Inveon rodent model scanner

PET imaging and data analysis were performed using the microPET/micro-CT Inveon rodent model scanner (Siemens Medical Solutions USA, Inc.) as previously described (21 (link),22 (link)). Each tumor-bearing mouse was intravenously injected with 5–10 MBq of ⁶⁴Cu-NOTA-RamAb, and static PET scans were obtained at 3, 24, and 48 h after injection. Tracer uptake was reported as the %ID/g (mean ± SD; ≥ 3 mice per group).
Receptor blocking studies were performed to evaluate the specificity of ⁶⁴Cu-NOTA-RamAb to VEGFR-2 in vivo. Briefly, 3 mice were each injected with 5–10 MBq of ⁶⁴Cu-NOTA-RamAb 24 h after the administration of a blocking dose (50 mg/kg) of RamAb. At the last time point (48 h), biodistribution studies were performed to validate the PET data. Radioactivity in tissues and organs was measured using a γ-counter (PerkinElmer).

PET scans were performed using a microPET/microCT Inveon rodent model scanner (Siemens Medical Solutions USA, Inc.). Procedures for image acquisition and reconstruction and region-of-interest (ROI) analysis of the PET data were described previously [24 (link)]. Quantitative PET data were presented as %ID/g. Hindlimb ischemia model mice received an intravenous injection of ~10 MBq of ⁶⁴Cu- RGO-IONP-PEG via tail vein before serial PET scans.

⁶⁴Cu was produced with an onsite cyclotron (GE PETtrace). ⁶⁴CuCl₂ (150 MBq) was diluted in 300 μL of 0.1 M sodium acetate buffer (pH 5.5) and mixed with 30 μL of FeSe₂/Bi₂Se₃-PEG (1 mg/mL). The reaction was conducted at 37 °C for 60 min with constant shaking. The labeling yield was determined by thin-layer chromatography (TLC) at different time points. The resulting ⁶⁴Cu-FeSe₂/Bi₂Se₃-PEG was purified by a PD-10 column using PBS as the mobile phase.
Serum stability study was carried out to ensure ⁶⁴Cu is stably attached on FeSe₂/Bi₂Se₃-PEG for in vivo PET imaging. ⁶⁴Cu-FeSe₂/Bi₂Se₃-PEG was incubated in PBS and complete serum at 37 °C for up to 24 h. At different time points, portions of the mixture were sampled and filtered through 100 kDa MWCO filters. The radioactivity that remained on the filter was measured after discarding the filtrate. The retained (i.e., intact) ⁶⁴Cu on FeSe₂/Bi₂Se₃-PEG was calculated using the equation (radioactivity on filter/total sampled radioactivity × 100%).
For PET imaging, 4T1 tumor-bearing mice (3 mice per group) post i.v injection of 5~10 MBq of ⁶⁴Cu-FeSe₂/Bi₂Se₃-PEG solution were performed using a microPET/microCT Inveon rodent model scanner (Siemens Medical Solutions USA, Inc.). Data acquisition, image reconstruction, and ROI analysis of the PET data were performed as described previously^{[32 (link)]}.

PET scans and image reconstruction (maximum a posteriori (MAP) algorithm, no attenuation or scatter correction) were performed using a microPET/microCT Inveon rodent model scanner (Siemens Medical Solutions USA, Inc.) at various time points postinjection (p.i.) as described previously.^{38 (link)–40 (link)} After each scan, region-of-interest (ROI) analyses were conducted in each mouse with Inveon Research Workplace. ⁶⁴Cu-labeled targeted or nontargeted micelles were injected into each tumor-bearing mouse via the tail vein at a dose of 5 to 10 MBq per mouse before 3 to 15 min static PET scans were performed. Quantitative data were shown as percent injected dose per gram of tissue (%ID/g). For the competitive binding studies, one mg of competing TRC105 antibody was preinjected into four 4T1 tumor-bearing mice one hour before the ⁶⁴Cu-labeled targeted micelles was administered to the mice in order to further assess the CD105 specificity in vivo. Biodistribution studies (mean ± SD) were conducted after the last PET scans using a gamma-counter (Perkin-Elmer) in order to validate the PET analysis.