Sequential KIM-1 targeting and SMOX-responsive release enhance renal delivery of dihydroartemisinin to restore endogenous Klotho and attenuate renal fibrosis.

Restoration of endogenous Klotho represents a promising strategy for suppressing renal fibrosis, but efficient delivery of Klotho-restoring therapeutics to injured renal tubules remains challenging. Here, we developed dihydroartemisinin (DHA)-loaded phosphatidylserine liposomes coated with spermine-modified chitosan (DHA-PSLPs@SC) to achieve sequential kidney injury molecule-1 (KIM-1) targeting and spermine oxidase (SMOX)-responsive release, thereby enhancing renal delivery of DHA to restore endogenous Klotho and attenuate renal fibrosis. Dioleoylphosphatidylserine (DOPS) was incorporated to facilitate uptake by KIM-1-expressing injured tubular epithelial cells, whereas the spermine-modified chitosan (SC) coating was designed to promote cargo release in the SMOX-enriched fibrotic cellular environment. The optimized DHA-PSLPs@SC exhibited a diameter of 102.3 nm, a zeta potential of 33.8 mV, and an encapsulation efficiency of 69.4%. In H₂O₂-injured HK-2 cells, DOPS-containing liposomes displayed enhanced uptake and colocalization with KIM-1, while KIM-1 antibody blockade markedly reduced their internalization. In TGF-β1-stimulated NRK-52E cells, the SC coating preserved cellular uptake and increased cumulative DHA release to 74.2% at 12 h, while DHA-PSLPs@SC more effectively restored Klotho and suppressed fibrotic markers than free DHA or non-targeted liposomes. In unilateral ureteral obstruction (UUO) mice, PSLPs@SC preferentially accumulated in obstructed kidneys, with approximately 2.7-fold higher renal accumulation than non-targeted liposomes at 2 h. DHA-PSLPs@SC improved renal function, reduced tubular injury and collagen deposition, restored renal Klotho, and suppressed fibrosis. These findings support sequential KIM-1 targeting and SMOX-responsive release as a strategy to enhance renal delivery and antifibrotic efficacy of DHA, restore endogenous Klotho, and attenuate renal fibrosis.