Redox-Responsive π-Conjugated Prodrug Nanoassemblies for Cancer Chemotherapy

<b>Background</b>: Redox-responsive prodrug nanoassemblies (NAs) have been extensively utilized in precise cancer therapy. But there is no research shedding light on the impacts of the π-π stacking interactions on the self-assembly capacity of redox-responsive prodrugs and the in vivo delivery fate of NAs. <b>Methods</b>: Three structurally engineered doxorubicin (DOX) prodrugs (FAD, FBD, and FGD) were developed through α-, β-, and γ-positioned disulfide linkages with π-conjugated Fmoc moieties. The NAs were comprehensively characterized for their self-assembly kinetics, redox-responsive drug release profiles, and physicochemical stability. Biological evaluations included cellular uptake efficiency, in vivo pharmacokinetics, and antitumor efficacy in tumor-bearing mouse models. <b>Results</b>: Systematic characterization revealed that π-conjugated disulfide bond positioning dictates prodrug self-assembly and inversely regulates reductive drug release relative to carbon spacer length. The FBD NAs demonstrated optimal redox-responsive release kinetics while maintaining minimal systemic toxicity, achieving 101.7-fold greater tumor accumulation (AUC) than DiR Sol controls. In 4T1 tumor-bearing models, FBD NAs displayed potent antitumor efficacy, yielding a final mean tumor volume of 518.06 ± 54.76 mm³ that was statistically significantly smaller than all comparator groups (<i>p</i> < 0.001 by ANOVA at a 99% confidence interval). <b>Conclusion</b>: These findings demonstrate that strategic incorporation of redox-sensitive disulfide bonds with different π-π stacking interactions in the prodrug structure effectively optimizes the delivery-release balance of DOX in vivo, ensuring both potent antitumor efficacy and reduced systemic toxicity.