Glutaraldehyde 50 solution

For the synthesis of Nic (niclosamide) loaded Chi (chitosan) nanoformulations, we followed desolvation method [19 (link)]. In brief, in a 10 ml chitosan solution (Sigma-Aldrich, USA) dissolved in dilute acetic acid (35 mM), Polysorbate 80 (Sigma-Aldrich, USA) (non-ionic surfactant: 230 µl) was added with constant stirring for 1 h at 440 r.p.m. After that, 350 µl of anti-cancer drug (Nic; stock: 1 mg ml⁻¹ in ethanol) (Sigma-Aldrich, USA) was added dropwise into 10 ml of 0.15% chitosan solution and left stirring for another 1.5 h. Following that, 200 µl of 20% sodium sulfate (Sigma-Aldrich, USA) solution was added dropwise with further stirring for another 1 h. In order to cross-link the formed nanoparticles 80 µl glutaraldehyde (50% solution) (Sigma-Aldrich, USA) was added to the solution and stirring continued for another 1 h. The concentration of glutaraldehyde and sodium sulfate has a great impact on the size of formed nanoparticles. Lastly, sodium metabisulfite (10%) (Sigma-Aldrich, USA) was added to the nanoparticulate solution and stirred for another 2–3 h. All reactions were carried out in cold conditions at 4°C followed by dialysis against water with two changes and against normal saline with two changes for the next 48 h. The solution was lyophilized and preserved at 4°C for further characterization and therapeutic studies.