**5. Conclusions**

We produced indirect DSPC-based microbubbles with 7, 10, 12, and 14 mol% cholesterol in the coating. Cholesterol reduced lipid phase separation in the microbubble coating, resulting in a single phase at 12 mol% where all components were miscible. Buckle formation was increased with the reduction of the LC phase area, suggesting increased spontaneous dissolution of the microbubbles. As the acoustic behavior of DSPC-based microbubbles made by the direct and indirect method was compared to that of indirect DSPC-based microbubbles with 12 mol% cholesterol, indirect DSPC microbubbles had the most uniform response to the ultrasound and were the most stable acoustically. They had a lower shell elasticity and higher shell viscosity than the direct DSPC microbubbles. The modified lateral molecular packing of indirect DSPC-cholesterol microbubbles resulted in the lowest shell elasticity and highest shell viscosity of all microbubble types. Direct DSPC microbubbles displayed more nonlinear acoustic behavior than the indirect DSPC and indirect DSPC-cholesterol microbubbles. Based on these results, we can conclude that both the lipid phase separation and lipid handling prior to microbubble production significantly affected the acoustic behavior of microbubbles. The indirect DSPC microbubbles had the most promising results with regard to stability and uniform ultrasound response. These are important traits for an ultrasound molecular imaging agent and for drug delivery applications, as the acoustic behavior of the microbubble must be predictable and controllable.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1999-492 3/13/1/119/s1, Figure S1: Normalization factors for quantitative analysis of 4Pi confocal microscopy data, Figure S2: Examples of buckle score based on confocal microscopy, Figure S3: Confocal slices from *z*-stack acquisition of indirect DSPC-cholesterol (12 mol%) microbubble, Figure S4: Relative oscillation amplitude of single microbubbles as a function of the initial diameter, Figure S5: Percentage of microbubbles with subharmonic responses and amplitudes of subharmonic response, Figure S6: Shell buckles versus oscillation amplitude, Figure S7: examples of LC domain size score based on confocal microscopy. Table S1. Concentration of indirect DSPC microbubbles (MBs).

**Author Contributions:** Conceptualization, S.A.G.L., I.B., N.d.J. and K.K.; methodology, S.A.G.L., I.B. and G.C.-L.; software, I.B.; validation, S.A.G.L., I.B. and G.C.-L.; formal analysis, S.A.G.L. and I.B.; investigation, S.A.G.L. and I.B.; resources, A.F.W.v.d.S. and K.K.; data curation, S.A.G.L.; writing original draft preparation, S.A.G.L.; writing—review and editing, S.A.G.L., I.B., G.C.-L., A.F.W.v.d.S., N.d.J. and K.K.; visualization, S.A.G.L.; supervision, N.d.J. and K.K.; project administration, S.A.G.L.; funding acquisition, A.F.W.v.d.S. and K.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded in part by the Phospholipid Research Center in Heidelberg, grant number KKO-2017-057/1-1, and in part by the Thorax Center of Erasmus University Medical Center in Rotterdam.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the complex nature and tailored analysis for the unique experimental set-ups used in this study.

**Acknowledgments:** The authors thank F. Mastik and R. Beurskens from the Department of Biomedical Engineering, Erasmus University Medical Center, Rotterdam, the Netherlands, for their technical assistance during the experiments. The authors thank the Erasmus Optical Imaging Centre of Erasmus MC for the use of their facilities and Gert van Cappellen and Alex Nigg for their help. Finally, the authors thank Lipoid GmbH for providing a free sample of DSPC phospholipid.

**Conflicts of Interest:** The authors declare no conflict of interest.
