Thermo-responsive copolymers grafted with N-isopropylacrylamide (NIPAm) are excellent candidates for drug release. Dextran sulfate (DS) acts as a specific ligand in inflamed regions, turning it highly useful as a target for drug delivery. DS was associated with NIPAm to produce amphiphilic graft copolymers prepared via free radicals. The molar ratio of feed reagents NIPAm/DS varied from 1 (DS-g-PNIPAm) to 4 (DS-g-4PNIPAm). The synthesis was confirmed by spectroscopic techniques (Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR)). All copolymers showed self-organization capacity in an aqueous medium in temperatures higher than 34 °C, and sizes less than 300 nm. DS-g-3PNIPAm exhibited stability in water and in phosphate buffer at pH 7.4. Scanning electron microscopy confirmed their spherical shape. This copolymer showed specificity to leukemic cells, and normal cells’ proliferation. Methotrexate (MTX) is a very low water-soluble drug used for rheumatoid arthritis and cancer. Unfortunately, MTX have severe collateral effects. MTX-loaded nanoparticles can overcome such issues as well as enhance bioactivity and stability. The MTX was encapsulated and delivered from the DS-g-3PNIPAm with potential target delivery due to the presence of DS. Comparison with MTX encapsulated in other nanoparticles reveals that the DS-g-PNIPam presents the best performance among the thermo-responsive and the second among the target MTX nanocarriers.