Using imaging modalities to predict nanoparticle distribution and treatment efficacy in solid tumors: The growing role of ultrasound.

Nanomedicine in oncology has not had the success in clinical impact that was anticipated in the early stages of the field's development. Ideally, nanomedicines selectively accumulate in tumor tissue and reduce systemic side effects compared to traditional chemotherapeutics. However, this has been more successful in preclinical animal models than in humans. The causes of this failure to translate may be related to the intra- and inter-patient heterogeneity of the tumor microenvironment. Predicting whether a patient will respond positively to treatment prior to its initiation, through evaluation of characteristics like nanoparticle extravasation and retention potential in the tumor, may be a way to improve nanomedicine success rate. While there are many potential strategies to accomplish this, prediction and patient stratification via noninvasive medical imaging may be the most efficient and specific strategy. There have been some preclinical and clinical advances in this area using MRI, CT, PET, and other modalities. An alternative approach that has not been studied as extensively is biomedical ultrasound, including techniques such as multiparametric contrast-enhanced ultrasound (mpCEUS), doppler, elastography, and super-resolution processing. Ultrasound is safe, inexpensive, noninvasive, and capable of imaging the entire tumor with high temporal and spatial resolution. In this work, we summarize the in vivo imaging tools that have been used to predict nanoparticle distribution and treatment efficacy in oncology. We emphasize ultrasound imaging and the recent developments in the field concerning CEUS. The successful implementation of an imaging strategy for prediction of nanoparticle accumulation in tumors could lead to increased clinical translation of nanomedicines, and subsequently, improved patient outcomes. This article is categorized under: Diagnostic Tools In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery Emerging Technologies.

Decorrelation Time Mapping as an Analysis Tool for Nanobubble-Based Contrast Enhanced Ultrasound Imaging.

Nanobubbles (NBs; ~100-500 nm diameter) are preclinical ultrasound (US) contrast agents that expand applications of contrast enhanced US (CEUS). Due to their sub-micron size, high particle density, and deformable shell, NBs in pathological states of heightened vascular permeability (e.g. in tumors) extravasate, enabling applications not possible with microbubbles (~1000-10,000 nm diameter). A method that can separate intravascular versus extravascular NB signal is needed as an imaging biomarker for improved tumor detection. We present a demonstration of decorrelation time (DT) mapping for enhanced tumor NB-CEUS imaging. In vitro models validated the sensitivity of DT to agent motion. Prostate cancer mouse models validated in vivo imaging potential and sensitivity to cancerous tissue. Our findings show that DT is inversely related to NB motion, offering enhanced detail of NB dynamics in tumors, and highlighting the heterogeneity of the tumor environment. Average DT was high in tumor regions (~9 s) compared to surrounding normal tissue (~1 s) with higher sensitivity to tumor tissue compared to other mapping techniques. Molecular NB targeting to tumors further extended DT (11 s) over non-targeted NBs (6 s), demonstrating sensitivity to NB adherence. From DT mapping of in vivo NB dynamics we demonstrate the heterogeneity of tumor tissue while quantifying extravascular NB kinetics and delineating intra-tumoral vasculature. This new NB-CEUS-based biomarker can be powerful in molecular US imaging, with improved sensitivity and specificity to diseased tissue and potential for use as an estimator of vascular permeability and the enhanced permeability and retention (EPR) effect in tumors.

Assessing Tumor Microenvironment Characteristics and Stratifying EPR with a Nanobubble Companion Nanoparticle via Contrast-Enhanced Ultrasound Imaging.

The tumor microenvironment is characterized by dysfunctional endothelial cells, resulting in heightened vascular permeability. Many nanoparticle-based drug delivery systems attempt to use this enhanced permeability combined with impaired lymphatic drainage (a concept known as the 'enhanced permeability and retention effect' or EPR effect) as the primary strategy for drug delivery, but this has not proven to be as clinically effective as anticipated. The specific mechanisms behind the inconsistent clinical outcomes of nanotherapeutics have not been clearly articulated, and the field has been hampered by a lack of accessible tools to study EPR-associated phenomena in clinically relevant scenarios. While medical imaging has tremendous potential to contribute to this area, it has not been broadly explored. This work examines, for the first time, the use of multiparametric dynamic contrast-enhanced ultrasound (CEUS) with a novel nanoscale contrast agent to examine tumor microenvironment characteristics noninvasively and in real-time. We demonstrate that CEUS imaging can: (1) evaluate tumor microenvironment features and (2) be used to help predict the distribution of doxorubicin-loaded liposomes in the tumor parenchyma. CEUS using nanobubbles (NBs) was carried out in two tumor types of high (LS174T) and low (U87) vascular permeability, and time-intensity curve (TIC) parameters were evaluated in both models prior to injection of doxorubicin liposomes. Consistently, LS174T tumors showed significantly different TIC parameters, including area under the rising curve (2.7x), time to peak intensity (1.9x) and decorrelation time (DT, 1.9x) compared to U87 tumors. Importantly, the DT parameter successfully predicted tumoral nanoparticle distribution (r = 0.86 ± 0.13). Ultimately, substantial differences in NB-CEUS generated parameters between LS174T and U87 tumors suggest that this method may be useful in determining tumor vascular permeability and could be used as a biomarker for identifying tumor characteristics and predicting sensitivity to nanoparticle-based therapies. These findings could ultimately be applied to predicting treatment efficacy and to evaluating EPR in other diseases with pathologically permeable vasculature.

Real-time imaging of nanobubble ultrasound contrast agent flow, extravasation, and diffusion through an extracellular matrix using a microfluidic model.

Lipid shell-stabilized nanoparticles with a perfluorocarbon gas-core, or nanobubbles, have recently attracted attention as a new contrast agent for molecular ultrasound imaging and image-guided therapy. Due to their small size (∼275 nm diameter) and flexible shell, nanobubbles have been shown to extravasate through hyperpermeable vasculature (e.g., in tumors). However, little is known about the dynamics and depth of extravasation of intact, acoustically active nanobubbles. Accordingly, in this work, we developed a microfluidic chip with a lumen and extracellular matrix (ECM) and imaging method that allows real-time imaging and characterization of the extravasation process with high-frequency ultrasound. The microfluidic device has a lumen and is surrounded by an extracellular matrix with tunable porosity. The combination of ultrasound imaging and the microfluidic chip advantageously produces real-time images of the entire length and depth of the matrix. This captures the matrix heterogeneity, offering advantages over other imaging techniques with smaller fields of view. Results from this study show that nanobubbles diffuse through a 1.3 µm pore size (2 mg mL-1) collagen I matrix 25× faster with a penetration depth that was 0.19 mm deeper than a 3.7 µm (4 mg mL-1) matrix. In the 3.7 µm pore size matrix, nanobubbles diffused 92× faster than large nanobubbles (∼875 nm diameter). Decorrelation time analysis was successfully used to differentiate flowing and extra-luminally diffusing nanobubbles. In this work, we show for the first time that combination of an ultrasound-capable microfluidic chip and real-time imaging provided valuable insight into spatiotemporal nanoparticle movement through a heterogeneous extracellular matrix. This work could help accurately predict parameters (e.g., injection dosage) that improve translation of nanoparticles from in vitro to in vivo environments.

Characterization of the interaction of nanobubble ultrasound contrast agents with human blood components.

Nanoscale ultrasound contrast agents, or nanobubbles, are being explored in preclinical applications ranging from vascular and cardiac imaging to targeted drug delivery in cancer. These sub-micron particles are approximately 10x smaller than clinically available microbubbles. This allows them to effectively traverse compromised physiological barriers and circulate for extended periods of time. While various aspects of nanobubble behavior have been previously examined, their behavior in human whole blood has not yet been explored. Accordingly, herein we examined, for the first time, the short and long-term effects of blood components on nanobubble acoustic response. We observed differences in the kinetics of backscatter from nanobubble suspensions in whole blood compared to bubbles in phosphate buffered saline (PBS), plasma, or red blood cell solutions (RBCs). Specifically, after introducing nanobubbles to fresh human whole blood, signal enhancement, or the magnitude of nonlinear ultrasound signal, gradually increased by 22.8 ± 13.1% throughout our experiment, with peak intensity reached within 145 s. In contrast, nanobubbles in PBS had a stable signal with negligible change in intensity (-1.7 ± 3.2%) over 8 min. Under the same conditions, microbubbles made with the same lipid formulation showed a -56.8 ± 6.1% decrease in enhancement in whole blood. Subsequent confocal, fluorescent, and scanning electron microscopy analysis revealed attachment of the nanobubbles to the surface of RBCs, suggesting that direct interactions, or hitchhiking, of nanobubbles on RBCs in the presence of plasma may be a possible mechanism for the observed effects. This phenomenon could be key to extending nanobubble circulation time and has broad implications in drug delivery, where RBC interaction with nanoparticles could be exploited to improve delivery efficiency.

Effects of shell-integrated Sudan Black dye on the acoustic activity and ultrasound imaging properties of lipid-shelled nanoscale ultrasound contrast agents.

SIGNIFICANCE: An effective contrast agent for concurrent multimodal photoacoustic (PA) and ultrasound (US) imaging must have both high optical absorption and high echogenicity. Integrating a highly absorbing dye into the lipid shell of gas core nanobubbles (NBs) adds PA contrast to existing US contrast agents but may impact agent ultrasonic response. AIM: We report on the development and ultrasonic characterization of lipid-shell stabilized C3F8 NBs with integrated Sudan Black (SB) B dye in the shell as dual-modal PA-US contrast agents. APPROACH: Perfluoropropane NBs stabilized with a lipid shell including increasing concentrations of SB B dye were formulated by amalgamation (SBNBs). Physical properties of SBNBs were characterized using resonant mass measurement, transmission electron microscopy and pendant drop tensiometry. Concentrated bubble solutions were imaged for 8 min to assess signal decay. Diluted bubble solutions were stimulated by a focused transducer to determine the response of individual NBs to long cycle (30 cycle) US. For assessment of simultaneous multimodal contrast, bulk populations of SBNBs were imaged using a PA and US imaging platform. RESULTS: We produced high agent yield (∼1011) with a mean diameter of ∼200 to 300 nm depending on SB loading. A 40% decrease in bubble yield was measured for solutions with 0.3 and 0.4 mg / ml SB. The addition of SB to the shell did not substantially affect NB size despite an increase in surface tension by up to 8 mN / m. The bubble decay rate increased after prolonged exposure (8 min) by dyed bubbles in comparison to their undyed counterparts (2.5-fold). SB in bubble shells increased gas exchange across the shell for long cycle US. PA imaging of these agents showed an increase in power (up to 10 dB) with increasing dye. CONCLUSIONS: We added PA contrast function to NBs. The addition of SB increased gas exchange across the NB shell. This has important implications in their use as multimodal agents.

Increasing Doxorubicin Loading in Lipid-Shelled Perfluoropropane Nanobubbles via a Simple Deprotonation Strategy.

Drug delivery to solid tumors using echogenic nanobubbles (NBs) and ultrasound (US) has recently gained significant interest. The approach combines attributes of nanomedicine and the enhanced permeation and retention (EPR) effect with the documented benefits of ultrasound to improve tumor drug distribution and treatment outcomes. However, optimized drug loading strategies, the drug-carrying capacity of NBs and their drug delivery efficiency have not been explored in depth and remain unclear. Here, we report for the first time on the development of a novel deprotonated hydrophobic doxorubicin-loaded C3F8 nanobubble (hDox-NB) for more effective US-mediated drug delivery. In this study, the size distribution and yield of hDox-NBs were measured via resonant mass measurement, while their drug-loading capacity was determined using a centrifugal filter technique. In vitro acoustic properties including contrast-imaging enhancement, initial echogenic signal, and decay were assessed and compared to doxorubicin hydrochloride loaded-NBs (Dox.HCl-NBs). In addition, in vitro therapeutic efficacy of hDox-NBs was evaluated by cytotoxicity assay in human ovarian cancer cells (OVCAR-3). The results showed that the hDox-NBs were small (300.7 ± 4.6 nm), and the drug loading content was significantly enhanced (2 fold higher) compared to Dox.HCl-NBs. Unexpectedly, the in vitro acoustic performance was also improved by inclusion of hDox into NBs. hDox-NB showed higher initial US signal and a reduced signal decay rate compared to Dox.HCl-NBs. Furthermore, hDox-NBs combined with higher intensity US exhibited an excellent therapeutic efficacy in human ovarian cancer cells as shown in a reduction in cell viability. These results suggest that hDox-NBs could be considered as a promising theranostic agent to achieve a more effective noninvasive US-mediated drug delivery for cancer treatment.

Pickering Bubbles as Dual-Modality Ultrasound and Photoacoustic Contrast Agents.

Microbubbles (MBs) stabilized by particle surfactants (i.e., Pickering bubbles) have better thermodynamic stability compared to MBs stabilized by small molecules as a result of steric hindrance against coalescence, higher diffusion resistance, and higher particle desorption energy. In addition, the use of particles to stabilize MBs that are typically used as an ultrasound (US) contrast agent can also introduce photoacoustic (PA) properties, thus enabling a highly effective dual-modality US and PA contrast agent. Here, we report the use of partially reduced and functionalized graphene oxide as the sole surfactant to stabilize perfluorocarbon gas bubbles in the preparation of a dual-modality US and PA agent, with high contrast in both imaging modes and without the need for small-molecule or polymer additives. This approach offers an increase in loading of the PA agent without destabilization and increased thickness of the MB shell compared to traditional systems, in which the focus is on adding a PA agent to existing MB formulations.