Molecular Ultrasound Imaging With Clinically Translatable cRGD-Coated Microbubbles to Assess αvβ3-Integrin Expression in Inflammatory Bowel Disease.

Abstract

Inflammatory bowel disease (IBD) subdivides into Crohn disease (CD) and ulcerative colitis (UC), and is characterized by unpredictable periods of inflammation and results in significant patient suffering and even death. Conventional diagnostic methods, for example, colonoscopy, computed tomography, or magnetic resonance imaging, have limitations such as invasiveness, patient discomfort, and limited sensitivity and accuracy. Therefore, we propose ultrasound molecular imaging (USMI) to detect and characterize IBD. First, we evaluated integrin-αvβ3 as a biomarker of IBD in human samples and then used clinically translatable cyclic Arg-Gly-Asp-D-Phe-Lys (cRGDfK)-coupled poly(butyl)cyanoacrylate microbubbles (cRGD-MB) to assess IBD in mice. Vascular integrin-αvβ3 expression in human colon tissue samples (healthy, CD and UC, n = 10 per group) was analyzed by immunofluorescence staining. In mice, acute colitis was induced by administration of 4% dextran sodium sulfate in drinking water for 5 days. On day 7, USMI with cRGD-MB was performed in colitis (n = 6) and healthy (n = 5) mice. The signal of bound cRGD-MB was assessed by the destruction-replenishment method. Ex vivo analysis of mouse colon tissue was performed to assess the degree of colitis by hematoxylin-eosin staining and the vascular expression of integrin-αv by immunofluorescence. Human samples showed a significantly higher vascular integrin-αvβ3 expression in CD and UC tissue, when compared with healthy samples (P < 0.005). In mice, a higher binding of cRGD-MB to inflamed colon was detected by USMI compared with healthy controls (P < 0.005). Immunofluorescence staining confirmed these findings, showing stronger integrin-αv expression in acute colitis, with a good correlation between USMI signal intensity and integrin-αv expression (r = 0.8, P = 0.0016). Integrin-αvβ3 on vessels is a suitable marker for IBD. USMI using cRGD-MB accurately detects this marker and correlates well with histology. These encouraging results support clinical translation of this imaging method as a noninvasive and cost-effective monitoring tool.