S. aureus were then serially diluted and spread-plated on nutrient agar. Bacterial viability was assessed by counting the number of colonies formed on the agar plate. The colony

count was normalized by considering the untreated colony (negative) as 100% of bacteria viability. The viability of E. coli and P. aeruginosa after 24 h was determined by turbidity measurements (OD600nm), taking into account background caused by the NPs themselves. Effect of NO/THCPSi NPs on established biofilms The reduction in total viable cells recovered from established S. epidermidis biofilms treated with NO/THCPSi NPs was compared to the control biofilms of the same species Quisinostat not treated with the NPs. Glass microscope slides were cut into pieces with surface areas of 24 mm2. The glass pieces were cleaned with 70% ethanol and dried. S. epidermidis was cultured at 37°C in TSB overnight and diluted to

106 CFU/mL. The 106 CFU/mL microbial suspension was then added to each tube containing the glass slide pieces. The vials containing bacteria, broth, and glass slide pieces were placed in a 37°C ACY-738 in vivo incubator for biofilm formation. After 24 h, the glass slide pieces were removed from the nutrient broth, rinsed twice in sterile PBS, and individually transferred into new Eppendorf tubes containing a fresh suspension 1 mL of 0.1 mg/mL NO/THCPSi NPs and THCPSi NPs (control) in PBS and returned to the 37°C incubator. After 24 h, the tubes MK-8931 in vitro containing glass slide pieces were sonicated in a 125-W ultrasonic cleaner for 5 min to remove the biofilm-forming cells from the slide. The resulting bacterial suspension was subjected to serial tenfold dilutions, and 100 μL of appropriate dilutions

was plated onto agar plates, which were then incubated at 37°C overnight. The total number of colonies that grew on each plate was counted, and the number of viable biofilm bacteria removed from each slide was determined. Mammalian cell viability assay The cytotoxicity of the NO/THCPSi NPs was evaluated using NIH/3T3 fibroblast cells. The cells were maintained in DMEM supplemented with 10% FBS and 2 mM l-glutamine, check details 100 U/mL penicillin, 100 μg/mL streptomycin, and incubated at 37°C with 5% CO2. All mentioned procedures for the preparation of NO/THCPSi NPs and glucose/THCPSi NPs were done under sterile conditions within a biological safety cabinet (Bio-cabinet, Aura 2000, Microprocessor Automatic Control, Firenze, Italy). The NIH/3T3 cells were trypsinized and then seeded into polystyrene 96-well plates (Nalge Nunc International, Penfield, NY, USA) at a density of 3 × 104 cells/mL and then after 24 h, the cultured cells were incubated with NO/THCPSi NPs, glucose/THCPSi NPs, and THCPSi NPs at four different concentrations from 0.05 to 0.2 mg/mL for 48 h. After the incubation period, the culture medium was separated from the cultured cells and subjected to a LDH assay that was carried out following the manufacturer’s instructions.