Molecular Beacon-Based Microrna Biosensor for Imaging EPC-Treated Cellular Therapy of Ischemia

Angiogenesis, the process of new blood vessel formation, is an important therapeutic target in cardiovascular and malignant diseases for diverse reasons. Molecular imaging for angiogenesis has been attracted due to the use of anti-angiogenic therapeutic drugs to treat tumours, and of therapeutic angiogenesis induction to treat vascular diseases. We developed a novel biosensor of imaging microRNA126 (mir126) expressed during angiogenesis using a miRNA Molecular Beacon (MB) composed of a stem loop-structured DNA complementary to mir126 and Cy5.5 (near infrared, NIR)-black hole quencher 2 (BHQ2) (mir126 NIR MB). Mir126 in cord blood-derived endothelial precursor cells (CB-EPCs) was highly expressed and more expressed after the wound healing. The quantitative and qualitative fluorescence intensity of the mir126 NIR MB was high in CB-EPC and significantly increased after the wound healing, showing a great specificity of sensing endogenous mir126. From the CB-EPC-treated hind limb ischemia, cellular morphology and immune histochemical analysis using antibodies of vWF and CD31 showed a successful induction of angiogenesis and vascularisation and fluorescence signals of the mir126 NIR MB was gradually increased during 6 days of the cellular therapy and much stronger than the signals of laser Doppler imaging. The mir126 NIR MB demonstrated that the mir126sensor will be useful for early diagnosis of cellular therapy of ischemia and non-invasively sensitive imaging for cellular developments, diagnosis of disease and cellular therapy related to the miRNA function.