Plasmid Transfection in Mammalian Cells Spatiotemporally Tracked by a Gold Nanoparticle

Abstract

Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to a AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to a AuNP allows spatiotemporal profiling of the transfection events, providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000 is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time-dependent expression of protein. The overall protein expression yield is enhanced when delivered on a AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape.

Virus Spike and Membrane-Lytic Mimicking Nanoparticles for High Cell Binding and Superior Endosomal Escape

“…As shown in Figure d, the interaction strength between 293T cells and Zn-DDAC or DDAC was quantified by quartz crystal microbalance (QCM). Zn-DDAC or DDAC is modified on the gold electrode of QCM via Au–S bond formation, and the frequency shift is proportional to the mass change on the QCM electrode. , As strong interaction exists between gold and cells, , the naked gold electrode exposed to cells resulted in a frequency shift of 29.9 Hz. However, the frequency shift decreased to 23.5 Hz after DDAC modification, which might be attributed to the spatial repulsive effect against cell adsorption.…”

Restoration of p53 Function in Ovarian Cancer Mediated by Gold Nanoparticle-Based EGFR Targeted Gene Delivery System

“…18 Gold-nanoparticle-based delivery systems (Aurimmune, gold-coated TNF-alpha; and Auroshell, gold-coated silica nanoparticles) are currently under phase II clinical trials. 19,20 Recent studies further point that gold nanoparticles mediate efficient transfection of nucleic acids into mammalian cells 21,22 and are hence utilized in the intervention of several cancers.…”

“…Because of their unique fundamental properties, (chemical, physical, electrical, and optical), gold nanoparticles (AuNPs) make ideal delivery vehicles, especially for varied biomedical applications . Gold-nanoparticle-based delivery systems (Aurimmune, gold-coated TNF-alpha; and Auroshell, gold-coated silica nanoparticles) are currently under phase II clinical trials. , Recent studies further point that gold nanoparticles mediate efficient transfection of nucleic acids into mammalian cells , and are hence utilized in the intervention of several cancers.…”

Enhanced Uptake of Luminescent Quantum Dots by Live Cells Mediated by a Membrane-Active Peptide

“…This can result in intracellular sequestration of the delivered nanomaterials within endosomal compartments. Alternative approaches that have been explored recently include use of chemical agents to disrupt endosomes following uptake or mechanical permeabilization of cell membranes (e.g., microinjection and electroporation). − However, these strategies require the use of exogenous reagents and stimuli that can induce NP aggregation, cell damage, and release of endosomal contents; these strategies are problematic or impractical to deploy in vivo. , …”