BOX-BEHNKEN OPTIMIZATION AND IN VITRO EVALUATION OF PIPERINE-LOADED CHITOSAN NANOPARTICLES FOR SUSTAINED RELEASE, ANTI-INFLAMMATORY ACTIVITY AND HT-29 CYTOTOXICITY

The present investigation aimed to develop and optimize piperine-loaded chitosan nanoparticles for enhanced
anti-inflammatory and anticancer activity against HT-29 human colorectal cancer cells. Piperine-loaded nanoparticles were prepared using ionic gelation technique employing chitosan as polymer and sodium tripolyphosphate as crosslinking agent. A Quality by Design-based Box-Behnken Design was utilized to optimize formulation variables including chitosan concentration,
TPP concentration, and Tween 80 concentration. The optimized nanoparticles were characterized for particle size, polydispersity index, zeta potential, entrapment efficiency, morphology, compatibility, crystallinity, and in vitro drug release behaviour. The
optimized formulation exhibited particle size of 176.4 nm, polydispersity index of 0.241, zeta potential of +38.2 mV, and
entrapment efficiency of 90.1%. FTIR, DSC and XRD studies confirmed successful encapsulation of piperine within the
chitosan matrix with reduced crystallinity. Sustained drug release was observed over 24 h following diffusion-controlled
kinetics. The optimized nanoparticles demonstrated significantly enhanced protein denaturation inhibition, nitric oxide
scavenging activity and cytotoxicity against HT-29 cells compared with pure piperine. The IC value ofoptimized nanoparticles
was markedly lower than that of pure piperine, indicating enhanced antiproliferative efficacy. The findings suggested that
chitosan-based nanoencapsulation significantly improved the pharmaceutical and biological performance ofpiperine for colorectal
cancer therapy.