Potent Delivery of Functional Proteins into Mammalian Cells in Vitro and in Vivo Using a Supercharged Protein

Abstract

The inability of proteins to potently penetrate mammalian cells limits their usefulness as tools and therapeutics. When fused to superpositively charged GFP, proteins rapidly (within minutes) entered five different types of mammalian cells with potency up to ∼100-fold greater than that of corresponding fusions with known protein transduction domains (PTDs) including Tat, oligoarginine, and penetratin. Ubiquitin-fused supercharged GFP when incubated with human cells was partially deubiquitinated, suggesting that proteins delivered with supercharged GFP can access the cytosol. Likewise, supercharged GFP delivered functional, nonendosomal recombinase enzyme with greater efficiencies than PTDs in vitro and also delivered functional recombinase enzyme to the retinae of mice when injected in vivo.

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

“…Further, we demonstrated that the integration of a bioreducible disulfide bond into the hydrophobic tail of the lipid enhances efficiency of small interfering RNA (siRNA) delivery (14), due to the improved endosomal escape and cargo release following lipid degradation in the reductive intracellular environment. Meanwhile, we engineered supercharged proteins shown to enhance protein delivery by fusing superpositively charged GFP to a protein of interest (15)(16)(17) and using cationic-lipid mediated delivery of supernegatively charged proteins (4). We hypothesized that combining cationic bioreducible lipids and supernegatively charged proteins would drive electrostatic selfassembly of a supramolecular nanocomplex to deliver the genome-editing protein (Fig.…”

Biodegradable double nanocapsule as a novel multifunctional carrier for drug delivery and cell imaging

“…36,37 Double nanocapsules designed to encapsulate proteins in their separated microsphere structures can protect the structural and functional integrities of proteins when they are transported into the cytosol and then redistributed within the cells. [38][39][40] Besides the transport of BSA into the cytosol, the study showed that the nanocapsules delivered NLS-GFP into the nucleus, indicating that the structure of nanocapsules did not interfere with the migration of proteins.…”

Electrostatics in Protein Engineering and Design

“…Currently, the leading strategy to improve cellular uptake is to express the target protein as a fusion with one of several polycationic amino acid sequences derived from natural cell-penetrating peptides (Heitz et al 2009). In a recent study, positively supercharged GFP was shown to be capable of entering a range of mammalian cells, and of delivering fused protein payloads more effectively than the standard cationic fusion tags Tat, Arg 10 , and penetratin (Cronican et al 2010). …”