To improve the efficiency of cargo delivery to cancer cells, pH-Low Insertion Peptide (pHLIP) variants were developed to insert into membrane at a pH range closer to tumor extracellular pH of 6.5-7.0 than the original pHLIP ('WT'). The Asp-to-Glu variants were based on the idea of increasing side-chain pKa value while preserving the protonation order of WT pHLIP insertion. The Thr19 variants represent efforts to influence the Pro20 cis-trans isomerization. The variants were characterized on POPC model membrane by following the pH range (pHinitial–pHfinal) where changes in Trp fluorescence signal occurred. Compared to WT pHLIP, which has a pHi–pHf range of 6.7–5.6, the D25E-D31E-D33E, D14E-D25E-D31E-D33E, and T19V-D25E variants demonstrated pHi–pHf range of 7.3–6.1, 7.2–6.3, and 7.8–5.8, respectively. To study the effect of cargo on pHLIP insertion, doxorubicin (Dox), a fluorescent antineoplastic drug, was conjugated to WT, D14E-D25E-D31E-D33E, and T19V-D25E pHLIP near the C-terminus (the inserting end). At pH 8, all pHLIP-Dox conjugates were more deeply embedded in the membrane than their pHLIP-only counterparts. The addition of Dox shifted pHi to higher pH values but did not alter the pHf of insertion. Interestingly, the pHi–pHf values obtained from C-terminal Dox fluorescence signals were largely consistent with those reported by N-terminal Trp residues. Despite all pHLIP-Dox conjugates seemingly reaching the final transmembrane state, the Dox fluorescence data suggested that the Dox cargo remained in the hydrophobic interior of the membrane after pHLIP insertion, which may impede drug release.

pH-Low insertion peptide variants that insert into membrane at higher pH values

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