Thiol-reacting toluidine blue derivatives: Synthesis, photophysical properties and covalent conjugation with human serum albumin

The development of improved photosensitizers for photodynamic therapy (PDT) is still a current research need. In this sense, third-generation photosensitizers employ different delivery vehicles to improve tumoral cells selectivity over healthy tissues. With this purpose in mind, we synthesized and characterized toluidine blue (TBO) derivates directed towards forming a covalent conjugation with human serum albumin (HSA) as a delivery vehicle under physiologically relevant conditions. We described herein the synthesis of 3-(2-pyridyldithio)propionate (PDP) and 6-maleimidocaproate (EMC) derivatives of TBO (TBOPDP and TBOEMC, respectively), their photophysical/photochemical properties and reactivity towards HSA. Hypsochromic shifts were observed in their absorption and emission bands, higher photostability and increased singlet oxygen generation compared to TBO. Moreover, these derivatives reacted with HSA at the cysteine 34 residue through disulfide exchange or thio-Michael additions. The HSA-TBOPDP and HSA-TBOEMC conjugates were analyzed by MALDI-TOF mass spectrometry, absorption/fluorescence emission spectroscopy and circular dichroism. Conjugation preserved the protein secondary structure and the photophysical properties of the photosensitizer, although the singlet oxygen generation was much lower due to the reaction with the vehicle protein. The potential of these new derivatives with covalent-conjugation to HSA for PDT are discussed.