2.14. In vitro cytotoxicity assay
To investigate the in vitro cytotoxicity of different nanomaterials, NIH-3T3 and HUV-EC cells (1×104) were seeded in a 96-well plate. After incubation for 24 h, the culture media was removed and fresh culture media comprising different concentrations of HMPB, ofloxacin or OHH NPs (0, 25, 50, 75, 100, 125 μg/mL). After 24 h incubation, the samples were washed with PBS three times and 100 μL of CCK-8 solution was added into each well. After incubation for 2 h, cell viability was measured by a microplate reader in the absorbance of 450 nm.
Hemolysis assay was performed using fresh Balb/c mouse blood. The fresh mouse blood cells were harvested after centrifugation (3500 rpm, 5 min, 4 °C). After that, 950 μL of PBS containing various concentrations of nanomaterials are mixed with 50 μL of red blood cells. After incubation for 2 h at 37 °C, the samples were centrifuged (3500 rpm, 5 min, 4 °C) and the optical density at 540 nm of the supernatant were determined on a microplate reader. Each sample was measured in triplicate. PBS and pure water were acted the role of a negative and positive control. The ratio of hemolysis was calculated using the following equation:
Hemolysis (%) = (Asample - Anegative)/(Apositive - Anegative)×100%;
Where Asample is the absorbance value of the supernatant after the addition of HMPB, ofloxacin and OHH NPs, respectively. Anegative and Apositive are the absorbance values after the addition of PBS and pure water, respectively.