Understanding stimulus-evoked cerebral hemodynamics is critical for elucidating brain function and neurological disease mechanisms. However, existing neuroimaging approaches are often limited by spatial or temporal resolution, imaging depth, sensitivity, or the ability to simultaneously capture multiple hemodynamic parameters. To address these challenges, we developed a hybrid functional ultrasound–photoacoustic (fUSPA) imaging platform integrating ultrafast ultrasound with multispectral photoacoustic techniques. This system enables real-time quantitative mapping of cerebral blood volume (CBV), cerebral blood flow (CBF), and blood oxygen saturation (SO2) at high spatiotemporal resolution using a compact head-mounted probe. In addition to intrinsic hemodynamic imaging, the platform supports microbubble-enhanced super-resolution ultrasound for microvascular flow measurements and photoacoustic contrast agent-based imaging, such as indocyanine green, for vascular perfusion assessment. Using fUSPA, we investigated brain-wide cerebrovascular reactivity (CVR) at single-vessel resolution by quantifying relative changes in CBV, CBF, and SO2 during hypercapnic stimulation. Functional analyses reveal distinct CVR responses between cortical arteries and veins, as well as anti-correlated CBV oscillations during resting conditions. We demonstrate the potential of multiparametric fUSPA imaging to interrogate complex cerebrovascular dynamics and advance studies of brain function and neurodiseases, such as brain cancer.

Kidney inflammation is a central driver of acute kidney injury (AKI) and its progression to chronic kidney disease (CKD). While several imaging and biomarker-based approaches are under development, clinically validated non-invasive methods to directly quantify renal inflammation remain limited. This study introduces a novel approach using contrast-enhanced ultrasound (CEUS) with Cy5-labeled nanobubbles (NBs) to address this critical knowledge gap. Using a murine ischemia-reperfusion injury (IRI) model, CEUS imaging enabled real-time visualization of inflammation-induced changes in kidney perfusion and vascular integrity. Parametric analyses of non-linear imaging revealed delayed time-to-peak (TTP) and increased area under the falling curve (AUfC) in IRI kidneys, suggesting impaired microvascular perfusion and NB retention. Decorrelation time (DT) mapping further identified prolonged NB retention in the IRI group, indicating increased capillary permeability and NB extravasation. These findings correlated with histological and immunofluorescent analyses, which confirmed the presence of tubular injury, extravascular Cy5 signal localization, and increased neutrophil infiltration in inflamed kidney tissues. This study is the first to establish CEUS with NBs as a non-invasive, quantitative method for measuring kidney inflammation. With strong correlations between imaging metrics and histologic injury scores, this technology provides an accessible and non-invasive tool for monitoring renal inflammation and reducing reliance on invasive renal biopsies.

Nanobubbles (NBs) are increasingly recognized as the next generation of ultrasound contrast agents that can provide alternate avenues for the advancement of cancer imaging and therapy. Compared to currently clinically viable microbubbles that are limited to the vasculature because of their size, nanobubbles with diameters ranging from 200 to 500 nm can potentially target the perivascular and possibly extravascular compartments. Their polymeric, lipid, or hybrid shells can be engineered to tune stability, increase circulation time, and support surface functionalization with ligands to facilitate receptor-directed binding. Preclinical studies show that NBs can extend ultrasound imaging windows, improve sensitivity, and increase tumor-to-background contrast across different types of cancers. NBs can also be formulated with secondary reporters that allow their combination with other imaging modalities, such as optical (photoacoustic and fluorescence) and magnetic resonance imaging. However, several challenges need to be overcome to allow the clinical translation of NBs, such as acoustic optimization and standardization, formulation and manufacturing reproducibility, and comprehensive safety characterization. Addressing these barriers will be essential to establishing NBs as clinically viable agents. Here, we summarize recent advances in NB design and functionalization, review key preclinical oncology applications, and discuss translational priorities to support their integration into precision oncology clinical workflows.

Tumors often exhibit an extracellular matrix with elevated stiffness due to excessive accumulation and cross-linking of proteins, particularly collagen. This elevated stiffness acts as a physical barrier, impeding the infiltration of immune cells and the effective delivery of various immunotherapeutic agents, such as lipid nanoparticle-based RNA therapeutics. Here, we investigate the ability of ultrasound-activated nanobubbles (US-NBs) to increase the permeability and immunogenicity of tumors. Our results show that US-NBs physically remodel the tumor tissue by decreasing its stiffness by 60% 5 days after a single treatment. US-NB-treated tumors display randomly oriented collagen with a 5.47-fold lower deposition compared to untreated tumors. This leads to the effective delivery and widespread distribution of lipid nanoparticles (LNPs) in the tumor. Importantly, when assisted by US-NB, LNPs exhibit superior gene-transfection efficiency across pan-immune cells and achieve efficient genetic modification of T cells directly in vivo. This combined approach engages both innate and adaptive immunity, enhancing tumor immunogenicity and boosting cytotoxic cell infiltration by 4-fold compared to LNPs alone. These results indicate that gentle mechanical stimulation of the tumor using US-NB offers a promising strategy to augment the delivery and efficacy of existing immunotherapies.

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.

Functional ultrasound (fUS) maps cerebral blood volume (CBV) but lacks molecular and neuronal specificity. By simultaneously integrating fUS with optical imaging, we show that fUS-derived CBV correlates with both optically measured hemoglobin and neuronal calcium activity in awake mice. We further derive hemodynamic response functions linking calcium activity to CBV during spontaneous and sensory-evoked activity. Application to a mouse glioblastoma model demonstrates utility for studying neurovascular dysfunction in complex neuropathologies.

We developed a non-invasive imaging method to track CAR-T cells using internalized nanobubble ultrasound contrast agents.

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.

Nanobubbles (NBs), consisting of a lipid shell surrounding a gas core, have gained significant interest as contrast agents for ultrasound molecular imaging. Their acoustic response is strongly influenced by size and shell properties, yet most prior work has focused on microbubble characterization. Building on insights from microbubble studies, this work investigates the viscoelastic properties of in-house synthesized phospholipid-coated submicron NBs (average diameters of 650-720 nm) using ultrasound bulk attenuation measurements. Three NB formulations with distinct shell compositions were examined. The results highlight the critical role of shell properties in determining NB resonance frequencies. Furthermore, pressure-dependent shifts in resonance revealed strong nonlinear behavior at higher acoustic driving pressures (up to 280 kPa). Comparison with microbubbles of identical shell types showed that shell stiffness and friction are size-dependent, likely due to shell properties and the shear-thinning behavior of phospholipids. These findings provide new insights into NB dynamics with potential implications for both diagnostic and therapeutic ultrasound applications.

CAR-T cell therapy has led to remarkable advances in the outcomes of patients with acute lymphoblastic leukemia (ALL), B cell lymphomas, and multiple myeloma. Given these successes in hematologic malignancies, extensive efforts are now focused on developing CAR-T cell therapies to treat solid tumors. The treatment of solid tumors poses significant hurdles with cell trafficking necessary to achieve efficacy and minimize off-tumor side effects. The development of simple, safe and inexpensive modalities for tracking CAR-T cell distribution in clinical use in vivo could provide critical insights to facilitate the development of improved CAR-T products for solid tumors. Here, we demonstrate a strategy to monitor CAR-T cells in vivo using ultrasound imaging of nanobubble (NB) labeled cells. NBs are ultrasound contrast agents composed of a lipid shell and a C4F10 gas core that can be efficiently internalized into cells. This approach enables us to image the CAR-T cells using nonlinear contrast-enhanced ultrasound (CEUS). Utilizing this method, we found that CAR-T cells can be visualized after injection into both tumor-bearing and non-tumor bearing mice. In summary, our ultrasound-based tracking approach can effectively monitor the trafficking of CAR-T cells in vivo, offering a valuable new strategy that can further enable the development of new CAR-T products and strategies to modulate cell trafficking.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies due to its dense stroma, which limits drug delivery and therapeutic efficacy. Ultrasound (US) mediated strategies using nanobubbles (NBs) offer a promising approach to enhance treatment, yet the biological effects of NB exposure and the timing of US application remain unclear. Here, we investigated how NB exposure with immediate (0h) or delayed (1h) US affects viability, proliferation, metabolism, and stress signaling in PANC-1 and BxPC-3 cells. Immediate US exposure in the presence of extracellular nanobubbles resulted in a greater reduction in cell viability at 24 h compared to delayed US application. Proliferation analysis showed that Ki67 positivity decreased following USNB treatments in both cell lines. Metabolically, NB treatment alone increased cellular activity, whereas combined USNB treatment reduced metabolic activity over time. Seahorse analysis revealed higher basal respiration in PANC-1 cells compared to BxPC-3 cells, consistent with a more glycolytic phenotype, while USNB treatment enhanced glycolytic responses, particularly in PANC-1. Moreover, stress responses were also more pronounced in PANC-1 cells, with HSP70 expression increasing up to 2-fold in NB incubated group and decreasing in USNB groups compared to untreated, whereas BxPC-3 cells exhibited only modest and opposite changes to PANC-1 in HSP70 expression decreasing with NB incubation. Treatment timing critically influenced outcomes, with immediate US producing stronger antiproliferative and cytotoxic effects, highlighting the importance of sequencing in USNB therapeutic strategies. Moreover, NBs alone stimulated metabolic and stress responses that may promote proliferation, whereas NBs combined with US induced stronger stress responses associated with metabolic reprogramming and reduced proliferation.

Clinical outcomes in aggressive breast cancer vary widely, in part because the tumor microenvironment is structured to exclude immune infiltration. Low antigen load, dysfunctional antigen-presenting cells, T cell exclusion and exhaustion, and a stiff extracellular matrix that physically restricts immune cell trafficking work together to form a suppressive barrier that current immunotherapies struggle to overcome. We addressed this barrier using ultrasound (US)-activated nanobubbles (NBs), a drug-free intervention based on perfluoropropane-filled nanoparticles. The size and deformable phospholipid shell enable NBs to achieve deep tumor penetration and a uniform distribution throughout the entire tumor. Upon ultrasound activation, NBs generate localized mechanical forces that restore extracellular matrix elasticity, disrupt tumor transport barriers, and drive HMGB1 release, re-engaging endogenous antitumor immunity without pharmacological agents. In a syngeneic triple-negative breast cancer model, US-NB treatment depleted immunosuppressive myeloid cells 3-fold within 3 hours, followed by a greater than 5-fold increase in the ratio of antigen-experienced to suppressive T cells at 48 hours. US-NB drives rapid infiltration of CD4 + and CD8 + T cells within 48 hours. US-NB treatment achieved an 85% cure rate in the D2A1 model; cured animals maintained durable systemic immune memory, rejecting both local and systemic tumor rechallenge. Consistent therapeutic benefit was observed in a luminal B-like mammary tumor model (E0771), supporting activity across breast cancer subtypes. These results establish US-NB mechanical immunomodulation as a drug-free therapeutic strategy capable of generating robust and durable antitumor immunity, acting through biophysical tissue properties rather than tumor-specific molecular targets. GRAPHICAL ABSTRACT:

Background: Type 1 diabetes (T1D) is an autoimmune disease where autoreactive T cells infiltrate pancreatic islets, resulting in beta-cell destruction. Antigen-specific immunotherapy with tolerogenic peptides to induce peripheral tolerance has shown promise in preclinical studies but has not shown clinical efficacy. Methods: Here, we develop peptide-nanobubbles (NBs) as an image-guided platform for induction of peripheral immune tolerance in mouse models of T1D. Sub-micron sized NB ultrasound contrast agents can passively accumulate in pancreatic islets of non-obese diabetic (NOD) mice during the development of diabetes as a result of increased microvascular permeability. We incorporated an insulin B:9-23 peptide mimotope into NBs to target peptides to pancreatic islets for expansion of islet-resident regulatory T cells. Results: NBs retained normal size distribution and acoustic properties following peptide incorporation. Peptide-NBs accumulated in islets of NOD mice and this accumulation could be visualized in real time using contrast enhanced ultrasound. This resulted in an increased proportion of islet insulin-reactive regulatory T cells. Further, peptide-NBs prepared with a hybrid insulin peptide (HIP) expanded islet HIP-reactive regulatory T cells and substantially delayed diabetes onset in an adoptive transfer mouse model of autoimmune diabetes. Conclusions: Peptide-NBs offer a promising 'theranostic' approach for induction of peripheral tolerance in T1D while monitoring delivery and action via ultrasound contrast.

In nonlinear contrast-enhanced ultrasound (CEUS) imaging, nanobubbles (NBs) offer a promising alternative for enhanced visualization of microvascular structures and molecular imaging. This study explores two amplitude-modulated (AM) techniques-cross amplitude modulation (xAM) and compound amplitude modulation (cAM)-to enhance the capabilities of NB-mediated CEUS imaging. Both methods were tested on the Vevo F2 ultrasound imaging system (Fujifilm VisualSonics Inc.) using the Vevo Advanced Data Acquisition (VADA) mode, allowing full customization of pulse sequences. The xAM technique utilized a three-event pulse sequence that transmits cross-propagating plane-wave beams from dual apertures. This method isolated nonlinear scattered waves from NBs, reducing background noise and enhancing image quality. In contrast, cAM achieved a high frame rate of 706 Hz, a valuable feature for tracking the NB vascular flow dynamics. cAM combined plane-wave compounding with amplitude modulation, transmitting two events (half- and full-amplitude), achieving high frame rates for velocity imaging at the expense of image quality. NBs at a concentration of 109 NBs/mL, intended to mimic estimated in vivo post-injection concentrations, were injected into custom-built tissue-mimicking vessel phantoms. Experiments demonstrated that xAM significantly improved the contrast-to-noise ratio (CNR) and contrast-to-tissue ratio (CTR) by over 10 times compared to B-mode imaging, especially at larger steering angles. Conversely, cAM's CNR and CTR were at least 50% lower than that of xAM, but it achieved a frame rate over 100 times faster than xAM. These results suggest xAM can enhance imaging clarity, while cAM offers high frame rates for velocity imaging, providing an imaging framework for preclinical and clinical applications.

This study presents an experimental investigation of the influence of MB concentration on the resonance frequency of lipid-coated microbubbles (MBs). Expanding on theoretical models and numerical simulations from previous research, this work experimentally investigates the effect of MB size on the rate of resonance frequency increase with concentration, a phenomenon observed across MBs with two different lipid compositions: propylene glycol (PG) and propylene glycol and glycerol (PGG). Employing a custom-designed ultrasound attenuation measurement setup, we measured the frequency-dependent attenuation of MBs, isolating MBs based on size to generate distinct monodisperse sub-populations for analysis. The resonance frequency of MBs was determined by identifying the attenuation peak in the broadband attenuation ultrasound attenuation measurements. Our experimental findings confirm that larger MBs (≈2.1μm) demonstrate a more significant shift in resonance frequency (≈ 5 MHz, ≈ 40%) as a function of MB concentration. In contrast, smaller MBs (≈1.3μm) show a minor shift in the resonant frequency (≈ 1.8MHz, ≈ 8%), underlining the importance of size in determining acoustic behavior compared to changes in the lipid shell properties. Additionally, we observed that resonance frequency increase with concentration reaching a saturation point at higher concentrations. This plateau occurs at higher concentrations for larger MBs (≈2.1μm), while smaller MBs (≈1.6μm and ≈1.3μm) reach this saturation point at lower concentrations. Furthermore, the study highlights the small effect of bubble-bubble interactions on the resonance frequency of MB populations, particularly at lower MB concentrations and for smaller MBs. This insight is important for applications utilizing MB clusters, such as contrast-enhanced ultrasound imaging and MB-mediated therapies. While both size and lipid shell composition influence resonance frequency, MB size has a more significant effect. In conclusion, our findings affirm the need to consider both MB size and concentration when utilizing MBs for clinical and industrial ultrasonic applications.

Prostate cancer is one of the most common malignancies among men globally. Early and accurate assessment of tumor aggressiveness is essential for guiding treatment decisions and improving patient outcomes. With growing clinical interest in combining optical, ultrasound, and photoacoustic imaging approaches for cancer detection, we systematically investigated the cellular uptake of three multimodal contrast agents─indocyanine green (ICG), ICG-loaded nanobubbles (ICG-NBs), and ICG-loaded microbubbles (ICG-MBs)─across prostate cancer cell lines with varying aggressiveness (PC-3M > PC-3 > DU-145 > LNCaP). Concentration- and time-dependent assays revealed that after 1 h of incubation, ICG-NBs exhibited 5-fold higher uptake (***p < 0.001) by highly metastatic PC-3M cells compared to the less aggressive and indolent cell lines. In contrast, ICG-MBs showed minimal uptake, detectable only after prolonged incubation (12 h) in PC-3M and PC-3 cells, while free ICG exhibited negligible uptake at 6 h, except in PC-3M cells, but increased over time in all cell lines. Validation across breast, colorectal, and pancreatic cancer cells further confirmed that ICG-NB uptake positively correlates with the metastatic potential of the cancer cell. Mechanistic studies identified macropinocytosis as the primary pathway for ICG-NB internalization, with additional contributions from clathrin-mediated endocytosis. These findings highlight that unconjugated ICG-NBs can selectively differentiate aggressive from indolent cancer phenotypes, offering a promising multimodal contrast agent for cancer theranostics.