臺灣博碩士論文加值系統

本研究為設計一具電場響應之生物相容性藥物載體模型，透過交流感應電場誘導帶電奈米粒子之電泳行為，進而加速藥物之釋放。利用生物可降解之高分子聚己內酯(Polycaprolactone diol，PCL diol)為基材，經末端官能基改質為聚己內酯(Polycaprolactone diacrylate，PCLDA)，由IR、NMR證實官能基成功改質。以乳化聚合法將聚己內酯(PCLDA)合成聚己內酯奈米粒子，並透過尼羅紅(Nile red)對其進行螢光標記，並利用Zeta-sizer及DLS探討不同參數下的粒徑與電位變化。由UV-vis測量得在尼羅紅200ug/ml的濃度下具有71%的藥物包覆率，並且在一個月內僅損失10%的藥物量。藉由CLSM觀察螢光包覆影像。對聚己內酯奈米粒子施加不同頻率及電壓之交流電場，利用OM觀察微觀下之變化，並藉由雷射去探討聚己內酯奈米粒子受電場響應之穿透率，其中以6V、6mHz之交流電穿透率最高。由UV-Vis觀察聚己內酯/尼羅紅奈米粒子在上述特定交流電場下之釋放率，在30分鐘釋放率可達30%。
將聚己內酯/尼羅紅奈米粒子與RAW264.7細胞共培養，再利用聚己內酯粒子之電響應特性，對其施加6V、6mHz交流電，利用CLSM觀察細胞攝取粒子情況與藥物釋放效果。本研究成功製備出具電場響應之聚己內酯奈米粒子模型，能透過交流電場誘導粒子泳動加速藥物之釋放。藉由調控粒徑與表面官能化修飾，電響應藥物遞送系統可以克服常規藥物製劑的缺點，它們能夠在特定的部位和時間以受控的方式遞送藥物，未來於癌症治療具有相當之潛力。

In this study, we prepared a biocompatible drug carrier model with electric-field response. First, we modified polycaprolactone diol’s terminal functional group to polycaprolactone diacrylate(PCLDA), which has been comfirmed by IR and NMR spectrum. Second, we used emulsion polymerization to synthesize the polycaprolactone nanoparticles with PCLDA and also labled with nile red via drug encapsulation. Using Zeta-sizer and DLS to explore particle size and potential changes under different concentration of SDS.Furthermore, a high loading efficiency 71% and high stability carrier that only loss approximately 10% drug amounts during one month nanocarrier was prepared, which is measured by UV-vis spectrum, and then we observed nanoparticles containing red fluorescence images by CLSM. Applying an alternating electric-field of different frequencies and voltages to the polycaprolactone nanoparticles, observing the change of nanoparticles under the electric-field by OM, and exploring the penetration rate of the polycaprolactone nanoparticles under the electric-field by laser, we found that the penetrationg rate is the highest under specific alternating electric-field, which is 6 volts and 6 mHz. The release rate reached 30% in 30 minutes under the alternating electric-field(6V、6mHz) was observed by UV-vis spectrum.
The polycaprolactone/Nile red nanoparticles were co-cultured with RAW264.7 cells. Owing to the characteristic of electric-field response of the polycaprolactone nanoparticles, we applied an alternating electric-field with 6 volts and 6 mHz, then using CLSM to observe the cellular uptake and drug release.
In this study, a polycaprolactone nanoparticle model with electric field response was successfully prepared. By controlling particle size and particle’s surface functionalization, the electric-field responsive drug delivery system can overcome the disadvantages of conventional pharmaceutical preparations. They are capable of delivering drugs in a controlled manner at specific sites and times, and have considerable potential for cancer treatments in the future.

摘要 I
Abstract III
致謝 V
目錄 VI
圖目錄 XI
表目錄 XV
第1章 前言 1
1.1 研究背景 1
1.2 研究動機與目的 3
第2章 理論與文獻回顧 5
2.1 奈米載體 5
2.1.1 奈米載體簡介 5
2.1.2 奈米載體種類 7
2.2 奈米載體應用於癌症治療之研究 12
2.2.1 奈米載體材料與抗癌藥物的結合 12
2.2.2 pH響應型奈米藥物載體 13
2.2.3 電場響應型奈米藥物載體 14
2.3 生物相容性高分子 15
2.3.1 聚己內酯(Polycaprolactone)簡介與應用 16
2.4 聚己內酯奈米粒子 18
2.4.1 乳化聚合法 18
2.4.2 奈米沉澱法(溶劑置換法) 22
2.5 交流電場誘導藥物釋放原理 23
2.5.1 電場 23
2.5.2 電泳理論 24
第3章 儀器原理 25
3.1 傅立葉轉換紅外線光譜儀(Fourier transform infrared spectrometer，FT-IR) 25
3.2 可見光紫外光分光光譜儀(Ultraviolet-visible spectroscopy，UV-vis) 30
3.3 核磁共振(Nuclear Magnetic Resonance，NMR) 32
3.4 動態光散射粒徑分析儀(Dynamic light scattering，DLS) 34
3.5 表面電位分析儀(Zeta-potential) 35
3.6 高解析度場發射掃描式電子顯微鏡(Field-emission scanning electron microscope，FE-SEM) 37
3.7 雷射掃描式共軛焦顯微鏡 (Laser scanning confocal microscope，LSCM) 39
3.8 陣列雷射光束分析儀(BeamMic) 40
第4章 實驗流程與方法 45
4.1 實驗流程圖 45
4.2 實驗藥品 46
4.3 實驗儀器 49
4.4 實驗步驟 51
4.4.1 聚己內酯末端官能基改質 51
4.4.1.2 聚己內酯末端官能基改質 51
4.4.2 聚己內酯奈米球製備 52
4.4.3 交流電場下，聚己內酯奈米球之變化與量測 53
4.4.4. 聚己內酯混摻尼羅紅之奈米粒子製備 55
4.4.5 聚己內酯混摻尼羅紅奈米載體穩定性測試 56
4.4.6 交流電場加速藥物釋放測試 57
4.4.7 生物體外實驗 57
第5章 結果與討論 61
5.1 聚己內酯官能基改質之定性分析 61
5.1.1 FT-IR光譜分析 61
5.1.2 NMR化學結構分析 62
5.2 PCL奈米粒子之形貌及定性分析 63
5.2.1 DLS粒徑分析 64
5.2.2 Zeta-potential表面電位分析 65
5.2.3 PCL奈米粒子影像型態分析 66
5.3 PCL粒子溶液之驅動電壓及頻率測試 70
5.3.1 PCL粒子溶液之驅動電壓測試 70
5.3.2 PCL粒子溶液之驅動頻率測試 72
5.3.3 PCL粒子在電場下之光學影像圖 75
5.4 PCL/Nile red 載體之形貌及定性分析 77
5.4.1 DLS粒徑分析 77
5.4.2 Zeta-potential表面電位分析 78
5.4.3 UV-vis光譜分析 79
5.4.4 CLSM雷射共軛焦螢光型態分析 85
5.5 生物體外實驗 86
5.5.1 細胞毒性測試 86
5.5.2 RAW264.7細胞攝取PCL/Nile red藥載之實驗 88
5.5.3 電場誘導RAW264.7細胞內藥物釋放之實驗 90
第6章 結論 94

Liang Zhaoa,1, Arjun Setha,1, Nani Wibowoa, Chun-Xia Zhaoa, Neena Mitterb, Chengzhong Yua, Anton P.J. Middelberga, Nanoparticle vaccines, Vaccine, 2014, 3, 27~337.

Guanying Chen, Indrajit Roy, Chunhui Yang, and Paras N. Prasad, Nanochemistry and Nanomedicine for Nanoparticle-based Diagnostics and Therapy, Chem. Rev., 2016, 116, 2826~2885.

Simona Mura, Julien Nicolas and Patrick Couvreur, Stimuli-responsive nanocarriers for drug delivery, NATURE MATERIALS, 2013, Vol. 12, 991~1003.

Ghosh Chaudhuri R, Paria S. Core/shell nanoparticles: classes, properties, synthesis mechanisms, characterization, and applications, Chem. Rev., 2012, 112, 2373-2433.

Erem Bilensoy, Can Sarisozen, Günes¸ Esendaglı, A. Lale Dogan, Yesim Aktas, Murat Sen, N. Aydın Mungan, Intravesical cationic nanoparticles of chitosan and polycaprolactone for the delivery of Mitomycin C to bladder tumors, International Journal of Pharmaceutics, 2009, 170–176.

Ying Zhang, Ren-xi Zhuo, Synthesis and in vitro drug release behavior of amphiphilictriblock copolymer nanoparticles based on poly (ethylene glycol) and polycaprolactone, Biomaterials, 2005, 6736–6742.

MATTEO CONTI, VALERIA TAZZARI, CESARE BACCINI, GIANNI PERTICI, LORENZO PIO SERINO AND UGO DE GIORGI, Anticancer Drug Delivery with Nanoparticles, in vivo, 2006, 20, 697-702.

Sarwar Hossen, M. Khalid Hossain, M.K. Basher, M.N.H. Mia, M.T. Rahman, M. Jalal Uddin. Smart nanocarrier-based drug delivery systems for cancer therapy and toxicity studies: A review, Journal of Advanced Research, 2018.
Arsalan Ahmed, Sen Liu, Yutong Pan, Shanmei Yuan, Jian He, and Yong Hu, Multicomponent Polymeric Nanoparticles Enhancing Intracellular Drug Release in Cancer Cells, ACS Appl. Mater. Interfaces, 2014, 6, 21316~21324.

Muhammad Sajid Hamid Akash, Kanwal Rehman & Shuqing Chen, Natural and Synthetic Polymers as Drug Carriers for Delivery of Therapeutic Proteins, Polymer Reviews, 2015, 55:3, 371~406.

Ge J, Neofytou E, Cahill TJ, Beygui RE, Zare RN. Drug release from electric-field-responsive nanoparticles. ACS Nano, 2012;6:227–33.

Han-Min Kim, Hak-Ryul Kim, Ching T. Hou, Beom Soo Kim, Biodegradable Photo-Crosslinked Thin Polymer Networks Based on Vegetable Oil Hydroxy Fatty Acids, J Am Oil Chem Soc, 2010, 87:1451–1459.

Klaus Strebhardt and Axel Ullrich, Paul Ehrlich’s magic bullet concept: 100 years of progress, Nature Rev. Cancer, 2008, 8, 473~480

Jorg Kreuter, Nanoparticles—a historical perspective, International Journal of Pharmaceutics, 2007, 331, 1~10.

Gurny, R., Peppas, N.A., Harrington, D.D., Banker, G.S., Development of biodegradable and injectable lattices for controlled release of potent drugs., Drug Dev. Ind. Pharm., 1981, 7, 1–25.

Birrenbach, G., Speiser, P.P., Polymerized micelles and their use as adjuvants in immunology., J. Pharm. Sci., 1976, 65, 1763–1766.

Khanna, S.C., Jecklin, T., Speiser, P., Bead polymerisation technique for sustained release dosage form., J. Pharm. Sci., 1970, 59, 614–618.

Perrault, S. D.; Chan, W. C. W., Synthesis and Surface Modification of Highly Monodispersed, Spherical Gold Nanoparticles of 50−200 nm, J. Am. Chem. Soc., 2009, 131 (47), 17042~17043.

Nikoobakht, B.; El-Sayed, M. A. Preparation and growth mechanism of gold nanorods (NRs) using seed-mediated growth method. Chem. Mater. 2003, 15 (10), 1957~1962.

Cui, Y.; Wei, Q. Q.; Park, H. K.; Lieber, C. M. Nanowire nanosensors for highly sensitive and selective detection of biological and chemical species. Science, 2001, 293 (5533), 1289~1292.

Altintas, O.; Vogt, A. P.; Barner-Kowollik, C.; Tunca, U., Constructing Star Polymers via Modular Ligation Strategies. Polym. Chem., 2012, 3, 34−45.

Prasad, P. N. Nanophotonics; Wiley-Interscience: Hoboken, NJ, 2004.

Burda, C.; Chen, X. B.; Narayanan, R.; El-Sayed, M. A. Chemistry and properties of nanocrystals of different shapes. Chem. Rev., 2005, 105 (4), 1025−1102.

Riehemann, K.; Schneider, S. W.; Luger, T. A.; Godin, B.; Ferrari, M.; Fuchs, H. Nanomedicine-Challenge and Perspectives., Angew. Chem., Int. Ed. 2009, 48 (5), 872−897.

Allen, T. M.; Cullis, P. R. Drug delivery systems: Entering the mainstream. Science, 2004, 303 (5665), 1818−1822.

Muller, R. H.; Mader, K.; Gohla, S. Solid lipid nanoparticles (SLN) for controlled drug delivery - a review of the state of the art. Eur. J. Pharm. Biopharm. 2000, 50 (1), 161−177.

Nicolas, J.; Mura, S.; Brambilla, D.; Mackiewicz, N.; Couvreur, P. Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery., Chem. Soc. Rev., 2013, 42 (3), 1147−1235.

McCarthy, J. R.; Weissleder, R. Multifunctional magnetic nanoparticles for targeted imaging and therapy. Adv. Drug Delivery Rev. 2008, 60 (11), 1241−1251.

Guanying Chen, Indrajit Roy, Chunhui Yang, and Paras N. Prasad, Nanochemistry and Nanomedicine for Nanoparticle-based Diagnostics and Therapy, Chem. Rev., 2016, 116, 2826~2885. [2]

João Conniot, Joana M. Silva, Joana G. Fernandes, Liana C. Silva, Rogério Gaspar, Steve Brocchini, Helena F. Florindo and Teresa S. Barata., Cancer immunotherapy: nanodelivery approaches for immune cell targeting and tracking., Frontiers in Chemistry, 2014, Vol. 2, Article 105., 1~27

Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M., Polymeric systems for controlled drug release., Chem. Rev., 1999, 99 (11), 3181~3198.

Yasuhiro Matsumura and Hiroshi Maeda, A New Concept for Macromolecular Therapeutics in Cancer Chemotherapy: Mechanism of Tumoritropic Accumulation of Proteins and the Antitumor Agent Smancs, cancer research, 1986, 46, 6387~6392

Guanying Chen, Indrajit Roy, Chunhui Yang, and Paras N. Prasad, Nanochemistry and Nanomedicine for Nanoparticle-based Diagnostics and Therapy, Chem. Rev., 2016, 116, 2826~2885.

Saina Yang, Feiyan Zhu, Qian Wang, Fuxin Liang, Xiaozhong Qu, Zhihua Gan and Zhenzhong Yang, Combinatorial targeting polymeric micelles for anti-tumor drug delivery, Journal of Materials Chemistry B, 2015, 3(19), 4043–4051.

Ruth Duncan, The dawning era of polymer therapeutics, Nature Reviews Drug discovery, 2003, Vol. 2., 347~360

LEIBLER, Ludwik; ORLAND, Henri; WHEELER, John C. Theory of critical micelle concentration for solutions of block copolymers. The Journal of chemical physics, 1983, 79(7), 3550~3557.

Ling Mei, Yayuan Liu, HuaJin Zhang, Zhirong Zhang, Huile Gao, and Qin He, Antitumor and Antimetastasis Activities of Heparin-based Micelle Served As Both Carrier and Drug, ACS Appl. Mater. Interfaces, 2016, 8, 9577~9589

SINGH, Jasvinder, et al. Diphtheria toxoid loaded poly-(ε-caprolactone) nanoparticles as mucosal vaccine delivery systems. Methods, 2006, 38.2: 96-105.

FLORINDO, H. F., et al. New approach on the development of a mucosal vaccine against strangles: systemic and mucosal immune responses in a mouse model. Vaccine, 2009, 27.8: 1230-1241.

FLORINDO, H. F., et al. The enhancement of the immune response against S. equi antigens through the intranasal administration of poly-ɛ-caprolactone-based nanoparticles. Biomaterials, 2009, 30.5: 879-891.

EMOTO, Kazunori; NAGASAKI, Yukio; KATAOKA, Kazunori. A Core− Shell Structured Hydrogel Thin Layer on Surfaces by Lamination of a Poly (ethylene glycol)-b-poly (d, l-lactide) Micelle and Polyallylamine. Langmuir, 2000, 16.13: 5738-5742.

RÖSLER, Annette; VANDERMEULEN, Guido WM; KLOK, Harm-Anton. Advanced drug delivery devices via self-assembly of amphiphilic block copolymers. Advanced drug delivery reviews, 2012, 64: 270-279.

Stephanie Tran1, Peter Joseph DeGiovanni1, Brandon Piel and Prakash Rai. Cancer nanomedicine: a review of recent success in drug delivery, Clin Trans Med, 2017 6:44.

Rizvi, S.A.A., Saleh, A.M., Applications of Nanoparticle Systems in Drug Delivery Technology, Saudi Pharmaceutical Journal, 2017

Ying-Jie Zhu and Feng, pH-Responsive Drug-Delivery, Chemistry - An Asian Journal,2014 , 284–305.

Elizabeth R. Gillies and Jean M. J. Fre´chet, pH-Responsive Copolymer Assemblies for Controlled Release of Doxorubicin, Bioconjugate Chem, 2005, 16, 361−368.
Ge J, Neofytou E, Cahill TJ, Beygui RE, Zare RN. Drug release from electric-field-responsive nanoparticles. ACS Nano, 2012;6:227–33.

Ge J, Neofytou E, Cahill TJ, Beygui RE, Zare RN. Drug release from electric-field-responsive nanoparticles. ACS Nano, 2012;6:227–33.

S. Ponsart, J. Coudane, and M. Vert, A Novel Route to Poly(E-caprolactone)-Based Copolymers via Anionic Derivatization, Biomacromolecules, 2000, 1, 275~281

A.G.A. Coombes, S.C. Rizzi, M. Williamson, J.E. Barralet, S. Downes, W.A. Wallace, Precipitation casting of polycaprolactone for applications in tissue engineering and drug delivery, Biomaterials, 2004, 25, 315~325.

Julien Nicolas, Simona Mura, Davide Brambilla, Nicolas Mackiewicz and Patrick Couvreur, Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery, Chem. Soc. Rev., 2013, 42, 1147

Fatemeh Bahadori, Aydan Dag, Hakan Durmaz, Nese Cakir, Hayat Onyuksel, Umit Tunca, Gulacti Topcu and Gurkan Hizal, Synthesis and Characterization of Biodegradable Amphiphilic Star and Y-Shaped Block Copolymers as Potential Carriers for Vinorelbine, Polymers, 2014, 6, 214~242.

Jorg Kreuter, Nanoparticles—a historical perspective, International Journal of Pharmaceutics, 2007, 331, 1~10.

Robert M. Fitch, Michael B. Prenosil, Karen J. Sprick, The mechanism of particle formation in polymer hydrosols. I. Kinetics of Aqueous Polymerization of Methyl Methacrylate, Journal of Polymer Science Part C: Polymer Symposia, Wiley Online Library, 1969, pp.95-118.

William D. Harkins, A General Theory of the Mechanism of Emulsion Polymerization, Journal of the American Chemical Society, 1947, 1428-1444.


H. Fessi, F. Puisieux, J.Ph. Devissaguet, N. Ammoury and S. Benita, Nanocapsule formation by interfacial polymer deposition following solvent displacement, International Journal of Pharmaceutics, 1989,55 R1-R4.

Miladi K., Sfar S., Fessi H., Elaissari A. Nanoprecipitation Process: From Particle Preparation to In Vivo Applications. In: Polymer Nanoparticles for Nanomedicines: A Guide for their Design, Preparation and Development (Vauthier C., Ponchel G. ed.). Springer International Publishing. 2016; pp 17–53.

Jong Min Sung. Dielectrophoresis and Optoelectronic Tweezers for Nanomanipulation. 2007.