Cell penetrating peptide-based drug delivery system for targeting mildly acidic pH

Properties of different arginine‐rich peptides, including net charge and charge distribution, were evaluated for their influence on surface binding, internalization, and intracellular localization. The peptides were radiolabeled and subsequently tested for surface binding and internalization in cells. Subcellular fractionation assays were performed to separate the amount of peptides associated with vesicles from those inside the cytosol. Net neutral charged peptides, YGR₆E₆ and YG(RE)₆, showed large decreases in both surface binding and cell uptake compared to their net positive charged counterparts, YGR₆G₆ and YG(RG)₆. The peptides with clustered arginine residues, YGR₆G₆ and YGR₆E₆, exhibited significantly higher binding and uptake than those with alternating arginine and glycine/glutamate residues, YG(RG)₆ and YG(RE)₆. The intracellular distribution analysis for all of the peptides tested showed that, regardless of the net uptake, the arginine‐rich peptides were preferentially localized in the cytosolic compartment of the cells. Both net positive charge and a clustered arginine sequence enhance the surface binding and cell uptake of peptides, however, the intracellular distribution does not change. These initial findings inspired the design of the following CPP‐based drug delivery systems. ❧ Citraconic anhydride was used to modify different CPPs in order to develop a pH‐sensitive delivery system. After the citraconylation, CA-MAP showed very low surface binding and cell uptake in cell assays. The binding and internalization could be recovered when the citracoyl groups in the peptide were removed at acidic pH (3-4). However, this acid facilitated process may not be very efficient inside endosomes with a mildly acidic pH (~6.5). The addition of folate to CA-MAP as targeting ligand could increase its cell uptake and nuclear internalization in folate receptor positive cells. Although several attempts involving chemical modification didn't lead to a feasible pH‐sensitive CPP for clinical application, the data from these attempts did prove the concept that the binding and uptake of CPP could be controlled through the manipulation of its net charge. ❧ Finally, a pH‐sensitive CPP, HE-MAP, was produced as a GST‐fusion protein in bacteria. Both radioactive and fluorescent assays showed pH‐dependent cell uptake for GST-HE-MAP in cells, which could be used for targeting mildly acidic pH. Cell uptake assays also showed that the pH‐dependent binding and uptake profile of GST-HE-MAP could be shifted when the repeat number of H/E co‐oligopeptide was changed. Compared with two other fusion proteins containing either (HE)₁₀ or MAP, only the fused peptide, HE-MAP, could effectively deliver the cargo GST protein to cells at pH 6.5 or below, while maintaining low delivery to cells at pH 7.0 and above. Using a xenograft mouse model of human breast cancer, fluorescent imaging showed that only HE-MAP could effectively target GST to the tumor site, while reducing non‐specific association of MAP in other organs. The in vitro and in vivo data have demonstrated the diagnostic and/or therapeutic potential of the fused peptide, HE-MAP, for targeting the acidic tumor microenvironment. The concise design of this pH‐sensitive peptide offers a simple way to overcome CPP's lack of selectivity, which could lead to increased application of CPPs and macromolecular therapeutics.