以高效能液相層析法應用於質體 DNA與聚合微膠混合體的測定

Determination of Plasmid DNA/Polymeric Micelles Complexes Using High-Performance Liquid Chromatography

李世翔(Shih-Hsiang Lee)

指導教授 ： 廖嘉鴻

臺北醫學大學/藥學院/藥學研究所/碩士(2011年)

摘要

先前本實驗室曾使用此三嵌段共聚合物當作質體 DNA的載體，證實可以增加轉殖基因的表現量，然而實際質體 DNA的包覆率 (encapsulation efficiency) 仍不清楚，因此本實驗目的為探討在使用微膠體當作載體包覆質體 DNA 後的包覆率。首先利用Zeta potential、 pH與膠體電泳分析對質體 DNA、聚合微膠體溶液、質體 DNA與聚合微膠混合體溶液三者的物理特性做探討，質體 DNA為對 pH 敏感的物質，而聚合微膠體溶液則相當穩定不會隨著 pH值改變，質體 DNA與聚合微膠混合體溶液則有兩種 Zeta 電位的分布；另外在膠體電泳分析實驗中，在應用聚合微膠體包覆質體 DNA後，有不同凝集 (aggregation) 現象出現。利用 HPLC 配合在 UV 260 nm偵測下，質體 DNA溶液在第9.3分有單一 peak形成，而在質體 DNA與聚合微膠混合體溶液，則在第6.3 分鐘中另一新形成之 peak，同時分別收集質體 DNA和質體 DNA與聚合微膠混合體在 HPLC分析下所形成 peak範圍，之後利用加入螢光染劑 Picogreen®、膠體電泳分析和即時定量聚合酶鏈鎖反應等方法對其定性及定量，做再次確認評估。
在利用 HPLC分析之圖譜計算包覆率後，經0.3%聚合微膠體包覆1.5、4.5、9 ng/μl質體 DNA後，其包覆量分別為1303.9 ± 139.75 ng、2541.7 ± 621.22 ng、2639.4 ± 555.08 ng，包覆率則各為37.76 ± 3.90%、23.96 ± 6.00%、13.62 ± 2.83%，與利用即時定量聚合酶鏈鎖反應所得包覆率各為30.97 ± 8.02、22.31 ± 8.97、13.77± 1.68 %，兩者結果類似；而經3%聚合微膠體包覆1.5、4.5、9 ng/μl質體 DNA後，其包覆量分別為1558.8 ± 86.75 ng、3561.2 ± 247.32 ng、4215.2 ± 356.67 ng，包覆率則各為44.84 ± 2.10%、33.71 ± 2.50%、22.09 ± 1.82%。而在藥物釋放過程中，質體 DNA與聚合微膠混合體 (10 ng/μl plasmid DNA/0.3%、3% polymeric micelles)的釋放速率為7.39、8.19 ng/min。

關鍵字

質體DNA ； 聚合物 ； 高效能液相層析法

並列摘要

Previously used the triblock copolymer as a carrier encapsulated of plasmid DNA, it could effectively enhance the gene expression. However, encapsulation of plasmid DNA still remains unsolved. The purpose of this study is determination encapsulation efficiency of plasmid DNA with polymeric micelles. Characterizations of plasmid DNA、polymeric micelles and plasmid DNA with polymeric micelles solutions by Zeta potential, pH effect and gel electrophoresis. The results showed that plasmid DNA was pH-sensitive with decreasing Zeta potential value, and plasmid DNA with polymeric micelles showed two Zeta potential peaks distribution, however polymeric micelles was stable under pH changed. Using electrophoresis observation, after application of polymeric micelles encapsulated plasmid DNA, the different aggregation were observed. Under High-Performance Liquid Chromatography and UV-VIS detector, the plasmid DNA was observed at 9.3 minutes, and plasmid DNA with polymeric micelles solutions had observed two peaks peak at 6.3 and 9.3 minutes. In addition, the fractions of these two peaks of plasmid DNA and plasmid DNA with polymeric micelles were consistently confirmed by Picogreen®, gel electrophoresis, and QPCR evaluation.
The three different plasmid DNA (1.5,4.5,9 ng/μl) with 0.3% polymeric micelles were found the encapsulated amount were 1303.9 ± 139.75 ng、2541.7 ± 621.22 ng、2639.4 ± 555.08 ng, respectively. The encapsulation efficiency were 37.76 ± 3.90%、23.96 ± 6.00%、13.62 ± 2.83%, respectively by HPLC analysis. When increasing polymeric micelles to 3% with 1.5,4.5,9 ng/ μl plasmid DNA, encapsulation were 1558.8 ± 86.75 ng、3561.2 ± 247.32 ng、4215.2 ± 356.67 ng, respectively. The encapsulation efficiency were 44.84 ± 2.10%、33.71 ± 2.50%、22.09 ± 1.82%, respectively. Furthermore, the in vitro release rates of 10 ng/μl plasmid with 0.3% 、3% polymeric micelles were 7.39、8.19 ng/ μl, respectively.

並列關鍵字

plasmid ； polymer ； HPLC

參考文獻

1. Batrakova EV, Kabanov AV. Pluronic block copolymers: evolution of drug delivery concept from inert nanocarriers to biological response modifiers. J Control Release 2008;130:98-106.

2. Mathot F, van Beijsterveldt L, Preat V, Brewster M, Arien A. Intestinal uptake and biodistribution of novel polymeric micelles after oral administration. J Control Release 2006;111:47-55.

3. Kabanov AV, Batrakova EV, Alakhov VY. Pluronic block copolymers as novel polymer therapeutics for drug and gene delivery. J Control Release 2002;82:189-212.

4. Zhang W, Shi Y, Chen Y, Ye J, Sha X, Fang X. Multifunctional Pluronic P123/F127 mixed polymeric micelles loaded with paclitaxel for the treatment of multidrug resistant tumors. Biomaterials 2011;32:2894-2906.

5. Agrawal S, Temsamani J, Galbraith W, Tang J. Pharmacokinetics of antisense oligonucleotides. Clin Pharmacokinet 1995;28:7-16.