KR100517072B1 - Polyol/polymer microcapsule and the stabilization method of enzyme using it - Google Patents
Polyol/polymer microcapsule and the stabilization method of enzyme using it Download PDFInfo
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- KR100517072B1 KR100517072B1 KR10-2002-0073433A KR20020073433A KR100517072B1 KR 100517072 B1 KR100517072 B1 KR 100517072B1 KR 20020073433 A KR20020073433 A KR 20020073433A KR 100517072 B1 KR100517072 B1 KR 100517072B1
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- Prior art keywords
- meth
- acrylate
- poly
- polymer
- polyol
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- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007127 saponification reaction Methods 0.000 description 1
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- 238000012546 transfer Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5015—Organic compounds, e.g. fats, sugars
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Abstract
본 발명은 효소를 폴리올계에서 근본적으로 안정화하고 이를 다시 고분자 마이크로캡슐로 궁극적으로 안정화하는 효소의 안정화 방법에 관한 것이다. 더욱 구체적으로는 효소를 저분자량 폴리올에 균일하게 분산시켜 효소를 안정화시키는 단계; 상기 안정화된 효소/폴리올을 고분자 용액에서 재분산시킨 후 유화과정을 거쳐 에멀젼을 수득하는 단계; 및 효소/폴리올/고분자 용액을 고화시켜 경질 고분자 마이크로캡슐로 회수하는 단계;를 포함한다.The present invention relates to a method for stabilizing enzymes which fundamentally stabilizes enzymes in the polyol system and ultimately stabilizes them again with polymer microcapsules. More specifically, uniformly dispersing the enzyme in a low molecular weight polyol to stabilize the enzyme; Redispersing the stabilized enzyme / polyol in a polymer solution to obtain an emulsion through emulsification; And solidifying the enzyme / polyol / polymer solution to recover the hard polymer microcapsules.
Description
본 발명은 제형에서의 사용시 안정성이 급격히 저하되는 특징을 지니는 효소를 효과적으로 안정화시킬 수 있는 고분자 마이크로캡슐의 제조 기술에 관한 것이다.The present invention relates to a technique for preparing polymer microcapsules capable of effectively stabilizing an enzyme having a characteristic of rapidly decreasing stability in use in a formulation.
바이오산업의 발전과 더불어, 다양한 효소가 치료/치유제로 사용되고 있다. 그러나, 대부분의 효소는 매우 짧은 반감기를 지니고 있어, 궁극적인 효능 발현 이전에 변성되는 경향이 있으며, 효소 성분의 이러한 낮은 안정도는 그 활용가치를 저하시키는 가장 큰 요인이 되고 있다. 따라서, 이러한 효소 성분의 응용을 확대하기 위하여 최근 적절한 전달시스템의 구축에 대한 연구가 활발히 진행되고 있다. 많은 효소 전달시스템 중에서 생분해성 고분자를 벽제로 활용한 연구는 장기 투여의 관점에서 많은 장점을 지니고 있다 (Y.Okawa, M. Yamamoto, H. Okada, T. Yashiki, T. Shimamoto, Chem. Pharmac. Bull. 5 (1988) 1095, H. Okada, Y. Doken, Y. Ogawa, H. Toguchi, Pharmac. Res. 11 (1994) 1143). 이러한 전달시스템 또한 제조 공정과 장기 보관의 관점에서는 안정도가 보장되어야 할 필요가 있다. 일반적으로 수-유-수 다중 에멀젼 방법이 효소 함유 마이크로캡슐의 제조를 위하여 자주 사용되지만, 수-유 계면에서 효소의 활성이 저하된다는 면에서 큰 문제를 지니고 있다 (P. Couvreur, M. J. Blanco-Proeto, F. Puisieux, B. Ropues, E. Fattal, Adv. Drug Del. Rev. 28 (1997) 85, H. Sah, J. Pharmac. Sci. 88 (1999) 1320). 따라서, 최근에는 고체-유-수 에멀젼 방법이 고체 상태에서 효소의 활성을 유지할 수 있다는 장점 때문에 보다 현실적으로 받아들여지고 있다 (T. Morita, Y. Sakamura, Y. Horikiri, T. Suzuki, H. Yoshino, J. Control. Rel. 69 (2000) 435). 그러나, 상기의 고체 상태의 효소를 캡슐화하는 방법들은 대부분 가열과 물리적인 힘을 가하는 제조 공정상의 특징 때문에 효소의 많은 부분에서의 변성을 초래한다.With the development of the bio industry, a variety of enzymes are being used for treatment / healing. However, most enzymes have a very short half-life, which tends to denature before the ultimate expression of efficacy, and this low stability of the enzyme component is the biggest factor degrading its utility value. Therefore, in order to expand the application of such an enzyme component, research on the construction of an appropriate delivery system has been actively conducted in recent years. Among many enzyme delivery systems, studies using biodegradable polymers as a wall have many advantages in terms of long-term administration (Y.Okawa, M. Yamamoto, H. Okada, T. Yashiki, T. Shimamoto, Chem. Pharmac. Bull. 5 (1988) 1095, H. Okada, Y. Doken, Y. Ogawa, H. Toguchi, Pharmac. Res. 11 (1994) 1143). This delivery system also needs to be assured in terms of manufacturing processes and long term storage. In general, the water-oil-water multiple emulsion method is frequently used for the preparation of enzyme-containing microcapsules, but has a big problem in that the activity of the enzyme is reduced at the water-milk interface (P. Couvreur, MJ Blanco-Proeto). , F. Puisieux, B. Ropues, E. Fattal, Adv. Drug Del. Rev. 28 (1997) 85, H. Sah, J. Pharmac. Sci. 88 (1999) 1320). Therefore, recently, the solid-oil-water emulsion method is more realistically accepted due to the advantage of maintaining the activity of the enzyme in the solid state (T. Morita, Y. Sakamura, Y. Horikiri, T. Suzuki, H. Yoshino, J. Control.Rel. 69 (2000) 435). However, these methods of encapsulating solid state enzymes often result in denaturation in many parts of the enzyme due to the nature of the manufacturing process, which exerts heating and physical force.
이에 본 발명자들은 종래의 이러한 효소의 변성을 막을 수 있는 안정화 시스템을 개발하고자 노력하였다. 그 결과 본 발명자들은 폴리올/고분자 마이크로캡슐이 효소의 안정화에 적합하다는 것을 발견하여 본 발명을 완성하였다. The present inventors have tried to develop a stabilization system that can prevent the denaturation of such enzymes in the prior art. As a result, the present inventors have found that polyol / polymer microcapsules are suitable for stabilizing enzymes and completed the present invention.
본 발명의 목적은 저분자량 폴리올, 고분자량 폴리올 및 고분자 용액을 이용한 폴리올/고분자 마이크로캡슐을 제공하는 것이다.It is an object of the present invention to provide polyol / polymer microcapsules using low molecular weight polyols, high molecular weight polyols and polymer solutions.
또한 본 발명의 목적은 저분자량 폴리올, 고분자량 폴리올 및 고분자 용액을 이용한 폴리올/고분자 마이크로캡슐의 제조 방법을 제공하는 것이다.It is also an object of the present invention to provide a method for producing polyol / polymer microcapsules using a low molecular weight polyol, a high molecular weight polyol and a polymer solution.
본 발명의 또다른 목적은 제약 또는 화장품 제형에 적용하기 위한 효소를 함유하는 효소 안정화 마이크로 캡슐 시스템을 제공하는 것이다.Another object of the present invention is to provide an enzyme stabilized microcapsule system containing enzymes for application in pharmaceutical or cosmetic formulations.
본 발명의 저분자량 폴리올, 고분자량 폴리올 및 고분자 용액을 이용한 폴리올/고분자 마이크로캡슐의 제조 방법은 아래와 같다.The production method of the polyol / polymer microcapsules using the low molecular weight polyol, the high molecular weight polyol and the polymer solution of the present invention is as follows.
먼저 저분자량 폴리올 및 고분자량 폴리올을 혼합하여 용매에 고분자 벽제를 용해하여 제조한 고분자 용액에 용해시킨다. 이 때 상기 저분자량 폴리올 및 고분자량 폴리올을 합한 중량과 고분자 용액의 고분자 벽제의 양은 벽제의 두께를 고려하며 조절 가능하나 동일한 양을 사용하는 것이 적절하다. 또한 상기 저분자량 폴리올 및 고분자량 폴리올을 합한 중량과 고분자 벽제의 양은 용매에 대하여 20 중량% 도입한다. 폴리올이 용해된 용액을 안정화제가 도입된 수용액에 넣고 호모게나이저를 이용하여 유화시킨다. 상기 유화액을 감압증발기에서 10∼120분간 감압교반하여 용매를 완전 제거한 후의 분산액을 여과하여 캡슐을 회수하고 그 외 수용성 물질은 모두 제거한다. 회수된 상기 캡슐은 빛이 차단된 실온 감압 건조기에서 24∼48 시간 동안 완전 건조한다. First, a low molecular weight polyol and a high molecular weight polyol are mixed and dissolved in a polymer solution prepared by dissolving a polymer wall in a solvent. At this time, the combined weight of the low molecular weight polyol and the high molecular weight polyol and the amount of the polymer wall of the polymer solution can be controlled considering the thickness of the wall, but it is appropriate to use the same amount. In addition, the combined weight of the low molecular weight polyol and the high molecular weight polyol and the amount of the polymer wall are 20 wt% based on the solvent. The solution in which the polyol is dissolved is placed in an aqueous solution in which a stabilizer is introduced and emulsified using a homogenizer. The emulsion was stirred under reduced pressure in a reduced pressure evaporator for 10 to 120 minutes to filter the dispersion after the solvent was completely removed to recover the capsules, and all other water-soluble substances were removed. The recovered capsules are completely dried for 24 to 48 hours in a room temperature depressurized dryer which is blocked from light.
또한, 상기의 방법으로 제조된 폴리올/고분자 마이크로캡슐을 효소 또는 단백질 안정화 마이크로캡슐로 활용할 수 있으며, 나아가 이 기술을 다른 효소 응용 기술에도 도입하는 것이 가능하다.In addition, the polyol / polymer microcapsules prepared by the above method can be utilized as enzyme or protein stabilizing microcapsules, and it is also possible to introduce this technique to other enzyme application techniques.
이를 위하여 효소가 내부 핵을 형성하고 이 효소는 소수성의 고분자량 폴리올에 의하여 코팅되어 있으며, 최종적으로 외각에 고분자가 벽제를 형성하는 구조를 지니고 있는 삼중 마이크로캡슐 구조가 가능하다.To this end, the enzyme forms an internal nucleus, which is coated with a hydrophobic high molecular weight polyol, and finally, a triple microcapsule structure having a structure in which the polymer forms a wall on the outer shell is possible.
상기의 삼중 마이크로캡슐의 제조 방법을 다음과 같다.The method for preparing the triple microcapsules is as follows.
즉, 효소를 저분자량 폴리올에 균일하게 분산시키는 단계; 상기 안정화된 효소/폴리올을 고분자 용액에서 재분산 시킨 후 유화과정을 거쳐 에멀젼을 수득하는 단계; 및 효소/폴리올/고분자 용액을 고화시켜 경질 고분자 마이크로캡슐로 회수하는 단계;를 포함한다.That is, uniformly dispersing the enzyme in a low molecular weight polyol; Redispersing the stabilized enzyme / polyol in a polymer solution to obtain an emulsion through an emulsification process; And solidifying the enzyme / polyol / polymer solution to recover the hard polymer microcapsules.
이하 본 제조 방법을 좀 더 구체적으로 살펴보면 다음과 같다.Hereinafter, the present manufacturing method will be described in more detail.
먼저, 저분자량 폴리올에 효소를 분산시킨다. 일반적으로 효소는 저분자량 폴리올에 대하여 부분적인 용해력을 지니고 있다. 이 경우, 효소는 저분자량 폴리올상에서 비교적 높은 젖음성으로 인하여 구형 분산체를 형성하고 외각층만 부분적으로 용해되어 효소/폴리올 혼합상을 형성한다. 이 효소/폴리올 분산액을 고분자량 폴리올이 녹아 있는 고분자 용액에 이어 분산시킨다. 상기 고분자 용액은 고분자량 폴리올, 고분자 벽제(벽제용 고분자) 및 용매를 함유한다. 이러한 공정을 거쳐 저분자량 폴리올에 의하여 보호되는 효소상은 고분자 용액의 용매에 대하여 영향을 받지 않고 안정하게 분산될 수 있다. 상기 고분자량 폴리올은 최종 마이크로캡슐에서 효소와 소수성 고분자 벽제(벽제용 고분자) 사이의 직접적인 접촉을 방지하는 완충작용을 한다. 이어지는 공정에선 효소/폴리올/고분자/용매로 구성되어 있는 액적에서 용매만 선택적으로 제거한다. 용매가 제거됨에 따라, 폴리올은 고분자에 대하여 비상용성을 지니고 있기 때문에, 상분리 현상이 발생한다. 이 상분리 과정에서 저분자량 폴리올은 높은 극성으로 인하여 약화된 외각 계면을 통하여 외수상으로 유출되고 마이크로캡슐 내부에는 고분자량 폴리올 상이 남게 된다. 결과적으로 마이크로캡슐은 효소가 내부 핵을 형성하고 이 효소는 고분자량 폴리올에 의하여 코팅되어 있고, 최종적으로는 외각에 고분자가 벽제를 형성하는 구조를 지니고 있는 삼중 마이크로캡슐 구조이다.First, the enzyme is dispersed in a low molecular weight polyol. In general, enzymes have partial solubility in low molecular weight polyols. In this case, the enzyme forms a spherical dispersion due to the relatively high wettability on the low molecular weight polyol and only the outer layer partially dissolves to form the enzyme / polyol mixed phase. This enzyme / polyol dispersion is dispersed following the polymer solution in which the high molecular weight polyol is dissolved. The polymer solution contains a high molecular weight polyol, a polymer wall (wall polymer) and a solvent. Through this process, the enzymatic phase protected by the low molecular weight polyol can be stably dispersed without being influenced by the solvent of the polymer solution. The high molecular weight polyols act as a buffer to prevent direct contact between enzymes and hydrophobic polymer walls (wall polymers) in the final microcapsules. In the following process, only the solvent is selectively removed from the droplet consisting of enzyme / polyol / polymer / solvent. As the solvent is removed, since the polyol is incompatible with the polymer, phase separation occurs. In this phase separation process, the low molecular weight polyol is discharged to the external water phase through the weakened outer interface due to the high polarity, and the high molecular weight polyol phase remains inside the microcapsules. As a result, the microcapsule is a triple microcapsule structure in which an enzyme forms an inner nucleus, which is coated by a high molecular weight polyol, and finally a polymer forms a wall on its outer shell.
즉, 본 발명에서는 기존의 연구들이 제안하는 단순한 캡슐화에서 탈피하여 효소 자체의 구조적 특징을 그대로 유지하고 외부 자극과의 상호작용을 근본적으로 차단하기 위하여, 저분자량 폴리올을 마이크로캡슐의 내부 기공형성 템플레이트(template) 및 효소의 분산매로 사용하고, 동시에 고분자량 폴리올을 효소의 소수성 분배화제 및 벽제를 형성하는 고분자의 내벽에 의한 변성을 차단하는 차단제로 도입함으로써, 생분해성 고분자 마이크로캡슐 내에서 제조 공정과 보관 중에 우수한 효소의 안정성을 유지할 수 있다.That is, in the present invention, in order to maintain the structural characteristics of the enzyme itself and to fundamentally block interaction with external stimuli by escaping from the simple encapsulation proposed by the existing studies, low molecular weight polyols are formed in the internal pore forming template of the microcapsule ( process and storage in biodegradable polymer microcapsules by using as a template and a dispersion medium of enzymes, and simultaneously introducing high molecular weight polyols as hydrophobic dispersing agents of enzymes and blocking agents that block denaturation by the inner walls of polymers forming walls. Stability of the enzyme can be maintained.
또한 본 발명에서 제조한 효소 안정화 마이크로캡슐은 제약 및 화장품 조성에서 유용하게 사용될 수 있을 것으로 기대된다.In addition, it is expected that the enzyme stabilized microcapsules prepared in the present invention may be usefully used in pharmaceutical and cosmetic compositions.
본 발명에서 적용 가능한 효소는 글루코오스옥시다아제, 크산틴옥시다아제, D-아미노산옥시다아제 등과 같은 산화환원효소류, 트랜스아미나아제, 핵소키나아제 등과 같은 전이효소류, 리파제, 아밀라아제, 펩신, 트립신, 우레아제, 아스파라기나아제, 파파인 등과 같은 가수분해효소류, 알도라아제, 푸마라아제, 펙틴리아제 등과 같은 리아제류, 락테이트 케탈 아이소메라아제, 락테이트 레세마아제, 유디피-디-글루코오스-4-에피메라아제 등과 같은 이성질화효소류, 아스파르테이트암모니아니카아제, DNA 리가아제 등과 같은 합성효소류 등의 효소를 포함한다. 상기 효소들은 하나 또는 둘 이상을 혼합하여 사용하는 것이 가능하다.Enzymes applicable in the present invention are redox enzymes such as glucose oxidase, xanthine oxidase, D-amino acid oxidase, transfer enzymes such as transaminase, nucleokinase, lipase, amylase, pepsin, trypsin, urease, asparaginana Hydrolyase such as papain, papain and the like, lyases such as aldorase, fumarase, pectinase, etc., lactate ketal isomerase, lactate resease, udapi-di-glucose-4-epimerase Enzymes such as isomerases such as isomers, aspartate ammonia kinase, and synthetic enzymes such as DNA ligase. It is possible to use one or more of these enzymes in combination.
본 발명에서는 고분자량 폴리올과 저분자량 폴리올을 혼합하여 사용한다. 저분자량 폴리올은 효소를 균일하게 분산시키고 캡슐 형성과정에서 캡슐외부로 유출되어 캡슐을 보다 효과적으로 형성시켜주는 템플레이트 역할을 수행한다. 사용 가능한 저분자량 폴리올은 1000g/mol 이하의 폴리에테르 형태의 고분자로서, 구체적으로는, 폴리에틸렌글리콜, 폴리프로필렌글리콜과 그들의 공중합체 및 유도체들, 부틸렌글리콜, 프로필렌글리콜, 글리세린 등을 포함하는 모든 저분자량의 알코올기를 포함하는 화합물이 해당된다. 사용량은 캡슐 총량 대비 0.1 ∼ 70 중량%가 적절하다. 0.1 중량% 이하의 농도에서는 효소를 효과적으로 분산시키기가 어렵고, 70 중량% 이상의 농도에서는 최종 마이크로캡슐의 수율이 낮아지는 문제점이 있다.In the present invention, a high molecular weight polyol and a low molecular weight polyol are mixed and used. The low molecular weight polyol uniformly disperses the enzyme and flows out of the capsule during capsule formation to serve as a template for forming the capsule more effectively. Low molecular weight polyols that can be used are polymers in the form of polyethers of 1000 g / mol or less, specifically, all low molecular weights including polyethylene glycol, polypropylene glycol and their copolymers and derivatives, butylene glycol, propylene glycol, glycerin, and the like. The compound containing the alcohol group of molecular weight corresponds. The amount of use is appropriately 0.1 to 70% by weight based on the total amount of the capsule. At concentrations of 0.1 wt% or less, it is difficult to effectively disperse the enzyme, and at a concentration of 70 wt% or more, the yield of the final microcapsules is lowered.
고분자량 폴리올은 캡슐화 과정에서도 외상으로의 유출 없이 캡슐 내부에 존재하여 효소에 대하여 소수성 분배효과를 부여하고 효소가 소수성 고분자 내벽에 의하여 변성되는 것을 차단하는 역할을 한다. 폴리올류의 분자량이 증가할 경우 폴리올 사슬의 소수성 또한 비례하여 증가한다. 따라서, 이러한 고분자량 폴리올이 효소와 소수성 고분자 내벽에 효과적으로 위치하였을 경우 소수성 분배 효과에 의하여 효소를 효과적으로 보호할 수 있다. 이러한 소수성 분배 효과를 유도하기 위해서는 폴리올의 분자량이 충분히 높아야만 한다. 적절한 폴리올은 분자량이 1000g/mol 이상의 왁스형태의 폴리에테르 고분자로서, 구체적으로는 폴리에틸렌글리콜, 폴리프로필렌글리콜과 그들의 공중합체 및 유도체들이 이에 해당한다. 사용량은 캡슐 총량 대비 0.1 ∼ 90 중량%가 적절하다. 0.1 중량% 이하의 농도에서는 효과적인 소수성 분배 효과를 기대하기 어렵고, 반면에 90 중량% 이상의 농도에서는 높은 함량으로 인한 경질 마이크로캡슐의 형성이 어렵다.The high molecular weight polyol is present in the capsule even without encapsulation into the trauma to impart hydrophobic partitioning effect to the enzyme and to prevent the enzyme from being denatured by the hydrophobic polymer inner wall. As the molecular weight of the polyols increases, the hydrophobicity of the polyol chains also increases proportionally. Therefore, when the high molecular weight polyol is effectively located on the inner wall of the enzyme and the hydrophobic polymer, the enzyme can be effectively protected by the hydrophobic partitioning effect. In order to induce this hydrophobic partitioning effect, the molecular weight of the polyol must be high enough. Suitable polyols are polyether polymers in the form of waxes having a molecular weight of 1000 g / mol or more, specifically polyethylene glycol, polypropylene glycol and their copolymers and derivatives. The amount of use is appropriately 0.1 to 90% by weight relative to the total amount of the capsule. At concentrations below 0.1% by weight it is difficult to expect effective hydrophobic dispensing effects, whereas at concentrations above 90% by weight it is difficult to form hard microcapsules due to their high content.
상기 과정 중, 마이크로캡슐의 벽제로 사용되는 고분자(고분자 벽제)는 생분해성 소수성 지방족 폴리에스테르로, 구체적으로는, 폴리-L-락트산, 폴리-D,L-글리콜산, 폴리-L-락트산-co-글리콜산, 폴리-D,L-락트산-co-글리콜산, 폴리카프로락톤, 폴리발레로락톤, 폴리하이드록시 부티레이트, 폴리하이드록시발러레이트, 폴리오르터에스테르 및 이들의 단량체로부터 제조된 공중합체를 포함한다. 또한, 마이크로 캡슐의 벽제로 사용한 가능한 고분자로(고분자 벽제)는 폴리스티렌, 폴리 p- 또는 m-메틸스티렌, 폴리 p- 또는 m-에틸스티렌, 폴리 p- 또는 m-클로로스티렌, 폴리 p- 또는 m-클로로메틸스티렌, 폴리 스티렌설포닉 엑시드, 폴리 p- 또는 m- 또는 t-부톡시스티렌, 폴리 메틸(메타)아크릴레이트, 폴리에틸(메타)아크릴레이트, 폴리프로필(메타)아크릴레이트, 폴리n-부틸(메타)아크릴레이트, 폴리이소부틸(메타)아크릴레이트, 폴리t-부틸(메타)아크릴레이트, 폴리2-에틸헥실(메타)아크릴레이트, 폴리 n-옥틸(메타)아크릴레이트, 폴리라우릴(메타)아크릴레이트, 폴리스테아릴(메타)아크릴레이트, 폴리2-히드록시에틸(메타)아크릴레이트, 폴리에틸렌 글리콜(메타)아크릴레이트, 메톡시폴리에틸렌글리콜(메타)아크릴레이트, 폴리글리시딜(메타)아크릴레이트, 폴리디메틸아미노에틸(메타)아크릴레이트, 폴리디에틸아미노에틸(메타)아크릴레이트, 폴리비닐아세테이트, 폴리비닐프로피오네이트, 폴리비닐부티레이트, 폴리비닐에테르, 폴리알릴부틸에테르, 폴리알릴글리시딜에테르, 폴리(메타)아크릴산, 폴리말레인산과 같은 불포화카르복시산, 폴리알킬(메타)아크릴아마이드, 폴리(메타)아크릴로니트릴 등을 포함하며, 이들은 혼용하여 사용할 수 있다. 이러한 고분자 벽제는 총 캡슐 함량 대비 1 ∼ 99.99 중량%가 적절하다. 1 중량% 이하의 농도에서는 마이크로캡슐 형성이 어렵고, 99.99 중량% 이상의 농도에서는 효소의 함량이 너무 낮아 효과 발현이 어렵다.In the above process, the polymer (polymer wall) used as a wall of the microcapsules is a biodegradable hydrophobic aliphatic polyester, specifically, poly-L-lactic acid, poly-D, L-glycolic acid, poly-L-lactic acid- air prepared from co-glycolic acid, poly-D, L-lactic acid-co-glycolic acid, polycaprolactone, polyvalerolactone, polyhydroxy butyrate, polyhydroxyvalorate, polyetherester and monomers thereof Include coalescing. In addition, possible polymer furnaces (polymer wall) used as the wall of the microcapsules include polystyrene, poly p- or m-methylstyrene, poly p- or m-ethylstyrene, poly p- or m-chlorostyrene, poly p- or m -Chloromethylstyrene, polystyrenesulphonic acid, poly p- or m- or t-butoxystyrene, poly methyl (meth) acrylate, polyethyl (meth) acrylate, polypropyl (meth) acrylate, polyn -Butyl (meth) acrylate, polyisobutyl (meth) acrylate, polyt-butyl (meth) acrylate, poly 2-ethylhexyl (meth) acrylate, poly n-octyl (meth) acrylate, polyla Uryl (meth) acrylate, polystearyl (meth) acrylate, poly2-hydroxyethyl (meth) acrylate, polyethylene glycol (meth) acrylate, methoxypolyethylene glycol (meth) acrylate, polyglycidyl (Meth) acrylate , Polydimethylaminoethyl (meth) acrylate, polydiethylaminoethyl (meth) acrylate, polyvinylacetate, polyvinylpropionate, polyvinylbutyrate, polyvinylether, polyallylbutyl ether, polyallyl glycidyl Ethers, poly (meth) acrylic acids, unsaturated carboxylic acids such as polymaleic acid, polyalkyl (meth) acrylamides, poly (meth) acrylonitrile, and the like, which may be used in combination. Such polymer walls are suitably in the range of 1-99.99% by weight relative to the total capsule content. It is difficult to form microcapsules at a concentration of 1% by weight or less, and the concentration of enzyme is too low at a concentration of 99.99% by weight or more, so that the effect is difficult to express.
마이크로캡슐 제조 중에 사용되는 용매는 선택되는 고분자와 유사한 용해도 파라미더를 지니는 모든 화합물로서, 구체적으로는, 헥산, 헵탄, 옥탄, 노난, 데칸 등과 같은 선형 알칸류, 부탄올, 선형 또는 가지형 펜탄올, 헥산올, 헵탄올, 옥탄올, 노난올, 테칸올 등과 같은 탄소수 4∼10의 알콜류, n-헥실 아세테이트, 2-에틸헥실 아세테이트, 메틸 올리에이트, 디부틸 세바케이트, 디부틸 아디베이트, 이부틸 카바메이트와 같은 탄소수 7 이상의 알킬 에스테르, 메틸이소부틸케톤, 이소부틸케톤과 같은 지방족 케톤, 벤젠, 톨루엔, o- 또는 p-크실렌과 같은 방향족 탄화수소, 메틸렌클로라이드, 클로로포름, 사염화탄소 등과 같은 염소화합물 등을 포함한다.The solvents used during the preparation of the microcapsules are all compounds having a solubility parameter similar to the polymer of choice, specifically, linear alkanes such as hexane, heptane, octane, nonane, decane, butanol, linear or branched pentanol, C4-C10 alcohols such as hexanol, heptanol, octanol, nonanol, tecanol, etc., n-hexyl acetate, 2-ethylhexyl acetate, methyl oleate, dibutyl sebacate, dibutyl adsorbate, ibutyl Alkyl esters having 7 or more carbon atoms such as carbamate, aliphatic ketones such as methyl isobutyl ketone, isobutyl ketone, aromatic hydrocarbons such as benzene, toluene, o- or p-xylene, chlorine compounds such as methylene chloride, chloroform, carbon tetrachloride, etc. Include.
마이크로캡슐 제조에 사용하는 안정화제는 수상에 도입하여 캡슐의 분산 안정성을 향상시킬 수 있는 모든 수용성 고분자들로서, 아라빅(arabic), 트래거캔스(tragacanth), 카라야 (karaya), 라치(larch), 가티(ghatti), 로커스트 빈(locust bean), 구아(guar), 아가(agar), 알지네이트(alginate), 카라기난(carrageenan), 퍼셀러랜(furcellaran), 펙틴(pectin), 젤라틴(gelatin), 스타치(starch) 및 그 유도체; 미생물 발효 합성법에 의하여 제조된 덱스트란(dextran), 잔탄 검(xanthan gum) 및 그 유도체; 및 라디칼 또는 개환중합법에 의하여 제조된 비닐고분자, 아크릴고분자, 폴리올 함유 공중합체 및 그 유도체를 포함하며, 이들 중에서 하나 이상을 선택하여 사용할 수 있다. 바람직하게는 폴리비닐알콜을 사용할 수 있다. 상기 분산 안정화제는 마이크로캡슐 분산 수용액의 질량에 대하여 0.01%∼30중량%를 첨가할 수 있다. 상기 농도 이하의 농도범위에서는 우수한 분산 안정성을 기대하지 어렵고, 그 이상의 농도범위에서는 겔화가 진행되어 마이크로캡슐의 제조공정을 진행하기가 어렵다.Stabilizers used in the manufacture of microcapsules are all water-soluble polymers that can be introduced into the water phase to improve the dispersion stability of the capsule, such as arabic, tragacanth, karaya, and lach. , Ghatti, locust bean, guar, agar, alginate, carrageenan, furcellaran, pectin, gelatin, Starch and its derivatives; Dextran, xanthan gum and derivatives thereof prepared by microbial fermentation synthesis; And vinyl polymers, acrylic polymers, polyol-containing copolymers and derivatives thereof prepared by radical or ring-opening polymerization, and one or more of them can be selected and used. Preferably polyvinyl alcohol can be used. The dispersion stabilizer may add 0.01% to 30% by weight based on the mass of the microcapsule dispersion aqueous solution. It is difficult to expect excellent dispersion stability in the concentration range below the concentration, it is difficult to proceed the manufacturing process of the microcapsules due to the gelation proceeds in the concentration range above.
이하 실시예 및 비교예를 통하여 본 발명을 보다 더 구체적으로 설명하지만, 본 발명이 이들 예로만 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited only to these examples.
<실시예 1> 폴리에틸렌글리콜 함유 폴리카프로락톤 마이크로캡슐의 제조<Example 1> Preparation of polyethylene glycol-containing polycaprolactone microcapsules
분자량 400, 4000, 8000, 12000 g/mol의 폴리에틸렌글리콜을 각각 고분자 벽제인 폴리카프로락톤 (80000g/mol 분자량)과 1:1의 중량비로 용매인 메틸렌클로라이드에 넣고 실온 교반하여 완전 용해시킨다. 폴리에틸렌글리콜과 폴리카프로락톤은 메틸렌클로라이드에 대하여 15 중량% 도입한다. 제조된 각각의 용액을 다시 1%의 폴리비닐알콜(평균 검화도 89%)이 녹아 있는 수용액에 넣고 기계식 호모게나이저를 이용하여 5000rpm에서 5분간 유화시킨다. 이 때, 폴리에틸렌글리콜/ 폴리카프로락톤/메틸렌클로라이드 용액은 수상에서 30 중량%의 농도를 갖는다. 유화가 끝난 후, 유화액은 감압증발기로 옮겨 실온에서 30분간 감압교반하여 용매인 메틸렌클로라이드를 완전 제거한다. 감압증발 공정이 끝난 후, 분산액은 여과지를 통과시켜 여과시켜 캡슐만 회수하고 물을 포함하는 모든 수용성 물질은 제거한다. 회수된 캡슐은 실온 감압 건조기에서 1일 동안 건조한다.Polyethylene glycols having a molecular weight of 400, 4000, 8000, and 12000 g / mol are respectively dissolved in polymprolactone (80000 g / mol molecular weight), which is a polymer wall, and in a methylene chloride, which is a solvent, in a weight ratio of 1: 1. Polyethylene glycol and polycaprolactone are introduced in an amount of 15% by weight based on methylene chloride. Each prepared solution was put in an aqueous solution of 1% polyvinyl alcohol (average degree of saponification of 89%) and emulsified at 5000 rpm for 5 minutes using a mechanical homogenizer. At this time, the polyethylene glycol / polycaprolactone / methylene chloride solution has a concentration of 30% by weight in the water phase. After the completion of the emulsification, the emulsion is transferred to a reduced pressure evaporator and stirred under reduced pressure at room temperature for 30 minutes to completely remove methylene chloride as a solvent. After completion of the vacuum evaporation process, the dispersion is filtered through filter paper to recover only the capsules and remove all water-soluble substances, including water. The recovered capsules are dried for 1 day in a room temperature reduced pressure dryer.
<실시예 2> 폴리에틸렌글리콜 함유 폴리메틸메타클리레이트 마이크로캡슐의 제조Example 2 Preparation of Polyethylene Glycol Microcapsules
분자량 400, 4000, 8000, 12000 g/mol의 폴리에틸렌글리콜을 각각 고분자 벽제인 폴리메틸메타크릴레이트(75000g/mol 분자량)와 1:1의 중량비로 용매인 메틸렌클로라이드에 넣고 실온 교반하여 완전 용해시킨다. 폴리에틸렌글리콜과 폴리메틸메타크릴레이트는 메틸렌클로라이드에 대하여 20 중량% 도입한다. 이하 과정은 실시예 1과 동일하게 진행하여 폴리에틸렌글리콜/폴리메틸메타크릴레이트 마이크로캡슐을 제조한다.Polyethylene glycols having a molecular weight of 400, 4000, 8000, and 12000 g / mol are respectively dissolved in polymethylene methacrylate (75000 g / mol molecular weight), which is a polymer wall, and in a weight ratio of 1: 1 to methylene chloride, which is a solvent, to be completely dissolved by stirring at room temperature. Polyethylene glycol and polymethyl methacrylate are introduced in an amount of 20% by weight based on methylene chloride. The procedure is the same as in Example 1 to prepare a polyethylene glycol / polymethyl methacrylate microcapsules.
<실시예 3> 폴리에틸렌글리콜 함유 폴리스티렌 마이크로캡슐의 제조Example 3 Preparation of Polyethylene Glycol-Containing Polystyrene Microcapsules
분자량 400, 4000, 8000, 12000 g/mol의 폴리에틸렌글리콜을 각각 고분자 벽제인 폴리스티렌 (100000g/mol 분자량)와 1:1의 중량비로 용매인 메틸렌클로라이드에 넣고 실온 교반하여 완전 용해시킨다. 폴리에틸렌글리콜과 폴리스티렌은 메틸렌클로라이드에 대하여 20 중량% 도입한다. 이하 과정은 실시예 1과 동일하게 진행하여 폴리에틸렌글리콜/폴리스티렌 마이크로캡슐을 제조한다.Polyethylene glycols having a molecular weight of 400, 4000, 8000, and 12000 g / mol are respectively dissolved in polystyrene (100000 g / mol molecular weight), which is a polymer wall, and in a methylene chloride, which is a solvent, in a weight ratio of 1: 1. Polyethylene glycol and polystyrene are introduced in an amount of 20% by weight based on methylene chloride. The procedure is the same as in Example 1 to prepare a polyethylene glycol / polystyrene microcapsules.
<실시예 4> 2종 폴리에틸렌글리콜 함유 폴리카프로락톤 마이크로캡슐의 제조Example 4 Preparation of Two Polyethylene Glycol-Containing Polycaprolactone Microcapsules
저분자량 폴리올인 분자량 400g/mol의 폴리에틸렌글리콜과 고분자량 폴리올인 8000 g/mol의 폴리에틸렌글리콜을 7:3, 5:5, 3:7의 중량비로 혼합하여 고분자 벽제와 용매로 이루어진 폴리카프로락톤/메틸렌클로라이드 고분자 용액에 도입한다. 이때 상기의 저분자량 및 고분자량 폴리에틸렌글리콜을 더한 중량과 고분자 벽제인 폴리카프로락톤의 중량은 동량을 사용한다. 또한 상기의 폴리에틸렌글리콜과 폴리카프로락톤은 용매인 메틸렌클로라이드에 대하여 20 중량% 도입한다. 이하 과정은 실시예 1과 동일하게 진행하여 폴리에틸렌글리콜/폴리카프로락톤 마이크로캡슐을 제조한다.Polycaprolactone / consisting of a polymer wall and a solvent by mixing polyethylene glycol having a molecular weight of 400 g / mol, which is a low molecular weight polyol, and polyethylene glycol having a high molecular weight, of 8000 g / mol, with a weight ratio of 7: 3, 5: 5, 3: 7. Introduced to methylene chloride polymer solution. At this time, the weight of the low molecular weight and high molecular weight polyethylene glycol plus the weight of the polycaprolactone which is a polymer wall is used in the same amount. In addition, the polyethylene glycol and polycaprolactone are introduced by 20% by weight based on the methylene chloride as a solvent. The following process was carried out in the same manner as in Example 1 to prepare a polyethylene glycol / polycaprolactone microcapsules.
<실시예 5> 파파인 함유 폴리에틸렌글리콜/폴리카프로락톤 마이크로캡슐의 제조Example 5 Preparation of Papain-Containing Polyethylene Glycol / Polycaprolactone Microcapsules
파파인을 모델 효소로 선정하여 사용한다. 파파인을 먼저 분자량 400 g/mol의 저분자량 폴리에틸렌글리콜에 분산시킨다. 이 때 파파인의 함량은 전체 캡슐 중량 대비 1, 3, 5 중량%로 각각 도입한다. 이어서, 분자량 8000g/mol의 고분자량 폴리에틸렌글리콜과 고분자 벽제인 폴리카프로락톤이 녹아있는 메틸렌클로라이드 수용액에 파파인/폴리에틸렌글리콜 분산체를 재분산시킨다. 본 과정에서 사용되는 성분들의 함량은 실시예 4의 조성을 따른다. 이하 과정은 실시예 1과 동일하게 진행하여 파파인 효소 함유 폴리에틸렌글리콜/폴리카프로락톤 마이크로캡슐을 제조한다.Papain is used as a model enzyme. Papain is first dispersed in low molecular weight polyethylene glycols having a molecular weight of 400 g / mol. At this time, the content of papain is introduced in 1, 3, 5% by weight relative to the total capsule weight. Subsequently, the papain / polyethylene glycol dispersion is redispersed in the methylene chloride aqueous solution in which a high molecular weight polyethylene glycol having a molecular weight of 8000 g / mol and a polycaprolactone as a polymer wall are dissolved. The content of the components used in this process is according to the composition of Example 4. The following procedure was carried out in the same manner as in Example 1 to prepare a papain enzyme-containing polyethylene glycol / polycaprolactone microcapsules.
<시험예 1> 폴리에틸렌글리콜/고분자 마이크로캡슐의 특성 분석Test Example 1 Characterization of Polyethylene Glycol / Polymer Microcapsules
실시예 1-3에서 제조된 폴리에틸렌글리콜/고분자 마이크로캡슐의 캡슐형태를 광학현미경으로 관찰하였다. 도 1에 나타내었듯이 제조된 폴리에틸렌글리콜/고분자 마이크로캡슐은 5 ∼ 20mm의 평균입자크기를 지니는 구형체이다. 각각의 구형체는 내부에 폴리에틸렌글리콜 도메인을 형성하고 있다. 하지만, 도입되는 폴리에틸렌글리콜의 함량은 분자량에 의존하는 결과를 나타내었다. 폴리에틸렌글리콜 분자량에 따른 고분자 마이크로캡슐 내 도입량 효과를 박층크로마토그래피 (Thin layer chromatography)를 이용하여 정량적으로 분석하여 표 1에 정리하였다.The capsule form of the polyethyleneglycol / polymer microcapsules prepared in Examples 1-3 was observed under an optical microscope. As shown in FIG. 1, the prepared polyethylene glycol / polymer microcapsules are spherical bodies having an average particle size of 5 to 20 mm. Each sphere forms a polyethyleneglycol domain therein. However, the content of polyethylene glycol introduced showed a result depending on the molecular weight. The effect of the amount introduced into the polymer microcapsules according to the molecular weight of polyethylene glycol was summarized in Table 1 by quantitative analysis using thin layer chromatography.
표 1에서 알 수 있듯이 폴리에틸렌글리콜 도입량은 분자량에 크게 의존하였다. 분자량이 증가할수록 폴리에틸렌글리콜 분자의 극성도가 감소하면서 도입량이 증가하는 결과를 얻었다. 그러나, 광학현미경 사진에서는 저분자량의 폴리에틸렌글리콜을 이용할 경우에도 내부에 도메인을 형성하는 결과를 얻었다. 이러한 마이크로캡슐 형성 거동은 폴리에틸렌글리콜이 용매의 제거 과정 중 캡슐 내부에서 상분리되어 특정 도메인을 형성하였다가 서서히 다시 캡슐 외부로 유출되기 때문이다. 이러한 저분자량의 폴리에틸렌글리콜의 거동은 캡슐 형성 과정 중 내부 도메인 형성을 유도하는 템플레이트 기능을 하는 것으로 판단된다. 고분자량의 폴리에틸렌글리콜은 공정에 상관없이 내부에 우수한 담지 결과를 보였다.As can be seen from Table 1, the amount of polyethylene glycol introduced was highly dependent on the molecular weight. As the molecular weight increased, the polarization of the polyethyleneglycol molecules decreased, and the amount of introduction increased. However, in the optical micrograph, even when low molecular weight polyethylene glycol was used, the result of forming a domain inside was obtained. This microcapsule formation behavior is because polyethylene glycol phase separates inside the capsule during the removal of the solvent to form a specific domain and then slowly flows out again. The low molecular weight polyethylene glycol is believed to have a template function to induce internal domain formation during capsule formation. High molecular weight polyethylene glycol showed excellent supporting results inside regardless of the process.
<시험예 2> 폴리에틸렌글리콜 혼합비에 따른 함량 분석Test Example 2 Content Analysis According to Polyethylene Glycol Mixing Ratio
폴리에틸렌글리콜 혼합비에 따른 캡슐 내 함유 거동을 확인하기 위하여 실시예 4에서 제조된 폴리에틸렌글리콜/폴리카프로락톤 마이크로캡슐을 박층크로마코그래피를 이용하여 정량 분석하여 그 결과를 표 2에 정리하였다. 폴리에틸렌글리콜 캡슐 내 도입량은 혼합비에 따라 검출되었다. 하지만, 도 2의 광학현미경 사진 분석에서는 캡슐 형태는 혼합비에 상관없이 관찰할 수 있었다. 이러한 결과는 저분자량의 폴리에틸렌글리콜은 캡슐 내부에서 도메인 형성을 위한 템플레이트 역할을 하고 고분자량의 폴리에틸렌글리콜은 그 템플레이트에 그대로 남아 있음을 의미한다.In order to confirm the behavior of the capsule contained in the polyethylene glycol mixing ratio, the polyethylene glycol / polycaprolactone microcapsules prepared in Example 4 were quantitatively analyzed using thin layer chromatography, and the results are summarized in Table 2. The amount introduced into the polyethylene glycol capsule was detected according to the mixing ratio. However, in the optical micrograph analysis of FIG. 2, the capsule form could be observed regardless of the mixing ratio. These results indicate that low molecular weight polyethylene glycol serves as a template for domain formation in the capsule and high molecular weight polyethylene glycol remains in the template.
<시험예 3> 폴리에틸렌글리콜/폴리카프로락톤 캡슐 내 파파인 도입량 분석<Test Example 3> Papain introduction amount analysis in the polyethylene glycol / polycaprolactone capsule
실시예 5에서 제조된 파파인 함유 폴리에틸렌글리콜/폴리카프로락톤 마이크로캡슐의 광학현미경 사진을 도 3에 나타내었다. 도 1과는 달리 제조된 마이크로캡슐 내부에 파파인 영역을 쉽게 관찰할 수 있다. 도 4에는 파파인 함유 마이크로캡슐의 주사전자현미경 사진을 나타내었다. 캡슐 외벽은 매끄러운 폴리카프로락톤으로 형성되어 있고 파파인 또는 폴리에틸렌글리콜의 캡슐 외부로의 유출은 전혀 관찰되지 않아, 파파인과 폴리에틸렌글리콜이 폴리카프로락톤 벽제에 의하여 효과적으로 포집되어 있음을 알 수 있다. 마이크로캡슐 내 파파인의 도입량은 다음과 같이 분석하였다. 먼저 파파인 함유 마이크로캡슐 50ml을 디메틸설폭사이드 1ml에 넣고 1시간 동안 인규베이션한 후, 0.05% 소듐도데실설페이트/0.01N 소듐하이드록사이드 용액 2ml를 첨가해 실온에서 1시간 방치해서 고분자와 효소를 완전 녹인다. 이어서 마이크로-BCA를 이용하여 단백질 정량한다. 이러한 분석법으로 정량 분석한 파파인 함량은 다음 표 3에 나타내었다. 표 3에서 알 수 있듯이, 본 발명에서 제안하는 방법은 효소를 효과적으로 포집함을 잘 나타내고 있다.An optical micrograph of the papain-containing polyethylene glycol / polycaprolactone microcapsules prepared in Example 5 is shown in FIG. 3. Unlike in FIG. 1, the papain region can be easily observed inside the manufactured microcapsules. 4 shows a scanning electron micrograph of papain-containing microcapsules. The capsule outer wall is formed of smooth polycaprolactone and no leakage of papain or polyethylene glycol to the outside of the capsule is observed, indicating that papain and polyethylene glycol are effectively collected by the polycaprolactone wall. The amount of papain introduced into the microcapsules was analyzed as follows. First, 50 ml of papain-containing microcapsules were added to 1 ml of dimethyl sulfoxide, incubated for 1 hour, and then 2 ml of 0.05% sodium dodecyl sulfate / 0.01 N sodium hydroxide solution was added and left at room temperature for 1 hour to completely polymer and enzyme. Dissolve. Protein is then quantified using micro-BCA. Papain content quantified by this method is shown in Table 3 below. As can be seen from Table 3, the method proposed in the present invention shows that the enzyme is effectively captured.
<시험예 4> 폴리에틸렌글리콜/폴리카프로락톤 캡슐 내 파파인 안정도 분석Test Example 4 Analysis of Papain Stability in Polyethyleneglycol / Polycaprolactone Capsules
실시예 5에서 제조된 폴리에틸렌글리콜/폴리카프로락톤 마이크로캡슐 내에서 파파인의 활성도를 저장 온도에 따라 확인하였다. 본 경우에는 파파인 3% 함유 마이크로캡슐을 선택하여 활성도를 측정하였다. 캡슐 내 파파인의 활성도는 다음과 같이 분석하였다. 아세톤 1ml에 파파인 함유 마이크로 캡슐 20ml을 넣고 10초간 초음파를 주사하였다. 이어서, 12000rpm에서 5분간 원심분리하였다. 이 과정을 3회 반복하여 순수 효소만을 얻었다. 얻어진 순수 효소는 카제인을 기질로 280nm에서 UV 흡광기를 이용하여 활성도를 측정하였다. 저장 조건에 따라 얻어진 파파인의 활성도를 다음 표 4에 나타내었다. 제조된 파파인 함유 폴리에틸렌글리콜/폴리카프로락톤 마이크로캡슐은 장기 저장 조건에서도 우수한 활성도를 나타내었다. 이러한 우수한 활성도는 폴리에틸렌글리콜과 같은 폴리올이 마이크로캡슐 내에서 고체형태의 효소를 효과적으로 안정화한다는 것을 의미한다. 효소와 폴리카프로락톤과 같은 고분자 벽제 사이에 폴리올이 적절하게 위치하여 소수성 분배 효과를 부여함으로써 효소와 고분자 간의 직접적인 상호작용을 차단하여 결과적으로 효소의 안정도를 향상시키는 것으로 해석된다.Papain activity in the polyethyleneglycol / polycaprolactone microcapsules prepared in Example 5 was confirmed according to storage temperature. In this case, papain 3% containing microcapsules were selected to measure the activity. Papain activity in the capsule was analyzed as follows. 20 ml of papain-containing microcapsules were placed in 1 ml of acetone, and ultrasonic waves were injected for 10 seconds. It was then centrifuged for 5 minutes at 12000 rpm. This process was repeated three times to obtain only pure enzyme. The obtained pure enzyme was measured for activity using a UV absorber at 280 nm as a casein substrate. The papain activity obtained according to the storage conditions is shown in Table 4 below. Papain-containing polyethyleneglycol / polycaprolactone microcapsules showed good activity even under long-term storage conditions. This good activity means that polyols such as polyethylene glycol effectively stabilize the enzyme in solid form in the microcapsules. It is interpreted that the polyol is properly positioned between the enzyme and the polymer wall such as polycaprolactone to impart hydrophobic partitioning effect, thereby blocking the direct interaction between the enzyme and the polymer and consequently improving the stability of the enzyme.
이상에서 상술한 바와 같이, 폴리올/고분자 마이크로캡슐은 내부에 효소성분을 효과적으로 도입할 수 있는 시스템이다. 특히, 폴리올의 분자량을 적절히 조절하여 캡슐 형성 중 템플레이트 역할과 효소 성분 도입 후 효소와 소수성 고분자 벽제 사이에서 소수성 분배 효과를 부여하여 효소의 변성을 막을 수 있다는 면에서 큰 의미를 찾을 수 있다. 또한, 제조된 효소함유 폴리올/고분자 마이크로캡슐은 담지 효소에 대한 우수한 안정도를 부여하여 실질적인 응용에 있어서 효소의 활용도를 더욱 높일 것으로 기대된다. 특히, 효소를 촉매로 사용하는 다양한 바이오엔지니어링 분야에서 반응 제어 시스템으로 활용 가능하고 효소를 분해 촉매로 사용하는 피부과학 분야에서는 각질 케어 및 타 유효성분 피부 흡수 가속화제로 의미 있게 활용될 것으로 기대된다.As described above, the polyol / polymer microcapsules are a system capable of effectively introducing an enzyme component therein. In particular, the molecular weight of the polyol can be appropriately controlled to provide a hydrophobic partition effect between the enzyme and the hydrophobic polymer wall after introduction of the enzyme component and the enzyme component during capsule formation. In addition, the prepared enzyme-containing polyol / polymer microcapsules are expected to give excellent stability to the supported enzyme to further increase the utilization of the enzyme in practical applications. In particular, it is expected to be used as a reaction control system in various bioengineering fields using enzymes as catalysts, and in dermatology fields using enzymes as decomposition catalysts.
도 1은 폴리에틸렌글리콜(400 g/mol)/폴리카프로락톤 마이크로캡슐의 광학현미경 사진이다.1 is an optical micrograph of polyethylene glycol (400 g / mol) / polycaprolactone microcapsules.
도 2는 폴리에틸렌글리콜 혼합비를 조절한 폴리에틸렌글리콜/폴리카프로락톤 마이크로캡슐의 광학현미경 사진이다(400 g/mol : 8000 g/mol = 5:5 (w/w)).Figure 2 is an optical micrograph of the polyethylene glycol / polycaprolactone microcapsules with a controlled polyethylene glycol mixing ratio (400 g / mol: 8000 g / mol = 5: 5 (w / w)).
도 3은 파파인 함유 폴리에틸렌글리콜/폴리카프로락톤 마이크로캡슐의 광학현미경 사진이다.3 is an optical micrograph of papain-containing polyethylene glycol / polycaprolactone microcapsules.
도 4는 파파인 함유 폴리에틸렌글리콜/폴리카프로락톤 마이크로캡슐의 주사전자현미경 사진이다.4 is a scanning electron micrograph of papain-containing polyethylene glycol / polycaprolactone microcapsules.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5385959A (en) * | 1992-04-29 | 1995-01-31 | Lever Brothers Company, Division Of Conopco, Inc. | Capsule which comprises a component subject to degradation and a composite polymer |
| WO1995028092A1 (en) * | 1994-04-18 | 1995-10-26 | Gist-Brocades B.V. | Stable water-in-oil emulsions |
| US5691060A (en) * | 1992-08-20 | 1997-11-25 | Coletica | Utilization of a transacylation reaction between an esterified polysaccharide and a polyaminated or polyhydroxylated substance for fabricating microparticles, microparticles thus obtained, methods and compositions containing them |
| KR0145846B1 (en) * | 1995-02-20 | 1998-08-17 | 한동근 | Capsule containing liquid crystal of polymer silicone gum and cosmetic composition comprising |
| WO2000048573A1 (en) * | 1999-02-19 | 2000-08-24 | Bioserv Ag | Biodegradable composite material for the production of microcapsules |
| US20020018812A1 (en) * | 2000-06-27 | 2002-02-14 | Patrick Busson | Process for preparing a pharmaceutical composition |
-
2002
- 2002-11-25 KR KR10-2002-0073433A patent/KR100517072B1/en active IP Right Grant
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5385959A (en) * | 1992-04-29 | 1995-01-31 | Lever Brothers Company, Division Of Conopco, Inc. | Capsule which comprises a component subject to degradation and a composite polymer |
| US5691060A (en) * | 1992-08-20 | 1997-11-25 | Coletica | Utilization of a transacylation reaction between an esterified polysaccharide and a polyaminated or polyhydroxylated substance for fabricating microparticles, microparticles thus obtained, methods and compositions containing them |
| WO1995028092A1 (en) * | 1994-04-18 | 1995-10-26 | Gist-Brocades B.V. | Stable water-in-oil emulsions |
| KR0145846B1 (en) * | 1995-02-20 | 1998-08-17 | 한동근 | Capsule containing liquid crystal of polymer silicone gum and cosmetic composition comprising |
| WO2000048573A1 (en) * | 1999-02-19 | 2000-08-24 | Bioserv Ag | Biodegradable composite material for the production of microcapsules |
| US20020018812A1 (en) * | 2000-06-27 | 2002-02-14 | Patrick Busson | Process for preparing a pharmaceutical composition |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101520642B1 (en) * | 2013-02-07 | 2015-05-18 | 주식회사김정문알로에 | Aloe gel microcapsule suspension, preparation thereof and cosmetic composition comprising same |
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