Ivis lumina s5

The amount of drug deposited onto a ZA/AF647-ZOL/PLGA-coated screw
was determined by measuring the fluorescence of the screw using an
IVIS Lumina S5 instrument (PerkinElmer, Waltham, MA) equipped with
Living Image software (Small Animal Imaging Core Facility; University
of Iowa). The fluorescence intensities of the ZA/AF647-ZOL/PLGA-coated
screws, uncoated screw, and remaining coating solution were determined
after the dip-coating process was completed. Since the screw was laid
on its side when being imaged, the value for the fluorescence intensity
was doubled to account for the fact that only half of the screw was
visible to the camera. These intensity values were compared to that
obtained from imaging AF647-ZOL in water solutions with known concentrations
ranging from 0.211 ng/mL to 0.132 μg/mL along with a blank containing
only Nanopure water. These samples were used as references against
which the screw and coating solution samples could be compared in
order to determine the amount of drug deposited onto the screw surface.
The absorbance wavelength used was 620 nm, and the emission wavelength
measured was 670 nm. The data was plotted using GraphPad Prism version
9.0.0.

All melanomas were tagged with stable luciferase expression, enabling the quantitation of metastatic disease burden by bioluminescence imaging. Five minutes before imaging, mice were injected intraperitoneally with 100 μL of PBS containing D-luciferin monopotassium salt (40 mg/mL) (Goldbio). Mice were anesthetized with isoflurane 2 min before imaging. All mice were imaged using an IVIS Lumina S5 (Perkin-Elmer) with Living Image software (Perkin-Elmer). After completion of whole-body imaging, mice were euthanized and individual organs (including the heart, lung, liver, pancreas, spleen, and kidney) were surgically removed and imaged. The exposure time ranged from 15 to 30 s, depending on the maximum signal intensity, to avoid saturation of the luminescence signal. To measure the background luminescence, a negative control mouse not transplanted with melanoma cells was imaged. The bioluminescence signal (total photon flux) was quantified with “region of interest” measurement tools in Living Image software. Metastatic disease burden was calculated as observed total photon flux from all organs in xenografted mice minus background total photon flux in negative control mice. Negative values were set to 1 for purposes of presentation and statistical analysis.

To map the in vivo biodistribution of liposomes, DiD-loaded liposomes (25 μg DiD per mouse) were intravenously injected into the mice. On day 1 after liposome injection, the bones were harvested for fluorescence imaging by the IVIS® Lumina S5™ (PerkinElmer).