Synthesis, characterization and stability of BSA-encapsulated microbubbles

Abstract

In this work, we present an account of experimental studies performed for the synthesis, shelf stability and in vitro stability of microbubbles made from perfluorobutane (PFB) gas and coated in a shell of Bovine Serum Albumin (BSA). These microbubbles were produced by probe sonication method using formulations containing BSA, caprylic acid (CA) and N-acetyl-DL-tryptophan (Tryp) in different combinations. The freshly prepared polydisperse (0.5–20 μm) microbubble samples were then size isolated by centrifugal differentiation to produce microbubble suspensions with a narrow size range of 3 to 5 μm. Among all the different combinations of BSA, CA and Tryp used, the formulation containing BSA and Tryp yields microbubbles with a maximum shelf life (of ∼8 months). This stability was observed when microbubbles were stored in an aqueous solution (80% v/v) consisting of the original solution [which contains BSA and Tryp dissolved in phosphate buffer saline (PBS)] used to make the microbubbles, 1,2-propane-diol (10% v/v) and glycerin (10% v/v). Fluorescence and Circular Dichroism (CD) spectroscopic analyses were carried out to study the effect of additives such as CA and Tryp, and heating and sonication on the secondary and tertiary structure of BSA. It was found that the use of Tryp in the formulation favors greater unfolding of BSA as compared to CA. This in addition to a lower diffusivity of PFB, and lower solubility of BSA in the solution containing Tryp leads to a relatively stable microbubble suspension. Microbubbles produced using different BSA formulations were mixed with PBS at 37 °C to check in vitro stability of the microbubbles. It was observed that microbubbles produced from BSA and Tryp persisted longer than other microbubbles. In addition to this, in vitro ultrasonography (USG) carried out using freshly prepared microbubbles and microbubbles stored for around 4 months also yielded promising results.