本研究使用兩種不同功能之雙親性嵌段共聚高分子(amphiphilic block copolymer) ，藥物接枝高分子DOX-hyd-PCL-SS-b-P(TEGMA-co-FA)以及螢光高分子PCL-b-(TEGMA-co-PPSEMA)於水溶液中自組裝形成具三重刺激響應性與螢光追蹤功能之高分子混合微胞系統，並針對其藥物釋放、細胞毒性以及生物螢光影像追蹤功能進行檢測與探討。
微胞組成包含疏水性核心poly(ε-caprolactone) (PCL)，並於PCL末端以酸鹼響應性的腙鍵(hydrazone, hyd)與疏水性藥物doxorubicin (DOX)鍵結以乘載藥物，親水性外殼則使用具溫度響應性之單體triethylene glycol methacrylate (TEGMA)，親疏水鏈段間之雙硫鍵(disulfide, SS)具有氧化還原響應性，聚合完成後再於親水鏈段上修飾具有酸鹼響應性以及主動標靶功能的葉酸(folic acid, FA)，最後形成藥物接枝高分子DOX-hyd-PCL-b-SS-P(TEGMA-co-FA)。另外將具有聚集誘發螢光(aggregation-induced emission, AIE)性質之螢光單體2-(1,2,3,4,5-pentaphenyl-1H-silol-yloxy) ethyl methacrylate) (PPSEMA)修飾於另一不具雙硫鍵之雙親性共聚高分子的親水鏈段上以得到PCL-b-P(TEGMA-co-PPSEMA)。
本研究藉由調整TEGMA、FA之聚合度，以及兩高分子DOX-hyd-PCL-b-SS-P(TEGMA-co-FA)、PCL-b-P(TEGMA-co-PPSEMA)於混合微胞中之比例，成功使此系統之最低臨界溶解溫度(lower critical solution temperature, LCST)符合於酸性環境低於體溫37 °C，於中性環境高於37 °C的條件，分別對應人體腫瘤細胞中溶酶體與血液之環境，以達到於血液中穩定乘載藥物，而於腫瘤細胞中因其親水外殼之對水溶解能力改變以及腙鍵斷鍵而迅速釋放藥物之目的；於親水外殼修飾FA使此奈米藥物載體可與腫瘤細胞上FA受體結合，促進胞吞作用以達到主動標靶的效果；連結親疏水鏈段之雙硫鍵具有氧化還原響應性，於腫瘤細胞的細胞質環境中有較高濃度還原劑榖光甘肽(GSH)進而促使其斷鍵，並造成微胞結構崩解； PPSEMA之螢光光譜與DOX吸收光譜有足夠重疊(overlap)，且當兩分子足夠接近時會發生螢光共振能量轉移(Förster Resonance Energy Transfer, FRET)現象，本研究即以FRET現象做為距離探針觀察藥物乘載與藥物於HeLa細胞之釋放情形。

In this research, we investigated the drug release, cytotoxicity and fluorescence bioimaging application to HeLa cells of the polymeric mixed micelle system. The triple stimuli-responsive mixed micelle system was co-assembled from two kinds of amphiphilic block copolymers, drug-conjugated polymer DOX-hyd-PCL-SS-b-P(TEGMA-co-FA) and PCL-b-P(TEGMA-co-PPSEMA) with fluorescent moieties.
In this micelle system, poly(ε-caprolactone) (PCL) formed the hydrophobic core of polymeric micelle. The hydrophobic anti-cancer drug doxorubicin (DOX) was connected with a pH-responsive linker hydrazone bond (hyd) to the chain end of PCL so as to load the drug with valent linkage stable in neutral environment but liable in acidic condition. Triethylene glycol methacrylate (TEGMA) was the thermo-responsive monomer and formed the hydrophilic shell of the micelle. The hydrophobic block (PCL) was connected with hydrophilic block (TEGMA) by the redox-responsive disulfide bond (SS). We also introduced the pH-responsive and targeting ligand folic acid (FA) to the hydrophilic block of copolymer, and finally formed the drug-conjugated polymer DOX-hyd-PCL-SS-b-P(TEGMA-co-FA). Besides, we introduced the monomer, 2-(1,2,3,4,5-pentaphenyl-1H-silol-yloxy) ethyl methacrylate) (PPSEMA) with aggregation-induced emission (AIE) characteristic as the fluorescent monomer, and were connected with another amphiphilic block copolymer to form PCL-b-P(TEGMA-co-PPSEMA).
To realize the desired lower critical solution temperature (LCST) of the mixed micelle system, we adjusted the degree of polymerization of TEGMA and FA in block copolymers and the mixing ratio of DOX-hyd-PCL-SS-b-P(TEGMA-co-FA) to PCL-b-P(TEGMA-co-PPSEMA) in mixed micelles. The LCST should be higher than 37 °C when in neutral environment but lower than 37 °C in acidic environment, which is corresponding to respectively human's blood and lysosomes of tumor cell environment, so the micelles can carry the drug in blood circulation stably and release drug in lysosomes. Since FA can be combined with the folate receptor which is overexpressed on the surfaces of certain types of tumor cells, the micelles can be effectively internalized by tumor cells via receptor-mediated endocytosis, and reach the goal of active targeting. Because of the higher concentration of glutathione in tumor cell cytoplasm than normal cell, the disulfide bond can be cleaved into thiol under the reductive environment, and cause the collapse of micelle structure. Because of the overlap of PPSEMA fluorescent and DOX absorption spectrum, Förster Resonance Energy Transfer (FRET) would occur if the donor (PPSEMA) was closed enough to the acceptor (DOX). In this research, the FRET phenomenon could be used as a distance probe to observe whether the drug is carried or released in HeLa cells.

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