2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/652326摘要：本計畫目的旨在開發研究一具有CD44 受體主動標的之微膠奈米遞輸載體。兩性高分子聚合物PLGA-PEG 所製備之微膠粒是目前倍受重視的奈米劑型，根據研究報告已知PLGA-PEG奈米劑型具有延長藥物在體內血液循環的時間及增加藥物聚集在腫瘤細胞之特性。CD44 受體廣泛表現在腫瘤細胞表面(例如乳癌及多形性膠質母細胞腦瘤等)；玻尿酸(hyaluronic acid;HA)和硫酸軟骨素(chondroitin sulfate; CS)為多醣類聚合物，已被證實與CD44 受體具有專一結合之特性，推測二者可能有作為主動標的配體之潛力，且從預試驗結果初步確認其可行性，但目前同時研究此兩種多醣類聚合物應用於微膠粒主動遞輸載體領域之相關研究有限。因此本計畫結合微膠粒奈米劑型和具有標的功能之多醣類配體所設計出的主動標的奈米遞輸載體，應有值得被開發之潛在意義。本研究計畫規劃以腫瘤細胞表面的CD44 作為奈米遞輸載體主動標的之受體，利用生物可分解之兩性高分子材料PLGA-PEG 所製備之微膠粒奈米載體，結合對腫瘤細胞表面CD44 受體具有專一結合的兩種多醣類聚合物，以期達到將DNA 主動標的至腫瘤細胞之目的。本研究規劃為三年期計畫，主要研究內容包括：(1)建立化學鍵結兩類多醣類配體與兩性高分子聚合物的合成條件、方法與步驟。結合的多醣類配體包括玻尿酸和硫酸軟骨素兩種，其中並選用高低兩種分子量的玻尿酸，以探討多醣類配體之種類及分子量對後續應用於遞送DNA 之奈米遞輸載體的影響；另外也採用不同的合成策略將PLGA-PEG 分別從多醣結構之尾端或側端，以不同化學鍵與之共價鍵結，藉由兩性高分子聚合物構型之改變探討其可能的影響。(2)將合成的多種多醣兩性高分子聚合物進行組成及物性確認；並定量其臨界微膠粒濃度、多醣類配體接枝率、及接枝後分子量；進而分析其對細胞之毒性。(3)探討所開發合成之多種多醣兩性高分子聚合物作為遞輸DNA 至CD44 受體過度表現癌細胞之潛力。針對包載DNA 之微膠粒奈米遞輸載體進行相關研究，包括：DNA包載率、DNA/微膠粒組成、生物相容性、安定性、體外釋出試驗、細胞毒性試驗、細胞基因遞送效率分析、及載體遞送基因之胞飲機制探討等實驗。以期藉由此三年期研究計畫，解析上述多個因子對此主動標的微膠粒奈米遞輸載體於遞送基因至腫瘤細胞的各方面影響，並達成開發一具有主動標的腫瘤CD44 受體之微膠粒奈米遞輸載體供基因遞送至腫瘤細胞之目的。<br> Abstract: The aim of this study is to develop polysaccharide-conjugated PLGA-PEG micelles possessingCD44 receptor targeting ability for gene delivery. The CD44 receptor is selected as the targetbinding receptor which is overexpressed in many kinds of tumors including human breast cancercells and glioblastoma cells etc. Two linear polysaccharides, hyaluronic acid (HA) and chondroitinsulfate (CD) are included in this study as CD44 receptor targeting moieties. Hyaluronic acid andchondroitin sulfate are endogenous polysaccharides existed in the extracellular matrix. It wasproved that both polysaccharides possess specific binding ability to CD44 receptor. It implies thatboth polysaccharides could be the potential candidates as CD44 receptor targeting moiety whichhas been confirmed from our preliminary study. However, the studies of polysaccharides as thetargeting moiety of nanocarriers for gene delivery are limited. This study is designed as athree-year project. There are several main issues planned to investigate and explore in this study:(1) The methods to synthesize polysaccharide-conjugated amphiphilic copolymers will bedeveloped based on the type (e.g., HA & CD), the molecular weight (e.g., low & high), and theconjugation site (e.g., linear type & branched type) of polysaccharides. (2) The synthesizedpolysaccharide-conjugated copolymers will be confirmed and characterized. Their polysaccharideconjugation ratio, the critical micelle concentration, and the corresponding molecular weight aredetermined. The cytotoxicity is performed to assure the safety of these synthesized copolymers. (3)The polysaccharide-conjugated copolymers will be further applied as nanocarriers for DNAdelivery. The effects of types of polysaccharide-conjugated micelles on biocompatibility, stability,in vitro release, cytotoxicity and cellular transfection are investigated. Finally, the endocytosismechanisms of polysaccharide-conjugated PLGA-PEG micelles are investigated. Through thisstudy, the polysaccharide-conjugated micelles with CD44 receptor targeting character for genedelivery will be elucidated.微膠粒CD44 受體多醣配體DNA細胞轉染micellespolysaccharideCD44 receptorDNAtransfection