Kundu Anup K, Iyer Swathi V, Chandra Sruti, Adhikari Amit S, Iwakuma Tomoo, Mandal Tarun K
Center for Nanomedicine and Drug Delivery, Xavier University College of Pharmacy, New Orleans, Louisiana, United States of America.
Department of Biology, Xavier University of Louisiana, New Orleans, Louisiana, United States of America.
PLoS One. 2017 Jun 21;12(6):e0179168. doi: 10.1371/journal.pone.0179168. eCollection 2017.
The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318-1, a murine osteosarcoma cell line.
The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP) and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency.
Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent.
This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.
肿瘤抑制因子p53在骨肉瘤的发展中起关键作用。本研究的主要目的是开发和优化基于脂质的纳米颗粒制剂，该制剂能够携带小干扰RNA（siRNA）并有效沉默小鼠骨肉瘤细胞系318-1中的突变型p53。
纳米颗粒由两种脂质（胆固醇和DOTAP）与聚乳酸-羟基乙酸共聚物（PLGA）或聚乳酸-羟基乙酸共聚物-聚乙二醇（PLGA-PEG）的混合物组成，使用EmulsiFlex-B3高压均质器制备。进行了一系列研究，包括使用不同的纳米颗粒、不同量的siRNA、细胞数量、孵育时间、转染培养基体积和储存温度，以优化基因沉默效率。
用PLGA或PLGA-PEG替代脂质可降低颗粒大小和总体细胞毒性。在所有脂质-聚合物纳米制剂中，当纳米颗粒与siRNA的比例等于6.8:0.66且使用75 nM siRNA时，含10%PLGA的纳米颗粒在保持较高细胞活力的同时，显示出最高的突变型p53敲低效率。长期储存时，突变型p53敲低效率下降幅度更大。
本研究值得在未来对该制剂在组织培养和动物模型中沉默突变型p53基因以治疗骨肉瘤的效率进行评估。
