Intracellular DNA Cargo Release from a Gold Nanoparticle Modulated by the Nature of the Surface Coupling Functionality.

Covalently coupling nucleic acids to a gold nanoparticle (AuNP) surface has been demonstrated for generating effective gene therapy agents to modify cellular protein expression. The therapeutic efficacy of the approach is anticipated to be impacted by the length of time the nucleic acid sequence resides in the endolysosomal pathway once transfected into a cell. It is believed that the dynamics of the processing should reflect the linkage chemistry of the DNA to the AuNP surface. In this manuscript the dynamics of nanotherapeutic uptake, nucleic acid release, and gene processing are investigated  in vitro for a AuNP-nucleic acid delivery platform transfected into A375 human melanoma cells, as a function of the nucleic acid-gold linkage chemistry. The dynamics of cell processing of the single monodentate thiol (SX), bidentate dual thiol (SS), or mixed bidentate thiol plus amine (SN) coordination of nucleic acids to the AuNP surface are evaluated using a multicolor nanosurface energy transfer bio-optical transponder (SET-BOT) technology. The use of live-cell fluorescence microscopy allows for the direct visualization of the uptake and localization of a lipofectamine-packaged SET-BOT using a dye (DL700) that is not quenched in the proximity of the AuNP, while fluorescence "turn on" of a dye that is proximally quenched by the AuNP (DL488) is used to report on the dynamics of release of the nucleic acid cargo within the cell. For protein expression following transcription of the gene, the emission signature of a red fluorescent protein, tdTomato, is monitored. The intracellular rates of DNA release from the AuNP surface once endosomally packaged within the A375 human melanoma cells were found to follow the binding activity series: bidentate thiol > bidentate thiol plus amine > monodentate thiol, consistent with the strength of multidentate chelation, paired with the stabilizing influence of π-backbonding of thiols compared to σ-donation in amines, when bound to a gold surface.

Selective Uptake Into Drug Resistant Mammalian Cancer by Cell Penetrating Peptide-Mediated Delivery.

Research over the past decade has identified several of the key limiting features of multidrug resistance (MDR) in cancer therapy applications, such as evolving glycoprotein receptors at the surface of the cell that limit therapeutic uptake, metabolic changes that lead to protection from multidrug resistant mediators which enhance degradation or efflux of therapeutics, and difficulty ensuring retention of intact and functional drugs once endocytosed. Nanoparticles have been demonstrated to be effective delivery vehicles for a plethora of therapeutic agents, and in the case of nucleic acid based agents, they provide protective advantages. Functionalizing cell penetrating peptides, also known as protein transduction domains, onto the surface of fluorescent quantum dots creates a labeled delivery package to investigate the nuances and difficulties of drug transport in MDR cancer cells for potential future clinical applications of diverse nanoparticle-based therapeutic delivery strategies. In this study, eight distinct cell penetrating peptides were used (CAAKA, HSV1-VP22, HIV-TAT, HIV-gp41, Ku-70, hCT(9-32), integrin-ß3, and K-FGF) to examine the different cellular uptake profiles in cancer versus drug resistant melanoma (A375 & A375-R), mesothelioma (MSTO & MSTO-R), and glioma (rat 9L and 9L-R, and human U87 & LN18) cell lines. The results of this study demonstrate that cell penetrating peptide uptake varies with drug resistance status and cell type, likely due to changes in cell surface markers. This study provides insight into developing functional nanoplatform delivery systems in drug resistant cancer models.

A Gold Nanoparticle Bio-Optical Transponder to Dynamically Monitor Intracellular pH.

A pH-sensitive bio-optical transponder (pH-BOT) capable of simultaneously reporting the timing of intracellular DNA cargo release from a gold nanoparticle (AuNP) and the evolving intracellular pH (pH i) during endosomal maturation is demonstrated. The pH-BOT is designed with a triple-dye-labeled duplex DNA appended to a 6.6 nm AuNP, utilizing pH-responsive fluorescein paired with DyLight405 as a surface energy transfer (SET) coupled dye pair to ratiometrically report the pH at and after cargo release. A non-SET-coupled dye, DyLight 700, is used to provide dynamic tracking throughout the experiment. The pH-BOT beacon of the cargo uptake, release, and processing was visualized using live-cell confocal fluorescent microscopy in Chinese hamster ovary cells, and it was observed that while maturation of endosomes carrying pH-BOT is slowed significantly, the pH-BOT is distributed throughout the endolysosomal system while remaining at pH ∼6. This observed decoupling of endosomal maturation from acidification lends support to those models that propose that pH alone is not sufficient to explain endosomal maturation and may enable greater insight into our understanding of the fundamental processes of biology.

Plasmid transfection in mammalian cells spatiotemporally tracked by a gold nanoparticle.

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.