聚(氮-異丙基丙烯醯胺)/聚乙烯醇衍生酸鹼/磁/熱敏感型奈米藥物載體於刺激藥物釋放之研究

Abstract

本研究的目的旨在設計以及製備以聚(氮-異丙基丙烯醯胺)，Poly(N-isopropylacrylamide) 或聚乙烯醇，Poly(vinyl alcohol)為主體之衍生酸鹼/磁/熱感應性高分子及其複合材料，並探討此複合材料作為腫瘤組織的醫療平台之適用性，以及是否能夠提供新穎及有效的診斷和治療。 本論文主文共分為三大部份。第一部份以可逆性分裂加成鏈轉移活性聚合法合成(Reversible Addition-Fragmentation chain Transfer polymerization, RAFT polymerization) 聚(氮-異丙基丙烯醯胺)以及聚(異丙基丙烯醯胺-丙烯酸)之嵌段共聚合物。第二部份將以第一部分所合成之嵌段共聚合物為主體，製備多功能性藥物載體，並透過體外及生物試驗獲得其生物醫學之性質。第三部份引入多重乳化法(double-emulsion)並以聚乙烯醇(Polyvinyl alcohol, PVA)為主體，製備具磁性之核殼型(core-shell)藥物載體。 第一部份挑選(S,S'-bis(a,a'-dimethyl-a'-acetic acid)trithiocarbonate, CMP)做為可逆性分裂加成鏈轉移活性聚合法(RAFT polymerization)之鏈轉移劑(chain transfer agent, CTA)。引入熱裂解型起始劑(azobisisobutyronitrile, AIBN)以及使用有機溶劑(methanol)於70度下反應後，可以製備出ABA三嵌段型高分子(poly(acrylic acid-block-N-isopropylacrylamide-block-acrylica acid), poly(AA-b-NIPAAm-b-AA)並能將準確的控制嵌段型高分子鏈段上NIPAAm與AA的鏈段比例。藉由改變不同的高分子鏈段比例、溶液酸鹼度、操作溫度、濃度等，觀察對於高分子自組裝的型態以及其物理性質之影響。 第二部份包含第三及第四章。第三章引入經表面改質過之磁性氧化鐵，結合自組裝後之高分子水膠，製備出具磁性控制、酸鹼、溫度感應型之藥物載體，並探討其藥物釋放之行為。第四章則是延續第三章之研究，將具備癌症細胞辨識性之標靶分子—葉酸(Folic acid)接枝於磁性藥物載體表面，同時也引入疏水型抗癌藥物—薑黃素(Curcumin)，製備出同時具有遠端操作(磁性控制)、溫度感應、標靶治療之藥物載體，並探討此藥物載體對於人體乳癌細胞株(MCF-7 cell line)之細胞選擇性以及毒殺效果。 第三部份則以第六章為主。以聚乙烯醇為主體，利用多重乳化法備製核殼型磁性藥物載體。實驗結果顯示此藥物載體對於抗癌藥物承載具有相當大之相容性，無論親、疏水型小分子藥物，胜肽型(peptide)巨分子藥物都能被穩定的包覆。接枝標靶分子(atherosclerotic plaque-specific peptide-1, AP-1)後，此藥物載體在細胞毒性測試中，也充分表現出對於小鼠直腸大腸癌細胞株(Mouse colon carcinoma cell line, CT26)的辨識能力。隨後進行動物實驗，對於刻意誘發之小鼠腫瘤組織進行磁性藥物載體之直接注射治療，發現在高週波磁場作用下不僅能夠抑制腫瘤組織的生長，磁性粒子所產生之高熱也同時能提供對腫瘤組織的熱治療。 附錄第一部分中藉由控制特定的溫度以及酸鹼值，將已自組裝後的遠嵌型高分子核殼奈米顆粒應用於藥物控制釋放系統。引入具有螢光特性之小分子(Rhodamine 6G, R6G)於此藥物載體之表面及內部後，探討在不同的環境及濃度下此螢光標籤型藥物載體之控制釋放行為。第二部份則收錄了利用脂類自組裝而形成的奈米圓盤，在引入具有酸降解性的雙胺(acid-degradable diamine)後，則可以製備出對癌症細胞感應型的藥物載體，並對此載體的物理、化學性質進行探討。至於附錄的第三部分則是使用高溫乳化法在以聚(氮-異丙基丙烯醯胺)為基質的水膠中引入磁性氧化鐵顆粒，並利用無電鍍法製備具有催化效果之鉑奈米粒子，未來此磁性催化複合材料應用時，便可以利用磁性來進行複材之控制及回收。

In this research, Poly(vinyl alcohol)- and Poly(N-isopropylacrylamide) based copolymers and their pharmatical composites were prepared as the medical platform with pH/magnetic/thermo properties for providing the novel and effective diagnosis and treatment toward tumor tissues. Three divided sections were contained in this thesis. In the first section, reversible addition-fragmentation chain transfer (RAFT) polymerization was performed and applied to synthesize ABA tri-block copolymers via living polymerization. In the second section, we fabricated several kinds of multi-functional nanomedicines as the extended employments of the as-synthesized block copolymers. Besides, their biomedical properties of nanomedicines were also obtained with in vitro and in vivo tests. In the third section, double-emulsion method was conducted for core-shell, PVA-based nanoparticles as well as the anti-cancer drugs’ encapsulation. Additionally, acid-cleavable, nanodiscs-based nanoscaffold was designed and prepared via EDC/NHS initiated crosslinking between the synthesized acid-degradable diamines and PEGylated nanodiscs. Platinum and Fe3O4 core-shell nanoparticles were designed and constructed by temperature-induced emulsion of the poly(N-isopropylacrylamide) following with electroless plating methods. The results were both shown in the appendix section. Chapter 2 was contained in the first section and RAFT polymerization with S,S'-bis(a,a'-dimethyl-a'-acetic acid)trithiocarbonate (CMP) as the chain transfer agent (CTA) were demonstrated. By using the thermal initiation following with the RAFT polymerization in methanol, ABA tri-block copolymers, poly(acrylic acid-block-N-isopropylacrylamide-block-acrylic acid), poly(AA-b-NIPAAm-b-AA) with precise control of the main chain composition to the repeating monomer units were prepared. Moreover, further discussions were studied toward the morphology and physical property changes resulting from the different composition ratio of NIPAAm to AA ratio and the system conditions involving pH values and temperatures. Various shapes of poly(AA-b-NIPAAm-b-AA)-based, self-assembly nanoparticles including core-shell nanoparticle, physical crosslinked nanogel, and inversed physically crosslinked nanogel formed at specific conditions. To the best of our knowledge, this was the first research that focus on the relationship between chemical structure and physical properties of poly(AA-b-NIPAAm-b-AA) tri-block copolymers systemically. The second section includes Chapter 3 and Chapter 4, in which different nanocarriers were fabricated and studied for bio-medical applications. In Chapter 3, physically crosslinked nanogels were employed into the preparation of anti-cancer nanomedicine. Magnetic iron oxide (Fe3O4) was introduced to achieve the remote control as well as the stability of magnetic nanocarriers. Furthermore, the intelligent functionality of nanocarriers was realized resulting from highly pH- and magnetically- sensitive quality because of the iron oxides and the functional groups (-COOH and -NH2) on its surface. Chapter 4 contains the following work related of Chapter 3, in which we further modified the magnetic nanocarriers with folic acid to gain the cancer cell-selectivity and replaced the model drug into the hydrophobic one, curcumin. Various release and pharmatical parameters such as biocompatibility, cytotoxicity, cell selectivity, and release behaviors were studied. Chapter 5 was contained in the third section where we paid more attention to develop another way of nanocarriers’ preparation including double-emulsion, sol-gel reaction, and following with targeting ligand immobilization. Both in vitro and in vivo experiments were brought out to give a full scope of pharmatical applications of the magnetic nanocarriers toward tumor therapy. In Appendix 1, we prepared the thermo- and pH-sensitive, fluorescent-labeled nanocarriers with two kinds of drug-loading process. Rhodamine 6G, a fluorescent dye, was used as the modeling drugs not only to demonstrate the release behavior but also for maintaining the spherical structure of self-assembled telechelic HOOC-PNIPAAm-COOH homopolymers. In Appendix 2, PEGylated lipids were mixed with long-chain and short-chain lipids to form carboxylic acids-exposed nanodiscs (a-discs). An acid-degradable diamine was synthesized and applied for stringing the nanodisc in order to enlarge their size for preventing the un-desired cellular uptake and providing controlled release as an anti-cancer platform. Attentions were concentrated on acquiring a stable, well-dispersed Fe3O4 and platinum composite for the purpose of reusable and recyclable catalysis in Appendix 3. Emulsion as well as the electroless plating synthesis with poly(N-isopropylacrylamide) as the matrix were utilized.