Production of Stable Monodisperse Phospholipid-coated Microbubbles at Room Temperature Using a Microfluidic Flow-focusing Device

Summary

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.

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 applications^1,2,3. Administered intravenously, these microbubbles remain confined in the vascular system, acting as blood pool agents^4,5. A stabilizing shell coats the microbubbles and reduces gas diffusion driven by Laplace pressure, thus extending the microbubble's lifespan^3,6. When exposed to ultrasound, microbubbles start to oscillate and produce strong echoes due t....

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 polydisperse^33,34 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....

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.......

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.......

Materials

List of materials used in this article

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