4.2. Preparation of the Photocatalysts for Deposition on Textiles
Amounts of 0.2 g photocatalyst powder and 0.006 g polyvinylpyrrolidone (PVP), respectively, were introduced in 400 mL of distilled water and stirred for 60 min in an ultrasonic bath. The final pH was adjusted to 8.5 with 1 N NaOH, which is relatively distant from pHpzc (point of zero charge) of TiO2, diminishing the repulsion forces between the particles and, consequently, their agglomeration. In addition, NaOH exhibits a swelling effect on textiles, especially on cotton, favoring the absorption of the photocatalysts.
PVP was used in formulating photocatalysts’ dispersions due to its ability to form molecular thin films on the surface of the particles, thereby preventing their agglomeration and sedimentation. Furthermore, PVP is a good moisturizer, absorbing over 40% of atmospheric moisture and, thus, contributing to the improvement of the photocatalytic effects.
4.3. Cotton Knit Treatment
4.3.1. Method P1
One hundred percent cotton knit (213 g/m2, 148/137.5 wales/courses; 1.08 mm thickness) was washed at 90 °C with water to remove any contaminants and detergent traces. The washed material was immersed in the above prepared photocatalyst dispersions, sonicated for 60 min at 40 °C and then dried in an oven at 100 °C for 60 min. It is important to note that a large amount of particles remained in the dispersion after this first treatment.
4.3.2. Method P2
In order to increase the degree of exhaustion of the treatment bath and to reduce the amount of TiO2 particles introduced into wastewaters, new textile materials were immersed in the remaining dispersion and treated in similar conditions as above. To fix the particles, the wet materials were immersed in 20 mL/L polyacrylic binder (ITOBINDER AG, self-cross linking aqueous acrylic copolymer emulsion acquired from LJ Specialities, Holmewood, UK, abbreviated ITO), maintained in an ultrasound bath for 30 min and then dried at 100 °C.
For comparisons, the fabric was treated with 400 mL solution containing 0.006 g PVP and 20 mL/L ITO.
The treated materials were noted: cotton ITO, knit treated with PVP and ITO; HT1, knit treated with TiO2-1% Fe-N (Sample 1); HT2, knit treated with TiO2-1% Fe-N (Sample 2); HT1 ITO, knit treated with TiO2-1% Fe-N (Sample 1) and ITO; HT2 ITO, knit treated with TiO2-1% Fe-N (Sample 2) and ITO.
4.4. Photocatalyst Characterization
The crystallization of photocatalysts obtained under hydrothermal conditions followed by thermal annealing at 400 °C for 2 h was highlighted by X-ray diffraction (XRD). A Bruker D8 Advance diffractometer (Bruker, Hamburg, Germany) using Cu Kα radiation (λ = 1.5406 Å) was used. The presence of iron in the TiO2 lattice was revealed by room temperature 57Fe Mössbauer transmission spectra recorded using a WissEL-ICE Oxford Mössbauer cryomagnetic system (Wissenschaftliche Elektronik GmbH, Starnberg, Germany, and ICE Innovative cryogenic system, Oxford, UK). The photocatalytic properties of Samples 1 and 2 (films on quartz) in the degradation of methylene blue, in both UV and visible regions, were investigated by means of the PCC2- ULVAC photocatalytic checker (ULVAC, Chigasaki, Kanagawa, Japan).
The morphology of the fabrics’ surfaces treated with photocatalysts and the amount of deposited metals were analyzed by SEM (Quanta 200, FEI, Eindhoven, The Netherlands) equipped with an EDX detector. Assessment of the photocatalytic effects was performed by staining the blank materials and those treated with TiO
2-1% Fe-N (Sample 1 and Sample 2) with 0.01 g/L MB solution and coffee (3 coffee teaspoons in 500 mL water). The materials were half covered with paper and were exposed to UV light (254 nm) in a completely closed cabinet and to visible light in a laboratory equipment, Xenotest. Chromaticity coordinates of exposed and non-exposed materials were measured by an UltraScan PRO Hunterlab UV-Vis spectrophotometer (Hunter Associates Laboratory, Reston, VA, USA). The color differences between the treated samples and the blank were measured via the CIE L*a*b* scale specified by the “Commission Internationale de l’Éclairage” (CIE) [
23], where:
L* represents lightness, a* signifies the red/green value and b* is the yellow/blue value;
dL* = L*sample − L*control; if dL* is positive, the sample is lighter than the control and, if dL* is negative, the sample is darker than the control;
da* = a*sample − a* control; if da* is positive, the sample is redder than the control. If da* is negative, the sample is greener than the control;
db* = b*sample − b* control; if db* is positive, the sample is yellower than the control. If db* is negative, the sample is bluer than the control;
dE* represents the total difference or distance on the CIELAB diagram as a single value for colour (da*, db*) and lightness (dL*) and, is calculated according to the following formula: dE* = .
4.5. Antimicrobial Activity Assay
The Gram-positive S. aureus ATCC 6538, E. faecalis ATCC 29212 and B. subtilis ATCC 6633 and the Gram-negative P. aeruginosa ATCC 27853 and E. coli ATCC 8739 were purchased from American Type Culture Collection (ATCC, Virginia, USA). Glycerol stocks were streaked on LB agar to obtain 24-h cultures to be used for all further studies.
Monospecific biofilm development was assessed at two different times of exposure, i.e., 15 min and 24 h. The textile materials were cut in equal circular samples of 8 mm and sterilized by autoclaving at 121 °C for 15 min. The sterile samples were then immersed in 1 mL of microbial suspensions of ~107 colony forming units (CFU)/mL performed in sterile saline and left in contact for 15 min and 24 h, respectively. After this interval, microbial suspensions incubated with the tested samples were vortexed and further serially ten-fold diluted, and 10 µL of each serial dilution were plated in triplicate on LB agar. After 24 h of incubation at 37 °C, viable cell counts were performed, and the number of CFU/mL for each sample was established.
4.6. In Vitro Biocompatibility Assessment
CCD-1070Sk normal human skin fibroblasts (purchased from ATCC, Cat. No. CRL-2091) were cultured at low passage in complete Eagle’s minimum essential medium (MEM; Gibco/Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco/Invitrogen) at 37 °C in a humidified atmosphere with 5% CO
2. Fibroblasts were grown to 70%–80% confluence within five to six days, then detached with 0.25% trypsin-0.03% EDTA and transferred to new culture flasks. The cells were seeded at a density of 2 × 10
4 cells per square centimeter in a 24-well plate and were left to adhere overnight. Then, cotton samples cut into 1 cm × 1 cm, sterilized at 120 °C for 20 min and exposed to a visible light source for 30 min, were soaked in culture medium and placed over the attached fibroblasts without disturbing the cells, as was previously reported for other materials [
24]. After 4 h of incubation, cytotoxicity tests were performed, and cell morphology and viability were evaluated. The untreated cotton sample (control) was used a control, and the results were expressed relative to this one.
The cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) assay (from the battery of cytotoxicity tests described in ISO 10993-5:2009, part 5) [
25] which is based on the quantification of mitochondrial succinate dehydrogenase activity in the viable cells. Briefly, the culture medium and cotton samples were removed at the end of the exposure time, and cells were incubated with 1 mg/mL MTT for 3 h at 37 °C and 5% CO
2. The purple formazan crystals formed in the viable cells were dissolved with 2-propanol (Sigma-Aldrich, St. Louis, MO, USA), and the absorbance was measured at 595 nm using a microplate reader (GENiosTecan, Salzburg, Austria).
The LDH release was assessed as a measure of cell membrane integrity using a commercial kit (TOX7, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Volumes of 50 µL of culture supernatants were incubated with 100 µL mix composed of equal parts of dye, substrate and cofactor for 30 min. The reaction was stopped by adding 15 µL of 1 N HCl, and the absorbance was read at 490 nm using a GENiosTecan microplate reader.
The level of nitric oxide (NO) released in the culture medium as an indicator of inflammation was determined using the Griess reagent. Culture supernatants were mixed with an equal volume of Griess reagent, a stoichiometric solution (v/v) of 0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% H3PO4. Absorbance was read at 550 nm using a GENiosTecan microplate reader, and the NO concentration was calculated on a NaNO2 standard curve.
Cell spreading and actin cytoskeleton morphology were investigated by fluorescence imaging using cells fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100-2% bovine serum albumin for 1 h. Images were captured using an inverse fluorescence microscope Olympus IX71 (Olympus, Tokyo, Japan). Filamentous actin (F-actin) was labelled with 20 µg/mL phalloidin conjugated with fluorescein isothiocyanate (FITC) (Sigma-Aldrich, Munich, Germany), and nuclei were stained with 2 µg/mL 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, Munich, Germany).