Targeted Intracellular Delivery of Proteins with Spatial and Temporal Control

Abstract

While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.

Light‐Triggered Genome Editing: Cre Recombinase Mediated Gene Editing with Near‐Infrared Light

“…The light‐activated Cre recombinase platform is based on the efficient cleavage of surface‐bound scaffolds from plasmonic nanoparticles and subsequent generation of nanobubbles for endosomal escape when exposed to ultrafast pulsed laser irradiation ( Scheme a) . To minimize off target light absorption we utilize HGN resonant at 800 nm NIR light to achieve maximal cell penetration . The particles are assembled in a bottom‐up approach as illustrated in Scheme b.…”

“…red fluorescent protein (DsRed) reporter that, upon DNA recombination of the upstream promoter region, begins expression of a fluorescent protein. We demonstrate that Cre recombinase is completely dependent on the exposure of the nanoshells to light to release the recombinase from the nanoparticle surface and endosomal escape by nanobubble generation . The present system offers a method to achieve unprecedented light control of gene regulation beyond siRNA knockdown.…”

“…More recently, CRISPR/Cas9 systems were engineered to respond to chemical inducers . While allowing temporal control of recombination events, there is an increasing level of interest in the ability to control genome editing with precise spatial control as well …”

Rational Design of Nanocarriers for Intracellular Protein Delivery

“…[157,158] With this system, any recombinant protein labelled a commonly used tag (e.g., His and GST) could be purified and loaded onto the aptamer-conjugated AuNPs. [159] The DNA layer provided terminal amine groups used to conjugate the Ni 2+ and protein chelator. Furthermore, epidermal growth factor (EGF), a target protein could be loaded onto the AuNP-(His-GST)-aptamer system with BIM protein together, showing significantly uptake in mice and growth inhibition of xenograft tumors without detectable systemic toxicity.…”

Light‐Patterned RNA Interference of 3D‐Cultured Human Embryonic Stem Cells

“…NIR light can be tightly focused threedimensionally (down to ≈1 fL) [27] and reach deep through tissues (up to 10 cm). [28,29] Small interfering RNAs (siRNAs) with thiol-modification are covalently attached to hollow gold nanoshells (HGNs) via quasicovalent AuS bonds, [30][31][32] followed by the functionalization of a cell-penetrating transactivating transcriptional activator (TAT) peptide. [33] Upon endosomal uptake, NIR light irradiation releases the RNAs from the…”