Charge/stiffness-tunable nano-microsphere overcomes intestinal mucosal barrier for rheumatoid arthritis treatment

Intestinal drug delivery is a crucial route for rheumatoid arthritis (RA) therapy. However, its effectiveness is often hampered by the viscosity gradient of the mucus layer and the selective degradation and efflux functions of the epithelial barrier. To address these challenges, we developed a nano-microsphere system featuring charge- and stiffness-tunable multilayered vesicles (MLVs) encapsulated within pH-sensitive microspheres. The MLVs are engineered to traverse the negatively charged, viscosity-gradient mucus by sequentially shedding their flexible shells and undergoing a positive-to-negative charge reversal. This exposes a rigid, neutral core that enables multimechanistic endocytosis with potential for transcellular transport. The dynamic tunability of the MLVs is attributed to the incorporation of di-artesunate-phosphatidylcholine (DAPC) in the vesicle shell. LC-MS/MS analysis reveals that DAPC undergoes terminal hydrophobic chain carboxylation <i>via</i> a non-classical hydrolysis pathway, facilitating the observed charge reversal. Encapsulation within pH-sensitive microspheres further protects the MLVs from premature degradation and ensures targeted intestinal delivery. Cryo-electron microscopy and <i>in vitro</i> studies confirmed the multilayered architecture, dynamic adaptability, and effective penetration of intestinal barriers by the MLVs and the nano-microspheres. <i>In vivo</i>, this system achieved a 1.6-fold increase in maximum blood concentration compared to conventional carriers, alongside significantly enhanced therapeutic efficacy for RA. In summary, we present a dynamically adaptive, intestinal barrier-penetrating nano-microsphere platform that offers a promising strategy for RA treatment.