Abstract
Protein delivery with cell-penetrating peptide is opening up the possibility of using targets inside cells for therapeutic or biological applications; however, cell-penetrating peptide-mediated protein delivery commonly suffers from ineffective endosomal escape and low tolerance in serum, thereby limiting in vivo efficacy. Here, we present an intracellular protein delivery system consisting of four modules in series: cell-penetrating peptide, pH-dependent membrane active peptide, endosome-specific protease sites and a leucine zipper. This system exhibits enhanced delivery efficiency and serum tolerance, depending on proteolytic cleavage-facilitated endosomal escape and leucine zipper-based dimerisation. Intravenous injection of protein phosphatase 1B fused with this system successfully suppresses the tumour necrosis factor-α-induced systemic inflammatory response and acetaminophen-induced acute liver failure in a mouse model. We believe that the strategy of using multifunctional chimaeric peptides is valuable for the development of cell-penetrating peptide-based protein delivery systems, and facilitate the development of biological macromolecular drugs for use against intracellular targets.
Highlights
Protein delivery with cell-penetrating peptide is opening up the possibility of using targets inside cells for therapeutic or biological applications; cell-penetrating peptidemediated protein delivery commonly suffers from ineffective endosomal escape and low tolerance in serum, thereby limiting in vivo efficacy
We further show that intravenous injection of eTAT-protein phosphatase 1B (Ppm1b) successfully suppresses the tumour necrosis factor-α-induced systemic inflammatory response and cures acetaminophen-induced acute liver failure in a mouse model
A series of recombinant proteins were designed and prepared with TAT (T)[17] and INF7 (I)[18] serving as a cell-penetrating peptides (CPPs) and pH-dependent membrane active peptides (PMAPs), respectively, and different cleavage sites recognised by endosomelocalised proteases, such as cathepsin L (CTSL)[19,20,21], cathepsin D (CTSD)[22] and furin[23,24], were inserted between the TAT-INF7 (TI) sequence and cargo (Fig. 1a)
Summary
Protein delivery with cell-penetrating peptide is opening up the possibility of using targets inside cells for therapeutic or biological applications; cell-penetrating peptidemediated protein delivery commonly suffers from ineffective endosomal escape and low tolerance in serum, thereby limiting in vivo efficacy. We present an intracellular protein delivery system consisting of four modules in series: cell-penetrating peptide, pH-dependent membrane active peptide, endosome-specific protease sites and a leucine zipper. This system exhibits enhanced delivery efficiency and serum tolerance, depending on proteolytic cleavage-facilitated endosomal escape and leucine zipper-based dimerisation. The efficient endosomal escape of fused cargos can be realised by removing fused cargo from CPP-PMAP in endosomes Another challenge involves low serum tolerance, evident by sharply decreased delivery efficiency in the presence of high concentrations of serum[11], which severely limits its application in situations in which serum exposure is unavoidable, for in vivo applications. We believe that the eTAT system or an improved version of it composed of multifunctional chimaeric peptides has the potential to become a major strategy enabling protein delivery in biological research and therapeutic applications
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