Date of Award

2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physics

First Advisor

Yana Reshetnyak

Abstract

Most drug molecules cannot freely diffuse across a cellular membrane because of the energetic barrier for transition across a hydrophobic lipid bilayer of a membrane is very high for polar molecules. Recently it was discovered that a moderately hydrophobic, water-soluble membrane peptide called pHLIP (pH (Low) Insertion Peptide) can insert into membranes and translocate molecules in a pH-dependent manner. This opens a unique opportunity to develop a novel concept in drug delivery, which is based on the use of a pH-sensitive single peptide molecular transporter. The main goal of this investigation is to elucidate mechanism of cargo translocation across a membrane by the single molecule transporter, pHLIP, and to demonstrate its utility for the translocation of function cargo molecules. We have carried out a variety of biophysical experiments and investigation on the cultured cells. As a functional cargo we selected a cyclic cell-impermeable polar peptide, phallatoxin (phalloidin or phallacidin) from the deadly Amanita phalloides mushroom. If it is delivered into a cell, it would bind tightly to actin filaments at nanomolar concentration and inhibits their depolymerization. Previously our lab has demonstrated that the pHLIP is capable of translocation of phalloidin-rhodamine across a cellular membrane. In the present study, we show that the pHLIP can move phalloidin across the membrane, when the hydrophobic facilitator (rhodamine) was attached to the peptide inserting end. The phalloidin translocated across a cellular membrane by the pHLIP induces > 90% inhibition of HeLa, JC and M4A4 cancer cell growth, their cytoskeletal immobilization and multinucleation, consistent with the expected binding of phalloidin to F-actin. The next step of our studies was tuning the hydrophobicity of polar cargo. We designed, synthesize and characterize three phallacidin cargoes, where the hydrophobicity of cargo was tuned by the attachment of diamines of different lengths of hydrophobic chains. The phallacidin cargo (phallC6) with a similar polarity to phallodin-rhodamine was conjugated to the pHLIP, and shown to selectively inhibit the proliferation of cancer cells at low pH. To elucidate the mechanism of cargo translocation across a membrane by the pHLIP and identify pHLIP variants for more efficient molecules translocation across a membrane, we carried out biophysical studies on several pHLIP variants conjugated with the cargoes of different polarity (biotin and biotin-Peg). We confirm that all pHLIP variants with attached cargo molecules preserve pH-dependent properties of interaction with membrane. While the equilibrium thermodynamics favor the binding and insertion of pHLIP-cargo constructs, kinetics was significantly slowed down. Our findings are valuable for the design of new delivery agents for the direct translocation of polar cargo across a membrane: to facilitate the different delivery needs for different applications the hydrophobicity of the cargo could be modified without affecting cargo‘s ability to bind to its cellular target and/or various peptides of the pHLIP family could be employed, which show different rates and pKa of the cargo translocation across cellular membranes. Thus, the maximum difference between the therapeutic effects at low pH versus at neutral pH could be achieved, thereby enhancing diseased-targeted delivery and reducing treatment side effects.

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