D-to-E and T19V Variants of the pH-Low Insertion Peptide and Their Doxorubicin Conjugates Interact with Membrane at Higher pH Ranges Than WT

To improve targeted cargo delivery to cancer cells, pH-Low Insertion Peptide (pHLIP) variants were developed to interact with the membrane at pH values higher than those of the WT. The Asp-to-Glu variants aim to increase side chain pK_a without disturbing the sequence of protonations that underpin membrane insertion. The Thr19 variants represent efforts to perturb the critical Pro20 residue. To study the effect of cargo on pHLIP insertion, doxorubicin (Dox), a fluorescent antineoplastic drug, was conjugated to selected variants near the inserting C-terminus. Variants and conjugates were characterized on a POPC membrane using Trp and Dox fluorescence methods to define the entire pH range of insertion (pH_initial-pH_final). Compared to WT with a pH_i-pH_f range of 6.7-5.6, D25E-D31E-D33E, D14E-D25E-D31E-D33E, and T19V-D25E variants demonstrated higher pH_i-pH_f ranges of 7.3-6.1, 7.3-6.3, and 8.2-5.4, respectively. The addition of Dox expanded the pH_i-pH_f range, mainly by shifting pH_i to higher pH values (e.g., WT pHLIP-Dox has a pH_i-pH_f range of 7.7-5.2). Despite the low Hill coefficient observed for the conjugates, D14E-D25E-D31E-D33E pHLIP-Dox completed insertion by a pH_f of 5.7. However, the Dox cargo remained in the hydrophobic membrane interior after pHLIP insertion, which may impede drug release. Finally, a logistic function can describe pHLIP insertion as a peripheral-to-TM (start-to-finish) two-state transition; wherever possible, we discuss data deviating from such sigmoidal fitting in support of the idea that pH-specific intermediate states distinct from the initial peripheral state and the final TM state exist at intervening pH values.