Mechanism of Anti-Cancer Activity of Benomyl Loaded Nanoparticles in Multidrug Resistant Cancer Cells.

Polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles loaded with benomyl as anticancer drug formulation against multidrug-resistant EMT6/AR1 cells were synthesized by amine-carboxylate reaction. Using transmission electron microscopy, the average size of chitosan-poly(D,L-lactide-co-glycolide) nanoparticles and benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles was estimated to be 155 ± 20 nm and 160 ± 25 nm, respectively. Fourier transform infrared spectroscopy revealed that poly(D,L-lactide-co-glycolide) and chitosan are linked by covalent bonds. Zeta potentials of benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles at pH 4, 7.2, and 10 were 30 ± 1.8, 19 ± 0.65, and -22 ± 0.15 mV, respectively, indicating the formation of stable, hydrophilic nanoparticles. The release of benomyl from benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles followed pH-dependent kinetics. The uptake of fluorescein isothiocyanate-labeled chitosan-poly(D,L-lactide-co-glycolide) nanoparticles was concentration-dependent in both MCF-7 and multidrug-resistant EMT6/AR1 cells. EMT6/AR1 cells showed 10-fold higher resistance to benomyl compared to MCF-7 cells; in contrast, benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles effectively inhibited proliferation of MCF-7 and EMT6/AR1 cells with a half-maximal inhibitory concentration of 4 ± 0.5 and 9 ± 0.5 pM, respectively. In the presence of a P-glycoprotein inhibitor, the activity of benomyl was increased, suggesting that benomyl is a substrate for P-glycoprotein. Further, benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles depoly-merized microtubules both in interphase and mitosis. It blocked cell cycle progression at G2/M and induced apoptosis in EMT6/AR1 cells, suggesting that benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles have chemotherapeutic activity against multidrug-resistant cancer cells.