To link Pen G to NPs, 20 μL/mL of carboxylated NPs or sulfated
NPs and 1 mg Pen G were added to 1 mL deionised (DI) water, mixed well and
incubated at 28 °C with gentle shaking (50 rpm/min.). Centrifugal
ultrafiltration (Amicon Ultra-0.5 tubes, 3000 MWCO, Invitrogen, Thermo Fisher
Scientific; 10,000 rpm; 10 min) was used to separate freely dissolved Pen G from
Pen G–NP complexes. Finally, the NP–Pen G complexes were recovered
from the centrifuged ultrafiltration device and placed into a clean
microcentrifuge tube for 2 min, 1000 rpm, 4 °C. The concentrated
complexes were suspended into 1 mL of DI water and stored at 4 °C until
further use.
The optimum times for incubation for Pen G–NP complexes were
analyzed by calculating the amount of free Pen G in supernatant using an
ultraviolet (UV) spectrophotometric method after determining traction times (0,
1, 2, 3 and 4 h). Complexes of bound versus unbound Pen G were quantified using
UV spectrophotometry at an absorbance of 220 nm (UV-Vis; Shimadzu, Tokyo). NPs
were quantified by fluorescence by constructing standard calibration curves. All
measurements were determined in triplicate. Optimal associations of Pen G to NPs
occurred after a 3-h incubation, which was used for all further experimental
incubations.
NPs and 1 mg Pen G were added to 1 mL deionised (DI) water, mixed well and
incubated at 28 °C with gentle shaking (50 rpm/min.). Centrifugal
ultrafiltration (Amicon Ultra-0.5 tubes, 3000 MWCO, Invitrogen, Thermo Fisher
Scientific; 10,000 rpm; 10 min) was used to separate freely dissolved Pen G from
Pen G–NP complexes. Finally, the NP–Pen G complexes were recovered
from the centrifuged ultrafiltration device and placed into a clean
microcentrifuge tube for 2 min, 1000 rpm, 4 °C. The concentrated
complexes were suspended into 1 mL of DI water and stored at 4 °C until
further use.
The optimum times for incubation for Pen G–NP complexes were
analyzed by calculating the amount of free Pen G in supernatant using an
ultraviolet (UV) spectrophotometric method after determining traction times (0,
1, 2, 3 and 4 h). Complexes of bound versus unbound Pen G were quantified using
UV spectrophotometry at an absorbance of 220 nm (UV-Vis; Shimadzu, Tokyo). NPs
were quantified by fluorescence by constructing standard calibration curves. All
measurements were determined in triplicate. Optimal associations of Pen G to NPs
occurred after a 3-h incubation, which was used for all further experimental
incubations.