含反譯寡核苷酸及化療藥物之固體脂質奈米粒子的製備

Preparation of Chemotherapy Agents and Oligonucleotides-Loaded Solid Lipid Nanoparticles

李沂瑾(Yi-Chin Lee)

指導教授 ： 邱士娟

臺北醫學大學/藥學院/藥學院生技製藥產業碩士專班/碩士(2013年)

摘要

固態脂質奈米粒子(SLN)具有高穩定性、易製備、毒性低等優點，且能保護被乘載的藥物不被降解，故被認為可作為乘載抗腫瘤藥物或治療基因的載體。本研究目標為開發由硬脂酸、DC-Chol、膽固醇及DOPE當作組成，且能同時乘載化療藥物 (阿黴素或太平洋紫杉醇) 及反譯寡核苷酸的奈米載體系統。
　　此新型帶正電奈米粒子使用溶劑乳化法製備，先形成包覆化療藥物 (阿黴素或太平洋紫杉醇) 的粒子，分別使用Zetasizer測定粒子大小及表面電荷，阿黴素奈米粒子由stearic acid： cholesterol： DC-Chol： DOPE為43：5：50：2 (w/w)組成，其粒子大小及電荷為273.93±49.30 nm、13.8±2.21 mV，太平洋紫杉醇奈米粒子則由stearic acid： cholesterol： DC-Chol： DOPE為35：5：50：10 (w/w)組成，其粒子大小及電荷為281.73±20.31 nm、26.03±8.41 mV。奈米粒子以穿透式電子顯微鏡觀察可得知為圓球形，藥物的包覆率利用高效液相層析法定量， 阿黴素奈米粒子及太平洋紫杉醇奈米粒子包覆率分別為62%±5%、88%±4%。阿黴素奈米粒子在4ºC環境下，能維持膠體安定性至少達7週。之後將含化療藥物的粒子與反譯寡核苷酸共同震盪，得到同時乘載化療藥物 (阿黴素或太平洋紫杉醇) 及反譯寡核苷酸的奈米載體，並使用凝膠阻滯實驗確認。證明了使用硬脂酸、DC-Chol、膽固醇及DOPE能形成正電性奈米粒子，且此粒子可同時乘載高量的化療藥物 (阿黴素或紫杉醇) 與反譯寡核苷酸。
　　在細胞毒性試驗中，將含不同量阿黴素的奈米粒子投予K562及CCRF-CEM細胞，結果發現在K562細胞中，培養48小時後，游離態阿黴素的IC50為729.35±209.75 nM，阿黴素奈米粒子的IC50為329.31±15.85 nM；在CCRF-CEM細胞中，經過24小時培養後，游離態阿黴素的IC50為380.87±29.57 nM，阿黴素奈米粒子的IC50為158.70±15.85 nM，證明以SLN當作阿黴素的載體時，能提高阿黴素低敏感度的K562或阿黴素高敏感的CCRF-CEM對於藥物的敏感度。
　　綜合以上，此研究證實帶正電性的固態脂質奈米粒子 (cSLN) 能作為同時乘載親脂性或親水性藥物與反譯寡核苷酸的載體。且發現使用SLN當作載體能提高人類慢性骨髓性白血病細胞K562及人類急性淋巴母細胞白血病細胞CCRF-CEM對阿黴素的敏感性。

關鍵字

固態脂質奈米粒子 ； 阿黴素 ； 太平洋紫杉醇 ； 溶劑乳化法

並列摘要

Solid lipid nanoparticles (SLNs) have attracted increasing attention as a potential anticancer drug or gene delivery carrier as a result of their physical stability, protection of labile drugs from degradation, easy preparation, and low toxicity. The purpose of this study is to develop a novel drug delivery system for antisense oligonucleotide (ODN) and chemotherapeutic agents (e.g., paclitaxel (PTX) or doxorubicin (DOX)).
The nano-sized solid lipid particles were composed of stearic acid, DC-Chol, cholesterol and DOPE. This cationic nanoparticle formulation was prepared by solvent emulsification method. Chemotherapeutic agents (PTX or DOX) were first incorporated into nanoparticles and characterized. The DOX-SLN made up of stearic acid: cholesterol: DC-Chol: DOPE (43: 5: 50: 2 (w/w)) had a mean diameter of 273.93±49.30 nm and zeta potential of 13.8±2.21 mV. PTX-SLN made up of stearic acid: cholesterol: DC-Chol: DOPE (355: 50: 10 (w/w)) had a mean diameter of 281.73±20.31 nm and zeta potential of 26.03 mV. The morphological characteristics were examined by transmission electron microscope. Encapsulation efficiency of DOX-SLN and PTX-SLN quantified by HPLC were 62%±5% and 88%±4%, repectively. Colloidal stability of all formulations developed can maintain for at least 7 weeks. ODN was incorporated onto DOX-SLN or PTX-SLN by vortexing. ODN-loaded cationic solid lipid nanoparticles (cSLN) were characterized by gel retardation assay. This study provides conceptual proof that stearic acid, DC-Chol, cholesterol and DOPE can form polycationic nano-sized particles. The formulation performs high entrapment efficiency of the capability of forming complex with ODN, and chemotherapeutic agent (DOX or PTX).
Significant reduction of cell viability was observed by the treatment of DOX-SLN in K562 or CCRF-CEM cells. In K562 cells, the IC50 of doxorubicin in DOX-SLN was decreased from 729.35±209.75 nM to 329.31±15.85 nM as compared with free doxorubicin after 48 hr incubation. In CCRF-CEM cells, the IC50 of free doxorubicin was 380.87±29.57 nM, and the IC50 of DOX-SLN was 158.70±15.85 nM after 24 hr incubation. The result in cytotoxicity studies indicated that DOX-SLN can increase the cytotoxic effect of doxorubicin in low-doxorubicin-sensitive K562 and high-doxorubicin-sensitive CCRF-CEM.
Taken together, the results in the current studies demonstrated the potential of cSLN for the development of co-delivery systems of various lipophilic or lipophobic anticancer drugs and therapeutic antisense agents. DOX-SLN can improve the sensitivity of doxorubicin in various leukemia-related cell lines.

並列關鍵字

solid lipid nanoparticles ； doxorubicin ； paclitaxel ； solvent emulsification

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