Departamento de Engenharia Química, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; CQE, Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Portugal.
Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal.
Mater Sci Eng C Mater Biol Appl. 2016 Jun;63:546-53. doi: 10.1016/j.msec.2016.03.006. Epub 2016 Mar 5.
Gene therapy stays on the cutting edge of biomedical research, being the design of the optimal gene delivery vector one of the key requests. Silica-based nanoparticles (NPs) have emerged as promising non-viral gene delivery vector, due to their high biocompatibility, nontoxicity, non-immunogenicity, biodegradability and enormous bioconjugation versatility. In this work a sol-gel methodology for the synthesis of amino-functionalized silica NPs (NH2-ORMOSIL NPs) was optimized, and NPs were characterized by TEM and FTIR. In a first step NH2-ORMOSIL NPs were bioconjugated with a plasmid DNA, pVAX1-GFP, assembling an ORMOPLEXE, confirmed by agarose gel electrophoresis. In a second step, in vitro studies have been performed with cultured CHO cells, where ORMOPLEXEs transfection was proved by CLSM. In vivo transfection efficiency and bio-distribution were performed in Zebrafish (Danio rerio) embryos, assessed by FM. Finally, NPs ecotoxicity was studied in zebrafish embryos by following the mortality and developmental endpoints.
基因治疗处于生物医学研究的前沿，设计最佳的基因传递载体是其中的关键要求之一。基于硅的纳米粒子（NPs）由于其高生物相容性、低毒性、无免疫原性、可生物降解性和巨大的生物缀合多功能性，已成为有前途的非病毒基因传递载体。在这项工作中，优化了一种用于合成氨基功能化硅纳米粒子（NH2-ORMOSIL NPs）的溶胶-凝胶方法，并通过 TEM 和 FTIR 对 NPs 进行了表征。在第一步中，NH2-ORMOSIL NPs 与质粒 DNA pVAX1-GFP 进行生物偶联，组装成 ORMOPLEXE，通过琼脂糖凝胶电泳得到证实。在第二步中，通过共聚焦显微镜（CLSM）证实了 ORMOPLEXE 在培养的 CHO 细胞中的转染。在斑马鱼（Danio rerio）胚胎中进行体内转染效率和生物分布研究，通过 FM 进行评估。最后，通过观察死亡率和发育终点，研究了 NPs 在斑马鱼胚胎中的生态毒性。
