A Delivery Method for Poly‐histidine Tagged Proteins and Peptides for Transient Protein Expression with Light Control via Hollow Gold Nanoshells: Successful delivery of CRISPR Cas9 and Apoptotic Peptide NuBCP with NIR light control.

Abstract

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.

Genetic and Covalent Protein Modification Strategies to Facilitate Intracellular Delivery

“…To limit the impact of mutagenesis on protein activity while maximizing changes that assist protein delivery, short sequences have been appended to the N- or C-terminus of native proteins. These polypeptide tags have been genetically added and can confer a range of properties including supercharging, − the ability to complex with divalent metal ions, − and specific cellular localization. ,− Charged tags have been added as an alternative to surface supercharging. Following the initial demonstration that supercationic GFP’s facilitate cytosolic delivery, GFP was supercharged via C-terminal fusion with an elastin-like polypeptide .…”

“…Nuclear localization sequences have been appended to cas9 to facilitate transport across the nuclear membrane after cytosolic delivery . Efficient delivery into the cytosol has been accomplished through a range of approaches, including encapsulation of anionically tagged cas9 in cationic nanoparticles, conjugation with CPPs, complexation with lipid nanoparticles, ,,, complexation with inorganic nanoparticles, ,,, fusion with a supercharged polypeptide, and encapsulation in biodegradable polymer nanocapsules . Approaches for cas9 delivery are similar to approaches for model proteins, but, the large size, sensitivity to pH, and loss of activity upon addition of point mutations represent additional complications for cas9 delivery.…”