Materials and methods Materials Chitosan MW of xA kDa
2. Materials and methods
Chitosan (MW of 80 kDa) with a degree of de-acetylation of 83% was purchased from Polysciences, Inc., USA. Hyaluronic acid, with MWs of 132 and 214 was purchased from Lifecore Biomedical. All other reagents were analytical grade and used without further purification.
2.2. Plasmid constructs and cell lines
A plasmid expressing enhanced green fluorescent protein driven by the cytomegalovirus TMC647055 (kindly provided by Jean Bennett, University of Pennsylvania, USA) was amplified in Top 10 bacteria and purified using a Plasmid Maxi kit (Qiagen, California, USA) following manufacturer guidelines. Plasmid DNA (DNA) was dissolved in TE buffer, and the concentration was evaluated using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA) at 260 nm.
Two cell lines were used for transfection and cytotoxicity evaluation: HEK 293T cells (kindly provided by Guilherme Ferreira, University of Algarve, Portugal) and a human retinal pigment epithelial cell line (ARPE-19, kindly provided by Francisco Ambrósio, University of Coimbra). All cell culture reagents were purchased from Sigma-Aldrich® (St. Louis, MO/USA).