製備應用於藥物釋放之新穎玻尿酸奈米粒

本研究發展製備新穎玻尿酸奈米粒子之藥物傳遞系統，提高玻尿酸奈米粒子在人體內的循環時間，並使抗癌藥物能集中在癌細胞周圍且有效的釋放藥物，增加癌細胞毒殺效率，並降低體內化療藥物毒性之影響。癌細胞因具有藥物抗性(multidrug resistance)，若能將高濃度藥物同時導入癌細胞，即可能增加毒殺效果。在本研究即設計玻尿酸奈米粒子使其能於癌細胞周圍自組裝以形成高藥物濃度。藉由癌細胞表面玻尿酸接受器CD44 之作用，將自組裝完畢攜帶高化療藥物濃度之玻尿酸奈米粒子胞飲(endocytosis)入細胞內，促成增強毒殺的效果。奈米粒子的自組裝是藉由biotin/neutravidin 表面修飾於玻尿酸奈米粒子而達成。此外將玻尿酸奈米粒子表面同時修飾polyethylene glacol (PEG)以減少玻尿酸奈米粒子與一般正常細胞表面CD44 作用及奈米粒子間非特異性(non-specific)的互相結合。實驗方法為酸降解高分子量(1,200 kDa)玻尿酸製備成不同分子量之玻尿酸，再將ADH (adipic acid dihydrazide)與分子量(25 kDa)之聚乳酸(polylactic acid,PLA) 鍵結。接續再將玻尿酸與PLA-ADH 聚合成共聚物(copolymer,HA-ADH-PLA)後，包覆螢光染劑或化療藥物(doxorubicin Dox)製備成奈米粒子，最後再進行表面修飾，以利於形成自聚集現象。在實驗結果中，將HA-ADH-PLA 包覆螢光染料之奈米粒子於癌細胞靶向測試。螢光顯微鏡下觀測，並用流式細胞儀定量分析下，MDA-MB-231(CD44+)與A549(CD44+)細胞株具有較佳之吞噬奈米粒子能力，而ZR-75-1(CD44-)細胞株則吞噬能力較弱。另外在肝星狀細胞中也發現到活化之星狀細胞會比未活化的具有較佳之奈米吞噬能力。因此本實驗結果有助於研發以玻尿酸受體為標靶導向之奈米粒子藥物(Doxorubicin, DOX)傳遞系統，以降低藥物毒性與副作用即可達到殺死癌細胞的目的。

關鍵字

玻尿酸；聚乳酸；奈米粒子；癌細胞； CD44

並列摘要

The objective of this study is to prepare self-assembled nanoparticles by using hyaluronic acid (HA), adipic acid dihydrazide (ADH), and polylactic acid (PLA) to carry anticancer drugs as drug delivery system. The preparation of HA-ADH-PLA nanoparticles (NP) employed the hydrophilic property of HA and the hydrophobicicity of PLA. The inward amphiphilic sides (PLA) of HA-ADH-PLA-NPs were able to load hydrophobic anticancer drugs. The outward hydrophilic side (HA) was able to recognize cancer cells by binding to the highly expressed cell surface marker, CD44. Additionally, the surfaces of these particles were modified with either biotin/metallo-matrixprotease substrate-polyethylene glycol (B/MMP-PEG, BNP) or neutravidin/metallo-matrix protease substratepolyethylene glycol (N/MMP-PEG, NNP) to enhance the specificity to cancer cells. The long carbon chain of PEG likely prevented HA-PLA-NP from self-assembling and the non-specific binding during circulation. These two surface-modified particles (B/N NPs) proceeded self-assembling via the interaction of biotin and neutravidin after excising the MMP substrates by cancer cells released MMPs. The formation of B/N NP complexes could provide higher drugs concentrations at both extra- and intra-sides of cells to promote the cytotoxic efficacy. The B/N NP complexes might also possibly overcome the cancer cell drug resistance, such as cancer stem cells, to further eradicate tumors. NMR and FTIR spectrum results indicated that HA, ADH and PLA were properly conjugated. The cellular binding efficiency of B/N NPs was examined on MDA-MB-231 (CD44+), A549 (CD44+), and ZR-75-1 (CD44-) cells. Data suggested that B/N NPs entrapping fluorescent dye (coumarin 6) demonstrated stronger fluorescent intensity on CD44+ cells rather than on CD44- counterpart,suggesting the binding efficacy was positively related to the abundance of cell surface CD44. Moreover, the sizes and stability of B/N NPs, and the modification rate of biotin/neutravidin on NPs were studied. Taken together, our B/N NPs might enhance the cytotoxcity to cancer cells which could help to reduce the drug dose and the side effects commonly encountered. The self-assembling effect might also benefit the development of HA based drug delivery system.