具有CD44受體標靶潛力之玻尿酸基因載體的研究

Abstract

本實驗目的為發展具生物可降解性、無毒性及選擇性標靶癌細胞的基因治療載體，並以幾丁聚醣(chitosan, CS)與具CD44標靶專一性的玻尿酸(hyaluronic acid, HA)作為主要材料。奈米複合體的基因治療效果牽涉到許多因子，包含奈米複合體的製備方法、表面電荷、安定性及聚合物的分子量，本實驗將詳細討論上述這些因子所造成的影響。另外，藉由GE11(2R)胜肽為表皮生長因子受體配體(ligand)的特性，實驗中將合成低分子量玻尿酸-聚乙二醇二胺-GE11(2R) (LHA-PEG-G)接枝聚合物，發展同時具CD44及表皮生長因子受體(epidermal growth factor receptor, EGFR)之新穎性雙重標靶載體。 第一部分實驗為利用去聚電質複合反應(polyelectrolyte complexation)將幾丁聚醣與表現加強型綠色螢光蛋白質體(pEGFP-N1 plasmids)、玻尿酸形成奈米複合體(nanocomplex)，並分析其相關物性包括粒子大小、表面電荷、奈米複合體吸附能力、質體DNA的構型(conformation)及細胞轉染效果。 第二部分實驗為合成低分子量玻尿酸-聚乙二醇二胺-GE11(2R)(LHA-PEG-G)接枝聚合物，首先於低分子量玻尿酸(LHA)上修飾不同接枝率的聚乙二醇二胺(PEG diamine)後，再修飾具EGFR標靶之GE11(2R)胜肽，並以使用核磁共振儀(1H-NMR)、傅立葉轉換紅外線光譜儀(FTIR)、膠體滲透層析儀(GPC)及連續式多功能微孔盤偵測系統，確認聚乙二醇接枝率、結構、分子量及GE11(2R)胜肽接枝率。並測試低分子量玻尿酸-聚乙二醇二胺(LHA-PEG)、低分子量玻尿酸-聚乙二醇二胺-GE11(2R)胜肽(LHA-PEG-G)所製成的奈米複合體其物性、安定性、細胞毒性、細胞轉染效果及血液凝集試驗。 實驗結果顯示，使用不同製備方法(A和B)製備奈米複合體，其粒徑、表面電荷無明顯差異。且去乙醯去聚合幾丁聚醣(DADPCS)形成的奈米複合體粒徑明顯小於幾丁聚醣的粒徑。 在MDA-MB-231細胞，由MTT試驗可確定本實驗所使用的CS、DADPCS、LHA和HHA具備低毒性。另外，在許多癌細胞株(例: A549, MDA-MB-231, SKOV-3, MCF-7, HepG2細胞)，因DADPCS有較低的分子量，導致DADPCS奈米複合體的轉染效率會大於CS奈米複合體轉染效率。從HA競爭實驗中可間接證明HA奈米複合體藉由CD44受體介導胞攝路徑(CD44 receptor-mediated endocytosis pathway)進入細胞。 由於PEG具有排斥力與電性遮蔽作用，伴隨著PEG接枝率上升，LHA衍生物奈米複合體的粒徑會下降且表面電荷強度逐漸中和。在同時具CD44受體與EGF受體表現的MDA-MB-468和MDA-MB-231細胞轉染實驗中，在含血清中給藥的轉染效果會較無血清給藥的轉染效果高，可能與粒子的安定性及細胞功能(如: 正向調節CD44受體)上升有關。另外，也觀察到在含血清的環境中，低分子量玻尿酸-聚乙二醇-GE11(2R)胜肽(LHA-PEG-G)奈米複合體可提升低分子量玻尿酸(LHA)奈米複合體細胞轉染效率。大部分負電的奈米複合體可避免血球凝集的發生，但正電的奈米複合體則無法避免。結論，低毒性的LHA-PEG-G奈米複合體可作為具標靶CD44受體與EGF受體潛力的基因遞送系統。

The study aims at developing the biodegradable, nontoxic and selectively targeting cancer cell gene delivery system by using chitosan and with CD44 targeting hyaluronic acid as main polymers. The gene delivery efficiency of nanocomplex may be influenced by many factors including preparation method, surface charge, stability and molecular weights of polymers. All of these factors will be discussed in this study. Moreover, because GE11(2R) is acted as a ligand of epidermal growth factor receptor (EGFR), we further synthesized GE11(2R)-conjugated low molecular weight hyaluronic acid-poly(ethylene glycol) bis(amine) (LHA-PEG-G) with CD44 and EGFR dual targeting novel delivery system. In the first part of the study, the nanocomplex composed of chitosan, pEGFP-N1 plasmid and hyaluronic acid was prepared by polyelectrolyte complexation method. The particle size, surface charge as well as adsorption capacity of nanocomplex, conformation of plasmid, and cell transfection efficiency were investigated. In the second part of the study, the LHA-PEG-G was synthesized. The LHA was conjugated with the different PEG graft ratios followed by further modification of GE11(2R) peptide as an epidermal growth factor receptor targeting ligand. The graft ratio of PEG, the structure, the molecular weight and the graft ratio of GE11(2R) peptide were analyzed by using nuclear magnetic resonance (1H-NMR), fourier transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC) and fluorescence spectroscopy, respectively. The nanocomplex of pDNA and LHA-PEG or LHA-PEG-G was prepared and their physicochemical properties, stability, cytotoxity, agglutination, and transfection were determined. The results showed that the particle size and surface charge of nanocomplex did not have significant difference between preparation methods A and B. Besides, the size of DADPCS nanocomplex was smaller than that of CS nanocomplex. In MDA-MB-231 cell, the MTT assay confirmed that CS, DADPCS, LHA, HHA were non-toxic materials. Moreover, because of the lower molecular weight of DADPCS than CS, DADPCS nanocomplex transfection efficiency was higher than CS nanocomplex in many cancer cell lines (e.g., A549, MDA-MB-231, SKOV-3, MCF-7, HepG2 cell). The cellular uptake of HA nanocomplex by CD44 receptor-mediated pathway was indirectly proven by HA competition assay. Due to repulsive and electrical shielding characteristics of PEG, the size of LHA-derived nanocomplexs was decreased and surface charge was neutralized with increasing PEG graft raios.The transfection efficiency of nanocomplex in CD44 and EGF receptor expression cells (e.g., MDA-MB-231 and MDA-MB-468) could be enhanced with FBS due to the increases of particle stability and cell function like up-regulated CD44 receptor. Furthermore, GE11(2R) conjugated LHA-PEG-G nanocomplex had higher transfection efficity than LHA nanocomplex in serum condition. All of the negative charge nanocomplexes can prevent red blood cell aggregation, but the positive charge nanocomplexes not. In conclusion, LHA-PEG-G nanocomplex with low toxicity can serve as a gene delivery system which possessed a specific targeting potential to CD44 and EGF receptors.