Non-Viral Gene Delivery Laboratory, Discipline of Biochemistry University of KwaZulu-Natal, School of Life Sciences, Private Bag X54001, Durban 4000, South Africa.
J Nanosci Nanotechnol. 2019 Apr 1;19(4):1959-1970. doi: 10.1166/jnn.2019.15798.
Gene therapy has opened doors for the treatment of genetic disorders such as cancer. However, it clinical application has been limited by safety-efficacy issues. Recently, dendritic stabilized metal nanoparticles have shown great potential as efficient non-viral modalities for plasmid DNA delivery. This study involved the synthesis, characterisation, evaluation of the cytotoxicity profiles, and ability of unmodified and folic acid modified poly(amidoamine) generation 5 dendrimer grafted gold nanoparticles to deliver plasmid DNA containing a luciferase gene to cancer cells Unmodified and folic acid modified poly(amidoamine) generation 5 dendrimer nanoparticles were also evaluated for comparative studies. Nanocomplexes prepared with folic acid unmodified/modified poly(amidoamine) grafted gold nanoparticles and plasmid DNA were characterized by transmission electron microscopy, nanoparticle tracking analysis, ultra-violet spectroscopy, nuclear magnetic resonance, band shift, dye displacement and nuclease protection assays. Cytotoxicity profiles and gene expression were evaluated in five mammalian cell lines, using the MTT cell viability and luciferase reporter gene assays respectively. Nanocomplexes at optimum / ratios of 5.2:1 and 6.0:1, protected the plasmid DNA against serum nucleases and were well tolerated by all cell lines. Transgene expression was higher with folic acid modified dendrimer grafted gold nanoparticles, in folic acid-receptor overexpressing tested cells, compared to that of the control dendrimer nanoparticles, decreasing significantly ( <0.05) in the presence of excess folic acid ligand, confirming nanocomplex uptake to be via receptor mediation. Overall, the transfection efficiency of the dendrimer modified gold nanocomplexes superseded that of the control dendrimer nanocomplexes indicating the importance of dendrimer modification and the significant role of gold nanoparticles in the formulation of these delivery systems.
基因治疗为癌症等遗传疾病的治疗开辟了道路。然而，其临床应用受到安全性和疗效问题的限制。最近，树突状稳定的金属纳米粒子作为有效的非病毒载体，在质粒 DNA 传递方面显示出巨大的潜力。本研究涉及合成、表征、评估细胞毒性谱，以及未修饰和叶酸修饰的聚（酰胺-胺）第五代树枝状大分子接枝金纳米粒子传递含有荧光素酶基因的质粒 DNA 的能力。还对未修饰和叶酸修饰的聚（酰胺-胺）第五代树枝状大分子纳米粒子进行了比较研究。通过透射电子显微镜、纳米粒子跟踪分析、紫外光谱、核磁共振、带位移、染料置换和核酸酶保护实验对未修饰/修饰叶酸的聚（酰胺-胺）接枝金纳米粒子与质粒 DNA 制备的纳米复合物进行了表征。采用 MTT 细胞活力测定和荧光素酶报告基因测定，在五种哺乳动物细胞系中评估了细胞毒性谱和基因表达。在最佳/比例为 5.2:1 和 6.0:1 时，纳米复合物可保护质粒 DNA 免受血清核酸酶的影响，且所有细胞系均能耐受。在叶酸受体过表达的测试细胞中，与对照树枝状大分子纳米粒子相比，叶酸修饰的树枝状大分子接枝金纳米粒子的转染效率更高，在存在过量叶酸配体的情况下，显著降低（<0.05），证实纳米复合物的摄取是通过受体介导的。总的来说，修饰后的树枝状大分子金纳米复合物的转染效率优于对照树枝状大分子纳米复合物，表明树枝状大分子修饰和金纳米粒子在这些递药系统中的重要性。
