RGD-Targeted Ultrasound Contrast Agent for Longitudinal Assessment of Hep-2 Tumor Angiogenesis In Vivo

Abstract

ObjectiveTo prepare arginine-glycine-aspartate (RGD)-targeted ultrasound contrast microbubbles (MBs) and explore the feasibility of their use in assessing dynamic changes in αvβ3 integrin expression in a murine model of tumor angiogenesis.MethodsRGD peptides were conjugated to the surfaces of microbubbles via biotin-avidin linkage. Microbubbles bearing RADfK peptides were prepared as controls. The RGD-MBs were characterized using an Accusizer 780 and optical microscopy. The binding specificity of the RGD-MBs for ανβ3-expressing endothelial cells (bEnd.3) was demonstrated in vitro by a competitive inhibition experiment. In an in vivo study, mice bearing tumors of three different stages were intravenously injected with RGD-MBs and subjected to targeted, contrast-enhanced, high-frequency ultrasound. Subsequently, tumors were harvested and sectioned for immunofluorescence analysis of ανβ3 expression.ResultsThe mean size of the RGD-MBs was 2.36 ± 1.7 μm. The RGD-MBs showed significantly higher adhesion levels to bEnd.3 cells compared to control MBs (P < 0.01). There was rarely binding of RGD-MBs to αvβ3-negative MCF-7 cells. Adhesion of the RGD-MBs to the bEnd.3 cells was significantly inhibited following treatment with anti-alpha(v) antibodies. The quantitative acoustic video intensity for high-frequency, contrast-enhanced ultrasound imaging of subcutaneous human laryngeal carcinoma (Hep-2) tumor xenografts was significantly higher in small tumors (19.89 ± 2.49) than in medium tumors (11.25 ± 2.23) and large tumors (3.38 ± 0.67) (P < 0.01).ConclusionsRGD-MBs enable noninvasive in vivo visualization of changes in tumor angiogenesis during tumor growth in subcutaneous cancer xenografts.

Advanced Microbubbles as a Multifunctional Platform Combining Imaging and Therapy

“…These RGD-MBs showed significantly higher adhesion to ανβ3-expressing endothelial cells compared to the controls. In vivo experiments verified that the quantitative acoustic intensity in Hep2-tumor xenografts showed significantly higher in small tumors (19.89 ± 2.49) than those in medium (11.25 ± 2.23) and large tumors (3.38 ± 0.67).The results concluded that RGD-MBs enabled non-invasive in vivo visualization of tumor angiogenesis during tumor growth in Hep2 cancers [40]. In addition to the targeted microbubbles have showed above to assess the tumor angiogenesis, the other targeted UCAs applied in animal experiments have been summarized in Table 2.…”

The principles and applications of avidin-based nanoparticles in drug delivery and diagnosis

“…[8] Microbubbles coupled with RGD peptide via avidin-biotin linkage were developed for the detection of Hep-2 related tumor angiogenesis. [9] Neutravidin conjugated superparamagnetic iron oxide nanoparticle has also been explored as an imaging agent for rhodopsin degeneration. [10] More recently, the avidin-based nanotechnology has found its applications in tissue engineering and cellular regeneration.…”

Molecular imaging of integrins in oncology

“…The authors argue that the secretion of pro-angiogenic growth factors and proteolytic enzymes in early fast growing tumors highly upregulates both sprouting and intussusceptive angiogenesis, while in larger stabilized tumors intussusceptive angiogenesis predominates. 60 All molecular imaging modalities used for the presented examinations have both advantages and disadvantages.…”