Plasmon-resonant nanoparticles provide unprecedented spatiotemporal control over the release of diverse cargoes into cells. Here we compare the loading, release and internalization efficiencies, and effectiveness of post transcriptional gene silencing of hollow gold nanoshells, hollow gold nanocages, and gold nanorods with plasmons tuned to absorb near-infrared light at 800 nm. The hollow gold nanoshells can be loaded with up to three times more siRNA cargo compared to nanocages and nanorods; however, nanorods exhibit the highest efficiency of release of attached siRNA strands when exposed to pulsed 800 nm laser excitation. In cellular treatments, all particles demonstrated efficient internalization into HeLa cells, but the nanoshells and nanocages display the highest downregulation of GFP expression 72 h after treatment. These results provide novel insights into the relative efficiencies of three structurally distinct types of gold nanoparticles as siRNA carriers and we examine different parameters that may influence their efficacy.

A light-activated genome editing platform based on the release of enzymes from a plasmonic nanoparticle carrier when exposed to biocompatible near-infrared light pulses is described. The platform relies on the robust affinity of polyhistidine tags to nitrilotriacetic acid in the presence of copper which is attached to double-stranded nucleic acids self-assembled on the gold nanoparticle surface. A protein fusion of the Cre recombinase containing a TAT internalization peptide sequence to achieve endosomal localization is also employed. High-resolution gene knock-in of a red fluorescent reporter is observed using a commercial two-photon microscope. High-throughput irradiation is described to generate useful quantities of edited cells.

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Tracking intracellular proteins in live cells has many challenges. The most widely used method, fluorescent protein fusions, can track proteins in their native cellular environment and has led to significant discoveries in cell biology. Fusion proteins add steric bulk to the target protein and can negatively affect native protein function. The use of exogenous probes such as antibodies or protein labels is problematic because these cannot cross the plasma membrane on their own and thus cannot label intracellular targets in cells. We developed a labeling platform, VIPER nano , for live cell imaging of intracellular proteins using a peptide fusion tag (CoilE) to the protein of interest and delivery of a fluorescently labeled probe peptide (CoilR). CoilR and CoilE form an αhelical heterodimer with the protein of interest, rendering a labeled protein. Delivery of CoilR into the cell uses hollow gold nanoshells (HGNs) as the primary delivery vehicle. The technology relies on the conjugation and light-activated release of the CoilR peptide on the surface of the HGNs. We demonstrate light-activated VIPER nano delivery and labeling with two intracellular proteins, localized either in the mitochondria or the nucleus. This technology has the ability to study intracellular protein dynamics and spatial tracking while lessening the steric bulk of tags associated with the protein of interest.

Intracellular delivery of biomolecules is hindered by the size and charge of the biomolecule. While a number of delivery methods are available for genetic material, few are available for larger biomolecules like peptides and proteins. The delivery of these biomolecules are often limited to viral transfections of the genetic material which can lead to off target gene editing due to prolonged expression of a protein within a cell. We have developed a delivery method for poly‐histidine tagged proteins and peptides of variable sizes and charges for transient protein expression with light control via hollow gold nanoshells (HGNs). The construct of our delivery system involves a thiolated DNA strand containing an NTA modification on the 3′ end attached to the gold surface. A his‐tagged protein or peptide of interest is attached to the HGN via copper‐NTA affinity. Endosomal uptake of these particles is mediated through an orthogonal strand containing a cell penetrating peptide (CPP). Protein release and endosomal disruption is achieved after irradiation with a focused femtosecond pulsed‐ laser of biologically benign near‐infrared light. With this technology we have successfully delivered multiple proteins of interest including the gene editing protein Cas9 for efficient knockout of GFP with light control as well as the Nur77 derived apoptotic peptide NuBCP which allowed for an increase in efficiency with our delivery method.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.