Enhanced Phlip (e-pHLIP) for Targeted Cytoplasmic Delivery of Polar Payloads
Abstract
Purpose
pH Low Insertion Peptide (pHLIP) technology is employed in clinics for targeting of tumors and delivery of imaging and therapeutic agents. Here we introduced the novel enhanced pHLIP (e-pHLIP) system for efficient cytoplasmic delivery of large polar payloads, including small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs).
Methods
We have designed and synthesized the e-pHLIP delivery system and tested it for pH-dependent interactions with membranes of liposomes and cultured cells, as well as tumor targeting and biodistribution in mouse tumor models. Fluorescently labeled siRNAs and ASOs for silencing of green fluorescent protein (GFP) were assessed in GFP-expressing HeLa cells using fluorescent microscopy. Cellular uptake and distribution of the fluorescent siRNAs and ASOs delivered by e-pHLIP were monitored, and the decay of GFP fluorescence over time was quantified.
Results
The e-pHLIP consists of two pHLIP peptides linked via a short PEG linker. The first pHLIP peptide serves as a membrane-anchoring domain and carries no payload molecule at its membrane-inserting terminus. This stabilizes membrane association for a second step where the second pHLIP peptide serves as a payload-transporting domain, which carries a payload at its membrane-inserting terminus for transport of a payload across a membrane. We demonstrated improved interactions of e-pHLIP with membranes, no cytoxicity, and enhanced targeting of acidic tumors by e-pHLIP (compared to pHLIPs) labeled with indocyanine green near infrared fluorescent dye, ICG. e-pHLIP exhibited successful cytoplasmic delivery of siRNAs and ASOs when tested on GFP-expressing HeLa cells.
Conclusion
We introduced the novel e-pHLIP delivery system comprised of one membrane insertion pHLIP peptide linked to another membrane insertion pHLIP peptide by a non-cleavable linker for cytoplasmic delivery of large polar payloads, which opens an opportunity for targeted delivery of gene-therapeutics.