Nanoparticles based on chitosan have been extensively studied for gene, drug and contrast agent delivery. The present work aimed to develop and characterize a glycol chitosan nanogel - also designated macromolecular micelles or hydrogel nanoparticles. The nanogel is obtained by chemically grafting hydrophobic chains on the hydrophilic glycol chitosan backbone, the amphiphilic structure obtained being capable of self-assembly in water, originating quite reproducible nanostructures with few hundred nanometers. The nanogel was decorated with folate conjugated polyethylene glycol, for active targeting purposes, as subsequently confirmed by in vitro assays. The positively charged nanogels exhibited colloidal stability up to at least four months. Considering their ability to complex siRNA, nanogels could represent promising vehicles for siRNA delivery. Moreover, their hydrophobic moieties could also carry hydrophobic drugs/imaging agents beyond nucleic acids, according to the theragnosis concept. The safety of the glycol chitosan nanogel as drug delivery system was comprehensively studied, since the biocompatibility of nanogels is still insufficiently reported. No cytotoxicity was detected in cell lines, RAW, 3T3, HMEC and HeLa, although a slight decrease on metabolic activity had been observed. Glycol chitosan nanogel didn’t induce cell membrane damage, cell death by apoptosis and/or necrosis, and cell cycling arresting (with exception to G1 arresting in RAW cells). Remarkably, glycol chitosan nanogel was poorly internalized by mouse bone marrow derived macrophages and does not trigger the activation of the complement system. Its blood compatibility was also confirmed through haemolysis and whole blood clotting time assays. The interaction of the nanogel with biological tissues, namely the endocytic mechanisms used by folate functionalized nanogels to entry HeLa cells, and the subsequent intracellular fate were studied using siRNA technology to deplete key proteins on regulation of each tested pathway. The nanogel cellular uptake is folate dependent, as expected since HeLa cells overexpress folate receptors. The internalization occurred mainly through clathrin and caveolin independent mechanisms, specifically by flotillin-1 and Cdc42-dependent endocytosis, as well as through micropinocytosis. Once internalized, after 7 h of incubation, approximately half of the nanogel was visualized in endolysosomal compartments, while the remaining was present in undefined regions of the cytoplasm. The biodistribution profile of the folate decorated glycol chitosan nanogel was assessed using in vivo near infrared fluorescence imaging as tool to track the nanogel over time in a mice model, after intravenous injection. Rapid nanogel whole body distribution was observed at early time points, and it is still detectable in a very wide distribution at least 6 h post-administration. The blood clearance occurred about 6 h post injection, with a blood half-life of approximately 2 h; surprisingly, the linear glycol chitosan seems to undergo a slower blood clearance. No accumulation in the organs was verified, the clearance from the body being observed apparently after a period of about 48h. In conclusion, the physicochemical features, the ability to complex nucleic acids and certain hydrophobic drugs, cell targeting ability, biocompatibility, internalization and intracellular trafficking, the fairly long blood circulation half-life and suitable body clearance, are pronounced hints of the engineered GC nanogel as a promising drug delivery system.