*3.2. Acoustical Characterization*

*3.2. Acoustical Characterization*  Based on the physicochemical characterization described above, indirect DSPCbased microbubbles with 12 mol% cholesterol were chosen for acoustical characterization because they had the most homogeneous ligand and lipid phase distribution. They were compared to the direct and indirect DSPC-based microbubbles without cholesterol, and for each type of microbubble, data were acquired from at least two separate batches. Figure 5 shows a typical example of a 3D confocal acquisition before and after ultrasound with the corresponding *R-t* curve obtained from the ultra-high-speed recording at 50 kPa PNP for a direct DSPC (top row), indirect DSPC (middle row), and indirect DSPC-cholesterol (bottom row) microbubble. The coating of direct and indirect DSPC microbubbles was phase-separated into dark LC domains with a bright interdomain region, while the coating of indirect DSPC-cholesterol microbubbles was in one homogeneous lipid phase. This was in line with the results obtained by 4Pi confocal microscopy. The direct DSPC Based on the physicochemical characterization described above, indirect DSPC-based microbubbles with 12 mol% cholesterol were chosen for acoustical characterization because they had the most homogeneous ligand and lipid phase distribution. They were compared to the direct and indirect DSPC-based microbubbles without cholesterol, and for each type of microbubble, data were acquired from at least two separate batches. Figure 5 shows a typical example of a 3D confocal acquisition before and after ultrasound with the corresponding *R-t* curve obtained from the ultra-high-speed recording at 50 kPa PNP for a direct DSPC (top row), indirect DSPC (middle row), and indirect DSPC-cholesterol (bottom row) microbubble. The coating of direct and indirect DSPC microbubbles was phase-separated into dark LC domains with a bright interdomain region, while the coating of indirect DSPC-cholesterol microbubbles was in one homogeneous lipid phase. This was in line with the results obtained by 4Pi confocal microscopy. The direct DSPC microbubble shown in Figure 5 had one bright spot present in the coating before and after the ultrasound, which was classified as a buckle. The coating of the indirect DSPC microbubble in Figure 5 had one large and several smaller LC phase domains. For the indirect DSPC-cholesterol microbubble in Figure 5, the maximum intensity projection of the confocal *z*-stacks resulted in more brightness near the edge of the microbubble than in the center. However, when looking at the separate *z*-slices, the fluorescent signal was homogeneous over the microbubble coating (Supplemental Figure S3).

tudes.

microbubble shown in Figure 5 had one bright spot present in the coating before and after the ultrasound, which was classified as a buckle. The coating of the indirect DSPC microbubble in Figure 5 had one large and several smaller LC phase domains. For the indirect DSPC-cholesterol microbubble in Figure 5, the maximum intensity projection of the confocal *z*-stacks resulted in more brightness near the edge of the microbubble than in the center. However, when looking at the separate *z*-slices, the fluorescent signal was homo-

Oscillation amplitudes at frequencies between 1 and 4 MHz and acoustic pressure of 50 kPa were obtained per microbubble from the *R-t* curves and fitted to the harmonic oscillator model at each *fT* (examples at 1.2, 1.6. and 2.0 MHz shown in Supplemental Figure S4). Resonance frequencies resulting from the fit to the harmonic oscillator model are presented in Figure 6, with the obtained shell elasticity and viscosity parameters listed in Table 1. The shell elasticity of direct DSPC microbubbles was the highest, while the shell elasticity of indirect DSPC-cholesterol microbubbles was close to that of an uncoated microbubble. The shell viscosity parameter is related to the damping of the oscillation and was lowest for the direct DSPC microbubbles, which had the highest oscillation ampli-

geneous over the microbubble coating (Supplemental Figure S3).

**Figure 5.** Maximum intensity projections of confocal *z*-stack from direct DSPC, indirect DSPC, and indirect DSPC-cholesterol (12 mol%) microbubbles with the LE phase in red, before and after ultrasound, with the microbubble radius as a function of time obtained from the Brandaris 128 ultrahigh-speed recordings during ultrasound (50 kPa peak negative pressure (PNP), 1.6 MHz). Scale bar is 1 µm and applies to all images. **Figure 5.** Maximum intensity projections of confocal *z*-stack from direct DSPC, indirect DSPC, and indirect DSPC-cholesterol (12 mol%) microbubbles with the LE phase in red, before and after ultrasound, with the microbubble radius as a function of time obtained from the Brandaris 128 ultra-high-speed recordings during ultrasound (50 kPa peak negative pressure (PNP), 1.6 MHz). Scale bar is 1 µm and applies to all images.

Oscillation amplitudes at frequencies between 1 and 4 MHz and acoustic pressure of 50 kPa were obtained per microbubble from the *R-t* curves and fitted to the harmonic oscillator model at each *f<sup>T</sup>* (examples at 1.2, 1.6. and 2.0 MHz shown in Supplemental Figure S4). Resonance frequencies resulting from the fit to the harmonic oscillator model are presented in Figure 6, with the obtained shell elasticity and viscosity parameters listed in Table 1. The shell elasticity of direct DSPC microbubbles was the highest, while the shell elasticity of indirect DSPC-cholesterol microbubbles was close to that of an uncoated microbubble. The shell viscosity parameter is related to the damping of the oscillation and was lowest for the direct DSPC microbubbles, which had the highest oscillation amplitudes.


**Table 1.** Microbubble (MB) spectroscopy results at 50 kPa.

<sup>1</sup> presented as median (IQR); <sup>2</sup> indirect DSPC-cholesterol microbubbles with 12 mol% cholesterol.

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 11 of 22

**Figure 6.** Resonance frequency (MHz) per initial diameter (µm) at 50 kPa of direct DSPC (green), indirect DSPC (blue), and indirect DSPC-cholesterol (red, 12 mol%) microbubbles. The dotted line represents the resonance frequency of uncoated microbubbles. The shaded areas indicate the range of individual microbubble resonance frequencies obtained by fitting at each *fT*. **Figure 6.** Resonance frequency (MHz) per initial diameter (µm) at 50 kPa of direct DSPC (green), indirect DSPC (blue), and indirect DSPC-cholesterol (red, 12 mol%) microbubbles. The dotted line represents the resonance frequency of uncoated microbubbles. The shaded areas indicate the range of individual microbubble resonance frequencies obtained by fitting at each *fT*. Direct DSPC 44 0.14 (0.12–0.15) 0.43 (0.38–0.61) 8.0 1.5 Indirect DSPC 49 0.03 (0.01–0.06) 0.99 (0.89–1.40) 4.5 0.6 DSPC-cholesterol 2 50 0.01 (0.01–0.02) 1.39 (0.97–1.55) 10.2 0.7 1 presented as median (IQR); 2 indirect DSPC-cholesterol microbubbles with 12 mol% cholesterol.

**Median IQR of Oscillation Amplitude (%)** 

**Table 1.** Microbubble (MB) spectroscopy results at 50 kPa. **MB Type** *N* **Shell Elasticity 1 (N/m) Shell Viscosity 1 (× 10−8 kg/s) Max IQR of Oscillation Amplitude (%) Median IQR of Oscillation Amplitude (%)**  Direct DSPC 44 0.14 (0.12–0.15) 0.43 (0.38–0.61) 8.0 1.5 Indirect DSPC 49 0.03 (0.01–0.06) 0.99 (0.89–1.40) 4.5 0.6 DSPC-cholesterol 2 50 0.01 (0.01–0.02) 1.39 (0.97–1.55) 10.2 0.7 Figure 7 illustrates the variability in acoustical response within the three types of microbubbles. The variability was quantified as the interquartile range (IQR) of the oscillation amplitude from different microbubbles of the same size at the same transmit frequency (*N* > 3 per bin). The maximum and median IQR values for each type of microbubble are listed in Table 1. Indirect DSPC-cholesterol microbubbles had the highest maximum IQR, while direct DSPC microbubbles had the highest median IQR. Overall, indirect DSPC microbubbles exhibited the lowest variability in acoustical response. Figure 7 illustrates the variability in acoustical response within the three types of microbubbles. The variability was quantified as the interquartile range (IQR) of the oscillation amplitude from different microbubbles of the same size at the same transmit frequency (*N* > 3 per bin). The maximum and median IQR values for each type of microbubble are listed in Table 1. Indirect DSPC-cholesterol microbubbles had the highest maximum IQR, while direct DSPC microbubbles had the highest median IQR. Overall, indirect DSPC microbubbles exhibited the lowest variability in acoustical response.

1 presented as median (IQR); 2 indirect DSPC-cholesterol microbubbles with 12 mol% cholesterol.

**Figure 7.** Variability in acoustic response represented as the IQR of the oscillation amplitude at 50 kPa of direct DSPC, indirect DSPC, and indirect DSPC-cholesterol (12 mol%) microbubbles of different sizes (3.5–6.5 µm) at different transmit frequencies (1–4 MHz). All bins are based on *N* > 3; bins with *N* < 3 are blank. **Figure 7.** Variability in acoustic response represented as the IQR of the oscillation amplitude at 50 kPa of direct DSPC, indirect DSPC, and indirect DSPC-cholesterol (12 mol%) microbubbles of different sizes (3.5–6.5 µm) at different transmit frequencies (1–4 MHz). All bins are based on *N* > 3; bins with *N* < 3 are blank.

**Figure 7.** Variability in acoustic response represented as the IQR of the oscillation amplitude at 50 kPa of direct DSPC, indirect DSPC, and indirect DSPC-cholesterol (12 mol%) microbubbles of different sizes (3.5–6.5 µm) at different transmit frequencies (1–4 MHz). All bins are based on *N* > 3; bins with *N* < 3 are blank. Figure 8 shows the deflation of the microbubble, quantified as the diameter decrease relative to the initial diameter, for direct DSPC, indirect DSPC, and indirect DSPCcholesterol microbubbles. At 50 kPa, direct DSPC microbubbles deflated significantly more than the indirect DSPC and DSPC-cholesterol microbubbles, while no statistically significant difference in deflation was found between the indirect DSPC and DSPC-cholesterol microbubbles. However, at 50 kPa, the direct DSPC microbubbles had higher oscillation amplitudes than the other two groups. When comparing the deflation of microbubbles with similar oscillation amplitudes, marked as a gray area in Figure 8B, no statistically significant differences were found. Therefore, the statistical differences found at 50 kPa can be explained by a difference in oscillation amplitude, not acoustical stability. At 150 kPa, all types of microbubbles deflated significantly more than at 50 kPa. Furthermore, the

indirect DSPC microbubbles deflated significantly less than both other groups, also when comparing only microbubbles with similar oscillation amplitudes (Figure 8C). No statistically significant difference in deflation was found between direct DSPC and indirect DSPC-cholesterol microbubbles. rect DSPC microbubbles deflated significantly less than both other groups, also when comparing only microbubbles with similar oscillation amplitudes (Figure 8C). No statistically significant difference in deflation was found between direct DSPC and indirect DSPC-cholesterol microbubbles.

Figure 8 shows the deflation of the microbubble, quantified as the diameter decrease relative to the initial diameter, for direct DSPC, indirect DSPC, and indirect DSPC-cholesterol microbubbles. At 50 kPa, direct DSPC microbubbles deflated significantly more than the indirect DSPC and DSPC-cholesterol microbubbles, while no statistically significant difference in deflation was found between the indirect DSPC and DSPC-cholesterol microbubbles. However, at 50 kPa, the direct DSPC microbubbles had higher oscillation amplitudes than the other two groups. When comparing the deflation of microbubbles with similar oscillation amplitudes, marked as a gray area in Figure 8B, no statistically significant differences were found. Therefore, the statistical differences found at 50 kPa can be explained by a difference in oscillation amplitude, not acoustical stability. At 150 kPa, all types of microbubbles deflated significantly more than at 50 kPa. Furthermore, the indi-

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 12 of 22

**Figure 8.** (**A**) Diameter decrease (%) for direct DSPC (green), indirect DSPC (blue), and indirect DSPC-cholesterol (red, 12 mol% cholesterol) microbubbles at 50 kPa (left panel) and 150 kPa (right panel). Boxplots represent the median and IQR. \*\* *p* < 0.01. \*\*\* *p* < 0.001. (**B**–**C**) Maximum oscillation (%) as a function of diameter decrease (%) for direct DSPC (green), indirect DSPC (blue), and DSPC-cholesterol (red, 12 mol% cholesterol) microbubbles at 50 kPa (**B**) and 150 kPa (**C**). Gray areas indicate the range of maximum oscillation values reached by all three types of microbubbles. **Figure 8.** (**A**) Diameter decrease (%) for direct DSPC (green), indirect DSPC (blue), and indirect DSPC-cholesterol (red, 12 mol% cholesterol) microbubbles at 50 kPa (left panel) and 150 kPa (right panel). Boxplots represent the median and IQR. \*\* *p* < 0.01. \*\*\* *p* < 0.001. (**B**,**C**) Maximum oscillation (%) as a function of diameter decrease (%) for direct DSPC (green), indirect DSPC (blue), and DSPC-cholesterol (red, 12 mol% cholesterol) microbubbles at 50 kPa (**B**) and 150 kPa (**C**). Grayareas indicate the range of maximum oscillation values reached by all three types of microbubbles.

The nonlinear behavior was studied by looking at the acoustic response at the subharmonic and second harmonic frequencies at 50 and 150 kPa. At subharmonic frequencies, all types of microbubbles had a low response rate, and no statistical differences were found between the groups (Supplemental Figure S5). The percentages of microbubbles with a response at the second harmonic frequency are presented in Figure 9A. At 50 kPa, the direct DSPC microbubbles exhibited the highest number of second harmonic responses (68%), while this number was considerably lower for the indirect DSPC (26%) and the indirect DSPC-cholesterol (38%) microbubbles. At 150 kPa, all three types had similar percentages of microbubbles with a second harmonic response, and all occurrences were higher than those at 50 kPa. The second harmonic amplitudes were similar for all microbubble types at 50 kPa (Figure 9B). At 150 kPa, however, the direct DSPC microbubbles had significantly higher second harmonic amplitudes than both other microbubble types. Additionally, the indirect DSPC-cholesterol microbubbles had a significantly higher second harmonic amplitude than the indirect DSPC microbubbles. The nonlinear behavior was studied by looking at the acoustic response at the subharmonic and second harmonic frequencies at 50 and 150 kPa. At subharmonic frequencies, all types of microbubbles had a low response rate, and no statistical differences were foundbetween the groups (Supplemental Figure S5). The percentages of microbubbles with a response at the second harmonic frequency are presented in Figure 9A. At 50 kPa, the direct DSPC microbubbles exhibited the highest number of second harmonic responses (68%), while this number was considerably lower for the indirect DSPC (26%) and the indirect DSPC-cholesterol (38%) microbubbles. At 150 kPa, all three types had similar percentages of microbubbles with a second harmonic response, and all occurrences were higher than those at 50 kPa. The second harmonic amplitudes were similar for all microbubble types at 50 kPa (Figure 9B). At 150 kPa, however, the direct DSPC microbubbles had significantly higher second harmonic amplitudes than both other microbubble types. Additionally, the indirect DSPC-cholesterol microbubbles had a significantly higher second harmonic amplitude than the indirect DSPC microbubbles.

Confocal *z*-stacks of each microbubble were manually scored for the presence of buckles (none, single, multiple, or extensive with examples provided in Supplemental Figure S2) before and after ultrasound insonification (Figure 10). Indirect DSPC microbubbles (*N* = 49 at 50 kPa; *N* = 39 at 150 kPa) had the lowest occurrence of buckles both before and after ultrasound insonification, which was comparable to that of the direct DSPC microbubbles (*N* = 44 at 50 kPa; *N* = 41 at 150 kPa). Indirect DSPC-cholesterol microbubbles (*N* = 50 at 50 kPa; *N* = 42 at 150 kPa) had a notably higher occurrence of buckles than both other groups at both 50 and 150 kPa. Further analysis did not reveal a direct correlation between the oscillation amplitude and the presence of buckles in the shell before ultrasound insonification (Supplemental Figure S6). The maximum oscillation amplitude was compared between microbubbles without buckles, with a single buckle, with multiple buckles, or with extensive buckles in the coating before ultrasound insonification. For all types of microbubbles, at 50 and 150 kPa, no statistically significant differences in oscillation amplitude were found between the groups. Next, the correlation between the change in microbubble coating upon ultrasound insonification and the maximum oscillation amplitude was evaluated, as shown in Figure 11. The median excursion amplitude of microbubbles that experienced a change, either by forming a buckle or by shedding lipids from the coating, was significantly larger (*p* < 0.001) than the excursion amplitude of unchanged microbubbles for all microbubble types. For direct DSPC microbubbles, the difference between changed and

unchanged coatings was the most explicit, with a threshold amplitude of approximately 20% above which most microbubbles were changed after ultrasound insonification. For indirect DSPC microbubbles, the threshold amplitude was similar, albeit less pronounced. The indirect DSPC-cholesterol microbubbles also exhibited the formation of buckles and shedding of lipid material in microbubbles oscillating with amplitudes <20%. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 13 of 22

**Figure 9.** (**A**) Percentage of direct DSPC (green), indirect DSPC (blue), and indirect DSPC-cholesterol (red, 12 mol% cholesterol) microbubbles with a second harmonic response at 50 kPa (left panel) and 150 kPa (right panel). (**B**) Second harmonic amplitude normalized to the fundamental (dB) of direct DSPC (green), indirect DSPC (blue), and indirect DSPCcholesterol (red, 12 mol% cholesterol) microbubbles at 50 kPa (left panel) and 150 kPa (right panel). Boxplots represent the median and interquartile range (IQR), with whiskers ranging from maximum to minimum. \*\* *p* < 0.01. \*\*\* *p* < 0.001. **Figure 9.** (**A**) Percentage of direct DSPC (green), indirect DSPC (blue), and indirect DSPC-cholesterol (red, 12 mol% cholesterol) microbubbles with a second harmonic response at 50 kPa (left panel) and 150 kPa (right panel). (**B**) Second harmonic amplitude normalized to the fundamental (dB) of direct DSPC (green), indirect DSPC (blue), and indirect DSPCcholesterol (red, 12 mol% cholesterol) microbubbles at 50 kPa (left panel) and 150 kPa (right panel). Boxplots represent the median and interquartile range (IQR), with whiskers ranging from maximum to minimum. \*\* *p* < 0.01. \*\*\* *p* < 0.001. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 14 of 22

tion amplitude were found between the groups. Next, the correlation between the change in microbubble coating upon ultrasound insonification and the maximum oscillation am-**Figure 10.** Percentage of microbubbles with buckles (none, single, multiple, or extensive) before and after insonification at 50 kPa (**left panel**) and 150 kPa (**right panel**). Indirect DSPC-cholesterol microbubbles contained 12 mol% cholesterol. **Figure 10.** Percentage of microbubbles with buckles (none, single, multiple, or extensive) before and after insonification at 50 kPa (**left panel**) and 150 kPa (**right panel**). Indirect DSPC-cholesterol microbubbles contained 12 mol% cholesterol.

plitude was evaluated, as shown in Figure 11. The median excursion amplitude of microbubbles that experienced a change, either by forming a buckle or by shedding lipids from the coating, was significantly larger (*p* < 0.001) than the excursion amplitude of unchanged microbubbles for all microbubble types. For direct DSPC microbubbles, the difference between changed and unchanged coatings was the most explicit, with a threshold amplitude of approximately 20% above which most microbubbles were changed after ultrasound insonification. For indirect DSPC microbubbles, the threshold amplitude was similar, albeit less pronounced. The indirect DSPC-cholesterol microbubbles also exhibited the formation of buckles and shedding of lipid material in microbubbles oscillating with amplitudes <20%. Finally, the correlation between LC domain size and oscillation amplitude was investigated for a limited size range of microbubbles, ruling out size-dependent differences in oscillation (Figure 12). Since the indirect DSPC-cholesterol microbubbles were lacking LC domains, they could not be scored for their LC domain size. Unscored microbubbles are shown as black dots in Figure 12. For the direct and indirect DSPC microbubbles of 4.5–6.0 µm (initial diameter), the lipid phase distribution was scored as "only large LC domains", "large and small LC domains", or "undefined" (Supplemental Figure S7). Both the direct (*N* = 11) and indirect (*N* = 14) DSPC microbubbles with large and small LC domains had a significantly higher oscillation amplitude than those with only large LC domains (direct: *N* = 4, indirect: *N* = 15).

Finally, the correlation between LC domain size and oscillation amplitude was inves-

oscillation (Figure 12). Since the indirect DSPC-cholesterol microbubbles were lacking LC domains, they could not be scored for their LC domain size. Unscored microbubbles are shown as black dots in Figure 12. For the direct and indirect DSPC microbubbles of 4.5– 6.0 µm (initial diameter), the lipid phase distribution was scored as "only large LC domains", "large and small LC domains", or "undefined" (Supplemental Figure S7). Both the direct (*N* = 11) and indirect (*N* = 14) DSPC microbubbles with large and small LC domains had a significantly higher oscillation amplitude than those with only large LC do-

**Figure 11.** Maximum oscillation amplitude (%) of single direct DSPC (**left**, *N* = 85), indirect DSPC (**middle**, *N* = 88), and indirect DSPC-cholesterol (**right**, 12 mol% cholesterol, *N* = 92) microbubbles as a function of the initial microbubble diameter (µm). Microbubbles imaged by confocal microscopy directly before and after insonification (1–4 MHz, 50 or 150 kPa)

were compared and scored as unchanged (gray), formed a buckle (red) or shed lipid material (blue).

mains (direct: *N* = 4, indirect: *N* = 15).

**Figure 10.** Percentage of microbubbles with buckles (none, single, multiple, or extensive) before and after insonification at 50 kPa (**left panel**) and 150 kPa (**right panel**). Indirect DSPC-cholesterol microbubbles contained 12 mol% cholesterol.

**Figure 11.** Maximum oscillation amplitude (%) of single direct DSPC (**left**, *N* = 85), indirect DSPC (**middle**, *N* = 88), and indirect DSPC-cholesterol (**right**, 12 mol% cholesterol, *N* = 92) microbubbles as a function of the initial microbubble diameter (µm). Microbubbles imaged by confocal microscopy directly before and after insonification (1–4 MHz, 50 or 150 kPa) were compared and scored as unchanged (gray), formed a buckle (red) or shed lipid material (blue). **Figure 11.** Maximum oscillation amplitude (%) of single direct DSPC (**left**, *N* = 85), indirect DSPC (**middle**, *N* = 88), and indirect DSPC-cholesterol (**right**, 12 mol% cholesterol, *N* = 92) microbubbles as a function of the initial microbubble diameter (µm). Microbubbles imaged by confocal microscopy directly before and after insonification (1–4 MHz, 50 or 150 kPa) were compared and scored as unchanged (gray), formed a buckle (red) or shed lipid material (blue). *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 15 of 22

**Figure 12.** Maximum oscillation amplitude (%) at 50 kPa (over 1–4 MHz) as a function of initial diameter (µm) (**A**,**B**) for single direct DSPC (left) and indirect DSPC (right) microbubbles of 4.5–6.0 µm in diameter with LC domain size score as undefined (gray), large and small (red) or only large LC domains (blue), and as boxplot (**C**,**D**) representing the median and IQR with whiskers ranging from maximum to minimum. In A and B, the unscored microbubbles outside the 4.5–6.0 µm range are shown as black dots. \* *p* < 0.05. \*\* *p* < 0.01. **Figure 12.** Maximum oscillation amplitude (%) at 50 kPa (over 1–4 MHz) as a function of initial diameter (µm) (**A**,**B**) for single direct DSPC (left) and indirect DSPC (right) microbubbles of 4.5–6.0 µm in diameter with LC domain size score as undefined (gray), large and small (red) or only large LC domains (blue), and as boxplot (**C**,**D**) representing the median and IQR with whiskers ranging from maximum to minimum. In A and B, the unscored microbubbles outside the 4.5–6.0 µm range are shown as black dots. \* *p* < 0.05. \*\* *p* < 0.01.

### **4. Discussion 4. Discussion**

The results of this study showed that cholesterol significantly affected the ligand and lipid phase distribution in DSPC-based phospholipid-coated microbubbles made by the indirect method. The lipid handling prior to microbubble production also affected the ligand distribution, as shown previously [16]. Both the addition of cholesterol and the lipid handling prior to microbubble production were shown to influence the acoustic behavior of the microbubbles, as reflected in the apparent elasticity and viscosity values and reso-The results of this study showed that cholesterol significantly affected the ligand and lipid phase distribution in DSPC-based phospholipid-coated microbubbles made by the indirect method. The lipid handling prior to microbubble production also affected the ligand distribution, as shown previously [16]. Both the addition of cholesterol and the lipid handling prior to microbubble production were shown to influence the acoustic

The first part of this study revolved around the production and physicochemical characterization of DSPC-based microbubbles with cholesterol. Results indicated that the mean size of the microbubbles decreased with increasing concentrations of cholesterol. In contrast, Kaur et al. found that microbubbles with DSPC and cholesterol (1:1 molar ratio) were not significantly different in size from microbubbles with DSPC only [25]. However, those microbubbles were air-filled and did not contain any emulsifier such as PEG40-stearate or DSPE-PEG2000 like the microbubbles investigated in the present study. In our study, the span value increased with increasing concentrations of cholesterol, indicating

microbubbles with 12 mol% cholesterol.

*4.1. Physicochemical Characterization* 

behavior of the microbubbles, as reflected in the apparent elasticity and viscosity values and resonance frequencies. Finally, the variability in acoustic response was enhanced for the microbubbles without lipid phase separation in the coating, namely the indirect DSPC-based microbubbles with 12 mol% cholesterol.
