生物可降解奈米載體之設計與合成及其在藥物傳遞及光驅動靶向藥物癌症治療之應用

Fabrication of biodegradable nanocarriers and their applications in drug delivery and light-activatable targeted cancer therapy

范念祖(Nien-Chu Fan)

指導教授 ： 黃賢達 ； 何佳安

國立清華大學/理學院/化學系/博士(2012年)

摘要

奈米科技於近十年來被廣泛的應用在許多科學領域中；而應用在醫學科學領域上的奈米技術被稱為奈米醫學。結合材料科學、化學與化學生物方面的基礎知識作為操控奈米尺度材料性質的依據，並利用其多元的物理化學特性，我們可將材料應用在癌症治療與臨床診斷上。本篇論文研究主要在合成三種不同的生物可降解奈米載體，包括微脂體 (liposomes)、聚乳酸聚甘醇酸 [poly(lactic－co－glycolic acid), PLGA] 奈米粒子及磷脂質和PLGA高分子混合的複合奈米粒子 (PLGA@Lipid NPs)，並探討其在in vitro及in vivo實驗組中對細胞及生物體的影響。本論文分為三大部份，(1) 以毛細管微胞電動之雷射誘導螢光層析法偵測阿黴素 (doxorubicin, DOX) 在中國倉鼠卵巢癌細胞 (CHO-K1) 內的分布情形：證實微脂體載體可顯著改善藥物傳遞的效率；(2) 發展以光觸發藥物載體進行主動式標記癌症細胞的治療癌症策略；(3) 探討標記有奈米粒子的幹細胞在活體動物中的分化情形。
第一部份的研究目的為監測以liposomal form及free form DOX被傳遞到細胞後之胞內分布情形。DOX為一種廣泛使用的蒽環類 (anthracycline) 抗癌藥物，可有效抑制許多惡性腫瘤的生長，如卵巢癌及乳腺癌等。不論是free form或經由載體裝載 (例如liposomal form) 的DOX進入癌細胞後皆可引發癌細胞的死亡。DOX毒殺癌細胞的作用機制被認為是與細胞核中DNA的複製有關，DOX抑制可解開雙股螺旋DNA的拓撲酶II (topoisomerase II) 的活性，使DNA無法順利進行複製而抑制了癌細胞的增生。DOX雖具有毒殺癌細胞的效果，但在治療的過程中常發現DOX藥物本身或其相關代謝物具有損害心臟與肝臟的毒性；此外，也發現治療過程中癌細胞可對DOX或其代謝物產生抗藥性。Liposomal DOX已被証實能降低被網狀內皮系統 (reticuloendothelial system, RES) 吞噬，並延長其在血液中的循環時間，因此具有減緩毒性副作用的產生。本研究發展一套毛細管電泳微胞電動層析/雷射誘導螢光法 (Micellar electrokinetic chromatography－Laser induced fluorescence, MEKC－LIF) 來分析生化樣品中DOX的含量。以10 mM硼酸鹽緩衝溶液 (borate buffer) 內含100 mM 陰離子型界面活性劑 (Sodium dodecyl sulfate, SDS) (pH = 9.3)　為遷移溶液(migration buffer)可有效率的分析樣品中的DOX。該系統對DOX的偵測範圍為11.3 ~ 725 ng/mL，定量極限 (the limit of quantitation, LOQ) 為43.1 ng/mL (S/N=10) ，偵測極限 (limit of detection, LOD) 為6.36 ng/mL (S/N=3) 。以此方法為基準，我們嘗試討論以25 µM的free DOX與liposomal DOX治療CHO-K1細胞後，DOX在細胞內的分布情形。被作用細胞在療程結束後以低張滲透及機械剪力的方式被打碎，再以不同離心力將細胞碎片分為三部分 ( < 1400 g vs. 1400~14000g vs. >1400g)，分別為富含細胞核部份 (< 1400 g)、富含胞器部份 (如粒腺體、溶酶體等) (1400~14000g) 及細胞質部份 (>1400g)。由liposomal form及free form的組別可觀察到DOX累積在細胞中達最大量的時間分別為6 h及12 h；這個定量的結果表示liposome可改善DOX被傳遞到細胞的能力。本研究成功的結合液相-液相萃取法 (liquid-liquid extraction method) 與MEKC－LIF，並應用於監測細胞內DOX的量分布；該系統亦可作為研究蒽環類抗癌藥物細胞毒性的研究。
第二部份研究的目標主要為解決現今化療過程中，投予藥物之理想劑量問題。一般說來，低劑量化療藥物對於抑制腫瘤生長的效果差；而高劑量卻容易造成病人難以負擔的毒性與副作用。本篇研究設計了一個新的抗癌藥物載體，主要為提高抗癌藥物在特定位置所累積的有效治療濃度。這裡採用了二個策略來完這個這目標：(1) 建構出可觸發 (stimuli-responsive)的藥物傳遞系統用來控制藥物的釋放；(2) 設計可進行主動的標靶傳遞系統。結合以上二個策略，將可降低傳統藥物的使用量，並促進藥物的治療效率。『主動式標靶』癌細胞需要使用特定的配體 [包括單株抗體、多肽及適體 (aptamer) ] 以辨認並結合癌細胞上特定的蛋白質標記分子或過度表現的表面抗原；本研究則選擇使用癌細胞表面過度表現的葉酸受器 (folate receptor, FR) 作為腫瘤標記物。我們製備表面攜帶葉酸分子的磷脂質/PLGA高分子複合奈米粒子 (folate/PLGA@Lipid NPs) 並以共價鍵修飾光敏感性的o-nitrobenzyl group (ONB) 於葉酸分子的-及-carboxylate groups上。藉光驅動觸發奈米載體表面的葉酸分子活化，進行主動式的標靶傳遞。本研究是首例將2-nitrobenzylamine (NBA) 修飾在生物可降解性奈米粒子 (folate/PLGA@Lipid NPs)上，並藉由光照觸發NBA分子的裂解，使folate分子回復原本可辨認葉酸受器的活性，續進行主動式標靶作用及持續性的藥物釋放。實驗之初，我們先將光敏感性的caged分子NBA及光觸發策略示範於表面具葉酸分子的13 nm 金奈米粒子 (folate/Au NPs) 系統上，並藉由光照証實其可回復folate分子的生物活性。接著我們製備裝載Taxol抗癌藥物的生物降解性脂質高分子混合的奈米粒子 (PLGA@Lipid NPs) 並在其表面修飾葉酸及NBA分子 (caged folate/PLGA@Lipid containing Taxol) ，以驗証光驅動藥物傳遞系統的概念。在細胞存活率實驗中，人類口腔癌細胞KB cell (FR-positive) 與裝載抗癌藥物Taxol (約21 ng) 的caged folate/PLGA@Lipid NPs共培養12小時後，移至4 oC下以汞燈曝照20分鐘以驅使 NBA之光解，讓葉酸分子恢復可以辨識葉酸受體的活性，細胞存活率降至51%，此結果足以驗証光觸發標靶的概念。本實驗的對照組則是於KB cells中施加21 ng的free Taxol (細胞存活率高達89%)。
第三部份的研究主要為探討奈米粒子是否會影響幹細胞在活體中的分化狀況。幹細胞已經被証實具治療神經退化性疾病、缺血及受損組織的潛力，為新興的細胞治療策略。運用成大化學系CS Yeh教授的團隊所發展的奈米沉澱法 (nanoprecipitation method)，在本實驗中，我們製備100 nm、且表面無穩定劑修飾的PLGA@QD655奈米粒子。在過去已發表的in vitro研究中已證實PLGA@QD655可標記間葉幹細胞 (mesenchymal stem cell, MSC)；若以高濃度的奈米粒子與細胞共培養長達四週後，並未觀察到明顯細胞毒性。此外，PLGA@QD655不會改變 MSCs的與生俱來的分化能力，他們仍然可以正常分化為脂肪細胞 (adipocyte)、骨細胞 (osteocyte) 及軟骨細胞 (chondrocyte)。而本研究中，我們深入探討已標記奈米粒子的間質幹細胞在進入動物體後，是否仍具有幹細胞的分化能力？實驗結果證實，我們可以成功地將PLGA@QD655標記在eGFP-MSCs上，並將標記後的eGFP-MSCs植入裸鼠皮下組織；且於四週之後以組織螢光染色法及IVIS螢光系統觀察MSCs的分化情形，所獲結果指出在活體老鼠中被標記PLGA@QD655奈米粒子的MSCs，其分化能力無顯著變化。

關鍵字

阿黴素 ； 聚乳酸甘醇酸高分子 ； 幹細胞

並列摘要

Nanomedicine is the medical application of nanotechnology. Combining the basic knowledge of the chemistry, material science and chemical biology, we are able to manipulate the nanoscale materials of interest and extend their uses in cancer treatment and clinical diagnosis. Three nanoparticles tentatively used in bioapplication include (1) liposome, (2) phospholipid－poly lactic acid－co－glycolic acid polymer hybrid nanoparticles (PLGA@Lipid NPs), and (3) quantum dot-conjugated PLGA hybrid nanoparticles (PLGA@QD NPs). Three studies are included in this dissertation: (1) Monitoring the subcellular Localization of doxorubicin in CHO-K1 using MEKC－LIF: liposomal carrier for enhanced drug delivery; (2) Photocontrolled targeted drug delivery: photocaged biologically active folic acid as a light-responsive tumor-targeting molecule; (3) In vivo cell fate tracking of mesenchymal stem cells using PLGA@QD nanoparticles.
The aim of the first study is to monitor the subcellular localization of doxorubicin (DOX) delivered in free form and liposomal form, respectively. DOX is an extensively used anthracycline that has proven to be effective against a variety of human malignant tumors, such as ovarian or breast cancer. While DOX was administered into cultured cancer cell targets (such as CHO-K1) in either free drug form or in drug carrier-associated form (i.e., DOX encapsulated in the drug delivery carrier), various action of mechanisms for DOX were initiated, among which, it has been long believed that DOX enters the nucleus, interacts with DNA in numerous ways, and finally halts cell proliferation. Aside from its therapeutic effect, regrettably DOX treatment may be accompanied by the occurrence of cardiac and liver toxicity and drug resistance that are attributed from cellular processes involving the parent drug or its metabolites. Liposomal formulation of DOX, known to be capable of attenuating direct uptake of reticuloendothelial system (RES) and prolonging the circulation time in blood, demonstrated reduced toxic side-effects. We herein report the development of a modified MEKC–LIF (Micellar electrokinetic chromatography－Laser induced fluorescence) method suitable for analyzing DOX in biological samples. The MEKC migration buffer, consisting of 10 mM borate, 100 mM sodium dodecyl sulfate (SDS) (pH 9.3), was found to provide an efficient and stable electrophoretic separation and analysis for DOX. Responses were linear in the range of 11.3–725 ng/mL; the limit of quantitation (LOQ) was found to be 43.1 ng/mL (S/N=10) (equivalent to 74.3 nM) and limit of detection (LOD) was calculated as 6.36 ng/mL (S/N=3) (equivalent to 11.0 nM). This approach was subsequently employed to compare the intracellular accumulation in three subcellular fractions of DOX-treated CHO-K1 cells. These fractions form a pellet at <1400g, 1400–14000g, and >14000g and are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosole components, respectively, resulting from treatment of CHO-K1 cells with 25 mM (equivalent to 14.5 mg/mL) of two DOX formats (in free drug form or liposomal form synthesized in current study) for different periods of time. Our results indicated that the most abundant DOX was found in the nuclear-enriched fraction of cells treated for 12 h and 6 h with free and liposomal DOX, respectively, providing direct evidence to confirm the enhanced efficiency of liposomal carriers in delivering DOX into the nucleus. The observations presented herein suggest that subcellular fractionation followed by liquid–liquid extraction and MEKC－LIF could be a powerful diagnostic tool for monitoring intracellular DOX distribution, which is highly relevant to cytotoxicity studies of anthracycline-type anticancer drugs.
The second study intends to solve a major problem in current chemotherapy, which is “how to determine the optimum drug dosage given to patients?” A low dosage of drug is ineffective in the treatment of a tumor, whereas a high dosage of chemotherapeutics is intolerable for patients due to toxicity and unwanted side effects. Therefore new designs for anticancer drugs are desirable to increase the local effective therapeutic concentration. Two promising strategies were primed herein to achieve this goal; one is to construct a stimuli-responsive drug delivery system for controlled drug release and the other is to formulate a system for actively targeting the delivery of the therapeutic agent. Both approaches can minimize adverse effect of cytotoxic drugs and improve the therapeutic efficacy of conventional pharmaceuticals. Active targeting that uses specific ligands including monoclonal antibodies, peptides, and aptamers that bind to specific proteins or surface antigens overexpressed on cancer cells is a practical way to enhance the local control of therapeutics. We herein designed the photocaged folate nanoconjugates that selectively target cancer cells upon irradiation with light. The folic acid is masked by a photocleavable o-nitrobenzyl (ONB) group through covalently binding to - and -carboxylate groups, which interact with folic acid receptors (FRs) on the cell surface. The obtained results revealed that caging and photouncaging can be applied to the FA to potentially improve its targeting specificity. Moreover the application of the caged folate for intracellular drug delivery was examined using a biodegradable PLGA@lipid hybrid nanoparticle. It was confirmed that the cytotoxicity of Taxol encapsulated in PLGA@lipid hybrid nanoparticles increased upon light irradiation.
In the final study, a novel PLGA@QD655 NPs were utilized to investigate whether the applied nanoparticles affect the in vivo differentiation of stem cells. Adult stem cells have been intensively studied for their potential use in cell therapies for neurodegenerative diseases, ischemia and traumatic injuries. It was reported previously that the mesenchymal stem cells (MSCs) could be labeled with poly(lactide－co－glycolide) nanoparticles (PLGA NPs) surface-conjugated quantum dots (QDs) (PLGA@QD655 NPs), which were found to exert no toxic effect on the human mesenchymal stem cell (hMSC) after at least 4-wk co-incubation. In addition, it was observed that no significant change of the PLGA@QD655 NPs-labeled hMSCs in their proliferation and differentiation capability toward the production of adipocytes, osteocytes, and chrondrocytes. In the current study, we adapted the synthetic approach previously described by Prof C.S. Yeh’s group at NCKU for the preparation of 100 nm PLGA@QD655 NPs and selected green fluorescence protein (GFP) transgenic mouse MSC (eGFP-MSC) as model stem cell. The morphology and the uptake efficiency of eGFP-MSC labeled with PLGA@QD655 NPs were investigated. The obtained results showed that nearly 66% of PLGA@QD655 NPs were uptaken by eGFP-MSCs that were injected subcutaneously into the flanks of mice, and it was also confirmed that the differentiation capability of eGFP-MSCs to produce adipocytes, osteocytes, and chrondrocytes in vivo remained unaffected.

並列關鍵字

doxorubicin ； poly(lactic－co－glycolic acid) ； stem cell

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