KR101352316B1 - Drug carrier comprising mineralized amphiphilic nanoparticles containing poly(ethylene glycol) - Google Patents

Drug carrier comprising mineralized amphiphilic nanoparticles containing poly(ethylene glycol) Download PDF

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KR101352316B1
KR101352316B1 KR1020110141737A KR20110141737A KR101352316B1 KR 101352316 B1 KR101352316 B1 KR 101352316B1 KR 1020110141737 A KR1020110141737 A KR 1020110141737A KR 20110141737 A KR20110141737 A KR 20110141737A KR 101352316 B1 KR101352316 B1 KR 101352316B1
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hyaluronic acid
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박재형
강영모
권익찬
김광명
한화승
윤홍열
유동길
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Abstract

본 발명은 무기화된 양친성 고분자 나노입자, 상기 나노입자에 약물이 봉입된 약물 전달체, 상기 약물 전달체를 포함하는 약학적 조성물 및 상기 나노입자에 형광체가 결합된 암 진단용 조영제에 관한 것이다. 상기 양친성 고분자는 폴리에틸렌 글리콜과 히알루론산을 함유하며, 인산칼슘으로 무기화될 수 있고, 소수성 약물의 봉입이 가능한 특징을 포함한다.
본 발명의 인산칼슘으로 무기화된 폴리에틸렌 글리콜 함유 양친성 히알루론산 나노입자는 내부에 소수성 약물의 봉입이 가능하고, 생체 내 정상조직의 중성 pH에서는 안정하여 약물을 서서히 방출하는 한편, 병변부위의 산성환경에서 인산칼슘이 용해되어 봉입된 약물을 효과적으로 빠르게 방출하므로 암조직 또는 암세포 내부로 효과적으로 암치료제를 전달할 수 있는 약물 전달체 조성물로 유용하게 사용될 수 있을 것이다. 또한, 같은 원리로 형광체를 화학적 반응을 통해 또는 물리적 상호작용에 의해 나노입자 내부에 봉입함으로써, 형광분광학에 의한 암진단용 조영제로서도 응용 가능하다.The present invention relates to inorganicized amphiphilic polymer nanoparticles, drug carriers in which drugs are enclosed in the nanoparticles, pharmaceutical compositions comprising the drug carriers, and contrast contrast agents for cancer diagnosis in which phosphors are bound to the nanoparticles. The amphiphilic polymer contains polyethylene glycol and hyaluronic acid, may be inorganicized with calcium phosphate, and includes a feature capable of encapsulating a hydrophobic drug.
Polyethylene glycol-containing amphiphilic hyaluronic acid nanoparticles inorganicized with calcium phosphate of the present invention are capable of encapsulating hydrophobic drugs therein, and are stable at neutral pH of normal tissues in vivo, releasing drugs slowly, and acidic environment of lesions. Since calcium phosphate is dissolved in and rapidly releases the encapsulated drug, it may be usefully used as a drug delivery composition that can effectively deliver a cancer therapeutic agent into cancer tissue or cancer cells. In addition, by encapsulating the phosphor inside the nanoparticles by chemical reaction or physical interaction, the same principle can be applied as a contrast agent for cancer diagnosis by fluorescence spectroscopy.

Description

무기화된 폴리에틸렌 글리콜 함유 양친성 나노입자를 포함하는 약물 전달체{Drug carrier comprising mineralized amphiphilic nanoparticles containing poly(ethylene glycol)}Drug carrier comprising mineralized amphiphilic nanoparticles containing poly (ethylene glycol)}

본 발명은 무기화된 양친성 고분자 나노입자, 상기 나노입자에 약물이 봉입된 약물 전달체, 상기 약물 전달체를 포함하는 약학적 조성물 및 상기 나노입자에 형광체가 결합된 암 진단용 조영제에 관한 것이다. 상기 양친성 고분자는 폴리에틸렌 글리콜과 히알루론산을 함유하며, 인산칼슘으로 무기화될 수 있고, 소수성 약물의 봉입이 가능한 특징을 포함한다.
The present invention relates to inorganicized amphiphilic polymer nanoparticles, drug carriers in which drugs are enclosed in the nanoparticles, pharmaceutical compositions comprising the drug carriers, and contrast contrast agents for cancer diagnosis in which phosphors are bound to the nanoparticles. The amphiphilic polymer contains polyethylene glycol and hyaluronic acid, may be inorganicized with calcium phosphate, and includes a feature capable of encapsulating a hydrophobic drug.

파크리탁셀, 도세탁셀, 시스플라틴, 캄토세신 등 약리학적으로 효과적인 항암제는 실제 임상적용에 있어서 세포 내 유전인자의 본체인 핵산의 합성을 억제하거나 핵산에 직접 결합하여 그 기능을 손상시킴으로서 효과를 나타낸다. 그러나 이들 항암제는 암세포뿐만 아니라 정상세포, 특히 세포분열이 활발한 정상 조직세포에도 손상을 입히기 때문에 골수 기능 저하, 위장관 점막손상, 탈모 등의 부작용을 보이며 약물의 심각한 독성 및 낮은 용해도로 인하여 기대와는 달리 획기적인 성과를 보여주지 못하였다. 따라서, 질병 치료에 사용되는 약물의 부작용을 최소화하기 위한 새로운 약물제형의 개발이 활발히 진행되어 왔다. 그 예로서, 약물의 독성을 최소화하면서 약물의 치료효능을 향상시킬 수 있는 나노입자, 마이셀, 미립구 등의 약물 전달체가 개발되어 왔다.Pharmacologically effective anticancer agents such as paclitaxel, docetaxel, cisplatin, and camptocecin are effective in inhibiting the synthesis of nucleic acids, which are the body of genetic factors in cells, or directly binding to nucleic acids and impairing their function in actual clinical application. However, these anticancer drugs damage not only cancer cells but also normal cells, especially normal tissue cells with active cell division, resulting in side effects such as decreased bone marrow function, damage to gastrointestinal tract, and hair loss. It did not show a breakthrough. Therefore, the development of new drug formulations for minimizing the side effects of drugs used to treat diseases has been actively progressed. For example, drug carriers such as nanoparticles, micelles, and microspheres have been developed that can improve the therapeutic efficacy of drugs while minimizing the toxicity of drugs.

양친성 고분자는 생체적합성이 우수하고 수성환경에서 자기집합 나노입자를 형성하며, 입자 내부에 소수성 약물의 봉입이 용이하기 때문에 상기 약물 전달체로 유용하게 사용될 수 있는 물질이다. 양친성 고분자는 친수성 고분자와 소수성 고분자가 화학적으로 결합된 형태의 공중합체로 제조하거나, 친수성 고분자를 저분자량의 소수성 물질로 개질하여 제조할 수 있다. 양친성 고분자는 다양한 친수성 고분자 및 소수성 고분자(저분자량의 소수성 물질)로 합성할 수 있으며, 양친성 고분자를 구성하는 성분의 구조 및 화학적 조성에 따라 다양한 기능성을 부여할 수 있다. 한편, 양친성 고분자는 수성환경에서 소수성 고분자(저분자량의 소수성 물질)간의 소수성 상호작용을 통해 형성되기 때문에 고분자의 농도가 일정 수준 이상되어야 나노입자의 형태를 유지한다. 따라서, 70% 이상이 물로 구성된 인체에 주입할 경우, 고분자의 농도가 낮아지는 효과로 인해 나노입자의 구조가 쉽게 붕괴된다. 이는 입자 내부에 봉입된 약물이 질병 부위에 도달하기 전에 방출되는 결과를 초래하기 때문에 약효가 떨어지게 된다. 따라서, 최근에는 양친성 고분자로 구성된 자기집합 나노입자의 안정성을 향상시키기 위하여, 나노입자 형성 후 화학적으로 가교시키는 방법 등이 개발되고 있으나, 제조 과정이 복잡하고 균일한 입자의 제조가 어려워 상용화에는 제한이 따르고 있다.Amphiphilic polymers have excellent biocompatibility, form self-assembling nanoparticles in an aqueous environment, and are easy to enclose hydrophobic drugs in the particles, and thus are useful as drug carriers. Amphiphilic polymers may be prepared by copolymerization of a form in which a hydrophilic polymer and a hydrophobic polymer are chemically bonded, or may be prepared by modifying a hydrophilic polymer with a low molecular weight hydrophobic material. Amphiphilic polymers can be synthesized into various hydrophilic polymers and hydrophobic polymers (low molecular weight hydrophobic materials), and can provide various functionalities according to the structure and chemical composition of the components constituting the amphiphilic polymer. On the other hand, since amphiphilic polymers are formed through hydrophobic interactions between hydrophobic polymers (low molecular weight hydrophobic materials) in an aqueous environment, the concentration of the polymer is maintained at a predetermined level or more to maintain the nanoparticle morphology. Therefore, when more than 70% is injected into the human body composed of water, the structure of the nanoparticles easily collapse due to the effect of lowering the concentration of the polymer. This is less effective because the drug encapsulated inside the particle is released before it reaches the disease site. Therefore, in recent years, in order to improve the stability of self-assembling nanoparticles composed of amphiphilic polymers, a method of chemically crosslinking after nanoparticle formation has been developed, but the manufacturing process is complicated and it is difficult to manufacture uniform particles. This is following.

또한, 암을 효과적으로 표적화하기 위하여 암세포를 특이적으로 인식하는 항체, 펩티드, 폴산, 히알루론산 등의 리간드로 나노입자의 표면을 개질한 표적 지향형 약물 전달체가 활발히 개발되어 왔다. 그러나, 상기한 표적 지향 리간드는 생체 조건 안에서 암세포뿐 아니라 간, 신장 등의 정상세포 표면의 수용체와도 강하게 결합하여, 질병 치료에 사용되는 약물의 부작용을 최소화하기 위해서는 항암제를 암조직에 선택적으로 투여할 수 있는 새로운 약물 제형의 개발이 요구되고 있다[Pharmacol . Rev ., 2001, 53(2): 283-318; Bioorgan . Med . Chem ., 2005, 13: 5043-5054; Nat . Rev . Drug Discov ., 2005, 4(2): 145-160].In addition, in order to target cancer effectively, target-directed drug delivery agents that have modified the surface of nanoparticles with ligands such as antibodies, peptides, folic acid, and hyaluronic acid that specifically recognize cancer cells have been actively developed. However, the target-directed ligand binds strongly to receptors on the surface of normal cells such as liver and kidney in cancer cells in vivo, and selectively administers anticancer drugs to cancer tissues in order to minimize side effects of drugs used to treat diseases. There is a need for development of new drug formulations that can be made [ Pharmacol . Rev. , 2001, 53 (2): 283-318; Bioorgan . Med . Chem . , 2005, 13: 5043-5054; Nat . Rev. Drug Discov . , 2005, 4 (2): 145-160.

그 중에서도 히알루론산은 인체 내에 존재하는 생체 고분자로서, D-글루크론산과 N-아세틸글루코사민으로 구성되는 고분자로서, 1934년 K. Meyer에 의하여 소의 눈의 유리체로부터 분리된 이당류이다. 이는 세포 외 기질의 주성분으로, 체내에서 면역반응을 일으키지 않으며 독성이 거의 없다는 점에서 매우 안전한 생체 재료로 알려져 있다.Among them, hyaluronic acid is a biopolymer existing in the human body, and is a polymer composed of D-glucuronic acid and N-acetylglucosamine, and is a disaccharide isolated from the vitreous of a cow's eye by K. Meyer in 1934. It is a major component of extracellular matrix and is known as a very safe biomaterial in that it does not cause an immune response in the body and has little toxicity.

특히, 난소, 결장, 위장 및 유방암 등 다양한 종양세포는 히알루론산 결합 수용체인 CD44 및 RHAMM을 과발현하는 것으로 알려져 있어, 종양과 관련된 다양한 분야에서 활발하게 연구되고 있다[Drug Deliv ., 2005, 12: 327-342].In particular, ovarian, colon, stomach, and various tumor cells such as breast cancer, it is known that the over-expression of hyaluronic acid binding receptors, CD44 and RHAMM, has been actively studied in various fields related to the tumor [Drug Deliv . , 2005, 12: 327-342.

하지만, 대부분의 히알루론산은 간에 존재하는 HARE 등의 세포막 수용체를 통하여 특이적으로 결합하기 때문에 정맥주사를 통하여 주사된 대부분의 나노입자가 간에 축적되는 것으로 알려져 있으며, 간장 및 림프선에 존재하는 HARE, LYVE-1 등의 수용체를 통한 세포 내로의 도입 및 히알루로니다아제에 의한 분해작용에 의해 빠르게 분해되므로, 암세포 선택적인 약물 전달체 조성물로 사용하기에 문제점이 있다.
However, since most of hyaluronic acid specifically binds to cell membrane receptors such as HARE present in the liver, most nanoparticles injected through intravenous injection are known to accumulate in the liver, and HARE and LYVE present in the liver and lymph glands. Since it is rapidly degraded by introduction into cells through receptors such as -1 and degradation by hyaluronidase, there is a problem in use as cancer cell selective drug delivery composition.

이에, 본 발명자들은 암병변 특이적 환경에서 선택적으로 빠르게 약물을 방출할 수 있는 나노입자 및 이를 포함하는 약물 전달체를 개발하기 위해 예의 노력한 결과, 폴리에틸렌 글리콜로 개질된 양친성 히알루론산 나노입자를 인산칼슘으로 무기화시킬 경우, 간의 세포망내피계를 피해 체내에서 장기간 순환하면서 암조직에 선택적으로 도달하고, 생체 내 정상조직의 중성 pH에서는 안정하여 약물을 서서히 방출하는 한편 병변부위의 약산성 환경에서는 선택적으로 효과적인 약물 방출이 가능함을 확인하고, 이에 본 발명을 완성하였다.
Accordingly, the present inventors have made intensive efforts to develop nanoparticles capable of selectively releasing drugs in a cancer lesion specific environment and drug carriers containing the same, and thus, amphiphilic hyaluronic acid nanoparticles modified with polyethylene glycol are calcium phosphate. In the case of mineralization, the cancer cells are selectively reached in cancer tissues while circulating in the body for a long period of time, avoiding the hepatic endothelial system. It was confirmed that drug release is possible, thereby completing the present invention.

본 발명의 하나의 목적은 폴리에틸렌 글리콜로 표면을 개질시킨 양친성 고분자 나노입자로서, 상기 양친성 고분자는 친수성 고분자와 소수성 물질이 결합된 것이며, 상기 나노입자의 표면은 무기화된 것을 특징으로 하는 나노입자를 제공하는 것이다.One object of the present invention is an amphiphilic polymer nanoparticles surface modified with polyethylene glycol, the amphiphilic polymer is a hydrophilic polymer and a hydrophobic material is combined, the nanoparticles surface characterized in that the inorganic particles To provide.

본 발명의 다른 목적은 상기 무기화된 양친성 고분자 나노입자에 약물이 봉입된 것을 특징으로 하는 약물 전달체를 제공하는 것이다.Another object of the present invention is to provide a drug carrier, characterized in that the drug is encapsulated in the inorganic amphiphilic polymer nanoparticles.

본 발명의 또 다른 목적은 상기 약물 전달체를 유효성분으로 함유하는 약학적 조성물을 제공하는 것이다.Still another object of the present invention is to provide a pharmaceutical composition containing the drug carrier as an active ingredient.

본 발명의 또 다른 목적은 상기 무기화된 양친성 고분자 나노입자에 형광체가 결합된 것을 특징으로 하는 암 진단용 조영제를 제공하는 것이다.
Still another object of the present invention is to provide a contrast agent for diagnosing cancer, wherein a phosphor is bound to the inorganicized amphiphilic polymer nanoparticle.

상기 목적을 달성하기 위한 하나의 양태로서, 본 발명은 폴리에틸렌 글리콜로 표면을 개질시킨 양친성 고분자 나노입자로서, 상기 양친성 고분자는 친수성 고분자와 소수성 물질이 결합된 것이며, 상기 나노입자의 표면은 무기화된 것을 특징으로 하는 나노입자를 제공한다.
As one embodiment for achieving the above object, the present invention is an amphiphilic polymer nanoparticles surface modified with polyethylene glycol, the amphiphilic polymer is a hydrophilic polymer and a hydrophobic material is combined, the surface of the nanoparticles is inorganic It provides a nanoparticles characterized in that.

본 발명의 용어 "나노입자"는 수 내지 수백 나노미터의 직경을 갖는 입자를 폭넓게 지칭한다. 제조 방법은 크게 물리적 방법인 탑-다운 접근법(top-down approach), 화학적 합성방법에 기초한 바텀-업 접근법(bottom-up approach) 및 자기조립법(self-assembly)의 세가지 방법으로 나뉜다. 마지막 자기조립법은 현재 생분자적 나노기법의 조립에 기초가 되는 것으로 이는 바텀-업 접근법의 일종이다. 입자의 구성요소들은 물리적, 화학적 및 구조적 성질에 의해 자발적으로 응집하여 나노입자를 형성한다. 이때, 입자의 크기는 반응 물질의 몰비를 조절하여 결정할 수 있다. 또한 형성된 나노입자는 표면을 개질하여 그 물성을 개선시켜 다양한 분야에 응용할 수 있다.The term "nanoparticle" of the present invention broadly refers to particles having a diameter of several to several hundred nanometers. The manufacturing method is largely divided into three methods, a physical method, a top-down approach, a bottom-up approach based on a chemical synthesis method, and a self-assembly method. The final self-assembly is currently based on the assembly of biomolecular nanotechnology, which is a kind of bottom-up approach. The components of the particles spontaneously aggregate to form nanoparticles by physical, chemical and structural properties. In this case, the size of the particles may be determined by adjusting the molar ratio of the reactant. In addition, the formed nanoparticles can be applied to various fields by improving the physical properties by modifying the surface.

본 발명의 용어 "양친성 고분자"는 친수성 고분자 물질에 소수성 물질이 결합된 화합물을 의미한다. 본 발명에서는 친수성인 고분자 물질에 소수성의 물질을 결합하여 양친성 고분자를 나노입자의 형태로 제공함으로써, 상기 나노입자는 수용액 상태에서도 안정한 구조를 가질 수 있다. 또한, 고분자 나노입자의 특성에 따라서 암조직 주변의 축적효율을 나타낼 수 있다. 암조직은 비정상적인 빠른 성장으로인해 혈관조직이 느슨하게 형성되고 높은 투과성을 보이므로, 고분자 나노입자 등은 암조직에 선택적으로 축적될 수 있으며, 이를 증가된 투과도 및 축적(enhanced permeability and retention; EPR) 효과라 한다. 따라서, 본 발명에서는 고분자로 이루어진 나노입자를 사용함으로써 약물의 생체 내 체류시간을 증가시킬 수 있었다.The term "amphiphilic polymer" of the present invention means a compound in which a hydrophobic material is bonded to a hydrophilic polymer material. In the present invention, by combining a hydrophobic material with a hydrophilic polymer material to provide an amphiphilic polymer in the form of nanoparticles, the nanoparticles can have a stable structure even in the aqueous solution state. In addition, depending on the characteristics of the polymer nanoparticles can represent the accumulation efficiency around the cancer tissue. Because cancer tissues are loosely formed and show high permeability due to abnormal rapid growth, polymer nanoparticles can be selectively accumulated in cancer tissues, and thus enhanced permeability and retention (EPR) effects. It is called. Therefore, in the present invention, it was possible to increase the residence time of the drug by using nanoparticles made of a polymer.

상기 친수성 고분자는 생체적합성을 갖는 모든 고분자가 사용될 수 있으며, 특히 암조직 축적효율이 높은 고분자를 사용할 수 있다. 상기 "생체적합성 고분자"는 생체조직 또는 혈액과 접촉하여 조직을 괴사시키거나 혈액을 응고시키지 않는 조직적합성(tissue compatibility) 및 항응혈성(blood compatibility)을 갖는 고분자를 의미한다. 상기 고분자의 우수한 생체적합성과 생분해성은 생체 안정성을 향상시켜 혈액 내에서의 생체 분포도를 높여 충분한 시간 동안 암조직에 지속적으로 축적될 수 있도록 한다. 이러한 친수성 고분자로는 덱스트란(dextran), 키토산(chitosan), 글리콜 키토산(glycol chitosan), 히알루론산(hyaluronic acid), 폴리-L-라이신(poly-L-lysine) 또는 폴리아스파르트산(poly-aspartic acid) 등의 생체 고분자, 또는 폴리(N-2-(하이드록시프로필)메타아크릴아마이드)(poly(N-2-(hydroxypropyl)methacrylamide)), 폴리(디비닐 에테르-코-말레익 언하이드라이드)(poly(divinyl ether-co-maleic anhydride)), 폴리(스틸렌-코-말레익 언하이드라이드)(poly(styrene-co-maleic anhydride)) 또는 폴리(에틸렌 글리콜)(poly(ethylene glycol)) 등의 합성 고분자가 사용될 수 있으며, 바람직하게는 히알루론산이 사용될 수 있으나, 이에 제한되지 않는다.As the hydrophilic polymer, any polymer having biocompatibility may be used, and in particular, a polymer having high cancer tissue accumulation efficiency may be used. The "biocompatible polymer" refers to a polymer having tissue compatibility and blood compatibility that does not necrolyse or coagulate tissue in contact with living tissue or blood. The excellent biocompatibility and biodegradability of the polymer improves the stability of the living body to increase the biodistribution in the blood to be continuously accumulated in cancer tissue for a sufficient time. Such hydrophilic polymers include dextran, chitosan, glycol chitosan, hyaluronic acid, poly-L-lysine or poly-aspartic acid. biopolymers such as acid) or poly (N-2- (hydroxypropyl) methacrylamide), poly (divinyl ether-co-maleic anhydride) (poly (divinyl ether-co-maleic anhydride)), poly (styrene-co-maleic anhydride) or poly (ethylene glycol) Synthetic polymers such as may be used, and preferably hyaluronic acid may be used, but is not limited thereto.

본 발명의 용어 "히알루론산(hyaluronic acid)"은 생체적합성 및 생분해성이 뛰어난 고분자 화합물로 암세포에 대한 높은 친화력을 갖는다. 상기 히알루론산은 인체 내 면역반응을 일으키지 않을 뿐 아니라 독성이 거의 없어 생체재료로 적합하고, 암세포 및 조직에 특이적으로 발현되는 CD44 수용체와 친화력이 높아 나노입자 제조시 암조직 축적효율이 매우 높으며, 히알루론산의 카르복실기는 소수성 물질과의 화학적 개질 유도를 용이하게 한다.The term "hyaluronic acid" of the present invention is a polymer compound having excellent biocompatibility and biodegradability and has a high affinity for cancer cells. The hyaluronic acid not only does not cause an immune response in the human body but also has little toxicity and is suitable as a biomaterial, and has a high affinity with CD44 receptors specifically expressed in cancer cells and tissues. The carboxyl groups of hyaluronic acid facilitate the induction of chemical modifications with hydrophobic materials.

상기 친수성 고분자에 도입되는 소수성 물질은 담즙산 유도체 또는 지방산 유도체일 수 있으며, 바람직하게는 디옥시콜산(deoxycholic acid), 타우로디옥시콜산(taurodeoxycholic acid), 타우로콜산(taurocholic acid), 글리코케노디옥시콜산(glycochenodeoxycholic acid), 타우로케노디옥시콜산(taurodeoxycholic acid), 스테아르산(stearic acid) 또는 올레산(oleic acid)일 수 있으나, 이에 제한되지 않는다. 본 발명에 의한 양친성 고분자 나노입자는 친수성 고분자에 소수성 물질이 결합되어 구성되므로 소수성과 친수성의 균형을 통하여 나노크기의 자기조립체(self-assembly) 또는 자기응집체(self-aggregate)를 형성할 수 있다. 또한, 고분자 나노입자의 특성을 개선하기 위한 표면개질이 용이하며, 근적외선 투시 등을 위한 형광체의 화학적 개질이 용이하여 근적외선 투시에 의해 암조직의 선택적 형광을 나타낼 수 있다.The hydrophobic material introduced into the hydrophilic polymer may be a bile acid derivative or a fatty acid derivative, preferably deoxycholic acid, taurodeoxycholic acid, taurocholic acid, taurocholic acid or glycokenodioxy. Glycochenodeoxycholic acid, taurodeoxycholic acid, tarodeoxycholic acid, stearic acid (stearic acid) or oleic acid (oleic acid), but is not limited thereto. Amphiphilic polymer nanoparticles according to the present invention is formed by combining a hydrophobic material to the hydrophilic polymer can form a nano-sized self-assembly or self-aggregate through the balance of hydrophobicity and hydrophilicity . In addition, it is easy to modify the surface to improve the characteristics of the polymer nanoparticles, and the chemical modification of the phosphor for near-infrared light and the like can be easily seen to show selective fluorescence of cancer tissue by near-infrared light.

상기 친수성 고분자와 소수성 물질이 결합되어 구성된 양친성 고분자 복합체의 표면에 폴리에틸렌 글리콜을 도입하였다. 본 발명의 "폴리에틸렌 글리콜(poly(ethylene glycol); PEG)"은 나노입자 표면의 친수성을 증가시키고 인체 내 면역기능으로 인한 빠른 분해를 방지하여 혈중 체류 시간을 향상시키기 위하여 도입된 고분자 물질이다. 이와 같이 폴리에틸렌 글리콜로 개질시키는 것을 페길화(pegylation)라 한다. 상기 페길화 과정을 통해 간축적이 감소되고 혈중 체류 시간이 증가된 양친성 고분자 나노입자를 제조할 수 있다. 즉, 나노입자 표면에 폴리에틸렌 글리콜을 도입함으로 입자의 친수성이 증가되고, 병원균, 노폐물 및 외부 유입 물질을 포식하고 소화시키는 인체 내의 대식세포(macrophage) 등을 포함하는 면역기능으로부터의 인식을 방지하는 소위 스텔스 효과(stealth effect)를 통한 신체 내에서의 빠른 분해가 방지될 수 있고, 나노입자의 혈중 체류 시간을 증가시킬 수 있다. 본 발명에서 사용되는 페길화는 히알루론산의 카르복실 그룹과 폴리에틸렌 글리콜의 아민 그룹의 결합에 의해 아미드 그룹을 형성하는 방법으로 수행될 수 있다. 그러나, 이에 제한되지 아니하며, 당업계에 공지된 다양한 방법에 의해 수행될 수 있다. 이때, 사용되는 폴리에틸렌 글리콜은 바람직하게는 100 내지 10,000 사이의 분자량을 가지며, 선형 또는 가지형 등의 다양한 구조를 가질 수 있다.Polyethylene glycol was introduced to the surface of the amphiphilic polymer composite formed by combining the hydrophilic polymer and the hydrophobic material. "Polyethylene glycol (PEG)" of the present invention is a polymer substance introduced to increase the hydrophilicity of the surface of nanoparticles and prevent rapid decomposition due to immune function in the human body to improve blood retention time. This modification with polyethylene glycol is called pegylation. Through the PEGylation process, amphipathic polymer nanoparticles having reduced accumulation and increased blood residence time can be prepared. That is, by introducing polyethylene glycol on the surface of nanoparticles, the hydrophilicity of the particles is increased, and the so-called microorganisms, which prevent recognition from immune functions, including macrophages in the human body that digest and digest pathogens, Rapid degradation in the body through the stealth effect can be prevented and the residence time of the nanoparticles in the blood can be increased. Pegylation used in the present invention can be carried out by a method of forming an amide group by combining a carboxyl group of hyaluronic acid with an amine group of polyethylene glycol. However, it is not limited thereto and may be performed by various methods known in the art. In this case, the polyethylene glycol used preferably has a molecular weight of between 100 and 10,000, and may have various structures such as linear or branched.

상기 폴리에틸렌 글리콜로 표면이 개질된 양친성 히알루론산 고분자 나노입자는 인산칼슘으로 무기화할 수 있다. 상기 과정을 통해 폴리에틸렌 글리콜로 개질된 양친성 히알루론산이 수성환경에서 자기집합 나노입자를 형성한 상태에서 질산칼슘용액과 인산암모늄용액을 첨가하여 나노입자의 히알루론산 주쇄에 인산칼슘을 물리적으로 침착시켜 나노입자를 얻는다. 상기 수득된 나노입자는 수용액에서 안정하며, 인산칼슘의 특성을 이용하여 정상세포의 환경에서의 약물방출 및 간에서의 축적을 최소화하고, 체내에서 장기간 순환하면서 암조직에 선택적으로 도달한 후 암 병변부위의 산성환경에서 인산칼슘이 용해됨으로 약물이 빠른 속도로 방출되는 특징을 갖는다. 따라서, 본 발명의 무기화된 폴리에틸렌 글리콜 함유 양친성 히알루론산 나노입자는 정맥 투여시 혈류 내에서 장시간 순환할 수 있으며, 병변부위에서 인산칼슘이 용해되어 노출되는 히알루론산에 의해 나타나는 암표적 지향성이 우수한 특징을 갖는다.
Amphiphilic hyaluronic acid polymer nanoparticles whose surface is modified with polyethylene glycol may be inorganicized with calcium phosphate. Amphiphilic hyaluronic acid modified with polyethylene glycol through the above process to form self-assembled nanoparticles in an aqueous environment by adding calcium nitrate solution and ammonium phosphate solution to physically deposit calcium phosphate on the hyaluronic acid backbone of the nanoparticles Obtain nanoparticles. The obtained nanoparticles are stable in aqueous solution, using the properties of calcium phosphate to minimize drug release in the environment of normal cells and accumulation in the liver, cancer lesions after selectively reaching the cancer tissue while circulating in the body for a long time The calcium phosphate dissolves in the acidic environment of the site, and the drug is rapidly released. Therefore, the inorganicized polyethylene glycol-containing amphiphilic hyaluronic acid nanoparticles of the present invention can circulate for a long time in the bloodstream upon intravenous administration, and have excellent cancer target directivity exhibited by hyaluronic acid exposed by dissolving calcium phosphate at the lesion site. Has

또 하나의 양태로서, 본 발명은 상기 무기화된 양친성 고분자 나노입자에 약물이 봉입된 것을 특징으로 하는 약물 전달체를 제공한다.
As another aspect, the present invention provides a drug carrier, characterized in that the drug is encapsulated in the inorganicized amphiphilic polymer nanoparticles.

또 하나의 양태로서, 본 발명은 상기 약물 전달체를 유효성분으로 함유하는 약학적 조성물을 제공한다.
As another aspect, the present invention provides a pharmaceutical composition containing the drug carrier as an active ingredient.

상기 약물을 봉입한 무기화된 양친성 고분자 나노입자는 생체 내 암 병변부위와 유사한 약산성의 환경에서 선택적으로 약물을 빠르게 방출한다. 이는 약산성 환경에서 나노입자를 무기화하기 위해 사용된 인산칼슘의 용해로 인한 것으로 상기 약물을 선택적으로 빠르게 방출하는 조건은 pH 4.0 내지 6.0일 수 있으나, 이에 제한되지 않는다.The inorganicized amphiphilic polymer nanoparticles encapsulating the drug selectively release the drug in a weakly acidic environment similar to the cancer lesion site in vivo. This is due to the dissolution of the calcium phosphate used to mineralize the nanoparticles in a weakly acidic environment, and the conditions for selectively releasing the drug may be pH 4.0 to 6.0, but is not limited thereto.

상기 무기화된 양친성 고분자 나노입자에 봉입되는 약물은, 이에 제한되지 않으나, 바람직하게 항암제일 수 있다. 상기 항암제는 도세탁셀(docetaxel), 시스플라틴(cis-platin), 캠토세신(camptothecin), 파클리탁셀(paclitaxel), 타목시펜(tamoxifen), 아나스테로졸(anasterozole), 글리벡(gleevec), 5-플루오로우라신(5-FU), 플록슈리딘(floxuridine), 류프로리드(leuprolide), 플로타미드(flutamide), 졸레드로네이트(zoledronate), 독소루비신(doxorubicin), 빈크리스틴(vincristine), 젬시타빈(gemcitabine), 스트렙토조신(streptozotocin), 카보플라틴(carboplatin), 토포테칸(topotecan), 벨로테칸(belotecan), 이리노테칸(irinotecan), 비노렐빈(vinorelbine), 히드록시우레아(hydroxyurea), 발루비신(valrubicin), 레티노익산(retinoic acid) 계열, 메토트렉세이트(methotrexate), 메클로레타민(meclorethamine), 클로람부실(chlorambucil), 부술판(busulfan), 독시플루리딘(doxifluridine), 빈블라스틴(vinblastin), 마이토마이신(mitomycin), 프레드니손(prednisone), 테스토스테론(testosterone), 미토산트론(mitoxantron), 아스피린(aspirin), 살리실레이트(salicylates), 이부프로펜(ibuprofen), 나프로센(naproxen), 페노프로펜(fenoprofen), 인도메타신(indomethacin), 페닐부타존(phenylbutazone), 시클로포스파미드(cyclophosphamide), 메클로에타민(mechloethamine), 덱사메타손(dexamethasone), 프레드니솔론(prednisolone), 셀렉콕시브(celecoxib), 발데콕시브(valdecoxib), 니메슐리드(nimesulide), 코르티손(cortisone) 또는 코르티코스테로이드(corticosteroid) 등에서 선택될 수 있다.The drug encapsulated in the inorganicized amphiphilic polymer nanoparticles is not limited thereto, but may preferably be an anticancer agent. The anticancer agent is docetaxel (docetaxel), cis-platin (cis-platin), camptothecin (camptothecin), paclitaxel (paclitaxel), tamoxifen (anasterozole), gleevec (gleevec), 5-fluorouracin ( 5-FU), floxuridine, leuprolide, flutamide, zledronate, doxorubicin, vincristine, gemcitabine Streptozosin, carboplatin, topotecan, belotecan, irinotecan, vinorelbine, hydroxyurea, valurecin, valrubicin, Retinoic acid family, methotrexate, mechloretamine, meclolorebumin, chlorambucil, busulfan, doxifluridine, doxifluridine, vinblastin, my Mitomycin, prednisone, testosterone rone, mitoxantron, aspirin, salicylates, ibuprofen, naproxen, fenoprofen, indomethacin, phenyl Phenylbutazone, cyclophosphamide, mechloethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nimesulide ), Cortisone or corticosteroid.

본 발명의 조성물은 암질환의 치료에 제한없이 사용될 수 있으며, 상기 조성물로 치료 가능한 암질환은 편평상피세포암, 자궁암, 자궁경부암, 전립선암, 두경부암, 췌장암, 뇌종양, 유방암, 간암, 피부암, 식도암, 고환암, 신장암, 대장암, 직장암, 위암, 방광암, 난소암, 담관암 또는 담낭암일 수 있다.The composition of the present invention can be used without limitation for the treatment of cancer diseases, cancer diseases treatable with the composition is squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, Esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, ovarian cancer, bile duct cancer or gallbladder cancer.

상기 나노입자에 봉입되는 항암제는 조성물 전체에 대하여 1 내지 60 중량%로 포함될 수 있으며, 바람직하게는 10 내지 30 중량%로 포함될 수 있다.
The anticancer agent encapsulated in the nanoparticles may be included in an amount of 1 to 60% by weight, and preferably 10 to 30% by weight, based on the total composition.

또 하나의 양태로서, 본 발명은 상기 무기화된 양친성 고분자 나노입자에 형광체가 결합된 것을 특징으로 하는 암 진단용 조영제를 제공한다.
In another aspect, the present invention provides a contrast agent for diagnosing cancer, characterized in that the phosphor is bound to the inorganicized amphiphilic polymer nanoparticles.

상기 형광체로는 방사선 동위원소, 양자점(quantum dot), MRI 조영제 등이 사용될 수 있고, 바람직하게는 근적외선 형광체를 사용할 수 있다. 이에 제한되지 않으나, 시아닌(cyanine), 플루오레신(fluorescein), 테트라메틸로다민(tetramethylrhodamine), 보디피(BODIPY) 및 알렉사(alexa) 등의 근적외선 형광체를 사용하여 비침투적 영상에 의한 암 진단이 가능하다.
As the phosphor, a radioisotope, a quantum dot, an MRI contrast agent, or the like may be used. Preferably, a near infrared phosphor may be used. Cancer diagnosis by non-invasive imaging using, but not limited to, near-infrared phosphors such as cyanine, fluorescein, tetramethylrhodamine, BODIPY, and alexa This is possible.

본 발명의 인산칼슘으로 무기화된 폴리에틸렌 글리콜 함유 양친성 히알루론산 나노입자는 내부에 소수성 약물의 봉입이 가능하고, 생체 내 정상조직의 중성 pH에서는 안정하여 약물을 서서히 방출하는 한편, 병변부위의 산성환경에서 인산칼슘이 용해되어 봉입된 약물을 효과적으로 빠르게 방출하므로 암조직 또는 암세포 내부로 효과적으로 암치료제를 전달할 수 있는 약물 전달체 조성물로 유용하게 사용될 수 있을 것이다. 또한, 같은 원리로 형광체를 화학적 반응을 통해 또는 물리적 상호작용에 의해 나노입자 내부에 봉입함으로써, 형광분광학에 의한 암진단용 조영제로서도 응용 가능하다.
Polyethylene glycol-containing amphiphilic hyaluronic acid nanoparticles inorganicized with calcium phosphate of the present invention are capable of encapsulating hydrophobic drugs therein, and are stable at neutral pH of normal tissues in vivo, releasing drugs slowly, and acidic environment of lesions. Since calcium phosphate is dissolved in and rapidly releases the encapsulated drug, it may be usefully used as a drug delivery composition that can effectively deliver a cancer therapeutic agent into cancer tissue or cancer cells. In addition, by encapsulating the phosphor inside the nanoparticles by chemical reaction or physical interaction, the same principle can be applied as a contrast agent for cancer diagnosis by fluorescence spectroscopy.

도 1은 제조된 나노입자들의 TEM 이미지를 나타낸 도이다. (A)는 PEG-HANP(polyethylene glycol-hyaluronic acid nanoparticle), (B)는 M-PEG-HANP(mineralized PEG-HANP) 및 (C)는 DOX-M-PEG-HANP(doxorubicin(DOX)-containing M-PEG-HANP)를 나타낸다. 삽입도는 확대된 이미지이다.
도 2는 제조된 나노입자의 특성을 나타낸 도이다. (A)는 M-PEG-HANP의 선택영역 전자회절(selected area electron diffraction; SAED) 패턴을, (B)는 M-PEG-HANP의 투과전자현미경-에너지분산분광(transmission electron microscopy-energy dispersive spectroscopy; TEM-EDS) 분석을, (C)는 나노입자들의 크기분포를 나타낸다.
도 3은 PEG-HANP 및 M-PEG-HANP의 특성을 나타낸 도이다. (A)는 FT-IR 스펙트럼을 (B)는 TGA 곡선을 나타내며, 화살표는 인산칼슘의 특성 피크를 나타낸다.
도 4는 다른 pH 조건에서 무기화된 나노입자로부터 DOX의 시간에 따른 시험관 내 누적 방출량을 나타낸 도이다.
도 5는 종양에서 무기화된 PEG-HANP 나노입자의 축적을 나타낸 도이다. (A)는 무기화된 PEG-HANP-Cy5.5 나노입자의 근적외선 이미지이며, (B)는 시간에 따른 종양자리에서의 형광세기를 나타낸다. (C)는 종양과 간 부위의 형광세기를 상대적으로 비교한 결과이다.
도 6은 종양 동물모델에 DOX(독소루비신) 및 DOX-M-PEG-HANP를 주입한 후, 종양의 크기변화를 나타낸 도이다.1 is a diagram showing a TEM image of the prepared nanoparticles. (A) is polyethylene glycol-hyaluronic acid nanoparticle (PEG-HANP), (B) is normalized PEG-HANP (M-PEG-HANP) and (C) is doxorubicin (DOX) -containing DOX-M-PEG-HANP M-PEG-HANP). Inset is an enlarged image.
2 is a view showing the characteristics of the prepared nanoparticles. (A) shows the selected area electron diffraction (SAED) pattern of M-PEG-HANP, and (B) shows the transmission electron microscopy-energy dispersive spectroscopy of M-PEG-HANP. TEM-EDS) analysis, and (C) shows the size distribution of the nanoparticles.
Figure 3 is a diagram showing the properties of PEG-HANP and M-PEG-HANP. (A) shows the FT-IR spectrum, (B) shows the TGA curve, and the arrow shows the characteristic peak of calcium phosphate.
Figure 4 shows the cumulative in vitro release of DOX over time from inorganicized nanoparticles at different pH conditions.
5 shows the accumulation of inorganicized PEG-HANP nanoparticles in tumors. (A) is a near infrared image of the inorganicized PEG-HANP-Cy5.5 nanoparticles, and (B) shows the fluorescence intensity at the tumor site with time. (C) is the result of relatively comparing the fluorescence intensity of the tumor and liver area.
Figure 6 is a diagram showing the change in tumor size after injecting DOX (doxorubicin) and DOX-M-PEG-HANP in the tumor animal model.

이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아닌 것은 당업자에게 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are for further illustrating the present invention and that the scope of the present invention is not limited to these embodiments.

실시예Example 1: 폴리에틸렌 글리콜( 1: polyethylene glycol ( polyethylenepolyethylene glycolglycol ; ; PEGPEG )-히알루론산() -Hyaluronic acid ( hyaluronichyaluronic acidacid ; ; HAHA )-) - 담즙산Bile acid (5-β-(5-β- cholaniccholanic acidacid ; ; CACA ) 복합체의 제조) Preparation of complex

600 mg의 히알루론산을 120 ml의 포름아마이드(formamide)에 녹이고 200 ml의 디메틸포름아마이드(dimethyl formamide)에 아미노에틸-5-β-콜라노아마이드(aminoethyl-5-β-cholanoamide) 199 mg을 녹여 글리콜 히알루론산 용액에 천천히 적하하였고, 364 mg의 1-에틸-3-(3-디메틸-아미노프로필) 카보디이미드(1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide; EDC)와 219 mg의 N-히드로숙시니미드(N-hydrosuccinimide; NHS)를 40 ml의 디메틸포름아마이드에 녹여 반응액에 가한 다음 상온에서 24시간 동안 교반하였다. 이후, 상기 반응액을 2일간 투석하여 미반응 물질을 제거한 후, 동결건조하여 담즙산-히알루론산 복합체를 제조하였다. 상기 제조된 담즙산-히알루론산 복합체 600 mg을 120 ml의 증류수에 녹이고 374 mg의 PEG-NH2, 57.3 mg의 EDC, 40.4 mg의 NHS를 첨가하여 상온에서 24시간 동안 교반하였다. 이후 상기 반응액을 2일간 투석하여 미반응 물질을 제거한 후, 동결건조하여 폴리에틸렌 글리콜-히알루론산-담즙산 복합체를 제조하였다.
Dissolve 600 mg of hyaluronic acid in 120 ml of formamide and 199 mg of aminoethyl-5-β-cholanoamide in 200 ml of dimethyl formamide. Slowly dropwise into the glycol hyaluronic acid solution, 219 mg with 364 mg of 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC) N-hydrosuccinimide (N-hydrosuccinimide; NHS) was dissolved in 40 ml of dimethylformamide, added to the reaction solution, and stirred at room temperature for 24 hours. Thereafter, the reaction solution was dialyzed for 2 days to remove unreacted material, and then lyophilized to prepare a bile acid-hyaluronic acid complex. 600 mg of the bile acid-hyaluronic acid complex prepared above was dissolved in 120 ml of distilled water, and 374 mg of PEG-NH 2 , 57.3 mg of EDC, and 40.4 mg of NHS were added and stirred at room temperature for 24 hours. Thereafter, the reaction solution was dialyzed for 2 days to remove the unreacted material, and then lyophilized to prepare a polyethylene glycol-hyaluronic acid-bile acid complex.

실시예Example 2: 인산칼슘으로 무기화된 나노입자의 제조 2: Preparation of Nanoparticles Mineralized with Calcium Phosphate

무기화를 위해 0.29 M 질산칼슘 용액과 0.24 M 인산암모늄 용액을 제조한 후, 각각 150 μl와 105μl를 10 ml의 탈이온수로 희석하였다. 상기 실시예 1에서 제조된 폴리에틸렌 글리콜(PEG)-히알루론산(HA)-담즙산(CA) 복합체를 탈이온수(Deionized water)에 2 mg/ml의 농도로 녹인 후, 초음파 처리를 하였다. 상기 반응액을 교반시키면서 칼슘용액을 넣어주고, 10분 후 인산염용액을 교대로 반복하여 첨가하였다. 이때, 첨가되는 칼슘용액 및 인산염용액의 양은 16 μl/mg이며, 교반속도는 300 rpm에서 시작하여 매 첨가시마다 10 rpm 씩 증가시켰다. 총 9회의 칼슘용액 및 인산염용액의 첨가가 이루어지면, 15분간 더 교반시켰다. 이때의 교반속도는 마지막 교반속도보다 100 rpm 정도 높게 조절하였다. 교반이 끝나면 탈이온수에서 1시간 정도 투석하여 미반응 물질을 제거하고, 동결건조하여, 최종적으로 무기화된 폴리에틸렌 글리콜-히알루론산-담즙산 복합체(이하 무기화된 폴리에틸렌 글리콜 함유 히알루론산 나노입자라 표기)를 제조하였다.
A 0.29 M calcium nitrate solution and a 0.24 M ammonium phosphate solution were prepared for mineralization, and then 150 μl and 105 μl were diluted with 10 ml of deionized water, respectively. The polyethylene glycol (PEG) -hyaluronic acid (HA) -bile acid (CA) complex prepared in Example 1 was dissolved in deionized water at a concentration of 2 mg / ml, followed by sonication. The calcium solution was added while stirring the reaction solution, and after 10 minutes, the phosphate solution was alternately added. At this time, the amount of added calcium solution and phosphate solution was 16 μl / mg, the stirring speed was started at 300 rpm and increased by 10 rpm for each addition. When nine total calcium and phosphate solutions were added, the mixture was further stirred for 15 minutes. The stirring speed at this time was adjusted to about 100 rpm higher than the last stirring speed. After stirring, the reaction product was dialyzed in deionized water for 1 hour to remove unreacted material, and lyophilized to finally prepare an inorganicized polyethylene glycol-hyaluronic acid-bile acid complex (hereinafter referred to as an inorganicized polyethylene glycol-containing hyaluronic acid nanoparticle). It was.

실시예Example 3: 나노입자의 크기 및 형태 분석 3: Analysis of size and shape of nanoparticles

실시예 1 내지 2에서 제조된 무기화된 폴리에틸렌 글리콜 함유 히알루론산 나노입자를 1 ml의 인산완충염용액(phosphate buffered saline; PBS, pH 7.4)에 녹인 후, 0.45 마이크로 필터로 여과하여 동적 광산란장치(dynamic laser scattering; DLS)와 투과전자현미경(field emission transmission electron microscopy; FE-TEM)으로 입자의 크기 및 형태를 관찰하였다(도 1B).The inorganicized polyethylene glycol-containing hyaluronic acid nanoparticles prepared in Examples 1 to 2 were dissolved in 1 ml of phosphate buffered saline solution (PBS, pH 7.4), and then filtered through a 0.45 micro filter. The particle size and shape were observed by laser scattering (DLS) and field emission transmission electron microscopy (FE-TEM) (FIG. 1B).

그 결과, 나노입자를 인산칼슘으로 무기화할 경우, 입자의 크기가 감소함을 확인하였으며, 이는 무기화과정에서 칼슘이 히알루론산의 카르복실산과 결합하면서 나노입자의 친수성을 감소시켰기 때문이다. FE-TEM 결과로부터 무기화된 나노입자는 구형이며, 특히 소수성 핵으로 구성된 부분과 무기화된 부분이 확연히 구별되는 것을 확인하였으며, 이는 인산칼슘이 히알루론산의 주쇄에 선택적으로 침착되기 때문이다.
As a result, it was confirmed that when the nanoparticles are mineralized with calcium phosphate, the size of the particles is reduced, since calcium is combined with the carboxylic acid of hyaluronic acid to reduce the hydrophilicity of the nanoparticles. The FE-TEM results confirmed that the inorganicized nanoparticles are spherical, and in particular, the distinction between the portion composed of the hydrophobic nucleus and the inorganicized portion is clearly distinguished because calcium phosphate is selectively deposited on the main chain of hyaluronic acid.

실시예Example 4: 나노입자의 특성 분석 4: Characterization of Nanoparticles

실시예 1 내지 2에서 제조된 나노입자를 이용하여 선택영역 전자회절(selected area electron diffraction; SAED) 패턴 및 적외선 분광분석법(FT-IR spectroscopy)을 이용하여 특성을 분석하였다. 이로부터 무기화된 나노입자의 전자회절고리를 발견하여 무결정 인산칼슘이 형성된 것을 확인하였다(도 2A).The nanoparticles prepared in Examples 1 and 2 were characterized using selected area electron diffraction (SAED) patterns and infrared spectroscopy (FT-IR spectroscopy). From this, the electron diffraction ring of the inorganicized nanoparticles was found to confirm that amorphous calcium phosphate was formed (FIG. 2A).

무기화된 나노입자의 적외선 분광분석법을 이용한 결과, P-O 비대칭신장(asymmertic stretching, 1000 cm-1)과 O-P-O 굽힘(bending, 600 cm-1)을 관찰할 수 있었으며, 이로부터 나노입자에 인산칼슘이 성공적으로 침착되었음을 확인할 수 있었다(도 3B). 무기화 전후의 나노입자 크기를 비교해보면, 무기화 과정을 거친 나노입자의 경우 나노입자의 크기가 60 nm 정도 감소하였으며, 또한 무기화 후의 나노입자의 표면전위는 인산칼슘 층에 의해 히알루론산의 카르복실기가 가려짐에 따라 표면전위 값이 증가함을 확인하였다(표 1).Infrared spectroscopy of inorganicized nanoparticles showed PO asymmetric stretching (1000 cm -1 ) and OPO bending (600 cm -1 ), from which calcium phosphate was successfully applied to the nanoparticles. It was confirmed that it was deposited (Fig. 3B). Comparing the nanoparticle size before and after the inorganicization, the nanoparticle size was reduced by about 60 nm in the case of the inorganicized nanoparticles, and the surface potential of the nanoparticles after the inorganicization was blocked by the carboxyl group of the hyaluronic acid by the calcium phosphate layer. It was confirmed that the surface potential value increased according to (Table 1).

Figure 112011103012407-pat00001
Figure 112011103012407-pat00001

실시예Example 5:  5: 독소루비신(doxorubicin; DOX)이Doxorubicin (DOX) 함유된 나노입자의 제조 및 특성 분석 Preparation and Characterization of Contained Nanoparticles

독소루비신(doxorubicin; DOX)이 함유된 나노입자를 하기의 방법으로 제조하였다. 50 mg의 폴리에틸렌 글리콜로 개질된 양친성 히알루론산 고분자를 25 ml의 탈이온수에 녹이고, 독소루비신 5.56 mg을 30 μl의 TEA(triethyl amine)을 포함한 클로로포름(chloroform) 용액 2.78 ml에 녹였다. 고분자 용액을 독소루비신 용액과 혼합하여 초음파 처리한 후, 12시간 동안 교반하였다. 이후, 상기 반응혼합액을 투석 및 필터로 여과한 후, 동결건조하여 독소루비신이 함유된 나노입자를 제조하였다(도 1C).Nanoparticles containing doxorubicin (DOX) were prepared by the following method. Amphiphilic hyaluronic acid polymer modified with 50 mg of polyethylene glycol was dissolved in 25 ml of deionized water, and 5.56 mg of doxorubicin was dissolved in 2.78 ml of chloroform solution containing 30 μl of triethyl amine (TEA). The polymer solution was mixed with the doxorubicin solution and sonicated and then stirred for 12 hours. Thereafter, the reaction mixture was filtered through dialysis and a filter, and then lyophilized to prepare nanoparticles containing doxorubicin (FIG. 1C).

상기 제조된 독소루비신이 봉입된 나노입자를 상기 실시예 2와 동일한 방법으로 무기화하였다. 상기 약물이 봉입된 나노입자의 크기를 동적 광산란장치로 분석하였으며, 약물의 봉입효율은 UV-Vis 분광법, 약물의 표면전위는 제타-전위 분석기를 이용하여 분석하였다. 무기화된 폴리에틸렌 글리콜 함유 히알루론산 나노입자의 약물 봉입효율 및 나노입자의 크기, 무기물의 함량, 표면전위 등의 제조된 나노입자의 특성분석 결과를 상기 표 1에 나타내었다.
The prepared doxorubicin-encapsulated nanoparticles were mineralized in the same manner as in Example 2. The size of the nanoparticles encapsulated in the drug was analyzed by a dynamic light scattering device, the encapsulation efficiency of the drug was analyzed by UV-Vis spectroscopy, the surface potential of the drug using a zeta-potential analyzer. The results of characterization of the prepared nanoparticles such as drug encapsulation efficiency and nanoparticle size, inorganic content, surface potential of the inorganicized polyethylene glycol-containing hyaluronic acid nanoparticles are shown in Table 1 above.

실시예Example 6:  6: 독소루비신이Doxorubicin 봉입된Enclosed 나노입자의 약물방출 특성 분석 Drug Release Characteristics of Nanoparticles

상기 제조된 무기화된 나노입자의 약물 방출 거동을 확인하기 위하여 독소루비신(항암제)이 봉입된 무기화된 나노입자를 다양한 pH의 완충용액에 녹인 후, 독소루비신의 방출량을 UV-Vis 분광법으로 정량분석하여 약물의 방출 거동을 확인하였다(도 4). 무기화하지 않은 독소루비신이 봉입된 나노입자의 경우 초기 24시간 동안 봉입된 약물의 70% 이상이 방출된 것을 확인하였으며, 무기화하지 않은 나노입자의 약물 방출 거동은 pH에 의한 영향이 나타나지 않았다. 반면, 독소루비신이 봉인된 나노입자에 무기화를 진행시킨 경우, pH 7.4에서는 방출속도가 현저히 낮았으며, pH가 낮아짐에 따라, 무기화된 나노입자로부터 독소루비신의 방출 속도가 증가됨을 알 수 있다. 특히, pH 5.0에서는 무기화를 진행한 나노입자와 그렇지 않은 나노입자의 약물 방출 거동이 동일함을 확인하였다. 이는 pH 5.0에서 인산칼슘이 빠른 속도로 용해됨을 의미하며, 상기 결과로부터 나노입자 표면에 인산칼슘으로 무기화한 경우 pH에 따라 약물 방출 속도를 조절할 수 있음을 확인하였다. 따라서, 본 발명의 나노입자는 주변 환경의 pH에 따라 약물 방출 속도를 제어할 수 있으며, 특히 산도가 강할수록 약물이 빠르게 방출되므로 병변부위의 약산성 환경에서 효과적인 약물전달 효과를 기대할 수 있다.
In order to confirm the drug release behavior of the prepared inorganicized nanoparticles, the inorganicized nanoparticles containing doxorubicin (an anticancer agent) are dissolved in buffers of various pHs, and the amount of doxorubicin is quantitatively analyzed by UV-Vis spectroscopy. Release behavior was confirmed (FIG. 4). In the case of non-mineralized doxorubicin-encapsulated nanoparticles, more than 70% of the encapsulated drug was released during the initial 24 hours, and the drug release behavior of the non-mineralized nanoparticles was not affected by pH. On the other hand, when inorganicization of the doxorubicin-sealed nanoparticles, the release rate was significantly lower at pH 7.4, it can be seen that the release rate of doxorubicin from the inorganicized nanoparticles increases as the pH is lowered. In particular, it was confirmed that at pH 5.0, the drug release behavior of the nanoparticles undergoing inorganicization and the nanoparticles that did not undergo the same mineralization were the same. This means that calcium phosphate dissolves rapidly at pH 5.0, and from the above results, it was confirmed that the drug release rate can be adjusted according to pH when inorganicized with calcium phosphate on the surface of the nanoparticles. Therefore, the nanoparticles of the present invention can control the drug release rate according to the pH of the surrounding environment, and in particular, since the drug is released faster as the acidity is stronger, an effective drug delivery effect can be expected in a weakly acidic environment of the lesion site.

실시예Example 7: 무기화된 폴리에틸렌 글리콜 함유 히알루론산 나노입자의 생체 내 거동 특성 평가 7: Evaluation of in vivo behavior of inorganicized polyethylene glycol-containing hyaluronic acid nanoparticles

상기 제조된 무기화된 폴리에틸렌 글리콜 함유 히알루론산 나노입자의 생체 내 거동을 평가하기 위하여, 무기화하지 않은 폴리에틸렌 글리콜 함유 히알루론산 나노입자와 비교실험을 진행하였다. 상기 나노입자들의 영상화를 위하여 형광 프로브인 Cy5.5를 나노입자에 표지하였다. 먼저 종양동물모델을 선정하기 위하여, 면역결핍된 5.5 주령 수컷 누드마우스의 좌측 옆구리에 60 μl의 배지에 분산시킨 1×106 개의 편평상피세포암(SCC7, Squamous cell carcinoma) 세포를 피하에 주입하였다. 편평상피세포암 세포를 접종한 뒤, 종양의 크기가 200 내지 250 mm3이 되었을 때, 근적외선 영상장치를 이용하여 형광이미지를 관찰하였다.In order to evaluate the in vivo behavior of the prepared inorganicized polyethylene glycol-containing hyaluronic acid nanoparticles, comparative experiments with non-mineralized polyethylene glycol-containing hyaluronic acid nanoparticles were conducted. For imaging of the nanoparticles, Cy5.5, a fluorescent probe, was labeled on the nanoparticles. First, in order to select a tumor animal model, 1 × 10 6 squamous cell carcinoma (SCC7) squamous cell carcinoma (SCC7) cells dispersed in 60 μl of medium in the left flank of immunodeficient 5.5-week old male nude mice were injected subcutaneously. . After inoculating squamous cell carcinoma cells, when the tumor size was 200 to 250 mm 3 , the fluorescence image was observed using a near infrared imaging apparatus.

각각의 물질을 종양동물모델에 투여하기 전, 각 물질을 1 mg/ml로 식염수에 분산시킨 후, UV-Vis 분광광도계를 이용하여 675 nm 파장에서의 Cy5.5 흡광도를 측정하여 그 값이 같아지도록 조절하였다. 형광물질의 양을 조절한 물질들을 종양동물모델의 꼬리혈관을 이용하여 정맥투여하고, 1, 3, 6, 9, 24, 48시간 경과 후 근적외선 형광이미지를 관찰하였다. 또한 48시간 경과 후, 동물모델의 장기를 적출하여 형광이미지를 수집하였으며, 종양과 간의 상대적인 형광세기를 측정하여 도 5C에 나타내었다.Before administering each substance to the tumor animal model, each substance was dispersed in saline at 1 mg / ml, and then measured by absorbing Cy5.5 at 675 nm using UV-Vis spectrophotometer. Adjusted to Substances in which the amount of fluorescent material was controlled were intravenously administered using tail blood vessels of tumor animal models, and near-infrared fluorescence images were observed after 1, 3, 6, 9, 24, and 48 hours. In addition, after 48 hours, the organs of the animal model were extracted, and fluorescence images were collected. The relative fluorescence intensity between the tumor and the liver was measured and shown in FIG. 5C.

그 결과, 도 5에 나타낸 바와 같이 근적외선 형광이미지를 바탕으로 폴리에틸렌 글리콜 함유 히알루론산 나노입자는 무기화 후에 암조직 주변으로의 형광량 축적이 증가하는 것을 확인하였으며, 결과적으로 무기화에 의해 나노입자의 안정성 및 체내 체류시간 증가에 의한 암세포에 대한 선택적 축적성이 증가함을 확인하였다. 뿐만 아니라 히알루론산 나노입자의 단점인 간축적을 효과적으로 저해함을 확인하였다. 상기 간축적 문제점을 해결하기 위하여 본 발명자들이 이전에 고안한 표면을 PEG로 개질한 히알루론산 나노입자와 비교하여, 이의 표면을 무기화시킨 본 발명의 무기화된 폴리에틸렌 글리콜 함유 히알루론산 나노입자에서 보다 높은 종양 대 간축적 비율을 나타내는 것을 확인하였다(도 5C).
As a result, as shown in FIG. 5, it was confirmed that the polyethylene glycol-containing hyaluronic acid nanoparticles increased the accumulation of fluorescence around the cancerous tissue after mineralization based on the near-infrared fluorescence image. It was confirmed that the selective accumulation of cancer cells by increasing the retention time in the body increases. In addition, it was confirmed that it effectively inhibits the accumulation, which is a disadvantage of hyaluronic acid nanoparticles. Compared to the hyaluronic acid nanoparticles of which the inventors have previously devised a surface to solve the contraction problem, compared to the hyaluronic acid nanoparticles modified with PEG, higher tumors in the inorganicized polyethylene glycol-containing hyaluronic acid nanoparticles of the present invention which inorganicized its surface. It was confirmed that the ratio represents the accumulation (Fig. 5C).

실시예Example 8:  8: 독소루비신이Doxorubicin 봉입된Enclosed 무기화 나노입자의 치료효능 평가 Evaluation of therapeutic efficacy of inorganic nanoparticles

상기 제조된 독소루비신이 봉입된 나노입자의 치료효과를 평가하기 위하여, 독소루비신이 함유된 무기화된 폴리에틸렌 글리콜 함유 히알루론산 나노입자 또는 독소루비신 주사제를 투여한 실험군, 및 약물을 투여하지 대조군에서 종양성장 비교실험을 진행하였다. 먼저 종양 동물모델을 설정하기 위하여, C3H/HeN 수컷 마우스의 좌측 허벅지에 60 μl의 배지에 분산시킨 1×106 개의 편평상피세포암(SCC7, Squamous cell carcinoma) 세포를 피하에 주입하였다. 편평상피세포암 세포를 접종한 뒤 12일 후, 각 실험군의 종양의 크기가 일정하도록 그룹을 지정하여 각각의 물질을 투여하였다.In order to evaluate the therapeutic effect of the prepared doxorubicin-encapsulated nanoparticles, comparison of tumor growth in the experimental group administered with doxorubicin-containing inorganic glycol glycol hyaluronic acid nanoparticles or doxorubicin injection, and the control group not administered the drug Proceeded. First, in order to establish a tumor animal model, 1 × 10 6 squamous cell carcinoma (SCC7) squamous cell carcinoma (SCC7) cells dispersed in 60 μl of medium were injected subcutaneously into the left thigh of a C3H / HeN male mouse. Twelve days after inoculation of squamous cell carcinoma cells, each substance was administered by assigning groups so that the tumor size of each experimental group was constant.

각 물질은 독소루비신의 양을 기준으로 하여 2 mg/kg을 200 μl의 식염수 또는 인산염완충용액(PBS, pH 7.4)에 분산시켜, 종양 동물모델의 꼬리정맥에 정맥투여하였다. 각각의 물질은 최초 투여일을 포함하여 3, 6, 9일 차에 걸쳐 추가적으로 투여하여 총 4회 투여를 진행하였다. 종양부피의 측정기간은 총 18일에 걸쳐 하루에 한번 동일한 시간에 측정하였다. 또한 측정한 지 18일째에, 종양을 적출하여 실제 종양부피를 확인하여 도 6에 나타내었다.Each material was dispersed 2 mg / kg in 200 μl saline or phosphate buffer solution (PBS, pH 7.4) based on the amount of doxorubicin and intravenously administered to the tail vein of the tumor animal model. Each substance was administered additionally over three, six, and nine days, including the initial administration date, for a total of four administrations. Tumor volume was measured at the same time once a day for a total of 18 days. In addition, 18 days after the measurement, the tumor was extracted and the actual tumor volume was shown in FIG. 6.

도 6에 나타난 바와 같이 종양모델의 종양부피를 바탕으로 독소루비신을 함유한 무기화된 폴리에틸렌 글리콜 함유 히알루론산 나노입자가 종양의 성장을 효과적으로 억제시키는 것을 확인하였다. 이는 독소루비신이 함유된 무기화된 폴리에틸렌 글리콜 함유 히알루론산 나노입자가 정맥투여 후 혈액 내에서 안정하게 장시간 순환하며, 정상부위가 아닌 종양부근의 약산성 pH 조건에서 무기물이 용해되어 종양부위에 특이적으로 독소루비신을 방출하였다는 것을 의미한다. 이러한 현상을 바탕으로 폴리에틸렌 글리콜 함유 히알루론산 나노입자에 무기화를 함으로써 생체 내에서 장시간 순환이 가능하고 약산성 환경을 갖는 질병부위에 효과적인 약물전달이 가능함을 확인하였다.As shown in FIG. 6, it was confirmed that inorganicized polyethylene glycol-containing hyaluronic acid nanoparticles containing doxorubicin effectively inhibit tumor growth based on the tumor volume of the tumor model. This is because inorganic mineralized polyethylene glycol-containing hyaluronic acid nanoparticles containing doxorubicin circulate stably in the blood after intravenous administration. It means that it was released. Based on these phenomena, it was confirmed that inorganicization of the polyethylene glycol-containing hyaluronic acid nanoparticles enables effective drug delivery to disease areas having a long cycle and a weakly acidic environment in vivo.

Claims (16)

폴리에틸렌 글리콜로 표면을 개질시킨 양친성 고분자 나노입자로서, 상기 양친성 고분자는 히알루론산(hyaluronic acid)과 담즙산 유도체가 결합된 것이며, 상기 나노입자의 표면은 인산칼슘으로 무기화된 것을 특징으로 하는 나노입자.
Amphiphilic polymer nanoparticles whose surface is modified with polyethylene glycol, wherein the amphiphilic polymer is a combination of hyaluronic acid and a bile acid derivative, and the surface of the nanoparticles is inorganicized with calcium phosphate. .
삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 제1항에 따른 인산칼슘으로 무기화된 양친성 고분자 나노입자에 약물이 봉입된 것을 특징으로 하는 약물 전달체.
Drug delivery system characterized in that the drug is encapsulated in the amphiphilic polymer nanoparticles inorganicized with calcium phosphate according to claim 1.
제7항에 있어서,
상기 무기화된 나노입자는 pH 4.0 내지 6.0에서 선택적으로 빠르게 약물을 방출하는 약물 전달체.
The method of claim 7, wherein
The inorganicized nanoparticles are drug delivery to release the drug selectively rapidly at pH 4.0 to 6.0.
제7항에 있어서,
상기 약물은 항암제인 것을 특징으로 하는 약물 전달체.
The method of claim 7, wherein
The drug carrier is characterized in that the anticancer agent.
제9항에 있어서,
상기 항암제는 도세탁셀(docetaxel), 시스플라틴(cis-platin), 캠토세신(camptothecin), 파클리탁셀(paclitaxel), 타목시펜(tamoxifen), 아나스테로졸(anasterozole), 글리벡(gleevec), 5-플루오로우라신(5-FU), 플록슈리딘(floxuridine), 류프로리드(leuprolide), 플로타미드(flutamide), 졸레드로네이트(zoledronate), 독소루비신(doxorubicin), 빈크리스틴(vincristine), 젬시타빈(gemcitabine), 스트렙토조신(streptozotocin), 카보플라틴(carboplatin), 토포테칸(topotecan), 벨로테칸(belotecan), 이리노테칸(irinotecan), 비노렐빈(vinorelbine), 히드록시우레아(hydroxyurea), 발루비신(valrubicin), 레티노익산(retinoic acid) 계열, 메토트렉세이트(methotrexate), 메클로레타민(meclorethamine), 클로람부실(chlorambucil), 부술판(busulfan), 독시플루리딘(doxifluridine), 빈블라스틴(vinblastin), 마이토마이신(mitomycin), 프레드니손(prednisone), 테스토스테론(testosterone), 미토산트론(mitoxantron), 아스피린(aspirin), 살리실레이트(salicylates), 이부프로펜(ibuprofen), 나프로센(naproxen), 페노프로펜(fenoprofen), 인도메타신(indomethacin), 페닐부타존(phenylbutazone), 시클로포스파미드(cyclophosphamide), 메클로에타민(mechloethamine), 덱사메타손(dexamethasone), 프레드니솔론(prednisolone), 셀렉콕시브(celecoxib), 발데콕시브(valdecoxib), 니메슐리드(nimesulide), 코르티손(cortisone) 및 코르티코스테로이드(corticosteroid) 중에서 선택되는 것을 특징으로 하는 약물 전달체.
10. The method of claim 9,
The anticancer agent is docetaxel (docetaxel), cis-platin (cis-platin), camptothecin (camptothecin), paclitaxel (paclitaxel), tamoxifen (anasterozole), gleevec (gleevec), 5-fluorouracin ( 5-FU), floxuridine, leuprolide, flutamide, zledronate, doxorubicin, vincristine, gemcitabine Streptozosin, carboplatin, topotecan, belotecan, irinotecan, vinorelbine, hydroxyurea, valurecin, valrubicin, Retinoic acid family, methotrexate, mechloretamine, meclolorebumin, chlorambucil, busulfan, doxifluridine, doxifluridine, vinblastin, my Mitomycin, prednisone, testosterone rone, mitoxantron, aspirin, salicylates, ibuprofen, naproxen, fenoprofen, indomethacin, phenyl Phenylbutazone, cyclophosphamide, mechloethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nimesulide ), A cortisone (cortisone) and a corticosteroid (corticosteroid).
제7항에 있어서,
상기 약물은 조성물 전체에 대하여 1 내지 60 중량%로 포함되는 것을 특징으로 하는 약물 전달체.
The method of claim 7, wherein
The drug carrier, characterized in that contained in 1 to 60% by weight based on the total composition.
제7항의 약물 전달체를 유효성분으로 함유하는 약학적 조성물.
A pharmaceutical composition comprising the drug carrier of claim 7 as an active ingredient.
제12항에 있어서,
상기 조성물은 암질환의 치료용 조성물인 것을 특징으로 하는 약학적 조성물.
The method of claim 12,
The composition is a pharmaceutical composition, characterized in that the composition for the treatment of cancer diseases.
제13항에 있어서,
상기 암질환은 편평상피세포암, 자궁암, 자궁경부암, 전립선암, 두경부암, 췌장암, 뇌종양, 유방암, 간암, 피부암, 식도암, 고환암, 신장암, 대장암, 직장암, 위암, 방광암, 난소암, 담관암 및 담낭암 중에서 선택되는 것을 특징으로 하는 약학적 조성물.
The method of claim 13,
The cancer diseases include squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, ovarian cancer, cholangiocarcinoma And gallbladder cancer.
제1항에 따른 인산칼슘으로 무기화된 양친성 고분자 나노입자에 형광체가 결합된 것을 특징으로 하는 암 진단용 조영제.
The contrast agent for diagnosing cancer, characterized in that the phosphor is bound to the amphiphilic polymer nanoparticles inorganicized with calcium phosphate according to claim 1.
제15항에 있어서,
상기 형광체는 시아닌(cyanine), 플루오레신(fluorescein), 테트라메틸로다민(tetramethylrhodamine), 보디피(BODIPY) 및 알렉사(alexa)로 이루어진 군으로부터 선택되는 근적외선 형광체인 것을 특징으로 하는 암 진단용 조영제.
16. The method of claim 15,
The phosphor is a contrast agent for cancer diagnosis, characterized in that the near-infrared phosphor selected from the group consisting of cyanine (fluorescein), tetramethylrhodamine (tetramethylrhodamine), BODIPY and Alexa (alexa).
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