkable functional group A 13.3% aqueous solution of methyl-4(4-formylstyryl)pyridinium) was prepared. The aqueous solution was electrospun for 10 minutes under conditions of a voltage of 17 kV and a flow rate of 0.25 ml/h at a height of 107 mm from a 1.8 mm×1.8 mm accumulator to prepare a nanofiber substrate.

In order to partially crosslink the nanofiber substrate, a photomask made of a polyester film material previously prepared by Auto CAD is placed on the nanofiber substrate, and the desired shape and size are irradiated with ultraviolet light for 30 seconds. Particles of 440 μm size were prepared.

Polyethyleneglycol-diacrylate (PEG-DA, M _w 575) monomer to 2-hydroxy-2-methylpropiophenone (HOMPP) photoinitiator to 2% by volume Mixed. A solution obtained by mixing iron oxide powder in distilled water to a concentration of 20 mg/ml was mixed with 10% by volume of the monomer/photoinitiator mixture solution to prepare a hydrogel precursor solution.

After 200 μl of a hydrogel precursor solution was applied on the particles, a photomask prepared in a 125 μm-thick ring shape was placed and irradiated with ultraviolet light for 1.5 seconds. Thereafter, the nanofiber substrate was washed with distilled water to obtain a scaffold having the same size and shape. An image of the completed scaffold was taken with an optical microscope and a scanning electron microscope (SEM), and is shown in FIG. 3.

After culturing the cells on the scaffold, the cell nucleus was stained with 4',6-diamidino-2-phenylindole (DAPI) and photographed by fluorescence microscopy. Thus, the image is shown in FIG. 4. Referring to FIG. 4, it was confirmed that cells selectively survived only on nanofibers, not on polyethylene glycol rings on the scaffold.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and the concept of equivalents thereof should be construed as being included in the scope of the present invention.

Claims (11)

Photo-crosslinked nanofiber substrate having a three-dimensional network structure; And
Containing, composite particles; a hydrogel coated and photocrosslinked on at least a portion of the surface of the substrate. The method of claim 1,
The nanofibers are chitosan, elastin, hyaluronic acid, alginate, gelatin, collagen, cellulose, polyethylene glycol, polyethylene oxide, polycaprolactone, polylactic acid, polyglycolic acid, poly[(lactic-co-(glycolic acid))], Poly[(L-lactide)-co-(caprolactone)], poly(ester urethane), poly[(L-lactide)-co-(D-lactide)], poly[ethylene-co-(vinyl) Alcohol)], polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, polystyrene, polyaniline, and a combination of two or more of them in a water-soluble polymer selected from the group consisting of a photo-crosslinkable moiety is introduced, composite particles. The method of claim 1,
The hydrogel is formed in a ring shape on the outside of the composite particle,
The nanofiber substrate is exposed on the inside of the annular shape, composite particles. The method of claim 1,
The hydrogel is polyethylene glycol, polyethylene oxide, polyhydroxyethyl methacrylate, poly(N-isopropylacrylamide), polycaprolactone, polylactic acid, polyglycolic acid, poly[(lactic-co-(glycolic acid) )], polydioxanone, poly[(L-lactide)-co-(caprolactone)], poly(ester urethane), poly[(L-lactide)-co-(D-lactide)], poly [Ethylene-co-(vinyl alcohol)], polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, gelatin, alginate, carrageenan, chitosan, hydroxyalkyl cellulose, alkyl cellulose, silicone, rubber, polyacrylic acetate, and among them Composite particles prepared from the introduction of a photocrosslinkable portion to a hydrophilic polymer selected from the group consisting of a combination of two or more. The method of claim 1,
The hydrogel comprises a magnetic material, a light emitting material, or a combination thereof, composite particles. The method of claim 1,
The composite particles have an average particle size of 500 μm or less,
The composite particles are injectable, composite particles. The method of claim 1,
The composite particle contains a drug, a biomaterial, or a cell. (a) preparing a nanofiber substrate by electrospinning a water-soluble polymer including a photocrosslinkable portion;
(b) selectively photocrosslinking the substrate;
(c) selectively photocrosslinking by applying a hydrogel precursor on the substrate; And
(d) washing the substrate with distilled water to obtain composite particles; including, a method for producing composite particles. The method of claim 8,
The selective photocrosslinking is performed by a photolithography method. The method of claim 8,
The manufacturing method does not use an organic solvent, a method of manufacturing composite particles. The method of claim 8,
The hydrogel precursor includes a magnetic material, a light-emitting material, or a combination thereof, a method for producing a composite particle.

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