Poly allylamine hydrochloride

Reagents. Quercetin (Qr), polystyrene sulfonate sodium, 70 kDa (PSS), polyallylamine hydrochloride, 58 kDa (PAH), chitosan of medium molecular weight, 450 kDa, degree of deacetylation 75-85% (CH), dextran sulfate, > 500 kDa (DexS), Dulbecco's modi ed Eagle's medium (DMEM), ethidium bromide (EB), practically all solvents, salts, and reagents were from Sigma-Aldrich. In addition, resveratrol (Res) from Biomol (Research Lab, Plymouth, MA), isotonic phosphate buffer pH 7.4 (PBS) from Lonza (Belgium), antibiotics from Gibco (USA), fetal bovine serum (FBS) from Capricorn (Poland) were used. Other reagents are mentioned herein below in the appropriate subsections.
Cell line. The immortalized human keratinocyte cell line HaCaT was a gift from N. E. Fusenig (Deutsches Krebsforschungszentrum, Heidelberg, Germany) and maintained in DMEM supplemented with 10% FBS, at 37°C in a humidi ed atmosphere containing 5% CO 2 .
Preparation of layer-by-layer shell on polyphenol microcrystals. A multilayer polyelectrolyte shell on polyphenol microcrystals was prepared using the layer-by-layer assembly method [27, 28] . Brie y, polyphenol powder was dispersed in distilled water to obtain a 10 mg/mL suspension. An aliquot of a 5 mg/mL PAH or chitosan solution was added in the amount of 0.2 mL per 1 mL of the initial suspension and thoroughly dispersed for 5 min in an ultrasonic bath. After the adsorption of the polymer layer for 15 min, the microcrystals were washed with water twice to remove unadsorbed polyelectrolyte and redispersed in a volume of water equal to the initial one. The next PSS or DexS layer was deposited on the surface of microcrystals in a similar way. The adsorption of positive and negative polymers was repeated n times to obtain n bilayers. Finally, shell-coated microcrystals were redispersed in 0.9% NaCl solution in a concentration of 10,0 mg/mL. The concentration of quercetin or resveratrol in a suspension of coated microcrystals (polyphenol/(CH/DexS) 4 and polyphenol/(PAH/PSS) 4 ) was determined spectrophotometrically, after extraction with ethanol.
UV irradiation. A germicidal lamp (G 30W, Sylvania), 95% of whose radiation is UV-C with a wavelength of 253.7 nm was used. The lamp was located at a distance of 10 cm from the cell plate, providing an irradiation intensity of 1.0 mW/cm 2 . Before irradiation, the medium was replaced with PBS. Immediately after irradiation, PBS was replaced with serum-free DMEM (Sham-irradiated control and UV-C series), native and particulate polyphenols at a dose of 50 µmol/L (UV-C + polyphenols series).
Analysis of cells viability. The study of the effect of UV-C on the viability of cultured cells was carried out in 96-well plates. After 20 h the viability of cells was determined using the PrestoBlueTM Reagent (Introvigen, USA) according to the instructions. The uorescence of resoru n was quanti ed on a microplate reader using an excitation of 560 nm and emission of 590 nm. The average uorescence intensity of wells containing control cells was taken as 100%.
Cell integrity was evaluated by lactate dehydrogenase (LDH) leakage. In these experiments, cells were grown in 24-well plates. The activity of LDH was measured using the direct spectrophotometric assay in 1 ml of phosphate buffer (pH 7.4) containing 30 µmol/L pyruvate and 30 µmol/L NADH. 100 µl of culture medium were added and changes in optical density were measured at 340 nm for 2 min. The percentage of LDH release was calculated by dividing the activity of LDH in the medium by the LDH activity measured after complete cell lysis. None of the detergents and drugs affected LDH activity, when added directly to the reaction mixture at the concentrations used in whole cell experiments.
Analysis of DNA damage by comet assay. Cells plated in 24-well plate were cultured for 2 h after UV irradiation. Alkaline Comet-assay was performed according to Singh and Tice [29] (link)[30] (link)[31] (link). Brie y, cells were trypsinized, 50 µl of the cell suspension of each experimental series was added to 300 µl of 0.7% lowmelting agarose, and the mixture was applied to glass slides pre-coated with normal-melting agarose. The preparations were placed in a lysis buffer and kept in the dark for 20 h (4°C). Slides were then incubated with alkaline electrophoresis buffer pH 13 (0.3 M NaOH and 1 mM EDTA) for 20 min.
Subsequently, electrophoresis was carried out for 20 min at 300 mA. The samples were washed twice in a neutralizing solution (pH 7.4, 4°C). Next, the slide was immersed in 70% ethanol for 5 min and 5 min in 96% ethanol then air dried, and stained with EB dye for 5 min. Comets were observed at 200 magni cation using a uorescence microscope Axiovert 25 (Zeiss, Germany) and documented using a digital camera. Percentage of DNA in the tail (damaged) was calculated for each comet using the histogram tool of Photoshop software. Data from three independent experiments were averaged for each experimental condition (n ≈ 150 cells).
Statistical analysis. The obtained data were tabulated and analyzed by Excel program. Results are presented as means ± standard deviation (SD). Since the data were normally distributed statistical signi cance was evaluated using a two-tailed unpaired by Student's t-test and P values < 0.05 were considered to be signi cant.