KR20240041172A - Micelle comprising polyphenol - Google Patents
Micelle comprising polyphenol Download PDFInfo
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- KR20240041172A KR20240041172A KR1020220120376A KR20220120376A KR20240041172A KR 20240041172 A KR20240041172 A KR 20240041172A KR 1020220120376 A KR1020220120376 A KR 1020220120376A KR 20220120376 A KR20220120376 A KR 20220120376A KR 20240041172 A KR20240041172 A KR 20240041172A
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- micelle
- micelles
- present
- drug delivery
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Abstract
본 발명은 양친매성 공중합체 및 폴리 페놀 화합물이 자기 조립되어 형성된 마이셀 입자, 이의 제조방법, 및 상기 마이셀 입자를 포함하는 약물 전달체 또는 약제학적 조성물에 관한 것이다. 본 발명의 마이셀 입자는 체내 체액에 의한 희석에도 높은 콜로이드 안정성을 가지고 있으며, 산화 및 온도 변화에도 일정하며, 높은 단백질 친화력으로 주변 체액에 존재하는 단백질을 억제할 수 있어 담지한 약물이 체액에 존재하는 여러 분해 효소에 저항할 수 있다. 따라서 소수성 약물 및 단백질 약물의 전달체 및 고지혈증 또는 이상지질혈증의 치료용 조성물로 유용하게 사용할 수 있다.The present invention relates to micelle particles formed by self-assembling an amphipathic copolymer and a polyphenol compound, a method for manufacturing the same, and a drug delivery vehicle or pharmaceutical composition containing the micelle particles. The micelle particles of the present invention have high colloidal stability even when diluted by body fluids, are constant even under oxidation and temperature changes, and can inhibit proteins present in surrounding body fluids with high protein affinity, thereby preventing the loaded drug from being present in body fluids. Can resist several degrading enzymes. Therefore, it can be usefully used as a carrier for hydrophobic drugs and protein drugs, and as a composition for treating hyperlipidemia or dyslipidemia.
Description
본 발명은 양친매성 공중합체 및 폴리 페놀 화합물이 자기 조립되어 형성된 마이셀 입자, 이의 제조방법, 및 상기 마이셀 입자를 포함하는 약물 전달체 또는 약제학적 조성물에 관한 것이다.The present invention relates to micelle particles formed by self-assembling an amphipathic copolymer and a polyphenol compound, a method for manufacturing the same, and a drug delivery vehicle or pharmaceutical composition containing the micelle particles.
고분자 마이셀(polymeric micelle)은 소수성 치료제(hydrophobic therapeutic agents)(예: 항암 및 항염증제)를 가용화하는 효과적인 약물 전달 시스템으로 널리 연구되어 왔다. 일반적으로 마이셀은 Pluronic® 및 폴리에틸렌 글리콜(polyethylene glycol, PEG) 및 폴리(락트산-코-글리콜산)(poly(lactic-co-glycolic acid)) 세그먼트와 함께 합성된 생체 적합성 공중합체와 같은 양친매성 이중 블록 또는 삼중 블록 공중합체의 자가 조립에 의해 생성된다. 대표적으로 두 개의 친수성 폴리에틸렌 옥사이드(polyethylene oxide, PEO) 블록과 소수성 폴리(프로필렌 옥사이드)(poly(propylene oxide), PPO) 블록으로 구성된 Pluronic®은 임계 마이셀 농도(critical micelle concentration, CMC) 이상의 비공유 소수성 상호작용으로 인해 가역적 마이셀 구조를 가지며, PPO 및 PEO의 온도와 길이에 따라 달라진다. 이러한 마이셀에 대한 손쉬운 제조 공정에도 불구하고, 특히 생체 내에서 시간에 따라 제어할 수 없는 응집 및 농도 의존적 해리와 같은 몇 가지 제한 사항이 있다. 예를 들어, 마이셀을 정맥 주사하면 체액(예: 혈액)에 의한 희석으로 인해 최종 농도가 감소하여 입자가 붕괴되어 결국 캡슐화된 약물이 손실된다. 따라서, 낮은 CMC 및 높은 안정성을 가지는 마이셀 개발이 필요한 실정이다.Polymeric micelles have been widely studied as an effective drug delivery system for solubilizing hydrophobic therapeutic agents (e.g., anticancer and anti-inflammatory agents). Typically, micelles are amphiphilic doublets such as Pluronic ® and biocompatible copolymers synthesized with polyethylene glycol (PEG) and poly(lactic-co-glycolic acid) segments. It is produced by self-assembly of block or triblock copolymers. Typically, Pluronic ® is composed of two hydrophilic polyethylene oxide (PEO) blocks and a hydrophobic poly(propylene oxide) (PPO) block, and has a non-covalent hydrophobic interaction above the critical micelle concentration (CMC). Due to its action, it has a reversible micelle structure and varies depending on the temperature and length of PPO and PEO. Despite the facile fabrication process for these micelles, there are some limitations, such as uncontrollable aggregation and concentration-dependent dissociation over time, especially in vivo. For example, when micelles are injected intravenously, dilution by body fluids (e.g. blood) reduces the final concentration, leading to particle collapse and ultimately loss of the encapsulated drug. Therefore, there is a need to develop micelles with low CMC and high stability.
본 발명자들은 상술한 문제를 해결하고 낮은 CMC 및 높은 안정성을 가지는 마이셀을 개발하고자 예의 연구 노력하였다. 그 결과 양친매성 고분자와 식물 추출물에서 발견되는 폴리 페놀 성분을 혼합하는 경우 마이셀의 CMC를 감소시키면서 높은 안정성을 제공함을 규명함으로써, 본 발명을 완성하게 되었다. The present inventors made extensive research efforts to solve the above-mentioned problems and develop micelles with low CMC and high stability. As a result, the present invention was completed by demonstrating that mixing amphiphilic polymers and polyphenol components found in plant extracts reduces the CMC of micelles while providing high stability.
따라서, 본 발명의 목적은 양친매성 공중합체 및 폴리 페놀 화합물이 자기 조립되어 형성된 마이셀 입자 및 상기 마이셀 입자의 제조방법을 제공하는 것이다. Therefore, the purpose of the present invention is to provide micelle particles formed by self-assembly of an amphipathic copolymer and a polyphenol compound, and a method for producing the micelle particles.
본 발명의 다른 목적은 상기 마이셀 입자를 포함하는 약물 전달체 또는 약제학적 조성물을 제공하는 것이다. Another object of the present invention is to provide a drug carrier or pharmaceutical composition containing the micelle particles.
본 발명의 일 양태에 따르면, 본 발명은 양친매성 공중합체 및 폴리 페놀 화합물이 자기 조립되어 형성된 마이셀 입자를 제공한다.According to one aspect of the present invention, the present invention provides micelle particles formed by self-assembly of an amphipathic copolymer and a polyphenol compound.
본 발명의 일 구현예에 있어서, 상기 양친매성 공중합체는 폴리(에틸렌옥사이드)-b-폴리(ε-카프로락톤) 공중합체(PEO-b-PCL), PE-bPCL (poly(ethylene)-b-poly(ε-caprolactone), PHBA-b-PEO (poly(3-hydroxybutyric acid)-b-poly(ethylene oxide), PEO-b-PAA (poly(ethylene oxide)-b-poly(acrylic acid), PEO-b-PGA (poly(ethylene oxide)-b-poly(glycolic acid), PMPC-b-PBMA (poly(2-methacryloyloxyethyl phosphorylcholine)-bpoly(butylmetacrylate), 및 PEO-b-PCL-b-PEO (poly (ethylene oxide)-b-poly(ε-caprolactone)-bpoly(ethylene oxide) 등으로 이루어진 그룹으로부터 선택된 적어도 하나일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the amphipathic copolymer is poly(ethylene oxide)-b-poly(ε-caprolactone) copolymer (PEO-b-PCL), PE-bPCL (poly(ethylene)-b -poly(ε-caprolactone), PHBA-b-PEO (poly(3-hydroxybutyric acid)-b-poly(ethylene oxide), PEO-b-PAA (poly(ethylene oxide)-b-poly(acrylic acid), PEO-b-PGA (poly(ethylene oxide)-b-poly(glycolic acid), PMPC-b-PBMA (poly(2-methacryloyloxyethyl phosphorylcholine)-bpoly(butylmetacrylate), and PEO-b-PCL-b-PEO ( It may be at least one selected from the group consisting of poly (ethylene oxide)-b-poly(ε-caprolactone)-bpoly(ethylene oxide), but is not limited thereto.
본 발명의 다른 구현예에 있어서, 상기 양친매성 공중합체는 폴리에틸렌옥사이드-폴리프로필렌옥사이드-폴리에틸렌옥사이드(PEO-PPO-PEO) 공중합체, PEO-b-PBLA (poly(ethylene oxide)-b-poly(β-benzyl-l-aspartate), PS-b-PEO (poly(styrene)-b-poly(ethylene oxide), PEO-b-PLGA-b-PEO ((poly (ethylene oxide)-b-poly(L-lactic acidcoglycolide)-b-poly(ethylene oxide) 등으로 이루어진 그룹으로부터 선택된 적어도 하나일 수 있으나, 이에 제한되는 것은 아니다.In another embodiment of the present invention, the amphipathic copolymer is polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) copolymer, PEO-b-PBLA (poly(ethylene oxide)-b-poly( β-benzyl-l-aspartate), PS-b-PEO (poly(styrene)-b-poly(ethylene oxide), PEO-b-PLGA-b-PEO ((poly (ethylene oxide)-b-poly(L It may be at least one selected from the group consisting of -lactic acidcoglycolide)-b-poly(ethylene oxide), etc., but is not limited thereto.
본 발명의 구체적인 구현예에 있어서, 상기 양친매성 공중합체는 폴리에틸렌옥사이드-폴리프로필렌옥사이드-폴리에틸렌옥사이드(PEO-PPO-PEO) 공중합체이다. In a specific embodiment of the present invention, the amphipathic copolymer is a polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) copolymer.
본 발명의 일 구현예에 있어서, 상기 마이셀이 포함하는 양친매성 공중합체 및 폴리 페놀 화합물의 중량비는 1-20:1 이다. 보다 구체적으로, 상기 마이셀이 포함하는 양친매성 공중합체 및 폴리 페놀 화합물의 중량비는 1-20:1, 1-18:1, 1-16:1, 1-15:1, 1-14:1, 1-13:1, 1-12:1, 1-11:1, 1-10:1, 1-9:1, 1-8:1, 1-7:1, 1-6:1, 1-5:1, 1-4:1, 1-3:1, 1-2:1, 20:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 또는 1:1일 수 있으나, 이에 한정되는 것은 아니다. In one embodiment of the present invention, the weight ratio of the amphipathic copolymer and the polyphenol compound contained in the micelle is 1-20:1. More specifically, the weight ratio of the amphipathic copolymer and polyphenol compound contained in the micelle is 1-20:1, 1-18:1, 1-16:1, 1-15:1, 1-14:1, 1-13:1, 1-12:1, 1-11:1, 1-10:1, 1-9:1, 1-8:1, 1-7:1, 1-6:1, 1- 5:1, 1-4:1, 1-3:1, 1-2:1, 20:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, It may be 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1, but is not limited thereto.
본 발명의 일 구현예에 있어서, 상기 폴리 페놀 화합물은 갈릭산 (gallic acid), 에피갈로카테친 갈레이트 (epigallocatechin gallate), 에피갈로카테킨 (epigallocatechin), 갈로카테친 갈레이트 (gallocatechin gallate), 에피카테친 갈레이트 (epicatechin gallate), 카테친 갈레이트 (catechin gallate), 알킬 갈레이트 (alkyl gallate), 부틸 갈레이트 (butyl gallate), 헥실 갈레이트 (hexyl gallate) 및 라우릴 갈레이트 (lauryl gallate)로 이루어진 군으로부터 선택되는 것이나, 이에 한정되는 것은 아니다. In one embodiment of the present invention, the polyphenol compound is gallic acid, epigallocatechin gallate, epigallocatechin, gallocatechin gallate, epi Composed of epicatechin gallate, catechin gallate, alkyl gallate, butyl gallate, hexyl gallate and lauryl gallate It is selected from the group, but is not limited to this.
본 발명의 구체적인 구현예에 있어서, 상기 폴리 페놀 화합물은 알킬 갈레이트이다. In a specific embodiment of the present invention, the polyphenol compound is an alkyl gallate.
본 발명의 보다 더 구체적인 구현예에 있어서, 상기 알킬 갈레이트는 옥틸 갈레이트이다.In a more specific embodiment of the present invention, the alkyl gallate is octyl gallate.
본 발명의 일 구현예에 있어서, 상기 마이셀 입자는 다음 특징을 가진다:In one embodiment of the present invention, the micellar particles have the following characteristics:
i) CMC(critical micelle concentration)가 양친매성 공중합체의 농도를 기준으로 5 내지 50 μM임;i) the critical micelle concentration (CMC) is 5 to 50 μM based on the concentration of the amphipathic copolymer;
ii) 30-40℃ 범위 내에서 수용액 내 마이셀 구조의 함량 변화가 20% 이내임; ii) the change in the content of the micelle structure in the aqueous solution within the range of 30-40℃ is within 20%;
iii) 단백질 친화성(affinity)을 나타냄;iii) indicates protein affinity;
iv) 산화제에 대한 산화 저항성을 나타냄; 또는iv) exhibits oxidation resistance to oxidizing agents; or
v) 이들의 조합.v) Combination of these.
본 발명의 일 구현예에 있어서, 본 발명의 양친매성 공중합체 및 폴리페놀 화합물을 포함하는 마이셀이 나타내는 상기 단백질 친화성, 산화 저항성은, 폴리페놀 화합물을 포함하지 않고 양친매성 공중합체를 포함하는 마이셀이 나타내는 것과 비교한 것이다. In one embodiment of the present invention, the protein affinity and oxidation resistance exhibited by micelles containing an amphipathic copolymer and a polyphenol compound of the present invention are determined by micelles containing an amphipathic copolymer but not a polyphenol compound. This is compared to what it represents.
본 발명의 일 실시예에 있어서, 상기 마이셀은 단백질 친화성을 나타내는 결과, 효소(예컨대, tPA (tissue plasminogen activator))에 흡착하여 효소에 의한 마이셀 및/또는 약물의 분해에 대한 저항성을 나타낸다. In one embodiment of the present invention, the micelles exhibit protein affinity and, as a result, adsorb to enzymes (e.g., tPA (tissue plasminogen activator)) and exhibit resistance to degradation of micelles and/or drugs by enzymes.
본 발명의 일 구현예에 있어서, 상기 산화제는 질산은(AgNO3), 과요오드산나트륨(NaIO4), 및 수산화나트륨(NaOH)을 포함하나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the oxidizing agent includes, but is not limited to, silver nitrate (AgNO 3 ), sodium periodate (NaIO 4 ), and sodium hydroxide (NaOH).
본 발명의 다른 일 양태에 따르면, 본 발명은 다음 단계를 포함하는, 양친매성 공중합체 및 폴리 페놀 화합물을 포함하는 마이셀 입자의 제조방법을 제공한다:According to another aspect of the present invention, the present invention provides a method for producing micelle particles containing an amphipathic copolymer and a polyphenol compound, comprising the following steps:
(a) 양친매성 공중합체 및 폴리페놀 화합물을 수성 용매에 첨가하여 혼합하는 단계; 및(a) adding and mixing an amphipathic copolymer and a polyphenol compound to an aqueous solvent; and
(b) 상기 결과물로부터 마이셀 입자를 분리 및 정제하는 단계.(b) separating and purifying micelle particles from the resulting product.
본 발명의 일 구현예에 있어서, 상기 수성 용매는 정제수, 탈이온수, 인산완충용액, 생리식염수 또는 이들의 조합일 수 있으나, 이에 한정되는 것은 아니다. In one embodiment of the present invention, the aqueous solvent may be purified water, deionized water, phosphate buffer solution, physiological saline, or a combination thereof, but is not limited thereto.
본 발명의 일 구현예에 있어서, 상기 (a) 단계의 혼합은 초음파 처리, 또는 고압유화기의 사용에 의하여 이루어지는 것이나, 이에 한정되는 것은 아니고 유화에 의하여 자가조립에 의한 마이셀을 만들 수 있는 방법이면 제한없이 사용될 수 있다.In one embodiment of the present invention, the mixing in step (a) is accomplished by ultrasonic treatment or the use of a high-pressure emulsifier, but is not limited to this, as long as it is a method that can produce micelles by self-assembly by emulsification. It can be used without restrictions.
상기 본 발명의 일 양태에 따른 마이셀 입자의 제조방법은 상술한 마이셀 입자와 그 구성을 같이하는 것이므로, 양 발명 사이에 공통되는 사항은 동일하게 적용이 가능하다.Since the method for producing micelle particles according to one aspect of the present invention has the same composition as the micelle particles described above, common matters between both inventions can be applied in the same way.
본 발명의 또 다른 일 양태에 따르면, 본 발명은 상술한 마이셀 입자를 포함하는 혈관 주사용 약물 전달체를 제공한다.According to another aspect of the present invention, the present invention provides a drug delivery system for vascular injection containing the above-described micelle particles.
본 발명의 상술한 마이셀 입자는 동적광산란(dynamic light scattering, DLS)에 의해 측정시 수력학적 직경(hydrodynamic diameter)이 10-200 ㎚인 것이 바람직하다. 10 ㎚에 이르지 못하면, 크기가 너무 작아 전달의 대상이 되는 약물의 포집에 문제가 발생한다. 200 ㎚를 초과하는 경우에는 혈관 내에 투여되었을 때에 응집체를 형성하거나 거대 분자 형성에 따른 혈전을 형성을 촉진할 수 있어 안전성에 문제가 있을 수 있다. The above-described micelle particles of the present invention preferably have a hydrodynamic diameter of 10-200 nm as measured by dynamic light scattering (DLS). If it does not reach 10 nm, the size is too small and problems arise in capturing the drug to be delivered. If it exceeds 200 nm, it may form aggregates when administered intravascularly or promote the formation of blood clots due to the formation of macromolecules, which may pose a safety problem.
본 발명의 일 구현예에 있어서, 상기 약물 전달체는 소수성 약물을 담지하는 것이다. 상기 소수성 약물에는 항암제, 항산화제, 항염증제, 진통제, 항관절염제, 진정제, 항우울증제, 항정신병 약물, 신경안정제, 항불안제, 항혈관신생 억제제, 면역억제제, 항바이러스제, 항생제, 식용억제제, 항히스타민제, 호르몬제, 항혈전제, 이뇨제, 항고혈압제, 심혈관질환 치료제 및 혈관 확장제 등을 들 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the drug carrier carries a hydrophobic drug. The hydrophobic drugs include anticancer agents, antioxidants, anti-inflammatory agents, analgesics, anti-arthritis agents, sedatives, antidepressants, antipsychotics, tranquilizers, anti-anxiety agents, anti-angiogenesis inhibitors, immunosuppressants, antivirals, antibiotics, food suppressants, and antihistamines. , hormones, antithrombotic agents, diuretics, antihypertensive agents, cardiovascular disease treatments, and vasodilators, but are not limited thereto.
본 발명의 일 구현예에 있어서, 상기 약물 전달체는 단백질 약물을 담지하는 것이다. 상기 단백질 약물은 융합단백질, 재조합 단백질, 항체 의약품, 항원, 호르몬 등을 포함하나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the drug carrier carries a protein drug. The protein drugs include, but are not limited to, fusion proteins, recombinant proteins, antibody drugs, antigens, hormones, etc.
본 발명의 또 다른 일 양태에 따르면, 마이셀 입자를 유효성분으로 포함하는 고지혈증 또는 이상지질혈증의 예방 또는 치료용 약제학적 조성물을 제공한다. According to another aspect of the present invention, a pharmaceutical composition for preventing or treating hyperlipidemia or dyslipidemia is provided, comprising micelle particles as an active ingredient.
본 명세서에서 용어 '고지혈증'이란 혈중에 콜레스테롤 및 중성지방이 증가된 상태를 의미한다. As used herein, the term 'hyperlipidemia' refers to a condition in which cholesterol and triglycerides are increased in the blood.
본 명세서에서 용어 '이상지질혈증'이란 혈중 콜레스테롤 및 중성지방이 증가되고, LDL-콜레스테롤 농도가 높거나 또는 HDL-콜레스테롤 농도가 낮은 상태를 의미한다. As used herein, the term 'dyslipidemia' refers to a state in which blood cholesterol and triglycerides are increased, LDL-cholesterol concentration is high, or HDL-cholesterol concentration is low.
본 발명의 조성물이 약제학적 조성물인 경우, 약제학적으로 허용되는 담체가 포함된다. 상기 약제학적으로 허용되는 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다.When the composition of the present invention is a pharmaceutical composition, a pharmaceutically acceptable carrier is included. The pharmaceutically acceptable carriers are commonly used in formulations and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Includes, but is not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.
본 발명의 약제학적 조성물은 비경구 투여가 가능하며, 예컨대 정맥내 투여, 복강내 투여, 근육내 투여, 피하투여 또는 국소 투여될 수 있다. 또한, 그 밖에도 경구투여, 직장투여, 흡입투여, 경비투여 등이 가능하다. 본 발명의 구체적인 구현예에 있어서, 상기 약제학적 조성물은 정맥내 투여용이다.The pharmaceutical composition of the present invention can be administered parenterally, for example, intravenously, intraperitoneally, intramuscularly, subcutaneously, or topically. Additionally, oral administration, rectal administration, inhalation administration, and nasal administration are possible. In a specific embodiment of the present invention, the pharmaceutical composition is for intravenous administration.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여방식, 환자의 연령, 체중, 성, 질병 증상의 정도, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 목적하는 치료에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. 본 발명의 바람직한 구현예에 따르면, 본 발명의 약제학적 조성물의 1일 투여량은 0.001-100 ㎎/㎏이다.The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate, and reaction sensitivity. And, usually, a skilled doctor can easily determine and prescribe an effective dosage for the desired treatment. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-100 mg/kg.
본 발명의 약제학적 조성물은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화됨으로써 단위 용량 형태로 제조되거나, 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성매질중의 용액, 현탁액 또는 유화액 형태일 수 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. Alternatively, it can be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, and may additionally contain a dispersant or stabilizer.
본 명세서에서 용어 "투여"는 임의의 적절한 방법으로 대상(subject)에게 소정의 물질을 제공하는 것을 의미한다. 본 발명의 조성물의 투여경로는 목적 조직에 도달할 수 있는 한 일반적인 모든 경로를 통하여 상술한 바와 같이 경구 또는 비경구 투여될 수 있다. 또한, 본 발명의 조성물은 유효성분을 표적 세포, 조직 또는 기관으로 전달할 수 있는 임의의 장치를 이용해 투여될 수도 있다.As used herein, the term “administration” means providing a predetermined substance to a subject by any suitable method. The route of administration of the composition of the present invention may be oral or parenteral administration as described above through all general routes as long as it can reach the target tissue. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells, tissues, or organs.
본 명세서에서 용어 "대상(subject)"은, 특별히 한정되는 것은 아니지만, 예를 들어, 인간, 원숭이, 소, 말, 양, 돼지, 닭, 칠면조, 메추라기, 고양이, 개, 마우스, 쥐, 토끼 또는 기니아 피그를 포함하고, 바람직하게는 포유류, 보다 바람직하게는 인간을 의미한다.The term “subject” herein is not particularly limited, but includes, for example, a human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, or Including guinea pigs, preferably mammals, more preferably humans.
본 명세서에서 용어, "예방"은 본 발명에 따른 조성물의 투여로 고지혈증 또는 이상지질혈증의 증상을 억제 또는 지연시키는 모든 행위를 의미한다.As used herein, the term “prevention” refers to any action that suppresses or delays the symptoms of hyperlipidemia or dyslipidemia by administering the composition according to the present invention.
본 명세서에서 용어, "치료"는 본 발명에 따른 조성물의 투여로 고지혈증 또는 이상지질혈증의 증상을 호전시키거나 완치시키는 모든 행위를 의미한다.As used herein, the term “treatment” refers to any action that improves or cures the symptoms of hyperlipidemia or dyslipidemia by administering the composition according to the present invention.
본 발명의 약제학적 조성물은 본 발명의 입자의 약제학적 유효량을 포함한다. 상기 약제학적 유효량은 상기한 입자가 약리학적 효과를 달성하는데 충분한 양을 의미한다.The pharmaceutical composition of the present invention comprises a pharmaceutically effective amount of the particles of the present invention. The pharmaceutically effective amount refers to an amount sufficient for the particles to achieve a pharmacological effect.
본 발명의 마이셀 입자는 체내 체액에 의한 희석에도 높은 콜로이드 안정성을 가지고 있으며, 산화 및 온도변화에도 일정하며, 높은 단백질 친화력으로 주변 체액에 존재하는 단백질을 억제할 수 있어 담지한 약물이 체액에 존재하는 여러 분해 효소에 저항할 수 있다. 따라서 소수성 약물 및 단백질 약물의 전달체 및 고지혈증 또는 이상지질혈증의 치료용 조성물로 유용하게 사용할 수 있다.The micelle particles of the present invention have high colloidal stability even when diluted by body fluids, are constant even under oxidation and temperature changes, and can inhibit proteins present in surrounding body fluids with high protein affinity, thereby preventing the loaded drug from being present in body fluids. Can resist several degrading enzymes. Therefore, it can be usefully used as a carrier for hydrophobic drugs and protein drugs, and as a composition for treating hyperlipidemia or dyslipidemia.
도 1은 플루로닉 및 옥틸 갈레이트를 용매에 교반하여 자가조립을 이용해 마이셀을 제작하는 모식도를 나타낸다.
도 2는 마이셀 농도(μM)에 따른 형광 세기 그래프를 나타낸다. 검정색 화살표는 CMC (critical micelle concetration)를 나타낸다. 도 2의 좌측 그래프는 플루로닉으로만 이루어진 마이셀에 대한 그래프이고, 도 2의 우측 그래프는 본 발명에 따른 플루로닉 및 옥틸 갈레이트를 포함하는 마이셀에 대한 그래프이다.
도 3은 9 μM, 20 μM, 또는 50 μM 농도의 마이셀의 30℃, 35℃, 또는 40℃ 온도에 따른 형광 세기 그래프를 나타낸다. 도 3의 좌측 그래프는 플루로닉으로만 이루어진 마이셀에 대한 그래프이고, 도 3의 우측 그래프는 본 발명에 따른 플루로닉 및 옥틸 갈레이트를 포함하는 마이셀에 대한 그래프이다.
도 4는 GA를 Plu에 포함시키기 전 후 마이셀의 유체역학적 직경의 변화를 나타낸 도이다.
도 5는 산화제(AgNO3 또는 NaIO4)에 의해 산화된 마이셀 사진, 수산화 나트륨 존재하에 산화된 마이셀 사진과 이의 색 변화를 흡광도로 정량 분석한 그래프, 및 산화제에 의한 마이셀의 크기변화 그래프를 나타낸다.
도 6은 피브리노겐(fibrinogen)을 포함하는 우무 젤(agar gel)에 마이셀 및 피브리노겐을 분해시키는 효소 tPA (tissue plasminogen activator)를 첨가한 경우 시간에 따라 젤이 분해되는 정도를 확인한 사진 및 tPA 활성(%) 그래프를 나타낸다.
도 7a는 본 발명의 Plu-GA 마이셀의 수불용성 분자(e.g. 콜레스테롤)에 대한 용해성을 확인하기 위한 실험의 개요를 나타낸 도이다.
도 7b는 본 발명의 Plu-GA 마이셀의 콜레스테롤 결정에 대한 가용화 정도를 나타낸 도이다.
도 8a 및 도 8b는 본 발명의 Plu-GA 마이셀의 Plu 농도에 따른 세포 생존율을 통하여 생물학적 안전성을 나타낸 도이다.
도 9는 본 발명의 Plu-GA 마이셀의 단백질(e.g. 알부민) 흡착 기능성을 확인한 결과를 나타낸 도이다.Figure 1 shows a schematic diagram of producing micelles using self-assembly by stirring pluronic and octyl gallate in a solvent.
Figure 2 shows a graph of fluorescence intensity according to micelle concentration (μM). Black arrow indicates critical micelle concentration (CMC). The left graph in Figure 2 is a graph for micelles made only of pluronic, and the right graph in Figure 2 is a graph for micelles containing pluronic and octyl gallate according to the present invention.
Figure 3 shows a graph of the fluorescence intensity of micelles at concentrations of 9 μM, 20 μM, or 50 μM as a function of temperature at 30°C, 35°C, or 40°C. The left graph in Figure 3 is a graph for micelles made only of pluronic, and the right graph in Figure 3 is a graph for micelles containing pluronic and octyl gallate according to the present invention.
Figure 4 is a diagram showing the change in hydrodynamic diameter of micelles before and after including GA in Plu.
Figure 5 shows a photograph of micelles oxidized by an oxidizing agent (AgNO 3 or NaIO 4 ), a photograph of micelles oxidized in the presence of sodium hydroxide, a graph quantitatively analyzing the color change using absorbance, and a graph of the size change of micelles due to an oxidizing agent.
Figure 6 is a photograph confirming the degree of gel decomposition over time when tPA (tissue plasminogen activator), an enzyme that decomposes micelles and fibrinogen, is added to agar gel containing fibrinogen and tPA activity (%) ) shows the graph.
Figure 7a is a diagram showing an outline of an experiment to confirm the solubility of Plu-GA micelles of the present invention to water-insoluble molecules (eg cholesterol).
Figure 7b is a diagram showing the degree of solubilization of Plu-GA micelles of the present invention with respect to cholesterol crystals.
Figures 8a and 8b are diagrams showing the biological safety of Plu-GA micelles of the present invention through cell viability according to Plu concentration.
Figure 9 is a diagram showing the results of confirming the protein (eg albumin) adsorption functionality of the Plu-GA micelle of the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 "%"는 별도의 언급이 없는 경우, 고체/고체는 (중량/중량) %, 고체/액체는 (중량/부피) %, 그리고 액체/액체는 (부피/부피) %이다.Throughout this specification, “%” used to indicate the concentration of a specific substance means (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.
실험방법 및 실험재료Experimental methods and experimental materials
1. 실험재료1. Experimental materials
옥틸갈레이트, 플루로닉 F127, 8-아닐리노-1-나프탈렌설폰산암모늄염(8-anilino-1-naphthalenesulfonic acid ammonium salt, ANS), 질산은(silver nitrate, AgNO3), 과요오드산나트륨(sodium (meta)periodate, NaIO4), 수산화나트륨(NaOH), 한천, 조직 플라스미노겐 활성제(tissue plasminogen activator, tPA), 인간 혈장의 피브리노겐, 인간 혈장의 트롬빈, 콜레스테롤 및 25-NBD 콜레스테롤은 Sigma-Aldrich(미국)에서 구입하였다. Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin, FBS(fetal bovine serum) 및 Dulbecco's phosphate- buffered saline (DPBS)는 Thermo Fisher Scientific(USA)에서 구입하였다. 무수 에틸 알코올은 삼전순약 (서울, 한국)에서 구입하였다.Octyl gallate, Pluronic F127, 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS), silver nitrate (AgNO 3 ), sodium periodate (sodium) (meta)periodate, NaIO 4 ), sodium hydroxide (NaOH), agar, tissue plasminogen activator (tPA), fibrinogen from human plasma, thrombin from human plasma, cholesterol and 25-NBD cholesterol were from Sigma-Aldrich. Purchased in (USA). Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin, fetal bovine serum (FBS), and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Thermo Fisher Scientific (USA). Anhydrous ethyl alcohol was purchased from Samjeon Pure Medicine (Seoul, Korea).
2. 플루로닉 F127 (Plu) 및 Plu-Ga 마이셀의 제작 (Preparation of Pluronic F127 (Plu) and Plu-GA micelles)2. Preparation of Pluronic F127 (Plu) and Plu-GA micelles
Plu 및 Plu-GA 마이셀은 탈이온수(deionised water, DW)에서 자가 조립을 통해 제작되었다. 플루로닉 F127 (Pluronic F127)은 Plu 마이셀화를 위해 3.9 mg mL-1의 농도로 용해되었다. Gallol 모이어티를 Plu 마이셀에 통합하기 위해 옥틸 갈레이트(octyl gallate, GA)를 Plu 대 GA 중량비 13에서 Pluronic 중합체와 함께 조립하였다.Plu and Plu-GA micelles were fabricated through self-assembly in deionized water (DW). Pluronic F127 was dissolved at a concentration of 3.9 mg mL -1 for Plu micellization. To incorporate the gallol moiety into Plu micelles, octyl gallate (GA) was assembled with Pluronic polymer at a Plu to GA weight ratio of 13.
3. CMC의 결정 (determination of critical micelle concentration)3. Determination of critical micelle concentration
각 마이셀의 CMC (critical micelle concentration)를 조사하기 위해 탈이온수에 고분자를 3.9 mg mL-1의 최종 농도로 용해하여 Plu-GA 및 Plu 스톡 용액을 준비하였다. 원액을 탈이온수로 희석하여 Plu 농도를 4에서 1323 μM로 조절한 다음, 희석된 용액 200 μL에 ANS(형광 프로브, 탈이온수에 용해시킨 10-5 M) 1 mL를 첨가하였다. 일련의 샘플 용액의 형광 방출(360 nm에서의 여기 및 460 nm에서의 방출)은 25℃에서 120초의 평형 후에 SYNERGY HTX 다중 모드 마이크로플레이트 판독기(BioTek, Winooski, VT, USA)를 사용하여 측정하였다. Plu 및 Plu-GA 마이셀의 CMC는 Plu 농도의 함수로 ANS 방출 곡선에서 두 선형 회귀 곡선의 절편을 계산하여 결정되었다. 형광 현미경(DMi8, Leica, Wetzlar, Germany)을 사용하여 10-5 M ANS 용액에서 Plu-GA 마이셀의 형광 이미지를 얻었다. 30~40℃의 배양 온도에 따른 마이셀 변화를 조사하기 위해 동일한 ANS 기반 기술이 사용되었다. 모든 샘플의 형광 방출(λExc = 360 nm 및 λEmi = 460 nm)은 평형 600초 후에 측정되었다(SYNERGY HTX 다중 모드 마이크로플레이트 판독기, BioTek, Winooski, VT, USA).To investigate the critical micelle concentration (CMC) of each micelle, Plu-GA and Plu stock solutions were prepared by dissolving the polymer in deionized water to a final concentration of 3.9 mg mL -1 . The stock solution was diluted with deionized water to adjust the Plu concentration from 4 to 1323 μM, and then 1 mL of ANS (fluorescent probe, 10 -5 M dissolved in deionized water) was added to 200 μL of the diluted solution. The fluorescence emission (excitation at 360 nm and emission at 460 nm) of a series of sample solutions was measured using a SYNERGY HTX multimode microplate reader (BioTek, Winooski, VT, USA) after 120 s of equilibration at 25°C. The CMC of Plu and Plu-GA micelles was determined by calculating the intercept of two linear regression curves from the ANS release curve as a function of Plu concentration. Fluorescence images of Plu-GA micelles in 10 -5 M ANS solution were obtained using a fluorescence microscope (DMi8, Leica, Wetzlar, Germany). The same ANS-based technique was used to investigate micelle changes with incubation temperature of 30–40°C. Fluorescence emission (λExc = 360 nm and λEmi = 460 nm) of all samples was measured after 600 s of equilibration (SYNERGY HTX multimode microplate reader, BioTek, Winooski, VT, USA).
4. 마이셀의 특성 확인4. Confirmation of micelle properties
동적 광산란(dynamic light scattering, DLS)(Nano-ZS90, Malvern, Worcestershire, UK)을 사용하여 마이셀의 유체역학적 직경과 제타 전위를 모니터링하였다. 각 마이셀은 Plu 및 Plu-GA에 대해 20 mg mL-1의 농도로 준비되었다(Plu의 경우 20 mg mL-1, [Plu]:[GA]의 5:1(w/w)). 모든 시료는 분석 직전에 0.22 μm 시린지 필터를 통해 여과하였으며, 측정은 25℃에서 120초 동안 평형시킨 후 수행하였다. Plu-GA(80 mg mL-1의 Plu, [Plu]:[GA]의 5:1(w/w)) 및 Plu (90 mg mL-1) 마이셀의 형태를 투과 전자 현미경(TEM)을 사용하여 평가하였다.The hydrodynamic diameter and zeta potential of the micelles were monitored using dynamic light scattering (DLS) (Nano-ZS90, Malvern, Worcestershire, UK). Each micelle was prepared at a concentration of 20 mg mL -1 for Plu and Plu-GA (20 mg mL -1 for Plu, 5:1 (w/w) of [Plu]:[GA]). All samples were filtered through a 0.22 μm syringe filter immediately before analysis, and measurements were performed after equilibrating for 120 seconds at 25°C. Morphology of Plu-GA (80 mg mL -1 of Plu, 5:1 (w/w) of [Plu]:[GA]) and Plu (90 mg mL -1 ) micelles using transmission electron microscopy (TEM). It was evaluated.
샘플 준비로는, 마이셀 용액의 분취량(5μL)을 탄소 코팅된 구리 그리드에 떨어뜨리고 20분 동안 인큐베이션하고 부드럽게 세척하였다. 그리드를 건조기(desiccator)에서 12시간 동안 공기 건조시키고 TALOS F200X 현미경(FEI, Hillsboro, USA)을 사용하여 관찰하였다.For sample preparation, an aliquot (5 μL) of the micelle solution was dropped onto a carbon-coated copper grid, incubated for 20 min, and gently washed. The grids were air dried in a desiccator for 12 hours and observed using a TALOS F200X microscope (FEI, Hillsboro, USA).
또한 마이셀에서 Plu와 GA 사이의 분자간 상호 작용을 조사하기 위하여, 푸리에 변환 적외선(Fourier transform infrared, FT-IR) 스펙트럼 분석(TENSOR27, Bruker, Bremen, Germany)을 수행하였다. Plu(7.5 mg mL-1), Plu-GA(Plu의 7.5 mg mL-1 [GA]:[Plu]의 비율 0.2, 0.5 및 0.8(w/w)) 및 GA 용액(7.5 mg mL-1) 물과 에탄올의 혼합용액(50:50 부피%)에 액화시켰다. 완전한 공기 건조 후, 샘플의 FT-IR 스펙트럼을 기록하였다.Additionally, to investigate the intermolecular interactions between Plu and GA in the micelles, Fourier transform infrared (FT-IR) spectral analysis (TENSOR27, Bruker, Bremen, Germany) was performed. Plu (7.5 mg mL -1 ), Plu-GA (7.5 mg mL -1 of Plu [GA]:[Plu] ratios 0.2, 0.5 and 0.8 (w/w)) and GA solution (7.5 mg mL -1 ) It was liquefied in a mixed solution of water and ethanol (50:50% by volume). After complete air drying, the FT-IR spectrum of the sample was recorded.
또한, 모든 마이셀의 흡수 스펙트럼(예: Plu(최종 농도 195 ㎍/mL), Plu-GA(Plu의 195 ㎍/mL, [Plu]:[GA] 비율 13:1) 및 GA (15 μg mL-1))는 UV-vis 분광 광도계 (Agilent 8453, Agilent, Santa Clara, CA, USA)를 사용하여 측정되었다.Additionally, the absorption spectra of all micelles, e.g., Plu (final concentration 195 μg/mL), Plu-GA (195 μg/mL of Plu, [Plu]:[GA] ratio 13:1), and GA (15 μg mL - 1 )) was measured using a UV-vis spectrophotometer (Agilent 8453, Agilent, Santa Clara, CA, USA).
5. Plu-GA 마이셀의 산화 (Oxidation of the Plu-GA micelles)5. Oxidation of the Plu-GA micelles
Plu-GA 마이셀에서 GA 산화를 유도하기 위해 세 가지 유형의 화학적 산화제인 NaIO4, AgNO3 또는 NaOH를 마이셀 용액에 첨가하였다. GA(0.3 mg mL-1) 및 Plu-GA(또는 Plu) 마이셀(3.9 mg mL-1)도 위에서 언급한 대로 준비되었다. NaIO4, AgNO3 또는 NaOH는 각각 0.05, 0.1, 0.1M의 최종 농도로 마이셀 용액에서 처리되었다.To induce GA oxidation in Plu-GA micelles, three types of chemical oxidants, NaIO 4 , AgNO 3 , or NaOH, were added to the micelle solution. GA (0.3 mg mL -1 ) and Plu-GA (or Plu) micelles (3.9 mg mL -1 ) were also prepared as mentioned above. NaIO 4 , AgNO 3 or NaOH were treated in micelle solutions at final concentrations of 0.05, 0.1, and 0.1 M, respectively.
이어서, 산화제/마이셀 용액(1mL)을 실온에서 16시간 동안 인큐베이션하였다. 또한, NaOH 처리군은 UV-vis 분광광도계(A450)를 사용하여 용액의 비색 변화를 감지하였다. DLS(Nano-ZS90, Malvern, UK)를 사용하여 산화제 처리 전후의 마이셀 크기 분포(0.22μm 주사기 필터로 여과)를 분석하였다.The oxidant/micelle solution (1 mL) was then incubated at room temperature for 16 hours. Additionally, in the NaOH treatment group, the colorimetric change of the solution was detected using a UV-vis spectrophotometer (A450). The micelle size distribution (filtered through a 0.22 μm syringe filter) before and after oxidizing agent treatment was analyzed using DLS (Nano-ZS90, Malvern, UK).
6. 섬유소 용해 활성 시험(Fibrinolytic activity test)6. Fibrinolytic activity test
마이셀과 혼합하기 전과 후에 tPA의 섬유소용해 활성을 평가하기 위해 1.25 mL 피브리노겐(pH 7.2 Tris 완충액에서 10 mg mL-1), 5 μL 트롬빈(mL당 250단위) 및 10 mL 한천 용액(15 mg mL-1)을 준비하고 37℃에서 유지하였다. 이어서, 한천 용액에 트롬빈을 혼합한 후, 피브리노겐 용액과 완전히 혼합하였다. 혼합 용액(1 mL)을 각 큐벳에 즉시 분배하고 37℃에서 2시간 동안 인큐베이션하여 탁한 한천-피브린 겔을 형성하였다. Plu/tPA(20 mg mL-1), Plu-GA/tPA(20 mg mL-1 lu, [Plu] : [GA]의 중량비 5:1) 및 PBS/tPA 용액은 동등한 농도의 tPA(20 μg mL-1)와 혼합하여 마이셀을 제작하였다. To evaluate the fibrinolytic activity of tPA before and after mixing with micelles, 1.25 mL fibrinogen (10 mg mL -1 in Tris buffer, pH 7.2), 5 μL thrombin (250 units per mL), and 10 mL agar solution (15 mg mL -1 1 ) was prepared and maintained at 37°C. Subsequently, thrombin was mixed with the agar solution and then thoroughly mixed with the fibrinogen solution. The mixed solution (1 mL) was immediately dispensed into each cuvette and incubated at 37°C for 2 hours to form a cloudy agar-fibrin gel. Plu/tPA (20 mg mL -1 ), Plu-GA/tPA (20 mg mL -1 lu, [Plu]:[GA] weight ratio 5:1) and PBS/tPA solutions were prepared with equivalent concentrations of tPA (20 μg mL-1) to produce micelles.
대조군으로 tPA가 없는 식염수를 준비하였다. 식염수(100μL), Plu/tPA, Plu-GA/tPA 및 PBS/tPA를 한천-피브린 겔에 첨가하고 37℃에서 0, 3, 6시간 동안 배양하였다. ImageJ 소프트웨어를 사용하여 겔의 투명 부피를 계산하여 각 샘플의 섬유소 용해 활성(%)을 평가하였다.As a control, a saline solution without tPA was prepared. Saline solution (100 μL), Plu/tPA, Plu-GA/tPA, and PBS/tPA were added to the agar-fibrin gel and incubated at 37°C for 0, 3, and 6 hours. The fibrinolytic activity (%) of each sample was evaluated by calculating the clear volume of the gel using ImageJ software.
7. 형광 콜레스테롤 결정의 제조(Preparation of fluorescent cholesterol crystals)7. Preparation of fluorescent cholesterol crystals
25-NBD 콜레스테롤과 콜레스테롤의 혼합물(1:50의 몰비)은 2 mg mL-1 농도의 에탄올에서 제조되었다. 탈이온수(30% v/v)의 첨가는 결정화를 유도하였고, 혼합물을 진공 오븐에서 완전히 건조시켜 형광성 콜레스테롤 결정을 얻었다.A mixture of 25-NBD cholesterol and cholesterol (molar ratio of 1:50) was prepared in ethanol at a concentration of 2 mg mL -1 . Addition of deionized water (30% v/v) induced crystallization, and the mixture was dried thoroughly in a vacuum oven to obtain fluorescent cholesterol crystals.
8. 콜레스테롤 결정의 가용화 (Solubilization of cholesterol crystals)8. Solubilization of cholesterol crystals
형광성 콜레스테롤 결정(PBS 중 500 μM)을 24시간 동안 다른 농도(0, 5 및 10 mg mL-1의 Plu, [Plu]:[GA]의 중량비 5:1)의 Plu 또는 Plu-GA 마이셀 용액에서 배양하였다. 마이셀 용액은 잔류 콜레스테롤 결정을 제거하기 위해 0.22 μm 필터를 사용하여 추가로 정제되었다. 이들 결정의 용해도는 SYNERGY HTX 다중모드 마이크로플레이트 판독기(BioTek, Winooski, VT, USA)를 사용하여 528 nm(λExc = 485 nm)의 파장에서 형광 방출을 측정하여 검출하였다.Fluorescent cholesterol crystals (500 μM in PBS) were incubated in Plu or Plu-GA micelle solutions at different concentrations (0, 5, and 10 mg mL -1 of Plu, [Plu]:[GA] weight ratio of 5:1) for 24 h. Cultured. The micelle solution was further purified using a 0.22 μm filter to remove residual cholesterol crystals. The solubility of these crystals was detected by measuring fluorescence emission at a wavelength of 528 nm (λExc = 485 nm) using a SYNERGY HTX multimode microplate reader (BioTek, Winooski, VT, USA).
9. 시험관 내 세포독성 시험(In vitro cytotoxicity test)9. In vitro cytotoxicity test
L929 마우스 섬유아세포를 미리 배양하여 마이셀의 세포적합성을 평가하였다. 세포를 배양하고 배양 배지(10% FBS 및 1% 페니실린/스트렙토마이신이 보충된 DMEM)에서 37℃, 5.0% CO2 가습 조건 하에서 배양하였다. 테스트를 위해 24웰 플레이트에 웰당 2.5×104 세포의 밀도로 세포를 시딩하고 24시간 동안 배양하였다. Plu-GA 또는 Plu를 포함하는 마이셀 용액의 분취액(50μL)을 각 웰에 추가하였다. 배양된 세포를 주변 광 및 실온에서 30분 동안 2 μM 칼세인 AM(살아있는 세포를 녹색으로 염색) 및 4 μM 에티듐 동종이량체(죽은 세포를 빨간색으로 염색)로 즉시 염색하였다. 형광현미경(DMi8, Leica, Wetzlar, Germany)을 사용하여 살아있는/죽은 형광 이미지를 관찰하였다. ImageJ 소프트웨어를 사용하여 무작위로 얻은 5개의 이미지에서 세포 수를 계산했으며, 빨간색 점은 죽은 세포를 나타내고 녹색 점은 살아있는 세포를 나타낸다. 세포 생존율(%)은 전체 모집단에서 살아있는 세포의 비율을 계산하여 정량화하였다.The cytocompatibility of the micelles was evaluated by pre-culturing L929 mouse fibroblasts. The cells were cultured in culture medium (DMEM supplemented with 10% FBS and 1% penicillin/streptomycin) at 37°C under 5.0% CO 2 humidified conditions. For testing, cells were seeded at a density of 2.5 × 10 4 cells per well in a 24-well plate and cultured for 24 hours. An aliquot (50 μL) of the micelle solution containing Plu-GA or Plu was added to each well. Cultured cells were immediately stained with 2 μM calcein AM (stains live cells green) and 4 μM ethidium homodimer (stains dead cells red) for 30 min in ambient light and room temperature. Live/dead fluorescence images were observed using a fluorescence microscope (DMi8, Leica, Wetzlar, Germany). Cell numbers were counted in five randomly acquired images using ImageJ software, with red dots representing dead cells and green dots representing live cells. Cell viability (%) was quantified by calculating the proportion of living cells in the total population.
실험결과Experiment result
실시예 1: 낮은 CMC 및 높은 안정성을 갖는 Plu-GA 마이셀의 형성 및 특성 확인Example 1: Formation and characterization of Plu-GA micelles with low CMC and high stability
안정하고 균질한 Plu-GA 마이셀은 물리적 자극(예: 열 또는 초음파 처리) 없이 수용액에서 양친매성 Plu와 GA의 자가 조립에 의해 생성되었다(도 1).Stable and homogeneous Plu-GA micelles were generated by self-assembly of amphiphilic Plu and GA in aqueous solution without physical stimulation (e.g., heat or sonication) (Figure 1).
Plu 또는 Plu-GA 마이셀의 CMC를 조사하기 위하여, 소수성 형광 프로브인 ANS가 사용되었다. ANS 분자는 극성 용매(예를 들어, 물과 에탄올)에 대해 소수성 마이셀 코어에 격리될 때 450 nm의 파장에서 높은 형광 방출을 나타낼 수 있다. 따라서 CMC는 분자의 형광 강도가 급격히 증가한 농도로 결정할 수 있다.To investigate the CMC of Plu or Plu-GA micelles, ANS, a hydrophobic fluorescent probe, was used. ANS molecules can exhibit high fluorescence emission at a wavelength of 450 nm when sequestered in hydrophobic micelle cores in polar solvents (e.g., water and ethanol). Therefore, CMC can be determined as the concentration at which the fluorescence intensity of the molecule rapidly increases.
대조군으로서 Plu의 CMC는 203 μM(도 2, 검은색 화살표)으로 이전에 보고된 79-556 μM 값과 상응하였다. As a control, the CMC of Plu was 203 μM (Figure 2, black arrow), corresponding to the previously reported value of 79-556 μM.
결과는 도 2에 나타내었다.The results are shown in Figure 2.
도 2에 나타낸 바와 같이, GA가 Plu에 첨가되었을 때, Plu 단독보다 약 22배 낮은 9 μM의 CMC에서 마이셀이 형성되었다(도 2, 검은색 화살표).As shown in Figure 2, when GA was added to Plu, micelles were formed at a CMC of 9 μM, approximately 22-fold lower than that of Plu alone (Figure 2, black arrow).
ANS를 포획하는 마이셀(예: Plu 농도 300 μM에서 Plu-GA)의 명확한 형성은 형광 현미경을 사용하여 관찰되었다(도 2). Plu-GA의 이러한 낮은 CMC는 혈관에 주사한 후에도 마이셀이 파괴되는 것을 방지할 것이다. Plu-GA의 낮은 CMC는 마이셀이 총 혈액량의 15%(예: 성인의 경우 5L, 마우스의 경우 2 mL)와 같은 최대 수준으로 주입될 때에도 예상대로 사용할 수 있을 것으로 예상된다.Clear formation of micelles that capture ANS (e.g., Plu-GA at a Plu concentration of 300 μM) was observed using fluorescence microscopy (Figure 2). This low CMC of Plu-GA will prevent the micelles from being destroyed even after intravascular injection. The low CMC of Plu-GA predicts that micelles can be used as expected even when injected at maximal levels, such as 15% of total blood volume (e.g., 5 L for adults, 2 mL for mice).
또한, Plu의 온도 반응성(temperature responsiveness)을 고려하여 Plu-GA의 열 안정성을 폴리머 단독의 열 안정성과 비교하였다.Additionally, considering the temperature responsiveness of Plu, the thermal stability of Plu-GA was compared with that of the polymer alone.
결과는 도 3에 나타내었다.The results are shown in Figure 3.
도 3에 나타낸 바와 같이, Pluronic®의 소수성 부분은 체온 37℃에서 자발적으로 얽혀 대량 규모에서 겔화를 유발하였다. 즉, Plu의 농도가 CMC보다 낮더라도 온도가 상승하면 고분자의 마이셀화 또는 응집이 발생할 수 있었다.As shown in Figure 3, the hydrophobic portion of Pluronic ® spontaneously entangled at body temperature of 37°C, causing gelation on a bulk scale. In other words, even if the concentration of Plu was lower than CMC, micellization or aggregation of the polymer could occur when the temperature increased.
도 3의 좌측 그래프에서 볼 수 있듯이, 40℃에서 Plu를 배양하면 CMC(검정색)보다 낮은 50 μM 농도에서도 마이셀이 형성되었다. 대조적으로, Plu와 동일한 농도(예: CMC 이상)의 Plu-GA는 온도의 함수로서 일정한 형광 방출을 나타내(도 3), 추가 응집 없이 안정적인 마이셀화를 나타냈다.As can be seen in the left graph of Figure 3, when Plu was incubated at 40°C, micelles were formed even at a concentration of 50 μM, which is lower than CMC (black). In contrast, Plu-GA at the same concentration as Plu (i.e., above the CMC) showed constant fluorescence emission as a function of temperature (Figure 3), indicating stable micellization without further aggregation.
한편, 본 발명자들은 GA를 Plu에 포함시킨 후 마이셀의 유체역학적 직경의 분포를 확인하였다. Meanwhile, the present inventors confirmed the distribution of hydrodynamic diameters of micelles after including GA in Plu.
결과는 도 4에 나타내었다.The results are shown in Figure 4.
도 4에 나타낸 바와 같이, GA를 Plu에 포함시킨 후 마이셀의 유체역학적 직경은 Plu 단독(도 4, 검은색)의 경우 9 nm에서 27 nm(도 4, 파란색)로 증가하였다. 이러한 결과는 GA가 Plu의 고분자 네트워크에 삽입될 수 있음을 시사한다. 마이셀화 동안, GA의 옥틸기는 Plu의 PPO와 소수성 상호작용을 경험할 수 있고, 상대적으로 친수성인 갈롤기가 마이셀 표면에 침착될 수 있고 중합체와의 분자간 상호작용을 통해 내부적으로 차폐될 수 있다.As shown in Figure 4, after incorporating GA into Plu, the hydrodynamic diameter of the micelles increased from 9 nm for Plu alone (Figure 4, black) to 27 nm (Figure 4, blue). These results suggest that GA can be inserted into the polymer network of Plu. During micellization, the octyl group of GA may experience hydrophobic interactions with the PPO of Plu, and the relatively hydrophilic gallol group may be deposited on the micelle surface and internally shielded through intermolecular interactions with the polymer.
실시예 2. Plu-GA 마이셀의 산화 저항성Example 2. Oxidation resistance of Plu-GA micelles
GA와 Plu의 결합은 갈롤기의 고유 산화에 영향을 줄 수 있다. GA의 3개의 수산기가 다중 수소 결합을 통해 Plu와 상호 작용하면 탈양성자화 후 갈로퀴논으로의 산화가 부분적으로 억제된다. The combination of GA and Plu can affect the intrinsic oxidation of the gallol group. When the three hydroxyl groups of GA interact with Plu through multiple hydrogen bonds, oxidation to galloquinone after deprotonation is partially inhibited.
본 발명자들은 Plu-GA 마이셀의 산화 저항성을 확인하고자, Plu, Plu-GA에 산화제인 질산은(AgNO3) 및 과요오드산나트륨(NaIO4)을 각각 첨가하고 16시간 후 이를 육안을 관찰 및 마이셀 입자의 사이즈 크기를 확인하였다. To confirm the oxidation resistance of Plu-GA micelles, the present inventors added silver nitrate (AgNO 3 ) and sodium periodate (NaIO 4 ), which are oxidizing agents, to Plu and Plu-GA, respectively, and observed them with the naked eye 16 hours later and observed the micelle particles. The size of was confirmed.
결과는 도 5에 나타내었다.The results are shown in Figure 5.
도 5에 나타낸 바와 같이, 예상대로 질산은(AgNO3) 및 과요오드산나트륨(NaIO4)과 같은 다양한 산화제가 Plu-GA에 첨가되었을 때 GA 산화 정도는 GA 단독의 산화 정도와 구별되었다. As shown in Figure 5, as expected, when various oxidizing agents such as silver nitrate (AgNO 3 ) and sodium periodate (NaIO 4 ) were added to Plu-GA, the degree of GA oxidation was distinct from that of GA alone.
첫 번째로, GA를 16시간 동안 질산은 (AgNO3)으로 처리하면 Gallol 그룹의 산화가 가속화되고 검은색 침전물이 생성되는데, 이는 빠른 은 환원 때문일 수 있다(도 5, 중간 사진 검은색 화살표). 대조적으로 같은 양의 GA를 포함하는 Plu-GA는 질산은 (AgNO3)을 첨가한 후에 갈색이 되어, 큰 응집체라기 보다는 은 나노입자를 형성하였다. First, treatment of GA with silver nitrate (AgNO 3 ) for 16 h accelerated the oxidation of Gallol groups and produced a black precipitate, which may be due to rapid silver reduction (Figure 5, black arrow in middle photo). In contrast, Plu-GA containing the same amount of GA turned brown after adding silver nitrate (AgNO 3 ), forming silver nanoparticles rather than large aggregates.
둘째로, GA 또는 Plu-GA에 과요오드산나트륨(NaIO4)을 첨가했을 때 침전물의 총량이 달랐다(도 5, 하단 사진). Plu-GA의 은 환원과 유사하게, GA 단독(검은색 화살표)과 비교하여 Plu-GA에서는 약간의 GA 응집만 관찰되었으며, 이는 감소된 갈롤의 산화를 나타낸다. Second, when sodium periodate (NaIO 4 ) was added to GA or Plu-GA, the total amount of precipitates was different (Figure 5, bottom photo). Similar to silver reduction in Plu-GA, only slight GA aggregation was observed in Plu-GA compared to GA alone (black arrow), indicating reduced oxidation of gallol.
또한 기본 조건(예: NaOH, 0.1M, pH 13)에서 GA 및 Plu-GA의 비색 변화를 모니터링하였다(도 5). 두 용액 모두 침전이 없었음에도 불구하고 산화적으로 가교된 갈롤 이량체에 해당하는 Plu-GA(0.4 ± 0.01; 도 3, 하늘색 막대)의 파장 450 nm에서의 흡광도는 GA (0.6 ± 0.01, 파란색 막대) 보다 낮았다.We also monitored the colorimetric changes of GA and Plu-GA under basic conditions (e.g., NaOH, 0.1 M, pH 13) (Figure 5). Despite the absence of precipitation in both solutions, the absorbance at a wavelength of 450 nm of Plu-GA (0.4 ± 0.01; Figure 3, light blue bars), corresponding to the oxidatively cross-linked gallol dimer, was significantly higher than that of GA (0.6 ± 0.01, blue bars). ) was lower than that.
Plu-GA에서 이러한 갈롤 산화는 갈롤 그룹의 공유 가교결합으로 인해 마이셀의 수축을 초래하여 과요오드산나트륨(NaIO4)(도 5, 우측 그래프, 빨간색)로 처리한 후 마이셀의 유체역학적 직경을 11.77 nm로 감소시키거나 염기성 pH(녹색)에서 7.59 nm로 감소시켰다. (예: 산화 없는 Plu-GA의 경우 24.72nm). This gallol oxidation in Plu-GA results in shrinkage of the micelles due to covalent cross-linking of the gallol groups, resulting in a hydrodynamic diameter of the micelles of 11.77 after treatment with sodium periodate (NaIO 4 ) (Figure 5, right graph, red). nm or reduced to 7.59 nm at basic pH (green). (e.g. 24.72 nm for Plu-GA without oxidation).
종합하면, 이러한 결과는 GA가 소수성 상호 작용 및 수소 결합을 통해 Plu와 상호 작용하고 이들의 산화적 가교가 마이셀을 더 조밀하고 작게 만들 수 있음을 나타낸다.Taken together, these results indicate that GA interacts with Plu through hydrophobic interactions and hydrogen bonds and that their oxidative cross-linking can make micelles more compact and smaller.
실시예 3: 외인성 효소 억제 및 콜레스테롤 용해에 있어서 Plu-GA 마이셀의 임상적 이점 (Clinical benefits of the Plu-GA micelles in inhibiting exogenous enzymes and dissolving cholesterol)Example 3: Clinical benefits of the Plu-GA micelles in inhibiting exogenous enzymes and dissolving cholesterol
식물 폴리페놀의 갈롤 모이어티는 소수성 상호작용과 수소 결합으로 인해 다양한 생체 거대분자, 특히 단백질에 대해 높은 친화성을 가지며, 그 결과 단백질의 고유한 생물학적 활성이 손실된다.The gallol moiety of plant polyphenols has high affinity for various biomacromolecules, especially proteins, due to hydrophobic interactions and hydrogen bonding, resulting in the loss of the intrinsic biological activity of proteins.
갈롤-접합 고분자 하이드로겔은 하이드로겔 표면에 외부 효소를 포획하기 때문에 효소 분해 저항성이 있다. 그 외에도 옥틸 갈레이트의 갈롤 부분이 활성 부위에 비특이적 결합하여, α-아밀라아제, 스쿠알렌 에폭시다아제(squalene epoxidase) 및 리폭시게나아제(lipoxygenase)의 효소 활성을 차단한다는 것이 입증된 바 있다. 따라서 Plu-GA 마이셀의 효소 저항성은 캡슐화된 소수성 약물을 보호하는 데 도움이 될 수 있을 것이다.Gallol-conjugated polymer hydrogels are resistant to enzymatic degradation because foreign enzymes are captured on the hydrogel surface. In addition, it has been proven that the gallol portion of octyl gallate binds non-specifically to the active site, blocking the enzymatic activities of α-amylase, squalene epoxidase, and lipoxygenase. Therefore, the enzyme resistance of Plu-GA micelles may be helpful in protecting the encapsulated hydrophobic drugs.
3-1. 외인성 효소 억제 실험3-1. Exogenous enzyme inhibition experiments
Plu-GA 마이셀의 효소적 억제 능력을 조사하기 위한 실험 환경에서, 본 발명자들은 혈전을 분해하는 모델 프로테아제로서, 조직 플라스미노겐 활성제(tissue-plasminogen activator, tPA)를 선택하고 체온에서 한천-피브리노겐 겔의 분해를 조사하여 활성을 평가하였다.In an experimental environment to investigate the enzymatic inhibition ability of Plu-GA micelles, the present inventors selected tissue-plasminogen activator (tPA) as a model protease to decompose blood clots and incubated it in an agar-fibrinogen gel at body temperature. The activity was evaluated by investigating the decomposition of .
tPA 용액(20 μg mL-1)을 각 마이셀 (Plu 또는 Plu-GA)과 혼합한 후, 이를 한천-피브리노겐 겔에 적용하고 겔의 분해된 부피(예: 투명 영역)를 관찰하였다(도 6의 노란색 선).After mixing the tPA solution (20 μg mL -1 ) with each micelle (Plu or Plu-GA), it was applied to the agar-fibrinogen gel and the resolved volume (e.g., transparent area) of the gel was observed (Figure 6). yellow line).
결과는 도 6에 나타내었다.The results are shown in Figure 6.
도 6에서 볼 수 있듯이 Plu-GA/tPA 그룹의 경우 한천-피브리노겐 겔의 분해가 억제되어 가장 작은 투명 영역(예: 6시간 처리 시 1200 mm3)이 생성되었다(도 6, A, 좌측 첫번째 하단 사진). 대조적으로, Plu/tPA 대조군의 경우 분해된 부피는 tPA 단독의 경우와 유사하였다(도 6, A, 두 번째 및 세 번째 하단 사진). tPA 활성의 정량적 분석을 위해, 분해된 부피를 활성의 100%를 나타내는 것으로 간주되는 tPA 단독의 부피와 비교하였다(도 6, B, 회색 막대). 그 결과, Plu-GA는 6시간 배양 후 tPA의 활성을 41% ± 7.6%로 감소시켰고(도 6, B, 파란색 막대), Plu는 94% ± 6.0%에서 단백질 활성을 유지하였다(도 6, B, 검은색 막대).As can be seen in Figure 6, in the case of the Plu-GA/tPA group, the decomposition of the agar-fibrinogen gel was suppressed, resulting in the smallest transparent area (e.g., 1200 mm 3 at 6 hours of treatment) (Figure 6, A, first bottom left) picture). In contrast, for the Plu/tPA control group, the decomposed volume was similar to that for tPA alone (Figure 6, A, second and third bottom photos). For quantitative analysis of tPA activity, the dissociated volume was compared to the volume of tPA alone, which was considered to represent 100% of activity (Figure 6, B, gray bars). As a result, Plu-GA reduced the activity of tPA to 41% ± 7.6% after 6 hours of incubation (Figure 6, B, blue bar), and Plu maintained protein activity at 94% ± 6.0% (Figure 6, B, black bars).
3-2. 콜레스테롤 용해 실험3-2. Cholesterol dissolution experiment
또한 본 발명자들은 Plu-GA와 Plu 마이셀이 수성 환경에서 수불용성 분자(예: 콜레스테롤)의 효율적인 용매화를 유도하는지 여부를 확인하였다.Additionally, the present inventors confirmed whether Plu-GA and Plu micelles induce efficient solvation of water-insoluble molecules (e.g. cholesterol) in an aqueous environment.
이를 입증하기 위해, 형광 콜레스테롤 결정을 293 K의 물에서 0.3 mg L-1 미만의 극도로 낮은 용해도를 갖는 모델 비극성 분자로 사용되었다. 구체적으로 Plu-GA 및 Plu 마이셀(Plu 농도 0, 5 및 10 mg mL-1)을 37℃에서 24시간 동안 콜레스테롤 결정과 함께 배양하고 형광 방출 정도를 확인하였다(도 7a).To demonstrate this, fluorescent cholesterol crystals were used as model nonpolar molecules with an extremely low solubility of less than 0.3 mg L −1 in water at 293 K. Specifically, Plu-GA and Plu micelles (Plu concentrations of 0, 5, and 10 mg mL -1 ) were incubated with cholesterol crystals at 37°C for 24 hours, and the degree of fluorescence emission was confirmed (Figure 7a).
결과는 도 7b에 나타내었다.The results are shown in Figure 7b.
도 7b에 나타낸 바와 같이, Plu-GA 및 Plu 마이셀(Plu 농도 0, 5 및 10 mg mL-1)을 37℃에서 24시간 동안 콜레스테롤 결정과 함께 배양하면 Plu 농도에 의존적으로 콜레스테롤이 용해되고, 콜레스테롤의 형광 방출을 향상시키는 것을 확인하였다. Plu-GA 및 Plu 마이셀 그룹 사이에서 향상된 형광 강도에는 큰 차이가 없었다. 따라서, 상기 결과로부터, Plu 마이셀에 GA가 추가된 본 발명의 Plu-GA 마이셀은 Plu 마이셀의 소수성 공동을 유지하면서도 포획된 약물을 효소 분해로부터 보호함을 알 수 있었다.As shown in Figure 7b, when Plu-GA and Plu micelles (Plu concentrations of 0, 5, and 10 mg mL -1 ) are incubated with cholesterol crystals at 37°C for 24 hours, cholesterol is dissolved and cholesterol is dissolved in a Plu concentration-dependent manner. It was confirmed that the fluorescence emission was improved. There was no significant difference in the enhanced fluorescence intensity between the Plu-GA and Plu micelle groups. Therefore, from the above results, it can be seen that the Plu-GA micelle of the present invention, in which GA is added to the Plu micelle, protects the captured drug from enzymatic degradation while maintaining the hydrophobic cavity of the Plu micelle.
실시예 4: Plu-GA 마이셀의 생물학적 안전성(Biological safety of the Plu-GA micelles)Example 4: Biological safety of the Plu-GA micelles
마지막으로 본 발명자들은, in vitro 세포독성 시험을 통해 Plu-GA 마이셀의 생물학적 안전성을 확인하였다. 실험 설정에서 본 발명자들은 [Plu]/[GA] 중량비 5로, GA를 포함하는 Plu-GA 마이셀을 사용했으며 샘플은 0, 250 및 500 μg mL-1의 Plu 농도에서 섬유아세포를 처리하는 데 사용되었다.Finally, the present inventors confirmed the biological safety of Plu-GA micelles through in vitro cytotoxicity tests. In the experimental setup, we used Plu-GA micelles containing GA at a [Plu]/[GA] weight ratio of 5 and samples were used to treat fibroblasts at Plu concentrations of 0, 250, and 500 μg mL -1 It has been done.
결과는 도 8a 및 도 8b에 나타내었다.The results are shown in Figures 8A and 8B.
도 8a에 나타낸 바와 같이, Plu 마이셀은 높은 세포 생존력을 나타내었지만, Plu-GA 마이셀의 세포 독성은 농도에 따라 달랐고, 97% ± 0.2%의 세포가 250 μg mL-1 미만의 Plu 농도로 Plu-GA 마이셀을 처리한 군에서 살아 있었다. As shown in Figure 8a, Plu micelles exhibited high cell viability, but the cytotoxicity of Plu-GA micelles was concentration-dependent, with 97% ± 0.2% of cells being infected with Plu-GA micelles at Plu concentrations below 250 μg mL -1 . The group treated with GA micelles was alive.
이 결과는 마이셀에 포함된 GA 분자의 독성 때문일 수 있다. 그러나 임상 시험에서 주입된 Pluronic®의 농도 (예: ~5L의 인간 혈액 부피에 희석된 2 wt%의 Pluronic F127 및 0.25 wt%의 Pluronic L61)를 고려할 때 최대 농도 250 μg mL-1 에서 생체이용이 가능할 것으로 사료된다. This result may be due to the toxicity of GA molecules contained in the micelles. However, considering the concentrations of Pluronic® injected in clinical trials (e.g., 2 wt% of Pluronic F127 and 0.25 wt% of Pluronic L61 diluted in ~5 L of human blood volume), bioavailability appears to be limited at a maximum concentration of 250 μg mL −1. It is believed to be possible.
실시예 5: Plu-GA 마이셀의 단백질(e.g. 알부민)의 흡착 기능Example 5: Adsorption function of Plu-GA micelles for proteins (e.g. albumin)
본 발명자들은 본 발명의 Plu-GA 마이셀이 단백질(e.g. 알부민)의 흡착 기능성도 가지고 있는지를 확인하였다. The present inventors confirmed whether the Plu-GA micelle of the present invention also has the function of adsorbing proteins (e.g. albumin).
구체적으로, 0.3 mg/mL의 octyl gallate, 3.9 mg/mL의 Pluronic F127을 pH 7.4 PBS에 녹여 제조하였다. 대조군인 Plu 마이셀은 octyl gallate를 제외하고 제조하였다. 여기에 270 μg의 Albumin-fluorescein isothiocyanate conjugate (FITC-BSA)를 첨가하고, 알부민 단백질이 흡착될 수 있도록 2시간 동안 인큐베이션하였다. 인큐베이션 후 형광현미경으로 형광 신호를 검출하였다.Specifically, 0.3 mg/mL of octyl gallate and 3.9 mg/mL of Pluronic F127 were prepared by dissolving them in PBS at pH 7.4. Control Plu micelles were prepared without octyl gallate. 270 μg of albumin-fluorescein isothiocyanate conjugate (FITC-BSA) was added and incubated for 2 hours to allow albumin protein to be adsorbed. After incubation, the fluorescence signal was detected using a fluorescence microscope.
결과는 도 9 및 표 1에 나타내었다. The results are shown in Figure 9 and Table 1.
표 1에 나타낸 바와 같이, Pluronic-gallol micelle의 경우 Pluronic micelle와 비교하여 형광이 덜 발현되는 것을 확인할 수 있었다. 이는 알부민 단백질이 마이셀에 흡착되면서, FITC-BSA의 3차원 분자구조가 바뀌어 형광을 더 이상 발현하지 못하기 때문이다.As shown in Table 1, it was confirmed that Pluronic-gallol micelle expressed less fluorescence compared to Pluronic micelle. This is because as the albumin protein is adsorbed to the micelle, the three-dimensional molecular structure of FITC-BSA changes and fluorescence can no longer be expressed.
결론conclusion
본 발명자들은 콜로이드 안정성과 단백질 친화도가 높으면서도, 낮은 CMC를 갖는 Plu-GA 마이셀을 개발하였다. Plu-GA의 손쉬운 제조는 안정화를 위한 후가교 없이 Gallol-tethered 계면활성제를 Plu에 간단히 통합함으로써 달성되었다. Gallol 그룹은 단백질에 대한 Gallol의 높은 친화도로 인해 Plu-GA 마이셀에 효소 분해 저항성을 부여하였다. 또한, Gallol이 마이셀에 혼입되었음에도 불구하고 Plu 마이셀의 원래 소수성 공동이 유지되어 외부 콜레스테롤 결정의 용해 및 포획이 가능하였다. 따라서 본 발명자들이 개발한 안정적인 마이셀 시스템은 기존 약물 담체의 생체 이용률을 향상시키는 데 유용하게 사용될 수 있을 것이다.The present inventors developed Plu-GA micelles with high colloidal stability and protein affinity, and low CMC. The facile preparation of Plu-GA was achieved by simply incorporating Gallol-tethered surfactant into Plu without post-crosslinking for stabilization. The Gallol group endowed Plu-GA micelles with enzymatic degradation resistance due to the high affinity of Gallol for proteins. In addition, despite the incorporation of Gallol into the micelles, the original hydrophobic cavity of the Plu micelles was maintained, enabling the dissolution and capture of external cholesterol crystals. Therefore, the stable micelle system developed by the present inventors may be useful in improving the bioavailability of existing drug carriers.
Claims (10)
Micelle particles formed by self-assembly of amphipathic copolymers and polyphenol compounds.
The micelle particles according to claim 1, wherein the weight ratio of the amphipathic copolymer and the polyphenol compound is 1-20:1.
The micelle particle of claim 1, wherein the polyphenol compound is an alkyl gallate compound.
i) CMC(critical micelle concentration)가 양친매성 공중합체의 농도를 기준으로 5 내지 50 μM임;
ii) 30-40℃ 범위 내에서 수용액 내 마이셀 구조의 함량 변화가 20% 이내임;
iii) 단백질 친화성(affinity)을 나타냄;
iv) 산화제에 대한 산화 저항성을 나타냄; 또는
v) 이들의 조합.
The micellar particle according to claim 1, having the following characteristics:
i) the critical micelle concentration (CMC) is 5 to 50 μM based on the concentration of the amphipathic copolymer;
ii) the change in the content of the micelle structure in the aqueous solution within the range of 30-40°C is within 20%;
iii) indicates protein affinity;
iv) exhibits oxidation resistance to oxidizing agents; or
v) Combination of these.
(a) 양친매성 공중합체 및 폴리페놀 화합물을 수성 용매에 첨가하여 혼합하는 단계; 및
(b) 상기 결과물로부터 마이셀 입자를 분리 및 정제하는 단계.
Method for preparing micelle particles comprising an amphipathic copolymer and a polyphenolic compound, comprising the following steps:
(a) adding and mixing an amphipathic copolymer and a polyphenol compound to an aqueous solvent; and
(b) separating and purifying micelle particles from the resulting product.
The method of claim 5, wherein the mixing in step (a) is performed by ultrasonic treatment or the use of a high-pressure emulsifier.
A drug delivery system for vascular injection comprising the micelle particle of any one of claims 1 to 4.
The drug delivery system according to claim 7, wherein the drug delivery system supports a hydrophobic drug.
The drug delivery system according to claim 7, wherein the drug delivery system carries a protein drug.
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| KR1020220120376A KR20240041172A (en) | 2022-09-22 | 2022-09-22 | Micelle comprising polyphenol |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101570300B1 (en) | 2014-10-01 | 2015-11-19 | 단국대학교 천안캠퍼스 산학협력단 | A drug delivery system comprising temperature-triggered micelles for multi-delivery of a drug and nucleic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101570300B1 (en) | 2014-10-01 | 2015-11-19 | 단국대학교 천안캠퍼스 산학협력단 | A drug delivery system comprising temperature-triggered micelles for multi-delivery of a drug and nucleic acid |
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