本研究藉由透析法製備成含有indomethacin的Polycaprolactone-Hyaluronic acid (PCL-HA)奈米微胞，並且摻混Poly(N-isopropylacrylamide)(PNIPAAm)使其具有溫感效應，再探討微胞特性及藥物釋放的效果。結果顯示，此PCL-HA高分子材料，其臨界微胞濃度(critical micelle concentration, CMC)為0.007mg/ml。對於含有indomethacin(IDM)的micelle，表面電位為-29.5±1.9mV，添加了PNIPPAm，表面電位為-27.7±2.7mV，不會影響到表面電位。含有PNIPAAm的micelle在25℃，粒徑為239±7.8nm，將此樣品升溫至37℃時，粒徑會縮小成210±9.5nm，且有可逆的現象；而不含PNIPAAm的micelle在25℃與37℃，粒徑大小為233±8.6nm與241±10.4nm，統計上無差別。藉由TEM觀察micelle形態為球形，其粒徑的大小約在50~150nm之間。以IDM當作包覆的藥物，在無添加PNIPAAm的micelle，包覆率為9.0±0.6%；添加了PNIPAAm後，其包覆率可以提升至16.3±3.3%。藥物釋放方面，同樣的樣品在25℃及37℃下，看不出釋放上的差異；但在25℃及37℃下，含有PNIPAAm的micelle皆會略高於不含PNIPAAm組。另外有利用QR作為螢光劑，再次確認以透析法製作出來的微胞有將大部分QR包覆在內。

In this study, preparing indomethacin-loaded Polycaprolactone-Hyaluronic acid (PCL-HA) polymeric micelles by the dialysis method and blending the PNIPAAm make it thermosensitive. Exploring the characteristics of the micelles and drug release effect. The experimental results show the critical micelle concentration is 0.007mg/ml. For the IDM-loaded micelle, the value of zeta potential is -29.5±1.9mV. After adding PNIPAAm the zeta potential of the micelle is -27.7±2.7mV. The partical size of NIPAAm-contained micelle is 239±7.8nm at 25℃. After the temperature raise to 37℃, the micelle shrink to 210±9.5nm reversible. The micelle without PNIPAAm at 25 ℃ and 37 ℃, the particle size was 233 ± 8.6nm and 241 ± 10.4nm, statistically no difference. Observed by TEM, the morphology of micelle is sphere, its particle size between 50~150nm.The encapsulating efficiency of micelle without PNIPAAm is9.0±0.6%; it raised to 16.3±3.3% by adding PNIPAAm. For drug release, there is no different between 25 ℃ and 37 ℃ in the same sample. But under 25 ℃ and 37 ℃, the micelle containing PNIPAAm are no PNIPAAm group will be slightly higher.

中文摘要 ........................................ i

ABSTRACT ...................................... ii

目錄 ......................................... iii

表目錄 .......................................... v

圖目錄 ......................................... vi

第一章. 緒論 .................................... 1

1-1 藥物控制釋放傳遞系統 .......................... 1

1-2 高分子微胞形成原理 ............................ 2

1-3高分子微胞藥物包覆原理 .......................... 3

1-4 常見的高分子微胞的應用 ......................... 5

1-4-1 酸鹼型應答型微胞 ............................ 7

1-4-2 溫度型應答型微胞 ............................ 8

1-5 異丙基丙烯醯胺 (PNIPAAm) ..................... 9

1-6 透明質酸（Hyaluronic Acid, HA）. ............ 10

1-7 聚己內酯(Poly ε-carprolactone, PCL) ........ 12

1-8 包覆藥物：吲哚美辛(Indomethacin, IDM) ........ 14

1-9奎納克林 (Quinacrine ;QR) ................... 15

1-10 研究動機 .................................. 16

第二章. 實驗設備與方法 ........................... 17

2-1實驗材料與藥品 ............................... 17

2-2實驗設備 .................................... 18

2-4材料製備與分析 ............................... 20

2-4-1 微胞之製備 ............................... 20

2-4-2 微胞之特性分析 ............................ 21

2-5 包覆效率 ................................... 27

2-6 微胞體外釋放 ................................ 28

2-7 濃縮QR微胞 ................................. 28

(1) PEG濃縮法 .................................. 28

(2)離心濃縮管法 ................................. 29

第三章. 結果與討論 ............................... 31

3-1 傅立葉轉換紅外線光譜儀FTIR .................... 31

3-2 臨界微胞濃度(CMC)鑑定 ........................ 31

3-3 粒徑分析(Particle size)及表面電位(Zeta potential) ................... 33

3-4 奈米藥物微胞之包覆效率鑑定 .................... 36

3-5 奈米藥物微胞之釋放行為探討 .................... 37

3-6 QR micelle 的濃縮 ......................... 39

第四章 結論 .................................... 41

附錄 .......................................... 42

粒徑整理 ....................................... 42

表面電位 ....................................... 43

包覆率 ......................................... 44

藥物釋放 ....................................... 45

IDM溶於氯仿中之檢量線 (n=3) ...................... 49

IDM溶於PBS中之檢量線 (n=4) ...................... 50

參考文獻 ....................................... 51

1. Khanna, S.C. and P. Speiser, Epoxy resin beads as a pharmaceutical dosage form. I. Method of preparation. J Pharm Sci, 1969. 58(9): p. 1114-7.

2. Chen, Y.C., et al., Pluronic block copolymers: novel functions in ultrasound-mediated gene transfer and against cell damage. Ultrasound Med Biol, 2006. 32(1): p. 131-7.

3. Papagiannaros, A., et al., Quantum dots encapsulated in phospholipid micelles for imaging and quantification of tumors in the near-infrared region. Nanomedicine: Nanotechnology, Biology and Medicine, 2009. 5(2): p. 216-224.

4. Jones, M.-C. and J.-C. Leroux, Polymeric micelles – a new generation of colloidal drug carriers. European Journal of Pharmaceutics and Biopharmaceutics, 1999. 48(2): p. 101-111.

5. Elworthy, P.H., A.T. Florence, and C.B. Macfarlane, Solubilization by surface-active agents and its applications in chemistry and the biological sciences. 1968: Chapman & Hall.

6. Gao, Z. and A. Eisenberg, A model of micellization for block copolymers in solutions. Macromolecules, 1993. 26(26): p. 7353-7360.

7. Remant Bahadur, K.C., et al., Novel amphiphilic triblock copolymer based on PPDO, PCL, and PEG: Synthesis, characterization, and aqueous dispersion. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2007. 292(1): p. 69-78.

8. Kataoka, K., et al., Spontaneous Formation of Polyion Complex Micelles with Narrow Distribution from Antisense Oligonucleotide and Cationic Block Copolymer in Physiological Saline. Macromolecules, 1996. 29(26): p. 8556-8557.

9. Kwon, G.S. and T. Okano, Polymeric micelles as new drug carriers. Advanced Drug Delivery Reviews, 1996. 21(2): p. 107-116.

10. Kataoka, K., Design of Nanoscopic Vehicles for Drug Targeting Based on Micellization of Amphiphiuc Block Copolymers. Journal of Macromolecular Science, Part A, 1994. 31(11): p. 1759-1769.

11. Savic, R., et al., Micellar nanocontainers distribute to defined cytoplasmic organelles. Science, 2003. 300(5619): p. 615-8.

12. Tonge, S.R. and B.J. Tighe, Responsive hydrophobically associating polymers: a review of structure and properties. Advanced Drug Delivery Reviews, 2001. 53(1): p. 109-122.

13. Torres-Lugo, M. and N.A. Peppas, Molecular Design and in Vitro Studies of Novel pH-Sensitive Hydrogels for the Oral Delivery of Calcitonin. Macromolecules, 1999. 32(20): p. 6646-6651.

14. Pinkrah, V.T., et al., Physicochemical Properties of Poly(N-isopropylacrylamide-co-4-vinylpyridine) Cationic Polyelectrolyte Colloidal Microgels. Langmuir, 2003. 19(3): p. 585-590.

15. Bae, Y., et al., Design of Environment-Sensitive Supramolecular Assemblies for Intracellular Drug Delivery: Polymeric Micelles that are Responsive to Intracellular pH Change. Angewandte Chemie International Edition, 2003. 42(38): p. 4640-4643.

16. Qiu, Y. and K. Park, Environment-sensitive hydrogels for drug delivery. Advanced Drug Delivery Reviews, 2001. 53(3): p. 321-339.

17. Chen, W.-Q., et al., Fabrication of star-shaped, thermo-sensitive poly(N-isopropylacrylamide)–cholic acid–poly(ɛ-caprolactone) copolymers and their self-assembled micelles as drug carriers. Polymer, 2008. 49(18): p. 3965-3972.

18. Muhlebach, A., S.G. Gaynor, and K. Matyjaszewski, Synthesis of Amphiphilic Block Copolymers by Atom Transfer Radical Polymerization (ATRP). Macromolecules, 1998. 31(18): p. 6046-6052.

19. Schild, H.G., Poly(N-isopropylacrylamide): experiment, theory and application. Progress in Polymer Science, 1992. 17(2): p. 163-249.

20. Maolin, Z., et al., Radiation preparation of PVA-g-NIPAAm in a homogeneous system and its application in controlled release. Radiation Physics and Chemistry, 2000. 57(3–6): p. 481-484.

21. Kohori, F., et al., Process design for efficient and controlled drug incorporation into polymeric micelle carrier systems. Journal of Controlled Release, 2002. 78(1–3): p. 155-163.

22. Chang, C., et al., Thermo-responsive shell cross-linked PMMA-b-P(NIPAAm-co-NAS) micelles for drug delivery. International Journal of Pharmaceutics, 2011. 420(2): p. 333-340.

23. Liu, Y., et al., Dual targeting folate-conjugated hyaluronic acid polymeric micelles for paclitaxel delivery. International Journal of Pharmaceutics, 2011. 421(1): p. 160-169.

24. Lin, Y., Y. Li, and C.P. Ooi, 5-Fluorouracil encapsulated HA/PLGA composite microspheres for cancer therapy. J Mater Sci Mater Med, 2012. 23(10): p. 2453-60.

25. De Campos, A.M., et al., The effect of a PEG versus a chitosan coating on the interaction of drug colloidal carriers with the ocular mucosa. Eur J Pharm Sci, 2003. 20(1): p. 73-81.

26. Zhang, L., et al., Camptothecin derivative-loaded poly(caprolactone-co-lactide)-b-PEG-b-poly(caprolactone-co-lactide) nanoparticles and their biodistribution in mice. Journal of Controlled Release, 2004. 96(1): p. 135-148.

27. Kang, S.W., et al., pH-triggered unimer/vesicle-transformable and biodegradable polymersomes based on PEG-b-PCL–grafted poly(β-amino ester) for anti-cancer drug delivery. Polymer, 2013. 54(1): p. 102-110.

28. Chaw, C.-S., et al., Thermally responsive core-shell nanoparticles self-assembled from cholesteryl end-capped and grafted polyacrylamides:: drug incorporation and in vitro release. Biomaterials, 2004. 25(18): p. 4297-4308.

29. Xin, J., et al., Study of branched cationic β-cyclodextrin polymer/indomethacin complex and its release profile from alginate hydrogel. International Journal of Pharmaceutics, 2010. 386(1–2): p. 221-228.

30. Li, N.-N., et al., New heparin–indomethacin conjugate with an ester linkage: Synthesis, self aggregation and drug delivery behavior. Materials Science and Engineering: C, 2014. 34(0): p. 229-235.

31. Fuchigami, T., et al., Synthesis and biological evaluation of radioiodinated quinacrine-based derivatives for SPECT imaging of Abeta plaques. Eur J Med Chem, 2013. 60: p. 469-78.

32. Wu, X., et al., Quinacrine Inhibits Cell Growth and Induces Apoptosis in Human Gastric Cancer Cell Line SGC-7901. Current Therapeutic Research, 2012. 73(1–2): p. 52-64.

33. Zhang, L., et al., Mitochondrial targeting liposomes incorporating daunorubicin and quinacrine for treatment of relapsed breast cancer arising from cancer stem cells. Biomaterials, 2012. 33(2): p. 565-82.

34. Wu, Y., Preparation of low-molecular-weight hyaluronic acid by ozone treatment. Carbohydrate Polymers, 2012. 89(2): p. 709-712.

35. Liu, C., et al., Synthesis and characterization of a thermosensitive hydrogel based on biodegradable amphiphilic PCL-Pluronic (L35)-PCL block copolymers. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2007. 302(1–3): p. 430-438.

36. Zhao, C.L., et al., Fluorescence probe techniques used to study micelle formation in water-soluble block copolymers. Langmuir, 1990. 6(2): p. 514-516.

37. Jae-Woon Nah, Young-Il Jeong, and C. Chong-Su, Polymeric Micelle Formation of Multiblock Copolymer Composed of Poly(r-benzyl L-glutamate) and Poly(ethylene oxide). Bulletin of Korean Chemical Society, 2000. 21(no. 4): p. 383.

38. Lee, H., C.H. Ahn, and T.G. Park, Poly[lactic-co-(glycolic acid)]-grafted hyaluronic acid copolymer micelle nanoparticles for target-specific delivery of doxorubicin. Macromol Biosci, 2009. 9(4): p. 336-42.

39. Teng T.M., et al.,”Development of a novel HA-based micelle material as a potent nanocarrier for targeting delivery”, Polymers at the Interface with Biology: Opportunities in Antimicrobial Materials, Immunology, Delivery, and Imaging - EVE Session,no.192, 244th ACS National Meeting, Sheration Philadelphia City, PA, U.S.A., 21 Aug. 2012