Living image

Cy5.5-labeled nanoparticles that were either preincubated with hMMP2 as described earlier or untreated were added to a streptavidin-coated 96 well plate for 1 h at room temperature. After incubation, the plate was washed three times with water, and the fluorescence from the wells was measured using a near-infrared imaging system (IVIS system, PerkinElmer) with excitation and emission wavelengths set at 640 and 700 nm, respectively. The Living Image (PerkinElmer) software was used for the analysis.

Mice were imaged using an IVIS Spectrum CT Pre-clinical in-vivo imaging system (PerkinElmer, MA, USA) with the following parameters: CT was set to 50ms, total projections were 720, voltage was set to 50kV while the current to 1mA the binning was determined to 2 and the X-ray filter was set to 440 A1. Hounsfield calibration was performed in air-0.004168 and water-0.5513. Two groups of tumors bearing mice (n=5 each) were divided to control and test group. The mice were weighed weekly and tumor progression was monitored by weekly IVIS scans (PerkinElmer). Epi-fluoresce scans were acquired under the following parameters: excitation 570nm, emission 620nm, at 3 seconds exposure. Therapeutic treatment began four weeks after the initial tumor cells injection. The test group received 300μl dose of collagozomes for two consecutive days, while the control group remained untreated. On the third day, both groups received a single dose of 10 mg/kg micellar paclitaxel.^{19 (link)} Tumor dimensions were assessed using the ROI tool in the IVIS imaging software (Living Image, PerkinElmer).

Mandible, radius, ulna, vertebra (3^rd lumbar, L3), distal
femur, and proximal tibia were assessed for whole bone fluorescence using
reflectance epi-fluorescence imaging (IVIS SpectralCT, PerkinElmer). Our lab and
others have previously used this method as an assay to measure
fluorescently-tagged bisphosphonate levels in tissues [17 (link),25 (link),27 (link),28 (link)]. Mandible, radius, and ulna were scanned whole whereas,
vertebrae processes were removed, and distal femur and proximal tibiae were cut
to 10 mm standard length sections. For each skeletal site, a bone from each
animal was run on a single plate to assume uniform scan setting. While this
allows comparison across animals within bone site, the optimization of settings
to accommodate for the size differences among bone sites restricts the ability
to compare levels across bone sites. Exposure time was approximately one second
per sample plate using broad emission spectral excitation and emission ranging
of 430–465nm and 500–540nm, respectively. FAM-ZOL signal was
distinguished from auto fluorescence and calcein blue signals by spectrally
unmixing the image series (Living Image, PerkinElmer). In all cases, bone
fluorescence levels are reported as average radiant efficiency
([photons/second]/[μW/cm2]).

hVNs/organs were imaged ex vivo with IVIS (Lumina Series III, PerkinElmer, MA) using the following settings: excitation 580–660 nm, emission 670–710 nm (NP-[CPP]-Alexa); excitation 480–560 nm, emission 620 nm (NP-[CPP]-Doxo). The fluorescence signal was analyzed using LivingImage (PerkinElmer, MA) and divided by the fluorescent signal obtained following saline injection. Animals were fed an AP Verified 75 IF Irradiated 5 V75 diet (ScottPharma, Marlborough, MA) prior to experimentation.