研發高血液穩定性酸鹼應答複合型奈米微胞及其在肺癌治療上之應用

Long Blood Circulation pH Responsive Mixed Micelle Developement and Application for Lung Cancer

張長榮

指導教授 ： 薛敬和

國立清華大學/工學院/化學工程學系/碩士(2010年)

摘要

摘要
近年來高分子微胞藥物載體在藥理上的開發與應淤備受期待，如何能使微胞更穩定於體內循環是現今重要課題之一。
為了將低高分子的臨界微胞濃度，以提升其在人體內穩定度，並且讓Enchanced Permeability and Retention Effect發揮最大功效，本研究設計與合成具有酸鹼應答、生物相容性及生物可降解之接枝共聚物(P(His-co-HPMA-co-HPMA-Lac2)-g-PLA)，以及具有免疫隱蔽性及生物可降解性之二團聯共聚物mPEG-PLA，共同製備具有高血液穩定性之複合型奈米微胞，以作為抗癌藥物Doxorubicin之藥物載體，並進而探討此劑型於肺癌治療之效用。
研究首先合成一系列不同比例(L0~L4)及(S0~S4)之接枝共聚物，並探討不同組成重量比下接枝共聚物/雙團聯共聚物對於複合型微胞粒徑及分佈之影響，結果顯示當接枝共聚物(L3)/雙團聯共聚物(B)重量比例為1:2時，以有機溶劑DMSO包覆藥物Doxorubicin，初期含水量為20%時，所形成複合型奈米微胞粒徑約243.8nm、粒徑分佈約為0.205且藥物包覆率為16.06%，適合用於動物體內之藥物傳輸。經由上述研究之最佳條件下，複合型奈米微胞導入Cy5.5-PEG-PLA以作為癌症組織之「顯影」功能，用以觀測癌細胞之毒殺與胞飲行為，以及動物實驗之微胞分佈與癌症治療情形。
經由體外藥物釋放模擬實驗證實複合型藥物微胞於中性環境pH7.4下可以穩定地將藥物包覆。而在酸性環境pH5.4、pH4.5下微胞結構對環境產生應答(膨潤)可將藥物釋放出，並呈穩定釋放的效果。在材料細胞毒性研究中可知，複合型奈米微胞由於結構組成皆為FDA 核可，故毒性較低。由藥物載體毒殺Hela、LL/2、ES2細胞之MTT結果，L3B之24小時和72小時之IC50差異很大，而S3B在24小時和72小時之IC50差異不大，証明L3B結構在24小時後鬆散釋放出藥物。
以共軛焦顯微鏡觀測藥物及複合型奈米微胞藥物分佈情形發現，複合型奈米藥物微胞隨著時間增加，藥物微胞經由進入細胞內慢慢累積在癌細胞，於酸性胞器中釋放藥物後，藥物則累積於細胞核部分。
在動物實驗中以optical imaging (2-D Near-IR)觀測複合型微胞在不同時間下於動物體內之累積量及分佈情形，實驗顯示複合型奈米藥物微胞經由EPR效應後其藥物微胞累積在腫瘤組織大於其他器官。
在動物實驗中觀測複合型奈米藥物微胞於動物體治療腫瘤情形，實驗得知以具酸鹼應答之複合型奈米微胞包覆Doxorubicine具有較低的毒性且相較於free dox及control實驗組有較好的腫瘤抑制效果。
綜合本研究所得之結果，複合型奈米藥物微胞不論於細胞毒性研究或動物實驗均證實極具癌症治療效果。未來可運用微胞具標識腫瘤組織的功能，診斷癌症組織之分佈位置，早期於癌細胞擴散前追蹤與治療，並且於治療時能更精準的殺死癌細胞，有效達到抗癌效果，促進人類醫療福祉，對癌症治療醫學上作出重大貢獻。

Abstract
Long-blood circulation mixed micelles that constructed from P(His-co-HPMA-co-HPMA-Lac2)-g-PLA and diblock copolymer PEG–PLA with was developed in this study. Stability analysis of the mixed micelles in bovine serum albumin (BSA) solution by DLS indicates that the diblock copolymer mPEG protected efficiently the BSA adsorption on the mixed micelles. Moreover, mixed micelles could keep the size and PI stable above 120 hr because the hydrophobic groups of graft copolymer could solidify inner core and mPEG offered the stability to the drug carrier as well as avoiding them recognized by mononuclear phagocytesystems (MPS). The mixed micelles had well defined core shell structure which was evaluated by TEM.
The functional inner core of P(His-co-HPMA-co-HPMA-Lac2)-g-PLA exhibited pH and hydrolysis stimulate to enable intracellular drug delivery and outer shell of PEG-b-PLA with functional moiety Cy5.5 for biodistribution diagnosis at the end of the polymer chain. The graft and diblock copolymer self assembled to empty nanospheres against water with an average diameter below 120 nm, and an average diameter of around 243.8nm when loaded with doxorubicin. From drug released study, a change in pH swollen and destroy the structure of the inner core from that of graft copolymer P(His-co-HPMA-co-HPMA-Lac2)-g-PLA, keeped the release of a significant quantity of doxorubicin (Dox) from mixed micelles.
Fluorescence images of multifunctional micelles by confocal laser scanning microscopy (CLSM) indicated that most of the anticancer drug and carriers were localized in nucleus and cytoplasm because of the colocalization of fluorescence with that from doxorubicin and Cy5.5. Encapsulation of doxorubicin in long-blood circulation mixed micelles increased its internalization by MDR LL/2 cells into lysosomes and enhanced cytotoxicity. The in vivo antitumor efficiency experiment revealed that high stable mixed micelles are effective in inhibiting tumor growth in the subcutaneous MDR LL/2 tumor model. To the best of our knowledge,, multifunctional micelles prepared from P(His-co-HPMA-co-HPMA-Lac2)-g-PLA, PEG–PLA and Cy5.5 PEG–PLA had great potential in cancer chemotherapy and diagnosis.

關鍵字

長效型藥物載體 ； 酸鹼應答 ； 複合型奈米微胞 ； LL/2 肺癌細胞 ； Doxorubicine

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