The inadequate transport of drugs into the tumor tissue caused by its abnormal vasculature is a major obstacle to the treatment of cancer. Anti-vascular endothelial growth factor (anti-VEGF) drugs can cause phenotypic alteration and maturation of the tumor's vasculature. However, whether this consistently improves delivery and subsequent response to therapy is still controversial. Clinical results indicate that not all patients benefit from antiangiogenic treatment, necessitating the development of criteria to predict the effect of these agents in individual tumors. We demonstrate that, in anti-VEGF-refractory murine tumors, vascular changes after VEGF ablation result in reduced delivery leading to therapeutic failure. In these tumors, the impaired response after anti-VEGF treatment is directly linked to strong deposition of fibrillar extracellular matrix (ECM) components and high expression of lysyl oxidases. The resulting condensed, highly crosslinked ECM impeded drug permeation, protecting tumor cells from exposure to small-molecule drugs. The reduced vascular density after anti-VEGF treatment further decreased delivery in these tumors, an effect not compensated by the improved vessel quality. Pharmacological inhibition of lysyl oxidases improved drug delivery in various tumor models and reversed the negative effect of VEGF ablation on drug delivery and therapeutic response in anti-VEGF-resistant tumors. In conclusion, the vascular changes after anti-VEGF therapy can have a context-dependent negative impact on overall therapeutic efficacy. A determining factor is the tumor ECM, which strongly influences the effect of anti-VEGF therapy. Our results reveal the prospect to revert a possible negative effect and to potentiate responsiveness to antiangiogenic therapy by concomitantly targeting ECM-modifying enzymes.

Localized and targeted drug delivery can be achieved by the combined action of ultrasound and microbubbles on the tumor microenvironment, likely through sonoporation and other therapeutic mechanisms that are not well understood. Here, we present a perfusable in vitro model with a realistic 3D geometry to study the interactions between microbubbles and the vascular endothelium in the presence of ultrasound. Specifically, a three-dimensional, endothelial-cell-seeded in vitro microvascular model was perfused with cell culture medium and microbubbles while being sonicated by a single-element 1 MHz focused transducer. This setup mimics the in vivo scenario in which ultrasound induces a therapeutic effect in the tumor vasculature in the presence of flow. Fluorescence and bright-field microscopy were employed to assess the microbubble–vessel interactions and the extent of drug delivery and cell death both in real time during treatment as well as after treatment. Propidium iodide was used as the model drug while calcein AM was used to evaluate cell viability. There were two acoustic parameter sets chosen for this work: (1) acoustic pressure: 1.4 MPa, pulse length: 500 cycles, duty cycle: 5% and (2) acoustic pressure: 0.4 MPa, pulse length: 1000 cycles, duty cycle: 20%. Enhanced drug delivery and cell death were observed in both cases while the higher pressure setting had a more pronounced effect. By introducing physiological flow to the in vitro microvascular model and examining the PECAM-1 expression of the endothelial cells within it, we demonstrated that our model is a good mimic of the in vivo vasculature and is therefore a viable platform to provide mechanistic insights into ultrasound-mediated drug delivery.

Despite advances in interventional procedures and chemotherapeutic drug development, hepatocellular carcinoma (HCC) is still the fourth leading cause of cancer-related deaths worldwide with a <30% 5-year survival rate. This poor prognosis can be attributed to the fact that HCC most commonly occurs in patients with pre-existing liver conditions, rendering many treatment options too aggressive. Patient survival rates could be improved by a more targeted approach. Ultrasound-induced cavitation can provide a means for overcoming traditional barriers defining drug uptake. The goal of this work was to evaluate preclinical efficacy of image-guided, cavitation-enabled drug delivery with a clinical ultrasound scanner. To this end, ultrasound conditions (unique from those used in imaging) were designed and implemented on a Philips EPIQ and S5-1 phased array probe to produced focused ultrasound for cavitation treatment. Sonovue ® microbubbles which are clinically approved as an ultrasound contrast agent were used for both imaging and cavitation treatment. A genetically engineered mouse model was bred and used as a physiologically relevant preclinical analog to human HCC. It was observed that imageguided and targeted microbubble cavitation resulted in selective disruption of the tumor blood flow and enhanced doxorubicin uptake and penetration. Histology results indicate that no gross morphological damage occurred as a result of this process. The combination of these effects may be exploited to treat HCC and other challenging malignancies and could be implemented with currently available ultrasound scanners and reagents.

A major challenge in developing blood-contacting medical devices is mitigating thrombogenicity of an intravascular device. Thrombi may interfere with device function or embolize from the device to occlude distant vascular beds with catastrophic consequences. Chemical interactions between plasma proteins and bioengineered surface occur at the nanometer scale; however, continuum models of blood predict local shear stresses that lead to platelet activation or aggregation and thrombosis. Here, an iterative approach to blood flow path design incorporating in silico, in vitro, and in vivo experiments predicted the occurrence and location of thrombi in an implantable hemofilter. Low wall shear stress (WSS) regions identified by computational fluid dynamics (CFD) predicted clot formation in vivo. Revised designs based on CFD demonstrated superior performance, illustrating the importance of a multipronged approach for a successful design process.

Ultrasound-mediated drug delivery using the mechanical action of oscillating and/or collapsing microbubbles has been studied in many different experimental platforms, both in vitro and in vivo; however, the mechanisms remain to be elucidated. Many groups use sterile, enclosed chambers, such as Opticells or Clinicells, to optimize acoustic parameters in vitro needed for effective drug delivery in vivo, as well as for mechanistic investigation of sonoporation, or the use to sound to permeate cell membranes. In these containers, cell monolayers are seeded on one side and the rest of the volume is filled with a solution containing microbubbles and a model drug. Ultrasound is then applied to study the effect of different parameters on model drug uptake in cell monolayers. Despite the simplicity of this system, the field has been unable to appropriately address what parameters and microbubble concentrations are most effective at enhancing drug uptake and minimizing cellular toxicity. In this work, a common in vitro sonoporation experimental setup was characterized through quantitative analysis of microbubble-dependent acoustic attenuation in combination with high frame rate and high resolution imaging of bubble activity during sonoporation pulse sequences. The goal was to visualize the effect that ultrasound parameters have on microbubble activity. It was observed that under literature-derived sonoporation conditions (0.1 to 1 MPa, 20 to 1000 cycles, and 10k to 10M microbubbles per mL), there is strong and nonlinear acoustic attenuation, as well as bubble destruction, gas diffusion, and bubble motion resulting in spatiotemporal pressure and concentration gradients. Ultimately, it was shown that the acoustic conditions in common in vitro sonoporation setups are much more complex and confounding than often assumed.