Targeted molecular imaging of VEGF receptors overexpressed in ischemic microvasculature using chitosan-DC101 conjugates

Expression of vascular endothelial growth factor receptors (VEGFRs) increases in ischemic muscles, and thus, VEGFR could potentially be used as marker to detect ischemia. Here, we investigated whether ^99mTc or Cy5.5-labeled chitosan-DC101 conjugates could identify VEGFR-2 overexpressed in ischemia. To this end, chitosan was conjugated with the DC101 antibody and Cy5.5, FITC, or the HYNIC chelator for ^99mTc-labeling. Targeting of the conjugate was evaluated in vitro and in vivo through cell-binding studies and gamma/optical imaging, respectively. A hindlimb ischemic mouse model was surgically created by femoral artery occlusion. The chitosan-DC101 conjugates exhibited VEGFR-selective cell binding properties as determined by both confocal microscopy and flow cytometry. At postoperative times of 2, 12, and 24 h, ^99mTc or Cy5.5-labeled chitosan-DC101 conjugates were intravenously injected into the mice, and gamma/optical imaging studies were conducted at 1 or 3 h. Both the gamma and optical imaging results indicated a significantly higher uptake in ischemic muscles when compared with the contralateral nonischemic muscle. Further, semiquantitative analysis of scintigraphic imaging data revealed that the ischemic to contralateral limb ratio was 4.5 ± 0.25 at 24 h postoperation. Western blotting analysis confirmed VEGFR expression in the ischemic muscle. In conclusion, we believe that ^99mTc or Cy5.5-labeled chitosan-DC101 conjugates have the potential to be useful as VEGFR-2-targeted imaging agents for monitoring ischemia.