Ultrasound is the second most common clinical imaging modality. Ultrasound image quality often suffers from poor contrast resolution and thus can greatly benefit from appropriate contrast agents. While micron-sized gas core particles (microbubbles) are clinically utilized in this space for applications such as echocardiography, a smaller agent could be more broadly applicable to enhance the sensitivity and specificity of disease detection. To this end, lipid-shelled nanobubbles have recently emerged as robust ultrasound contrast agents for both diagnostic and therapeutic purposes. They have been used in preclinical applications ranging from oncology to endocrinology and are notably relevant in diseases that involve pathological vasculature. Owing to their shell composition, nanobubbles are versatile and can be functionalized with fluorophores, targeting agents and therapeutic moieties. Here we provide the steps needed to formulate plain, fluorophore-conjugated, ligand-conjugated, hydrophilic dye-integrated and drug-loaded nanobubbles. The typical formulation for lipid-shelled nanobubbles takes place via self-assembly driven by mechanical agitation, followed by differential centrifugation. The process is deceptively simple, yet there are many nuances in the formulation process that must be followed to produce consistently successful nanobubble batches without contamination from microbubbles. Even minor deviation from the standard protocol can substantially affect nanobubble yield, stability, acoustic performance and batch-to-batch reproducibility. This procedure also details common pitfalls and their potential consequences for bubble quality and performance. The procedure requires 3 h to complete the formulation and activation of the plain nanobubbles by users with basic laboratory expertise.

The localization of prostate cancer by ultrasound remains limited by the lack of B-mode conspicuity and the confinement of clinically approved microbubbles (MBs) to the vasculature. This precludes differentiating viable tumor, necrotic tissue, and margin-associated disease. We investigated prostate-specific membrane antigen (PSMA)-targeted lipid-shelled perfluorocarbon nanobubbles (PSMA-NBs) in an orthotopic rabbit model using a clinical contrast-enhanced ultrasound (CEUS) system. We implanted PSMA-positive PC3pip-GFP tumors into the prostates of immunosuppressed New Zealand White rabbits and performed transabdominal imaging with PSMA-NBs, MBs, and Plain-NBs using identical protocols. To address tumor heterogeneity and ultrasound boundary ambiguity, regions of interest were defined from baseline B-mode images and segmented into the tumor core, rim, and a peritumoral area. Pixel-wise parametric and decorrelation time (DT) maps were generated and compared with whole-slide histology (H&E) and, in an exploratory and non-specific analysis, with PSMA IHC. Compared to MBs at the doses used, PSMA-NBs exhibited higher peak intensities in the tumor core and rim (1.60-fold and 1.50-fold, respectively) and improved retention (mean transit time [MTT]: 4.20 to 5.40-fold higher) for up to 10 min in the tumor and peritumoral areas. In an exploratory analysis constrained by cohort size, PSMA-NB kinetics, notably MTT, tracked histology-defined tumor viability, and DT mapping showed spatially heterogeneous retention at the tumor periphery. Compared to Plain-NBs, PSMA-NBs also exhibited improved retention (MTT +21% overall) in the rim and peritumoral areas. This study demonstrates the potential of PSMA-NBs to characterize prostate cancer by molecularly targeted CEUS beyond that achieved with MBs at the doses used.

The term nanobubble (NB) includes a wide range of gas core, submicron particles. A subgroup of NBs consists of phospholipid-shelled (or coated) nanoparticles stabilizing a perfluorocarbon gas core which have gained recent interest as ultrasound (US) contrast agents. Several methods are available to produce coated NBs. Among these, amalgamation driven self-assembly has been the most utilized. Amalgamation (also referred to as mechanical agitation) is a simple technique currently used for production of commercial and clinically relevant microbubble suspensions. When combined with size-isolation steps, it can also generate submicron NB suspensions with a narrow size distribution. While this technique has been used extensively, no prior work has systematically examined the critical manufacturing parameters needed to produce the optimal coated NB formulation. In this work, we investigate how the precursor lipid dispersion, perfluorocarbon gas to lipid ratio, and pressurized size isolation affect the formation and size isolation of stable, uniform NBs. Results show that the precursor lipid dispersions exhibiting a monomodal size distribution produced the most stable NBs. Additionally, perfluorocarbon volume in excess of lipid dispersion volume is required to form high concentration, stable NBs. Finally, pressurized size isolation resulted in high concentration, US stable NBs. These findings establish the understanding of the key process parameters which affect uniform size and stable NB production via mechanical amalgamation.

Near-infrared photoacoustic imaging (NIR-PAI) integrates optical excitation with ultrasound detection to enable high-resolution, deep-tissue imaging by taking advantage of reduced light scattering and absorption in this spectral window. Despite its potential, clinical translation of contrast-enhanced NIR-PAI is limited by the scarcity of effective contrast agents. Indocyanine green (ICG), an FDA-approved NIR dye, is a strong candidate due to its biocompatibility and photoacoustic efficiency. However, its concentration-dependent aggregation, lack of facile targeting strategies, instability in aqueous environments, and low photostability result in variable signal, high background noise, and reduced reliability in vivo. To address these challenges, we developed three biocompatible ICG-based nanoprobe platforms amenable to facile, scalable synthesis: 5-arm DNA-ICG nanostructures (5-arm DNA-ICG), lipid-shelled ICG nanobubbles (ICG-NBs), and Azide-modified ICG J-aggregates (JAAZ). These platforms are designed to preserve ICG monomers or control aggregation, enabling enhanced NIR-PAI performance. Spectroscopic and photoacoustic analyses revealed consistent absorbance and photoacoustic profiles , showing enhanced signals compared to free ICG. The greatest improvement was observed for JAAZ, followed by ICG-NBs and 5-arm DNA-ICG. Photostability studies showed that JAAZ aggregation protects ICG from light-induced photodegradation, whereas monomer preservation in 5-arm DNA-ICG and ICG-NBs provides less protection and moderate signal stability. All three probes demonstrated stable performance under physiological conditions, achieved strong signal-to-noise ratios at depth and under tissue-mimicking conditions, and required markedly reduced probe concentrations to generate robust signals. Their modular architectures allow incorporation of targeting ligands, offering molecular specificity and multimodal functionality. Collectively, these contrast agent platforms provide noninvasive, deep-tissue molecular imaging and biosensing, with strong potential for future preclinical and clinical translation, and represent a promising alternative to free ICG for biomedical applications. ### Competing Interest Statement S.S., P.V.C and R.V. have a patent pending on the JAAZ nanoprobes. All other authors declare no competing interest. National Science Foundation, 2128821 Virginia Innovation Partnership Corporation, CCF23-0092-HE Office of Research, Innovation, and Economic Impact (ORIEI) at George Mason University, G00002563

Purpose To evaluate the use of prostate-specific membrane antigen-targeted nanobubbles (PSMA-NBs) for contrast-enhanced ultrasound (CEUS) in a rabbit model, aiming to enhance prostate cancer imaging and guide clinical translation. Materials and Methods PSMA-NBs were formulated using lipid encapsulation and PSMA-targeting ligands. Human PSMA-positive PC3pip-GFP cells were injected into the prostates of immunosuppressed rabbits to establish tumors. Tumor growth was monitored via B-mode ultrasound (US) and MRI. CEUS was conducted with PSMA-NBs and commercial microbubbles (MBs). Time-intensity curve (TIC) analysis, parametric mapping, and post-mortem histological correlation were performed. Results PSMA-NBs demonstrated 1.60-fold (p = 0.013) and 1.50-fold (p = 0.016) higher peak signal intensities in the tumor core and rim, respectively, compared to MBs, with significantly longer mean transit times (MTTs) in the core (4.20-fold; p = 0.001) and rim (4.50-fold; p < 0.001). At 10 minutes, PSMA-NBs retained detectable signals in tumor rim (7.0 ± 3.0 a.u.), core (3.0 ± 1.0 a.u.), and surrounding tissues (12.0 ± 5.0 a.u.), unlike MBs. Larger tumors showed prolonged MTTs in the rim (3.70 ± 0.50 min) and surrounding tissues (4.60 ± 0.50 min) compared to the core (2.10 ± 0.40 min, p < 0.001). TIC parameters (MTT, AUCwo) correlated with tumor viability, emphasizing PSMA-NBs’ ability to delineate viable regions. Conclusion PSMA-NBs significantly enhanced prostate cancer imaging, correlating with tumor viability and outperforming MBs. These findings support their potential to improve diagnostic precision and guide targeted therapy in prostate cancer. ### Competing Interest Statement A. Exner is a founding member of Visano Theranostics. The authors report no additional affiliations or financial interests with any entities that could be perceived as having a financial stake or conflict related to the subject matter discussed in this manuscript, beyond those already disclosed. Wallace H. Coulter Foundation, https://ror.org/04cmszv87 National Institutes of Health, R01-EB025741, T32GM007250, F30HL160111 Hospital Israelita Albert Einstein, https://ror.org/04cwrbc27

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.

This study explores the challenges associated with nanoparticle-based drug delivery to the tumor parenchyma, focusing on the widely utilized enhanced permeability and retention effect (EPR). While EPR has been a key strategy, its inconsistent clinical success lacks clear mechanistic understanding and is hindered by limited tools for studying relevant phenomena. This work introduces an approach that employs multiparametric dynamic contrast-enhanced ultrasound (CEUS) with a nanoscale contrast agent for noninvasive, real-time examination of tumor microenvironment characteristics. We demonstrate that CEUS imaging can: (1) evaluate tumor microenvironment features, (2) be used to help predict the distribution of doxorubicin-loaded liposomes in the tumor parenchyma, and (3) be used to predict nanotherapeutic efficacy. CEUS using nanobubbles (NBs) was carried out in two tumor types of high (LS174T) and low (U87) vascular permeability. LS174T tumors consistently showed significantly different time intensity curve (TIC) parameters, including area under the rising curve (AUCR, 2.7x) and time to peak intensity (TTP, 1.9x) compared to U87 tumors. Crucially, a recently developed decorrelation time (DT) parameter specific to NB CEUS dynamics successfully predicted the distribution of doxorubicin-loaded liposomes within the tumor parenchyma (r = 0.86 +/- 0.13). AUCR, TTP, and DT were used to correlate imaging findings to nanotherapeutic response with 100% accuracy in SKOV-3 tumors. These findings suggest that NB-CEUS parameters can effectively discern tumor vascular permeability, serving as a biomarker for identifying tumor characteristics and predicting the responsiveness to nanoparticle-based therapies. The observed differences between LS174T and U87 tumors and the accurate prediction of nanotherapeutic efficacy in SKOV-3 tumors indicate the potential utility of this method in predicting treatment efficacy and evaluating EPR in diseases characterized by pathologically permeable vasculature. Ultimately, this research contributes valuable insights into refining drug delivery strategies and assessing the broader applicability of EPR-based approaches.

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 Medical imaging modalities, especially those with companion nanoparticle capabilities, can be used to analyze tumors and separate patients into potential positive and negative responders to nanotherapeutics. Ultrasound is an excellent modality for this purpose due to its versatility through its many functions including contrast-mode, B-mode, doppler, elastography, and more. image

Contrast-enhanced ultrasound is currently used worldwide with clinical indications in cardiology and radiology, and it continues to evolve and develop through innovative technological advancements. Clinically utilized contrast agents for ultrasound consist of hydrophobic gas microbubbles stabilized with a biocompatible shell. These agents are used commonly in echocardiography, with emerging applications in cancer diagnosis and therapy. Microbubbles are a blood pool agent with diameters between 1 and 10 μm, which precludes their use in other extravascular applications. To expand the potential use of contrast-enhanced ultrasound beyond intravascular applications, sub-micron agents, often called nanobubbles or ultra-fine bubbles, have recently emerged as a promising tool. Combining the principles of ultrasound imaging with the unique properties of nanobubbles (high concentration and small size), recent work has established their imaging potential. Contrast-enhanced ultrasound imaging using these agents continues to gain traction, with new studies establishing novel imaging applications. We highlight the recent achievements in nonlinear nanobubble contrast imaging, including a discussion on nanobubble formulations and their acoustic characteristics. Ultrasound imaging with nanobubbles is still in its early stages, but it has shown great potential in preclinical research and animal studies. We highlight unexplored areas of research where the capabilities of nanobubbles may offer new advantages. As technology advances, this technique may find applications in various areas of medicine, including cancer detection and treatment, cardiovascular imaging, and drug delivery.

Nanobubbles (NBs) have demonstrable potential for ultrasound imaging and therapeutic applications. Recent studies have even shown their capacity for cellular internalization, which has important implications for their in-vivo stability and bioaccumulation. Traditional methods for observing NBs often involve fluorescence labelling, which can influence NB behaviour. Moreover, these methods are unsuitable for detecting intact (acoustically active) NBs within a cellular environment. This study introduces a label-free approach employing optical coherence tomography (OCT) to investigate the temporal variations in speckle intensity of the OCT backscatter signal of cells interacting with NBs. The temporal variations in the signal intensity of cell aggregates result from the motion of subcellular scatterers within the cellular environment. In this work, we investigate whether internalized NBs modify the temporal variations in the signal intensity. For our experimental imaging set-up, we used a Thorlabs MEMS-VCSEL Swept Source OCT system with a central wavelength of 1300 nm to acquire M-Mode and B-Mode acquisitions. PC3 prostate cancer cells and in-house lipid-shelled NBs were used. The sensitivity of the speckle decorrelation technique was tested on our system using an intensity autocorrelation function on polystyrene microspheres and diluted NBs. Our study demonstrates that speckle decorrelation OCT can effectively detect NBs within a compact cell pellet under specific conditions and was verified using contrast-enhanced ultrasound. This approach provides an additional optical method for NB detection within cellular environments and holds the potential for broader applications in detecting NBs in in-vivo applications.

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.

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.

Lipid-shell C3F8 nanobubble (NB) ultrasound contrast agents have demonstrated an extended in vivo lifespan compared to traditional microbubble contrast agents. One potential explanation behind this extended lifespan is non-covalent interactions between NBs and red blood cells (RBCs). This has been observed in vitro where interactions increased contrast intensity and signal stability over time. However, the mechanism of this interaction and the factors influencing it have not been fully elucidated. In this study, we investigate the role of NB shell stiffness and solution temperature on signal stability in human whole blood and PBS in vitro. We used three NB shell stiffnesses and a fourth formulation with the addition of a targeting ligand and assessed extent of RBC interaction using autocorrelation analysis. Results demonstrate a clear dependence of RBC interaction on shell stiffness. The stiffest NB formulation had a decorrelation time ~2x faster than all other formulations and showed no effect on signal change over time. Less stiff NBs resulted in an increase in intensity over time and increased decorrelation time, consistent with prior studies. No such effects were observed in PBS. Furthermore, the presence of a targeting ligand did not appear to play a role in contrast enhancement over time or decorrelation time. Decorrelation time in whole blood decreased with increasing temperature. This work examined the effect of shell stiffness and solution temperature on NB signal stability and interactions with RBCs in human whole blood in vitro. Understanding why NBs interact with RBCs could improve NB formulation optimization for extended circulation time in vivo.

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.

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 (similar to 10(11)) with a mean diameter of similar to 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. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License.

Nanobubble ultrasound contrast agents (NBs) offer enhanced access to deep tumor tissue by extrapolation from vasculature. When combined with targeting moieties, shell-stabilized nanobubbles can outperform other agents used in molecular ultrasound imaging due to their small size. The visualization and decoupling of extravasated and intravascular NBs is critical to furthering nanobubble molecular imaging with ultrasound. Spatiotemporal processing of echo data, usually via singular value decomposition (SVD), enables enhanced decoupling of signals of stationary from moving scatterers and therefore provides a novel tool for analysis of NB extravasation into the parenchyma. Instead of trivial solutions, we have developed non-negative SVD (nnSVD) methods applicable to most commercial scanners without requiring the raw data. Additionally, our formulation allows addition of proper regularizers, such as sparsity, to further expand the decomposition dimensions. We validate these methods in phantom models and present how the suggested decompositions can distinctly decouple flowing from extravasated NBs in in vivo tumour models with enhanced permeability. Our results show the successful tracking of nanobubble accumulation in tumour tissue and can serve as a relative metric for molecular targeting of prostate cancer. Further exploration of these methods could be used to improve long-term imaging and drug delivery capabilities of NBs.

In this work, we describe applications of nanobubbles in superresolution imaging. Conventional ultrasound localization microscopy (ULM) techniques require sparse scatterers in the form of echogenic microbubbles (MB, ∼1–10 μm diameter). ULM enables imaging of capillaries larger than ∼50 μm in diameter but becomes too time consuming in smaller capillaries due to insufficient MB density. A viable alternative for microvascular imaging may be the use of submicron bubbles, or nanobubbles (NBs). NBs are ∼100i–500 nm in diameter and have a particle density 5 orders of magnitude higher than MBs without associated attenuation artifacts. In addition to strong nonlinear response, under some conditions NBs can produce SNR linear signals stronger than erythrocytes. This allows for formulating a superresolution imaging problem using computational approaches suitable for dense sources instead of relying on physical sparsity. With much smaller sizes compared to MBs, NBs can access the smallest capillaries at much higher concentrations (∼1011), offering enhanced detection and faster superresolution acquisition. In addition, NB nonlinear signals can be decomposed to elucidate intravascular and extravasation into tissues with vascular hyperpermeability, such as tumors. Thus, when combined with appropriate processing, NBs can provide information about vascular architecture and extravascular transport which is not achievable with MBs.

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.

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.