JPH01168337A - Coated microcapsule and manufacture thereof - Google Patents

Abstract

PURPOSE:To raise the total adaptability and improve structural stability, by forming high-molecular electrolyte complex in the suspension of microcapsules, coating thereby the surfaces of the microcapsules with high-molecular electrolyte. CONSTITUTION:Enzyme-treated collagen is subjected to thermal denaturation in the region of temperature 37-90 deg.C and mixed with mucopolysaccharide in an aqueous solvent, the mixture of which is stirred, while pH2-7, preferably pH3.5-5.0 being held, to form coacervate so that microcapsules are obtained. Thereafter, the microcapsules are suspended in a suitable solvent, e.g., water, wherein high-molecular electrolyte complex is formed in said suspension so that the coating of high-molecular electrolyte complex is formed over the surfaces of the microcapsules. As mucopolysaccharide stated above, acidic mucopolysaccharide of contorting saluted, Hyperion sulfate, termagant sulfate, acid, etc., are desirable.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a coated microcapsule and a method for producing the same. The microcapsules of the present invention are made of biologically derived materials that are harmless to living organisms, and are used in fields such as pharmaceuticals, cosmetics, and foods.

[Prior Art] Conventionally, microcapsules have been produced by a coacervation method using gelatin-gum arabic or the like, or a liposome method using phospholipids. this house,
Microcapsules using gelatin-gum arabic have been the most studied and have been used as effective microcapsules since the beginning because they have been recognized for their ability to incorporate substances into coacervates. This type of gelatin is made by industrially decomposing the animal protein collagen, but because the fibrous collagen is randomly cut and formed into random coils, antigenic groups and other parts are not removed. Because it is structurally unstable, it is not necessarily suitable for operations such as implantation into a living body. Microcapsules are attracting attention for their use as unique reaction sites or drug carriers, but in any case, there remains a problem in their structural stability that needs to be improved.

[Problems to be Solved by the Invention] An object of the present invention is to provide highly biocompatible and structurally stable microcapsules.

[Means for Solving the Problems] In order to solve the above problems, the present invention has the following configuration.

(1) A coated microcapsule in which the surface of the microcapsule is coated with a polymer electrolyte complex.

(2) The coated microcapsule according to item 1, wherein the microcapsule is composed of heat-denatured collagen and mucopolysaccharide and forms a coacervate structure.

(3) The coated microcapsule according to item 2, wherein the heat-denatured collagen is atelocollagen from which the terminal antigenic group telopeptide has been removed with protase or pepsin.

(4) The coated microcapsule according to item 1, wherein the polymer electrolyte complex comprises heat-denatured collagen and mucopolysaccharide.

(5) The coated microcapsule according to item 4, wherein the mucopolysaccharide is an acidic mucopolysaccharide.

(6) The coated microcapsule according to item 5, wherein the acidic mucopolysaccharide is chondroitin sulfate, heparan sulfate, dermatan sulfate, hyaluronic acid, or heparin.

(7) A method for producing coated microcapsules, characterized in that the surface of the microcapsules is coated with a polymer electrolyte by forming a polymer electrolyte complex in a suspension of microcapsules.

(8) The method for producing coated microcapsules according to item 1, wherein the microcapsules are composed of heat-denatured collagen and mucopolysaccharides and form a coacervate structure.

(9) The method for producing coated microcapsules according to item 7, wherein the formation of the polyelectrolyte complex is carried out by a reaction between reconstituted collagen and mucopolysaccharide.

(10) Reconstituted collagen is a collagen aqueous solution of 37~
10. The method for producing coated microcapsules according to item 9, wherein the coated microcapsules are partially denatured by heat denaturation at 90° C. and left at room temperature for 3 hours or more to undergo molecular reorganization.

There are no particular limitations on the microcapsules to be coated in the present invention; conventionally known ones can be used.

Particularly preferably, microcapsules obtained by adding mucopolysaccharide to heat-denatured collagen to form coacervate are used.

The formation of the above coacervate was discovered by the present inventors. Collagen is derived from bovine dermis,
It is preferable to use enzyme-treated collagen (atelocollagen) in which fibrous collagen obtained by acid or alkali treatment is treated with protase or pepsin to digest and remove the telopeptide of the antigenic group at the terminal of the molecule. Atelocollagen is swollen with water and thermally denatured when kept at 60°C for several hours. The mucopolysaccharides are preferably acidic mucopolysaccharides such as chondroitin sulfate, heparan sulfate, dermatan sulfate, hyaluronic acid, and heparin.

Chondroitin sulfate is particularly preferred. Coacervate formation is achieved by heat denaturing enzyme-treated collagen at a temperature range of 37-90°C, mixing it with mucopolysaccharide in an aqueous solvent, and maintaining the pl+ at 2-7, preferably poa, 5-5.0. This is done by stirring.

The concentrations of collagen and mucopolysaccharide were each 5 (W/
V) S or higher. The content of the mucopolysaccharide in the microcapsules is 50 (v/v) S or more and 5 (w/v) S or more. When the coacervate thus obtained is treated with formalin or the like and hardened, the microcapsules are less likely to adhere to each other, so it is easy to form a structure in which one microcapsule is coated. On the other hand, without curing treatment, a structure in which multiple microcapsules are often scattered in a polymer matrix is often obtained. These microcapsules can be selected depending on the usage.

The microcapsules thus obtained are suspended in a suitable solvent such as water, and a polyelectrolyte complex is formed in the suspension, thereby coating the surface of the microcapsule with the polyelectrolyte complex.

The term "polyelectrolyte complex" refers to a complex of a polymer compound having a dissociative group, and natural protein complexes, particularly complexes consisting of collagen and mucopolysaccharide, are suitable. Collagen is an aqueous solution of atelocollagen that has undergone the above enzyme treatment.
Particularly desirable is reconstituted collagen, which is partially thermally denatured at a temperature of 90° C. and left at room temperature for 3 hours or more to partially undergo molecular rearrangement. In reconstituted collagen, the helical structure consisting of three polypeptide chains of the collagen molecule is once decomposed by heating, and then the triple helical structure is partially restored by cooling. On the other hand, in heat-denatured collagen used for coacervate formation, the triple helical structure is completely decomposed by heating, resulting in a -layered structure.

The mucopolysaccharide used to form the polyelectrolyte complex can be the same as that used to form the coacervate.

Next, the present invention will be explained in more detail by showing reference examples, examples, and test series.

Reference Example 1 Preparation of microcapsules Oxygen solubilized collagen (atelocollagen) was mixed with 0.3 (
The atelocollagen aqueous solution is obtained by swelling with distilled water at 4°C overnight while maintaining the concentration of v, /v)%.

In order to make this aqueous solution react quickly to the temperature increase caused by heating,
When left at room temperature (24°C) for 1 to 2 hours and then heated in a constant temperature bath kept at 60°C,

Due to its known properties, collagen becomes heat-denatured collagen whose viscosity rapidly decreases at temperatures above 37°C. Stir as is in a constant temperature bath at 60°C for about 2 hours. This heat-denatured collagen is kept at a temperature above the denaturation temperature until the mixing operation.

On the other hand, mucopolysaccharides are usually chondroitin-6-sulfate H
Prepare a + type one. Chondroitin-6-sulfate is usually commercially available as Na-type sodium chondroitin-6-sulfate, but this is replaced with H-type in solution using a cation exchange resin. Thus 1(ν/v
) Obtain an aqueous chondroitin-6-6A acid solution of 96. The heat-denatured collagen and chondroitin-6-sulfuric acid aqueous solution are each passed through a 0.45 μm filter to remove impurities, and then mixed at a temperature above the denaturation temperature. On this occasion,
Chondroitin-6 per 1000 volumes of denatured collagen
- Mix 75 volumes of sulfuric acid so that the proportion of chondroitin-6-sulfuric acid to the total amount is 20 (v/v)%.

After thorough mixing, the pH is adjusted using 1N HCl2. When pl+ falls to around 4.0, cloudiness occurs after mixing and coacervate is obtained. The thus obtained coacervate is hardened by adding 1% formalin to form microcapsules.

Reference Example 2 Preparation of Microcapsules During the operation of Reference Example 1, the temperature of the constant temperature bath was raised to 90°C,
Although the mixture was stirred in a constant temperature bath for 15 hours, the same coacervate was still obtained. However, when the mixture was stirred in a constant temperature bath at 90° C. for 24 hours or more, coacervate was no longer formed. Also, when treated at 90°C for 15 hours, the region of coacervate formation was narrower than when treated at 60°C. The thus obtained coacervate was cured to form microcapsules in the same manner as in Reference Example 1.

Example 1 Atelocollagen was dissolved in 1mM hydrochloric acid solution, and 0.3
% solution. 20 ml of this atelocollagen solution was mixed with 20 ml of microcapsules obtained in Reference Example 1 in an equal amount, and 0.68 ml of 1% chondroitin-6-sulfuric acid solution was added.
By adding m1, coated microcapsules coated with a polyelectrolyte complex are obtained. The optical micrograph is shown in Fig. 1.

Example 2 Atelocollagen was dissolved in 1111M hydrochloric acid solution, and 0
．． Make a 3% solution. After stirring the atelocollagen solution in a constant temperature bath at 60°C for 2 hours, at room temperature or at a low temperature (4°C)
Leave it for several hours. The atelocollagen solution thus obtained has a partially helical structure, and molecular rearrangement has partially occurred. 20ml of this reconstituted atelocollagen solution was added to 20ml of the microcapsules obtained in Reference Example 1.
1 and further add 0.86 ml of 1% chondroitin-6-sulfuric acid solution to obtain coated microcapsules coated with a polyelectrolyte complex.

Example 3 The coacervate obtained as in Reference Example 1 was heated at low temperature (
4° C.) for several hours to several days to harden and form microcapsules. This microcapsule is made up of a plurality of microcapsules stuck together.

Atelocollagen was dissolved in 111M hydrochloric acid solution and 0.
Make a 3% solution. 20 ml of this atelocollagen solution was mixed with 20 ml of the above microcapsules in an equal amount, and 1
% chondroitin-6-sulfate. (If i[iml is added, a coated microcapsule in which a plurality of microcapsules are coated with a polyelectrolyte complex is obtained.

The optical micrograph is shown in FIG. 2.

Test Example 1 Stability of coated microcapsules When coated microcapsules prepared as in Example 1 were centrifuged (15,000 p, m, 20 minutes), the microcapsules phase separated and remained in the lower layer. . Discard the supernatant solution and
Add H7,4 phosphate buffer solution. The stability is tested by quantifying the amount of protein eluted by the biuret method in a constant temperature bath at 37°C. The results are shown in Figure 3.

In FIG. 3, the vertical axis indicates protein elution ff1 (%) from the microcapsules, which is responsible for the solubility of the capsules. The horizontal axis indicates time (hours). In the figure, -〇- indicates the uncoated microcapsules, -∆- indicates the solubility of the coated microcapsules of the present invention obtained in Example 2, and -Rho indicates the solubility of the coated microcapsules of the present invention obtained in Example 1. shows.

Test Example 2 Sustained release of multilayer-coated microcapsules containing hydrocortiscin When hydrocortiscin dissolved in ethanol in advance was added to the coated microcapsules prepared as in Example 1, hydrocortiscin-containing coated microcapsules are obtained.

The microcapsules were centrifuged (15,00 Or, p
, Ill, , 20 minutes), the phase separates and remains in the lower layer.

The supernatant solution is discarded, physiological saline is added, and the content of hydrocortiscin eluted over time is determined. Figure 4 shows the content of hydrocortiscin eluted from the microcapsules.

In FIG. 4, the vertical axis shows the elution amount (μg/ml) of hydrocortiscin from the microcapsules, indicating the sustained release property of hydrocortiscin. The horizontal axis indicates time (hours). In the figure, -0-1-Δ- and 1 row have the same meaning as in FIG.

[Effects of the Invention] Since the surface of the microcapsule of the present invention is coated with a polymer electrolyte complex, the strength of the microcapsule is increased.

In the present invention, when the microcapsule is composed of heat-denatured atelocollagen and mucopolysaccharide to form a coacervate structure, and the polyelectrolyte complex is composed of reconstituted atelocollagen and mucopolysaccharide, the microcapsules are highly effective especially in transdermal areas. It has good affinity and can be incorporated into artificial coatings, ointments, cosmetics, etc., and because it does not have a fibroblast culture substrate or antigenicity, it can be used by implanting it into the treatment site in the body. Moreover, it is harmless because it is a bioabsorbable material. In addition, the manufacturing method is simple and allows capsules to be obtained easily and reliably, and depending on the purpose of use, formalin treatment prevents microcapsules from adhering to each other, so the surface of each microcapsule is coated with a polymer electrolyte complex. Multilayer-coated microcapsules with a structured structure can be obtained. Furthermore, without formalin treatment, it is possible to obtain multilayer-coated microcapsules having a structure in which a plurality of microcapsules are scattered in a polymer matrix.

[Brief explanation of the drawing]

FIGS. 1 and 2 are optical micrographs of coated microcapsules of the present invention obtained in Examples 1 and 3, respectively. FIG. 3 and FIG. 4 are graphs showing the elution amount of protein or hydrocortiscin in Test Examples 1 and 2, respectively. Time 01 Figure 3 Time (hours) Figure 4 Procedural amendment (method) % formula % Display of the case 1988 Patent Application No. 32731, Name of the invention Covered microcapsules and manufacturing method thereof 3, Person making the amendment Case Relationship with Patent applicant Address: 2-44-1 Hatagaya, Shibuya-ku, Tokyo Name: Chill-7 Co., Ltd.

Claims (1)

[Claims] (1) A coated microcapsule in which the surface of the microcapsule is coated with a polymer electrolyte complex. (2) The coated microcapsule according to claim 1, wherein the microcapsule is composed of heat-denatured collagen and mucopolysaccharide and forms a coacervate structure. (3) The coated microcapsule according to claim 2, wherein the heat-denatured collagen is atelocollagen from which the terminal antigenic group telopeptide has been removed using protase or pepsin. (4) The coated microcapsule according to claim 1, wherein the polymer electrolyte complex comprises reconstituted collagen and mucopolysaccharide. (5) The coated microcapsule according to claim 4, wherein the mucopolysaccharide is an acidic mucopolysaccharide. (6) The coated microcapsule according to claim 5, wherein the acidic mucopolysaccharide is chondroitin sulfate, heparan sulfate, dermatan sulfate, hyaluronic acid, or heparin. (7) A method for producing coated microcapsules, which comprises forming a polymer electrolyte complex in a microcapsule suspension to coat the surface of the microcapsule with the polymer electrolyte. (8) The method for producing coated microcapsules according to claim 7, wherein the microcapsules are composed of heat-denatured collagen and mucopolysaccharides and form a coacervate structure. (9) The method for producing coated microcapsules according to claim 7, wherein the formation of the polyelectrolyte complex is carried out by a reaction between reconstituted collagen and mucopolysaccharide. (10) Reconstituted collagen is a collagen aqueous solution of 37~
10. The method for producing coated microcapsules according to claim 9, wherein the coated microcapsules are thermally denatured at 90° C. and left at room temperature for 3 hours or more to partially undergo molecular rearrangement.