Direct Functional Protein Delivery with a Peptide into Neonatal and Adult Mammalian Inner Ear In Vivo

Abstract

The aim of this study was to study an antimicrobial peptide (AMP), aurein 1.2, which substantially increased protein delivery directly into multiple mammalian inner-ear cell types in vivo . Different concentrations of aurein 1.2 with superpositively charged GFP (+36 GFP) protein fused with Cre recombinase were delivered to postnatal day 1-2 (P1-2) and adult cochleae of Cre reporter transgenic mice with various delivery methods. By cochleostomy at different concentrations of aurein 1.2–+36 GFP (1 μM, 5 μM, 22.5 μM, and 50 μM, respectively), the tdTomato (tdT) expression was observed in outer hair cells (OHCs; 20.77%, 23.02%, 76.36%, and 92.47%, respectively) and inner hair cells (IHCs; 14.90%, 44.50%, 89.59%, and 96.13%, respectively) in the cochlea. The optimal concentration was 22.5 μM with the highest transfection efficiency and the lowest cytotoxicity. Wide-spread tdT signals were detected in the cochlear-supporting cells, utricular-supporting cells, auditory nerve, and spiral ligament in neonatal and adult mice. Compared to cochleostomy, injection through the round window membrane (RWM) also produced highly efficient tdT+ labeled cells with less cell loss. In summary, the peptide aurein 1.2 fused with +36 GFP dramatically expanded the target cells with increased efficiency in direct protein delivery in the inner ear. Aurein 1.2–+36 GFP has the potential to be developed as protein-based therapy in regeneration and genome editing in the mammalian inner ear.

Improved transfer efficiency of supercharged 36 + GFP protein mediate nucleic acid delivery

“…Previous studies suggested that supercharging with cationic Lys/Arg/His residues allow supercharging proteins to control their properties such as temperature resistance, unusual resistance to aggregation and catalytic activity (Prasse et al., 2014 ; Ma et al., 2020 ). Although published reports indicated that 36 + GFP can enter the cultured cells, and mediate fusion protein and siRNA fragment (McNaughton et al., 2009 ) in vitro and in vivo (Li et al., 2015 ; Zhang et al., 2020 ), we noted that the efficiency of 36 + GFP based delivery was not high, which may be entrapped in the endosome. Moreover, even though cationic peptide fused with 36 + GFP can facilitate the endosomal escape, it is still limited (Li et al., 2015 ).…”

“…All engineered supercharged green fluorescent proteins (GFPs, net theoretical charges: −30 to +48) exhibit nearly identical emission and excitation spectrum as starting GFP. References suggested that 36 + GFP can enter the cells and mediate fusion protein (Motevalli et al., 2018 ) and siRNA (McNaughton et al., 2009 ) delivery into cultured cells in vitro and the inner ear of live mice in vivo (Li et al., 2015 ; Zhang et al., 2020 ); however, we noted that the efficiency of 36 + GFP based delivery was very low and not well-distributed in cytosol (Wu et al., 2015 ). Moreover, the majority of 36 + GFP protein was entrapped in the endosome (Thompson et al., 2012 ); although, the Aurein 1.2 as an antimicrobial peptide (AMP) can facilitate the endosomal escape of a variety of proteins fused to 36 + GFP in vitro , it only achieves 50% efficiency (Li et al., 2015 ).…”

Protein Corona Inhibits Endosomal Escape of Functionalized DNA Nanostructures in Living Cells

“…Recently a 13-residue peptide, termed aurein 1.2 (GLFDIIKKIAESF), was discovered that enhances endolysosomal escape and improved the cytosolic delivery of proteins it was appended to by up to ∼5-fold. , In fact, this peptide can disrupt endolysosomal membranes and in such a way trigger the escape of cargo to cytosol. ,, Importantly, aurein facilitates endolysosomal escape without concomitant disruption of the cell membrane and does not exhibit cytotoxicity. ,, Therefore, we used this peptide to electrostatically coat DNs (Figure c) for potential enhancement of endolysosomal escape.…”

“…59,102 In fact, this peptide can disrupt endolysosomal membranes and in such a way trigger the escape of cargo to cytosol. 59,102,103 Importantly, aurein facilitates endolysosomal escape without concomitant disruption of the cell membrane and does not exhibit cytotoxicity. 59,102,103 Therefore, we used this peptide to electrostatically coat DNs (Figure 1c) for potential enhancement of endolysosomal escape.…”

Protein Delivery to Insect Epithelial Cells In Vivo: Potential Application to Functional Molecular Analysis of Proteins in Butterfly Wing Development

“…However, the obstacle that proteins are often incorporated into a cell by endocytosis and are confined within endosomes has not been completely overcome. Several different peptides and their modified versions have been tested for more efficient endosomal escape [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. Due to the technological demands for quick protein delivery to cells with fewer technical requirements, membrane-lytic peptides are receiving more attention from researchers in the twenty-first century.…”