KR100720056B1 - Preparation of nanospheres coated with growth factor and microspheres and its use as a neural differentiation - Google Patents

Abstract

The present invention relates to the regeneration of nerves by the growth factor attached to the microspheres or microbeads, more specifically, the cation is formed by electrostatic bonding of polyethyleneimine consisting of cations to the microspheres or microbeads with the carboxyl group out The nanoparticles consisting of heparin / polylysine containing fibroblast growth factor (bFGF), which is a growth factor in the outer polyethyleneimine, are coated by electrostatic reactions to induce differentiation of neurons. Thus, microspheres or microbeads for nerve tissue reconstruction can be used as an injectable microfabrication scaffold.

Microspheres, microbeads, growth factors, nanoparticles, neuronal differentiation, neuronal regeneration

Description

A method for producing microsphere coated with nanoparticle containing growth factor and the using method for neuronal differentiation hence}

1 is a diagram observed under a microscope (× 400) in microspheres containing growth factors and microspheres without growth factors in order to observe the differentiation of PC12 cells by the nerve cell culture method performed in Experimental Example 1-2. ,

FIG. 2 shows the expression of β-3 tubulin according to the concentration of growth factors in order to observe the differentiation of PC12 cells by the culturing method of neurons performed in Experimental Example 1-2 using a confocal laser microscope. Degrees,

Figure 3 shows the expression of β-3 tubulin of PC12 cells with time for stimulation of growth factors in order to observe the differentiation of PC12 cells by the method of culturing neurons carried out in Experimental Example 1-2. It is the degree observed with a focal point laser microscope,

4 is a diagram illustrating the differentiation of PC12 cells by scanning microscope in order to observe the differentiation of PC12 cells by the culturing method of neurons carried out in Experimental Example 1-2.

Recently, many studies have been conducted to improve the therapeutic effect and reduce systemic side effects of drugs by adjusting the concentration and duration of drugs at the site of action using biodegradable polymer matrix containing drugs. Spherical biodegradable particles have been of great interest because they have a large area useful for drug release, can be easily formulated into injections and do not need to be removed from the body after drug release. Biodegradable polymers are synthetic or natural polymers that are degraded by enzymes or other processes in vivo to produce biocompatible or nontoxic metabolites. Important classes of biodegradable polymers are lactic acid and glycolic acid. Preparation of microspheres controlled by drug release using polyester of glycocoic acid and poly (d, l-lactic-co-glycolide) and their in vitro release pattern Many studies have been conducted on (Matusmoto et al, J. Control.Rel. 18 , pp 172-180, 2005).

There are several methods used to make microspheres of biodegradable polymers, and the choice of suitable methods for the preparation of microspheres depends on the nature of the drug and the polymer and has a great influence on the morphology of the microspheres and the pattern of drug release from the microspheres. Can give Solvent evaporation and solvent extraction are commonly used methods for preparing microspheres of biodegradable polymers (Fu et al, J. Microencapsul., 22 , pp 57-66, 2005).

Research of microspheres made of biodegradable polymer materials for biotissue engineering has been widely conducted. Research on the formation of artificial biological tissues (eg cartilage) by injecting tissue cells onto microspheres has been reported to have partial success cases at home and abroad (Kang et al., Tissue Engineering, 11 , pp 438-447, 2005). . The treatment and rehabilitation has become a very important factor in modern medicine for various diseases that cause damage to body tissues. The recovery of damaged tissues is reported to be prolonged with age and low cure rate. Therefore, in the case of adults who lead the production activities of society, the contraction of social activities due to the long healing period is mentioned.

Recently, biotissue engineering research using stem cells has been actively conducted. In order to successfully reconstruct damaged tissue using biological tissue regeneration material, it is important to maintain the unique differentiation function of tissue cells. Most microsphere supports simply serve to provide a framework for inducing tissue formation in three-dimensional structures.

In addition, the trend of recent research is focused on the release of various bioactive substances that induce and promote differentiation of tissue cells from the microspheres in a sustained release form or by immobilization on the surface of the polymer to achieve surface adhesion and differentiation of tissue cells. ought. (Hong et al., Biomaterials, 32 , pp.6305-6313, 2005)

Therefore, the present inventors found that the adhesion of neurons to the surface of the microspheres of the bioactive substance can be smoothly adhered to the cells, and induction of the differentiation and neurites of the neurons on the surface of the microspheres in the presence of the culture medium may induce nerve regeneration. The present invention was completed.

An object of the present invention is to provide a neural tissue reconstruction through the induction of differentiation of neurons using microspheres or microbeads attached to the surface of the polymer electrolyte complex containing a bioactive material.

The present invention provides microspheres or microbeads for inducing neuronal differentiation and neuronal regeneration by attaching nanoparticles composed of a polymer electrolyte complex containing growth factors to the surface.

 Growth factors as defined herein include fibroblast growth factor (bFGF). Also included is a microsphere or microbead characterized in that the concentration of the growth factor is 0.1 ~ 10 ㎍ / ㎖.

Polyelectrolyte complexes as defined herein include microspheres or microbeads that are heparin / poly L-lysine.

Microspheres or microbeads as defined herein may further comprise dexamethasone or dehydroepiandrosterone (DHEA).

In addition, the present invention provides a pharmaceutical composition for inducing neuronal differentiation and neuronal regeneration comprising microspheres or microbeads and pharmaceutically acceptable excipients attached to the surface nanoparticles consisting of a polymer electrolyte complex containing growth factors to provide.

Pharmaceutical compositions as defined herein include those in the form of injectables.

In another aspect, the present invention provides a medical material for inducing neuronal differentiation and neuronal regeneration including microspheres or microbeads attached to the surface of the nanoparticles consisting of a polymer electrolyte complex containing a growth factor.

The medical material includes a neural conduit, a catheter, and an antiadhesion membrane.

Hereinafter, a method of obtaining microspheres or microbeads will be described in detail.

The biodegradable polymer is dissolved in 5-20 times, preferably 10 times, organic solvent, and the solution is 50 to 300 ml of polyvinyl alchol (Mw 13,000 to 23,000) 3% aqueous solution, preferably Dropwise to 200 ml and emulsified at 500 to 20000 rpm, preferably 1,000 rpm for at least 30 seconds, preferably 5 minutes using a biomixer. It was then stirred using a mechanical stirrer at constant speed for 30 minutes to 24 hours, preferably 3 hours at room temperature for solvent evaporation. The suspension is centrifuged at 500 to 2000 rpm, preferably at 1,000 rpm for 30 seconds to 30 minutes, preferably 5 minutes and 10 to 200 ml of distilled water, preferably 100 ml to remove the remaining surfactant. After washing three times or more, preferably three times, and freeze-drying, it is possible to obtain a microsphere having a carboxyl group.

The organic solvent is one or more selected from the group consisting of acetic acid, lactic acid, formic acid, acetone, acetonitrile, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, dioxane, N-methylpyrrolidone and aqueous solutions containing them It features.

In addition, PLGA (dispersion phase) dissolved in various concentrations in dichloromethane (DCM) was put into a syringe, and then the solution was discharged through a flat needle 27G using gravity to release polyvinyl chloride at various concentrations. In a distilled water (continuous phase) in which alcohol (PVA) is dissolved, a drop having a diameter of about 0.5 to 1 mm is made, pulverized by mechanical stirring using a magnetic bar, and then pulverized. The microbeads can be obtained by leaving DCM for 6 hours to remove DCM and lyophilize.

The pharmaceutical composition comprising the microspheres or microbeads of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the microspheres or microbeads of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

 It can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like oral formulations, external preparations, suppositories, and sterile injectable solutions. Carriers, excipients and diluents that may be included in the composition comprising wood vinegar include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention comprises the first step of combining the growth factor of each concentration with heparin; A second step of dropping polylysine while stirring after heparin binding; Provided is a method of coating a nanosphere containing microparticles or growth particles containing a growth factor comprising three steps of binding the resulting nanoparticles to a microsphere or microbead surface coated with polyethyleneimine.

The present invention also provides a method for culturing neurons inducing neuronal differentiation in the presence of a culture medium after adhering stem cells to microspheres or microbeads attached to the surface of the polymer electrolyte complex containing a growth factor.

 Combining the growth factors of each concentration with heparin; A second step of dropping polylysine while stirring after heparin binding; Binding the resulting nanoparticles to a microsphere or microbead surface coated with polyethyleneimine; Spreading microspheres or microbeads containing growth factors on plates and adhering nerve cells; After stabilizing the adhered cells for 2-3 days, the method comprises culturing under culture.

The conditions of the culture include the addition of 10% horse serum and 5% fetal calf serum to the RPMI1640 culture medium for 3-7 days.

The nerve cell culture is preferably carried out up to 10 days containing the fibroblast growth factor in the concentration of 0.1 ~ 10 ㎍ / ㎖, and the fibroblast growth factor (bFGF) less than 0.01 ㎍ / ㎖ nanoparticles When contained in, the stimulation of growth factors is weak and does not affect the culture of neurons, 10 ㎍ / ㎖ or more than 10 ㎍ / ㎖ compared to the concentration of the stimulation received by the cell is an undesirable concentration .

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention.

Example  1. Having a carboxyl group Microspheres  Produce

First, 500 mg of a biodegradable polymer is dissolved in 5 ml of an organic solvent, and this solution is added dropwise to 200 ml of a 3% aqueous solution of polyvinyl alchol (Mw 13,000 to 23,000) and a biomixer is used. Emulsification for 5 minutes at 1,000 rpm. It was then stirred using a mechanical stirrer at constant speed for 3 hours at room temperature for solvent evaporation. The suspension was centrifuged at 1,000 rpm for 5 minutes and washed with 100 ml of distilled water three times to remove the remaining surfactant, followed by freeze drying to prepare a microsphere having a carboxyl group.

Example  2. Microbead  Produce

First, the PLGA (dispersion phase) dissolved in various concentrations in dichloromethane (DCM) was put into a syringe, and then polyvinyl alcohol of various concentrations was released by using gravity to discharge the solution through a flat needle (27G). (PVA) is dissolved in distilled water (continuous phase) dissolved in a diameter of approximately 0.5 ~ 1 mm, and then pulverized by mechanical stirring using a magnetic bar, and then at room temperature Leave for 6 hours to remove DCM and lyophilize to obtain microbeads.

Example  3. Nanoparticle coating containing fibroblast growth factor

The fibroblast growth factor at each concentration (0.1, 1, 10 μg / ml) was combined with heparin and the polylysine was dropped while stirring. The nanoparticles thus produced are combined with microspheres (1 g) coated with polyethyleneimine.

Experimental Example  1. Surface observation and cell culture

1-1. Scanning Microscope

In order to examine the particle shape and surface structure, the prepared microspheres were observed with a scanning electron microscope. The sample was placed in a gold evaporator and the surface of the particle was coated under vacuum conditions of 0.15 torr and 6-7 mA to prepare a sample for imaging. The sample was then fixed in the scanning microscope body and the particles were observed at an appropriate rate at 15 kv.

1-2. culture

PC12 cell lines were cultured in collagen coated tissue culture dishes under RPMI1640 culture under 10% horse serum and 5% fetal calf serum.

Cultured PC12 cells are cultured by dispensing the microspheres (10 mg) containing the growth factor of Example 3 at a density of 2 × 10 ⁵ cells (cells). After incubation, observe the neurites of PC12 cells using each microsphere.

Experimental Example  2. contained at a concentration of 5 μg / ml In the microsphere  Differentiation of PC12 Cells by Stimulation of Growth Factors

In order to observe the differentiation of PC12 cells by the neuronal cell culture method of the above example, microspheres containing growth factors and microspheres without growth factors were observed under a microscope (× 400).

As shown in Figure 1, fibroblast growth factor (bFGF) can be seen that the brightness increases with increasing concentration. At the highest concentration (D), the fluorescence intensity of the secondary antibody combined with the monoclonal antibody was the brightest, and in the control (A) without the fibroblast growth factor (bFGF), Fluorescence could not be observed. Thus, it was demonstrated that heparin / polylysine nanoparticles contained fibroblast growth factor (bFGF).

Experimental Example  3. β-3 Tubulin  Observation of the used neurites

In order to observe the differentiation of PC12 cells by the culturing method of neurons carried out in Experimental Example 1-2, the expression of β-3 tubulin according to the growth factor concentration was observed by a confocal laser microscope. PC12 cells adhered to microspheres without growth factor were used as controls.

As shown in Figure 2, fibroblast growth factor (bFGF) it can be seen that the effect on the differentiation of cells with increasing concentration. In microspheres containing 0.1 μg / ml fibroblast growth factor (bFGF), cell differentiation was hardly observed, and when the cells were increased to 1 μg / ml, cell differentiation increased. In addition, the fibroblast growth factor (bFGF) increased to 5 ㎍ / ㎖ can be seen that the size of the neurites of the cell increases. As a result, it can be seen that external stimulation is essential for the differentiation of neurons, and that the external stimulus does not induce differentiation of cells unless the external stimulus becomes a certain degree. However, even when the fibroblast growth factor (bFGF) is 50 ㎍ / ml or more compared with 5 ㎍ / ml was observed that no significant difference.

Experimental Example  4. Stimulation of growth factors over time PC12  Differentiation of cells

4-1. Collection Point Laser Microscope  observe

In order to observe the differentiation of PC12 cells by the culturing method of neurons carried out in Experimental Example 1-2, the expression of β-3 tubulin of PC12 cells over time with respect to growth factor stimulation It was observed under a microscope.

As shown in Figure 3, the neurites of the neurons in the microspheres contained at a concentration of 5 ㎍ / ml was observed after one day, it can be said that the size is insufficient. After three days, longer neurites were observed in PC12 cells than in one day, and the longer neurites were observed in PC12 cells observed after five days. After 7 days, the longest neurites were observed among the observed neurites. As a result, in the microspheres coated with fibroblast growth factor (bFGF), PC12 cells did not show an immediate response, but the cells responded to the growth factor over time.

4-2. Scanning Microscopy

In order to observe the differentiation of PC12 cells by the culturing method of neurons carried out in Experimental Example 1-2, the differentiation of PC12 cells was observed by scanning microscope. PC12 cells adhered to microspheres containing no growth factor were used as comparative examples.

As shown in Figure 4, it was found that the neurites of the neurons in the microspheres contained at a concentration of 5 ㎍ / ㎖ extend longer than the control.

Formulation example  1. Preparation of Injection

Microsphere of Example 1 10 mg

Sodium Metabisulfite ........................ 3.0mg

Methylparaben ............... 0.8 mg

Propylparaben ....................... 0.1mg

Sterile Distilled Water for Injection ...

The above ingredients are mixed and made into 2 ml by a conventional method, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation example  2. Pilled  Produce

Microbead of Example 2 ...... 120 mg

Corn starch ... 100 mg

Sterilized Distilled Water ...............

The above components are mixed and refilled in a 0.3 cm diameter by a conventional pill manufacturing method to prepare pills.

Formulation example  3. Preparation of Tablets

Microsphere of Example 1 ... 200 mg

Lactose ... 100 mg

Starch ..................... 100 mg

Magnesium Stearate ...............

A tablet is prepared by mixing and tableting the above components according to a conventional tablet manufacturing method.

Formulation example  4. Preparation of Capsules

Microbead of Example 2 ... 100 mg

Lactose ... .50 mg

Starch ..................... .50 mg

Talc .................. ..2 mg

Magnesium stearate .....................................

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation example  5. Liquid  Produce

Microspheres of Example 1 ............. 1000 mg

Sugar................................................. ... 20 g

Isomerized sugar ... 20 g

Lemon flavor Quantity

Purified water was added to make a total of 1000 ml. According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Although the composition ratio is mixed with a relatively suitable component in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and use purpose.

As described above, the present invention by coating the nanoparticles consisting of a polymer electrolyte complex containing a growth factor by an electrostatic reaction, induce the differentiation of neurons, microspheres or microbeads for nerve tissue reconstruction It can be used as a microfabrication scaffold.

Claims (9)

Induction of neuronal differentiation and neuronal regeneration by attaching nanoparticles composed of heparin / polylysine (bFGF) containing 0.1 to 10 μg / ml of fibroblast growth factor (bFGF) to the surface Microspheres or microbeads. delete delete The microsphere or microbead according to claim 1, further comprising dexamethasone or dehydroepiandro sterone (DHEA). delete Microspheres or microbeads attached to the surface of nanoparticles composed of heparin / poly L-lysine containing fibroblast growth factor (bFGF) at a concentration of 0.1 to 10 μg / ml and pharmaceutical Injectable pharmaceutical composition for the prevention and treatment of spinal cord injury or Parkinson's disease for spinal cord or brain tissue neuronal differentiation and neuronal regeneration comprising an acceptable excipient. delete Nerves containing microspheres or microbeads having nanoparticles composed of heparin / poly L-lysine containing fibroblast growth factor (bFGF) at a concentration of 0.1 to 10 μg / ml attached to the surface Neural conduits, catheters or anti-adhesion membranes for cell differentiation and nerve regeneration. delete

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