Targeted microbubbles for imaging tumor angiogenesis: assessment of whole-body biodistribution with dynamic micro-PET in mice

Abstract

Purpose: To evaluate in vivo whole-body biodistribution of microbubbles (MBs) targeted to tumor angiogenesis-related vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) by using dynamic micro-positron emission tomography (PET) in living mice.

Materials and methods: Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Lipid-shell perfluorocarbon-filled MBs, targeted to VEGFR2 via anti-VEGFR2 antibodies, were radiolabeled by conjugating the radiofluorination agent N-succinimidyl-4-[(18)F]fluorobenzoate (SFB) to the anti-VEGFR2 antibodies. These MBs were then injected intravenously into nude mice (n = 4) bearing angiosarcomas, and the whole-body biodistribution of these probes was assessed for 60 minutes by using dynamic micro-PET. Results were compared with ex vivo gamma counting (n = 6) and immunofluorescence staining (n = 6). Control studies in angiosarcoma-bearing mice were performed with injection of the radiolabeled antibodies alone (n = 3) or free SFB (n = 3). A mixed-effects regression of MB accumulation on fixed effects of time and tissue type (tumor or muscle) and random effect of animal was performed.

Results: VEGFR2-targeted MBs rapidly cleared from the blood circulation (50% blood clearance after approximately 3.5 minutes) and accumulated in the liver (mean, 33.4% injected dose [ID]/g +/- 13.7 [standard deviation] at 60 minutes) and spleen (mean, 9.3% ID/g +/- 6.5 at 60 minutes) on the basis of micro-PET imaging. These findings were confirmed with ex vivo gamma counting. Uptake of targeted MBs was significantly higher (P < .0001) in tumor than in adjacent skeletal muscle tissue. Immunofluorescence staining demonstrated accumulation of the targeted MBs within hepatic Kupffer cells and splenic macrophages. Biodistribution of the radiolabeled antibodies and free SFB differed from the distribution of the targeted MBs.

Conclusion: Dynamic micro-PET allows assessment of in vivo biodistribution of VEGFR2-targeted MBs.

Figure 1:

In vivo biodistribution of intravenously injected ¹⁸F-labeled targeted MBs measured by using region-of-interest–based analysis of dynamic micro-PET data sets in nude mice (n = 4). Rapid uptake and retention of targeted MBs were observed in both liver and spleen. Targeted MBs cleared quickly from blood, with almost background levels observed 30 minutes after intravenous administration of imaging agent. Error bars were omitted for better visibility.

Figure 2:

In vivo biodistribution of intravenously injected ¹⁸F-labeled targeted MBs in lung, gut, brain, muscle, and tumor measured by using region-of-interest–based analysis of dynamic micro-PET data sets in nude mice (n = 4). Retention of tumor angiogenesis–targeted MBs was higher in tumor tissue than in surrounding muscle tissue. Error bars were omitted for better visibility.

Figure 3a:

Coronal micro-PET images showed different whole-body biodistribution patterns among (a) radiolabeled targeted MBs, (b) radiolabeled antibodies alone, and (c) free SFB in living mouse. (a) Targeted MBs accumulated primarily in liver (arrow) and spleen (arrowhead) at 60 minutes after intravenous injection. (b) Radiolabeled antibodies alone showed high activity retained in blood pool (thin arrows) even after 60 minutes. Uptake in supraaortic vessels (arrowheads) and liver (thick arrow) were observed. (c) Free SFB cleared through kidneys (arrows).

Figure 3b:

Coronal micro-PET images showed different whole-body biodistribution patterns among (a) radiolabeled targeted MBs, (b) radiolabeled antibodies alone, and (c) free SFB in living mouse. (a) Targeted MBs accumulated primarily in liver (arrow) and spleen (arrowhead) at 60 minutes after intravenous injection. (b) Radiolabeled antibodies alone showed high activity retained in blood pool (thin arrows) even after 60 minutes. Uptake in supraaortic vessels (arrowheads) and liver (thick arrow) were observed. (c) Free SFB cleared through kidneys (arrows).

Figure 3c:

Coronal micro-PET images showed different whole-body biodistribution patterns among (a) radiolabeled targeted MBs, (b) radiolabeled antibodies alone, and (c) free SFB in living mouse. (a) Targeted MBs accumulated primarily in liver (arrow) and spleen (arrowhead) at 60 minutes after intravenous injection. (b) Radiolabeled antibodies alone showed high activity retained in blood pool (thin arrows) even after 60 minutes. Uptake in supraaortic vessels (arrowheads) and liver (thick arrow) were observed. (c) Free SFB cleared through kidneys (arrows).

Figure 4:

Traditional ex vivo biodistribution studies in nude mice at 4 minutes (n = 3; teal bars) and 60 minutes (n = 3; red bars) after intravenous administration of targeted MBs. Ex vivo biodistribution confirmed trends seen in in vivo biodistribution study, with highest retention of targeted MBs in both liver and spleen. Bars are means; error bars are standard deviations.

Figure 5:

Immunofluorescence staining of liver tissue for MBs (arrows) and Kupffer cells (arrowheads). Targeted MBs were located within Kupffer cells as soon as 4 minutes after intravenous administration. For MB staining, slices were incubated with Texas red–conjugated rabbit antistreptavidin primary antibody (red). For Kupffer cell staining, slices were incubated with primary monoclonal rat antimouse F4/80 antibody and secondary fluorescein isothiocyanate–labeled polyclonal goat antirat IgG2b antibody (green). (Original magnification, ×400.)

Figure 6:

Immunofluorescence staining of spleen 4 minutes after intravenous administration of targeted MBs. Most of MBs (red areas, arrows) are localized in splenic macrophages (green areas); some MBs remain free in splenic sinusoids (arrowheads). MBs were stained with Texas red–conjugated rabbit antistreptavidin primary antibody and macrophages were visualized with primary monoclonal rat antimouse F4/80 antibody and secondary fluorescein isothiocyanate–labeled polyclonal goat antirat IgG2b antibody. (Original magnification, ×400.)