Kim Byungji, Sailor Michael J
Materials Science and Engineering Program, University of California, San Diego;
Materials Science and Engineering Program, University of California, San Diego; Department of Chemistry and Biochemistry, University of California, San Diego.
J Vis Exp. 2019 Apr 16(146). doi: 10.3791/59440.
With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic porous silicon nanoparticles (F-pSiNPs) have recently demonstrated high in vivo gene silencing efficacy due to its high oligonucleotide loading capacity and unique cellular uptake pathway that avoids endocytosis. The synthesis of F-pSiNPs is a multi-step process that includes: (1) loading and sealing of oligonucleotide payloads in the silicon pores; (2) simultaneous coating and sizing of fusogenic lipids around the porous silicon cores; and (3) conjugation of targeting peptides and washing to remove excess oligonucleotide, silicon debris, and peptide. The particle's size uniformity is characterized by dynamic light scattering, and its core-shell structure may be verified by transmission electron microscopy. The fusogenic uptake is validated by loading a lipophilic dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), into the fusogenic lipid bilayer and treating it to cells in vitro to observe for plasma membrane staining versus endocytic localizations. The targeting and in vivo gene silencing efficacies were previously quantified in a mouse model of Staphylococcus aureus pneumonia, in which the targeting peptide is expected to help the F-pSiNPs to home to the site of infection. Beyond its application in S. aureus infection, the F-pSiNP system may be used to deliver any oligonucleotide for gene therapy of a wide range of diseases, including viral infections, cancer, and autoimmune diseases.
随着基因治疗的出现，开发一种有效的体内核苷酸载体递送系统变得同等重要。由于其高寡核苷酸负载能力和避免内吞作用的独特细胞摄取途径，融合性多孔硅纳米颗粒（F-pSiNPs）最近已显示出高体内基因沉默功效。F-pSiNPs的合成是一个多步骤过程，包括：（1）在硅孔中加载和密封寡核苷酸载体；（2）在多孔硅核周围同时包覆融合性脂质并调整尺寸；（3）连接靶向肽并洗涤以去除过量的寡核苷酸、硅碎片和肽。通过动态光散射表征颗粒的尺寸均匀性，其核壳结构可通过透射电子显微镜进行验证。通过将亲脂性染料1,1'-二辛基-3,3,3',3'-四甲基吲哚碳菁高氯酸盐（DiI）加载到融合性脂质双层中并在体外处理细胞以观察质膜染色与内吞定位，来验证融合性摄取。靶向和体内基因沉默功效先前已在金黄色葡萄球菌肺炎小鼠模型中进行了量化，其中靶向肽有望帮助F-pSiNPs归巢到感染部位。除了其在金黄色葡萄球菌感染中的应用外，F-pSiNP系统还可用于递送任何寡核苷酸，用于多种疾病的基因治疗，包括病毒感染、癌症和自身免疫性疾病。
