KR20050008323A - Chitosan complex having hydrophobic moiety and preparation method thereof - Google Patents
Chitosan complex having hydrophobic moiety and preparation method thereof Download PDFInfo
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- KR20050008323A KR20050008323A KR1020030048240A KR20030048240A KR20050008323A KR 20050008323 A KR20050008323 A KR 20050008323A KR 1020030048240 A KR1020030048240 A KR 1020030048240A KR 20030048240 A KR20030048240 A KR 20030048240A KR 20050008323 A KR20050008323 A KR 20050008323A
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- C—CHEMISTRY; METALLURGY
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
Abstract
Description
본 발명은 소수성기를 함유한 키토산 복합체 및 그의 제조방법에 관한 것이다.The present invention relates to a chitosan complex containing a hydrophobic group and a method for producing the same.
친수성과 소수성을 동시에 가지고 있는 양친성 고분자는 수용액상에서 계면에너지의 안정화를 위해 소수성 블록간의 상호작용을 통해 교질입자(micelle) 또는자기집합체(self-aggregate)를 형성하게 된다. 양친성 고분자에 의해 형성된 고분자 교질입자는 친수성 및 소수성의 정도에 따라 입자의 크기, 분포, 유동학적 성질, 및 열역학적 안정성 등이 다르게 나타나는 것으로 알려져 있다. 또한, 최근 이러한 교질입자 내부의 소수성 영역(hydrophobic domain)에 여러 가지 소수성 약물을 봉입하여 약물의 선택적 및 효과적인 전달을 유도할 수 있다고 보고되어 있다(Adv. Drug. Deliv. Rev. 2001,47, 113-131).Amphiphilic polymers having both hydrophilicity and hydrophobicity form a microelle or self-aggregate through interaction between hydrophobic blocks to stabilize interfacial energy in an aqueous solution. Polymeric colloidal particles formed by amphiphilic polymers are known to exhibit different sizes, distributions, rheological properties, and thermodynamic stability, depending on the degree of hydrophilicity and hydrophobicity. In addition, it has recently been reported that various hydrophobic drugs can be enclosed in the hydrophobic domain inside these colloids to induce selective and effective delivery of drugs ( Adv. Drug. Deliv. Rev. 2001 , 47 , 113 -131).
일본 동경대의 Kataoka 교수팀은 폴리에틸렌옥사이드(poly(ethylene oxide))와 폴리아스파르트산(poly(aspartic acid))으로 구성된 블록형 고분자 공중합체에 항암제인 아드리아마이신(adriamycin)을 화학적으로 결합시켜 항암제 함유 교질입자를 개발하였으며(J. Control. Rel. 2001,74, 295-302), 상기 제제의 항암효과가 입증되어 현재 일본에서 임상 2상 단계에 진입해 있다.Professor Kataoka of Tokyo University in Japan chemically binds adriamycin, an anticancer agent, to a block-type polymer copolymer composed of poly (ethylene oxide) and poly (aspartic acid). Particles have been developed ( J. Control. Rel. 2001 , 74 , 295-302) and the anticancer effects of the formulations have been demonstrated and are currently entering phase II clinical trials in Japan.
Okano 등은 N-아이소프로필아크릴아마이드(N-isopropylacrylamide)와 스티렌 (styrene)으로 구성된 양친성 블록 공중합체를 제조하였는데, 이는 수용액상에서 상온일때는 약 20㎚ 크기의 교질입자를 형성하고, 32℃ 이상의 온도에서는 교질입자간의 상호작용에 의해 커다란 집합체를 이루는 특징이 있어 온도에 따른 교질입자의 거동을 이용한 표적형 약물전달체로서의 가능성을 보고하였다(J. Control. Rel. 1997,48, 157-164).Okano et al. Prepared an amphiphilic block copolymer composed of N-isopropylacrylamide and styrene, which formed colloidal particles having a size of about 20 nm at room temperature in an aqueous solution, and at least 32 ° C. At the temperature, there is a characteristic that large aggregates are formed by the interaction between the colloidal particles, and thus the possibility of targeting drug carriers using the behavior of the colloidal particles with temperature has been reported ( J. Control. Rel. 1997 , 48 , 157-164).
Sunamoto 등은 교질입자를 형성하는 콜레스테롤(cholesterol) 함유 다당류를 개발하여 단백질 약물 전달체로서의 응용가능성을 보고하였다(J. Am. Chem. Soc. 1996,118, 6110-6115).Sunamoto et al. Developed cholesterol-containing polysaccharides that form colloids and reported their applicability as protein drug carriers ( J. Am. Chem. Soc. 1996 , 118 , 6110-6115).
그 이외에도 다양한 친수성 및 소수성 고분자들을 조합하여 수용액상에서 교질입자를 형성하는 화합물들의 개발이 시도되고 있다.In addition, the development of compounds that form colloidal particles in an aqueous solution by combining various hydrophilic and hydrophobic polymers has been attempted.
한편, 키토산은 자연계에서 셀룰로오스(cellulose) 다음으로 풍부한 천연 고분자 재료인 키틴(chitin)을 탈아세틸화(deacetylation)하여 얻어지는 화합물을 총칭하는 것으로, 2-아미노-2-디옥시-β-D-글루코피라노오스(2-amino-2-deoxy-β-D-glucopy ranose)로 구성된 다당류(polysaccharide)이다. 키토산은 자연계에 존재하는 다른 다당류와 달리 주쇄에 1차 아민을 함유하고 있어 매우 독특한 성질을 나타내며, 환경, 농업, 의약 등 여러 분야에서 응용되고 있고, 특히 생체적합성 및 생분해성이 우수하여 유전자 및 약물 전달체, 조직공학을 위한 스케폴드, 주입형 하이드로젤 등의 구성요소로서 의약분야에서 집중적 연구대상이 되고 있다(Polym. Int.1999, 48, 732~734).On the other hand, chitosan is a generic term for compounds obtained by deacetylating chitin, a natural polymer material rich in cellulose after nature, and 2-amino-2-dioxy-β-D-glu It is a polysaccharide consisting of 2-amino-2-deoxy-β-D-glucopy ranose. Chitosan, unlike other polysaccharides in nature, contains primary amines in its main chain and has very unique properties.It is applied in various fields such as environment, agriculture, and medicine.In particular, chitosan has excellent biocompatibility and biodegradation, so that genes and drugs As a component of delivery vehicles, scaffolds for tissue engineering, injectable hydrogels, etc., it has been the subject of intensive research in the pharmaceutical field ( Polym. Int. 1999, 48, 732 ~ 734).
기존의 합성고분자로 구성된 대부분의 고분자 교질입자는 얻을 수 있는 입자크기의 범위가 제한적이며, 대부분 유기용매에서 반응해야 하므로 독성을 나타낼 우려가 있고, 생체적합성에 문제가 있어 각종 염증반응을 유도할 수 있으며, 생분해성이 없는 경우 몸에서 제거하기가 쉽지 않다. 또한, 수용액상에서의 안정성이 부족하여 수시간에서 수일 이내에 형성된 교질입자의 구성이 엉성해지거나 침전되는 단점이 있다.Most polymer colloidal particles composed of existing synthetic polymers have a limited range of particle sizes that can be obtained, and most of them must be reacted in an organic solvent, which may cause toxicity, and biocompatibility may cause various inflammatory reactions. It is not easy to remove from the body if it is not biodegradable. In addition, there is a disadvantage in that the composition of the colloidal particles formed within several hours to several days due to the lack of stability in the aqueous solution is loose or precipitated.
이에, 본 발명자들은 생체적합성 및 생분해성이 우수하며 자연계에 매우 풍부한 천연 고분자인 키토산에 담즙산과 같은 소수성 그룹을 화학적으로 결합시켜 양친성이 부여된 유도체를 만들 경우 소수성 그룹간의 상호작용에 기인한 나노자기집합체의 형성이 가능하다는 것에 착안하여 본 발명을 완성하였다.Therefore, the present inventors chemically bond hydrophobic groups such as bile acids to chitosan, a natural polymer having excellent biocompatibility and biodegradability, and are very rich in nature, to make amphiphilic derivatives resulting from the interaction between hydrophobic groups. The present invention has been completed by focusing on the possibility of forming self-assembly.
본 발명은 수용액상에서 안정적이며, 나노자기집합체를 형성하는 소수성기를 함유한 키토산 복합체 및 그의 제조방법을 제공하고자 한다.The present invention is to provide a chitosan composite containing a hydrophobic group which is stable in an aqueous solution and forms a nanomagnetic aggregate, and a method for preparing the same.
도 1은 본 발명의 키토산 복합체 제조시 키토산과 담즙산의 반응 몰비에 따른 담즙산 치환도를 나타낸 도이다.1 is a diagram showing the degree of bile acid substitution according to the reaction mole ratio of chitosan and bile acid when the chitosan complex of the present invention is prepared.
도 2는 본 발명의 키토산 복합체의1H-NMR 스펙트럼을 나타낸 도이다.Figure 2 is a diagram showing the 1 H-NMR spectrum of the chitosan complex of the present invention.
도 3은 본 발명의 키토산 복합체로 형성된 나노자기집합체의 입자모양을 투과전자현미경으로 관찰한 도이다.Figure 3 is a view of the particle shape of the nano-magnetic assembly formed of the chitosan composite of the present invention by transmission electron microscope.
본 발명은 소수성기를 함유한 키토산 복합체를 제공한다.The present invention provides a chitosan complex containing a hydrophobic group.
본 발명의 키토산 복합체는 키토산의 아미노기와 소수성 담즙산의 카르복실기가 결합하여 수용액상에서 안정적이며, 다양한 크기의 나노자기집합체(nanosized self-aggregate)를 형성하는 것을 특징으로 한다.The chitosan complex of the present invention is characterized in that the amino group of chitosan and the carboxyl group of hydrophobic bile acid are stable in an aqueous solution and form nanosized nano-aggregates of various sizes.
또한, 본 발명은 상기 키토산 복합체의 제조방법을 제공한다.The present invention also provides a method for preparing the chitosan complex.
본 발명의 키토산 복합체의 제조방법은 1) 키토산에 1-에틸-3-(3-디메틸아미노프로필)카보디이미드 염산염[1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; EDC] 및 N-하이드로숙신이미드(N-hydrosuccinimide; NHS)를 첨가하여 키토산 용액을 제조하는 단계, 및 2) 상기 키토산 용액에 담즙산이 녹아있는 메탄올 또는 에탄올 용액을 천천히 적하시킨 후 교반하여 반응시키는 단계로 이루어진다.The preparation method of the chitosan complex of the present invention comprises 1) 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride in chitosan; EDC] and N-hydrosuccinimide (NHS) are added to prepare a chitosan solution, and 2) methanol or ethanol solution in which bile acid is dissolved in the chitosan solution is slowly added dropwise and stirred to react. Consists of steps.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 키토산 복합체에 사용되는 키토산은 평균 분자량이 수천에서 수백만이고, 아세틸화도(degree of acetylation)가 0 ~ 70% 이며, 생체적합성과 생분해성이 우수하다. 본 발명에 사용되는 키토산은 수용성 글리콜 키토산, 키토산 올리고머, 50% 아세틸화된 키토산 중에서 선택된 것을 사용하며, 수용성 글리콜 키토산이 바람직하다.The chitosan used in the chitosan complex of the present invention has an average molecular weight of thousands to millions, a degree of acetylation of 0 to 70%, and excellent biocompatibility and biodegradability. The chitosan used in the present invention is one selected from water-soluble glycol chitosan, chitosan oligomer, and 50% acetylated chitosan, and water-soluble glycol chitosan is preferable.
본 발명의 키토산 복합체에 사용되는 소수성 담즙산은 친수성 키토산에 소수성을 부여하여 양친성 키토산 복합체를 얻기 위하여 사용한 것으로, 키토산과 화학적 결합을 유도할 수 있는 카르복실산을 함유하고 있는 콜릭산(cholic acid), 키노디옥시콜릭산(chenodeoxycholic acid), 디옥시콜릭산(deoxycholic acid), 리소콜릭산(lithocholic acid), 7-옥소-리소콜릭산(7-oxo-lithocholic acid), 5β-콜라닉산 (5β-cholanic acid) 등의 일차, 이차 및 삼차 담즙산으로 이루어진 군으로부터 선택된 1종 이상을 포함하며, 5β-콜라닉산이 바람직하다.The hydrophobic bile acid used in the chitosan complex of the present invention is used to obtain an amphiphilic chitosan complex by imparting hydrophobicity to the hydrophilic chitosan and contains a carboxylic acid that can induce chemical bonds with chitosan (cholic acid). , Chenodeoxycholic acid, deoxycholic acid, lithocholic acid, 7-oxo-lithocholic acid, 5β-cholic acid (5β -cholanic acid) and at least one selected from the group consisting of primary, secondary and tertiary bile acids, and 5β-colonic acid is preferred.
이들 담즙산은 화학적 구조가 서로 다르며 특히 종류에 따라 소수성의 정도가 달라, 이들을 키토산에 결합시킬 경우 담즙산의 종류에 따라 나노자기집합체 형성 거동을 달리하고 입자크기 또한 다르게 나타난다.These bile acids have different chemical structures, and in particular, the degree of hydrophobicity varies depending on the type. When the bile acids are combined with chitosan, the formation of nano-self aggregates varies depending on the type of bile acid, and particle sizes are also different.
담즙산은 체내에 존재하는 간의 콜레스테롤로부터 생산되는 유기물로서 수용액상에서 교질입자를 형성하여 지방(fat)과 소수성 단백질(lipophilic vitamin)의 유상화(emulsification) 및 용해를 용이하게 하여 그들의 체내흡수를 돕는 것으로 알려져 있다.Bile acids are organic substances produced from cholesterol in the liver that are known to help their absorption into the body by forming colloidal particles in aqueous solution to facilitate emulsification and dissolution of fats and lipophilic vitamins. have.
본 발명에 따른 키토산 복합체의 제조방법은 다음과 같다.The preparation method of the chitosan complex according to the present invention is as follows.
글리콜 키토산을 증류수에 0.5~2 w/v%로 녹인후 1-에틸-3-(3-디메틸아미노프로필)카보디이미드 염산염[1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; EDC] 및 N-하이드로숙신이미드(N-hydrosuccinimide; NHS)를 적정량 첨가한다. 이때 EDC 및 NHS는 담즙산의 카르복실기를 활성화시켜 키토산의 아미노기와 아미드결합(amide bond)을 유도하는 역할을 하며, 첨가량은 향후 부가될 담즙산의 1.5 ~ 2배에 해당하는 몰비로 한다.Glycol chitosan was dissolved in distilled water at 0.5-2 w / v%, and then 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride; EDC] and N-hydrosuccinimide (NHS) are added in an appropriate amount. At this time, EDC and NHS activates the carboxyl group of bile acid to induce the amide bond with the amino group of chitosan, and the addition amount is 1.5 to 2 times the molar ratio of bile acid to be added in the future.
상기 키토산 용액에 적정량의 담즙산이 녹아있는 메탄올 또는 에탄올 용액을 천천히 적하시킨 후 24시간 동안 교반하여 반응시킨다. 이때 담즙산의 종류, 적하 양 및 용매의 양을 달리하여 담즙산의 치환도를 조절할 수 있다.The methanol or ethanol solution in which the appropriate amount of bile acid is dissolved is slowly added dropwise to the chitosan solution, followed by stirring for 24 hours. At this time, the degree of substitution of the bile acid can be adjusted by changing the type of bile acid, the dropping amount and the amount of the solvent.
본 발명에 따른 소수성기를 함유한 키토산 복합체의 제조방법은 하기 반응식 1로 나타낸다.The method for preparing a chitosan complex containing a hydrophobic group according to the present invention is shown in Scheme 1 below.
본 발명의 키토산 복합체에서 담즙산의 치환도는 담즙산의 몰비, 담즙산의종류 및 반응용매에 의존한다.The degree of substitution of bile acid in the chitosan complex of the present invention depends on the mole ratio of bile acid, the type of bile acid, and the reaction solvent.
또한 본 발명의 키토산 복합체는 치환된 담즙산의 종류 및 치환도에 따라 수용액상에서 형성되는 나노자기집합체의 임계회합농도(critical aggregation concentration, CAC) 및 입자크기 조절이 가능하며, 본 발명에서는 임계회합농도가 21 ~ 4 × 10-2㎎/㎖의 범위에 분포하고 있으며, 치환도가 클수록 임계회합농도가 감소한다. 또한 본 발명의 키토산 복합체의 입자크기는 직경이 수용액상에서 100 ~ 1,000㎚ 가 바람직하며, 200 ~ 850㎚ 가 더욱 바람직하다.In addition, the chitosan complex of the present invention is capable of controlling the critical aggregation concentration (CAC) and particle size of the nanomagnetic aggregate formed in the aqueous solution according to the type and degree of substitution of the bile acid, and in the present invention, the critical association concentration It is distributed in the range of 21 ~ 4 × 10 -2 mg / ㎖, the larger the degree of substitution, the lower the critical association concentration. In addition, the particle size of the chitosan composite of the present invention is preferably 100 ~ 1,000nm, more preferably 200 ~ 850nm in diameter in the aqueous solution.
본 발명의 키토산 복합체에 의해 수용액상에서 형성된 나노자기집합체의 입자모양은 대부분 구형이며, 입자 크기는 균일하다.The particle shape of the nanomagnetic aggregate formed in the aqueous solution by the chitosan composite of the present invention is mostly spherical, and the particle size is uniform.
본 발명의 소수성기를 함유한 키토산 복합체는 크기가 다양한 나노자기집합체로서 제조할 수 있고, 각종 질병에 대하여 최적화된 나노자기집합체의 입자크기를 사용하여 선택적 약물전달이 가능하여 향후 질병치료를 위한 약물전달체 및 기타 의료용 재료로서 다양한 응용이 가능하다.The chitosan complex containing the hydrophobic group of the present invention can be prepared as nanomagnetic aggregates of various sizes, and can be selectively delivered using nanoparticles optimized for various diseases, thereby enabling drug delivery for future disease treatment. And various other medical materials.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.
실시예 1Example 1 : 5β-콜라닉산 함유 글리콜 키토산 복합체의 제조: Preparation of 5β-Colanic Acid-Containing Glycol Chitosan Complex
글리콜 키토산 0.5g(2.00×10-6㏖, 단량체 몰수 = 2.38×10-3㏖)을 60㎖의 증류수에 용해시키고 60㎖(또는 180㎖)의 메탄올로 희석한 후, EDC 및 NHS를 첨가될 5β-콜라닉산에 대한 몰비를 기준으로 1.5배에 해당하는 양인 6.8㎎(3.57×10-5㏖) 및 4.1㎎(3.57×10-5㏖)을 용해시켰다. 소량의 메탄올에 용해시킨 5β-콜라닉산 8.6㎎(2.38×10-5㏖)을 천천히 적하한 후 상온에서 24시간 교반하였다.0.5 g (2.00 x 10 -6 mol, monomer moles = 2.38 x 10 -3 mol) of glycol chitosan was dissolved in 60 ml of distilled water and diluted with 60 ml (or 180 ml) of methanol, followed by addition of EDC and NHS. 6.8 mg (3.57 × 10 −5 mol) and 4.1 mg (3.57 × 10 −5 mol), 1.5 times the amount based on the molar ratio to 5β-colonic acid, were dissolved. 8.6 mg (2.38 × 10 -5 mol) of 5β-collonic acid dissolved in a small amount of methanol was slowly added dropwise, followed by stirring at room temperature for 24 hours.
상기 용액을 50%(또는 75%) 메탄올에서 투석하여 동결건조하고, 본 발명의 키토산 복합체를 제조하였다.The solution was lyophilized by dialysis in 50% (or 75%) methanol to prepare a chitosan complex of the present invention.
실시예 2Example 2 : 디옥시콜릭산 함유 글리콜 키토산 복합체의 제조: Preparation of Deoxycholic Acid-Containing Glycol Chitosan Complex
글리콜 키토산 0.5g을 60㎖의 증류수에 용해시키고 60㎖의 메탄올로 희석한 후, EDC 및 NHS를 첨가될 디옥시콜릭산에 대한 몰비를 기준으로 1.5배에 해당하는 양인 6.8㎎(3.57×10-5㏖) 및 4.1㎎(3.57×10-5㏖)을 용해시켰다. 소량의 메탄올에 용해시킨 디옥시콜릭산 9.4㎎(2.38×10-5㏖)을 천천히 적하한 후 상온에서 24시간 교반하였다.0.5 g of glycol chitosan was dissolved in 60 ml of distilled water and diluted with 60 ml of methanol, and then 6.8 mg (3.57 × 10 −) , equivalent to 1.5 times the molar ratio of the deoxycholic acid to which EDC and NHS were added. 5 mol) and 4.1 mg (3.57 x 10 -5 mol) were dissolved. 9.4 mg (2.38 × 10 -5 mol) of dioxycholic acid dissolved in a small amount of methanol was slowly added dropwise, followed by stirring at room temperature for 24 hours.
상기 용액을 50% 메탄올에서 투석하여 동결건조하고, 본 발명의 키토산 복합체를 제조하였다.The solution was lyophilized by dialysis in 50% methanol to prepare a chitosan complex of the present invention.
실험예 1Experimental Example 1 : 담즙산의 치환도 측정: Measurement of substitution degree of bile acid
본 발명의 키토산 복합체에 담즙산이 치환된 정도를 알아보기 위하여, 콜로이달 적정법(colloidal titration method)을 이용하여 측정하였다.In order to determine the degree of bile acid substitution in the chitosan complex of the present invention, it was measured using a colloidal titration method.
상기 실시예 1, 2에서 제조한 키토산 복합체 약 5㎎을 각각 2% 아세트산 수용액 5㎖에 녹인 후 5㎖의 메탄올로 희석하여 0.1% 지시약(toluidine blue)을 20㎕ 첨가 후 N/400 폴리비닐설페이트(polyvinyl sulfate) 용액으로 적정하였다.About 5 mg of the chitosan complexes prepared in Examples 1 and 2, respectively, were dissolved in 5 ml of 2% acetic acid aqueous solution, diluted with 5 ml of methanol, and 20 µl of 0.1% indicator (toluidine blue) was added, followed by N / 400 polyvinyl sulfate. Titration with (polyvinyl sulfate) solution.
결과는 도 1에 나타내었다.The results are shown in FIG.
도 1에 나타난 바와 같이, 본 발명의 키토산 복합체에서 담즙산의 치환도는 전반적으로 담즙산의 몰비가 증가할수록 높게 나타나고 있으며, 디옥시콜릭산이 5β-콜라닉산에 비해 반응 몰비와 상관 없이 담즙산의 치환도가 높게 나타남을 알 수 있다. 또한, 5β-콜라닉산의 경우 반응용매인 75% 메탄올이 50% 메탄올보다 높은 치환도를 나타내고 있어, 반응용매의 양에 따라 치환도가 다르게 나타남을 알 수 있다.As shown in Figure 1, in the chitosan complex of the present invention, the degree of substitution of bile acid is generally higher as the mole ratio of bile acid is increased, and the degree of substitution of bile acid regardless of the reaction molar ratio of deoxycholic acid compared to 5β-cholic acid It can be seen that high. In addition, in the case of 5β-cholonic acid, 75% methanol, which is a reaction solvent, shows a higher degree of substitution than 50% methanol, and thus, the degree of substitution may be different depending on the amount of the reaction solvent.
따라서, 담즙산의 치환도는 담즙산의 종류 및 반응용매에 의존함을 알 수 있으며, 이는 글리콜 키토산과 각 담즙산의 서로 다른 용해도에 기인한다고 사료된다.Therefore, it can be seen that the substitution degree of bile acid depends on the type of bile acid and the reaction solvent, which is attributed to the different solubility of glycol chitosan and each bile acid.
실험예 2Experimental Example 2 : 나노자기집합체의 물성 측정: Measurement of physical properties of nano magnetic assembly
본 발명의 키토산 복합체로부터 수용액상에서 형성된 나노자기집합체의 구조 및 입자크기를1H NMR과 DLS(dynamic light scattering)를 이용하여 측정하였고, 나노자기집합체를 형성할 수 있는 임계회합농도(CAC)를 형광 강도 측정기(fluorometer)를 이용하여 관찰하였다.The structure and particle size of the nanomagnetic aggregates formed in aqueous solution from the chitosan complex of the present invention were measured by 1 H NMR and dynamic light scattering (DLS), and the threshold association concentration (CAC) that can form the nanomagnetic aggregates was fluorescent. Observation was made using a fluorometer.
DLS는 키토산 복합체를 PBS용액에 1 ㎎/㎖로 녹인후 측정하였다.DLS was measured after the chitosan complex was dissolved in PBS solution at 1 mg / ml.
CAC는 키토산 복합체를 PBS용액에 1.0 × 10-4㎎/㎖부터 5.0㎎/㎖까지 다양한 용액을 만들어 파이렌(pyrene)을 첨가한 후 나노자기집합체가 형성되어 파이렌이 포집되기 시작하는 농도를 형광 강도 측정기로 관찰하여 계산하였다.CAC prepared chitosan complex in PBS solution ranging from 1.0 × 10 -4 mg / ml to 5.0 mg / ml, and added pyrene. Then, nano magnetic aggregate was formed and pyrene concentration was started. Calculated by observing with a fluorescence intensity meter.
수용액상에서 형성된 나노자기집합체의 구조는 도 2에 나타내었으며, 물성치는 표 1에 나타내었다.The structure of the nanomagnetic assembly formed in the aqueous solution is shown in Figure 2, the physical properties are shown in Table 1.
도 2에 나타난 바와 같이, 본 발명의 5β-콜라닉산에 의한 치환도가 12%인 키토산 복합체는 D2O/CD3OD 용매에서는 키토산과 5β-콜라닉산(화살표로 표시)의 피크가 모두 관찰된 반면, D2O 용매에서는 5β-콜라닉산의 피크가 없어짐을 확인할 수 있다. 이는 본 발명의 키토산 복합체가 D2O에 노출되었을 때 소수성 5β-콜라닉산이 서로 응집하여 나노자기집합체의 내부에 소수성 영역을 형성하기 때문이다.As shown in FIG. 2, in the chitosan complex having a substitution degree of 12% by 5β-cholonic acid of the present invention, both peaks of chitosan and 5β-collonic acid (indicated by arrows) were observed in a D 2 O / CD 3 OD solvent. On the other hand, it can be seen that the peak of 5β-cholic acid disappears in the D 2 O solvent. This is because when the chitosan complex of the present invention is exposed to D 2 O, the hydrophobic 5β-cholaric acid aggregates with each other to form a hydrophobic region inside the nanomagnetic assembly.
또한 표 1에 나타난 바와 같이, 본 발명의 키토산 복합체의 임계회합농도는 담즙산의 종류 및 치환도에 따라 임계회합농도가 21 ~ 4 × 10-2㎎/㎖의 범위에 분포하고 있으며, 치환도가 클수록 임계회합농도가 감소함을 알 수 있다. 일반적인 저분자량 계면활성제(surfactant)가 교질입자를 이루는 CMC(critical micell concentration)가 1㎎/㎖ 이상이므로, 본 발명의 키토산 복합체가 수용액상에서 매우 안정적인 나노자기집합체를 형성함을 알 수 있다.In addition, as shown in Table 1, the critical association concentration of the chitosan complex of the present invention, the critical association concentration is distributed in the range of 21 ~ 4 × 10 -2 mg / ㎖ depending on the type and degree of substitution of the bile acid, It can be seen that the larger the critical association concentration decreases. Since the CMC (critical micell concentration) of which a general low molecular weight surfactant (surfactant) forms a colloidal particle is 1 mg / ml or more, it can be seen that the chitosan complex of the present invention forms a very stable nanomagnetic aggregate in an aqueous solution.
또한, DLS로 측정한 나노자기집합체의 입자크기는 지름 200~850 ㎚ 범위의 다양한 크기를 가지고 있으며, 분산계수가 모두 0.05 미만의 값을 나타내므로 입자크기의 분포가 매우 적음을 알 수 있다.In addition, the particle size of the nano-magnetic assembly measured by the DLS has a variety of sizes in the range of 200 ~ 850 nm in diameter, the dispersion coefficient shows a value of less than 0.05 it can be seen that the distribution of the particle size is very small.
실험예 3Experimental Example 3 : 나노자기집합체의 입자모양 관찰: Observation of Particle Shape of Nano Magnetic Assembly
본 발명의 키토산 복합체에 의해 수용액상에서 형성된 나노자기집합체의 입자모양을 투과전자현미경(transmission electron microscopy; TEM)을 이용하여 관찰하였다.The particle shape of the nanomagnetic aggregate formed in the aqueous solution by the chitosan complex of the present invention was observed using a transmission electron microscopy (TEM).
상기 실시예 1, 2에서 제조한 키토산 복합체를 각각 2 ㎎/㎖씩 증류수에 녹인 후 약 5 ㎕를 탄소로 도포된 구리기판 위에 골고루 퍼뜨려 대기상에서 건조시킨 후, 2% 우라닐아세테이트(uranyl acetate) 용액으로 염색후 투과전자현미경으로 나노자기집합체의 입자모양을 관찰하였다.After dissolving the chitosan complexes prepared in Examples 1 and 2 in distilled water at 2 mg / ml, respectively, about 5 μl of the chitosan complex was evenly spread on a copper-coated copper substrate and dried in air, followed by 2% uranyl acetate. After staining with solution, the particle shape of the nanomagnetic assembly was observed by transmission electron microscope.
결과는 도 3에 나타내었다.The results are shown in FIG.
도 3에 나타난 바와 같이, 본 발명의 5β-콜라닉산으로 치환된 키토산 복합체에 의해 수용액상에서 형성된 나노자기집합체의 입자모양은 대부분 구형이었으며, 입자의 크기는 균일하였다.As shown in FIG. 3, the particle shape of the nanomagnetic aggregates formed in the aqueous solution by the chitosan complex substituted with 5β-cholic acid of the present invention was mostly spherical, and the size of the particles was uniform.
실험예 4Experimental Example 4 : 키토산 복합체의 농도에 따른 나노자기집합체의 입자크기: Particle size of nanomagnetic aggregates according to the concentration of chitosan complex
본 발명의 키토산 복합체에 의해 형성된 나노자기집합체의 입자크기를 조절하기 위하여 유기용매를 사용하여 관찰하였다.In order to control the particle size of the nanomagnetic aggregate formed by the chitosan complex of the present invention was observed using an organic solvent.
본 발명의 5β-콜라닉산에 의한 치환도가 5%인 키토산 복합체를 0.3, 0.5, 1.0, 2.0 ㎎/㎖로 각각 PBS용액에 녹인 후 용해된 고분자의 1 wt.%에 해당하는 클로로포름을 넣고 10분간 초음파 처리후 DLS로 입자의 크기를 측정하였다.The chitosan complex having 5% substitution by 5β-colonic acid of the present invention was dissolved in PBS solution at 0.3, 0.5, 1.0 and 2.0 mg / ml, respectively, and chloroform corresponding to 1 wt.% Of the dissolved polymer was added thereto. After sonication for minutes, the particle size was measured by DLS.
결과는 표 2에 나타내었다.The results are shown in Table 2.
표 2에 나타난 바와 같이, 본 발명의 5β-콜라닉산에 의한 치환도가 5%인 키토산 복합체는, 클로로포름 없이 PBS상에서 형성된 나노자기집합체의 크기가 302㎚(표 1 참조) 였으나, 클로로포름 첨가시에는 키토산 복합체의 농도에 따라 약 100~250㎚의 범위에서 입자크기의 조절이 가능하였다. 또한 입자형성후 클로로포름을 제거하여도 크기에는 변함이 없었으므로, 본 발명의 키토산 복합체는 클로로포름에 크게 영향을 받지 않음을 알 수 있다.As shown in Table 2, the chitosan complex having a degree of substitution of 5% by 5β-cholonic acid of the present invention was 302 nm (see Table 1) in the size of the nanomagnetic aggregate formed on PBS without chloroform. Depending on the concentration of the chitosan complex was able to control the particle size in the range of about 100 ~ 250nm. In addition, even after removing chloroform after particle formation, the size did not change, and thus the chitosan composite of the present invention was not significantly affected by chloroform.
본 발명의 키토산 복합체는 수용액상에서 수백 나노미터 크기의 나노자기집합체를 형성하므로, 나노자기집합체 내부에 소수성 영역이 형성되어 다양한 소수성 약물 및 치료용 단백질을 봉입할 수 있어, 암과 같은 혈관신생과 관련된 질병에 선택적 전달이 가능하여 의료용 약물전달체로서 널리 응용될 수 있다.Since the chitosan complex of the present invention forms nanomagnetic aggregates of hundreds of nanometers in an aqueous solution, hydrophobic regions are formed inside the nanomagnetic aggregates to encapsulate various hydrophobic drugs and therapeutic proteins, which are associated with angiogenesis such as cancer. Selective delivery to diseases is possible, so that it can be widely applied as a medical drug carrier.
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Cited By (8)
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|---|---|---|---|---|
| WO2006075881A1 (en) * | 2005-01-14 | 2006-07-20 | Korea Institute Of Science And Technology | Cholanic acid-chitosan complex forming self-aggregates and preparation method thereof |
| WO2007086651A1 (en) * | 2006-01-26 | 2007-08-02 | Jae-Woon Nah | Anti-cancer agent loaded hydrophobic bile acid conjugated hydrophilic chitosan oligosaccharide nanoparticles and preparation method thereof |
| KR100809046B1 (en) * | 2006-03-10 | 2008-03-03 | 가톨릭대학교 산학협력단 | Self assembled nanoparticle having pullulan and methods of using thereof |
| WO2009145594A3 (en) * | 2008-05-29 | 2010-03-11 | 남정선 | Drug delivery carrier |
| CN101810564A (en) * | 2010-03-29 | 2010-08-25 | 中山大学 | Method for preparing water-soluble nanometer preparation by insoluble traditional Chinese medicine containing ring structures |
| CN102260357A (en) * | 2011-07-14 | 2011-11-30 | 济南大学 | Amphipathic chitosan-bile acid derivatives and preparation method thereof |
| KR101323102B1 (en) * | 2011-01-18 | 2013-10-30 | 씨제이제일제당 (주) | Nanoparticles formed by encapsulating an anticancer drug into glycolchitosan-cholanic acid complex and a process for the preparation thereof |
| CN114702608A (en) * | 2022-03-19 | 2022-07-05 | 浙江大学 | Esterase response polymer and application thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2006075881A1 (en) * | 2005-01-14 | 2006-07-20 | Korea Institute Of Science And Technology | Cholanic acid-chitosan complex forming self-aggregates and preparation method thereof |
| WO2007086651A1 (en) * | 2006-01-26 | 2007-08-02 | Jae-Woon Nah | Anti-cancer agent loaded hydrophobic bile acid conjugated hydrophilic chitosan oligosaccharide nanoparticles and preparation method thereof |
| KR100762954B1 (en) * | 2006-01-26 | 2007-10-04 | 나재운 | Anti-cancer Agent Loaded Hydrophobic Bile Acid Conjugated Hydrophilic Chitosan Oligosaccharide Nanoparticles and Preparation Method Thereof |
| KR100809046B1 (en) * | 2006-03-10 | 2008-03-03 | 가톨릭대학교 산학협력단 | Self assembled nanoparticle having pullulan and methods of using thereof |
| WO2009145594A3 (en) * | 2008-05-29 | 2010-03-11 | 남정선 | Drug delivery carrier |
| EP2319503A2 (en) * | 2008-05-29 | 2011-05-11 | Soon-Chang Kwon | Drug delivery carrier |
| EP2319503A4 (en) * | 2008-05-29 | 2012-12-05 | Kwon Soon Chang | Drug delivery carrier |
| CN101810564A (en) * | 2010-03-29 | 2010-08-25 | 中山大学 | Method for preparing water-soluble nanometer preparation by insoluble traditional Chinese medicine containing ring structures |
| KR101323102B1 (en) * | 2011-01-18 | 2013-10-30 | 씨제이제일제당 (주) | Nanoparticles formed by encapsulating an anticancer drug into glycolchitosan-cholanic acid complex and a process for the preparation thereof |
| CN102260357A (en) * | 2011-07-14 | 2011-11-30 | 济南大学 | Amphipathic chitosan-bile acid derivatives and preparation method thereof |
| CN114702608A (en) * | 2022-03-19 | 2022-07-05 | 浙江大学 | Esterase response polymer and application thereof |
| CN114702608B (en) * | 2022-03-19 | 2024-01-26 | 浙江大学 | Esterase response polymer and application thereof |
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