Dynamic supramolecular assembly and pH-responsive doxorubicin release in zwitterionic hyaluronate-quercetin...

Here, a novel pH-responsive, supramolecular self-assembled zwitterionic nanocarrier based on quaternized hyaluronate conjugated with quercetin (QHQ) for enhanced intracellular delivery of the anticancer drug doxorubicin (Dx) is presented. Hyaluronic acid, a biocompatible and non-toxic natural polysaccharide, was first quaternized with Quat-188 to introduce stable cationic sites, and then conjugated with the hydrophobic flavonoid quercetin (QT) to enable π-π stacking and hydrophobic interactions. The amphiphilic QHQ conjugates spontaneously self-assembled in aqueous media and showed a high drug-loading capacity, pH-responsive release, and improved cellular uptake. At an optimized Dx : QHQ mass ratio of 1 : 8 (DxQHQ1 : 8), the encapsulation efficiency of Dx reached 75%. Increasing the Dx content reduced the nanoparticle (NP) size, as Dx-QT interactions competitively disrupted QT-QT stacking within the supramolecular core. QHQ alone was non-cytotoxic towards human HaCaT, NIH-3T3, and WI-38 cell lines below 125 µg mL^-1, whereas DxQHQ1 : 8 NPs showed high cytotoxicity toward HeLa and A549 cancer cell lines relative to free Dx. DxQHQ1 : 8 remained colloidally stable at pH 7.4 for at least 64 h, while drug release was substantially accelerated under acidic conditions (pH 4.0 and 5.0) consistent with endosomal environments. The release kinetics were well described by a newly proposed Muangsin-Moya model (R² = 0.984 over 48 h), consistent with diffusion and supramolecular disassembly. Flow cytometry confirmed higher intracellular Dx accumulation in cells treated with DxQHQ1 : 8 compared with free Dx. These results show that DxQHQ1 : 8 NPs serve as a pH-responsive platform for the delivery of hydrophobic anticancer drugs, with selective activation under acidic conditions, such as those in tumor and endosomal microenvironments.