Introduction: The utility of recombinant proteins as potential therapeutics and cellular tools are largely impeded by endosome entrapment and susceptibility of delivered proteins in lysositic environments and degradative enzymes. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis, and deliver the cargo to the cytosol by light triggered endosomal escape in a timely manner. The delivery platform is based on plasmonic hollow gold nanoshells (HGN) assembled with a cargo linking layer based on nickel affinity capable of supporting virtually any soluble poly-histidine tagged proteins. Transport into endosomes is mediated by the fusion of cell-penetrating peptides onto the protein of interest or by presenting an orthogonal internalizing handle. Once internalized, cytosolic localization of the protein of interest is achieved by excitation of nanocarriers with pulsed near-infrared (NIR) light to trigger protein release and disruption of the endosome by nanobubble formation. We demonstrate this method has unprecedented spatial and temporal control of protein release at the single cell level that can be monitored in real time.
Materials and Methods: HGN are synthesized by the galvanic exchange of a sacrificial silver nanoparticle template and decorated with affinity handles for recombinant protein attachment by self-assembly of robust thiol scaffolds to gold surfaces. Particles are internalized into human cancer cell lines and then exposed to 800 nm NIR light using a commercial two-photon confocal microscope for high resolution release of protein inside cells.
Results and Discussion: Using the HGN carrier system we achieved spatial and temporally controlled release of the green fluorescent protein (GFP), the pro-apoptotic peptide (KLAKLAK)2, and the DNA editing enzyme, Cre recombinase. The method requires significantly less protein than the transduction of free protein, has minimal deleterious effects on cell viability, and most notably, cells in the periphery of region irradiated by light remain unaffected by treatment clearly showing the spatial resolution of this technology (Figure 1).
Figure 1. Left: Hollow gold nanoshell (HGN) platform designed for intracellular protein delivery modeled with GFP. Right: NIR light release of GFP in PPC-1 prostate cancer cells using a commercial two-photon microscope. Before irradiation, the GFP is sequestered in endosomes. Only after irradiation does the GFP release into the cytosol. Cells outside the red box that were not excited by NIR light showing no change in GFP fluorescence profile.
Conclusion: In summary we demonstrate with unprecedented spatial and temporal control recombinant protein release inside mammalian cell lines and can monitor its activity in real-time. This technique opens the possibility of delivering various His-tagged proteins to the cytosol of cells, individually or in concert, and could enable optical control for stem cell differentiation, selective apoptosis, or cell specific gene editing.