環境應答性複合型奈米微胞及其在癌症治療之應用

Mixed Micellar Systems based on Environmental-Responsive Copolymers for Application in Cancer Therapy

陳奕君(Chen, Yi Chun)

指導教授 ： 薛敬和

國立清華大學/工學院/化學工程學系/博士(2012年)

摘要

高分子微胞被認為是目前最有潛力的藥物載體，其優點包括降低細胞毒性、保護藥物在體內不易被酵素分解、增加藥物在體內的溶解度而提高藥物效率、延長體內循環時間而使藥物能累積在適當的組織，達到局部釋放的效果、躲避巨噬細胞與內質網的功能、在高分子微胞表面接上特定的功能性官能基等。本研究主要藉由合成生物可分解性多功能性高分子，製備奈米載體並應用藥物傳遞及其在癌症顯影及治療之研究。
本研究所製備之奈米載體具有以下優點：一、高分子在生物分解後不具毒性。二、可增加細胞與腫瘤傳輸藥物效率。三、具備生物體內腫瘤的標的性與顯影性。結合奈米科技、螢光與放射顯影的技術，並評估嶄新奈米載體對腫瘤治療之效益，以及未來做為癌症診斷試劑之可行性。本研究分為三個主題分別論述內容如下：
第一部分、雙重應答微胞在腫瘤組織的累積
本研究合成具有酸鹼應答性與溫度應答性的團聯高分子mPEG-b-P(HPMA-Lac-co-His) 以及具有低微胞臨界濃度的mPEG-b-PLA，以熱衝擊法製備雙重應答微胞，雙重應答微胞是藉由溫度及酸鹼敏感型微胞控制藥物釋放，加入臨界微胞濃度的雙性團聯共聚物，增加進入血液循環中藥物載體的穩定，使微胞至具有弱酸性環境的腫瘤病理部位將藥物釋放，具備免疫迴避性的智慧型功能之外殼，雙重應答微胞同時具有環境應答性、生物可分解性與生物造影性及藥物釋放功能，在病理位置溢出血管，且微胞尺寸改變並滯留於腫瘤組織中，藉由螢光標記觀察活體影像，奈米微胞以被動標的方式大量累積於腫瘤組織，實驗證明載體具有癌症治療潛力並達到降低藥物副作用之目的。
第二部分、應用雙重應答微胞輸送疏水性藥物Rapamycin
Rapamycin研究發現有抗生素、免疫抑制及抗腫瘤的作用，然而Rapamycin在應用於治療上有所限制，Rapamycin為疏水性的藥物，在人體血液中為不穩定易聚集的狀態，Rapamycin在水中溶解度僅2.6 μg/mL，對於進行靜脈注射為一大受限，且經口服途徑藥物也僅有15%的生物利用度(bioavailability)，因此將疏水性藥物Rapamycin包覆於親水性的載體為一大利多。本章應用第一部分之複合型高分子微胞，具有雙重應答與完整的殼核結構，並藉由親水性的表面保護疏水性藥物Rapamycin。此微胞是藉由溫度及酸鹼敏感型微胞進一步控制藥物釋放，使微胞至具有弱酸性環境的腫瘤部位將藥物釋放，藉以準確達到毒殺大腸癌細胞的效果，但repamycin藥物本身引發自體吞噬形成雙層膜的autophagosomes的產生，接下來以與lysosomes融合後形成autophagolysosome，然而對於某些癌細胞而言，autophagosomes的發生會抑制細胞凋亡途徑，因此對於癌細胞的抑制為雙面刃，共軛焦顯微鏡影像發現以複合型奈米微胞進入細胞後引發形成autophagolysosome，在酸性環境下將藥物釋出，藉以對於細胞毒殺有控制成效，應用大腸癌模式裸鼠證實達到治療大腸癌的成果，並使腫瘤切片中的癌細胞凋亡。
第三部分、接枝與團聯高分子製備高穩定性奈米載體及其在抗藥性腫瘤之應用
本研究應用接枝共聚物PLA-g-P(HPMA-Lac-co-His)與團聯共聚物mPEG-b-PLA混合自組裝形成複合型高分子奈米微胞， PLA-g-P(HPMA-Lac-co-His)為在HPMA主鏈修飾Lactate增加其穩定性，並以PLA側鏈穩定藥物的內核，並應用團聯共聚物mPEG-b-PLA藉以增加其在小牛血清蛋白溶液中的穩定性，在酸性條件下(~pH 6.0)，即藥物載體位於secondary lysosome，複合型奈米微胞可藉由此酸鹼值的改變達到藥物釋放的效果，細胞實驗證實高穩定性複合型奈米微胞可更有效率將藥物傳輸至細胞核內，動物活體影像發現複合型奈米微胞可經由EPR效應大量累積於腫瘤部位，且由於增加穩定性的奈米載體，可增加藥物在腫瘤的累積量，可克服肺癌細胞LL/2產生的抗藥性，動物模式腫瘤切片觀察證明累積較多的藥物在細胞核中，並具有較佳的治療肺癌腫瘤的效果以及提高存活率，此結果可證實高穩定複合性微胞可做為抗藥性腫瘤治療之應用。

關鍵字

高分子微胞 ； 酸鹼應答性 ； 溫度應答性 ； 藥物傳輸 ； 癌症治療

並列摘要

For anticancer drug delivery systems, many systems have been discussed and exemplified regarding as traditional systems such as polymer-based therapeutics, liposomes, and inorganic particles. Polymer therapeutic is considered to be a potential candidate displaying well bioavailability and high molecular manipulation for use in cancer treatment. The term polymer therapeutics describes several distinct classes of agent, including polymer-drug conjugates, micelles and mixed micelles that have now entered clinical development because of their intrinsic physical properties and their abilities to target specific locations. Much research has recently been focused on the study of mixed micelles as drug carriers in the hunt for improved cancer therapy. The potential advantages of mixed micelles as potential drug carriers include 1) the fact that they can be degraded into nontoxic substances that may be readily excreted by the body; 2) the possibility of modulating the micellar structure to improve intracellular drug delivery; and 3) the possibility of modifying the polymers for in vivo cancer targeting and imaging. Despite such potential advantages, in vivo studies on mixed micelles as potential anticancer drug delivery systems remain scanty. The major problem that limits the wider application of mixed micelles as a drug carrier is the uncertainty about the structure of micelles during micellization in individual and mixed micellar systems. Therefore, my major is focus on the mixed micellar systems for the in vivo application, as follows:
(1) The Accumulation of Dual pH and Temperature Responsive Micelles in Tumors
An optimized, biodegradable, dual temperature- and pH- responsive micelle system prepared from methoxy poly(ethylene glycol)-block-poly(N- (2-hydroxypropyl) methacrylamide dilactate)-co-(N-(2-hydroxypropyl) methacrylamide-co-histidine) (mPEG-b-P(HPMA-Lac-co-His)) copolymer and methoxy poly(ethylene glycol)-block-poly(D,L-lactide) (mPEG-b-PLA) copolymer conjugated with functional group Cy 5.5 was prepared in order to enhance tumor accumulation. Anticancer drug, doxorubicin was incorporated into the inner core of micelle by hot shock protocol. The size and stability of the micelle were controlled by the copolymer composition and is fine tuned to extracellular pH of tumor. The mechanism then caused pH change and at body temperature which induce doxorubicin release from micelles and have strong effects on the viability of HeLa, ZR-75-1, MCF-7 and H661 cancer cells. Our in vivo results revealed a clear distribution of Doxorubicin-loaded mixed micelle (Dox-micelle) and efficiency targeting tumor site with particles increasing size in the tumor interstitial space, and the particles could not diffuse throughout the tumor matrix. In vivo tumor growth inhibition showed that Dox-micelle exhibited excellent antitumor activity and a high rate of anticancer drug in cancer cells by this strategy.
(2) Rapamycin Encapsulated in Dual-Responsive Micelles for Intracellular Drug Delivery
Rapamycin has been developed as a potential anticancer drug for treatment in rapamycin-sensitive cancer models, but its poor water solubility greatly hampers the application to cancer therapy. This study investigated the preparation, release profiles, uptake and in vitro/in vivo study of a dual responsive micellar formulation of rapamycin. Rapamycin-loaded micelles (rapa-micelles) measured approximately ca. 150 nm with narrow size distribution and high stability in bovine serum albumin solution. It was shown that rapamycin could be loaded efficiently in mixed micelles up to a concentration of 1.8 mg/mL by a hot shock protocol. Rapamycin release kinetic studies demonstrated that this type of micellar system could be applied in physiological conditions under varied pH environments. Confocal and pH-topography imaging revealed a clear distribution of rapa-micelles, and visible intracellular pH changes which induced encapsulated rapamycin to be released and then induced autophagolysosome formation. In vivo tumor growth inhibition showed that rapa-micelles exhibited excellent antitumor activity and a high rate of apoptosis in HCT116 cancer cells. These results indicated that dual responsive mixed micelles provided a suitable delivery system for the parenteral administration of drugs with poor water solubility, such as rapamycin, in cancer therapy.
(3) Graft and Diblock Micelles with Enhanced Stability for Overcoming Multidrug Resistance in Cancer
A graft and diblock polymeric micelles, self-assembling from poly(N-(2-hydroxypropyl) methacrylamide dilactate)-co-(N-(2-hydroxypropyl) methacrylamide-co-histidine)-graft-poly(D,L-lactide) graft copolymers and methoxyl/functionalized-PEG-b-PLA diblock copolymers, as an anticancer drug doxorubicin carrier for cancer targeting, imaging, and overcoming multidrug resistance in cancer therapy. This high stability nanoparticle exhibited a pH-dependent drug release behavior, owning to the pH-sensitive structure of imidazole of histidine, to release doxorubicin in acidic surroundings (intracellular endosomes) and to capsulate doxorubicin in neutral surroundings (blood circulation or extracellular matrix). The in vitro results revealed released doxorubicin from mixed micelles was more effective accumulation into the nuclei than free doxorubicin. Imaging by in vivo image system showed that high stability ensures a high intratumoral accumulation due to EPR effect. Mixed micelles with enhanced stability inherently overcome a certain degree of multidrug resistance by tumor cells, since such vesicles can deliver between more drug to solid lesions when compared with the administered drug in its free drug. In vivo tumor growth inhibition shows that nanoparticles exhibited excellent antitumor activity and a high rate of apoptosis in cancer cells. The results indicate that the high stbility carriers with a pH-dependent drug release can be allowed to accurately deliver to targeted tumors for multidrug-resistant cancer therapy.

並列關鍵字

Polymeric micelles ； pH-responsive ； Temperature-responsive ； Drug delivery ； Cancer therapy

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