The objective of the present study was to prepare and characterized de-esterified tragacanth-chitosan nanoparticles (DET-CS NPs) as a novel carrier for methotrexate, with a view to improve drug efficacy and target ability. The preparation process was optimized using Taguchi design. NPs were characterized for size, zeta potential, morphology, thermal stability, loading efficiency, cytotoxicity and cellular uptake. Taguchi design indicated that the molecular weight of chitosan possessed the most effect on the zeta potential, PDI, and zeta deviation, and the size of nanoparticles was significantly affected by the DET concentration. The size and zeta potential of drug loaded nanoparticles at optimum condition were 322.9â±â26ânm and 17.3â±â5.73âmV, and thermal analysis indicated ionic bond between DET and CS in NPs. The loading efficiency was 20.32%â±â2.01, and the sustained release was observed within nine days. The IC was 280â”g/mL in HT-29, and the mitochondrial membrane potential in HT-29 was reduced more than that in MCF-7. The uptake of NPs in HT-29 was higher than that in MCF-7, and active endocytosis was the key mechanism of uptake. These phenomena altogether make DET-CS NPs a proper choice for targeted drug delivery to cells containing asialoglycoprotein receptors.