Ivis instrument

For in vivo imaging, after the tumor reached ~5 mm in long diameter, the mice were injected with 100 μl saline, 100 μg ICG in 100 μl saline, HC60S-DI nanoparticles (DOX: 100 μg and ICG: 100 μg for both low and high drug loading content nanoparticles) in 100 μl saline. In vivo fluorescence images were taken at 6 h after intravenous injection via the tail vein using a PerkinElmer (Waltham, MA, USA) IVIS instrument with excitation at 780 nm and an 831 nm filter to collect the fluorescence emission of ICG. After in vivo imaging, the mice were sacrificed and the tumor, liver, kidney, lung, spleen, and heart were removed and collected for further ex vivo fluorescence imaging of ICG using the same IVIS instrument.

Female Nod-SCID mice were purchased from Jackson Labs at 9 weeks of age. At 12 weeks of age, mice were injected by tail vein with 1×10⁶ Ba/F3 cells transduced with pMIG-BCR-ABL1^T315I/H396R and pMMP-luciferase-hygromycin (Ridges et al., 2012 (link)) (kindly provided by Dr. Michael Engel, The University of Utah). Oral drug delivery was initiated 72 hr later. Mice received 50 μL of drug in solvent SCIE (66.25 mM sucrose, 13.25 mM citric acid, 40% isopropanol, and 6.7% ethanol; see below). Animals were dosed with asciminib (30 mg/kg), ponatinib (25 mg/kg), or the combination once daily. Luciferase was imaged on days 14 and 21 post-injection on a PerkinElmer (Waltham, MA) IVIS instrument. Dosing was held if mice lost >10% of body weight and maintained this loss on two consecutive days or if a mouse lost 4 grams in 24 hours. Mice were re-enrolled if they gained two grams or more. Mice were euthanized upon exhibiting signs of suffering, generally including lack of response to stimuli and/or lethargy.

SKH1E nude immunocompetent mice were used to assess the release behavior of LNHs containing labeled IgG and IL-12. Mice were subcutaneously injected in the hind flank with a 100 μL volume of either a bolus solution or a liposomal hydrogel. Each injection administered 10 μg of mouse IgG antibody and 12.5 μg of human IL-12. Isotype control IgG and human IL-12 were chosen for this study because both cargo were expected to have minimal immunoactivity, which would simplify our experimental system. Mice were imaged sequentially using a PerkinElmer IVIS instrument using automatic exposure acquisition settings. Mice were imaged immediately before injection, 30 minutes after injection, 1 hour after injection, 7 hours after injection, and then daily thereafter. Data were analyzed in the LivingImage software. Analysis consisted of defining a region of interest over the injection site or hydrogel, and exporting the total radiant efficiency measured in that region. Data were normalized to the maximum signal recorded during the study and then plotted in PRISM. Sample was n=3 for the bolus condition and n=4 for the liposomal hydrogel condition.