Lipid monolayer composition and production efficiency in DSPC/PEG₄₀St microbubbles for ultrasound applications

Lipid-coated microbubbles are widely used as ultrasound contrast agents (UCAs) and are being developed as carriers for drug and gene delivery. These microbubbles typically consist of an inert gas core and a stabilizing monolayer shell of phospholipid and a PEGylated emulsifier. In practice, a 9:1 M ratio of DSPC (a saturated phospholipid) to PEG-40-stearate (PEG₄₀St) is conventionally used, under a long-standing assumption that the final composition of the microbubble shell is identical to the initial mixture composition. In this study, we tested that assumption over a wide range of DSPC/PEG₄₀St ratios. Using sonication-based fabrication, we prepared microbubble suspensions with PEG₄₀St fractions from 10 % up to 90 %. We then quantified the shell composition by proton nuclear magnetic resonance (¹H NMR) and measured microbubble yield. Contrary to expectation, the PEG₄₀St content in the bubble shells lower than PEG₄₀St added, indicating selective exclusion or "squeezing out" of PEG₄₀St during formation. Only about 4-6 % of the total lipid mixture ended up in the bubble shells and the rest remained as excess in the sub-phase. Thus, 94-96 % of the costly lipid/emulsifier was wasted in the production process. These results overturn the conventional assumption and highlight a critical inefficiency such that substantial amounts of lipid and PEG₄₀St were lost during production, and the bubble yields were low. The findings have important implications for microbubble manufacturing, suggesting that alternative formulations or other production methods are needed to improve efficiency, and thus reduce costs.