酸鹼與溫度應答複合型奈米微胞之研發及其在細胞內藥物傳輸之應用

隨著科技的進步，奈米技術的觸角已延伸到各個領域，尤其在生醫與生技的潛力更凸顯其重要性。近幾年發展中，奈米技術已從單一組成奈米微胞發展至複合型奈米微胞，揉合了各組成奈米微胞之優勢於一身，因此倍受矚目可說是日後奈米技術發展新星。本研究主要以開發新系統、組成及製程製備複合型奈米微胞為主軸，首先成功將兩性質迥異高分子，一為具有臨界微胞濃度 (CMC) 之雙性團聯共聚物mPEG-b-PLA，另一則為具有臨界微胞溫度 (CMT) 之溫度/酸鹼應答型團聯共聚物mPEG-b-P(NnPAAm-co-VIm)，以熱衝擊程序 (Hot shock protocol) 使其自我組裝 (self-assembly) 形成具有核殼結構 (core shell structure) 且微胞粒徑較小 (70~100nm) 及分佈均一 (PI＜0.2) 之複合型奈米微胞。兩團聯共聚物在複合型奈米微胞中各扮演了舉足輕重的角色。mPEG-b-PLA使mPEG-b-P(NnPAAm-co-VIm)的結構更加穩定，不會因為溫度的下降而使結構迅速瓦解；此外，藉由環境酸鹼值的改變使mPEG-b-P(NnPAAm-co-VIm)在人類體內溫度37℃即可讓結構完全破壞，同時在短時間內瓦解mPEG-b-PLA結構，改善其在體內降解緩慢之缺點。為了取得最佳複合型奈米微胞組成比例，本研究以動態光散射粒徑分析儀量測混合後之微胞粒徑大小與分佈，並以螢光光譜儀量測螢光染劑pyrene發散波長強度I1/I3的變化，觀察在不同酸鹼值環境下結構破壞的情形，最後將複合型奈米微胞溶於含有4wt% BSA緩衝溶液，模擬複合型奈米微胞於體內循環之穩定性。本研究係以製備出的複合型奈米微胞包覆疏水性抗癌藥物－Doxorubicin做為細胞內藥物釋放之用。以靜脈注射方式使複合型奈米微胞載體進入體內，經由EPR效應 (Enhanced Permeability and Retention effect) 累積於腫瘤組織後，利用包飲作用 (endocytosis) 吞噬高分子載體進入細胞，因胞內酸性胞器的影響使得imidazole group被質子化 (protonation) 而改變PNnPAAm低溫臨界溶液溫度 (LCST) 達到結構破壞而釋放出藥物。本研究以熱衝擊程序所製備之複合型奈米藥物微胞，其藥物包覆率均達30%，於體外模擬藥物釋放當所處環境pH5.0時其藥物釋放率可達60%以上，且處於環境pH7.0時因為藥物突釋 (initial burst) 的關係藥物釋放率保持在10%左右。此外，將包覆藥物之奈米微胞與人類子宮頸癌HeLa細胞共同培養72小時後可發現在高濃度下 (100μg/mL) 與未包覆之藥物具相同細胞毒殺性。並由共軛焦顯微鏡可觀察到藥物在細胞內分佈與釋放之行為乃經由包飲作用後進入細胞，使藥物累積於細胞質中而後進入細胞核。綜合以上所述，可知複合型奈米微胞兼具環境應答、生物可分解與低毒性等優點，能於體內循環時保持穩定的結構而在胞內因酸性胞器釋放出藥物達到適時、適時之控釋效果，因此在藥物傳輸上極具開發之潛力。

關鍵字

臨界微胞濃度；臨界微胞溫度；熱衝擊程序；自我組裝；核殼結構； EPR效應；包飲作用；細胞內藥物釋放

並列摘要

A novel mixed micelles composed of methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-b-PLA) and methoxy poly(ethylene glycol)-b-poly(N-n-propylacrylamide-co-vinylimidazole) (mPEG-b-P(Nn- PAAm-co-VIm)) diblock copolymers, having critical micelle concentration (CMC) and critical micelle temperature (CMT) properties, were successful prepared in this study. Such nanostructures have well biocompatibility and thermal/pH sensitivity owing to their component natures. Based on these advantages, mixed micelles had a candidate as drug carriers for cancer therapy. The thermal/pH responsive properties of mixed micelles can be measured by UV/vis spectrometer at 542 nm. The lower critical solution temperature (LCST) occurred at 21.5℃~35℃ and gradually increased with reducing environment pH, and therefore we utilized its special behavior to prepare well-mixed micelles that exhibited a uniform size about 75~100nm and narrow polydispersity about 0.1~0.2 under 37℃ buffer solution via hot-shock protocol. Furthermore, the use of pyrene as probe for fluorescence spectroscopic measurement could observe that the I1/I3 decreased with raising surrounding temperature, indicating that the probe diffused from aqueous phase to the core of mixed micelles. The characteristics and morphologies of mixed micelles were analyzed from 1H-NMR, DLS, zeta potential, fluorescence spectrometer, AFM, and TEM. Additionally, doxorubicin (Dox) was incorporated into mixed micelles for cancer therapy. In neutral surroundings, the release of Dox from mixed micelles was less. In contrast, a significant release of Dox was observed in acidic surroundings about pH＜6. The amount of drug released from mix micelles was isolated from mixed micelles buffer solution by ultrafiltration and measured by UV/vis spectrometer at 485 nm in a time-course procedure. The result of CLSM observation indicated the loading drug successfully released in the acidic organelles due to the deformation of the micelle structure. Above all, the release of drug from mixed micelles could be strongly controlled by pH changes. From these results, the novel mixed micelles showed high potential for drug carriers in intracellular drug delivery.

並列關鍵字

CMC ； CMT ； LCST ； Intracellular drug delivery ； mPEG-b-P(NnPAAm-co-VIm)

參考文獻

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酸鹼與溫度應答複合型奈米微胞之研發及其在細胞內藥物傳輸之應用

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