Nanobubble Ultrasound-Contrast Agents as a Strategy to Assess Tumor Microenvironment Characteristics and Nanoparticle Extravasation

In many chronic inflammatory diseases, the vascular endothelium becomes pathologically permeable due to conditions like angiogenesis and production of growth factors and inflammatory cytokines (e.g., histamine, bradykinin, etc.). In cancer, this process can be exploited for delivery of nanoparticles to tumors via the enhanced permeability and retention (EPR) effect. However, nanoparticle-based therapeutics reliant on the EPR effect have led to inconsistent results in patients. This is due to many factors, with a significant one being heterogeneous tumor vascular architecture and morphology both between patients and within a single tumor. Transport of the nanoparticle to the tumor and into the parenchyma is complicated by uptake by the immune system, ineffective margination, and inefficient extravasation. Guidance is needed to inform clinicians on what therapies may be most effective for each patient. Effective guidance could reduce health-care costs and negative side effects of medication. An inexpensive, safe, non-invasive, and real-time imaging method that has high temporal and spatial resolution may be capable of categorizing the extent of vascular permeability in tumors and once validated, personalize therapeutic regimens for patients. Such a tool could be used not only for tumors, but for all diseases involving pathologically permeable vasculature. With this goal in mind, the objective of this thesis is to work toward development of a real-time method for evaluating vascular permeability over the entire tumor using novel nanobubble (NB)-based contrast-enhanced ultrasound (CEUS).This work builds upon dynamic CEUS protocols used clinically with microbubbles (MBs). NBs, which are 100-400 nm in diameter, are approximately 10x smaller than MBs and have been shown to extravasate into the tumor interstitium. To reach the final objective of this work, NB dynamics from intravenous injection to retention in the tumor must be studied. To this aim, in vitro studies concerning NB backscatter and kinetics based on their cellular environment were first performed. These include NB interactions with human whole blood (i.e., NB-red blood cell adsorption) and the difference in NB-CEUS generated data when comparing NBs flowing through a vessel versus those diffusing through an extracellular matrix. The results from the in vitro work were then applied to an in vivo mouse tumor model where time-intensity curve and decorrelation time parameters were used to differentiate tumors with high and low potential for nanoparticle extravasation. Decorrelation time was compared to the intratumoral distribution of a doxorubicinloaded liposome and found that it was a potential parameter to assess nanoparticle extravasation and retention. The work in this thesis represents a new strategy for companion nanoparticles using NB-CEUS to characterize tumoral microenvironment characteristics like vascular permeability and nanoparticle retention to predict the heterogeneous distribution of therapeutic nanoparticles. These findings could lead to the use of personalized medicine for all patients receiving a nanomedicine treatment and could be potentially applicable to other pathologies involving pathologically permeable vasculature.