Gel Chromatography Essay

2. Experimental Part
2.1 Materials
Starting deacetylated chitosans (Mws, 129.4 and 236.7 kDa) were prepared as described previously by Tiera et al. [27] from commercial chitosan (degree of deacetylation (DD) 86 %) purchased from Polymar (Fortaleza, Brazil). 2-Chloro-N,N-diethylethylamine hydrochloride (DEAE), folic acid, sodium acetate, acetic acid, dicyclohexyl carbodiimide (DCC), N- hydroxylsuccinimide (NHS), dimethylsulfoxide (DMSO) were purchased from Aldrich Chemical Co. All solvents were reagent grade and used as received. Spectra/Pore membranes (Spectrum) were employed for dialysis. Sjogren syndrome antigen (SSB, GenBank accession number NM_009278)-targeted siRNA was provided by Merck and Co, Inc, (West Point, USA). TNF--targeted siRNA

Solutions for GPC analysis were prepared by dissolving the samples in an acetic acid buffer solution (0.3 mol L-1) / sodium acetate (0.2 mol L-1) at pH 4.5, to achieve a concentration of 5.0 mg.mL-1. Solutions were kept under stirring for 3 days until complete solubilization and then filtered using a 0.45 μm membrane before analysis. Two columns in series: SB-803 HQ and SB-805-HQ (Shodex, Tokyo, Japan) with sizes of 8 mm x 300 mm and flow rate of 0.8 mL min-1 were used for the analysis of the samples. Standards of pullulan with molecular weight in the range of 6.2 kDa to 805 kDa were used to build the calibration

After 4 hours, the culture medium was removed and cells were treated with 200 μL of nanoparticles containing an amount of 5 μg of siRNA-TNF-  plus 200 μL of incomplete AMEM medium added without FBS and PS. Cells treated with 200 μL of free siRNA were used as a negative control. After 3 hours of incubation at 37 °C, the medium was removed and the cells received 1 ml of complete medium with 10 % FBS and 1 % PS and incubated for 48 hours. After this time, the medium was removed and a new complete medium containing 100 ng / ml lipopolysaccharide (LPS) was added [30]. After 24 hours, the medium was removed and stored for TNF-α protein concentration analysis using the Elisa quantikine immunoassay kit for mouse TNF-α (R & D Systems, Minneapolis, USA ), following the manufacturer's instructions. Cells were washed with cold phosphate buffered saline solution (PBS) and lysed with lysis buffer for the determination of total protein concentration using the bicinchoninic acid-based BCA kit (Thermo Fisher Scientific, Waltham, USA), with bovine serum albumin (BSA) standard according to the manufacturer's instructions. The transfection efficiency is given by the ratio of the concentration of TNF-α (in pg) to the total proteins (in