Hydrophobic Gas Bubble Formation in Definity®: A Freeze Fracture Electron Microscopy Study

Freeze fracture‐electron microscopy (FF‐EM) was used to study the structure of the colloidal components present in Definity® and to determine changes that occur when the pharmaceutical is “activated” by shaking–a process by which the hydrophobic gas present in the headspace of the vial is incorporated into micro‐bubbles. Photomicrographs of the initial pharmaceutical show the presence of small colloidal structures which are also present after the formulation is shaken. While the product initially contains mainly small and some large gas bubbles, they mostly disappear upon shaking to be replaced by larger, more regular micro‐bubbles. This more regular distribution of larger micro‐bubbles is responsible for the functioning of the product as an ultrasonic contrast enhancement agent. The structure of the gas‐liquid interface is found to consist of a mixed phospholipid monolayer and not the previously hypothesized ordered interfacial liposome assembly.