酸鹼開關複合型奈米藥物傳輸系統之研發及其在癌症治療上之應用

Abstract

本研究係利用一具有酸鹼應答行為、生物相容性及生物可降解性之Poly(D,L-lactide)-g-Poly(N-vinylimidazole-co-N-vinyl-2-pyrrolidone)(PLA-g-P(NVI-co-NVP))接枝共聚物以及具有免疫隱蔽性及生物可降解性之methoxy poly(ethylene glycol)-b-poly(D,L-lactide)(mPEG-PLA)二團聯共聚物，利用自我組裝設計出一同時具有環境酸鹼應答行為、可逆開關行為及免疫隱蔽性之接枝/複合型奈米微胞。mPEG可隱蔽內核結構的強正電性與疏水特性進而增加於體內循環時之穩定性與細胞吞噬量；NVI可藉由其酸鹼應答行為，待藥物載體被細胞吞噬後，可因正電排斥力造成結構的膨潤進而釋放藥物;PLA則具有包覆輸水抗癌藥物的能力。

研究中我們首先探討接枝型奈米微胞之形成機制並得到製備時的最佳化條件，而後我們依PLA-g-P(NVI-co-NVP)與mPEG-PLA臨界微胞濃度之不同，分別以不同重量比製備出各種組合之複合型奈米微胞，並從中篩選出較佳的組成進一步探討NVI本身之酸鹼應答行為對微胞型態之影響，結果指出接枝/複合型奈米微胞皆有良好的On-Off酸鹼應答行為。除此之外，我們更利用TEM與AFM等電子顯微鏡証實奈米微胞於不同環境下之核殼結構與表面型態變化。

藥物載體相關研究方面，我們將抗癌藥物doxorubicin包覆於PLA疏水內核，並利用免疫隱蔽性之外殼與具有酸鹼應答之特性進行「適時」、「適地」之藥物控制釋放。其中我們探討接枝/複合型藥物微胞之藥物包覆最適化條件(藥物包覆率高達約40%)，並觀察藥物載體於不同酸鹼環境下之On-Off藥物釋放情形，發現於pH5.0可得到快速且穩定的藥物釋放曲線；而於pH7.4時卻可將藥物確實地包覆於疏水內核。進一步地我們將藥物載體分別與Hs68、HeLa、HepG2等正常/癌細胞共同培養，結果指出於IC50附近，材料不具有任何毒性可言。最後，我們以共軛焦電子顯微鏡證實藥物載體於細胞酸性胞器內進行藥物制放，而後藉由擴散作用至細胞核內將細胞毒殺。

A novel mixed micelle comprised of poly(D,L-lactide)-g-poly(N-vinylimidazole-co-N-vinyl-2-pyrrolidone) (PLA-g-P(NVI-co-NVP)) graft copolymer with methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-PLA) diblock copolymer was successfully developed for application in cancer therapy. The mixed micelle had an biocompatibility, biodegradab, pH-triggered inner core of P(NVI-co-NVP)-g-PLA to enable intracellular drug delivery and an extended hydrophilic outer shell of mPEG to hide the inner core. PNVI in backbone of graft copolymer exhibited pH-triggered property, when pH<6.0, the nanoparticle swelled (about 50%) depend on composition of NVI in backbone but not deformed its conformation. Otherwise, the conformation went back to initial state when pH>6.0, if micelle capsulated hydrophobic anticancer drug, this phenomenon would be called as “On-Off” controlled drug release.

In this study, we investigated the effect of mixed micelle with different critical micellar concentration (CMC) of diblock copolymer on comicellization. The results indicated that the CMC of diblock copolymer decreasing, the stability of mixed micelles increasing. Furthermore, the average size and polydipersity index(PI) of graft/mixed micelle can be measure by dynamic light scattering with the sample in phosphate buffer saline (PBS) at pH 7.4, it exhibited uniform size(about 70~100 nm) and narrow distribution(about 0.8~1.2).Besides, the core-shell structure of graft/mixed micelle at pH5.0 or pH7.4 can be prove by transmission electron microscopy (TEM).

The hydrophobic doxorubicin(DOX) was capsulated into the inner core of graft/mixed micelle by hydrophobic segment PLA for application in cancer therapy, and the capsulated efficiency can be as high as approximately 40 wt%. Graft/mixed micelle both exhibited high releasing rate in the initial 24 hr and the releasing behavior remained constant after 168 hr in the acidic surroundings(pH 5.0).Furthermore, there were rare initial burst releasing of graft/mixed micelle in neutral surroundings(pH 7.4). It means that the “On-Off” controlled drug release had successfully developed by altered pH value. In addition to above study, the efficiency of screening feature of mixed micelle can be distinguished from graft micelle in BSA/PBS stabe test and cytotoxicity, it means that mixed micelle exhibited better drug activity and lower material cytotoxitity. Finally, the free DOX and DOX-graft/mixed micelle distribution in cancer cell can be easily confirmed by confocal laser scanning microscopy(CLSM).