*4.2. Acoustical Characterization*

Microbubble spectroscopy was performed on direct DSPC, indirect DSPC, and indirect DSPC-cholesterol (12 mol%) microbubbles to characterize their acoustic behavior. The shell parameters found here can be directly compared to a previous study by van Rooij et al. [11], which used a similar method and included the same direct DSPC microbubbles as the current study. The shell elasticity found in the present study (0.14 (0.12–0.15) N/m) (median (IQR)) was slightly lower and the shell viscosity (0.43 (0.38–0.61) <sup>×</sup> <sup>10</sup>−<sup>8</sup> kg/s) slightly higher than previously published (0.26 <sup>±</sup> 0.13 N/m, mean <sup>±</sup> SD; 1.0 (0.7) <sup>×</sup> <sup>10</sup>−<sup>8</sup> kg/s, median (IQR)), however, still within the error margins. The indirect DSPC microbubbles had a shell elasticity approaching that of an uncoated microbubble, similar to the DPPCbased microbubbles studied by van Rooij et al. [11]. As the shell elasticity was lower, the resonance frequency was also lower (Equation (2)). Both the indirect DSPC and the indirect DSPC-cholesterol microbubbles had a higher shell viscosity than the direct DSPC microbubbles. This was reflected in the oscillation amplitudes at 50 kPa, which were higher for the direct DSPC microbubbles than for the other groups, indicating lower damping and, therefore, lower viscosity (Equation (1)).

The influence of lipid phase distribution and lipid handling on the variability in acoustic response was assessed by comparing microbubbles with lipid phase separation, i.e., indirect DSPC, to those without lipid phase separation, i.e., indirect DSPC-cholesterol (12 mol%), and to those made with a different way of lipid handling prior to microbubble production, i.e., direct DSPC. While indirect DSPC-cholesterol microbubbles had the

highest maximum variability in response, the median variability was highest for the direct DSPC microbubbles. These results suggest that lipid handling prior to microbubble production can reduce the variability in response and that although the maximum variability was highest in the indirect DSPC-cholesterol microbubbles, the difference in lipid phase separation did not affect the variability in acoustic response overall. Due to their more uniform response, the indirect DSPC microbubbles would be the most suitable candidate for drug delivery applications. Two maxima can be observed in the variability in the response ofdirect and indirect DSPC microbubbles to ultrasound insonification. While this may be explained as a size-dependent effect, it is not a distinct trend and perhaps more likely due to the limited sample size. Apart from differences between the microbubble types, all microbubbles exhibited the highest variability in response at the resonance frequency. Thus, to insonify microbubbles at a frequency other than their resonance frequency could be a new strategy to achieve a more uniform response to ultrasound, although monodisperse microbubbles are needed for this strategy to yield a uniform and predictive response of a bulk of microbubbles.

Acoustical stability was studied using the decrease in diameter after ultrasound, i.e., deflation. For this analysis, the mean microbubble diameter of 128 frames without ultrasound was regarded as the initial diameter. Since the final diameter was determined based on the last recording of each microbubble, i.e., the recording of ultrasound insonification at 4 MHz, only the last 10 frames were used to calculate the mean microbubble diameter. The microbubble size in these last frames was stable, and the difference in sample size is therefore not expected to influence the results. At 50 kPa, statistical differences in deflation could be explained by differences in the oscillation amplitude. At 150 kPa, however, the indirect DSPC microbubbles were significantly more stable than the direct DSPC and indirect DSPC-cholesterol microbubbles. Since no statistical differences were found between the direct DSPC and indirect DSPC-cholesterol microbubbles, the difference in acoustical stability was not caused by a difference in coating microstructure, which is in accordance with previous studies [40]. As the composition of direct and indirect DSPC microbubbles was exactly the same, the difference in acoustical stability must have been caused by the difference in lipid handling prior to microbubble production, which is known to alter the ligand distribution, synonymous to the distribution of DSPE-PEG2000 [16].

The nonlinear behavior of microbubbles is imperative for successful contrast-enhanced ultrasound imaging and ultrasound molecular imaging. At 50 kPa, the direct DSPC microbubbles had a more frequent second harmonic response than the other types of microbubbles. At 150 kPa, the majority of all types of microbubbles had a second harmonic response. The differences in variability in the acoustic response between the types of microbubbles, as presented in Figure 7, are also reflected in the range of the second harmonic response at 150 kPa, presented in Figure 9B. Indirect DSPC microbubbles had the lowest variability in second harmonic amplitude at 150 kPa, while the response at 50 kPa was comparable to the other types. This could be due to the low amplitudes of the second harmonic response at 50 kPa, which translates to a larger experimental error. The percentage of direct DSPC microbubbles with a nonlinear response, subharmonic or second harmonic, at 50 kPa was lower than published before [11]. This may be explained by the different pulse lengths (8 cycle- instead of 10-cycle pulse) or the fact that as a lower amount of light reaches the Brandaris 128 camera in the current imaging system, the noise level is slightly higher. More experiments focused on nonlinear behavior are needed for a comprehensive assessment of indirect DSPC microbubbles for ultrasound molecular imaging. However, this lies outside the scope of the present study.
