  Understanding the pressure dependence of the nonlinear behavior of
ultrasonically excited phospholipid (PL)-stabilized NBs is important for
optimizing US exposure parameters for implementations of contrast enhanced
ultrasound, critical to molecular imaging. The viscoelastic properties of the
shell can be controlled by introduction of membrane additives, such as
propylene glycol as a membrane softener or glycerol as a membrane stiffener. We
report for the first time, the production of high yield NBs with narrow
dispersity and different shell properties. Through precise control over size
and shell structure, we show how these shell components interact with the
phospholipid membrane, change its structure, affect their viscoelastic
properties, and consequently change their acoustic response. A two-photon
microscopy technique through a polarity-sensitive fluorescent dye, C-laurdan,
was utilized to gain insights on the effect of membrane additives to the
membrane structure. We report how the shell stiffness of NBs affects the
pressure threshold (Pt) for the sudden amplification in the scattered acoustic
signal from NBs. For narrow size NBs with 200 nm mean size, we find Pt to be
between 123-245 kPa for the NBs with the most flexible membrane as assessed
using C-Laurdan, 465-588 kPa for the NBs with intermediate stiffness and
588-710 kPa for the NBs with stiff membranes. Numerical simulations of the NB
dynamics are in good agreement with the experimental observations confirming
the dependence of acoustic response to shell properties, thereby substantiating
further the development in engineering the shell of UCAs. The viscoelastic
dependent threshold behavior can be utilized for significantly and selectively
enhancing the diagnostic and therapeutic ultrasound applications of potent
narrow size NBs.
