Effect of PEG-Induced Liquid–Liquid Phase Separation on DNA–Topotecan Interactions

Understanding the impact of liquid-liquid phase separation (LLPS) on drug and biological molecules is key to advancing drug delivery and biomolecular engineering. This study investigates how polyethylene glycol (PEG)-induced LLPS modulates the photophysical behavior of topotecan (TPT) and the structural organization of calf thymus DNA (ctDNA) in binary (PEG:TPT and PEG:ctDNA) and ternary (PEG:TPT:ctDNA) systems. In the PEG:TPT system, LLPS induced a shift from the emissive Z-form to the more stable C-TPT form, with enhanced fluorescence and a longer ∼4.0 ns lifetime, suggesting reduced water accessibility in the PEG-rich environment. The suppression of excited-state proton transfer (ESPT) further indicates the role of molecular crowding in modifying photophysical properties. In the PEG:ctDNA system, PEG-induced phase separation caused significant structural changes in ctDNA, including quenching of B-form DNA signals and a blue shift in absorption spectra. The ternary PEG:TPT:ctDNA system showed similar behavior to the binary PEG:TPT system, but DNA binding partially attenuated PEG's modulation of TPT's excited states. Microscopic analysis revealed altered condensate morphologies, with both spherical and elongated structures. Molecular dynamics (MD) simulation studies corroborated the experimental findings, revealing PEG-induced crowding around TPT and enhanced hydrogen bonding between the TPT-DNA complex under crowded conditions compared to dilute environments. These findings underscore the ability of PEG-mediated LLPS to tune the excited-state dynamics of small molecules and the structural organization of nucleic acids, offering insights into their potential applications in complex molecular environments.