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Molecules 2019, 24, 1388 11 of 14 Molecules 2019, 24, x 11 of 14 inclduedveidceaibnlculuedLeEdDaabrlruaeyLcoEmDpaorsreadyocofm80pLoEseDdsocfen80teLreEdDastc4e2n5tenrmed,atco4o25lingmf,aancsoyostlienmgtfoanprseyvsetenmt to oveprhrevate,natnodvaershpeacitfi,ca-nddesaigsnpeedccifaisce-d.eTshiegnileludmciansaet.ioTnhearielaluwmaisndateisoingnaerdeatowbaesadblesitgonaecdcotmombeodaabtleto theasctcaonmdamrdod9a6t-ewthelel sptlantdesarudse9d6-iwnecllelpl-labtaeseudsaesdsainysc.elAl-bmasoevdaabslseapylsa.tAfomrmovinabstlaelpleldatfinortmheindsetavlilceed in allotwhedetvhiecediasltlaonwcedbtehtwe deeisntatnhceeLbEeDtwaerernaythaenLdEtDheairlrlauymaindatethdeairlleuam(tihneatteidssaureeacu(ltthueretispsluaetec)utloture be apdlajutes)tetdo bfreoamdj0ustote1d8frcomin0 tinoc1r8emcmenitns ionfc4re.5mcemnt.sTof d4.e5tecrm.iTnoe tdheeteerfmfeicnteotfhtehefdfeicstaonfcteheonditshtaence irraodnianthceeirmraitdtieadnbceyethmeitLtEedDbayrrathy,ethLeEiDrradrrianyc,etsheweirreamdieaanscuersedwaetrenimneasspuortesdonathneinilelusmpointsatoionnthe areaillfuomreiancahtioincraermeaefnotroefadchistiannccrem. entofdistance. Figure 5. Photograph of the illumination device (left) and the 10 × 8 blue LED array (right). Figure 5. Photograph of the illumination device (left) and the 10 × 8 blue LED array (right). 3.6. Cell Culture 3.6. Cell Culture The immortalized HaCaT human keratinocyte line was obtained from American Type Culture The immortalized HaCaT human keratinocyte line was obtained from American Type Culture Collection (Manassas, VA). The cells were cultured in CM, comprised of Dulbecco’s modified Eagle’s Collection (Manassas, VA). The cells were cultured in CM, comprised of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin-streptomycin at 37 ◦C in a humidified atmosphere of 5% CO2/95% air. penicillin-streptomycin at 37 °C in a humidified atmosphere of 5% CO2/95% air. 3.7. Induction of Proliferation of HaCaT Cells by TNF-α 3.7. Induction of Proliferation of HaCaT Cells by TNF-α The HaCaT cells in CM were seeded in 96-well plates at a density of 5 × 103 cells per well and The HaCaT cells in CM were seeded in 96-well plates at a density of 5 × 103 cells per well and incubated at 37 ◦C in a humidified atmosphere of 5% CO2/95% air for 24 h. The cultured cells were incubated at 37 °C in a humidified atmosphere of 5% CO2/95% air for 24 h. The cultured cells were then washed with serum free CM and TNF-α in serum free CM was added to 10 ng/mL and the cells then washed with serum free CM and TNF-α in serum free CM was added to 10 ng/mL and the cells were incubated for 24 h. were incubated for 24 h. 3.8. Cell Treatment and Photo-Irradiation 3.8. Cell Treatment and Photo-Irradiation Control and TNF-α-induced HaCaT cells were used to investigate the individual and combined Control and TNF-α-induced HaCaT cells were used to investigate the individual and combined effects of curcumin, Cur-CS/Alg NPs, and blue LED photo-irradiation on keratinocyte proliferation. effects of curcumin, Cur-CS/Alg NPs, and blue LED photo-irradiation on keratinocyte proliferation. Free curcumin dissolved in DMSO and Cur-CS/Alg NPs were added to the cells at final curcumin Free curcumin dissolved in DMSO and Cur-CS/Alg NPs were added to the cells at final curcumin concentrations of 0.05 and 0.1 μg/mL, while DMSO and CS/Alg NPs at 0.5% (v/v) and 1% (v/v), concentrations of 0.05 and 0.1 μg/mL, while DMSO and CS/Alg NPs at 0.5% (v/v) and 1% (v/v), respectively, were used as controls. The treated cells were incubated for 1 h and then the plate was respectively, were used as controls. The treated cells were incubated for 1 h and then the plate was exposed to the blue LED light for 5 min. A control plate not subjected to photo-irradiation was also exposed to the blue LED light for 5 min. A control plate not subjected to photo-irradiation was also prepared. The number of viable cells after treatment and photo-irradiation was then assessed by the prepared. The number of viable cells after treatment and photo-irradiation was then assessed by the MTT assay to determine the number of viable cells relative to that for the untreated control culture MTT assay to determine the number of viable cells relative to that for the untreated control culture (Section 3.9). (Section 3.9). 3.9. Cell Viability AssayPDF Image | Synergistic Effects of Photo-Irradiation and Curcumin-Chitosan
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