Search Results (55)

The aim of this prospective study was to evaluate contrast-enhanced urosonography (CE-VUS) as an alternative to radiographic retrograde cystourethrography (RCUG) in the detection of vesicoureteral reflux (VUR) in young dogs. A total of 62 dogs, ranging in age from 6 weeks to 12 months, and 124 ureterorenal units (UUs) were investigated for VUR using RCUG and CE-VUS. After a baseline gray-scale ultrasound (US) of the urinary tract, a second-generation ultrasound contrast agent (UCA), diluted in a bottle of normal saline, was instilled into the urinary bladder via a catheter and CE-VUS was performed. VUR was detected when microbubbles were observed in the ureter and/or in the renal pelvis. In addition, RCUG was performed on the same day. The findings obtained by CE-VUS and RCUG were concordant in 117/124 UUs (94.35%). No reflux was detected in 101/124 UUs (81.45%) using both methods. With RCUG as the standard of reference, the sensitivity of CE-VUS was 94.12%, and the specificity was 94.39%. The positive predictive value was 72.73%, and the negative predictive value was 99.02%. CE-VUS is highly sensitive for the detection of VUR in young dogs. Full article

Objective: In previous studies, echogenic liposomes with liquid and gas cores were analyzed as alternative carriers of drug molecules and cavitation nuclei for sonoporation. The possibility of small interfering RNA (si-RNA) encapsulation has also been presented. In this study, the usability of echogenic liposomes as drug carriers and cavitation seeds was evaluated using an in vivo model. Methods: A doxorubicin-loaded echogenic liposome was synthesized as a drug carrier. The size distribution and the number of formed echogenic liposomes were measured. Five comparative in vivo experiments were conducted with and without doxorubicin-loaded echogenic liposomes, and the results were statically analyzed. Results: Sonoporation with doxorubicin-loaded echogenic liposomes at 3.05 W/cm² of ISPTA ultrasound sonication and 0.98 MHz results in an average tumor volume growth of less than 25% of that following the simple administration of doxorubicin. Considering the p-value between the two groups is approximately 0.03, doxorubicin-loaded echogenic liposomes were effectively applicable as cavitation nuclei for sonoporation. Conclusions: Although further studies are needed to clarify the responses to incident ultrasound fields, the proposed echogenic liposome appears to be a promising alternative cavitation nuclei/carrier for sonoporation. Full article

Oncolytic virotherapy has shown great promise in mediating targeted tumor destruction through tumor-selective replication and induction of anti-tumor immunity; however, obstacles remain for virus candidates to reach the clinic. These include avoiding neutralizing antibodies, preventing stimulation of the adaptive immune response during intravenous administration, and inducing sufficient apoptosis and immune activation so that the body’s defense can work to eradicate systemic disease. We have developed a co-formulation of oncolytic viruses (OVs) with Imagent^® lipid-encapsulated, perfluorocarbon microbubbles (MBs) to protect the OVs from the innate and adaptive immune system. Once inside the MB, the viral particles become acoustically active such that external ultrasound can target the delivery of the virus locally within the tumor. Humanized NSG female mice (Hu-CD34⁺ NSG-SGM3) engrafted in their flanks with MDA-MB-231-Luc triple-negative breast cancer (TNBC) cells were transduced with MB/OVs, with or without adjuvant Pembrolizumab treatment, and tumor sizes and tumor necrosis were assessed. The presence of CD8⁺ (cytotoxic T-cells), CD4⁺ (helper T-cells), and CD25⁺ (Tregs) tumor-infiltrating lymphocytes (TILs) was quantified in the tumor samples by immunohistochemistry. In an in vivo model of humanized mice engrafted with a human immune system, we observed significantly greater tumor necrosis and smaller tumor mass in human TNBC xenografts systemically treated with MB/OV complexes in the presence or absence of pembrolizumab adjuvant treatment, compared to controls. Additionally, we observed a low ratio of CD4⁺/CD8⁺ TILs and a high ratio of CD8⁺/CD25⁺ TILs in the MDA-MB-231 xenografts treated with MB/OVs complexes with or without pembrolizumab adjuvant treatment, compared to controls. Our study demonstrated the feasibility of using MBs to target OVs to TNBC through diagnostic ultrasound, which decreased tumor mass by increasing tumor necrosis and stimulated a local and systemic antitumoral immune response by increasing intratumoral CD8⁺ T-cytotoxic lymphocyte infiltration and decreasing CD25⁺ Treg cells. Full article

Background: The aim of this study was to evaluate the performance and the impact of contrast-enhanced intraoperative ultrasound (CE-IOUS) on intraoperative decision-making, as there is still no standardized protocol for its use. Therefore, we retrospectively analyzed multiple CE-IOUS performed in hepato-pancreatic-biliary surgery with respect to pre- and postoperative imaging and histopathological findings. Methods: Data of 50 patients who underwent hepato-pancreatic-biliary surgery between 03/2022 and 03/2024 were retrospectively collected. CE-IOUS was performed with a linear 6–9 MHz multifrequency probe connected to a high-resolution device. The ultrasound contrast agent used was a stabilized aqueous suspension of sulphur hexafluoride microbubbles. Results: In total, all 50 lesions indicated for surgery were correctly identified. In 30 cases, CE-IOUS was used to localize the primary lesion and to define the resection margins. In the remaining 20 cases, CE-IOUS identified an additional lesion. Fifteen of these findings were identified as malignant. In eight of these cases, the additional malignant lesion was subsequently resected. In the remaining seven cases, CE-IOUS again revealed an inoperable situation. In summary, CE-IOUS diagnostics resulted in a high correct classification rate of 95.7%, with positive and negative predictive values of 95.2% and 100.0%, respectively. Conclusions: CE-IOUS shows excellent performance in describing intraoperative findings in hepato-pancreatic-biliary surgery, leading to a substantial impact on intraoperative decision-making. Full article

Administering intraluminal fluid can improve the acoustic window for the visualization of the lumen and wall layers in the cavitary organs. Microbubbles in ultrasound contrast agents can also be used for intracavitary applications to enhance visualization of the lesion in human patients. However, there was no literature extending the clinical application of intraluminal contrast-enhanced ultrasonography (CEUS) to patients with naturally occurring diseases in veterinary medicine. This case series aims to describe the detailed application and diagnostic value of intraluminal CEUS in six clinical cases with naturally occurring gastrointestinal (GI) and urinary tract diseases. Full article

Contrast-enhanced ultrasound could assess whether cancer chemotherapeutic agents work in days, rather than waiting 2–3 months, as is typical using the Response Evaluation Criteria in Solid Tumors (RECIST), therefore avoiding toxic side effects and expensive, ineffective therapy. A total of 40 mice were implanted with human colon cancer cells: treatment-sensitive mice in control (n = 10, receiving saline) and treated (n = 10, receiving bevacizumab) groups and treatment-resistant mice in control (n = 10) and treated (n = 10) groups. Each mouse was imaged using 3D dynamic contrast-enhanced ultrasound with Definity microbubbles. Curvature learning, an unsupervised learning approach, quantized pixels into three classes—blue, yellow, and red—representing normal, intermediate, and high cancer probability, both at baseline and after treatment. Next, a curvature learning score was calculated for each mouse using statistical measures representing variations in these three color classes across each frame from cine ultrasound images obtained during contrast administration on a given day (intra-day variability) and between pre- and post-treatment days (inter-day variability). A Wilcoxon rank-sum test compared score distributions between treated, treatment-sensitive mice and all others. There was a statistically significant difference in tumor score between the treated, treatment-sensitive group (n = 10) and all others (n = 30) (p = 0.0051). Curvature learning successfully identified treatment response, detecting changes in tumor perfusion before changes in tumor size. A similar technique could be developed for humans. Full article

The prostate is the only sexual gland of the male dog, and dihydrotestosterone (DHT) regulates its growth. In intact dogs, constant DHT stimulation results in benign prostatic hyperplasia (BPH) that can be treated with osaterone acetate (OSA). This study describes the effects of OSA treatment, detected by contrast-enhanced ultrasonography (CEUS), highlighting prostatic vascularization with a contrast agent composed of gas microbubbles. Fifteen dogs (2–8 years) of different sizes and breeds (4–30 kg) diagnosed with BPH are involved in the study. Before treatment (D₀), CPSE is measured (294.05 ± 115.97 ng/mL), and a B-mode ultrasound is performed (Vratio = 2.80 ± 1.85), confirming BPH. CEUS highlights the length of the wash-in (11.93 ± 2.08 s) and wash-out (42.20 ± 6.99 s) phases of the contrast agent in the prostate and the presence of cysts and parenchymal alteration. Dogs are treated with OSA (0.5 mg/kg for 7 days) and reassessed after 21 days (D₁): CPSE and prostate volume are significantly (p < 0.001) reduced. The length of the wash-in (14.73 ± 2.54 s) and wash-out (51.13 ± 6.03 s) phases are significantly (p < 0.001) increased. The results confirm the effectiveness of the treatment, particularly the reduction in prostatic perfusion, confirmed by the increase in diffusion times of the contrast. Although preliminary, these findings are promising for the use of CEUS in monitoring dogs with BPH. Full article

Gas-filled microbubbles are well-established contrast agents for ultrasound imaging and widely studied as delivery systems for theranostics. Herein, we have demonstrated the promising potential of the hydrophobin HFBII—a fungal amphiphilic protein—in stabilizing microbubbles with various fluorinated core gases. A thorough screening of several experimental parameters was performed to find the optimized conditions regarding the preparation technique, type of core gas, HFBII initial concentration, and protein dissolution procedure. The best results were obtained by combining perfluorobutane (C₄F₁₀) gas with 1 mg/mL of aqueous HFBII, which afforded a total bubble concentration higher than 10⁹ bubbles/mL, with long-term stability in solution (at least 3 h). Acoustic characterization of such microbubbles in the typical ultrasound frequency range used for diagnostic imaging showed the lower pressure resistance of HFBII microbubbles, if compared to conventional ones stabilized by phospholipid shells, but, at the same time, revealed strong non-linear behavior, with a significant harmonic response already at low acoustic pressures. These findings suggest the possibility of further improving the performance of HFBII-coated perfluorinated gas microbubbles, for instance by mixing the protein with other stabilizing agents, e.g., phospholipids, in order to tune the viscoelastic properties of the outer shell. Full article

Optimising drug delivery to tumours remains an obstacle to effective cancer treatment. A prerequisite for successful chemotherapy is that the drugs reach all tumour cells. The vascular network of tumours, extravasation across the capillary wall and penetration throughout the extracellular matrix limit the delivery of drugs. Ultrasound combined with microbubbles has been shown to improve the therapeutic response in preclinical and clinical studies. Most studies apply microbubbles designed as ultrasound contrast agents. Acoustic Cluster Therapy (ACT^®) is a novel approach based on ultrasound-activated microbubbles, which have a diameter 5–10 times larger than regular contrast agent microbubbles. An advantage of using such large microbubbles is that they are in contact with a larger part of the capillary wall, and the oscillating microbubbles exert more effective biomechanical effects on the vessel wall. In accordance with this, ACT^® has shown promising therapeutic results in combination with various drugs and drug-loaded nanoparticles. Knowledge of the mechanism and behaviour of drugs and microbubbles is needed to optimise ACT^®. Real-time intravital microscopy (IVM) is a useful tool for such studies. This paper presents the experimental setup design for visualising ACT^® microbubbles within the vasculature of tumours implanted in dorsal window (DW) chambers. It presents ultrasound setups, the integration and alignment of the ultrasound field with the optical system in live animal experiments, and the methodologies for visualisation and analysing the recordings. Dextran was used as a fluorescent marker to visualise the blood vessels and to trace drug extravasation and penetration into the extracellular matrix. The results reveal that the experimental setup successfully recorded the kinetics of extravasation and penetration distances into the extracellular matrix, offering a deeper understanding of ACT’s mechanisms and potential in localised drug delivery. Full article

Polypodium aureum, a fern, possesses a specialized spore-releasing mechanism like a catapult induced by the quick expansion of vaporized bubbles. This study introduces lipid-coated perfluorocarbon droplets to enable repeatable vaporization–condensation cycles, inspired by the repeatable vaporization of Polypodium aureum. Lipid-perfluorocarbon droplets have been considered not to exhibit repeatable oscillations due to bubble collapse of the low surface tension of lipid layers. However, a single lipid-dodecafluoropentane droplet with a diameter of 9.17 µm shows expansion–contraction oscillations over 4000 cycles by changing lipid composition and applying a low-power 1.7 MHz ultrasound to induce the partial vaporization of the droplets. The optimal combinations of shell composition, droplet fabrication, and acoustic conditions can minimize the damage on shell structure and promote a quick recovery of damaged shell layers. The highly expanding oscillatory microbubbles provide a new direction for fuel-free micro- or nanobots, as well as biomedical applications of contrast agents and drug delivery. Full article

There is an urgent need to realize precise clinical ultrasound with ultrasound contrast agents that provide high echo intensity and mechanical index tolerance. Graphene derivatives possess exceptional characteristics, exhibiting great potential in fabricating ideal ultrasound contrast agents. Herein, we reported a facile and green approach to synthesizing reduced graphene oxide with ellagic acid (rGO-EA). To investigate the application of a graphene derivative in ultrasound contrast agents, rGO-EA was dispersed in saline solution and mixed with SonoVue (SV) to fabricate SV@rGO-EA microbubbles. To determine the properties of the product, analyses were performed, including ultraviolet–visible spectroscopy (UV–vis), Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy, transmission electron microscopy (TEM), thermal gravimetric analysis (TGA), X-ray photoelectron spectrum (XPS), X-ray diffraction analysis (XRD) and zeta potential analysis. Additionally, cell viability measurements and a hemolysis assay were conducted for a biosafety evaluation. SV@rGO-EA microbubbles were scanned at various mechanical index values to obtain the B-mode and contrast-enhanced ultrasound (CEUS) mode images in vitro. SV@rGO-EA microbubbles were administered to SD rats, and their livers and kidneys were imaged in CEUS and B-mode. The absorption of rGO-EA resulted in an enhanced echo intensity and mechanical index tolerance of SV@rGO-EA, surpassing the performance of SV microbubbles both in vitro and in vivo. This work exhibited the application potential of graphene derivatives in the field of ultrasound precision medicine. Full article

Standard imaging cannot reliably predict the nature of renal tumors. Among malignant renal tumors, clear cell renal cell carcinoma (ccRCC) is the most common histological subtype, in which the vascular endothelial growth factor 2 (VEGFR-2) is highly expressed in the vascular endothelium. BR55, a contrast agent for ultrasound imaging, consists of gas-core lipid microbubbles that specifically target and bind to the extracellular portion of the VEGFR-2. The specific information provided by ultrasound molecular imaging (USMI) using BR55 was compared with the vascular tumor expression of the VEGFR-2 by immunohistochemical (IHC) staining in a preclinical model of ccRCC. Patients’ ccRCCs were orthotopically grafted onto Nod-Scid-Gamma (NSG) mice to generate patient-derived xenografts (PdX). Mice were divided into four groups to receive either vehicle or axitinib an amount of 2, 7.5 or 15 mg/kg twice daily. Perfusion parameters and the BR55 ultrasound contrast signal on PdX renal tumors were analyzed at D0, D1, D3, D7 and D11, and compared with IHC staining for the VEGFR-2 and CD34. Significant Pearson correlation coefficients were observed between the area under the curve (AUC) and the CD34 (0.84, p < 10⁻⁴), and between the VEGFR-2-specific signal obtained by USMI and IHC (0.72, p < 10⁻⁴). USMI with BR55 could provide instant, quantitative information on tumor VEGFR-2 expression to characterize renal masses non-invasively. Full article

Ultrasound contrast agents are clinically used for diagnosis of internal organs, but ultrasound contrast agents are rarely applied clinically in musculoskeletal disorders. Our study aims to comparatively analyze the differences between ultrasonographic images through peri-injury injection of the clinically used microbubble and researched nanoparticle contrast agents in various muscular injury models. To compare contrast-enhanced images in different muscle injury models, we prepared groups of rats with sham, laceration, punch, contusion, and toxin injection injuries. We measured H₂O₂ levels using the Amplex Red assay by extracting tissue from the damaged area. As comparative contrast agents, SonoVue^®, a commercially available microbubble contrast agent, and poly(vanillinoxalte) (PVO) nanoparticles, which are H₂O₂-responsive nanoparticles, were used. The difference in contrast between the two contrast agents was recorded as an ultrasound movie, and J-image software 1.53p was used to quantify and analyze the maximum and minimum echogenicity values of the images after contrast enhancement. In the Amplex red assay for the highest H₂O₂ level in each muscle injury model, the maximum level showed 24 h after the modeling. In the sham rats, PVO injection showed no increased echogenicity except at the needle insertion site, but SonoVue^® injection showed increased echo signal throughout the injected muscle immediately after injection. One day after the preparation of the lesion, PVO and SonoVue^® were injected into the lesion site and ultrasound was performed on the lesion site. After the injection of PVO nanoparticles, contrast enhancement was observed at the lesion site immediately. SonoVue^® injections, on the other hand, showed a widespread pattern of echo signals and an increase in echo retention only at the lesion site over time, but this was not clear. There were statistically significant differences between the highest and lowest echogenicity in PVO and SonoVue^® contrast-enhanced images in all models. Contrast enhancement lasted more than 3 h in the PVO injection, but disappeared within 3 h in the SonoVue^® injection. PVO nanoparticles showed the possibility of physiologic contrast by CO₂ generated by conjugation with H₂O₂ generated by muscle injuries, and SonoVue^® injection observed the possibility of microbubble contrast as a contrast agent with a pooling effect that lasts longer on the lesion. Further research is needed to investigate the use of various ultrasound contrast agents, including nanoparticles, in musculoskeletal disorders, as well as the potential for further utilities of microbubble contrast agents. Full article

The main aim of this study is to synthesize contrast microbubbles (MB) functionalized with engineered protein ligands using a microfluidic device to target breast cancer specific vascular B7-H3 receptor in vivo for diagnostic ultrasound imaging. We used a high-affinity affibody (ABY) selected against human/mouse B7-H3 receptor for engineering targeted MBs (TMBs). We introduced a C-terminal cysteine residue to this ABY ligand for facilitating site-specific conjugation to DSPE-PEG-2K-maleimide (M. Wt = 2.9416 kDa) phospholipid for MB formulation. We optimized the reaction conditions of bioconjugations and applied it for microfluidic based synthesis of TMBs using DSPE-PEG-ABY and DPPC liposomes (5:95 mole %). The binding affinity of TMBs to B7-H3 (MB_B7-H3) was tested in vitro in MS1 endothelial cells expressing human B7-H3 (MS1_B7-H3) by flow chamber assay, and by ex vivo in the mammary tumors of a transgenic mouse model (FVB/N-Tg (MMTV-PyMT)634Mul/J), expressing murine B7-H3 in the vascular endothelial cells by immunostaining analyses. We successfully optimized the conditions needed for generating TMBs using a microfluidic system. The synthesized MBs showed higher affinity to MS1 cells engineered to express higher level of hB7-H3, and in the endothelial cells of mouse tumor tissue upon injecting TMBs in a live animal. The average number (mean ± SD) of MB_B7-H3 binding to MS1_B7-H3 cells was estimated to be 354.4 ± 52.3 per field of view (FOV) compared to wild-type control cells (MS1_WT; 36.2 ± 7.5/FOV). The non-targeted MBs did not show any selective binding affinity to both the cells (37.7 ± 7.8/FOV for MS1_B7-H3 and 28.3 ± 6.7/FOV for MS1_WT cells). The fluorescently labeled MB_B7-H3 upon systemic injection in vivo co-localized to tumor vessels, expressing B7-H3 receptor, as validated by ex vivo immunofluorescence analyses. We have successfully synthesized a novel MB_B7-H3 via microfluidic device, which allows us to produce on demand TMBs for clinical applications. This clinically translatable MB_B7-H3 showed significant binding affinity to vascular endothelial cells expressing B7-H3 both in vitro and in vivo, which shows its potential for clinical translation as a molecular ultrasound contrast agent for human applications. Full article

The noninvasive estimation of interstitial fluid pressure (IFP) using ultrasound contrast agent (UCA) microbubbles as pressure sensors will provide tumor treatments and efficacy assessments with a promising tool. This study aimed to verify the efficacy of the optimal acoustic pressure in vitro in the prediction of tumor IFPs based on UCA microbubbles’ subharmonic scattering. A customized ultrasound scanner was used to generate subharmonic signals from microbubbles’ nonlinear oscillations, and the optimal acoustic pressure was determined in vitro when the subharmonic amplitude reached the most sensitive to hydrostatic pressure changes. This optimal acoustic pressure was then applied to predict IFPs in tumor-bearing mouse models, which were further compared with the reference IFPs measured using a standard tissue fluid pressure monitor. An inverse linear relationship and good correlation (r = −0.853, p < 0.001) existed between the subharmonic amplitude and tumor IFPs at the optimal acoustic pressure of 555 kPa, and pressure sensitivity was 1.019 dB/mmHg. No statistical differences were found between the pressures measured by the standard device and those estimated via the subharmonic amplitude, as confirmed by cross-validation (mean absolute errors from 2.00 to 3.09 mmHg, p > 0.05). Our findings demonstrated that in vitro optimized acoustic parameters for UCA microbubbles’ subharmonic scattering can be applied for the noninvasive estimation of tumor IFPs. Full article