Surface Morphology of Lipid-Shelled DSPC:PEG ₄₀ St Microbubbles

Lipid-coated microbubbles (MBs) are widely used as ultrasound contrast agents and drug delivery vehicles, yet the microscale organization of their shell constituents remains poorly understood. In this study, we investigated how lipid shell composition and environmental factors influence the formation of domains in the shell of microbubbles composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and polyoxyethylene-40-stearate (PEG₄₀St) from 9:1 to 1:9 molar ratios. Fluorescence microscopy images revealed that these shells formed polymorphic, heterogeneous domain structures rather than uniform monolayers. We systematically vary the fluorescent probe type, probe concentration, solution pH, and PEG₄₀St content and found that each factor influenced the shell microstructure. Labeling MBs with FITC-DHPE and NBD-PC fluorophores showed complementary laterally segregated domains. Notably, increasing the PEG₄₀St fraction or the solution pH accentuated the heterogeneity among individual microbubbles. Three-dimensional z-stack reconstructions revealed clustered protrusions at the boundaries of these domains. Time-lapse imaging showed that MBs gradually developed bright rims as DSPC domains condensed and fluorophores migrated toward the edges of domains by diffusion. Fluorescence intensity differences among MBs, also within the same batch, indicated heterogeneous shell compositions. These findings demonstrated that DSPC/PEG₄₀St microbubble shells do not remain homogeneously mixed; instead, they segregate into compositionally distinct domains. Understanding the shell microstructure may lead to different ultrasound responses of microbubbles, their acoustic stability, drug loading, and conjugations, which may result in the design of more effective and reliable contrast agents for biomedical applications.