Programa de Pós-Graduação em Ciências Farmacêuticas da Universidade Federal do Rio Grande do Sul (UFRGS), Faculdade de Farmácia, Av. Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil; Centro de Terapia Gênica - Hospital de Clinicas de Porto Alegre, R. Ramiro Barcelos 2350, 90035-903 Porto Alegre, RS, Brazil.
Programa de Pós-Graduação em Genética e Biologia Molecular da Universidade Federal do Rio Grande do Sul (UFRGS), Campus do Vale, Av. Bento Gonçalves, 9500, 91501-970 Porto Alegre, RS, Brazil; Centro de Terapia Gênica - Hospital de Clinicas de Porto Alegre, R. Ramiro Barcelos 2350, 90035-903 Porto Alegre, RS, Brazil.
J Colloid Interface Sci. 2018 Nov 15;530:243-255. doi: 10.1016/j.jcis.2018.06.058. Epub 2018 Jun 22.
In this study, we investigated the effects of the association of a single plasmid or its co-complexation along with an oligonucleotide on the physicochemical properties of cationic nanoemulsions and liposomes intended for gene editing. Formulations composed of DOPE, DOTAP, DSPE-PEG (liposomes), MCT (nanoemulsions), and water were obtained by microfluidization. DSPE-PEG was found to play a crucial role on the size and polydispersity index of nanocarriers. Nucleic acids were complexated by adsorption at different charge ratios. No significant differences were noticed in the physicochemical properties of nanocarriers (i.e. droplet size, polydispersity index, or zeta potential) when a single plasmid or both plasmid and oligonucleotide were adsorbed to the formulations. Transmission electron microscopy photomicrographs suggested round nanostructures with the nucleic acids and DSPE-PEG enfolding the surface. Complexes at +4/-1 charge ratio protected nucleic acids against DNase I degradation. The oligonucleotide seemed to be released from the liposomal complexes, while nanoemulsions only released the plasmid after 24 and 48 h of incubation in DMEM supplemented or not. In vitro experiments demonstrated that complexes were highly tolerable to human fibroblasts, Hep-G2, and HEK-293 cells, demonstrating also an uptake ability of about 30%, 30%, and 90%, respectively, no matter what the formulation or the combination of nucleic acids used. Transfection efficiency of the formulations was around 25% in human fibroblasts, 32% in HEK-293, and 15% in Hep-G2 cells. The overall results demonstrated the behavior of liposomes and nanoemulsions complexed with a plasmid or a mixture of a plasmid and an oligonucleotide, and demonstrated that the association with one or two nucleic acids sequences of different length does not seem to interfere in the physicochemical characteristics of complexes or in the uptake capacity by three different types of cells.
在这项研究中，我们研究了单个质粒或其与寡核苷酸共复合对用于基因编辑的阳离子纳米乳液和脂质体的物理化学性质的影响。通过微流化法获得由 DOPE、DOTAP、DSPE-PEG（脂质体）、MCT（纳米乳液）和水组成的制剂。发现 DSPE-PEG 对纳米载体的大小和多分散指数起着至关重要的作用。核酸通过不同的电荷比进行吸附复合。当单一质粒或质粒和寡核苷酸被吸附到制剂中时，纳米载体的物理化学性质（即粒径、多分散指数或 Zeta 电位）没有明显差异。透射电子显微镜照片表明，纳米结构呈圆形，表面包裹着核酸和 DSPE-PEG。在+4/-1 电荷比下，复合物可保护核酸免受 DNase I 降解。寡核苷酸似乎从脂质体复合物中释放出来，而纳米乳液仅在补充或不补充 DMEM 的情况下孵育 24 和 48 小时后才释放质粒。体外实验表明，复合物对人成纤维细胞、Hep-G2 和 HEK-293 细胞具有高度耐受性，分别显示约 30%、30%和 90%的摄取能力，无论制剂或使用的核酸组合如何。制剂在人成纤维细胞中的转染效率约为 25%，在 HEK-293 中的转染效率为 32%，在 Hep-G2 细胞中的转染效率为 15%。总体结果表明，与质粒或质粒和寡核苷酸混合物复合的脂质体和纳米乳液的行为，并且表明与一个或两个不同长度的核酸序列的关联似乎不会干扰复合物的物理化学特性或三种不同类型细胞的摄取能力。
