This article describes a method to produce monodisperse phospholipid-coated microbubbles using a microfluidic flow-focusing device at room temperature, including how to prepare the phospholipid coating formulation.
Method Article
Production of Stable Monodisperse Phospholipid-coated Microbubbles at Room Temperature Using a Microfluidic Flow-focusing Device
In This Article
Summary
Abstract
Monodisperse microbubbles have demonstrated significant potential in enhancing the efficacy of ultrasound contrast agents for both imaging and therapeutic applications. These microbubbles can be directly produced using a microfluidic flow-focusing method, which allows precise control over their size. However, preventing coalescence during production remains a critical challenge. While elevated production temperatures (e.g., 55 °C) can suppress coalescence, such conditions complicate microfluidic device design and may be incompatible with targeting agents and drug conjugates. This protocol outlines a method for producing monodisperse phospholipid-coated microbubbles at room temperature, with the microbubbles' stability and monodispersity maintained for at least 7 days. The protocol also describes the fabrication of reusable PDMS microfluidic chips and preparation of the phospholipid coating formulation containing the surfactant Pluronic F68. Our findings show that the microbubbles produced using this method maintain their size and stability over 7 days, supporting their future applicability in clinical settings where stable and controllable ultrasound contrast agents are essential.
Introduction
Ultrasound contrast agents (UCA) are widely used in clinical echography and therapeutic applications1,2,3. Administered intravenously, these microbubbles remain confined in the vascular system, acting as blood pool agents4,5. A stabilizing shell coats the microbubbles and reduces gas diffusion driven by Laplace pressure, thus extending the microbubble's lifespan3,6. When exposed to ultrasound, microbubbles start to oscillate and produce strong echoes due t....
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Protocol
1. Making lipid films
NOTE: The lipid mixture for the monodisperse microbubble production is formulated with a binary mixture of DSPC and DPPE-PEG5000 at a molar ratio of 90:10. DSPC was chosen as the main lipid because it has been shown to provide superior stability against gas dissolution in both polydisperse33,34 and monodisperse microbubbles31. This enhanced stability is hypothesized to result from its relatively high gel-to-liquid crystalline phase transition temperature of 55 °C35, which is higher than t....
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Results
In this protocol, we present a method for producing stable, monodisperse microbubbles at room temperature using a flow-focusing microfluidic chip. The PDMS chip is fabricated by casting from a master mold, as shown in Figure 1. To bond the PDMS chip to a glass slide, plasma treatment is employed. Plasma treatment is crucial as it alters the PDMS surface from hydrophobic to hydrophilic. This hydrophilicity is essential for maintaining the shape and position of the gas thread during microbubbl.......
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Discussion
This protocol describes in detail how to produce and store stable monodisperse microbubbles at room temperature using a microfluidic flow-focusing chip. The monodisperse microbubble can be used for contrast-enhanced ultrasound imaging18, non-invasive pressure sensing19, and microbubble-mediated drug delivery22, both in vitro and in vivo.
The stabilization of monodisperse microbubbles post-production has p.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded in part by the European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Program (Grant Agreement 805308) and in part by the Applied and Engineering Sciences TTW (VIDI-project 17543), part of NWO, both awarded to K.K. The Horizon Microfluidic Platform was developed with funding from EPSRC grants (Grant Nos. EP/I000623 and EP/K023845). The authors thank the Microbubble Consortium (http://www.microbubbles.leeds.ac.uk/) at the University of Leeds for useful discussions as well as Stuart Weston and Andrew Price for their help in conceptualizing the instrument and production of parts for the Horizon Microfluidi....
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 1 mm biopsy punch | Kai Medical | 143831 | |
| 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine (DiD) | Thermo Fisher Scientic | D307 | a lipophilic carbocyanine dye |
| 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carbonyl-methoxypolyethylene glycol] (DPPE-PEG5000) | Lipoid | CAS-No. 384835-61-4 | |
| 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) | Lipoid | CAS-No. 816-94-4 | |
| 100 mL glass measuring cylinder | BLAUBRAND | ||
| 2 mL volumetric glass flasks | BLAUBRAND | ||
| 250 mL glass Erlenmeyer | BLAUBRAND | ||
| 30 mL glass vial | DWK Life Sciences | SCERC40752700D0C2 | |
| 70% ethanol | |||
| 75 °C hot plate | |||
| Adhesive tape | |||
| Aluminum cap | Sigma-aldrich | 27016 | |
| Argon gas | Linde Gas Benelux | ||
| C4F10 gas | F2 Chemicals | ||
| Chloroform | Merck KGaA | 67-66-3 | |
| Clinicallly available ultrasound contrast agent (i.e., microbubble) | Bracco, Plan-LesOuates, Switzerland | SonoVue | Figure 6A. |
| Collection and ventilation 19G needle | 100 Sterican | 4657799 | |
| Coulter Counter Multisizer 3 | Beckman Coulter | Particle characterization machine | |
| Custom-made ultrasound contrast agent (i.e., monodisperse microbubble) | NA | F1-10PF | Figure 6A. Produced as described by: Wang, Y. et al. Influence of Pluronic F68 on Size Stability and Acoustic Behavior of Monodisperse Phospholipid-Coated Microbubbles Produced at Room Temperature. ACS Appl. Mater. Interfaces 2025, 17, 8976−8986. (2025). |
| Custom-made ultrasound contrast agent (i.e., monodisperse microbubble) | NA | F2-10 PF | Figure 6A. Produced as described by: Wang, Y. et al. Influence of Pluronic F68 on Size Stability and Acoustic Behavior of Monodisperse Phospholipid-Coated Microbubbles Produced at Room Temperature. ACS Appl. Mater. Interfaces 2025, 17, 8976−8986. (2025). |
| Demi water | |||
| External LED light | GSVITEC | GS02370 | |
| Freeze dryer | Mertin Christ GmbH | Alpha 1–2 LD plus | |
| Function waveform generator | Agilent | 33220A | |
| Gas tight glass vial | Sigma-aldrich | Z113964-288EA | |
| Glass slide | VWR | 631-1552 | |
| High-speed camera | Photron | Nova S16 | |
| Horizon microfluidic platform | University of Leeds, England | The platform is commercially available and described in detail in: Abou-Saleh, R. H. et al. Horizon: Microfluidic platform for the production of therapeutic microbubbles and nanobubbles. Review of Scientific Instruments. 92 (7), 074105 (2021). | |
| Matlab | The MathWorks | R2023a | |
| Methanol | Merck KGaA | 67-56-1 | |
| Milli-Q water | |||
| PDMS gel | SYLGARD | SYLGARD® 184 | |
| Phosphate-buffered saline (PBS 1x) | Gibco | 10010023 | |
| Plasma cleaner | Henniker Scientific | HPT-100 | |
| Pluronic F68 | Thermo Fisher Scientific | 24040032 | |
| Rubber stopper | Sigma | Z166065-100EA | |
| Tubing | Masterflex | MFLX06407-41 | PTFE, 1/32" ID x 1/16 " OD; 25 ft |
| Tweezer | |||
| Vortex | Sigma-aldrich | Z654779 | |
| Water bath sonicator | EMAG | EMMI 30HC |
References
- Kooiman, K., et al. Ultrasound-responsive cavitation nuclei for therapy and drug delivery. Ultrasound Med Biol. 46 (6), 1296-1325 (2020).
- Langeveld, S. A. G., Meijlink, B., Kooiman, K. Phospholipid-coated targeted microbubbles for ultrasou....
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