Novel Generation of PHLIP Peptides for Targeted Delivery of Imaging and Therapeutic Payloads to Tumors
Abstract
Purpose
pHLIP (pH (Low) Insertion Peptides) technology is used in preclinical and clinical studies for targeted delivery of imaging and therapeutic agents to acidic tumors. pHLIPs sense low pH at the surface of cancer and immune cells, insert into cellular membrane and position payload either at cell surface or translocate payload across membrane into cytoplasm. Here we introduce new generation of pHLIPs with improved properties for targeted delivery of payloads to acidic solid tumors.
Methods
Eleven Gen2.0 pHLIP peptides were synthesized and tested on liposomes, cultured cells and in vivo for tumor targeting and biodistribution in mice. The pK of pHLIP insertion into a lipid bilayer of membrane was explored using tryptophan fluorescence spectroscopy along with circular dichroism. The rate of peptide insertion and exit from the membrane was measured using stopped-flow fluorescence kinetics. Peptides were conjugated with indocyanine green (ICG) fluorescent dye to obtain pHLIP-ICG conjugates, which were tested for binding with human serum albumin, cell viability and tumor targeting and biodistribution. Stability of the selected pHLIP peptides were assessed in mice and human plasma.
Results
All peptides exhibited pHLIP-like properties of pH-dependent membrane-associated folding, while individual parameters of pKs and rates of insertion into membrane were different. All pHLIP conjugates were not cytotoxic and demonstrated binding to human serum albumin. Ex vivo imaging data indicated tumor targeting and favorable biodistribution and clearance of the agents from major organs. Agents were stable in mice and human plasma.
Conclusion
Var3 of the first generation (Gen1.0) of pHLIP peptides demonstrates excellent tumor targeting of imaging and cytotoxic therapeutic agents for visualization and treatment of tumors in humans. We identified the best Gen2.0 pHLIP peptide with improved parameters for potential clinical development and translation for targeted intracellular delivery of therapeutic payloads.