Nonspherical ultrasound microbubbles.

Surface tension provides microbubbles (MB) with a perfect spherical shape. Here, we demonstrate that MB can be engineered to be nonspherical, endowing them with unique features for biomedical applications. Anisotropic MB were generated via one-dimensionally stretching spherical poly(butyl cyanoacrylate) MB above their glass transition temperature. Compared to their spherical counterparts, nonspherical polymeric MB displayed superior performance in multiple ways, including i) increased margination behavior in blood vessel-like flow chambers, ii) reduced macrophage uptake in vitro, iii) prolonged circulation time in vivo, and iv) enhanced blood-brain barrier (BBB) permeation in vivo upon combination with transcranial focused ultrasound (FUS). Our studies identify shape as a design parameter in the MB landscape, and they provide a rational and robust framework for further exploring the application of anisotropic MB for ultrasound-enhanced drug delivery and imaging applications.

Ultrasound-mediated gene delivery specifically targets liver sinusoidal endothelial cells for sustained FVIII expression in hemophilia A mice.

The ability to target the native production site of factor VIII (FVIII)-liver sinusoidal endothelial cells (LSECs)-can improve the outcome of hemophilia A (HA) gene therapy. By testing a matrix of ultrasound-mediated gene delivery (UMGD) parameters for delivering a GFP plasmid into the livers of HA mice, we were able to define specific conditions for targeted gene delivery to different cell types in the liver. Subsequently, two conditions were selected for experiments to treat HA mice via UMGD of an endothelial-specific human FVIII plasmid: low energy (LE; 50 W/cm2, 150 µs pulse duration) to predominantly target endothelial cells or high energy (HE; 110 W/cm2, 150 µs pulse duration) to predominantly target hepatocytes. Both groups of UMGD-treated mice achieved persistent FVIII activity levels of ∼10% over 84 days post treatment; however, half of the HE-treated mice developed low-titer inhibitors while none of the LE mice did. Plasma transaminase levels and histological liver examinations revealed minimal transient liver damage that was lower in the LE group than in the HE group. These results indicate that UMGD can safely target LSECs with a lower-energy condition to achieve persistent FVIII gene expression, demonstrating that this novel technology is highly promising for therapeutic correction of HA.

Assembly of Metal-Phenolic Networks onto Microbubbles for One-Step Generation of Functional Microcapsules.

The one-step assembly of metal-phenolic networks (MPNs) onto particle templates can enable the facile, rapid, and robust construction of hollow microcapsules. However, the required template removal step may affect the refilling of functional species in the hollow interior space or the in situ encapsulation of guest molecules during the formation of the shells. Herein, a simple strategy for the one-step generation of functional MPNs microcapsules is proposed. This method uses bovine serum albumin microbubbles (BSA MBs) as soft templates and carriers, enabling the efficient pre-encapsulation of guest species by leveraging the coordination assembly of tannic acid (TA) and FeIII ions. The addition of TA and FeIII induces a change in the protein conformation of BSA MBs and produces semipermeable capsule shells, which allow gas to escape from the MBs without template removal. The MBs-templated strategy can produce highly biocompatible capsules with controllable structure and size, and it is applicable to produce other MPNs systems like BSA-TA-CuII and BSA-TA-NiII . Finally, those MBs-templated MPNs capsules can be further functionalized or modified for the loading of magnetic nanoparticles and the pre-encapsulation of model molecules through covalence or physical adsorption, exhibiting great promise in biomedical applications.

Spatiotemporal analysis of contrast-enhanced ultrasound for differentiating between malignant and benign breast lesions.

OBJECTIVES: The aim of this study was to apply spatiotemporal analysis of contrast-enhanced ultrasound (CEUS) loops to quantify the enhancement heterogeneity for improving the differentiation between benign and malignant breast lesions. MATERIALS AND METHODS: This retrospective study included 120 women (age range, 18-82 years; mean, 52 years) scheduled for ultrasound-guided biopsy. With the aid of brightness-mode images, the border of each breast lesion was delineated in the CEUS images. Based on visual evaluation and quantitative metrics, the breast lesions were categorized into four grades of different levels of contrast enhancement. Grade-1 (hyper-enhanced) and grade-2 (partly-enhanced) breast lesions were included in the analysis. Four parameters reflecting enhancement heterogeneity were estimated by spatiotemporal analysis of neighboring time-intensity curves (TICs). By setting the threshold on mean parameter, the diagnostic performance of the four parameters for differentiating benign and malignant lesions was evaluated. RESULTS: Sixty-four of the 120 patients were categorized as grade 1 or 2 and used for estimating the four parameters. At the pixel level, mutual information and conditional entropy present significantly different values between the benign and malignant lesions (p < 0.001 in patients of grade 1, p = 0.002 in patients of grade 1 or 2). For the classification of breast lesions, mutual information produces the best diagnostic performance (AUC = 0.893 in patients of grade 1, AUC = 0.848 in patients of grade 1 or 2). CONCLUSIONS: The proposed spatiotemporal analysis for assessing the enhancement heterogeneity shows promising results to aid in the diagnosis of breast cancer by CEUS. CLINICAL RELEVANCE STATEMENT: The proposed spatiotemporal method can be developed as a standardized software to automatically quantify the enhancement heterogeneity of breast cancer on CEUS, possibly leading to the improved diagnostic accuracy of differentiation between benign and malignant lesions. KEY POINTS: • Advanced spatiotemporal analysis of ultrasound contrast-enhanced loops for aiding the differentiation of malignant or benign breast lesions. • Four parameters reflecting the enhancement heterogeneity were estimated in the hyper- and partly-enhanced breast lesions by analyzing the neighboring pixel-level time-intensity curves. • For the classification of hyper-enhanced breast lesions, mutual information produces the best diagnostic performance (AUC = 0.893).

Clinical sonochemotherapy of inoperable pancreatic cancer using diagnostic ultrasound and microbubbles: a multicentre, open-label, randomised, controlled trial.

OBJECTIVES: Sonochemotherapy, which uses microbubble (MB)-assisted ultrasound (US) to deliver chemotherapeutic agents, has the potential to enhance tumour chemotherapy. The combination of US and MB has been demonstrated to prolong the survival of patients with pancreatic cancer. This phase 2 clinical trial aimed to determine the clinical efficacy and safety of sonochemotherapy for inoperable pancreatic ductal adenocarcinoma by using US and MB. METHODS: Eighty-two patients with stage III or IV pancreatic cancer were recruited from July 2018 to March 2021 and followed up until September 2022. US treatment was performed with a modified diagnostic US scanner for 30 min after chemotherapeutic infusion. The primary endpoint was overall survival (OS), and the secondary endpoints were Eastern Cooperative Oncology Group (ECOG) status < 2, progression-free survival (PFS), disease control rate (DCR), and adverse events. RESULTS: Seventy-eight patients were randomly allocated (40 to chemotherapy and 38 to sonochemotherapy). The median OS was longer with sonochemotherapy than with chemotherapy (9.10 vs. 6.10 months; p = 0.037). The median PFS with sonochemotherapy was 5.50 months, compared with 3.50 months (p = 0.080) for chemotherapy. The time of ECOG status < 2 was longer with sonochemotherapy (7.20 months) than with chemotherapy (5.00 months; p = 0.029). The DCR was 73.68% for sonochemotherapy compared with 42.50% for the control (p = 0.005). The incidence of overall adverse events was balanced between the two groups. CONCLUSIONS: The use of sonochemotherapy can extend the survival and well-being time of stage III or IV pancreatic cancer patients without any increase in serious adverse events. TRIAL REGISTRATION: ChineseClinicalTrials.gov ChiCTR2100044721 CLINICAL RELEVANCE STATEMENT: This multicentre, randomised, controlled trial has proven that sonochemotherapy, namely, the combination of diagnostic ultrasound, microbubbles, and chemotherapy, could extend the overall survival of patients with end-stage pancreatic ductal adenocarcinoma from 6.10 to 9.10 months without increasing any serious adverse events. KEY POINTS: • This is the first multicentre, randomised, controlled trial of sonochemotherapy for clinical pancreatic cancer treatment using ultrasound and a commercial ultrasound contrast agent. • Sonochemotherapy extended the median overall survival from 6.10 (chemotherapy alone) to 9.10 months. • The disease control rate increased from 42.50% with chemotherapy to 73.68% with sonochemotherapy.

Luminal Delivery of Pectin-Modified Oxygen Microbubbles Mitigates Rodent Experimental Intestinal Ischemia.

INTRODUCTION: Ischemic gut injury is common in the intensive care unit, impairs gut barrier function, and contributes to multiorgan dysfunction. One novel intervention to mitigate ischemic gut injury is the direct luminal delivery of oxygen microbubbles (OMB). Formulations of OMB can be modified to control the rate of oxygen delivery. This project examined whether luminal delivery of pectin-modified OMB (OMBp5) can reduce ischemic gut injury in a rodent model. METHODS: The OMBp5 formulation was adapted to improve delivery of oxygen along the length of small intestine. Adult Sprague-Dawley rats (n = 24) were randomly allocated to three groups: sham-surgery (SS), intestinal ischemia (II), and intestinal ischemia plus luminal delivery of OMBp5 (II + O). Ischemia-reperfusion injury was induced by superior mesenteric artery occlusion for 45 min followed by reperfusion for 30 min. Outcome data included macroscopic score of mucosal injury, the histological score of gut injury, and plasma biomarkers of intestinal injury. RESULTS: Macroscopic, microscopic data, and intestinal injury biomarker results demonstrated minimal intestinal damage in the SS group and constant damage in the II group. II + O group had a significantly improved macroscopic score throughout the gut mucosa (P = 0.04) than the II. The mean histological score of gut injury for the II + O group was significantly improved on the II group (P ≤ 0.01) in the proximal intestine only, within 30 cm of delivery. No differences were observed in plasma biomarkers of intestinal injury following OMBp5 treatment. CONCLUSIONS: This proof-of-concept study has demonstrated that luminal OMBp5 decreases ischemic injury to the proximal small intestine. There is a need to improve oxygen delivery over the full length of the intestine. These findings support further studies with clinically relevant end points, such as systemic inflammation and vital organ dysfunction.

Noninvasive Evaluation of Breast Tumor Response to Combined Ultrasound-Stimulated Microbubbles and Hyperthermia Therapy Using Quantitative Ultrasound-Based Texture Analysis Method.

OBJECTIVES: Quantitative ultrasound (QUS) is a noninvasive imaging technique that can be used for assessing response to anticancer treatment. In the present study, tumor cell death response to the ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) treatment was monitored in vivo using QUS. METHODS: Human breast cancer cell lines (MDA-MB-231) were grown in mice and were treated with HT (10, 30, 50, and 60 minutes) alone, or in combination with USMB. Treatment effects were examined using QUS with a center frequency of 25 MHz (bandwidth range: 16 to 32 MHz). Backscattered radiofrequency (RF) data were acquired from tumors subjected to treatment. Ultrasound parameters such as average acoustic concentration (AAC) and average scatterer diameter (ASD), were estimated 24 hours prior and posttreatment. Additionally, texture features: contrast (CON), correlation (COR), energy (ENE), and homogeneity (HOM) were extracted from QUS parametric maps. All estimated parameters were compared with histopathological findings. RESULTS: The findings of our study demonstrated a significant increase in QUS parameters in both treatment conditions: HT alone (starting from 30 minutes of heat exposure) and combined treatment of HT plus USMB finally reaching a maximum at 50 minutes of heat exposure. Increase in AAC for 50 minutes HT alone and USMB +50 minutes was found to be 5.19 ± 0.417% and 5.91 ± 1.11%, respectively, compared to the control group with AAC value of 1.00 ± 0.44%. Furthermore, between the treatment groups, ΔASD-ENE values for USMB +30 minutes HT significantly reduced, depicting 0.00062 ± 0.00096% compared to 30 minutes HT only group, showing 0.0058 ± 0.0013%. Further, results obtained from the histological analysis indicated greater cell death and reduced nucleus size in both HT alone and HT combined with USMB. CONCLUSION: The texture-based QUS parameters indicated a correlation with microstructural changes obtained from histological data. This work demonstrated the use of QUS to detect HT treatment effects in breast cancer tumors in vivo.

Optimal Treatment Parameters for Ultrasound-Stimulated Microbubbles in Upregulating Proliferation and Stemness of Bone Marrow Mesenchymal Stem Cells.

OBJECTIVES: Ultrasound-targeted microbubble disruption (UTMD) is a widely used technique to improve the differentiation and proliferation capacity of mesenchymal stem cells (MSCs), but the optimal therapeutic parameters for UTMD are unclear. In this study, we aimed to find the appropriate peak negative pressure (PNP), which is a key parameter for enhancing the stemness properties and proliferation of MSCs. METHODS: Experiments were performed in UTMD group, ultrasound (US) group under different PNP exposure conditions (0.5, 1.0, and 1.5 MPa), and control group. Apoptosis safety was analyzed by flow cytometry and MSC proliferation was measured at 12, 24, and 36 hours after irradiation by cell counting kit 8. The expression of the stemness genes NANOG, OCT-4, and SOX-2 were determined by enzyme-linked immunosorbent assay (ELISA) or reverse transcription polymerase chain reaction. RESULTS: The results showed that the 1.5 MPa UTMD-treated group had the highest proliferation capacity of MSCs at 24 hours. ELISA or quantitative reverse transcription polymerase chain reaction results showed that UTMD treatment of the 1.5 MPa group significantly upregulated the expression of the stemness genes NANOG, SOX-2, and OCT-4. CONCLUSIONS: In conclusion, the appropriate peak PNP value of UTMD was 1.5 MPa, and 1.5 MPa-mediated UTMD group obviously promoted MSCs proliferation and maintained stemness by upregulating the expression of stemness genes.

Nanotargeted Cationic Lipid Microbubbles Carrying HSV-TK Gene Inhibit the Development of Subcutaneous Liver Tumor Model After HIFU Ablation.

OBJECTIVES: High-intensity focused ultrasound (HIFU) has been widely used in clinical settings and has achieved suitable results in the treatment of many cancerous or noncancerous diseases. However, in the treatment of liver cancer, because the tumor is located deep within the liver tissue, when ultrasound penetrates the tissue, it will inevitably produce sound energy attenuation. This attenuation limits the reliability of HIFU treatment, reduce the efficacy of HIFU, and increase the risk of tumor recurrence. METHODS: Cationic microbubbles (CMB) were successfully linked with GPC3 and HSV-TK plasmids, and targeted gene-carrying CMB were successfully constructed. Moreover, the gene-targeted cation microbubbles had suitable targeting and can specifically bind with liver cancer cells. RESULTS: The HSV-TK transfection efficiency was high and had a significant inhibitory effect on the proliferation and invasion of liver cancer cells. After the gene-carrying cation microbubbles entered the animal body, they had a great targeting effect in vivo. They transfected the target genes into liver cancer cells, and the HSV-TK/GCV system initiated cell death, demonstrating that these targeted microbubbles, enhanced HIFU treatment. CONCLUSIONS: Overall, CMB combined with a GPC3 antibody and HSV-TK plasmid can target residual subcutaneous liver tumor cells under the guidance of GPC3 antibody, and kill residual subcutaneous liver tumor cells under the action of ultrasound, thus enhancing the therapeutic effect of HIFU on liver cancer.

Interaction Between Microbubbles and Microwave Ablation: A Phantom and Rabbit Model.

OBJECTIVES: This study aimed to explore the interactions between microbubbles and microwave ablation (MWA). METHODS: The study employed custom-made phantoms (in vitro) and white New Zealand rabbits (in vivo). MWA was performed with or without microbubbles in the phantoms (2 × 105 particles mL-1) and rabbit livers (intravenous injection of 0.05 mL/kg SonoVue). During the MWA, K-type thermocouple probes were used to monitor the MWA-induced temperature increase. Contrast-enhanced ultrasound imaging (CEUS) was used to monitor and analyze the microbubbles signal intensity. After MWA, the ablation-zone volumes were evaluated and compared between the groups with and without microbubbles. RESULTS: In both the phantom models and rabbits, microbubbles showed no significant influence on MWA, including the ablation range and MWA-induced temperature increase. In phantoms and rabbit livers filled with microbubbles, MWA caused the formation of a gradually expanding microbubble-defect region over the ablation time. An increase in the temperature caused microbubble destruction. CONCLUSIONS: Microbubbles had no significant influence on MWA. However, MWA induced the destruction of microbubbles in a temperature-dependent manner. Thus, the poor thermotolerance of microbubbles is a non-negligible barrier when using CEUS to monitor the ablation range during MWA in real-time.

Super-Resolution Ultrasound Imaging for Monitoring the Therapeutic Efficacy of a Vascular Disrupting Agent in an Animal Model of Breast Cancer.

OBJECTIVE: Evaluate the use of super-resolution ultrasound (SRUS) imaging for the early detection of tumor response to treatment using a vascular-disrupting agent (VDA). METHODS: A population of 28 female nude athymic mice (Charles River Laboratories) were implanted with human breast cancer cells (MDA-MB-231, ATCC) in the mammary fat pad and allowed to grow. Ultrasound imaging was performed using a Vevo 3100 scanner (FUJIFILM VisualSonics Inc) equipped with the MX250 linear array transducer immediately before and after receiving bolus injections of a microbubble (MB) contrast agent (Definity, Lantheus Medical Imaging) via the tail vein. Following baseline ultrasound imaging, VDA drug (combretastatin A4 phosphate, CA4P, Sigma Aldrich) or control saline was injected via the placed catheter. After 4 or 24 hours, repeat ultrasound imaging along the same tumor cross-section occurred. Direct intratumoral pressure measurements were obtained using a calibrated sensor. All raw ultrasound data were saved for offline processing and SRUS image reconstruction using custom MATLAB software (MathWorks Inc). From a region encompassing the tumor space and the entire postprocessed ultrasound image sequence, time MB count (TMC) curves were generated in addition to traditional SRUS maps reflecting MB enumeration at each pixel location. Peak enhancement (PE) and wash-in rate (WIR) were extracted from these TMC curves. At termination, intratumoral microvessel density (MVD) was quantified using tomato lectin labeling of patent blood vessels. RESULTS: SRUS images exhibited a clear difference between control and treated tumors. While there was no difference in any group parameters at baseline (0 hour, P > .09), both SRUS-derived PE and WIR measurements in tumors treated with VDA exhibited significant decreases by 4 (P = .03 and P = .05, respectively) and 24 hours (P = .02 and P = .01, respectively), but not in control group tumors (P > .22). Similarly, SRUS derived microvascular maps were not different at baseline (P = .81), but measures of vessel density were lower in treated tumors at both 4 and 24 hours (P < .04). An inverse relationship between intratumoral pressure and both PE and WIR parameters were found in control tumors (R2 > .09, P < .03). CONCLUSION: SRUS imaging is a new modality for assessing tumor response to treatment using a VDA.

Tornado-inspired acoustic vortex tweezer for trapping and manipulating microbubbles.

Spatially concentrating and manipulating biotherapeutic agents within the circulatory system is a longstanding challenge in medical applications due to the high velocity of blood flow, which greatly limits drug leakage and retention of the drug in the targeted region. To circumvent the disadvantages of current methods for systemic drug delivery, we propose tornado-inspired acoustic vortex tweezer (AVT) that generates net forces for noninvasive intravascular trapping of lipid-shelled gaseous microbubbles (MBs). MBs are used in a diverse range of medical applications, including as ultrasound contrast agents, for permeabilizing vessels, and as drug/gene carriers. We demonstrate that AVT can be used to successfully trap MBs and increase their local concentration in both static and flow conditions. Furthermore, MBs signals within mouse capillaries could be locally improved 1.7-fold and the location of trapped MBs could still be manipulated during the initiation of AVT. The proposed AVT technique is a compact, easy-to-use, and biocompatible method that enables systemic drug administration with extremely low doses.

Localized blood-brain barrier opening in infiltrating gliomas with MRI-guided acoustic emissions-controlled focused ultrasound.

Pharmacological treatment of gliomas and other brain-infiltrating tumors remains challenging due to limited delivery of most therapeutics across the blood-brain barrier (BBB). Transcranial MRI-guided focused ultrasound (FUS), an emerging technology for noninvasive brain treatments, enables transient opening of the BBB through acoustic activation of circulating microbubbles. Here, we evaluate the safety and utility of transcranial microbubble-enhanced FUS (MB-FUS) for spatially targeted BBB opening in patients with infiltrating gliomas. In this Phase 0 clinical trial (NCT03322813), we conducted comparative and quantitative analyses of FUS exposures (sonications) and their effects on gliomas using MRI, histopathology, microbubble acoustic emissions (harmonic dose [HD]), and fluorescence-guided surgery metrics. Contrast-enhanced MRI and histopathology indicated safe and reproducible BBB opening in all patients. These observations occurred using a power cycling closed feedback loop controller, with the power varying by nearly an order of magnitude on average. This range underscores the need for monitoring and titrating the exposure on a patient-by-patient basis. We found a positive correlation between microbubble acoustic emissions (HD) and MR-evident BBB opening (P = 0.07) and associated interstitial changes (P < 0.01), demonstrating the unique capability to titrate the MB-FUS effects in gliomas. Importantly, we identified a 2.2-fold increase of fluorescein accumulation in MB-FUS-treated compared to untreated nonenhancing tumor tissues (P < 0.01) while accounting for vascular density. Collectively, this study demonstrates the capabilities of MB-FUS for safe, localized, controlled BBB opening and highlights the potential of this technology to improve the surgical and pharmacologic treatment of brain tumors.

The Effect of Mixing Iodinated Contrast Media and Ultrasound Contrast Agents on Subharmonic Signals.

Subharmonic aided pressure estimation (SHAPE) is a technique that utilizes subharmonic signals from microbubble contrast agents for pressure estimation. Validation of the SHAPE technique relies on synchronous measurements of in vivo pressures using contrast microbubbles and a pressure catheter (reference standard). For the guidance and placement of pressure catheter in vivo, iodinated contrast is used with fluoroscopy. Therefore, during data acquisition for validation studies of the SHAPE technique, both contrast microbubbles and iodinated contrast are present simultaneously within the vasculature. This study aims to elucidate the effects of iodinated contrast (Visipaque, GE HealthCare) on subharmonic signal amplitude from contrast microbubbles (Definity, Lantheus Medical Imaging, Inc.). In an acrylic water tank, 0.06 mL of Definity and varied amounts of Visipaque (0.14, 0.43, 0.85, and 1.70 mL) were added to 425 mL of deionized water. Ultrasound scanning was performed with a SonixTablet scanner (BK Medical Systems) using optimized parameters for SHAPE with Definity (ftransmit/receive = 3.0/1.5 MHz; chirp down pulse). Subharmonic data was acquired and analyzed at 9 different incident acoustic outputs (n = 3). Results showed an increase in subharmonic signal amplitude from Definity microbubbles in the presence of 0.14 mL Visipaque by 2.8 ± 1.3 dB (p < .001), no change with 0.85 mL Visipaque (0.7 ± 1.2 dB; p = .09) and a decrease in subharmonic amplitude in the presence of 1.70 mL Visipaque by 1.9 ± 0.7 dB (p < .001). While statistically significant effect on subharmonic signal amplitude of Definity microbubbles was noted due to the mixture, the magnitude of the effect was minimal (~2.8 dB) and unlikely to impact in vivo SHAPE measurements.

Bubble Printing of Ti3C2TX MXene for Patterning Conductive and Plasmonic Nanostructures.

MXenes represent a novel class of 2D materials with unique properties and have great potential for diverse applications in sensing and electronics; however, their directed assembly at interfaces has not yet been achieved. Herein, the plasmonic heating of MXenes was exploited to achieve the controlled deposition of MXene assemblies via a laser-directed microbubble. The influence of various factors such as solvent composition, substrate surface chemistry, MXene concentration, and laser fluence was investigated, establishing the optimal conditions for rapid patterning with good fidelity. Printed MXene assemblies showed good electrical conductivity and plasmonic sensing capabilities and were able to meet or exceed the state of the art without additional postprocessing steps. This represents the first study of a directed approach for microfabrication using MXenes and lays the foundation for future work in optically directed assembly of MXenes and MXene-based nanocomposites at interfaces toward sensors and devices.

Ultrasound-Mediated Lysozyme Microbubbles Targeting NOX4 Knockdown Alleviate Cisplatin-Exposed Cochlear Hair Cell Ototoxicity.

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein plays an essential role in the cisplatin (CDDP)-induced generation of reactive oxygen species (ROS). In this study, we evaluated the suitability of ultrasound-mediated lysozyme microbubble (USMB) cavitation to enhance NOX4 siRNA transfection in vitro and ex vivo. Lysozyme-shelled microbubbles (LyzMBs) were constructed and designed for siNOX4 loading as siNOX4/LyzMBs. We investigated different siNOX4-based cell transfection approaches, including naked siNOX4, LyzMB-mixed siNOX4, and siNOX4-loaded LyzMBs, and compared their silencing effects in CDDP-treated HEI-OC1 cells and mouse organ of Corti explants. Transfection efficiencies were evaluated by quantifying the cellular uptake of cyanine 3 (Cy3) fluorescein-labeled siRNA. In vitro experiments showed that the high transfection efficacy (48.18%) of siNOX4 to HEI-OC1 cells mediated by US and siNOX4-loaded LyzMBs significantly inhibited CDDP-induced ROS generation to almost the basal level. The ex vivo CDDP-treated organ of Corti explants of mice showed an even more robust silencing effect of the NOX4 gene in the siNOX4/LyzMB groups treated with US sonication than without US sonication, with a marked abolition of CDDP-induced ROS generation and cytotoxicity. Loading of siNOX4 on LyzMBs can stabilize siNOX4 and prevent its degradation, thereby enhancing the transfection and silencing effects when combined with US sonication. This USMB-derived therapy modality for alleviating CDDP-induced ototoxicity may be suitable for future clinical applications.

Ultrasound-mediated HGF Gene Microbubbles Mitigate Hyperkinetic Pulmonary Arterial Hypertension in Rabbits.

AIM: Hyperkinetic pulmonary arterial hypertension (PAH) is a complication of congenital heart disease. Gene therapy is a new experimental treatment for PAH, and ultrasound-mediated gene-carrying microbubble targeted delivery is a promising development for gene transfer. METHODS: This study successfully established a hyperkinetic PAH rabbit model by a common carotid artery and jugular vein shunt using the cuff style method. Liposome microbubbles carrying the hepatocyte growth factor (HGF) gene were successfully constructed. An in vitro experiment evaluated the appropriate intensity of ultrasonic radiation by Western blots and 3H-TdR incorporation assays. In an in vivo experiment, after transfection of ultrasound-mediated HGF gene microbubbles, catheterisation was applied to collect haemodynamic data. Hypertrophy of the right ventricle was evaluated by measuring the right ventricle hypertrophy index. Western blot and immunohistochemistry analyses were used to detect the expression of human (h)HGF and angiogenic effects, respectively. RESULTS: The most appropriate ultrasonic radiation intensity was 1.0 W/cm2 for 5 minutes. Two weeks after transfection, both systolic pulmonary arterial pressure and mean pulmonary arterial pressure were attenuated. Hypertrophy of the right ventricle was reversed. hHGF was transplanted into the rabbits, resulting in a high expression of hHGF protein and an increase in the number of small pulmonary arteries. Ultrasound-mediated HGF gene microbubble therapy was more effective at attenuating PAH and increasing the density of small pulmonary arteries than single HGF plasmid transfection. CONCLUSIONS: Ultrasound-mediated HGF gene microbubbles significantly improved the target of gene therapy in a rabbit PAH model and enhanced the tropism and transfection rates. Thus, the technique can effectively promote small pulmonary angiogenesis and play a role in the treatment of PAH without adverse reactions.

Target tumor therapy in human gastric cancer cells through the combination of docetaxel-loaded cationic lipid microbubbles and ultrasound-triggered microbubble destruction.

It is well accepted that ultrasound-induced microbubble (USMB) cavitation is a promising method for drug delivery. Ultrasound-targeted destruction of cytotoxic drug-loaded lipid microbubbles (LMs) is used to promote the treatment of cancer. This study aimed to investigate the antitumor effects from a combination of docetaxel-loaded cationic lipid microbubbles (DLLM+) and ultrasound (US)-triggered microbubble destruction (UTMD) on gastric cancer (GC). It was found that the functional dose of DOC in this study was 1 × 10-9 mol/L. We found that DLLM combined with the UTMD group showed greater growth inhibition of the cultured human gastric cancer cells (HGCCs) when compared with the other five groups by arresting the G2/M phase in the cell cycle. However, DLLM+ combined with UTMD showed a higher inhibition rate of tumor growth than DLLM combined with UTMD and that of the RC/CMV-p16 combined with UTMD in vitro and in vivo experiments. DLLM+ combined with UTMD significantly suppressed proliferation and promoted the apoptosis of HGCCs with more cells arrested in the G2/M phase. In addition, DLLM+ combined with UTMD suppressed the proliferation and induced apoptosis by arresting cells in the G2/M phase, which led to a great inhibition of GC progression. Thus, our results indicated that the combination of DLLM+ and UTMD might represent a novel and promising approach to chemotherapy for GC.

Toward a theranostic device for gliomas.

BACKGROUND: In the surgical management of glioblastoma, a highly aggressive and incurable type of brain cancer, identification and treatment of residual tissue is the most common site of disease recurrence. Monitoring and localized treatment are achieved with engineered microbubbles (MBs) by combining ultrasound and fluorescence imaging with actively targeted temozolomide (TMZ) delivery. METHODS: The MBs were conjugated with a near-infrared fluorescence probe CF790, cyclic pentapeptide bearing the RGD sequence and a carboxyl-temozolomide, TMZA. The efficiency of adhesion to HUVEC cells was assessed in vitro in realistic physiological conditions of shear rate and vascular dimensions. Cytotoxicity of TMZA-loaded MBs on U87 MG cells and IC50 were assessed by MTT tests. RESULTS: We report on the design of injectable poly(vinyl alcohol) echogenic MBs designed as a platform with active targeting ability to tumor tissues, by tethering on the surface a ligand having the tripeptide sequence, RGD. The biorecognition of RGD-MBs onto HUVEC cells is quantitatively proved. Efficient NIR emission from the CF790-decorated MBs was successfully detected. The conjugation on the MBs surface of a specific drug as TMZ is achieved. The pharmacological activity of the coupled-to-surface drug is preserved by controlling the reaction conditions. CONCLUSIONS: We present an improved formulation of PVA-MBs to achieve a multifunctional device with adhesion ability, cytotoxicity on glioblastoma cells and supporting imaging.

Long-Term Retention Microbubbles with Three-Layer Structure for Floating Intravesical Instillation Delivery.

Intravesical instillation is an effective treatment for bladder cancer. However, clinical anticancer agents always suffer rapid excretion by periodic urination, leading to low therapeutic efficacy. Prolonging the retention time of drugs in the bladder is the key challenge for intravesical instillation treatment. Herein, a facile and powerful surface cross-linking-freeze drying strategy is proposed to generate ultra-stable albumin bovine air microbubbles (BSA-MBs) that can float and adhere to the bladder wall to overcome the excretion of urination and exhibit a remarkable property of long-term retention in the bladder. More noteworthy, BSA-MBs are endowed with a specific three-layer structure, namely, the outer membrane, middle drug loading layer and inner air core, which makes them have a low density to easily float and possess a high drug loading capacity. Based on their unique superiorities, the therapeutic potential of doxorubicin (DOX)-loaded BSA-MBs (DOX-MBs) is exemplified by intravesical instillation for bladder cancer. After injection into the bladder, DOX-MBs can remain in the bladder for a long time and sustain the release of DOX in urine, exhibiting potent anticancer efficacy. Consequently, the prolonged retention of BSA-MBs in the bladder renders them as an effective floating drug delivery system for intravesical instillation therapy.