*2.4. Antibiotic Activity against Staphylococcus aureus and Pseudomonas aeruginosa*

The antibiotic activity of TC in the in vitro release study was assessed against a Gram-positive bacterial strain, *Staphylococcus aureus*, and a Gram-negative bacterial strain, *Pseudomonas aeruginosa*, using the antibiotic elution samples from a turbidity assay. TC (500 μL) eluated during the in vitro release study or from PBS (as the control group) was added to the inoculum containing 500 μL of the 2× nutrient broth solution and 100 μL of bacteria in 5-mL test tubes. After 24 h of incubation, the optical density (OD) of the culture medium was measured using a spectrophotometer at a wavelength of 600 nm. The relative antibacterial activity (R%) was calculated as follows:

$$\text{R\%} = \text{[(OD}\_{\text{growth medium}} - \text{OD}\_{\text{released tetraycline}}) / \text{OD}\_{\text{growth medium}}] \times 100\%$$
