Ultrasound-mediated Microbubble Research Articles

Long-pulsed ultrasound-mediated microbubble thrombolysis in a rat model of microvascular obstruction.

In up to 30% patients who experience acute myocardial infarction, successful recanalization of the epicardial coronary artery cannot provide adequate microvascular reperfusion. In this study, we sought to determine whether long-pulsed ultrasound (US)-mediated microbubble (MB) cavitation was useful for the treatment of microvascular obstruction, and the therapeutic effects were compared within different long-pulse-length and short-pulsed US. Microvascular obstruction model was established by injecting micro-thrombi into common iliac artery of a rat's hind limb. About 1 MHz US with different long pulse lengths (ranging from 100 to 50,000 cycles) was delivered, compared to short pulse (5 cycles). The control group was given MB only without therapeutic US. Contrast perfusion images were performed at baseline, emboli, and 1, 5, 10 min post-embolization, and peak plateau video intensity (A) was obtained to evaluate the therapeutic effects. Long-tone-burst US showed better thrombolytic effects than short-pulsed US (1,000, 5,000 cycles >500 cycles, >5 cycles, and control) (P < 0.01). 1,000 cycles group showed the optimal thrombolytic effect, but microvascular hemorrhage was observed in 50,000 cycles group. In conclusion, long-tone-burst US-enhanced MB therapy mediated successful thrombolysis and may offer a powerful approach for the treatment for microvascular obstruction within a certain pulse length.

Potentiation of anti-angiogenic eNOS-siRNA transfection by ultrasound-mediated microbubble destruction in ex vivo rat aortic rings.

Nitric oxide (NO) regulates vascular homeostasis and plays a key role in revascularization and angiogenesis. The endothelial nitric oxide synthase (eNOS) enzyme catalyzes NO production in endothelial cells. Overexpression of the eNOS gene has been implicated in pathologies with dysfunctional angiogenic processes, such as cancer. Therefore, modulating eNOS gene expression using small interfering RNAs (siRNAs) represents a viable strategy for antitumor therapy. siRNAs are highly specific to the target gene, thus reducing off-target effects. Given the widespread distribution of endothelium and the crucial physiological role of eNOS, localized delivery of nucleic acid to the affected area is essential. Therefore, the development of an efficient eNOS-siRNA delivery carrier capable of controlled release is imperative for targeting specific vascular regions, particularly those associated with tumor vascular growth. Thus, this study aims to utilize ultrasound-mediated microbubble destruction (UMMD) technology with cationic microbubbles loaded with eNOS-siRNA to enhance transfection efficiency and improve siRNA delivery, thereby preventing sprouting angiogenesis. The efficiency of eNOS-siRNA transfection facilitated by UMMD was assessed using bEnd.3 cells. Synthesis of nitric oxide and eNOS protein expression were also evaluated. The silencing of eNOS gene in a model of angiogenesis was assayed using the rat aortic ring assay. The results showed that from 6 to 24 h, the transfection of fluorescent siRNA with UMMD was twice as high as that of lipofection. Moreover, transfection of eNOS-siRNA with UMMD enhanced the knockdown level (65.40 ± 4.50%) compared to lipofectamine (40 ± 1.70%). Silencing of eNOS gene with UMMD required less amount of eNOS-siRNA (42 ng) to decrease the level of eNOS protein expression (52.30 ± 0.08%) to the same extent as 79 ng of eNOS-siRNA using lipofectamine (56.30 ± 0.10%). NO production assisted by UMMD was reduced by 81% compared to 67% reduction transfecting with lipofectamine. This diminished NO production led to higher attenuation of aortic ring outgrowth. Three-fold reduction compared to lipofectamine transfection. In conclusion, we propose the combination of eNOS-siRNA and UMMD as an efficient, safe, non-viral nucleic acid transfection strategy for inhibition of tumor progression.

Caveolin Delivered by Ultrasound-Mediated Microbubble Destruction Prevents Endothelial Cell Proliferation.

The nitric oxide synthase (eNOS) is an important regulator of vascular homeostasis. eNOS is modulated by intracellular mechanisms that include protein-protein interaction with Caveolin-1 (Cav). Cav binds to and impairs eNOS activation reducing vascular permeability and angiogenesis. Blocking of eNOS by Cav has been proposed as therapeutic antiangiogenic approach. However, the efficient and controlled delivery of the peptide requires to be solved. The effect of antennapedia (AP)-Cav loaded into microbubbles (MBs) and delivered by ultrasound-mediated microbubble destruction (UMMD) into brain endothelial cells (bEnd.3 cells) was evaluated on NO production using DAF2-DA, cell migration assessed by the wound healing assay, cell proliferation with BrdU, and ex-vivo angiogenesis in rat aortic rings. An enhanced inhibitory effect of AP-Cav was observed on cells treated with UMMD. MBs and ultrasound disruption delivery of AP-Cav increased acetylcholine-induced NO release, wound healing, cell proliferation, and angiogenesis inhibition on bEnd.3 cells, compared to free AP-Cav administration. We demonstrated that the delivery of Cav via AP-Cav-loaded MBs and UMMD may be an administration method for Cav that would increase its therapeutic potential by enhancing efficacy and cellular specificity.

High‐Performance Delivery of a CRISPR Interference System via Lipid‐Polymer Hybrid Nanoparticles Combined with Ultrasound‐Mediated Microbubble Destruction for Tumor‐Specific Gene Repression (Adv. Healthcare Mater. 10/2023)

CRISPR Interference Systems The main objective of this study in article 2203082 by Yujin Zong, Mingxi Wan, and co-workers, is to develop novel pH-responsive lipid-polymer hybrid nanoparticles (PLPNs) to achieve optimal CRISPRi system delivery via ultrasound-mediated microbubble destruction (UMMD) technology, thereby inducing efficient target gene repression.

Ultrasound-Triggered Microbubbles: Novel Targeted Core-Shell for the Treatment of Myocardial Infarction Disease.

Myocardial infarction (MI) is known as a main cardiovascular disease that leads to extensive cell death by destroying vasculature in the affected cardiac muscle. The development of ultrasound-mediated microbubble destruction has inspired extensive interest in myocardial infarction therapeutics, targeted delivery of drugs, and biomedical imaging. In this work, we describe a novel therapeutic ultrasound system for the targeted delivery of biocompatible microstructures containing basic fibroblast growth factor (bFGF) to the MI region. The microspheres were fabricated using poly(lactic-co-glycolic acid)-heparin-polyethylene glycol- cyclic arginine-glycine-aspartate-platelet (PLGA-HP-PEG-cRGD-platelet). The micrometer-sized core-shell particles consisting of a perfluorohexane (PFH)-core and a PLGA-HP-PEG-cRGD-platelet-shell were prepared using microfluidics. These particles responded adequately to ultrasound irradiation by triggering the vaporization and phase transition of PFH from liquid to gas in order to achieve microbubbles. Ultrasound imaging, encapsulation efficiency cytotoxicity, and cellular uptake of bFGF-MSs were evaluated using human umbilical vein endothelial cells (HUVECs) in vitro. In vivo imaging demonstrated effective accumulation of platelet- microspheres injected into the ischemic myocardium region. The results revealed the potential use of bFGF-loaded microbubbles as a noninvasive and effective carrier for MI therapy.

High-Performance Delivery of a CRISPR Interference System via Lipid-Polymer Hybrid Nanoparticles Combined with Ultrasound-Mediated Microbubble Destruction for Tumor-Specific Gene Repression.

The dCas9-based CRISPR interference (CRISPRi) system efficiently silences genes without causing detectable off-target activity, thus showing great potential for the treatment of cancer at the transcriptional level. However, due to the large size of the commonly used CRISPRi system, effective delivery of the system has been a challenge that hinders its application in the clinic. Herein, a combination of pH-responsive lipid-polymer hybrid nanoparticles (PLPNs) and ultrasound-mediated microbubble destruction (UMMD) is used for the delivery of the CRISPRi system. The core-shell structure of PLPNs can effectively be loaded with the CRISPRi plasmid, and increases the time spent in the circulating in vivo, and "actively target" cancer cells. Moreover, the combination of PLPNs with UMMD achieves a higher cellular uptake of the CRISPRi plasmid in vitro and retention in vivo. Furthermore, when PLPNs loaded with a CRISPRi plasmid that targets microRNA-10b (miR-10b) are used in combination with UMMD, it results in the effective repression of miR-10b in breast cancer, simultaneous disturbance of multiple cell migration and invasion-related signaling pathways, and a significant inhibition of lung metastasis. Thus, the established system presents a versatile, highly efficient, and safe strategy for delivery of the CRISPRi system both in vitro and in vivo.

Dual-Modality Molecular Imaging of Tumor via Quantum Dots-Liposome-Microbubble Complexes.

Molecular imaging has demonstrated promise for evaluating the expression levels of biomarkers for the early prediction of tumor progression and metastasis. However, most of the commonly used molecular imaging modalities are relatively single and have difficulties imaging complex biological processes. Here, we fabricated αvβ3-integrin-targeted quantum-dots-loaded liposome-microbubble (iRGD-QDLM) complexes that combined ultrasound imaging with optical imaging. The resulting iRGD-QDLM has excellent binding capability to 4T1 breast cancer cells. Ultrasound molecular imaging of 4T1 tumors demonstrated that significantly enhanced ultrasound molecular signals could be observed in comparison with non-targeted QDLM. Importantly, our study also suggested that iRGD-QDL on the surface of microbubbles could be delivered into a tumor by ultrasound-mediated microbubble destruction and adhered to αvβ3 integrin on breast cancer cells, achieving transvascular fluorescent imaging. Our study provides a novel approach to dual-modality molecular imaging of αvβ3 integrin in the tumor tissue.

Targeting nanoparticle-conjugated microbubbles combined with ultrasound-mediated microbubble destruction for enhanced tumor therapy

The stress of the abnormal stromal matrix of solid tumors is a major limiting factor that prevents drug penetration. Controlled, accurate, and efficient delivery of theranostic agents into tumor cells is crucial. Combining ultrasound with nanocarrierbased drug delivery systems have become a promising approach for targeted drug delivery in preclinical cancer therapy. In this study, to ensure effective tumor barrier penetration, access to the tumor microenvironment, and local drug release, we designed targeted nanoparticle (NP)-conjugated microbubbles (MBs); ultrasound could then help deliver acoustic energy to release the NPs from the MBs. The ultrasound-targeted MB destruction (UTMD) system of negatively charged NPs was conjugated with positively charged MBs using an ionic gelation method. We demonstrated the transfer of targeted NPs and their entry into gastric cancer cells through ligand-specific recognition, followed by enhanced cell growth inhibition owing to drug delivery-induced apoptosis. Moreover, the UTMD system combining therapeutic and ultrasound image properties can effectively target gastric cancer, thus significantly enhancing antitumor activity, as evident by tumor localization in an orthotopic mouse model of gastric cancer. The combination of ultrasound and NP-based drug delivery systems has become a promising approach for targeted drug delivery in preclinical cancer therapy.

Ultrasound‐targeted microbubbles destruction assists dual delivery of beta‐amyloid antibody and neural stem cells to restore neural function in transgenic mice of Alzheimer's disease

To explore the feasibility, efficacy and safety of ultrasound targeted microbubbles destruction (UTMD) assisted dual delivery of beta-amyloid (Aβ) antibody loaded by microbubbles (MBAβ ) and neural stem cells (NSCs) on Alzheimer's disease (AD). 27 APP/PS1 double transgenic mice (Tg mice) and 33 wild-type mice were used. Wild-type mice were insonated by diagnostic ultrasound with microbubbles (MB) for 5 min to observe the blood brain barrier (BBB) opening. The survival situation of engrafted NSCs crossing the opened BBB mediated by UTMD in AD mice was evaluated by in vivo imaging system. We further explored the combination therapy of UTMD mediated Aβ antibody and NSCs dual delivery. Tg mice in each group were exposed to diagnostic ultrasound for 5 min once a week for four times, with MB, MBAβ , and/or NSCs administration. Cognition and memory functions were explored by Morris water maze test, Aβ plaques deposition was evaluated by immunohistochemical, and brain-derived neurotrophic factor (BDNF) and synaptophysin (SYN) expression were detected by western blot and immunofluorescence. BBB was opened mediated by diagnostic ultrasound with MB and the duration of opening was about 10 h. The transplanted NSCs survived in Tg mice for no more than 72 h. Compared with control group, the mice in combined delivery of NSCs and Aβ antibody by UTMD group improved memory function and spatial learning with shorter latency to find the platform, longer distance traveled and longer time spent in targeted quadrant, and more crossing times (P < 0.05). Besides, the combination delivery group promoted the clearance of Aβ plaques compared with control group both in hippocampus (P < 0.01) and cortex (P < 0.05). Moreover, the expression of BDNF in combination delivery group was significantly higher than that in control group and US mediated MB group (P < 0.05). No significant change of SYN was observed in each group. UTMD assisted dual delivery of Aβ antibody and NSCs to AD mice brain could help to clear Aβ plaques, increase the expression of BDNF and restore the impaired neural function. This finding may offer potential insight into treatment of AD. This article is protected by copyright. All rights reserved.

Applications of Ultrasound-Mediated Drug Delivery and Gene Therapy.

Gene therapy has continuously evolved throughout the years since its first proposal to develop more specific and effective transfection, capable of treating a myriad of health conditions. Viral vectors are some of the most common and most efficient vehicles for gene transfer. However, the safe and effective delivery of gene therapy remains a major obstacle. Ultrasound contrast agents in the form of microbubbles have provided a unique solution to fulfill the need to shield the vectors from the host immune system and the need for site specific targeted therapy. Since the discovery of the biophysical and biological effects of microbubble sonification, multiple developments have been made to enhance its applicability in targeted drug delivery. The concurrent development of viral vectors and recent research on dual vector strategies have shown promising results. This review will explore the mechanisms and recent advancements in the knowledge of ultrasound-mediated microbubbles in targeting gene and drug therapy.

A Comprehensive Review on Microbubble Concept, Development and Its Application in Therapeutic Drug Delivery and Clinical Management of Disease.

The general purpose of this review was to briefly describe the Gastro-Retentive Drug Delivery System (GRDDS) and to primarily focus on mechanisms of flotation for gastric retention. The principle mechanism of Floating Drug Delivery Systems (FDDS) is mainly based on hydro-dynamically controlled low-density system, which gets swollen when comes in contact with the gastric environment and remains buoyant for an extended period of time with improved bioavailability and therapeutic efficacy. The main perspective of this review was to focus on microbubble drug delivery due to its wide range of applications. Microbubble is a new, economically viable, and non-invasive technique that is alternative to conventional techniques. The size of microbubbles ranges from 1-100 micrometers, which contain oxygen or air and remain suspended for prolonged drug release. Due to its great potential, microbubble is used in the therapeutic delivery of drug molecules or genetic material at their specific targeted site in various diseases. The recent research also suggests that ultrasound-mediated microbubble is more frequently used in the biomedical field for imaging of site-specific molecule targeting for the detection of disease and site-specific drug delivery. Gastro-retentive floating drug delivery system was considered as the most favorable approach for site-specific drug delivery. But nowadays, microbubble is gaining more popularity for its potential applications in the biomedical field. In recent years, there is much progress in microbubble for site-specific delivery of a drug or genetic material. Recent research also suggests that the ultrasound- mediated microbubble has shown a positive impact on the diagnosis and clinical management of various diseases such as liver cirrhosis, brain tumor, ocular, and urinary tract infection.

Ultrasound-mediated microbubbles cavitation enhanced chemotherapy of advanced prostate cancer by increasing the permeability of blood-prostate barrier

Although chemotherapy is an important treatment for advanced prostate cancer, its efficacy is relatively limited. Ultrasound-induced cavitation plays an important role in drug delivery and gene transfection. However, whether cavitation can improve the efficacy of chemotherapy for prostate cancer remains unclear. In this study, we treated RM-1 mouse prostate carcinoma cells with a combination of ultrasound-mediated microbubble cavitation and paclitaxel. Our results showed that combination therapy led to a more pronounced inhibition of cell viability and increased cell apoptosis. The enhanced efficacy of chemotherapy was attributed to the increased cell permeability induced by cavitation. Importantly, compared with chemotherapy alone (nab-paclitaxel), chemotherapy combined with ultrasound-mediated microbubble cavitation significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice in an orthotopic mouse model of RM-1 prostate carcinoma, indicating the synergistic effects of combined therapy on tumor reduction. Furthermore, we analyzed tumor-infiltrating lymphocytes and found that during chemotherapy, the proportions of CTLA4+ cells and PD-1+/CTLA4+ cells in CD8+ T cells slightly increased after cavitation treatment.

Release of vascular agonists from liposome-microbubble conjugate by ultrasound-mediated microbubble destruction: effect on vascular function.

The endothelium is a single cell layer of the vessel wall and a key regulator of blood flow in vascular beds. Local and systemic pathologies have been associated with alterations in endothelial function. However, targeting the endothelium with vasoconstrictor or vasodilator drugs is often accompanied by systemic effects. Here, we evaluated a liposome-microbubble delivery system as a vascular hydrophilic agonist carrier. Phenylephrine (Phe) or acetylcholine (Ach)-loaded liposomes were conjugated to microbubbles. The drug release was triggered by ultrasound (US), and the vascular response was assessed in rat aortic rings using an isolated organ chamber. Aortic rings incubated with Phe-liposome-microbubble conjugate, exposed to US showed a marked contractile response (0.79±0.04g) compared to empty liposomes conjugated to microbubbles, aortic rings exposed only to US, and Phe-liposome-microbubble conjugate without US exposure that elicited a minimal or no response. Expressed as %, contractile responses were 85.24±4.31% and 12.62±3.23% for Phe-Chol-liposome-microbubble conjugate and empty Chol-liposome-microbubble conjugate exposed to US, respectively. Addition of 1×10-5M Ach to pre-contracted aortic rings decreased the contraction response from 1 to 0.21g. The addition of Ach-liposome conjugate and exposure to US decreased the contraction response to 0.32g. Additionally, the ED50 values for Phe and Ach released by US from liposome-microbubble conjugates were 3.6×10-8M±2.8×10-9M for Phe and 2.0×10-8M±1.8×10-9M. In conclusion, we evaluated a hybrid delivery system that consisted of loaded liposomes conjugated to microbubbles to deliver and release vascular agonists using UMMD.

Ultrasound-Mediated Microbubble Destruction Inhibits Skin Melanoma Growth by Affecting YAP1 Translation Using Ribosome Imprinting Sequencing.

Cutaneous melanoma (CMM) is a skin tumor with a high degree of malignancy. BRAF resistance imposes great difficulty to the treatment of CMM, and partially contributes to the poor prognosis of CMM. YAP is involved in the growth and drug resistance of a variety of tumors, and mechanical signals may affect the activation of YAP1. As a novel ultrasound treatment technology, ultrasound-mediated microbubble destruction (UMMD) has been reported to have a killing effect on isolated CMM cells. In this study, the tumor tissue samples were collected from 64 CMM patients. We found that YAP1 mRNA expression was irrelevant to the clinicopathological characteristics and prognostic survival of the CMM patients. The drug-resistant cell line was constructed and subcutaneously implanted into nude mice, which were further separately treated with UMMD, ultrasound (US), and microbubbles (MB). The result showed that UMMD significantly inhibited the growth of tumor tissues. Ribosome imprinting sequencing (Ribo-seq) is a genetic technology for studying protein translation at genetic level. Ribo-seq, RNA-seq, and RT-qPCR were applied to detect YAP1 expression in CMM mouse tumor tissues. Ribo-seq data revealed that UMMD greatly up-regulated the expression of YAP1, interestingly, the up-regulated YAP1 was found to be negatively correlated with the weight of tumor tissues, while no significant change in YAP1 expression was detected by RNA-seq or RT-qPCR assay. These results indicated that UMMD could inhibit the tumor growth of drug-resistant CMM by affecting the translation efficiency of YAP1, providing a strong basis for the clinical treatment of UMMD in CMM.

Localized Delivery of Caveolin-1 Peptide Assisted by Ultrasound-Mediated Microbubble Destruction Potentiates the Inhibition of Nitric Oxide-Dependent Vasodilation Response

In the endothelium, nitric oxide synthase (eNOS) is the enzyme that generates nitric oxide, a key molecule involved in a variety of biological functions and cancer-related events. Therefore, selective inhibition of eNOS represents an attractive therapeutic approach for NO-related diseases and anticancer therapy. Ultrasound-mediated microbubble destruction (UMMD) conjugated with cell-permeable peptides has been investigated as a drug delivery system for effective delivery of anticancer molecules. We investigated the feasibility of loading antennapedia-caveolin-1 peptide (AP-Cav), a specific eNOS inhibitor, onto microbubbles to be delivered by UMMD in rat aortic endothelium. AP-Cav-loaded microbubbles (AP-Cav-MBs) and US parameters were characterized. Aortas were treated with UMMD for 30 s with 1.3 × 108 MBs/mL AP-Cav (8 μM)-MBs at 100-Hz pulse repetition frequency, 0.5-MPa acoustic pressure, 0.5 mechanical index and 10% duty cycle. NO-dependent vascular responses were assessed using an isolated organ system, 21 h post-treatment. Maximal relaxation response was inhibited 61.8% ± 1.6% in aortas treated with UMMD-AP-Cav-MBs, while in aortas treated with previously disrupted AP-Cav-MBs and then US, the inhibition was 31.6% ± 1.6%. The vascular contractile response was not affected. The impact of UMMD was evaluated in aortas treated with free AP-Cav; 30 μM of free AP-Cav was necessary to reach an inhibition response similar to that obtained with UMMD-AP-Cav-MBs. In conclusion, UMMD enhances the delivery and potentiates the effect of AP-Cav in the endothelial layer of rat aorta segments.

Ultrasound-Mediated Microbubble Cavitation Transiently Reverses Acute Hindlimb Tissue Ischemia through Augmentation of Microcirculation Perfusion via the eNOS/NO Pathway

Ultrasound-mediated microbubble cavitation improves perfusion in chronic limb and myocardial ischemia. The purpose of this study was to determine the effects of ultrasound-mediated microbubble cavitation in acute limb ischemia and investigate the mechanism of action. The animal with acute hindlimb ischemia was established using male Sprague-Dawley rats. The rats were randomly divided into three groups: intermittent high-mechanical-index ultrasound pulses combined with microbubbles (ultrasound [US] + MB group), US alone (US group) and MB alone (MB group). Both hindlimbs were treated for 10 min. Contrast ultrasound perfusion imaging of both hindlimbs was performed immediately and 5, 10, 15, 20 and 25 min after treatment. The role of the nitric oxide (NO) pathway in increasing blood flow in acutely ischemic tissue was evaluated by inhibiting endothelial nitric oxide synthase (eNOS) with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME). In the US + MB group, microvascular blood volume and microvascular blood flow of the ischemic hindlimb were significantly increased after treatment (both p values <0.05), while the microvascular flux rate (β) increased, but not significantly (p > 0.05). The increases were observed immediately after treatment, and had dissipated by 25 min. Changes in the US and MB groups were minimal. Inhibitory studies indicated cavitation increased phospho-eNOS concentration in ischemic hindlimb muscle tissue, and the increase was significantly inhibited by L-NAME (p < 0.05). Ultrasound-mediated microbubble cavitation transiently increases local perfusion in acutely ischemic tissue, mainly by improving microcirculatory perfusion. The eNOS/NO signaling pathway appears to be an important mediator of the effect.

Abstract 14150: Long-pulsed Ultrasound Mediated Microbubble Thrombolysis: Is There an Upper Limit?

Introduction: Long-pulsed ultrasound (US) mediated microbubble (MB) has shown successful thrombolystic effects in recent years. Compared to traditional short-pulse US(&lt;5 cycles), long-pulsed US induced more sustained cavitation followed by more complete thrombolysis. It was found more micro-thrombi were dissolved when pulse lengths were increased. However, taking the US safety into consideration, the upper limit of long pulse in vivo remains unknown. Hypothesis: Long-tone-burst US is able to cause successful thrombolysis through microbubble cavitation, and there is an upper limit for the safe long-pulse length in vivo. Methods: Different long- pulse lengths (5,100,500,1000, 5000, 50,000 cycles) were used and their thrombolytic effects in a rat model were observed. About 200μm micro-thrombi were injected into common iliac artery on rats hindlimb to make an embolization model. 1MHz long-pulsed US were delivered at mechanical index 1.8 every 3 seconds (pulse repetition frequency 0.33). The control group was given MBs only without therapeutic US. Contrast echocardiography was performed at three stages: baseline, embolization and 10 minutes post embolization. Peak plateau video intensity ( A ) were obtained for assessment of thrombolytic effects. Finally muscular samples were taken for pathology for assessment of both beneficial and potential detrimental effects from long-tone-burst US. Results: 100 and 500 cycles long-pulsed US showed successful thrombolysis and peak plateau video intensity ( A ) was significantly elevated post US treatment ( P&lt;0.01 vs. baseline ). Pathology revealed 1000 and 5000 cycles groups were the best parameters to induce optimal sonothrombolysis ( P&lt;0.01 vs. other groups), possibly by more sustained cavitation. However, 50,000 cycles showed endothelial injury and tissue bleeding that may be detrimental and hazard. Conclusions: Long-tone-burst US with more than 100 cycles were proved to induce very good micro-thrombi resolution in a rat model of embolization. It seems there is an upper limit for the cycle length to generate beneficial biomechanical effects, and detrimental effect may occur if it was exceeded.

Ultrasound-microbubble cavitation facilitates adeno-associated virus mediated cochlear gene transfection across the round-window membrane.

The round window of the cochlea provides an ideal route for delivering medicines and gene therapy reagents that can cross the round window membrane (RWM) into the inner ear. Recombinant adeno‐associated viruses (rAAVs) have several advantages and are recommended as viral vectors for gene transfection. However, rAAVs cannot cross an intact RWM. Consequently, ultrasound‐mediated microbubble (USMB) cavitation is potentially useful, because it can sonoporate the cell membranes, and increase their permeability to large molecules. The use of USMB cavitation for drug delivery across the RWM has been tested in a few animal studies but has not been used in the context of AAV‐mediated gene transfection. The currently available large size of the ultrasound probe appears to be a limiting factor in the application of this method to the RWM. In this study, we used home‐made ultrasound probe with a decreased diameter to 1.5 mm, which enabled the easy positioning of the probe close to the RWM. In guinea pigs, we used this probe to determine that (1) USMB cavitation caused limited damage to the outer surface layer or the RWM, (2) an eGFP‐gene carrying rAAV could effectively pass the USMB‐treated RWM and reliably transfect cochlear cells, and (3) the hearing function of the cochlea remained unchanged. Our results suggest that USMB cavitation of the RWM is a good method for rAAV‐mediated cochlear gene transfection with clear potential for clinical translation. We additionally discuss several advantages of the small probe size.

Acoustic Radiation or Cavitation Forces From Therapeutic Ultrasound Generate Prostaglandins and Increase Mesenchymal Stromal Cell Homing to Murine Muscle.

Non-ablative ultrasound (US)-based techniques to improve targeted tropism of systemically infused cell therapies, particularly mesenchymal stromal cell (MSC), have gained attention in recent years. Mechanotransduction following targeted US sonications have been shown to modulate tissue microenvironments by upregulating cytokines, chemokines, and trophic factors in addition to vascular cell adhesion molecules (CAM) that are necessary to promote tropism of MSC. While numerous US treatment parameters have demonstrated increased MSC homing, it remains unclear how the different mechanical US forces [i.e., acoustic radiation forces (ARF) or cavitation forces] influence tissue microenvironments. This study sonicated murine muscle tissue with pulsed focused ultrasound (pFUS) at 0.5 or 1.15 MHz each over a range of US intensities. Following sonication, tissue was assayed for the prostaglandins (PG) PGH2 and PGE2 as indicators of microenvironmental changes that would support MSC tropism. PGH2 and PGE2 levels were correlated to physical pFUS parameters and acoustic emissions measured by hydrophone. While ARF (pFUS with absence of cavitation signatures) was sufficient to increase PGH2 and PGE2, non-linear curve fitting revealed a frequency-independent relationship between prostaglandin production and mechanical index (MI), which accounts for increased cavitation probabilities of lower frequencies. The prostaglandin data suggested molecular changes in muscle would be particularly sensitive to cavitation. Therefore, low-intensity pulsed ultrasound (LIPUS) at 1 MHz was administered with low ARF (MI = 0.2) in combination with intravenous (IV) infusions of microbubble (MB) contrast agents. This combination upregulated prostaglandins and CAM without ultrasound-mediated microbubble destruction and ultimately promoted tropism of IV-infused MSC. This study revealed that accentuating non-destructive MB cavitation by US using parameters similar to diagnostic US contrast imaging increased MSC homing. Such approaches are particularly attractive to overcome clinical translation barriers of many still-experimental US parameters used in previous stem cell tropism studies.

Functional Activity and Endothelial-Lining Integrity of Ex Vivo Arteries Exposed to Ultrasound-Mediated Microbubble Destruction

Ultrasound-mediated microbubble destruction (UMMD) is a promising strategy to improve local drug delivery in specific tissues. However, acoustic cavitation can lead to harmful bioeffects in endothelial cells. We investigated the side effects of UMMD treatment on vascular function (contraction and relaxation) and endothelium integrity of ex vivo Wistar rat arteries. We used an isolated organ system to evaluate vascular responses and confocal microscopy to quantify the integrity and viability of endothelial cells. The arteries were exposed for 1–3 min to ultrasound at a 100 Hz pulse-repetition frequency, 0.5 MPa acoustic pressure, 50% duty cycle and 1%–5% v/v microbubbles. The vascular contractile response was not affected. The acetylcholine-dependent maximal relaxation response was reduced from 78% (control) to 60% after 3 min of ultrasound exposure. In arteries treated simultaneously with 1 min of ultrasound exposure and 1%, 2%, 3% or 5% microbubble concentration, vascular relaxation was reduced by 19%, 58%, 80% or 93%, respectively, compared with the control arteries. Fluorescent labeling revealed that apoptotic death, detachment of endothelial cells and reduced nitric oxide synthase phosphorylation are involved in relaxation impairment. We demonstrated that UMMD can be a safe technology if the correct ultrasound and microbubble parameters are applied. Furthermore, we found that tissue-function evaluation combined with cellular analysis can be useful to study ultrasound–microbubble–tissue interactions in the optimization of targeted endothelial drug delivery.