Page 94 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Chapter 4
presence of MB and on cancer/epithelial cells. These studies may therefore not translate to our study and it needs further investigation whether the amount of cell confluence influences the response to tMB-treatment.
It was previously shown [38] that microbubbles with SPIO incorporated in their coating in combination with ultrasound could lead to an about three fold increase of SPIO labeling of tumor cells in vitro, without compromising cell viability. A polymer coated microbubble was used, which has been shown to behave differently than a lipid coated microbubble when exposed to ultrasound. Polymer microbubbles have a stiff coating, which can respond to ultrasound exposure under a high MI (>1) by cracking and releasing the encapsulated gas. On the contrary, lipid coated microbubbles will oscillate at low MI and can also fuse [26]. They can therefore have a more prolonged interaction with cells than polymer microbubbles. This could explain why we found a ~12 fold increase in SPIO labeling compared to the control treatment.
Clinical implications
From the in vivo perspective, using tMB instead of non-tMB is preferable for endothelial cell labeling and drug delivery since tMB can be specifically targeted to diseased endothelial cells [84]. In addition, when ultrasound is applied to bound tMB, the vibrations of the ultrasound-activated tMB will have a direct effect on the cell membrane. This may be the reason why tMB have been shown to be up to ~5 times more effective in stimulating cellular uptake of therapeutics in vivo [30]. CD31 used as target in this proof of concept study was chosen as model ligand because it is constitutively expressed on endothelial cell membranes. It can therefore be used to label endothelial cells with SPIO in tissue-engineered valves or vascular grafts in vitro. However, CD31 cannot be used in vivo as it is expressed throughout the entire vasculature tree [85]. For targeting tMB to tumor vasculature, αvβ3 or vascular endothelial growth factor receptor 2 (VEGFR2) can be used [84]. SPIO uptake by ultrasound-activated tMB in vivo is expected as we recently reported that tMB bound to αvβ3 can stimulate endothelial cell drug uptake in vivo [50]. VEGFR2 is another biomarker of interest for tMB. BR55, a tMB against VEGFR2, has recently successfully been used in clinical trials for prostate, breast, and ovarian cancer [86, 87]. Our future studies will focus on in vivo SPIO labeling as well as in vivo MRI tracking of the labeled endothelial cells. For the in vivo studies, our in vitro acoustic settings will need to be extrapolated taking into account that ultrasound is attenuated
92