Service of Development of Medicines (SDM), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain; Department of Molecular Biology, Institute of Parasitology and Biomedicine "López-Neyra" (IPBLN-CSIC), Granada, Spain; Institute of Research in Microbiology and Medical Parasitology (IMPaM), Faculty of Medicine, University of Buenos Aires-CONICET, Buenos Aires, Argentina.
Service of Development of Medicines (SDM), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain; Department of Molecular Biology, Institute of Parasitology and Biomedicine "López-Neyra" (IPBLN-CSIC), Granada, Spain.
Colloids Surf B Biointerfaces. 2019 Dec 1;184:110533. doi: 10.1016/j.colsurfb.2019.110533. Epub 2019 Sep 29.
Nanoparticle-mediated plasmid delivery is considered a useful tool to introduce foreign DNA into the cells for the purpose of DNA vaccination and/or gene therapy. Cationic solid-lipid nanoparticles (cSLNs) are considered one of the most promising non-viral vectors for nucleic acid delivery. Based on the idea that the optimization of the components is required to improve transfection efficiency, the present study aimed to formulate and characterize cholesteryl oleate-containing solid-lipid nanoparticles (CO-SLNs) incorporating protamine (P) to condense DNA to produce P:DNA:CO-SLN complexes as non-viral vectors for gene delivery with reduced cytotoxicity and high cellular uptake efficiency. For this purpose, CO-SLNs were used to prepare DNA complexes with and without protamine as DNA condenser and nuclear transfer enhancer. The main physicochemical characteristics, binding capabilities, cytotoxicity and cellular uptake of these novel CO-SLNs were analyzed. Positively charged spherical P:DNA:CO-SLN complexes with a particle size ranging from 330.1 ± 14.8 nm to 347.0 ± 18.5 nm were obtained. Positive results were obtained in the DNase I protection assay with a protective effect of the genetic material and 100% loading efficiency was achieved at a P:DNA:CO-SLN ratio of 2:1:7. Transfection studies in human embryonic kidney (HEK293T) cells showed the versatility of adding protamine to efficiently transfect cells, widening the potential applications of CO-SLN-based vectors, since the incorporation of protamine induced almost a 200-fold increase in the transfection capacity of CO-SLNs without toxicity. These results indicate that CO-SLNs with protamine are a safe and effective platform for non-viral nucleic acid delivery.
纳米颗粒介导的质粒传递被认为是将外源 DNA 导入细胞的有用工具，目的是进行 DNA 疫苗接种和/或基因治疗。阳离子固体脂质纳米颗粒（cSLN）被认为是最有前途的核酸传递非病毒载体之一。基于优化成分以提高转染效率的理念，本研究旨在制备并表征含有胆固醇油酸的固体脂质纳米颗粒（CO-SLNs），并将其与鱼精蛋白（P）结合，用于压缩 DNA，以产生 P:DNA:CO-SLN 复合物，作为具有降低细胞毒性和高细胞摄取效率的非病毒载体进行基因传递。为此，使用 CO-SLNs 制备了具有和不具有鱼精蛋白的 DNA 复合物，鱼精蛋白用作 DNA 凝聚剂和核转移增强剂。分析了这些新型 CO-SLNs 的主要理化特性、结合能力、细胞毒性和细胞摄取。得到了粒径为 330.1 ± 14.8nm 至 347.0 ± 18.5nm 的带正电的球形 P:DNA:CO-SLN 复合物。在 DNA 酶 I 保护实验中得到了阳性结果，遗传物质具有保护作用，并且在 P:DNA:CO-SLN 比例为 2:1:7 时达到了 100%的载药效率。在人胚肾（HEK293T）细胞中的转染研究表明，添加鱼精蛋白可有效地转染细胞，从而拓宽了基于 CO-SLN 载体的潜在应用，因为鱼精蛋白的掺入在没有毒性的情况下诱导 CO-SLN 的转染能力增加近 200 倍。这些结果表明，载有鱼精蛋白的 CO-SLNs 是一种安全有效的非病毒核酸传递平台。
