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Development and Characterization of Polymer-based Nanocarriers for Poorly Soluble Materials

난용성 물질의 가용화를 위한 고분자를 이용한 나노 운반체의 개발 및 특성화

  • 안주영 (Jooyoung An, 대학원 약학과 약물과학전공)

초록/요약

Natural products exhibit excellent properties such as chemical diversity, specificity, low toxicity, chemical and biological properties. However, More than 40% of the New Chemicals (NCEs) developed in the pharmaceutical industry are almost insoluble in water. These poorly soluble active materials or drugs with low absorption have low bioavailability, lack of solubility for IV administration, development problems leading to increased cost and time, and transfer of burden to patients (frequently high doses) In this study, Polymeric micelles were used as carrier to solubilize natural products for antiwrinkle cosmetic agents. Polymeric micelles are prepared with di-block or tri-block copolymers which can form a hydrophobic inner core and hydrophilic outer shell. Also, the multilayer nanoparticle technology which can improve stability of polymeric micelle nanoparticles is also used for the solubilization of the anticancer drug. The multilayer is formed with lipid encapsulating polymeric micelle nanoparticles. Using these nano-carrier preparation technologies, docetaxel, a drug for cancer therapy, propolis extract, an antioxidant and antiaging material, oleanolic acid, an antiwrinkle material were solubilized and applied to several in-vitro and in-vivo tests. The aim of the study is to improve the efficacy of poorly soluble materials and drugs and to develop an alternative drug delivery system of these materials and drugs for the safety and efficient application in cosmetic and pharmaceutical products. The solubility of poorly soluble materials and drugs was evaluated in solubilizers, surfactants, and polymers. The particle sizes and shapes of polymeric micelle nanoparticle and multilayer nanoparticles were evaluated by electrophoretic light scattering spectrophotometer and scanning electron microscopy or transmission electron microscopy. Encapsulation efficiency were measured by HPLC. Stability of the developed nanoparticles was evaluated by visual observation, particle size measurement, and content measurement. Several in-vitro and in-vivo tests were conducted with polymeric micelle nanoparticles and multilayer nanoparticles. Clinical trials for anti-wrinkle efficacy were conducted with those polymeric micelle nanoparticles in cosmetic products. These products were applied around the eyes of test subjects for 8 weeks. Firstly, polymeric micelles of oleanolic acid with a particle size of less than 100 nm were prepared using Capryol 90® and poloxamer. The skin permeation rate of the oleanolic acid in the polymeric micelle was higher than that in the other solutions made of oleanolic acid dispersed in 2 different surfactants. No significant changes in particle size, color, or oleanolic acid content were observed, and the polymeric micelles stored at 40 °C for 3 months did not undergo phase separation. After 8 weeks of application, skin irritation had not developed and all five parameters evaluated by optical profilometry as well as the visual evaluation scores were significantly improved. This study showed that the polymeric micelles of oleanolic acid prepared in this study were stable and effective at alleviating wrinkles in humans as the principal active ingredient. Polymeric nanoparticles containing propolis extract, polyethylene glycol 400, and poloxamer 407 were prepared via a temperature-induced phase transition method. The particle size of the polymeric nanoparticles was approximately 20.75 nm. The results of an in vitro procollagen type I carboxy-terminal peptide assay and a matrix metalloproteinase-1 inhibition assay showed that the polymeric nanoparticles increased collagen production by 19.81%–24.59% compared to blank (p < 0.05), and significantly reduced intracellular collagenase activity by 7.46%–31.52% compared to blank (p < 0.05). In a clinical trial, polymeric nanoparticles in a cosmetic formulation were applied around the eyes of 24 female subjects for 8 weeks. Five skin parameters were significantly improved after the application of the test ampoule. Visual evaluation using the Global Photo Damage Score showed a significant reduction in wrinkles after the application of the test ampoules (p < 0.001). Finally, docetaxel was encapsulated into polymeric micelle nanoparticles using a triblock copolymer, a biocompatible polymer, and the internal pH of micelles was adjusted to prevent decomposition of docetaxel. In addition, after preparing liposomes composed of a lipid bilayer using egg lecithin, micelle nanoparticles containing docetaxel were encapsulated therein to enhance their stability. The average particle size of the prepared DTX-LNPs was 130±30 nm and the internal structure of DTX-LNPs measured by Cryo-TEM was confirmed that micelle nanoparticles were encapsulated inside the lecithin shell. The particle size over time was measured to confirm that they were sufficiently stable even in aqueous solution. In vivo anticancer efficacy test was conducted by intravenous injection of DTX-LNPs and Taxotere® into BALB/c nude mice transplanted with A549 lung cancer cell line and MDA-MB-231 breast cancer cell line and was confirmed that the efficacy was significantly equivalent to that of the commercially used Taxotere® . The average blood concentration of DTX-LNPs was measured by conducting an in vivo pharmacokinetic test, the half-life of docetaxel in the body increased by 1.5 times and AUC by about 25% compared to Taxotere® , and the average Cmax increased by about 2 times compared to Taxotere® . It was confirmed that the residence time and bioavailability in the body were increased compared to Taxotere® at the same dose. Based on these findings, it is expected that polymeric micelles nanoparticles can be widely used in large spectrum of industrial field, from treatment of disease to cosmetic products.

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초록/요약

천연물은 우수한 화학적 다양성, 특이성 및 저독성 화학적 및 생물학적 특성과 같은 우수한 특성을 나타낸다. 그러나 제약 산업에서 개발된 천연물 기반 NCE(New Chemicals)의 40% 이상이 물에 거의 녹지 않는다. 이러한 낮은 난용성 약물은 낮은 흡수율과 생체 이용률, IV 투여 시 혈중 농도의 문제, 개발 비용 및 시간 증가로 이어지는 개발 문제, 환자에게 부담 전가(고용량 필요)가 있다. 본 연구에서 고분자 마이셀 나노입자 기술을 천연물을 가용화하기 위한 운반체로 사용하였다. 고분자 마이셀 나노입자는 소수성 내부 코어와 친수성 외부 쉘을 형성할 수 있는 이중 블록 또는 삼중 블록 공중합체로 만들어진다. 또한, 고분자 마이셀 나노입자의 안정성을 향상시키기 위해 지질-고분자 하이브리드 나노입자로 알려진 지질-고분자 다층 나노입자 기술또한 사용하였다. 이러한 다양한 고분자 기반 나노 입자 제조 기술을 사용하여 암 치료제인 도세탁셀, 항산화 및 노화 방지 물질인 프로폴리스 추출물, 주름 개선 물질인 올레아놀릭산을 가용화하여 여러 in-vitro 및 in-vivo 시험을 시행하였다. 본 연구의 목적은 난용성 물질 및 약물의 효능 발현 개선하고 화장품 및 의약품에 안전하고 효율적인 적용을 위해 약물의 대체 약물 전달 시스템을 개발하는 것이다. 난용성 물질 및 약물은 가용화제, 계면활성제와의 적합성을 먼저 확인하여 조성을 선정한다. 고분자 마이셀 나노입자와 다층 나노입자의 입자 크기와 모양을 동적 광산란 분광광도계와 주사전자현미경 또는 투과전자현미경으로 평가하였다. 캡슐화 효율은 HPLC 로 측정하였다. 개발된 나노입자의 안정성은 육안 관찰, 입도 측정 및 함량 측정을 통해 평가하였다. 고분자 마이셀 나노입자와 다층 나노입자를 사용하여 여러 가지 in-vitro 및 in-vivo 테스트를 진행하였다. 고분자 마이셀 나노 입자를 적용한 화장품에 대한 주름 개선 효능 임상 시험 또한 진행하였다. 먼저, Capryol 90® 및 poloxamer 를 사용하여 100 nm 미만의 입자 크기를 갖는 oleanolic acid 의 고분자 마이셀을 제조하였다. 고분자 마이셀에서 oleanolic acid 의 피부 투과율은 2 가지 다른 계면활성제에 분산시킨 oleanolic acid 샘플보다 더 높았다. 입자 크기, 색상 또는 oleanolic acid 함량 변화, 상분리, 침전 형성 등의 유의한 변화는 관찰되지 않았으며 40°C 에서 3 개월 동안 안정하였다. 도포 8 주 후, 피부 자극은 발생하지 않았으며, 임상시험 8 주 후 oleanolic acid 고분자 마이셀 소재가 포함된 화장품을 도포한 시험자에게서 유의하게 주름이 개선되었다. 프로폴리스 추출물, 폴리에틸렌 글리콜 400 및 폴록사머 407 을 포함하는 고분자 나노 입자는 온도 기인성 상전이 방법을 통해 제조되었다. 고분자 나노입자의 입자 크기는 약 20.75 nm 였다. In-vitro procollagen type I carboxy-terminal peptide assay 와 matrix metalloproteinase1 inhibitors assay 를 통한 in-vitro 효능 시험 결과 고분자 나노입자가 콜라겐 생성을 19.81%- 24.59% 증가시켰고 세포내 7.46%–31.52%의 억제율로 콜라게나아제를 유의하게 감소시켰다. 소재를 화장품에 적용한 후 임상 시험에서 눈 주위에 동안 도포한 결과 4 주 8 주 후 주름이 현저히 감소한 것으로 나타났다. 마지막으로 생체적합성 고분자인 트리블록 공중합체를 이용하여 고분자 마이셀 나노입자로 도세탁셀을 봉입하고 마이셀 내부 pH 를 조절하여 도세탁셀의 분해를 방지하였다. 또한, 난황 레시틴을 이용하여 지질 이중층으로 구성된 리포솜을 제조한 후, 도세탁셀을 포함하는 마이셀 나노입자를 봉입하여 안정성을 향상시켰다. 제조된 DTXLNP 의 평균 입경은 130±30 nm 였으며 Cryo-TEM 으로 측정한 DTX-LNP 의 내부 구조는 마이셀 나노입자가 레시틴 쉘 내부에 봉입되어 있음을 확인하였다. 시간 경과에 따른 입자 크기를 측정하여 수용액에서도 충분히 안정함을 확인하였다. A549 폐암 세포주와 MDAMB-231 유방암 세포주가 이식된 BALB/c 누드마우스에 DTX-LNPs 와 Taxotere® 를 정맥 주사하여 in-vivo 항암 효능 시험을 진행하였고, 같은 농도에서는 Taxotere® 과 유의하게 동등한 효능을 나타내었고 농도 의존적으로 효능이 높아지는 것도 확인하였다. DTXLNPs 의 평균 혈중 농도를 in-vivo 약동학 시험을 통해 측정한 결과, Taxotere® 대비 도세탁셀의 체내 반감기는 1.5 배, AUC 는 약 25% 증가하였으며, 평균 Cmax 는 약 2 배 증가하였다. 동일한 용량의 Taxotere® 에 비해 체내 체류시간 및 생체이용률이 증가함을 확인하였다. 고분자 마이셀로 제조된 화장품 제형은 효능 발현 및 피부 투과율이 증가하였으며 임상시험에서도 효능 발현을 확인할 수 있었다. Taxotere® 에서 사용되는 용매가 다양한 부작용을 일으켜 이를 이용하지 않은 대체 약물전달시스템으로 다층나노입자를 개발하였고 유의하게 동일한 효능을 확인하였고 약동학 시험 결과를 통하여 생체이용률이 증가한 것으로 확인되었다. 이러한 연구 내용을 바탕으로 고분자 마이셀 나노입자는 질병의 치료에서 화장품에 이르기까지 광범위한 산업분야에서 폭넓게 사용될 수 있을 것으로 기대된다

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목차

Introduction 16
1. Solubility improvement technology 16
2. Polymeric micelle nanoparticles 18
Part 1. Development of Polymeric Micelles of Oleanolic Acid and Evaluation of Their Clinical Efficacy 21
1. Introduction 21
2. Materials and methods 23
2.1. Materials 23
2.2. Methods 24
2.2.1. HPLC Analysis 24
2.2.2. Solubility and Formulation Optimization Study 24
2.2.3. Preparation of Polymeric Micelles of Oleanolic Acid 25
2.2.4. Staining Test 29
2.2.5. Particle Size Measurement 29
2.2.6. Scanning Electron Cryomicroscopy (Cryo-SEM) Analysis 29
2.2.7. Encapsulation Efficiency 30
2.2.8. Stability of oleanolic acid polymeric micelles 30
2.2.9. In Vitro Skin Permeation Test 30
2.2.10. Statistics of the Study 31
2.2.11. Ampoule Preparation with PMO-H 31
2.2.12. Human Skin Irritation Study 32
2.2.13. Clinical Trial for Wrinkle Improvement 32
3. Results and discussion 34
3.1. Determination of Solubilizer, Surfactant, and Polymer 34
3.2. Characteristics of the Polymeric Micelles of Oleanolic Acid 36
3.3. In Vitro Skin Permeation Study of PMO 43
3.4. Stability of Liquid Oleanolic Acid Polymeric Micelles 45
3.5. Clinical Test 47
3.5.1 Human Irritation Test 47
3.5.2 Clinical Trial 47
4. Conclusions 57
Part 2. Clinical Anti-aging Efficacy of Propolis Polymeric Nanoparticles Prepared by a Temperature-induced Phase Transition Method 58
1. Introduction 58
2. Materials and methods 60
2.1. Materials 60
2.2. Methods 61
2.2.1. High-performance liquid chromatography (HPLC) analysis 61
2.2.2. Solubility and formulation optimization study of propolis extract 64
2.2.3. Preparation of polymeric nanoparticles 66
2.2.4. Encapsulation efficiency 68
2.2.5. Transmission electron microscopy (TEM) 68
2.2.6. Particle size measurement 68
2.2.7. Differential scanning calorimetry (DSC) 68
2.2.8. Stability test 69
2.2.9. In vitro cell viability test. 69
2.2.10. In vitro anti-wrinkle test 70
2.2.11. Procollagen type i c peptide (pip) contents in hdfs 70
3. Results and discussion 75
3.1. Determination of solubilizer and polymer 75
3.2. Characterization of polymeric nanoparticles containing the propolis extracts 76
3.3. Encapsulation efficiency of the polymeric nanoparticle containing propolis extract 83
3.4. Stability of polymeric nanoparticle containing propolis extract 86
3.5. In vitro cell viability test and In vitro anti-wrinkle efficacy test 89
3.6. Human irritation test 95
3.7. Clinical trial 95
4. Conclusions. 103
Part 3. Preparation and characterization of docetaxel loaded biocompatible multilayer nanoparticles 104
1. Introduction 104
2. Materials and methods 107
2.1. Materials 107
2.2. Methods 107
2.2.1. Preparation of docetaxel loaded multilayer nanoparticles 107
2.2.2. Particle size analysis 108
2.2.3. Cryo-TEM analysis 108
2.2.4. Particle size stability test 109
2.2.5. Anticancer efficacy test of multilayer nanoparticles containing docetaxel in lung cancer cells 109
2.2.6. Anticancer efficacy test of nanoparticles containing docetaxel in breast cancer cells 110
2.2.7. Pharmacokinetic study of nanoparticles containing docetaxel 111
2.2.8. Statistical Analysis 112
3. Results and discussion 113
3.1. Physical Characterization of Nanoparticles Containing Docetaxel 113
3.1.1. Particle Size and Distribution Analysis 113
3.1.2. Particle cross-sectional structure analysis Cryo-TEM 118
3.2. Stability test 120
3.3. Evaluation of In vivo Anticancer Effect of Docetaxel Nanoparticles 121
3.3.1. Evaluation of anticancer efficacy in lung cancer 121
3.3.2. Evaluation of anticancer efficacy in breast cancer 128
3.4. In vivo pharmacokinetic study 135
4. Conclusions 138
References 140
국문초록 153

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