Lipid Microbubbles Research Articles

Engineered microbubbles decorated with red emitting carbon nanoparticles for efficient delivery and imaging

Altering the route of uptake by the cells is an attractive strategy to overcome drug-receptor adaptation problems. Carbon nanoparticles (CNPs) with emission beyond tissue autofluorescence for imaging biological tissues were used to study the phenomenon of uptake by the cells. In this regard, red-emitting carbon nanoparticles (CNPs) were synthesized and incorporated onto lipid microbubbles (MBs). The CNPs showed red emissions in the range of 640 nm upon excitation with 480 nm wavelength of light. Atomic force microscopic and confocal microscopic images showed the successful loading of CNPs onto the MB. Carbon nanoparticle loaded microbubbles (CNP-MBs) were treated with NIH 3 T3 cells at different concentrations. Confocal microscopic imaging studies confirm the presence of CNPs inside the treated cells. Cytotoxicity studies revealed that the CNPs showed minimal toxicity towards cells after loading onto MBs. The CNPs are usually taken up by the cells through the clathrin-mediated (CME) pathway, but when loaded onto MBs, the mechanism of uptake of CNPs is altered, and the uptake by the cells was observed even in the presence of inhibitors for the CME pathway. Loading CNPs onto MBs resulted in the uptake of CNPs by the cell through micropinocytosis and sonophoresis in the presence of ultrasound. The in vivo uptake CNP-MBs were performed in Danio rerio (Zebrafish larvae). This study provides insights into altering the uptake pathway through reformulation by loading nanoparticles onto MBs.

Abstract 2888: Sensitization of radiation resistant colon cancer - The oxygenated microbubble hydrogel approach

Abstract Background: Hypoxia, a state of reduced oxygen that develops within the tumor due to its irregular vasculature, could result in treatment resistance. Mounting evidence suggests that reoxygenation of the tumor could improve outcomes of conventional cancer therapies. Our approach combats hypoxia using oxygenated lipid microbubbles (OMB) loaded in a temperature sensitive gel. The OMB would carry the oxygen while the gel would sustain its release into the tumor microenvironment. Objectives: To formulate an oxygen loaded MB dispersed in a temperature sensitive poloxamer hydrogel for intratumoral administration. Methods: OMB were generated by first preparing a liposomal mixture DSPC:DSPE-PEG2000, which was dispersed in a thermosensitive poloxamer hydrogel (P407:P188). This was pressurized with oxygen and vortexed in a gas-tight vial. Characterizations included OMB size distribution, injectability, thermosensitivity, oxygen loading and release, and the impact of the formulation on efficacy of radiotherapy against a colon cancer cell line (HCT116) was evaluated. Results: DSPC:DSPE-PEG2000 liposomes at a molar ratio of 94:6 dispersed in P407:P188 (21:6.5 %w/w) generated OMB with a size distribution in the acceptable range of 0.8-8 µm. Formulations were within the established limits of injectability (F<38N) at room temperature, and gelled near physiological temperatures enabling a sustained release of oxygen.. The formulation co-treatment enhanced radiotherapy significantly as evidenced by the remarkable reductions of clonogenic survival rates in both hypoxic and normoxic HCT116 cells (reduction by 78% vs 68% compared to just their respective radiated controls; p<0.0001 for both cases). Conclusion: Reoxygenation with a newly developed OMB hydrogel formulation effectively sensitized HCT116 to radiotherapy in vitro. Studies are underway to explore the importance of reoxygenation rate and extent for optimal tumor sensitization. Citation Format: Ashok David Jose, Celine Hui-Ning Chong, Jagdish Jaiswal, Zimei Wu, Sachin Sunil Thakur. Sensitization of radiation resistant colon cancer - The oxygenated microbubble hydrogel approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2888.

Mediation of long-pulsed ultrasound enhanced microbubble recombinant tissue plasminogen activator thrombolysis in a rat model of platelet-rich thrombus.

Ultrasound (US)-enhanced microbubble (MB) therapy has been investigated as a therapeutic technique to facilitate the thrombolysis for the treatment of pericardial and microvascular obstruction. This study sought to assess the therapeutic effects of long-pulsed US-assisted MB-mediated recombinant tissue plasminogen activator (rt-PA) thrombolysis in a rat model of platelet-rich thrombus. Ferric chloride (10%) was used to induce total arterial occlusion before formation of platelet-rich thrombi. Therapeutic long-tone-burst US (1 MHz, 0.6 MPa, 1,000-µs pulse length) was used, and 2.9×109/mL of lipid MBs and 1 mg/mL of rt-PA were infused. Subsequently, 42 Sprague-Dawley (SD) male rats were randomly divided into seven groups: (I) control; (II) rt-PA; (III) high duty cycle US + MB; (IV) low duty cycle US + rt-PA; (V) high duty cycle US + rt-PA; (VI) low duty cycle US + rt-PA + MB; and (VII) high duty cycle US + rt-PA + MB. The recanalization grades were evaluated after 20 minutes' treatment. Compared to the control, there was significant improvement in recanalization in the US + rt-PA groups (P=0.01 vs. control), US (low duty cycle) + rt-PA + MB (P=0.003 vs. control) and US (high duty cycle) + rt-PA + MB (P<0.001 vs. control) groups, in which recanalization was successfully achieved in all rats. Long-pulsed US-enhanced MB-mediated rt-PA thrombolysis offered a powerful approach in the treatment of platelet-rich thrombus.

Fibrin-targeted phase shift microbubbles for the treatment of microvascular obstruction.

Rationale: Microvascular obstruction (MVO) following percutaneous coronary intervention (PCI) is a common problem associated with adverse clinical outcomes. We are developing a novel treatment, termed sonoreperfusion (SRP), to restore microvascular patency. This entails using ultrasound-targeted microbubble cavitation (UTMC) of intravenously administered gas-filled lipid microbubbles (MBs) to dissolve obstructive microthrombi in the microvasculature. In our prior work, we used standard-sized lipid MBs. In the present study, to improve upon the efficiency and efficacy of SRP, we sought to determine the therapeutic efficacy of fibrin-targeted phase shift microbubbles (FTPSMBs) in achieving successful reperfusion of MVO. We hypothesized that owing to their much smaller size and affinity for thrombus, FTPSMBs would provide more effective dissolution of microthrombi when compared to that of the corresponding standard-sized lipid MBs. Methods: MVO in the rat hindlimb was created by direct injection of microthrombi into the left femoral artery. Definity MBs (Lantheus Medical Imaging) were infused through the jugular vein for contrast-enhanced ultrasound imaging (CEUS). A transducer was positioned vertically above the hindlimb for therapeutic US delivery during the concomitant administration of various therapeutic formulations, including (1) un-targeted MBs; (2) un-targeted phase shift microbubbles (PSMBs); (3) fibrin-targeted MB (FTMBs); and (4) fibrin-targeted PSMBs (FTPSMBs). CEUS cine loops with burst replenishment were obtained at baseline (BL), 10 min post-MVO, and after each of two successive 10-minute SRP treatment sessions (TX1, TX2) and analyzed (MATLAB). Results: In-vitro binding affinity assay showed increased fibrin binding peptide (FBP) affinity for the fibrin clots compared with the untargeted peptide (DK12). Similarly, in our in-vitro model of MVO, we observed a higher binding affinity of fluorescently labeled FTPSMBs with the porcine microthrombi compared to FTMBs, PSMBs, and MBs. Finally, in our hindlimb model, we found that UTMC with FTPSMBs yielded the greatest recovery of blood volume (dB) and flow rate (dB/sec) following MVO, compared to all other treatment groups. Conclusions: SRP with FTPSMBs achieves more rapid and complete reperfusion of MVO compared to FTMBs, PSMBs, and MBs. Studies to explore the underlying physical and molecular mechanisms are underway.

P407 hydrogel loaded with nitric oxide microbubbles promotes angiogenesis and functional improvement in testicular transplantation.

Prepubertal male patients with cancer have decreased fertility after treatment, but there are currently no suitable means for fertility rescue. Testicular transplantation seems to be a promising treatment. The short-term insufficiency of blood supply after transplantation is the key problem that needs to be solved. In this research, nitric oxide (NO), a gas and small molecule transmitter with the effect of promoting angiogenesis, acted at the site of testicular transplantation. Herein, poloxamer-407 (P407) and lipid microbubble materials served as transport carriers for NO and helped NO to function at the transplant site. P407 hydrogel loaded with NO microbubbles (PNO) slowly released NO in vitro. The three-dimensional space of the hydrogel provided a stable environment for NO microbubbles, which is conducive to the continuous release of NO. In this study, 25% PNO (w/v) was selected, and the gelling temperature was 19.47 °C. The gelling efficiency was relatively high at body temperature. Rheological experiments showed that PNO, at this concentration, had stable mechanical properties. The results from in vivo experiments demonstrated that testicular grafts in the PNO group exhibited a notably accelerated blood flow recovery compared to the other groups. Additionally, the PNO group displayed a significant improvement in reproductive function recovery. In conclusion, PNO exhibited slow release of NO, and a small amount of NO promoted angiogenesis in testicular grafts and restored reproductive function.

Development of an In Vitro Model to Study Mechanisms of Ultrasound-Targeted Microbubble Cavitation–Mediated Blood–Brain Barrier Opening

Ultrasound-targeted microbubble cavitation (UTMC)-mediated blood-brain barrier (BBB) opening is being explored as a method to increase drug delivery to the brain. This strategy has progressed to clinical trials for various neurological disorders, but the underlying cellular mechanisms are incompletely understood. In the study described here, a contact co-culture transwell model of the BBB was developed that can be used to determine the signaling cascade leading to increased BBB permeability. This BBB model consists of bEnd.3 cells and C8-D1A astrocytes seeded on opposite sides of a transwell membrane. Pulsed ultrasound (US) is applied to lipid microbubbles (MBs), and the change in barrier permeability is measured via transendothelial electrical resistance and dextran flux. Live cell calcium imaging (Fluo-4 AM) is performed during UTMC treatment. This model exhibits important features of the BBB, including endothelial tight junctions, and is more restrictive than the endothelial cell (EC) monolayer alone. When US is applied to MBs in contact with the ECs, BBB permeability increases in this model by two mechanisms: UTMC induces pore formation in the EC membrane (sonoporation), leading to increased transcellular permeability, and UTMC causes formation of reversible inter-endothelial gaps, which increases paracellular permeability. Additionally, this study determines that calcium influx into ECs mediates the increase in BBB permeability after UTMC in this model. Both transcellular and paracellular permeability can be used to increase drug delivery to the brain. Future studies can use this model to determine how UTMC-induced calcium-mediated signaling increases BBB permeability.

Ultrasound-Induced Microbubble Cavitation for Targeted Delivery of MiR-29b Mimic to Treat Cardiac Fibrosis.

Cardiac fibrosis contributes to adverse ventricular remodeling and is associated with loss of miR-29b. Overexpression of miR-29b via plasmid or intravenous injection of microRNA mimic has blunted fibrosis, but these are inefficient and non-targeted delivery strategies. In this study, we tested the hypothesis that delivery of microRNA-29b (miR-29b) using ultrasound-targeted microbubble cavitation (UTMC) of miR-29b-loaded microbubbles would attenuate cardiac fibrosis and preserve left ventricular (LV) function. Lipid microbubbles were loaded with miR-29b mimic (miR-29b-MB) or negative control (NC) mimic (NC-MB), placed with cardiac fibroblasts (CFs) and treated with pulsed ultrasound. Cells were harvested to measure downstream fibrotic mediators. Mice received angiotensin II (ANG II) infusion causing afterload increase and direct ANG II-induced cardiac fibrosis. UTMC of miRNA-loaded microbubbles was administered to the heart at days 0, 3 and 7. Serial echocardiography was performed, and hearts were harvested on day 10. UTMC treatment of CFs with miR-29b-MB increased miR-29b and decreased fibrotic transcripts compared with NC-MB treatment. In vivo UTMC+NC-MB led to increased LV mass, reduction in cardiac function and increase in fibrotic markers, demonstrating ANGI II-induced adverse cardiac remodeling. Mice treated with UTMC+miR-29b-MB had preservation of cardiac function, downregulation of cardiac fibrillin and trends of lower COL1A1, COL1A2 and COL3 mRNA and decreased cardiac α-smooth muscle protein. UTMC-mediated delivery of miR-29b mimic blunts expression of fibrosis markers and preserves LV function in ANG II-induced cardiac fibrosis.

Controlled Tempering of Lipid Concentration and Microbubble Shrinkage as a Possible Mechanism for Fine-Tuning Microbubble Size and Shell Properties.

The acoustic response of microbubbles (MBs) depends on their resonance frequency, which is dependent on the MB size and shell properties. Monodisperse MBs with tunable shell properties are thus desirable for optimizing and controlling the MB behavior in acoustics applications. By utilizing a novel microfluidic method that uses lipid concentration to control MB shrinkage, we generated monodisperse MBs of four different initial diameters at three lipid concentrations (5.6, 10.0, and 16.0 mg/mL) in the aqueous phase. Following shrinkage, we measured the MB resonance frequency and determined its shell stiffness and viscosity. The study demonstrates that we can generate monodisperse MBs of specific sizes and tunable shell properties by controlling the MB initial diameter and aqueous phase lipid concentration. Our results indicate that the resonance frequency increases by 180-210% with increasing lipid concentration (from 5.6 to 16.0 mg/mL), while the bubble diameter is kept constant. Additionally, we find that the resonance frequency decreases by 260-300% with an increasing MB final diameter (from 5 to 12 μm), while the lipid concentration is held constant. For example, our results depict that the resonance frequency increases by ∼195% with increasing lipid concentration from 5.6 to 16.0 mg/mL, for ∼11 μm final diameter MBs. Additionally, we find that the resonance frequency decreases by ∼275% with increasing MB final diameter from 5 to 12 μm when we use a lipid concentration of 5.6 mg/mL. We also determine that MB shell viscosity and stiffness increase with increasing lipid concentration and MB final diameter, and the level of change depends on the degree of shrinkage experienced by the MB. Specifically, we find that by increasing the concentration of lipids from 5.6 to 16.0 mg/mL, the shell stiffness and viscosity of ∼11 μm final diameter MBs increase by ∼400 and ∼200%, respectively. This study demonstrates the feasibility of fine-tuning the MB acoustic response to ultrasound by tailoring the MB initial diameter and lipid concentration.

BR55 Ultrasound Molecular Imaging of Clear Cell Renal Cell Carcinoma Reflects Tumor Vascular Expression of VEGFR-2 in a Patient-Derived Xenograft Model.

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.

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

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. 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. 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. 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.

Preparation of Lipid Microbubble/Paclitaxel Nanoparticle Complex and Its In Vitro Antigastric Tumor Effect Mediating the STAT3-NF-κB Pathway

In this research, lipid microbubbles (MBs) were prepared first, and then Tat peptide, carboxylated heparin, amino biotin, and amino folic acid were successively synthesized. After a certain amount of paclitaxel was added, paclitaxel nanoparticles (NPs) with a double-ligand were obtained through reaction. The lipid MBs prepared above were added to streptavidin. After washing and purification, the lipid MBs and double-ligand paclitaxel NPs were added. After incubation on ice, the lipid MB-double-ligand paclitaxel NP complex was obtained. In addition to the physical characterization of the materials, human breast cancer cells MDA-MB-231 and lung cancer cells A549 were first utilized to test the biological properties of the NP complex In Vitro and then utilized to study the effects of gastric cancer (GC) cells. The results revealed that the lipid MBs were uniformly distributed and did not aggregate. The concentration of the NP complex reached 7.75±0.93×108 NPs/mL, and the particle size was 2.23±0.68 μm. At various radiation intensities, blue fluorescently stained MDA-MB-231 cells and A549 cells showed greener fluorescently labeled double-ligand paclitaxel NPs around and inside the nucleus of Hoechst 33342. According to the prepared products and byproducts, they were grouped to compare different prepared products. The fluorescence uptake of the two cells at 4 h was the highest under the condition of the NP complex combined with ultrasonic radiation, and the destruction of cancer cells (MDA-MB-231 and A549) was the strongest under the condition of the NP complex combined with ultrasonic radiation. In GC cells, NP complexes inhibited cell migration and invasion relative to the other groups (P &lt;0.05), the level of Bax protein increased (P &lt;0.05), while that of Bcl-2, pSTAT3/STAT3, and phosphorylation of NF-kappa B (PNF-κB)/NF-κB protein were markedly decreased (P &lt;0.05).

In Vivo Validation of Modulated Acoustic Radiation Force-Based Imaging in Murine Model of Abdominal Aortic Aneurysm Using VEGFR-2-Targeted Microbubbles.

The objective of this study is to validate the modulated acoustic radiation force (mARF)-based imaging method in the detection of abdominal aortic aneurysm (AAA) in murine models using vascular endothelial growth factor receptor 2 (VEGFR-2)-targeted microbubbles (MBs). The mouse AAA model was prepared using the subcutaneous angiotensin II (Ang II) infusion combined with the β-aminopropionitrile monofumarate solution dissolved in drinking water. The ultrasound imaging session was performed at 7 days, 14 days, 21 days, and 28 days after the osmotic pump implantation. For each imaging session, 10 C57BL/6 mice were implanted with Ang II-filled osmotic pumps, and 5 C57BL/6 mice received saline infusion only as the control group. Biotinylated lipid MBs conjugated to either anti-mouse VEGFR-2 antibody (targeted MBs) or isotype control antibody (control MBs) were prepared before each imaging session and were injected into mice via tail vein catheter. Two separate transducers were colocalized to image the AAA and apply ARF to translate MBs simultaneously. After each imaging session, tissue was harvested and the aortas were used for VEGFR-2 immunostaining analysis. From the collected ultrasound image data, the signal magnitude response of the adherent targeted MBs was analyzed, and a parameter, residual-to-saturation ratio ( Rres - sat ), was defined to measure the enhancement in the adherent targeted MBs signal after the cessation of ARF compared with the initial signal intensity. Statistical analysis was performed with the Welch t test and analysis of variance test. The Rres - sat of abdominal aortic segments from Ang II-challenged mice was significantly higher compared with that in the saline-infused control group ( P < 0.001) at all 4 time points after osmotic pump implantation (1 week to 4 weeks). In control mice, the Rres - sat values were 2.13%, 1.85%, 3.26%, and 4.85% at 1, 2, 3, and 4 weeks postimplantation, respectively. In stark contrast, the Rres - sat values for the mice with Ang II-induced AAA lesions were 9.20%, 20.6%, 22.7%, and 31.8%, respectively. It is worth noting that there was a significant difference between the Rres - sat for Ang II-infused mice at all 4 time points ( P < 0.005), a finding not present in the saline-infused mice. Immunostaining results revealed the VEGFR-2 expression was increased in the abdominal aortic segments of Ang II-infused mice compared with the control group. The mARF-based imaging technique was validated in vivo using a murine model of AAA and VEGFR-2-targeted MBs. Results in this study indicated that the mARF-based imaging technique has the ability to detect and assess AAA growth at early stages based on the signal intensity of adherent targeted MBs, which is correlated with the expression level of the desired molecular biomarker. The results may suggest, in very long term, a pathway toward eventual clinical implementation for an ultrasound molecular imaging-based approach to AAA risk assessment in asymptomatic patients.

Enhanced bone regeneration by low-intensity pulsed ultrasound and lipid microbubbles on PLGA/TCP 3D-printed scaffolds

BackgroundTo investigate the effect of low-intensity pulsed ultrasound (LIPUS) combined with lipid microbubbles on the proliferation and bone regeneration of bone marrow mesenchymal stem cells (BMSCs) in poly (lactic-glycolic acid copolymer) (PLGA)/α-tricalcium phosphate (TCP) 3D-printed scaffolds.MethodsBMSCs were irradiated with different LIPUS parameters and microbubble concentrations, and the best acoustic excitation parameters were selected. The expression of type I collagen and the activity of alkaline phosphatase were detected. Alizarin red staining was used to evaluate the calcium salt production during osteogenic differentiation.ResultsBMSCs proliferation was the most significant under the condition of 0.5% (v/v) lipid microbubble concentration, 2.0 MHz frequency, 0.3 W/cm2 sound intensity and 20% duty cycle. After 14 days, the type I collagen expression and alkaline phosphatase activity in the scaffold increased significantly compared to those in the control group, and alizarin red staining showed more calcium salt production during osteogenic differentiation. After 21 days, scanning electron microscopy experiments showed that osteogenesis was obvious in the PLGA/TCP scaffolds.ConclusionLIPUS combined with lipid microbubbles on PLGA/TCP scaffolds can promote BMSCs growth and bone differentiation, which is expected to provide a new and effective method for the treatment of bone regeneration in tissue engineering.

Dynamic Behavior of Polymer Microbubbles During Long Ultrasound Tone-Burst Excitation and Its Application for Sonoreperfusion Therapy.

Ultrasound (US)-targeted microbubble (MB) cavitation (UTMC)-mediated therapies have been found to restore perfusion and enhance drug/gene delivery. Because of the potentially longer circulation time and relative ease of storage and reconstitution of polymer-shelled MBs compared with lipid MBs, we investigated the dynamic behavior of polymer microbubbles and their therapeutic potential for sonoreperfusion (SRP) therapy. The fate of polymer MBs during a single long tone-burst exposure (1 MHz, 5 ms) at various acoustic pressures and MB concentrations was recorded via high-speed microscopy and passive cavitation detection (PCD). SRP efficacy of the polymer MBs was investigated in an in vitro flow system and compared with that of lipid MBs. Microscopy videos indicated that polymer MBs formed gas-filled clusters that continued to oscillate, fragment and form new gas-filled clusters during the single US burst. PCD confirmed continued acoustic activity throughout the 5-ms US excitation. SRP efficacy with polymer MBs increased with pulse duration and acoustic pressure similarly to that with lipid MBs but no significant differences were found between polymer and lipid MBs. These data suggest that persistent cavitation activity from polymer MBs during long tone-burst US excitation confers excellent reperfusion efficacy.

Contrast-enhanced ultrasound combined targeted microbubbles for diagnosis of highly aggressive papillary thyroid carcinoma.

Accurate diagnosis of highly aggressive papillary thyroid cancer (PTC) may greatly help avoid overdiagnosis and overtreatment of PTC. However, there is still a lack of a convenient and accurate method. Targeted microbubbles, an emerging ultrasound contrast agent, have the potential to accurately diagnose highly aggressive PTC. To design and prepare a targeted microbubble for specific contrast-enhanced ultrasound (CEUS) imaging of highly invasive PTC. Using β-galactoside-binding protein galectin-3 (Gal-3) overexpressed on the surface of highly invasive PTC cells as a target, C12 polypeptide (ANTPCGPYTHDCPVKR) with high affinity and specificity for Gal-3 was coupled to the surface of lipid microbubbles to prepare targeted microbubbles (Gal-3-C12@lipo MBs). The targeted microbubbles were prepared by thin-film hydration method and mechanical shaking method. The morphology, diameter, concentration and stability of microbubbles were investigated by fluorescence microscopy and an AccuSizer. The biosafety of microbubbles was studied using BCPAP cells through CCK8 assay. Confocal laser scanning microscope and flow cytometry were applied to research the cellular uptake of microbubbles to investigate the targeting ability to highly aggressive PTC. Finally, the specific contrast-enhanced ultrasound imaging of microbubbles in highly invasive PTC was validated on the mice bearing subcutaneous BCPAP tumor model via a clinically ultrasound imaging system. Gal-3-C12@lipo MBs were successfully prepared which showed a well-defined spherical morphology with an average diameter of 1.598 ± 0.848 μm. Gal-3-C12@lipo MBs showed good stability without rupture within 4 hours after preparation. At the cellular level, Gal-3-C12@lipo MBs exhibited favorable biosafety and superior targeting ability to BCPAP cells, with 2.8-fold higher cellular uptake than non-targeted lipid microbubbles (Lipo MBs). At the animal level, Gal-3-C12@lipo MBs significantly improved the quality of contrast-enhanced ultrasound imaging in highly invasive PTC, with an echo intensity of tumor significantly higher than that of Lipo MBs. We designed and fabricated a novel targeted microbubble for the specific ultrasound imaging diagnosis of highly aggressive PTC. The targeted microbubbles have good stability, superior biosafety and high targeting specificity, which can significantly improve the tumor signal-to-noise ratio of highly invasive PTC, and have the potential to facilitate and accurately diagnose highly invasive PTC.

Understanding the mechanisms of ultrasound-targeted microbubble cavitation-mediated blood brain barrier opening

Ultrasound-targeted microbubble cavitation (UTMC) transiently opens the blood brain barrier (BBB). We previously determined that UTMC induces BBB hyperpermeability through an influx of calcium. As activation of RhoA is a calcium-dependent pathway that causes cytoskeletal reorganization, leading to the breakdown of tight junctions, we tested the hypothesis that UTMC-induced activation of RhoA leads to BBB hyperpermeability. We utilized a transwell model with brain endothelial cells and astrocytes on opposite sides of a support membrane. Ultrasound (1 MHz, 250 kPa, 10 μs pulse duration, 10 ms pulse interval) was applied in the presence of lipid microbubbles for 20 s. BBB permeability was assessed using dextran flux and transendothelial electrical resistance (TEER) measured across the membrane. Integrity of tight junctions was evaluated by staining for ZO-1. UTMC reduced TEER (p &amp;lt; 0.05), confirming reduced barrier integrity. One hour after UTMC, there was a significant decrease in ZO-1 mean pixel intensity (p &amp;lt; 0.05). Treatment of cells with Rho inhibitor II (Y16) significantly reduced UTMC-induced dextran flux across the BBB (p &amp;lt; 0.05) and UTMC-induced decrease in TEER. In our co-culture model of the BBB, UTMC induces hyperpermeability through modulation of tight junctions, at least in part through a RhoA-dependent mechanism.

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 × 10-9mol/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.

Abstract 15305: The Dynamic Behavior of Polymer Microbubbles During Long Ultrasound Tone-Burst Excitation and Its Application for Sonoreperfusion Therapy

Objective: Ultrasound (US)-targeted microbubble (MB) cavitation (UTMC) mediated therapies have been shown to restore perfusion and enhance drug/gene delivery. Due to the potentially longer circulation time and relative ease of storage and reconstitution of polymer shelled MBs compared with lipid MBs, we investigated the dynamic behavior of polymer MBs and its therapeutic potential for sonoreperfusion (SRP) therapy. Methods: We explored the fate of polymer MBs during a single long tone-burst exposure (1 MHz, 5 ms) at various acoustic pressures (0.25-1.5 MPa) and MB concentrations (2х10 6 -2х10 8 MB/mL) via direct high-speed microscopy observation and passive cavitation detection (PCD) and compared with lipid MBs. SRP efficacy of these MBs was investigated in an in vitro constant flow system at various pulse durations (1 MHz, 1-5 ms every 3 sec for 20 min) and acoustic pressures (0.6-1.5 MPa). The pressure differential across a porous mesh partially occluded with microthrombi was recorded as a function of time during treatment and used as a measure of thrombus burden. The terminal pressure drop and the initial rate of pressure drop were derived and compared. Results: Both polymer and lipid MBs first underwent stable or inertial cavitation depending on the acoustic pressure, and then formed gas-filled clusters that continued to oscillate, fragment, and form new gas-filled clusters during the single US burst. PCD confirmed continued acoustic activity throughout the 5-ms US excitation. SRP efficacy with polymer MBs increased with the pulse duration and acoustic pressure ( p &lt;0.05, ANOVA), similarly to that of lipid MBs, but no significant differences were found between polymer and lipid MBs ( Figure 1 ). Conclusion: Under the acoustic conditions tested, both polymer and lipid MBs demonstrate similar thrombolytic effects in vitro suggesting that persistent cavitation activity from polymer MBs during long tone-burst US confers excellent reperfusion efficacy.

Abstract 15915: Ultrasound Targeted Microbubble Cavitation-Induced Endothelial Hyperpermeability is Calcium-Dependent and Regulated by Enos Signaling

Introduction: Ultrasound ( US )-targeted microbubble ( MB ) cavitation ( UTMC ) facilitates delivery of cell-impermeant drugs across the endothelial barrier; mechanisms are poorly understood. We reported that UTMC causes inter-endothelial gaps in cultured endothelial cell ( EC ) monolayers, which could increase barrier permeability, and that UTMC increases Ca 2+ influx into ECs. Given that increased intracellular Ca 2+ activates eNOS, and nitric oxide (NO) release promotes microvascular permeability, we hypothesized that UTMC increases intracellular Ca 2+ through activation of mechanosensitive Piezo-1 Ca 2+ channels, leading to NO synthesis and endothelial barrier hyperpermeability. Methods: Transwells were endothelialized with human coronary artery ECs. Lipid MBs were added, and US was delivered for 10 s (1 MHz, 250 kPa, 10 cycles, 10 ms interval). Permeability was measured using dextran transfer and transendothelial electrical resistance ( TEER ). GsMTx4 and L-NAME were used to inhibit Piezo1 and eNOS, respectively; Fluo-4 AM to visualize Ca 2+ influx; DAF-FM to detect NO. S-nitrosylation was measured with biotin switch assay. Results: UTMC reduced TEER (1.7 fold, p&lt;0.05 ) and increased dextran flux across the transwell (2-fold, p&lt;0.01 for 70 kDa dextran, 1.6 fold, p&lt;0.05 for 10 kDa dextran), which was abrogated by L-NAME. UTMC significantly increased Ca 2+ influx into ECs and tripled NO ( p&lt;0.05 ), but these increases and UTMC-mediated hyperpermeability were blunted by Piezo1 channel inhibition ( p&lt;0.0001) . UTMC increased S-nitrosylation of proteins (1.7 fold, p&lt;0.01 ) and was associated with s-nitrosylation of VE-cadherin, its redistribution into focal adhesion junctions, and increased stress fiber formation, which were abolished by L-NAME. Conclusions: UTMC causes endothelial barrier hyperpermeability through a Ca 2+ -dependent mechanism, where Ca 2+ influx into ECs, mediated in part by mechanosensitive Piezo1 channels, modulates eNOS signaling. Our data suggest that eNOS regulates UTMC-induced hyperpermeability, possibly through s-nitrosylation of VE-cadherin. Further studies to examine mechanisms regulating UTMC bioeffects will aid in clinical translation and optimization of UTMC for delivery of cell-impermeant drugs.

Phospholipid-mimicking block, graft, and block-graft copolymers for phase-transition microbubbles as ultrasound contrast agents.

Background: Lipid and polymer microbubbles (MBs) are widely used as ultrasound contrast agents in clinical diagnosis, and possess great potential in ultrasound-mediated therapy due to their drug loading function. However, overcoming the limitations of stability and echo enhancement of MBs are still a considerable challenge. Methods: A series novel block, graft and block-graft copolymers was proposed and prepared in this work, and these copolymers were used as shells to encapsulate perfluoropentane as ultrasound contrast agents. First, block, graft and block-graft copolymers with different topological structures were prepared. Then, these copolymers were prepared into block copolymer phase-transition MBs, graft copolymer phase-transition MBs, and block-graft copolymer phase-transition MBs, respectively. Finally, the dexamethasone was used for drug-loaded phase-transition microbubbles model to explore the potential of theranostic microbubbles. Results: Finally, these three resulting copolymer MBs with average size of 4–5 μm exhibited well enhancement of ultrasound imaging under the influence of different frequencies and mechanical index, and they exhibited a longer contrast-enhanced ultrasound imaging time and higher resistance to mechanical index compared with SonoVue in vitro and in vivo. In vitro drug release results also showed that these copolymer MBs could encapsulate dexamethasone drugs, and the drug release could be enhanced by ultrasonic triggering. These copolymer MBs were therapeutic MBs for targeted triggering drug release. Conclusion: Therefore, the feasibility of block, graft, and block-graft copolymers as ultrasonic contrast agents was verified, and their ultrasonic enhancement performance in vitro and in vivo was compared. The ultrasound contrast agents developed in this work have excellent development potential in comprehensive diagnosis and treatment.