KR100831391B1 - Chitosan Complexes Containing a WH-sensitive Imidazole Group and Methods for Manufacturing the Same - Google Patents
Chitosan Complexes Containing a WH-sensitive Imidazole Group and Methods for Manufacturing the Same Download PDFInfo
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Abstract
본 발명은 pH 민감성 이미다졸 그룹을 함유하는 키토산 복합체 및 그 제조방법에 관한 것으로, 더욱 상세하게는 글리콜 키토산의 아미노기와 중성의 영역에서는 소수성을 갖으나 약한 산성의 영역에서는 친수성을 갖는 히스티딘을 결합하여 중성의 수용액 상에서 안정적인 pH 민감성 이미다졸 그룹을 함유하는 키토산 복합체 및 그 제조방법에 관한 것이다. 본 발명에 따른 키토산 복합체는 항암제를 포함한 다양한 소수성 약물 및 유전자를 효과적으로 키토산 복합체의 내부로 물리적인 봉입이 가능하여 의료용 약물전달체, 분자 영상학적으로 사용이 가능한 추적자 및 유전자 전달체로서 유용하게 사용될 수 있다.The present invention relates to a chitosan complex containing a pH sensitive imidazole group and a method for preparing the same. More specifically, the amino group of glycol chitosan combines histidine having hydrophobicity in the neutral region but hydrophilicity in the weak acid region. The present invention relates to a chitosan complex containing a stable pH sensitive imidazole group in a neutral aqueous solution and a method for preparing the same. The chitosan complex according to the present invention can effectively encapsulate various hydrophobic drugs and genes including an anticancer agent into the chitosan complex, and thus can be usefully used as a medical drug carrier, a tracer and a gene transporter capable of molecular imaging.
pH 민감성, 나노자기집합체, 글리콜 키토산, 히스티딘, 이미다졸 pH Sensitivity, Nanomagnetic Aggregates, Glycol Chitosan, Histidine, Imidazole
Description
도 1은 본 발명의 키토산 복합체의 세포 내 거동을 나타내는 모식도이고;1 is a schematic diagram showing the intracellular behavior of the chitosan complex of the present invention;
도 2는 본 발명의 일 실시예에 따른 키토산 복합체로 형성된 나노자기집합체의 입자 크기를 동적광산란법(dynamic light scattering, DLS)을 이용하여 측정한 결과이고;2 is a result of measuring the particle size of the nano-magnetic aggregate formed of the chitosan complex according to an embodiment of the present invention using dynamic light scattering (DLS);
도 3은 본 발명의 일 실시예에 따른 키토산 복합체에 항암제인 파클리탁셀을 봉입한 후 암세포주에 처리하여 암세포의 세포주기를 유세포분석기(Fluorescence Activated Cell Sorting, FACS)로 측정한 결과이고;Figure 3 is a result of measuring the cell cycle of cancer cells by treating the cancer cell line after encapsulating paclitaxel as an anticancer agent in chitosan complex according to an embodiment of the present invention by Fluorescence Activated Cell Sorting (FACS);
도 4는 본 발명의 일 실시예에 따른 키토산 복합체를 방사선 동위원소(131I)로 표지한 후, 종양을 갖고 있는 실험용 쥐의 꼬리 정맥을 통해 주사하여 키토산 복합체의 종양 선택성을 나타낸 도이다. Figure 4 is a diagram showing the tumor selectivity of the chitosan complex by injecting the chitosan complex according to an embodiment of the present invention with a radioisotope ( 131 I), and then injected through the tail vein of a rat having a tumor.
본 발명은 pH 민감성 이미다졸 그룹을 함유하는 키토산 복합체 및 그 제조방법에 관한 것이다.The present invention relates to chitosan complexes containing pH sensitive imidazole groups and methods for their preparation.
많은 약물들이 질환 부위에 적절한 약효를 나타내기 위해 필요로 하는 양보다 많은 양을 투약하여 약효를 나타내지만, 적절치 않은 약물의 양으로 인해 그 이외의 부분에서는 부작용을 일으키기도 한다. 이로 인해, 많은 제약회사들은 상기 부작용을 줄이고, 약효를 극대화하도록 하는 제형에 대한 필요성을 갖게 되었다.Many drugs show efficacy by administering more than necessary amount to show the proper effect at the disease site, but due to the inappropriate amount of drug, other side effects may occur. As a result, many pharmaceutical companies have a need for formulations that reduce the side effects and maximize the efficacy.
상기 문제점을 해결하기 위해 약물전달시스템(drug delivery system, 이하 DDS)에 대한 활용도가 높아지고 있는데, 상기 DDS란, 기존 의약품의 부작용을 최소화하고 효능 및 효과를 극대화시키는 방법이며, 이것은 이전보다 낮은 농도 또는 투약하기 곤란한 약물의 선택성을 개선하는데 유용하다. 투여되는 약물이 물리화학적으로나 약동학적으로 특별한 특성, 예를 들면 높은 수용성, 높은 지용성, 불용해성 등을 보인다면, 특별한 전달체계의 사용이 요구된다.In order to solve the problem, the use of a drug delivery system (drug delivery system, hereinafter DDS) is increasing, the DDS is a method of minimizing the side effects and maximizing efficacy and effectiveness of the existing medicine, which is lower than It is useful for improving the selectivity of drugs that are difficult to administer. If the drug to be administered exhibits physicochemical or pharmacokinetic specific properties, for example high water solubility, high fat solubility, insolubility, use of a special delivery system is required.
또한, 특별한 요구사항을 갖는 약물들, 예를 들면, 독성이 심한 일회 투여되는 주사용 약물, 세포독성을 갖는 불안정 약물, 고청소율을 갖는 약물, 생체내에서 쉽게 불활성화되는 약물, 국소적 적용이 요구되는 약물 등은 적절한 DDS를 갖도록 고려되어야 한다. 상기 DDS기술은 70년대부터 선진국들이 첨단기술을 동원해 활발히 연구되고 있는 분야이다. 87년 물질특허제도가 도입됨에 따라 위기의식을 느낀 국내 제약회사들이 평균 15년의 기간과 2억 달러 이상의 비용이 소요되는 신약개발보다는 기존 약물의 단점을 개선한 DDS을 응용한 제품 개발이 새로운 신약개발에 필요한 기간과 비용이 약 1/3로 단축되고 성공 확률도 매우 높다는 것을 알게 되었고, 이에 많은 제약회사에서 DDS에 대한 연구가 본격화되었다. 국내에서는 90년부터 본격적인 연구가 시작되었으며, 이와 관련된 특허출원은 92년 이후 지속적인 증가세를 보이고 있다.In addition, drugs with special requirements, such as once-injectable drugs that are highly toxic, labile drugs with cytotoxicity, drugs with high cleaning rates, drugs that are readily inactivated in vivo, and topical applications are also possible. Drugs required and the like should be considered to have appropriate DDS. The DDS technology is a field that advanced countries have been actively researching with advanced technology since the 1970s. The introduction of a new patented drug developed by applying the DDS that improved the shortcomings of the existing drug rather than the development of a new drug that took an average of 15 years and a cost of more than $ 200 million for Korean pharmaceutical companies that felt the crisis due to the introduction of the material patent system in 1987. We found that the time and cost required for development was reduced to about one-third and that the probability of success was very high, which led many pharmaceutical companies to study DDS. In Korea, full-fledged research began in 1990, and related patent applications have been continuously increasing since 92.
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-glucopyranose)로 구성된 다당류(polysaccharide)이다. 키토산은 자연계에 존재하는 다른 다당류와 달리 주쇄에 1차 아민을 함유하고 있으며, 이로 인해 매우 독특한 성질을 나타내어, 환경, 농업, 의약 등 여러 분야에서 응용되고 있고, 특히 생체적합성 및 생분해성이 우수하여 유전자 및 약물전달체, 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 composed of copyranose (2-amino-2-deoxy-β-D-glucopyranose). Unlike other polysaccharides present in nature, chitosan contains primary amines in its main chain, and because of this, it has very unique properties and is applied in various fields such as environment, agriculture, and medicine. Especially, it has excellent biocompatibility and biodegradability. As a component of genes and drug carriers, scaffolds for tissue engineering, injectable hydrogels, etc., they have been intensive research in the pharmaceutical field ( Polym . Int . 1999 , 48, 732 ~ 734 ).
나노는 수십 ~ 수백 나노미터를 가리키는 것으로서, 화학적 요소, 올리고 핵산, siRNA, DNA, 단백질 등과 같은 치료학적으로 유효한 성분들을 나노 크기의 전달체를 사용하여 세포내로 전달시키는 시스템에 대한 연구가 진행되고 있으며, 이 들을 이용한 효과가 분명하게 나타나기 위해서는 세포막 사이에 특이적인 경로 또는 비특이적 경로를 거쳐 세포막의 지질 이중층을 통과하여 세포질 내에 약물의 방출이 이루어져야 한다.Nano refers to tens to hundreds of nanometers, and research into a system for delivering therapeutically effective components such as chemical elements, oligo nucleic acids, siRNAs, DNA, proteins, etc. into cells using nano sized carriers, In order for these effects to be evident, the drug must be released into the cytoplasm through the lipid bilayer of the cell membrane through specific or non-specific pathways between the cell membranes.
이에, 친수성과 소수성을 동시에 가지고 있는 양친성 고분자는 수용액 상에서 계면에너지의 안정화를 위해 소수성 블록간의 상호작용을 통해 교질입자(micelle) 또는 자기집합체(self-aggregate)를 형성하게 된다. 양친성 고분자에 의해 형성된 고분자 교질입자는 친수성 및 소수성의 정도에 따라 입자의 크기, 분포, 유동학적 성질, 열역학적 안정성 등이 다르게 나타나는 것으로 알려져 있다. 최근에는 이러한 교질입자 내부의 소수성 영역(hydrophobic domain)에 여러 가지 소수성 약물을 봉입하여 약물의 선택적 및 효과적인 수송을 유도할 수 있다는 보고가 있다(Adv. Drug. Deliv. Rev. 2001, 47, 113-131). Thus, amphiphilic polymers having both hydrophilicity and hydrophobicity form a colloidal particle or a 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 particle sizes, distributions, rheological properties, and thermodynamic stability depending on the degree of hydrophilicity and hydrophobicity. Recently, it has been reported that various hydrophobic drugs can be enclosed in the hydrophobic domain inside the colloid particles to induce selective and effective transport of drugs (Adv. Drug. Deliv. Rev. 2001, 47, 113- 131).
이를 응용한 약물 전달 시스템의 종래 기술에는, 일본 동경대의 카타오카 교수 팀은 폴리에틸렌옥사이드(polyethyleneoxide)와 폴리아스파르트산(polyaspartic acid)으로 구성된 블록형 고분자 공중합체에 항암제인 아드리아마이신(adriamycin)을 화학적으로 결합시켜 항암제 함유 교질입자를 개발하였다(J. Control . Release . 2001, 74, 295-302).In the prior art of the drug delivery system using this, a team of Prof. Kataoka of Tokyo University of Japan chemically used adriamycin, an anticancer agent, in a block polymer copolymer composed of polyethylene oxide and polyaspartic acid. The anti-cancer agent-containing colloids were developed ( J. Control . Release . 2001, 74, 295-302 ).
또한, 오카노 등은 N-이소프로필아크릴아마이드(N-isopropylacrylamide)와 스티렌(styrene)으로 구성된 양친성 블록 공중합체를 제조하였는데, 이는 수용액 상에서 상온일 때는 약 20 nm 크기의 교질입자를 형성하고, 32 ℃ 이상의 온도에서 는 교질입자간의 상호작용에 의해 커다란 집합체를 이루는 특징이 있어 온도에 따른 교질입자의 거동을 이용한 표적형 약물전달체로서의 가능성을 보고하였다(J. Control. Release . 1997, 48, 157-164).In addition, Okano et al. Prepared an amphiphilic block copolymer composed of N-isopropylacrylamide and styrene, which forms colloidal particles having a size of about 20 nm at room temperature in an aqueous solution. At temperatures above ℃, it is characterized by the formation of large aggregates by the interaction between the colloidal particles and reported the potential as a targeted drug carrier using the behavior of the colloidal particles with temperature ( J. Control. Release . 1997 , 48, 157- 164 ).
한편, 수나모토 등은 교질입자를 형성하는 콜레스테롤(Cholesterol) 함유 다당류를 개발하여 단백질 약물전달체로서의 응용가능성을 보고하였다(J. Am . Chem . Soc. 1996, 118, 6110-6115).Meanwhile, Sunamoto et al . Developed cholesterol-containing polysaccharides that form colloidal particles and reported their applicability as protein drug carriers ( J. Am . Chem . Soc. 1996 , 118, 6110-6115 ).
그러나 상기 종래 기술들에는 생체내 같은 기관에서 다른 pH 분포를 보이는, 예를 들면, 암과 같은, 부위에 선택적으로 약물을 전달할 수 있는 방법에 대해서 보고되어 있지 않다.However, these prior arts do not report a method of selectively delivering a drug to a site, such as cancer, which has a different pH distribution in the same organ in vivo.
따라서, 본 발명은 생체적합성 및 생분해성이 우수할 뿐만 아니라 중성 혹은 염기성에서는 나노자기집합체가 형성되고 산성에서는 나노자기집합체가 해체되면서 그 내부에 봉입된 약물이 세포내로 방출될 수 있는 pH 민감성 이미다졸 그룹을 함유한 키토산 복합체 및 그 제조방법을 제공하고자 함에 그 목적이 있다.Accordingly, the present invention is not only excellent in biocompatibility and biodegradability, but also in a neutral or basic form, the nanomagnetic assembly is formed, and in acidic, the nanomagnetic assembly is disassembled, so that the drug encapsulated therein can be released into the cell. It is an object to provide a chitosan complex containing a group and a method for producing the same.
상기 목적을 달성하기 위하여, 본 발명은 수용성 키토산의 사슬에 소수성기로서 pH 민감성 이미다졸 그룹을 도입하여 나노자기집합체를 형성하는 키토산 복합 체를 제공한다.In order to achieve the above object, the present invention provides a chitosan complex which forms a nano-self aggregate by introducing a pH-sensitive imidazole group as a hydrophobic group in the chain of water-soluble chitosan.
또한, 본 발명은 수용성 키토산에 pH 민감성 이미다졸 그룹을 부가하여 아마이드 결합을 형성하는 단계를 포함하는 키토산 복합체의 제조방법을 제공한다.In addition, the present invention provides a method for preparing a chitosan complex comprising the step of adding an pH-sensitive imidazole group to a water-soluble chitosan to form an amide bond.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 구형 나노자기집합체를 형성하는 pH 민감성 이미다졸 그룹을 함유한 키토산 복합체를 제공한다.The present invention provides chitosan complexes containing pH sensitive imidazole groups that form spherical nanomagnetic aggregates.
본 발명의 키토산 복합체는 수용성 키토산의 아미노기와 상기 pH 민감성 이미다졸 그룹의 카복실기가 결합하여 나노자기집합체를 형성한 것이다.The chitosan complex of the present invention combines the amino group of the water-soluble chitosan and the carboxyl group of the pH-sensitive imidazole group to form a nanomagnetic aggregate.
또한, 상기 수용성 키토산은 수용성 글리콜 키토산, 키토산 올리고머, 아세틸화된 키토산 등을 사용할 수 있다. In addition, the water-soluble chitosan may be a water-soluble glycol chitosan, chitosan oligomer, acetylated chitosan and the like.
더욱이, 상기 pH 민감성 이미다졸 그룹은 히스티딘, N-아세틸 히스티딘, 폴리히스티딘 중에서 1종 이상을 선택하여 사용할 수 있으나, 이에 한정되지 않는다.Furthermore, the pH sensitive imidazole group may be selected from one or more of histidine, N-acetyl histidine, and poly histidine, but is not limited thereto.
본 발명에 따른 상기 pH 민감성 이미다졸 그룹은 키토산 단량체에 대하여 1 내지 30%의 범위로 치환된 것이 바람직하다. 상기 한 범위에서 DDS 약물을 봉입하기 위한 최적의 나노자기집합체가 형성될 수 있기 때문이다. The pH sensitive imidazole group according to the present invention is preferably substituted in the range of 1 to 30% relative to the chitosan monomer. This is because optimal nanomagnetic aggregates can be formed to encapsulate DDS drugs in the above range.
본 발명의 pH 민감성 이미다졸 그룹을 함유한 키토산 복합체는 pH 민감성 이미다졸 그룹의 소수성기와 키토산의 친수성기에 의한 양친성으로 인하여 수계에서 구형 나노자기집합체를 형성할 수 있다.Chitosan complexes containing the pH sensitive imidazole group of the present invention can form spherical nanomagnetic aggregates in water due to the amphipathicity of the hydrophilic groups of chitosan with hydrophobic groups of the pH sensitive imidazole group.
상기 키토산 복합체의 크기는 직경이 50 ㎚ 내지 500 ㎚의 범위가 될 수 있 으며, 상기 키토산 복합체의 크기는 형성된 키토산 복합체가 표적세포로 이동하기에 적절하다.The size of the chitosan complex may be in the range of 50 nm to 500 nm in diameter, the size of the chitosan complex is suitable for the formed chitosan complex to move to the target cell.
또한, 본 발명은 수용성 키토산의 사슬에 소수성 기로서 pH 민감성 이미다졸 그룹을 도입하여 나노자기집합체가 형성된 키토산 복합체에 파클리탁셀이 봉입된 파클리탁셀 함유 나노자기집합체를 제공한다. The present invention also provides a paclitaxel-containing nanomagnetic aggregate in which paclitaxel is encapsulated in a chitosan complex in which a nanomagnetic aggregate is formed by introducing a pH-sensitive imidazole group as a hydrophobic group in a chain of water-soluble chitosan.
상기 파클리탁셀 함유 나노자기집합체는 세포이물흡수(endocytosis) 단계를 거치는 동안 엔도좀(endosome) 내 pH 환경이 산성으로 변하여 자기집합체의 히스티딘이 소수성에서 친수성 성질로 바뀌게 된다. 이에 친수성 및 소수성 균형이 무너지게 되면서, 내부에 봉입된 파클리탁셀을 방출하게 된다. 이는 기존의 자기집합체가 세포내로 전달은 되지만, 약물 방출이 너무 느리거나 방출되기 어렵다는 문제점을 pH 민감성 이미다졸 그룹을 도입함으로서 해결한 것이다. The paclitaxel-containing nanomagnetic aggregate undergoes an endocytosis step, and the pH environment in the endosome is changed to acid so that histidine of the aggregate is changed from hydrophobic to hydrophilic. The hydrophilic and hydrophobic balance is broken, thereby releasing paclitaxel encapsulated therein. This is solved by introducing a pH-sensitive imidazole group that the existing self-assembly is delivered into the cell, but drug release is too slow or difficult to release.
상기 파클리탁셀의 봉입효율은 30 내지 95%일 수 있다. 이러한 봉입 효율의 증가에 따른 상기 파클리탁셀이 봉입된 파클리탁셀 함유 나노자기집합체는 증가된 항암효과를 가질 수 있다.The encapsulation efficiency of the paclitaxel may be 30 to 95%. According to the increase of the encapsulation efficiency, the paclitaxel-containing nanomagnetic aggregate encapsulated may have an increased anticancer effect.
이외에도 상기 본 발명의 pH 민감성 이미다졸 그룹을 함유한 키토산 복합체의 내부에 물리적으로 포함될 수 있는 항암제로서 모든 종류의 소수성 항암제가 사용될 수 있으며, 구체적인 예로는 아드리아마이신, 시스-플라틴(cis-platin), 미토마이신-C, 다우노마이신(daunomycin), 5-플루오로우라실(5-fluorouracil) 등을 들 수 있다.In addition, all kinds of hydrophobic anticancer agents may be used as anticancer agents that may be physically included in the chitosan complex containing the pH-sensitive imidazole group of the present invention. Specific examples include adriamycin and cis-platin. , Mitomycin-C, daunomycin, 5-fluorouracil and the like.
일반적으로 자기집합체(self-aggregate)는 친수성 및 소수성을 모두 가진 양친성 분자가 수용액 상에서 회합하여 형성하는 구형집합체로서 친수성기는 구형 집합체의 외부에 소수성기는 내부에 모이게 된다[Adv. Drug Deliv. Rev., 1996, 21, 107]. 따라서 소수성을 가지는 여러 항암제를 효율적으로 전달하는 체계로 널리 이용되고 있다. 이러한 자기집합체를 형성하는 양친성 고분자를 이용한 약물 전달 방법은 표적세포에 대한 선택성을 충분히 나타내면서 정상세포에의 독성을 현저히 줄이고, 장기간 약물이 지속적으로 방출되도록 하는 약물 전달체계로서 암과 같은 심각한 질환을 치료하는 새로운 개념의 치료법의 연구에 사용될 수 있다.In general, self-aggregates are spherical aggregates formed by the formation of amphiphilic molecules having both hydrophilicity and hydrophobicity in an aqueous solution. Hydrophilic groups are collected outside the spherical aggregates and hydrophobic groups are collected in the interior [Adv. Drug Deliv. Rev., 1996, 21, 107]. Therefore, it is widely used as a system for efficiently delivering various anticancer drugs with hydrophobicity. Drug delivery methods using amphiphilic polymers that form these self-assemblies exhibit sufficient selectivity for target cells, significantly reduce toxicity to normal cells, and allow drug to be released for long periods of time. Can be used to study new concepts of treatment.
따라서, 본 발명의 pH 민감성 이미다졸 그룹을 함유한 키토산 복합체는 일반 저분자량의 항암제보다 투과 보유성 효과(enhanced permeability and retention, 이하 EPR)에 의하여 암조직에 대한 선택성이 높아, 더 많은 양이 암조직에 축적되어 효과적으로 항암 작용을 발휘할 수 있는 장점이 있으며, 주쇄로 사용되어진 친수성 키토산과 결합된 pH 민감성 히스티딘에 의하여 수용액 상에서 수십 ~ 수백 나노미터 크기의 구형 나노자기집합체를 자발적으로 형성하여 항암제의 전달체로서 표적 지향적이며, 지속적 약물 방출이 가능한 높은 항암 효과를 발휘할 수 있는 약제로서 암 등의 질환의 치료에 유용하게 사용될 수 있다.Therefore, the chitosan complex containing the pH-sensitive imidazole group of the present invention has higher selectivity for cancer tissues by enhanced permeability and retention (EPR) than a general low molecular weight anticancer agent, and a greater amount of cancer It has the advantage of being effective in anticancer activity by accumulating in tissues, and spontaneously forming spherical nano-self aggregates of several tens to hundreds of nanometers in aqueous solution by pH sensitive histidine combined with hydrophilic chitosan used as a main chain to deliver an anticancer carrier. As a target-oriented, drug that can exert a high anticancer effect capable of sustained drug release can be usefully used in the treatment of diseases such as cancer.
또한, 본 발명은 수용성 키토산에 pH 민감성 이미다졸 그룹을 부가하여 아마이드 결합을 형성하는 단계를 포함하는 키토산 복합체의 제조방법을 제공한다.In addition, the present invention provides a method for preparing a chitosan complex comprising the step of adding an pH-sensitive imidazole group to a water-soluble chitosan to form an amide bond.
상기 pH 민감성 이미다졸 그룹의 부가시에 1-에틸-3-(3-디메틸아미노프로필)카보디이미드 염산염(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 이하 EDC) 및 N-하이드록시숙신이미드(N-hydroxysuccinimide, 이하 NHS)를 첨가하여 상기 아마이드 결합의 유도를 더욱 활성화시킬 수 있다.1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy upon addition of the pH sensitive imidazole group Succinimide (N-hydroxysuccinimide, hereinafter NHS) can be added to further activate the induction of the amide bond.
더욱이, 상기 pH 민감성 이미다졸 그룹은 히스티딘, N-아세틸 히스티딘, 폴리히스티딘 중에서 1종 이상을 선택하여 사용할 수 있으나, 이에 한정되지 않는다.Furthermore, the pH sensitive imidazole group may be selected from one or more of histidine, N-acetyl histidine, and poly histidine, but is not limited thereto.
본 발명에 따른 상기 수용성 키토산은 인산완충식염수(phosphate buffered saline)에 녹여 사용할 수 있다.The water-soluble chitosan according to the present invention can be used by dissolving in phosphate buffered saline.
또한, 상기 pH 민감성 이미다졸 그룹은 히스티딘 계열의 화합물에 포함된 것으로 인산완충식염수에 녹여 사용할 수 있다.In addition, the pH-sensitive imidazole group is contained in the histidine-based compound can be used dissolved in phosphate buffered saline.
이때, 상기 pH 민감성 이미다졸 그룹은 키토산 단량체에 대하여 1 내지 30%의 범위로 치환될 수 있다. 상기 범위 내에서 치환되는 pH 민감성 이미다졸에 의해 나노자기집합체의 형성이 용이하게 이루어질 수 있다.In this case, the pH sensitive imidazole group may be substituted in the range of 1 to 30% with respect to the chitosan monomer. The formation of nanomagnetic aggregates can be easily achieved by the pH sensitive imidazole substituted within the above range.
본 발명에 따른 상기 반응액에 파클리탁셀을 부가하여 상기 파클리탁셀이 상기 키토산 복합체의 소수성기인 pH 민감성 이미다졸 그룹으로 이루어진 소수성 영역에 봉입되게 하는 단계를 포함할 수 있다.Adding paclitaxel to the reaction solution according to the present invention may include the step of encapsulating the paclitaxel in a hydrophobic region consisting of a pH sensitive imidazole group which is a hydrophobic group of the chitosan complex.
이상 살펴본 바와 같은 본 발명의 키토산 복합체는 항암제를 포함한 다양한 소수성 약물 및 유전자를 효과적으로 나노자기집합체 내부로 물리적으로 봉입시킬 수 있으므로 약물전달체로서 유용하게 이용될 수 있다.As described above, the chitosan complex of the present invention can be effectively used as a drug carrier because it can effectively encapsulate various hydrophobic drugs and genes including anticancer agents into the nanomagnetic aggregates.
이하, 본 발명을 실시예 및 실험예에 의해 더욱 상세히 설명한다. 단, 하기의 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기의 실시예 및 실험예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.
<실시예 1> 히스티딘을 함유 키토산 복합체의 제조Example 1 Preparation of Histidine-Containing Chitosan Complex
글리콜 키토산(Mw: 250 kDa) 0.5 g을 100 ㎖의 인산완충식염수에 용해시킨 후, 상기 용액에 글리콜 키토산의 단량체 100 개에 대하여 히스티딘이 5 ~ 30 개가 치환될 수 있는 양을 천천히 적하하였다. EDC 및 NHS는 상기 용액에 첨가된 히스티딘에 대한 몰 비를 기준으로 3 배에 해당하는 양을 천천히 적하시킨 후 상온에서 24 시간 교반하였다. 반응이 완료 후 반투과막을 사용하여 탈 이온수에서 4 일간 투석(dialysis)을 진행한 후 동결 건조하여 본 발명의 히스티딘이 함유된 키토산 복합체의 나노자기집합체를 제조하였다.After dissolving 0.5 g of glycol chitosan (Mw: 250 kDa) in 100 ml of phosphate buffered saline, the solution was slowly added dropwise an amount of 5 to 30 histidine to 100 monomers of glycol chitosan. EDC and NHS were slowly added dropwise an amount of 3 times based on the molar ratio of histidine added to the solution, followed by stirring at room temperature for 24 hours. After completion of the reaction, dialysis (dialysis) was performed in deionized water for 4 days using a semi-permeable membrane, followed by freeze-drying to prepare a nanomagnetic aggregate of the histidine-containing chitosan complex.
<실시예 2> N-아세틸 히스티딘 함유 키토산 복합체의 제조Example 2 Preparation of N-acetyl Histidine-Containing Chitosan Complex
N-아세틸 히스티딘을 상기용액의 글리콜 키토산의 단량체 100 개당 5 ~ 30 개를 치환시킬 양 및 그에 따른 N-아세틸 히스티딘 첨가량의 3배에 해당하는 EDC 및 NHS를 첨가한 것을 제외하고는 상기 실시예 1과 동일한 방법으로 N-아세틸 히스티딘이 함유된 키토산 복합체의 나노자기집합체를 제조하였다.Example 1 except that N-acetyl histidine was added in an amount of 5 to 30 substitutions per 100 monomers of glycol chitosan in the solution, and EDC and NHS corresponding to three times the amount of N-acetyl histidine added thereto were added. In the same manner as in the nanomagnetic assembly of the chitosan complex containing N-acetyl histidine was prepared.
<실시예 3> 생체내 추적용 방사성 동위원소가 도입 가능한 히스티딘이 함유된 키토산 복합체Example 3 Chitosan Complex Containing Histidine Incorporated with Radioisotopes for In Vitro Tracking
상기 실시예 1에 의해 제조된 키토산 복합체 0.5 g을 탈 이온수 100 ㎖에 용해시킨 후, N-아세틸 티로신(N-acetyl tyrosine)을 상기용액의 글리콜 키토산의 단량체 100 개당 1 ~ 20 개를 치환시킬 양을 적하하고, 방사선 동위원소 131I을 도입하였다. 반응 완료 후 반투과막을 사용하여 탈 이온수에서 4일간 투석한 후, 동결건조하여, 방사선 동위원소의 도입이 가능한 히스티딘이 함유된 키토산 복합체의 나노자기집합체를 제조하였다.After dissolving 0.5 g of the chitosan complex prepared in Example 1 in 100 ml of deionized water, N-acetyl tyrosine was substituted for 1 to 20 per 100 monomers of glycol chitosan in the solution. Was added dropwise and radioisotope 131 I was introduced. After completion of the reaction, the dialysis membrane was dialyzed in deionized water for 4 days, and then lyophilized to prepare a nanomagnetic aggregate of a histodine-containing chitosan complex capable of introducing radioisotopes.
<실시예 4> 생체내 추적용 방사성 동위원소가 도입 가능한 N-아세틸 히스티딘 함유 키토산 복합체<Example 4> N-acetyl histidine-containing chitosan complex which can introduce radioisotope for in vivo tracking
실시예 2에 의해 제조된 키토산 복합체를 사용하는 것을 제외하고는, 상기 실시예 3과 동일한 방법으로 방사선 동위원소 131I을 도입하여 N-아세틸 히스티딘 함유된 키토산 복합체의 나노자기집합체를 제조하였다.Except for using the chitosan complex prepared in Example 2, the radioisotope 131 I was introduced in the same manner as in Example 3 to prepare a nano-self aggregate of the chitosan complex containing N-acetyl histidine.
<실험예 1> 키토산 복합체의 N-아세틸 히스티딘의 치환도 측정Experimental Example 1 Measurement of Substitution of N-acetyl Histidine in Chitosan Complex
본 발명의 키토산 복합체의 N-아세틸 히스티딘 함량을 측정하기 위하여 하기와 같은 실험을 하였다. In order to measure the N-acetyl histidine content of the chitosan complex of the present invention was performed as follows.
상기 실시예 2에서 제조한 키토산 복합체를 중수(D2O)/디메틸술폭사이 드(dimethylsulfoxide)(d-6)(1/4, v/v) 혼합용매에 1 ㎎/㎖의 농도로 녹여 1H-NMR로 측정한 후, 7 ~ 8 ppm 영역의 히스티딘 특성 피크 및 3.0 ~ 3.3 ppm 영역의 키토산의 C-2 수소의 특성 피크로부터 N-아세틸 히스티딘 치환도를 측정하였다. The chitosan complex prepared in Example 2 was dissolved in a concentration of 1 mg / mL in a mixed solvent of heavy water (D 2 O) / dimethylsulfoxide (d-6) (1/4, v / v) 1 After measurement by H-NMR, the degree of N-acetyl histidine substitution was measured from the histidine characteristic peak in the 7 to 8 ppm region and the characteristic peak of C-2 hydrogen in the chitosan in the 3.0 to 3.3 ppm region.
그 결과는 하기 표 1에 나타내었다.The results are shown in Table 1 below.
상기 표 1에서 나타난 바와 같이, 상기 N-아세틸 히스티딘 함유 키토산 복합체의 합성시에 공급되는 N-아세틸 히스티딘의 비율이 증가함에 따라 키토산 복합체의 N-아세틸 히스티딘의 치환도가 증가함을 알 수 있었다.As shown in Table 1, it was found that the degree of substitution of N-acetyl histidine in the chitosan complex increased as the ratio of N-acetyl histidine supplied in the synthesis of the N-acetyl histidine-containing chitosan complex increased.
<실험예 2> pH에 따른 키토산 복합체의 형태 변화Experimental Example 2 Changes in the Form of Chitosan Complex According to pH
pH의 변화에 따른 키토산 복합체의 수용액 상에서 형성된 나노자기집합체의 입자 크기를 측정하기 위하여 하기와 같은 실험을 하였다.In order to measure the particle size of the nano-magnetic aggregate formed on the aqueous solution of the chitosan complex with the change of pH was carried out as follows.
상기 실시예 2에 따른 N-아세틸 히스티딘 함유 키토산 복합체를 수용액 상에 녹인 후, 형성되는 나노자기집합체의 크기를 관찰하기 위하여 키토산 복합체를 pH 7.4인 인산완충식염수에 1 ㎎/㎖의 농도로 녹이고, 동적광산란법(dynamic light scattering, 이하 DLS)을 이용하여 중성 영역에서 형성된 나노자기집합체의 크기를 측정하였다. 그 결과를 도 2에 나타내었다.After dissolving the N-acetyl histidine-containing chitosan complex according to Example 2 on an aqueous solution, the chitosan complex was dissolved in a phosphate buffered saline solution having a pH of 7.4 in order to observe the size of the formed nanomagnetic aggregate, Dynamic light scattering (DLS) was used to measure the size of the nanomagnetic aggregates formed in the neutral region. The results are shown in FIG.
도 2에 나타난 바와 같이, pH 7.4 중성의 수용액에서 형성된 나노자기집합체가 보였다. 이에, 상기 나노자기집합체는 중성 영역에서 형성됨을 확인할 수 있었다.As shown in FIG. 2, nanomagnetic aggregates were formed in a neutral aqueous solution of pH 7.4. Thus, it could be seen that the nanomagnetic assembly is formed in the neutral region.
<실험예 3> 항암제가 함유된 나노자기집합체의 암세포 독성 실험(in vitro)Experimental Example 3 Cancer Cell Toxicity Test of Nano Magnetic Assembly Containing Anticancer Agent (In vitro)
항암제가 함유된 나노자기집합체의 암세포 독성을 알아보기 위하여 하기와 같은 실험을 하였다.In order to determine the cancer cell toxicity of the nano-self aggregate containing the anticancer agent, the following experiment was performed.
항암제인 파클리탁셀(paclitaxel)을 실시예 2의 나노자기집합체 내부에 봉입시킨 후, 이를 인간 폐암세포주(A549) 및 인간 유방암세포주(MDA-MB231)에 처리하였다. 유세포분석기(FACS, Fluorescence activated cell sorter)를 이용하여 시간에 따른 세포주기를 관찰하여 상기 나노자기집합체의 항암효과를 평가하였다. 그 결과는 도 3에 나타내었다.Paclitaxel, an anticancer agent, was encapsulated inside the nanomagnetic aggregate of Example 2 and then treated with human lung cancer cell line (A549) and human breast cancer cell line (MDA-MB231). The anticancer effect of the nanomagnetic aggregate was evaluated by observing the cell cycle with time using a flow cytometer (FACS, Fluorescence activated cell sorter). The results are shown in FIG.
도 3에서 나타난 바와 같이, 항암제가 함유된 나노자기집합체를 인간 폐암세포주 및 인간 유방암세포주에 처리했을시, 1시간 내에 G0/G1기는 대조군과 큰 차이를 보이지 않았다. 이후, 6시간부터는 G2/M기가 증가하는 경향을 보이며, 24시간 후에는 G2/M기가 크게 증가한 것을 알 수 있었다. 이는 항암제가 함유된 나노자기집합체가 암세포 내로 전달되어, 낮은 pH를 갖는 상기 암세포에서 나노 자기집합체가 해체되면서 항암제가 세포내에 방출되었으며, 이로 인해 세포주기 정지(cell cycle arrest)로 암세포의 괴사를 유도하였음을 알 수 있었다.As shown in FIG. 3, when treated with a human lung cancer cell line and a human breast cancer cell line containing a nano-self aggregate containing an anticancer agent, G 0 / G 1 phase within 1 hour did not show a significant difference from the control group. After 6 hours, the G 2 / M group showed a tendency to increase, and after 24 hours, the G 2 / M group was found to increase significantly. This is because the nanoself aggregate containing the anticancer agent is transferred into the cancer cell, and the anticancer agent is released into the cell as the nano self aggregate is disassembled in the cancer cells having a low pH, thereby inducing the necrosis of the cancer cell by cell cycle arrest It can be seen that.
이에, 상기 나노자기집합체는 매우 뛰어난 전달체인 것을 확인하였다.Thus, it was confirmed that the nanomagnetic assembly is a very excellent carrier.
<실험예 4> 나노자기집합체의 종양 선택성 실험(in vivo)Experimental Example 4 Tumor Selectivity Experiment of Nanomagnetic Aggregates (in vivo)
나노자기집합체의 종양 선택성을 알아보기 위해서 하기와 같은 실험을 진행하였다.In order to determine the tumor selectivity of the nano-self aggregates, the following experiment was conducted.
상기 실시예 2를 암세포가 이식된 실험용 쥐의 꼬리 정맥으로 실시예 4에 따른 생체내 추적용 방사선 동위원소가 도입된 N-아세틸 히스티딘 함유 키토산 복합체를 주사하고, 감마카메라 영상을 통한 핵의학 영상화 기술로 나노자기집합체의 종양 선택성을 관찰하고, 그 결과를 도 4에 나타내었다.In Example 2, the N-acetyl histidine-containing chitosan complex into which the in vivo tracking radioisotope according to Example 4 was introduced into a tail vein of a rat in which cancer cells were implanted, and a nuclear medicine imaging technique using a gamma camera image Tumor selectivity of the nano-self aggregates was observed, and the results are shown in FIG. 4.
도 4에 나타난 바와 같이, 나노자기집합체에 방사선 동위 원소(131I)를 표지한 상기 나노자기집합체를 감마카메라로 촬영했으며, 이를 통하여 종양이 분포하는 부분에서 상기 방사선 동위원소가 집중적으로 분포하는 것을 확인할 수 있었다. 이에, 상기 나노자기집합체는 매우 뛰어난 분자 영상학적으로 사용이 가능한 추적자임을 알 수 있었다.As shown in FIG. 4, the nanomagnetic assembly labeled with the radioisotope ( 131 I) on the nanomagnetic assembly was photographed with a gamma camera, thereby intensively distributing the radioisotope in the tumor distribution area. I could confirm it. Thus, the nanomagnetic assembly was found to be a tracer that can be used in a very good molecular imaging.
본 발명은 pH 민감성 이미다졸 그룹을 함유하는 키토산 복합체, 이의 제조방 법에 관한 것으로, 본 발명의 키토산 복합체는 생체 적합성(biocompatibility) 및 생분해성(biodegradability)이 우수하며, 수용액 상에서 표면이 친수성 글리콜 키토산으로 구성되고 내부가 소수성 히스티딘으로 이루어진 수십 ~ 수백 나노미터 크기의 구형 집합체로, 항암제를 포함한 다양한 소수성 약물, 추적자 및 유전자를 효과적으로 나노자기집합체 내부로 물리적인 봉입이 가능하여 의료용 약물전달체, 분자영상학적으로 사용 가능한 추적자, 유전자 전달체로서 유용하게 사용될 수 있다.The present invention relates to a chitosan complex containing a pH-sensitive imidazole group, a method for preparing the same, and the chitosan complex of the present invention is excellent in biocompatibility and biodegradability, and the surface of the hydrophilic glycol chitosan in an aqueous solution is excellent. It is composed of tens to hundreds of nanometers of spherical aggregate composed of hydrophobic histidine inside, and it is possible to effectively encapsulate various hydrophobic drugs, tracers and genes including anticancer drugs into nano magnetic aggregates, and thus, medical drug carriers, molecular imaging It can be usefully used as a tracer and gene carrier.
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