Comparative study of poly (lactic-co-glycolic acid)-poly ethyleneimine-plasmid DNA microparticles prepared using double emulsion methods
Comparative study of poly (lactic-co-glycolic acid)-poly ethyleneimine-plasmid DNA microparticles prepared using double emulsion methods
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DOI：
10.1080/02652040701659347
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发表时间：
2008-01-01
影响因子：
3.9
通讯作者：
Salem, Aliasger K.
中科院分区：
文献类型：
--
作者：
Zhang, Xue-Qing;Intra, Janjira;Salem, Aliasger K.
从可生物降解的聚乳酸-羟基乙酸（PLGA）微粒中控制释放质粒DNA （pDNA）具有增强转基因表达的潜力。然而，这种方法的障碍包括有限的封装效率，制造过程中的pDNA损伤和吞噬溶酶体腔室内微粒的限制。将PLGA与聚乙烯亚胺（PEI）结合可以改善制备过程中对pDNA的保护，提高包封效率，并赋予PLGA微粒逃离吞噬溶酶体腔室的能力。本研究比较了制备PLGA PEI pDNA微粒的三种有前途的配方方法，并对缓冲能力、细胞摄取、转染效率和毒性进行了评价。在第一种方法中，采用双乳液油包水溶剂蒸发技术（PA）将pDNA包埋在混合PLGA/PEI中制备PLGA PEI pDNA微粒。在第二种方法中，制备PEI-pDNA多聚物，然后使用双乳液溶剂蒸发法（PB）将其包裹在PLGA微粒中。然后利用碳二亚胺化学（PC）将PA和PB制备的微粒子与PEI偶联表面的PLGA微粒子进行比较；经鉴定，表面活性剂的最佳浓度为0.5% PVA，转染效率最高。每组的制备均选用氮磷比为5和10。凝胶电泳表明，所有的PLGA微粒都具有较强的pDNA结合能力。MTT试验表明，PLGA PEI微颗粒的体外细胞毒性明显低于PEI单独。在COS7和HEK293细胞中，PLGA PEI pDNA微粒介导更高的细胞摄取效率，因此比未修饰的PLGA微粒介导更高的转基因表达。在PLGA微粒（PB）包埋之前制备PEI-pDNA多聚体可获得最高的pDNA负载。这比未修饰的PLGA微粒中的pDNA负载高2.5倍。采用PB方法制备的PLGA PEI pDNA微粒子转染效率最高，在HEK293细胞中的转染效率比未修饰的PLGA pDNA微粒子高500倍，在COS-7细胞中的转染效率高1800倍。使用PB方法制备的微粒在N:P比为5时产生的转染效率最高。
Controlled release of plasmid DNA (pDNA) from biodegradable poly lactic-co-glycolic acid (PLGA) microparticles has the potential to enhance transgene expression. However, barriers to this approach include limited encapsulation efficiency, pDNA damage during fabrication and confinement of the microparticles inside phagolysosomal compartments. Combining PLGA with poly ethyleneimine (PEI) can improve protection of pDNA during fabrication, increase encapsulation efficiencies and impart the PLGA microparticles with the capacity to escape the phagolysosomal compartments. This study compares three promising formulation methods for preparing PLGA PEI pDNA microparticles and evaluates for buffering capacity, cellular uptake, transfection efficiency and toxicity. In the first method, PLGA PEI pDNA microparticles are prepared by entrapping pDNA in blended PLGA/PEI using the double emulsion water-in-oil-in-water solvent evaporation technique (PA). In a second approach, PEI-pDNA polyplexes are prepared and then entrapped in PLGA microparticles using a double emulsion solvent evaporation method (PB). Microparticles prepared using formulation methods PA and PB are then compared against PLGA microparticles with PEI conjugated to the surface using carbodiimide chemistry (PC); 0.5% PVA is identified as the optimum concentration of surfactant for generating the strongest transfection efficiencies. N:P ratios of 5 and 10 are selected for preparation of each group. Gel electrophoresis demonstrates that all PLGA microparticle formulations have strong pDNA binding capacity. An MTT assay shows that in vitro cytotoxicity of PLGA PEI microparticles is significantly lower than PEI alone. PLGA PEI pDNA microparticles mediate higher cellular uptake efficiency and consequently higher transgene expression than unmodified PLGA microparticles in COS7 and HEK293 cells. Preparing PEI-pDNA polyplexes prior to entrapment in PLGA microparticles (PB) results in the highest pDNA loading. This is 2.5-fold higher than pDNA loading in unmodified PLGA microparticles. PLGA PEI pDNA microparticles prepared using method PB generates the strongest transfection efficiencies, which are 500-fold higher than unmodified PLGA pDNA microparticles in HEK293 cells and 1800-fold higher in COS-7 cells. The highest transfection efficiencies generated from microparticles prepared using method PB is achieved using an N:P ratio of 5.