Charge-engineered microparticles for targeted delivery of RhoA activator to nucleus pulposus

Intervertebral disc degeneration (IVDD) involves dysregulated mechanobiology of nucleus pulposus (NP) cells, marked by disrupted F-actin organization and inflammation-driven extracellular matrix (ECM) catabolism. Restoring actomyosin contractility and cytoskeletal integrity can blunt ECM degradation. Employing a constitutive Rho activator, CN03, reinstates F-actin organization via the RhoA-ROCK pathway and rescues NP cells from cytokine-driven degeneration. However, its constitutive RhoA activity is short-lived, which demands sustained, spatially targeted NP delivery. In this study, an engineered poly-(lactic-co-glycolic) acid microparticle was designed with a viscous inner core ( e PLGA-CN03) to encapsulate hydrophilic CN03, minimizing burst and achieving a sustained release profile while preserving bioactivity. In 2D and 3D NP cultures, released CN03 elevated intracellular Ca 2+ , increased GTP-RhoA, and pMLC to levels comparable to free CN03 and restored F-actin architecture under TNFα stimulation, reducing cell spreading to near control levels. To enhance NP spatial targeting and retention, e PLGA-CN03 was functionalized with cationic avidin (Av- e PLGA-CN03), leveraging electrostatic affinity for the highly anionic, aggrecan-rich NP matrix. Av- e PLGA-CN03 reversed the negative surface charge to cationic, exhibited strong tissue binding, and achieved month-long intra-NP spatial localization in both healthy and degenerated explants, outperforming unmodified particles. Av- e PLGA-CN03 maintained sustained release and remained bioactive. Safety assessments showed no adverse effects on NP swelling properties or cell viability at the effective dose, supporting biocompatibility of avidin functionalization. Collectively, these data demonstrate charge-guided, avidin-modified PLGA depots as a modular approach for sustained, spatially targeted, intra-NP delivery of CN03 and other hydrophilic disease-modifying biologics, supporting future translation evaluation in models of IVD degeneration. Statement of significance This study engineers a charge-mediated, avidin-anchored, engineered poly-(lactic-co-glycolic) acid microparticle system (Av- e PLGA) designed to selectively bind the aggrecan-rich proteoglycans of the nucleus pulposus (NP), addressing long-standing challenges in tissue-specific drug delivery within the intervertebral disc. This platform achieves spatially confined distribution and month-long retention within the NP while minimizing leakage to adjacent disc regions. The tunable Av- e PLGA carrier accommodates hydrophilic protein biologics, and here we demonstrate efficient encapsulation and sustained release of the Rho-GTP activator CN03 without loss of bioactivity in NP cells. Importantly, surface-anchored cationic charges do not disrupt NP electromechanical integrity. Together, these findings position Av- e PLGA as a promising strategy for spatially targeted, long-acting biologic delivery to the NP.