Page 91 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

enhanced permeability of the cell membrane [26]. As was expected, the total duration of insonification also showed an effect on the SPIO uptake (Fig 3A). It has been reported that at 1 MHz frequency at a low mechanical index (MI < 0.1), a lipid-coated microbubble can repeatedly oscillate for thousands of cycles; while at higher MI microbubbles are destroyed within about 100 μs (i.e. 100 cycles) irrespective of pulse length [62]. We indeed observed microbubbles still present up to 30 s at 80 kPa PNP (MI=0.08) (Fig 2). The improved uptake with prolonged insonification may be related with the persistent effect produced by microstreaming generated by microbubble vibrations as formulated earlier. Moreover, we noticed displacement of tMB with subsequent microbubble clustering and merging driven by secondary radiation force over the prolonged burst, as illustrated by Fig 2. These findings are in line with our previous study where tMB bound to endothelial cells also displaced, clustered, and merged by insonification at 1 MHz, albeit for a single burst of up to 50,000 cycles [63]. Detachment of bound lipid-coated tMB has been reported to be due to the attractive secondary Bjerknes force between two tMB [64, 65]. Aggregation of detached tMB forming bigger microbubble clusters may have influenced their oscillation dynamics as larger microbubbles have a lower resonance frequency than individual small microbubbles [66]. At a low driving frequency (for example 1 MHz as applied in this study), microbubble clusters are expected to have a higher amplitude of oscillation as they will be closer to resonance, which could have contributed to the enhanced SPIO uptake.
SPIO uptake
In our study, HUVECs showed ~1% natural uptake after 1 h of incubation, and this value increased to ~5% after 3 h (Fig 4A). Although the natural uptake of SPIO by HUVECs was previously reported by van Tiel et al. [67], this percentage of labeled cells is not sufficient for cell tracking. Treatment of HUVECs with ultrasound and tMB led to a dramatic increase of ~10-fold in SPIO uptake after 1 h incubation (Fig 4A). On the other hand, cell viability decreased between 5 min and 1 h incubation time (Fig 4B), suggesting that instantaneous cell death (i.e. irreversible sonoporation due to large pores) is less prominent than induced cell death. Induced cell death could occur via the apoptotic pathway, a process that takes time [68, 69], that can be activated by ultrasound and microbubbles as previously reported by others [70-72]. We also observed that the SPIO labeling efficiency was influenced by the SPIO addition time in
SPIO cell labeling using ultrasound
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