UC Santa Barbara

Size and charge of proteins can hinder the intracellular delivery of biomolecules. Larger biomolecules, like proteins and peptides, have fewer delivery methods than those available for genetic material, which have a number of different delivery methods. This often limits the delivery of these larger biomolecules to viral transfections of the desired genetic material, but these can lead to gene editing of non-desired targets, due to the extended expression of the protein within cells. We’ve created and studied a delivery method into cells using proteins and peptides of various sizes and charges with poly-histidine tags for light-controlled delivery with hollow gold nanoshells (HGNs). The design of our delivery system includes a thiolated linker that is attached to the gold’s surface and that has a nitrilotriacetic acid (NTA), biotin, or carboxylic acid (COOH) functional group on the end with polyethylene glycol (PEG) in between. Through copper-NTA affinity or 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (EDC) chemistry, a histidine-tagged peptide or protein of interest is coupled to the HGN. Cell-penetrating peptides (CPP) on an orthogonal linker are used to accomplish endosomal uptake of the HGN. After irradiation with a pulsed femtosecond laser with non-damaging near-infrared light (NIR), endosomal disruption and protein release is accomplished. This thesis covers the construction and visualization of protein constructs using live and fixed cell imaging with DLCPT (Direct Live Cell Protein Tracking).