Page 92 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Chapter 4
respect to the time of treatment with tMB and ultrasound (Fig 5). We observed the highest efficacy when SPIO were added with the tMB (0 min) for acoustic PNPs up to 80 kPa. When SPIO were added 5 or 15 min after treatment, SPIO uptake was lower, but still significantly higher (more than fivefold at 5 min) than natural uptake. This may suggest stimulated endocytosis as uptake mechanism rather than sonoporation, since resealing of pores created by ultrasound activated microbubbles has been reported on a relatively short time scale of up to a minute [28, 73]. On the other hand, our results may also suggest that both uptake by stimulated endocytosis and pore formation occurred when SPIO were added 5 min before or just before (0 min) treatment (Fig 5A). This is supported by four earlier ultrasound contrast agent studies [46, 47, 74, 75] as they reported uptake by both pore formation and endocytosis using similar (1 MHz [46, 47, 74, 75]) and different (0.3 MHz [74]) acoustic settings. Meijering et al. [46] and De Cock et al. [47] also addressed the influence of particle size on the uptake route: for dextrans larger than ~17 nm in radius, the primary uptake route was endocytosis instead of pore formation. On the other hand, pores of 1 nm in size [76] to > 100 μm2 in area [28] have been observed. However, induced pores >100 μm2 do not tend to reseal [28] which likely leads to cell death. This could explain why higher acoustic pressures (i.e., ≥ 80 kPa) in our study corresponded to increased cell death. Pores < 100 μm2 can still reseal, according to a recent study [28]. The SPIO colloid with low molecular weight dextran coating in EndoremTM, as used in our study, are 120 – 180 nm in size [77] so they could enter cells via resealable pores. The reason why Meijering et al. [46] observed dextran particles larger than ~17 nm mainly to be taken up by endocytosis, could be the different type of microbubble used (SonoVue versus tMB in our study) and/or the type of cells studied (bovine aortic endothelial cells versus HUVECs in our study).
The amount of SPIO positive cells was almost two times higher when SPIO were added 5 min instead of 15 min after insonification. This suggests a relatively short temporal window when the therapeutic agent can be actively taken up after microbubble insonification, especially when compared to the study by Yudina et al. who reported a temporal window of 24 h [78]. The difference could be due to the therapeutic compound (small 600 Da molecule Sytox Green versus the 120 – 180 nm SPIO in our study), the type of microbubble (SonoVue versus tMB in our study), the acoustic settings (ultrasound frequency
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