Lipid-shelled nanobubbles (NBs) are emerging as potential
dual
diagnostic and therapeutic agents. Similar to their micron-scale counterparts,
microbubbles (1–10 μm), they can act as ultrasound contrast
agents as well as locally enhance therapeutic uptake. Recently, it
has been shown that the reduced size of NBs (<1 μm) promotes
increased uptake and accumulation in tumor interstitial space, which
can enhance their diagnostic and therapeutic performance. However,
accurate characterization of NB size and concentration is challenging
and may limit their translation into clinical use. Their submicron
nature limits accuracy of conventional microscopy techniques, while
common light scattering techniques fail to distinguish between subpopulations
present in NB samples (i.e., bubbles and liposomes). Due to the difficulty
in the characterization of NBs, relatively little is known about the
influence of size on their therapeutic performance. In this study,
we describe a novel method of using a commercially available nanoparticle
tracking analysis system, to distinguish between NBs and liposomes
based on their differing optical properties. We used this technique
to characterize three NB populations of varying size, isolated via
centrifugation, and subsequently used this to assess their potential
for enhancing localized delivery. Confocal fluorescence microscopy
and image analysis were used to quantify the ultrasound enhanced uptake
of fluorescent dextran into live colorectal cancer cells. Our results
showed that the amount of localized uptake did not follow the expected
trends, in which larger NB populations out-perform smaller NBs, at
matched concentration. To understand this observed behavior, the stability
of each NB population was assessed. It was found that dilution of
the NB samples from their stock concentration influences their stability,
and it is hypothesized that both the total free lipid and interbubble
distance play a role in NB lifetime, in agreement with previously
proposed theories and models.