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

## TL;DR

A tunable nano-microsphere system was developed to overcome intestinal barriers for more effective rheumatoid arthritis treatment.

## Contribution

A dynamically tunable nano-microsphere system with charge and stiffness adaptability for intestinal drug delivery is introduced.

## Key findings

- The nano-microspheres achieved a 1.6-fold increase in maximum blood concentration compared to conventional carriers.
- MLVs demonstrated effective penetration of intestinal barriers and multimechanistic endocytosis potential.
- DAPC in the vesicle shell enables charge reversal via non-classical hydrolysis, confirmed by LC-MS/MS analysis.

## Abstract

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 via 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 in vitro studies confirmed the multilayered architecture, dynamic adaptability, and effective penetration of intestinal barriers by the MLVs and the nano-microspheres. In vivo, 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.