Our objective was to determine the lowest diagnostically effective dose for E-selectin-targeted poly n-butyl cyanoacrylate (PBCA)-shelled microbubbles and to apply it to monitor antiangiogenic therapy effects.
PBCA-shelled microbubbles (MBs) coupled to an E-selectin-specific peptide were applied in mice carrying MLS or A431 carcinoma xenografts scaling down the MB dosage to the lowest level where binding could be examined with a 18-MHz small animal ultrasound transducer. Differences in E-selectin expression in the two carcinoma xenografts were confirmed by enzyme-linked immunosorbent assay (ELISA). In addition, MLS tumor-bearing mice under antiangiogenic therapy were monitored using E-selectin-targeted MBs at the lowest applicable dose. Therapy effects on tumor vascularization were verified by immunohistological analyses.
The minimally required dosage was 7 × 107 MBs/kg body weight. This dosage was sufficient to enable E-selectin detection in high E-selectin-expressing MLS tumors, while low E-selectin-expressing A431 tumors required almost 2.5-fold higher doses. At the dose of 7 × 107 MBs/kg body weight, a decrease in E-selectin MB binding under antiangiogenic therapy could be assessed (being significant after 3 days of treatment; p < 0.0001), which was in line with the significant drop in E-selectin-positive area fractions that was found histologically (p < 0.05).
Molecular ultrasound imaging with our E-selectin-targeted MB and therapy monitoring was possible down to a dose of 7 × 107 MBs/kg body weight (equates to 66 μg PBCA/kg and 4.6 mg PBCA/70 kg). Improvements in choice of targets, MB composition, and other MB detection methods may improve sensitivity and lead to reliable detection results of clinically transferrable MBs at even lower dosage levels.
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This work was supported by the German Research Foundation (DFG), grant numbers KI 1072/5-1, KI1072/11-1, and SCHM1171/3-1, the DAAD project PHC Procope 31017PH, “Development of multimodal imaging probes for intravascular molecular imaging of tumors and inflammation,” and the Portuguese Fundação para a Ciência e a Tecnologia (FCT), grant number PTDC/QEQ-MED/2656/2012.
Conflict of Interest
The authors declare that they have no competing interests.
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Images showing single PBCA MBs in a gelatin matrix. B-mode (a) and contrast-mode-specific (b) ultrasound images were recorded shortly before (pre) and after (post) a destructive pulse (ultrasound imaging protocol correspondent to the experimental setup mentioned in the “Material and Methods” section). Through dilution of the MB suspension up to 2,750 MBs/ml, the calculated average amount of MBs in the ultrasound section was 6.25. The B-mode image shows signal from six similar particles before the destructive pulse, while the captured image after the pulse shows sole background signal. Hence, it is very likely that the Vevo 2100 with 18 MHz can detect single PBCA MBs in B-mode, while imaging with amplitude modulation showed no signal from MBs in the matrix at this level of dilution (PDF 821 kb)
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Spivak, I., Rix, A., Schmitz, G. et al. Low-Dose Molecular Ultrasound Imaging with E-Selectin-Targeted PBCA Microbubbles. Mol Imaging Biol 18, 180–190 (2016). https://doi.org/10.1007/s11307-015-0894-9
- Contrast-enhanced ultrasound
- Molecular imaging