KR20100037443A - Manufacturing method for spherical and elastic chitosan microsphere having regular size - Google Patents

Abstract

PURPOSE: A method for preparing chitosan microsphere is provided to produce chitosan microsphere having improved elasticity and flexibility. CONSTITUTION: A method or preparing chitosan microsphere comprises: a step of making chitosan solution; a step of dropping chitosan solution to upper layer of organic solvent and lower layer of alkali solution or cationic solution; and a step of cross-linking the microsphere. The organic solvent which is not soluble with water is petroleum ether or ethel ether. Dropping is performed using nitrogen gas. The cross-linking is performed by reacting with genipin.

Description

Manufacturing method for spherical and elastic chitosan microsphere having regular size

The present invention relates to a method for producing chitosan microspheres which are elastic, spherical and of constant size.

The invention also relates to a method of controlling the elasticity and swelling properties of chitosan microspheres.

Microspheres of biocompatible or biodegradable polymers are not only used for embolization for the treatment of tumors by controlling bleeding or blocking oxygen / nutrient supply vessels around tumors, but also mainly researched as carriers of drugs, genes, etc. have. The microspheres are preferably uniform in size in order to maintain a constant effect in embolization and use of the drug as a carrier, and are preferably spherical in shape for enveloping drugs and preventing vascular irritation. . In addition, it is preferable that the polymer microspheres are not broken by an external impact, and the shape of the polymer microspheres is preferably elastic so that a small passage can be easily passed.

Chitosan is a linear polysaccharide consisting of β- (1-4) -linked D -glucosamine (deacetylation unit) and N -acetyl- D -glucosamine (acetylation unit). Such chitosan is abundant, has excellent biocompatibility, biodegradability, and low toxicity, so it is widely used in the medical and pharmaceutical fields.

Attempts have been made to produce medical or pharmaceutical films using such biodegradable polymers, chitosan, but films made of pure chitosan have weaknesses due to their poor tensile strength and elasticity (S. Wittaya-areekul, C.). Prahsarn, Int. J. Pharm. 313 (2006) 123). In addition, attempts have been made to produce spherical microspheres having a constant size using such chitosan, but using chitosan as a biodegradable polymer, the size is uniform, that is, the size distribution is narrow, and spherical microspheres are prepared. It was not easy to do.

Therefore, the technical problem to be achieved by the present invention is to provide a method for producing a microsphere of uniform size and spherical shape using chitosan.

Another object of the present invention is to provide a method for producing chitosan microspheres having excellent swelling properties and improved elasticity / flexibility.

In order to achieve the above technical problem, the present invention is to prepare a (S1) chitosan solution; (S2) dropping the chitosan solution to an upper layer of an organic solvent which is not mixed with water and a lower layer which is an aqueous alkali solution or an anionic solution; And (S3) provides a method for producing a chitosan microspheres comprising the step of crosslinking the formed microspheres. The present invention is based on the surprising discovery that this manufacturing method allows the production of chitosan microspheres that are uniform in size and close to a complete sphere.

In the manufacturing method according to the present invention, the organic solvent upper layer which does not mix with water, which does not mix with the aqueous solution of chitosan dropping, but does not mix with the water to serve as a sphere form of a hydrocarbon of C _4-9 , C _10-30 of aliphatic alcohols and aromatic alcohols, C _10-30 aliphatic esters and aromatic esters, C _10-30 aliphatic ethers and an aromatic ether, ketone, aldehyde, ether, halohydrocarbons, glycol ether ester or a phthalic ray bit (phthalate of Solvents may be used, more specifically ethyl ether including petroleum ether, isopropyl acetate, n-propyl acetate, isobutyl acetate, n-butyl acetate, isobutyl isobutyrate, 2-ethylhexyl acetate, ethylene glycol diacetate , C ₉ acetate, C ₁₀ acetate, methyl ethyl ketone, methyl isobutyl ketone, methylisobutyl Wheat ketone, methyl n-amyl ketone, dibutyl ketone, cyclohexanone, isophorone, acetaldehyde, n-butylaldehyde, crotonaldehyde, 2-ethylhexaaldehyde, isobutylaldehyde, propionaldehyde, ethyl 3-ethoxypro Cypionate, toluene, xylene, trichloroethane, propylene glycol monomethyl ethyl acetate, ethylene glycol monoethyl ether acetate, ethylene glycol monobutyl ether, diethylene glycol monobutyl ether acetate, dibutyl phthalate, ethyl phthalate, dimethyl phthalate , Dioctyl phthalate, dioctyl terephthalate, butyl octyl phthalate, butyl benzene phthalate, dioctyl adipate, triethylene glycol di-2-ethylhexanoate, trioctyl trimethylate, glyceryl triacetate, glyceryl / tri Propionine, 2,2,4-trimethyl-1,3-penta Nediol diisobutylate and the like may be used alone or in combination. Among the organic solvents which are not mixed with various kinds of water, petroleum ether or ethyl ether is preferable in terms of forming particles into spheres.

The chitosan or chitosan / PEO particles having passed through the organic solvent upper layer pass through the lower layer, which is an aqueous alkali solution or an anionic solution, to form cations of the chitosan and form precipitates. Such alkaline aqueous solutions include aqueous NaOH, aqueous KOH, or mixtures thereof As the anionic aqueous solution, an aqueous pyrophosphate solution, tripolyphosphate solution, tetrapolyphosphate solution, octapolyphosphate solution, hexametaphosphate solution, octyl sulfate salt solution, lauryl sulfate salt solution, hexadecyl sulfate salt solution, cetyl Stearylsulfate aqueous solution, alginate aqueous solution, carrageenan aqueous solution and the like may be used alone or in combination. As the material for forming a salt with the anion, calcium, sodium, ammonium, iron (divalent or trivalent), potassium, magnesium, barium, hydrogen, zinc, copper, titanium and the like may be used. Among these alkaline aqueous solutions or anionic aqueous solutions, NaOH aqueous solution is more preferable in terms of making the chitosan particles spherical.

Moreover, in the manufacturing method of this invention, it is preferable that ethanol is mixed in the alkali aqueous solution or anion aqueous solution lower layer which is a coacervation medium. Ethanol is effective in preventing droplets from passing between organic and aqueous solutions by reducing the difference in specific gravity between the upper and lower layers, which in turn helps to form spherical microspheres.

Moreover, in the manufacturing method of this invention, it is preferable to perform dropping of the said chitosan aqueous solution using gas. As such a gas, an inert gas such as nitrogen gas, helium gas, argon gas, carbon dioxide, or the like may be used, and nitrogen gas is preferable.

In the preparation method of the present invention, the crosslinking of chitosan or chitosan and PEO microspheres is carried out using formaldehyde, glutaraldehyde, dialdehyde starch, epoxy compound, glutaraldehyde Synthetic crosslinking agents such as glyoxal, glyoxal, 2,2-dimethoxy-2-phenylacetophenone, tripolyphosphate sodium salt, diacetaldehyde PEG, scleraldehyde, diethylsquarate, epichlorohydrin And may be performed using a natural crosslinking agent such as genipin (genipin), but is not limited thereto. Among the various crosslinking agents, genipine is preferred over other crosslinking agents in terms of safety, biocompatibility, slower degradation rate of the crosslinked particles in vivo, and the like.

It is more preferable that the manufacturing method of the present invention further includes the step (S4) of washing the remaining crosslinking agent without reaction and the step (S5) drying the formed microspheres.

The present invention also provides a method of controlling the swelling and elasticity of the chitosan microspheres, characterized in that the preparation of the microspheres by mixing chitosan and polyethylene oxide (PEO).

More preferably, the present invention comprises the steps of preparing a solution in which (S1) chitosan and polyethylene oxide are dissolved; (S2) dropping the solution into an upper layer of an organic solvent which is not mixed with water and a lower layer which is an aqueous alkali solution or an anionic solution; And (S3) provides a method of controlling the swelling and elasticity of the chitosan microspheres comprising the step of crosslinking the formed microspheres.

That is, in the above-described method for producing spherical chitosan microspheres, adding PEO to the chitosan aqueous solution does not affect the mechanical strength of the microspheres or increases the mechanical strength of the chitosan microspheres while increasing the mechanical strength. Can be increased.

The molecular weight of such PEO can be used 100,000 to 8,000,000, it is preferable that the 3,000,000 to 5,000,000. When using a relatively low molecular weight PEO such as PEO having a molecular weight of 1,000,000, it was more difficult to maintain particle shape and secure elasticity as compared to when using a PEO having a molecular weight of 4,000,000, and relatively high as a PEO having a molecular weight of 8,000,000. When the molecular weight of PEO was used, the viscosity was very high, and when dropped using nitrogen gas, there was a fear that tailing particles were produced.

The content of PEO having a molecular weight of 3,000,000 to 5,000,000 is preferably 1 to 10% by weight relative to the weight of chitosan, but if it is higher than this, the viscosity is high and it is difficult to control the discharge rate of the chitosan / PEO solution from the syringe. It may be somewhat difficult to produce, and the high content of the hydrophilic polymer may shorten the decomposition time of the particles in the body and may weaken the strength of the particles.

The present invention provides a method for producing microspheres of uniform size and spherical shape using chitosan, more preferably uniform size, spherical shape, excellent swelling property, and improved elasticity / flexibility. Provided are methods for preparing chitosan microspheres (microspheres).

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example 1 Preparation of Chitosan Microspheres

Chitosan (about 85% diacetylated) was dissolved in 1% (v / v) acetic acid aqueous solution to prepare a chitosan solution at a concentration of 1.5% (w / v). The viscous chitosan solution was mixed with a 1% aqueous solution of polyethylene oxide (PEO, molecular weight 4,000,000 g / mol) in various weight ratios. The percentage ratios of PEO to chitosan (%, w / w) were 0, 1, 2.5 and 5%, respectively. The chitosan solution or chitosan / PEO solution was filled in a 1 ml syringe (31 G) and discharged from the syringe at a rate of 600 ul / h and at the same time coacervation medium (ethanol) with nitrogen gas (1-2 kgf / cm ² ). : 20% (w / v) aqueous NaOH solution: petroleum ether = 50: 5: 5 v / v) (see FIG. 1). The coaceravated microspheres (45 mg) formed were then dispersed and cross-linked in 10 ml of 0.05% genipin ethanol solution. The crosslinking reaction was carried out at room temperature for 5, 10, 15, 30, 60, 120 and 240 minutes respectively. The crosslinked chitosan microspheres were then washed with ethanol to remove the remaining genipine and then washed three times with acetone. The washed microspheres were dried at 80 ° C.

Example 2 Evaluation of Microsphere Manufacturing Conditions

To prepare microspheres with uniform size and spherical shape, the type of coacervation medium and the interfacial tension between the organic solvent and the aqueous polymer solution added were evaluated. Pure chitosan or chitosan / PEO aqueous solution was loaded onto various coacervation media. Coacervation media is a mixture of ethanol and 20% (w / v) aqueous NaOH solution (50: 5 v / v, 55 ml) or a mixture of 20% (w / v) sodium lauryl sulfate (SLS) solution (50: 5 v / v, 55 ml) was used.

In addition, petroleum ether having a low specific gravity (0.64 at 20 ° C.) and an organic solvent not mixed with water were added, and an upper layer was formed on the coacervation medium as shown in FIG. 1. The dropped aqueous polymer solution interacts with the upper organic solvent layer and then precipitates with a water-soluble coacervation medium.

The morphology of the prepared chitosan microspheres was evaluated by light microscopy. The dried microspheres were immersed in the medium and the fully swelled microspheres were placed on the slide glass and the morphological characteristics of the microspheres were observed. The size and size distribution of the chitosan microspheres were measured by image analysis software (Optimas 6.1, VSG, U.K.).

(1) Impact assessment results according to types of coacervation media

Morphological features of chitosan microspheres coacervated with NaOH-based coacervation medium or sodium lauryl sulfate-based coacervation medium as surfactants are shown in FIGS. 2A and 2B, respectively. The chitosan microspheres precipitated by NaOH were homogeneous and spherical (FIG. 2A), but the shape of the chitosan particles obtained using SLS, a polymer ion, was very irregular (FIG. 2B).

Low specific gravity (0.813 at 20 ° C) and water miscible ethanol solutions reduce the difference in specific gravity between petroleum ether (0.640 at 20 ° C) and aqueous coacervation solution (> 1.00 at 20 ° C). It was added to prevent the enemy from passing through, and this mixing was effective in forming a microsphere having a uniform size and spherical shape.

As a result, a mixture of ethanol and 20% (w / v) NaOH aqueous solution was preferred as coacervation medium, and the mixture was used in subsequent evaluation.

(2) Effect evaluation result by addition of petroleum ether layer

Chitosan microspheres prepared after interaction with the organic solvent layer, petroleum ether, were almost completely spherical (FIG. 3A), whereas chitosan microspheres prepared without this petroleum ether layer were elongated ovals (FIG. 3B). From these results it can be seen that the presence of the petroleum ether layer plays a very positive role in the spherical preparation of chitosan particles. This is thought to be because the interfacial tension between the organic solvent and the aqueous chitosan solution is so large that it can overcome the energy to modify the shape of the spherical droplets and offset the gravitational effect that the shape can be broken to some extent.

(3) Result of impact evaluation by adding polyethylene oxide

The forms of pure chitosan microspheres and PEO 5% -mixed chitosan microspheres are shown in FIGS. 4A and 4B, respectively. Both microspheres were almost spherical and showed little difference.

The size distribution immediately after preparation of the pure chitosan microspheres and PEO 5% -mixed chitosan microspheres and after drying is shown in FIG. 5. The chitosan microspheres before drying were both over 1000 um and had a very narrow distribution. That is, at least about 90% of the prepared microspheres were 1200-1400 um in size. The size of the microspheres was also reduced to about 350-380 um after drying, which is believed to mean that the droplets of the aqueous chitosan solution were rapidly hardened by the coacervation medium in a fully swollen state.

Chitosan microspheres were able to maintain a size small enough to be easily administered after drying even after the inclusion of the polymer PEO. Specifically, the particle sizes after drying of pure chitosan microspheres and PEO mixed microspheres were 384 and 350.5 um, respectively.

Example 3 Cross-linking Density Measurement

The crosslinking density of the prepared chitosan microspheres was measured by ninhydrin assay (FL Mi, YC Tan, HC Liang, RN Huang, HW Sung, J. Biomat. Sci-Polym.E. 12 (8) ( 2001) 835). This method measures the percentage of free amino groups remaining in the chitosan microspheres after crosslinking. Ninhydrin solution was prepared as follows. Solution A was prepared by mixing 1.05 g citric acid, 10 ml (1 M) NaOH and 0.04 g SnCl ₂ .2H ₂ O, then adding deionized water to 25 ml. Solution B was prepared by adding 1 g ninhydrin to 25 ml ethylene glycol monomethyl ether. The solutions A and B were mixed and stirred for 45 minutes. 1 ml ninhydrin solution was added to 1.5 mg of dry chitosan microspheres and heated to 100 ° C. in a water bath for 40 minutes. The solution was cooled to room temperature and diluted with 50% isopropanol (9 ml), then the absorbance of the solution was evaluated at 570 nm. The amount of free amino groups in the sample detected after heating with ninhydrin is proportional to the absorbance of the solution. The crosslink density of the sample was calculated as follows.

Crosslink density (%) = [absorbance _0h -absorbance _t ) / absorbance _0h ] × 100

In Equation 1, absorbance _0h means absorbance of the uncrosslinked sample, and absorbance _t means absorbance of the crosslinked sample for t hours.

6 shows the results of evaluating the increase in the degree of crosslinking with time using a constant concentration of genipin. The degree of crosslinking increased up to about 26% by 240 minutes. The degree of crosslinking of the microspheres was 1.68, 13.13, 17.15, 20.08, 22.38, 24.61 and 26.33% at 5, 10, 15, 30, 60, 120 and 240 minutes, respectively. However, the degree of crosslinking did not increase significantly after about 30 minutes.

The chitosan microspheres crosslinked with genipine according to the present invention are expected to exhibit high biocompatibility, and the degradation of chitosan microspheres is delayed compared to chitosan microspheres crosslinked with other crosslinking agents, thereby maintaining vascular occlusion for a long time.

Example 4 Swelling Evaluation

The swelling ratio of chitosan microspheres was measured. Chitosan microspheres were added to the 5% dextrose solution at room temperature and evaluated by gentle stirring. Chitosan microspheres with different PEO ratios and crosslinking densities were evaluated to assess the effect of PEO mixing and crosslinking density on the swelling properties of chitosan microspheres. The degree of swelling was evaluated using the extent to which the diameter of the microspheres increases. Microspheres were withdrawn from the medium at predetermined times and the diameters of the microspheres on the slide glass were measured by an image analysis program. The degree of swelling was measured as in Equation 2 below.

Swelling degree (%) = (D ₂ -D ₁ ) / D ₁ × 100

In Equation 2, D ₁ refers to the initial diameter immediately after preparation, and D ₂ refers to the diameter of the swollen chitosan microspheres.

The size swelling ratio of the chitosan microspheres is shown in FIG. 7. The dried chitosan microspheres hydrated quickly in water and after crosslinking the hydrophilicity of the microspheres changed and consequently the swelling properties. The addition of PEO also significantly increased the swelling properties of chitosan microspheres. As 0, 1, 2.5 and 5% of PEO were added, the size swelling ratio increased to 23, 67, 129 and 160%.

Example 5 Compression and Flexibility Evaluation Results

The compressibility and flexibility of the microspheres were evaluated using a texture analyzer. Microspheres were placed in ample holders 7-10 mm in diameter. Surrounding water was carefully removed by wicking. The diameter of the probe was 5.0 mm and the crosshead speed was 0.2 mm / sec for 2 seconds. The microspheres were pressed at various pressures, and the breaking force of the microspheres was measured under a microscope. All experiments were performed at room temperature.

The force required to break the crosslinked chitosan microspheres for 5, 15, 30 and 60 minutes respectively was measured and shown in FIG. 8. As seen previously, the addition of PEO dramatically increased the swelling properties of the chitosan microspheres, but also increased the strength to break the mixed microspheres when PEO was crosslinked for a certain period of time. These results indicate that when PEO is mixed with chitosan microspheres, both mechanical strength and flexibility can be increased compared to microspheres made only of chitosan.

1 is a view showing the manufacturing process of the chitosan microspheres for thrombosis according to the present invention.

2 is a comparative photograph (50 times) showing the shape of chitosan microspheres according to coacervation medium. (a) is a picture of chitosan microspheres coacervated with NaOH, and (b) is a picture of chitosan microspheres coacervated with sodium lauryl sulfate.

Figure 3 is a comparative photograph (50 times) showing the shape of chitosan microspheres with and without petroleum ether layer. (a) is a microsphere photograph prepared in the presence of a petroleum ether layer, and (b) is a chitosan microsphere photograph prepared without a petroleum ether layer.

Figure 4 is a comparative photograph (50 times) showing the form of chitosan microspheres with or without the addition of PEO. (a) is a photograph of chitosan microspheres prepared without adding PEO, and (b) PEO is Chitosan microspheres prepared by adding 5%.

5 is a graph showing the average diameter of chitosan microspheres measured immediately after preparation and after drying. In Figure 5, A is the result measured immediately after preparation, and B is the result measured after drying. The results are expressed as mean ± S.D. (N = 5).

FIG. 6 is a graph showing the degree to which chitosan microspheres are crosslinked by genipin at a predetermined concentration (0.05%). Results are expressed as mean ± S.D. (N = 4).

7 is a graph showing the size swelling ratio of chitosan microspheres prepared according to the present invention. Results are expressed as mean ± S.D. (N = 4).

8 is a result of measuring the force required to break the chitosan microspheres according to the present invention measured by a texture analyzer (texture analyzer). Results are expressed as mean ± S.D. (N = 4).

Claims (11)

(S1) preparing a chitosan solution; (S2) dropping the chitosan solution to an upper layer of an organic solvent which is not mixed with water and a lower layer which is an aqueous alkali solution or an anionic solution; And (S3) cross-linking the formed microspheres Method for producing a chitosan microspheres of uniform and spherical size, characterized in that it comprises a. The method of claim 1, wherein the organic solvent that is not mixed with water is a method for producing chitosan microspheres, characterized in that the petroleum ether or ether ether. The method of claim 1, wherein the dropping is carried out using a gas. The method of claim 3, wherein the gas is nitrogen gas. The method of claim 1, wherein the crosslinking is a method for producing chitosan microspheres, characterized in that by reacting with genipin (genipin). The method of claim 1, wherein the manufacturing method further comprises (S4) washing the remaining crosslinking agent without reacting, and (S5) drying the formed microspheres. The method of claim 1, wherein the alkaline aqueous solution is a method for producing chitosan microspheres, characterized in that the aqueous NaOH solution. The method of claim 1, wherein the anionic aqueous solution is pyrophosphate aqueous solution, tripolyphosphate aqueous solution, tetrapolyphosphate aqueous solution, octapolyphosphate aqueous solution, hexametaphosphate aqueous solution, octyl sulfate salt aqueous solution, lauryl sulfate salt aqueous solution, hexadecyl sulfate salt aqueous solution, Cetyl stearyl sulfate salt solution, alginate solution and carrageenan aqueous solution of any one selected from the group consisting of aqueous solution. The method of claim 1, wherein the lower layer is a mixture of ethanol and an aqueous solution. A method of controlling the swelling and elasticity of chitosan microspheres, characterized in that to produce a microsphere by mixing chitosan and polyethylene oxide. The method of claim 10, wherein the method comprises: (S1) preparing a solution in which chitosan and polyethylene oxide are dissolved; (S2) dropping the solution into an upper layer of an organic solvent which is not mixed with water and a lower layer which is an aqueous alkali solution or an anionic solution; And (S3) crosslinking the formed microspheres. A method of controlling swelling and elasticity of chitosan microspheres.

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