From single DNA molecule to nanoparticle formation: mechanistic basis for monocationic aromatic drug-induced DNA frameworks

We investigated the interaction between the monocationic aromatic drug propranolol (PPL) and double-stranded DNA (dsDNA) to elucidate how small molecules can drive higher-order DNA frameworks and nanoparticles (NPs) formation. Single-molecule force spectroscopy with optical tweezers revealed that, at concentrations below 4 mM, PPL interacts with dsDNA through an intercalation-like mode, altering contour length, persistence length, and stretch modulus. At higher concentrations, PPL induced dsDNA compaction, corroborated by atomic force microscopy imaging of condensed structures. Multimolecular assays supported these findings: electrophoretic mobility shift assays revealed progressive mobility loss with increasing PPL concentrations, consistent with aggregate formation, while UV-vis spectroscopy confirmed intercalation-like behavior and strong binding affinity (Kb=1.67 × 10⁶ M⁻¹). At millimolar PPL/DNA ratios (10–14), NPs formulations were obtained with hydrodynamic diameters of 120–244 nm, low polydispersity (0.19–0.30), negative zeta potential (-25 to -35 mV), and particle concentrations up to 5.26 × 10¹¹ NPs/mL. These NPs exhibited very high drug loading (59–72%) and stability under both biological and storage conditions. Collectively, our results demonstrate that PPL engages dsDNA through intercalation-like behavior, compaction, aggregation, and stabilization processes, uncovering a previously unreported mechanism for a monocationic aromatic drug and allowing the efficient formation of NPs. This work expands the current understanding of small molecule-DNA interactions and may be extended to other hydrophilic aromatic drugs, positioning DNA as a versatile building block and ultimately for the development of nucleic acid-based nanomedicines.