Ultrasound protein-copolymer microbubble library engineering through poly(vinylpyrrolidone-co-acrylic acid) structure

While albumin-coated microbubbles are routine contrast agents for ultrasound imaging, their short duration of contrast enhancement limits their use, yet can be improved by incorporating protein-copolymer hybrids into microbubble shells. The incorporation of N-vinyl-2-pyrrolidone and acrylic acid copolymer (P(VP-AA)) has been shown to enhance the performance of bovine serum albumin (BSA)-coated microbubbles. However, the impact of the copolymer structural properties on key microbubble characteristics (i.e., concentration, mean diameter and acoustic response) remains poorly understood. Therefore, we hypothesize that the copolymer structure affects its capacity to form micelle-like nanoaggregates, protein-copolymer hybrids, and microbubble shells, ultimately influencing the physicochemical and acoustic properties of the microbubbles.

Here we evaluate the production and performance of BSA@P(VP-AA) microbubbles synthesized using a series of P(VP-AA) copolymers with -C₈H₁₇ and -C₁₈H₃₇ end groups and molecular weight cutoffs between 3.5 and 15 kDa. Both simulation and experimental data demonstrate that interactions between BSA and the copolymers significantly influence the performance of the resulting microbubbles across the library of 60 formulations.

The introduction of -C₈H₁₇ terminated copolymers into microbubble shells resulted in up to 200-fold higher concentration, 7-fold greater acoustic response, and 5-fold longer ultrasound contrast enhancement compared to plain BSA microbubbles. The enhanced acoustic performance was sustained during in vivo cardiac ultrasound imaging, without altering liver accumulation after copolymer introduction. These findings underscore how optimizing copolymer structure (specifically the terminal end group and molecular weight) can tailor the formation and performance of protein-copolymer-coated microbubbles, offering valuable insights for designing ultrasound contrast agents.