Direct insights into synthesis, protein integrity, and blood microrheology of albumin microbubbles.

Albumin microbubbles (MB) were among the first ultrasound (US) contrast agents used clinically. However, they are believed to contain denatured protein motifs, which compromise their stability, hence, limit their diagnostic and therapeutic utility. This study investigates the protein integrity, cavitation dynamics during US-assisted synthesis, and blood microrheology of albumin MB to better understand and improve their performance. Using bovine serum albumin as the shell material, we found that complexation with either a small molecule or macromolecular additive increased MB yield and enhanced acoustic stability. Spectroscopic analysis showed that MB shell formation favors protein structures close to the native state, while more severely altered fractions remain excluded from the shell. High-speed imaging and cavitation activity profiling revealed that additive-containing solutions suppressed cavitation activity while promoting the formation of sub-50 µm MB precursors under synthesis-mimicking conditions, leading to higher MB concentrations. Blood microrheology tests confirmed that albumin-copolymer MB had minimal impact on red blood cell deformability, aggregation, and critical shear stress, while in vivo cardiac US imaging showed their strong echogenicity lasting over 5 min post-injection. Together, these findings highlight how fine-tuning MB shell composition ‒ combined with structural and functional evaluation ‒ advances the understanding needed to improve albumin MB application potential. STATEMENT OF SIGNIFICANCE: This work provides integrated analysis of albumin-coated microbubbles, correlating protein structural integrity, synthesis dynamics, and blood microrheology. By combining spectroscopy, high-speed imaging, and rheological profiling, we demonstrate that rational additive selection enables microbubble formulations with enhanced acoustic stability, supported by in vivo cardiac ultrasound imaging. Notably, we show that microbubble formation favors albumin molecules retaining structures close to the native state, challenging the prevailing assumption that albumin shells are irreversibly denatured during synthesis. These findings provide a basis for designing structurally stable protein-coated microbubbles for effective ultrasound use.