Abstract
摘要:本計劃擬將可分解性之高分子 poly(D, L-lactides) (PLA) 與poly(D,L-lactide-co-glycolide)(PLGA) 顆粒遞送系統以自行組裝複合物表面修飾後,研究其抗原傳輸之免疫效應與機制。實驗部分將以雙乳劑法 (double emulsion method) 製備含ovalbumin (OVA) 之poly(D,L-lactides) (PLA)顆粒性遞送系統,並以各種不同分子量之高分子聚合物以layer-by-layer 之技術作表面修飾化包覆,再測其物理化學性質,包括粒子大小,界面電位,並以掃描式電子顯微術觀察。經界面修飾之PLA 顆粒,將先進行細胞毒性試驗。擬將小鼠巨噬細胞與樹突細胞以此顆粒傳輸系統處理培養後,以MTT 法測存細胞存活率,再以樹突細胞以此顆粒遞送系統處理後,檢視其對於共刺激分子,包括CD40, CD80 與CD86 之活化狀況。這些顆粒在遞送至細胞內後之行徑,將以螢光顯微鏡觀察之。同時擬以B3Z 細胞進行antigen presentationassay,並研究其所引起之免疫反應。有關 in vivo 方面之基因傳輸與免疫效應的實驗,將於接種EL4 或EG7-OVA 小鼠之腫瘤模式進行。擬將已經自行組裝表面修飾技術修飾含抗原之PLA 或PLGA 顆粒遞送系統打至六至八週之小鼠,使產生免疫效應,再測其T 細胞之proliferation 以及CD8+ T 細胞之細胞毒殺效果。同時擬以ELISA 檢測其所產生細胞激素等之humoral response,並將以 real timeRT-PCR 與流式細胞儀測其淋巴組織proinflammatory cytokines 之表現情況。此研究之實驗結果將顯示經自行組裝表面修飾過之可分解性顆粒性含高分子傳輸系統在抗原至抗原呈現細胞之影響與作用,以及此製備法對於活體內腫瘤細胞生長之抑制效果。
Abstract: This study attempts to examine the effect of surface modification by self-assembly techniqueon biodegradable polymeric delivery of vaccines. Poly(D,L-lactides) (PLA) andpoly(D,L-lactide-co-glycolide) (PLGA) microparticles will be prepared by the double-emulsion andsolvent evaporation method, using ovalbumin as the model antigen. The particles will beencapsulated with alternating layers of the polyelectrolytes, including protamine and dextran sulfateof various molecular weights. Physicochemical properties, including size and zeta potentials, willbe characterized, and examined by scanning electron microscopy.Viability assays will be performed to determine the effect of surface modification by thelayer-by-layer technique on the viability of the cells. Mouse macrophage cells and dendritic cellswill be treated with the surface-modified polymeric particles. Murine bone marrow-deriveddendritic cells will be incubated with nanoencapsulated polymeric microparticles for determinationof activation of costimulatory molecules, including CD40, CD80, and CD86. Intracellulartrafficking of polymeric particles will be examined by fluorescence miscroscopy. B3Z cells willbe employed for the antigen presentation assay and correlate with the physicochemical properties ofthe polymeric particulates.The in vivo studies on the effect of surface modified polymeric particles will be carried out inthe EL4 thymoma tumor models. Tumors will be induced in the adult 6-8-wk old C57BL/6 miceby inoculation with EL4 or EG7-OVA cells. Animals will then be inoculated with theantigen-containing polymeric particles, with or without surface modification, followed by T cellsproliferation and antigen-specific CD8+ cytotoxicity lymphocyte (CTL) assays. Stimulation of thecostimulatory molecules will be examined in the antigen presenting cells in the secondary lymphoidorgans. ELISA assays will be carried out to characterize the humoral response, and real timeRT-PCR will be employed to examine the expression of proinflammatory cytokines in thesecondary lymphoid organs after immunization with the surface-modified polymeric particles.Expression of several chemokine receptors will be examined by flow cytometry. Results obtainedin this study are expected to demonstrate the effect of surface modification of biodegradableparticulates by self-assembly on the delivery of antigens to the antigen-presenting cells, and thetherapeutic effect on the tumor growth.
Keyword(s)
高分子
自行組裝
傳輸polymers
self-assembly
delivery