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.

Delivery of Anticancer Drugs Using Microbubble-Assisted Ultrasound in a 3D Spheroid Model.

Tumor spheroids are promising three-dimensional (3D) in vitro tumor models for the evaluation of drug delivery methods. The design of noninvasive and targeted drug methods is required to improve the intratumoral bioavailability of chemotherapeutic drugs and reduce their adverse off-target effects. Among such methods, microbubble-assisted ultrasound (MB-assisted US) is an innovative modality for noninvasive targeted drug delivery. The aim of the present study is to evaluate the efficacy of this US modality for the delivery of bleomycin, doxorubicin, and irinotecan in colorectal cancer (CRC) spheroids. MB-assisted US permeabilized the CRC spheroids to propidium iodide, which was used as a drug model without affecting their growth and viability. Histological analysis and electron microscopy revealed that MB-assisted US affected only the peripheral layer of the CRC spheroids. The acoustically mediated bleomycin delivery induced a significant decrease in CRC spheroid growth in comparison to spheroids treated with bleomycin alone. However, this US modality did not improve the therapeutic efficacy of doxorubicin and irinotecan on CRC spheroids. In conclusion, this study demonstrates that tumor spheroids are a relevant approach to evaluate the efficacy of MB-assisted US for the delivery of chemotherapeutics.

Facilitating New Chromium Reducing Microbes to Enhance Hexavalent Chromium Reduction by In Situ Sonoporation-Mediated Gene Transfer in Soils.

Chromium (Cr) is a heavy metal with a high toxicity and pathogenicity. Microbial reduction is an effective strategy to remove Cr(VI) at contaminated sites but suffers from the low populations and activities of Cr-reducing microorganisms in soils. This study proposed an in situ sonoporation-mediated gene transfer approach, which improved soil Cr(VI) reduction performance by delivering exogenous Cr-transporter chrA genes and Cr-reducing yieF genes into soil microorganisms with the aid of ultrasound. Besides the increasing populations of Cr-resistant bacteria and elevated copy numbers of chrA and yieF genes after sonoporation-mediated gene transfer, three new Cr-reducing strains were isolated, among which Comamonas aquatica was confirmed to obtain Cr-resistant capability. In addition, sonoporation-mediated gene transfer was the main driving force significantly shaping soil microbial communities owing to the predominance of Cr-resistant microbes. This study pioneered and evidenced that in situ soil sonoporation-mediated gene transfer could effectively deliver functional genes into soil indigenous microbes to facilitate microbial functions for enhanced bioremediation, e.g., Cr-reduction in this study, showing its feasibility as a chemically green and sustainable remediation strategy for heavy metal contaminated sites.

Pentacyclic Triterpenoid Phytochemicals with Anticancer Activity: Updated Studies on Mechanisms and Targeted Delivery.

Pentacyclic triterpenoids (TTs) represent a unique family of phytochemicals with interesting properties and pharmacological effects, with some representatives, such as betulinic acid (BA) and betulin (B), being mainly investigated as potential anticancer molecules. Considering the recent scientific and preclinical investigations, a review of their anticancer mechanisms, structure-related activity, and efficiency improved by their insertion in nanolipid vehicles for targeted delivery is presented. A systematic literature study about their effects on tumor cells in vitro and in vivo, as free molecules or encapsulated in liposomes or nanolipids, is discussed. A special approach is given to liposome-TTs and nanolipid-TTs complexes to be linked to microbubbles, known as contrast agents in ultrasonography. The production of such supramolecular conjugates to deliver the drugs to target cells via sonoporation represents a new scientific and applicative direction to improve TT efficiency, considering that they have limited availability as lipophilic molecules. Relevant and recent examples of in vitro and in vivo studies, as well as the challenges for the next steps towards the application of these complex delivery systems to tumor cells, are discussed, as are the challenges for the next steps towards the application of targeted delivery to tumor cells, opening new directions for innovative nanotechnological solutions.

Landscape of Cellular Bioeffects Triggered by Ultrasound-Induced Sonoporation.

Sonoporation is the process of transient pore formation in the cell membrane triggered by ultrasound (US). Numerous studies have provided us with firm evidence that sonoporation may assist cancer treatment through effective drug and gene delivery. However, there is a massive gap in the body of literature on the issue of understanding the complexity of biophysical and biochemical sonoporation-induced cellular effects. This study provides a detailed explanation of the US-triggered bioeffects, in particular, cell compartments and the internal environment of the cell, as well as the further consequences on cell reproduction and growth. Moreover, a detailed biophysical insight into US-provoked pore formation is presented. This study is expected to review the knowledge of cellular effects initiated by US-induced sonoporation and summarize the attempts at clinical implementation.

Sonoporation enables high-throughput loading of trehalose into red blood cells.

Despite recent advances in biostabilization, clinical blood supplies still experience shortages and storage limitations for red blood cells (RBCs) have not yet been sufficiently addressed. Storing RBCs in a frozen or dried state is an appealing solution to address storage limitations, but many promising cryoprotectants, including the non-reducing sugar trehalose, are impermeant to mammalian cell membranes and cannot be utilized effectively using currently available compound-loading methods. We found that transient pore formation induced by ultrasound and microbubbles (sonoporation) offers an effective means of loading trehalose into RBCs to facilitate long-term storage in a frozen or desiccated state. The protective potential of trehalose loading was demonstrated by freezing processed RBCs at -1 °C/min to -80 °C, then either storing the cells at -80 °C or lyophilizing them. RBCs were either thawed or rehydrated after 42 days of storage and evaluated for membrane integrity and esterase activity to estimate recovery and cell viability. The intracellular concentration of trehalose reached 40 mM after sonoporation and over 95% of treated RBCs were recovered after loading. Loading of trehalose was sufficient to maintain RBC morphology and esterase activity in most cells during freezing (>90% RBC recovery) and to a lower degree after lyophilization and rehydration (>20% recovery). Combining sonoporation with an integrated fluidics device allowed for rapid loading of up to 70 mM trehalose into RBCs. These results demonstrate the potential of sonoporation-mediated trehalose loading to increase recovery of viable RBCs, which could lead to effective methods for long-term stabilization of RBCs.

Modifications of Plasma Membrane Organization in Cancer Cells for Targeted Therapy.

Modifications of the composition or organization of the cancer cell membrane seem to be a promising targeted therapy. This approach can significantly enhance drug uptake or intensify the response of cancer cells to chemotherapeutics. There are several methods enabling lipid bilayer modifications, e.g., pharmacological, physical, and mechanical. It is crucial to keep in mind the significance of drug resistance phenomenon, ion channel and specific receptor impact, and lipid bilayer organization in planning the cell membrane-targeted treatment. In this review, strategies based on cell membrane modulation or reorganization are presented as an alternative tool for future therapeutic protocols.

Development of an ultrasound triggered nanomedicine-microbubble complex for chemo-photodynamic-gene therapy.

Recently, combination therapy has received much attention because of its highly therapeutic effect in various types of cancers. In particular, chemo-photodynamic combination therapy has been considered as an outstanding strategy. However, an abnormal increase in tumor angiogenesis caused by reactive oxygen species (ROS) generated during photodynamic therapy (PDT) has been reported. In this study, the complex of doxorubicin (DOX)-encapsulating anti-angiogenic small interfering RNA (siRNA) nanoparticle and chlorin e6 (Ce6)-encapsulating microbubble has been developed to suppress tumor angiogenesis. The first compartment, doxorubicin-encapsulating siRNA nanoparticle, was electrostatically coated using two biocompatible polymers to prevent the damage of genetic materials. The other part, Ce6-encapsulating microbubble, serves as an ultrasound-triggered local delivery system as well as a drug carrier. Both the in vitro and in vivo experimental results demonstrate successful inhibition of angiogenesis with a minimized damage of siRNAs caused by ROS as well as improved therapeutic effect by chemo-photodynamic-gene triple combination therapy using ultrasound-triggered local delivery.

Enhanced Radiosensitization for Cancer Treatment with Gold Nanoparticles through Sonoporation.

We demonstrate the megavoltage (MV) radiosensitization of a human liver cancer line by combining gold-nanoparticle-encapsulated microbubbles (AuMBs) with ultrasound. Microbubbles-mediated sonoporation was administered for 5 min, at 2 h prior to applying radiotherapy. The intracellular concentration of gold nanoparticles (AuNPs) increased with the inertial cavitation of AuMBs in a dose-dependent manner. A higher inertial cavitation dose was also associated with more DNA damage, higher levels of apoptosis markers, and inferior cell surviving fractions after MV X-ray irradiation. The dose-modifying ratio in a clonogenic assay was 1.56 ± 0.45 for a 10% surviving fraction. In a xenograft mouse model, combining vascular endothelial growth factor receptor 2 (VEGFR2)-targeted AuMBs with sonoporation significantly delayed tumor regrowth. A strategy involving the spatially and temporally controlled release of AuNPs followed by clinically utilized MV irradiation shows promising results that make it worthy of further translational investigations.

Enhancing Nab-Paclitaxel Delivery Using Microbubble-Assisted Ultrasound in a Pancreatic Cancer Model.

A combination of microbubbles (MBs) and ultrasound (US) is an emerging method for noninvasive and targeted enhancement of anti-cancer drug uptake. This method showed an increase local drug extravasation in tumor tissue while reducing the systemic adverse effects in various tumor models. The present study aims to evaluate the effectiveness of this approach for Nab-paclitaxel delivery in a pancreatic tumor model. US and MBs of different types in combination with Nab-paclitaxel showed a loss in cell viability of pancreatic cancer cells in comparison with Nab-paclitaxel treatment alone in in vitro scenario. The in vivo data revealed that US and MBs in combination with Nab-paclitaxel induced a significant decrease in the tumor volume in a subcutaneous pancreatic adenocarcinoma mouse model in comparison to tumors treated with Nab-paclitaxel alone. The postmortem anatomopathological analyses of tumor tissues partially confirmed these results. In conclusion, this study demonstrates that MB-assisted US is a relevant technology to increase the therapeutic effectiveness of Nab-paclitaxel in a pancreatic cancer model.

Biophysical insight into mechanisms of sonoporation.

This study presents a unique approach to understanding the biophysical mechanisms of ultrasound-triggered cell membrane disruption (i.e., sonoporation). We report direct correlations between ultrasound-stimulated encapsulated microbubble oscillation physics and the resulting cellular membrane permeability by simultaneous microscopy of these two processes over their intrinsic physical timescales (microseconds for microbubble dynamics and seconds to minutes for local macromolecule uptake and cell membrane reorganization). We show that there exists a microbubble oscillation-induced shear-stress threshold, on the order of kilopascals, beyond which endothelial cellular membrane permeability increases. The shear-stress threshold exhibits an inverse square-root relation to the number of oscillation cycles and an approximately linear dependence on ultrasound frequency from 0.5 to 2 MHz. Further, via real-time 3D confocal microscopy measurements, our data provide evidence that a sonoporation event directly results in the immediate generation of membrane pores through both apical and basal cell membrane layers that reseal along their lateral area (resealing time of ∼<2 min). Finally, we demonstrate the potential for sonoporation to indirectly initiate prolonged, intercellular gaps between adjacent, confluent cells (∼>30-60 min). This real-time microscopic approach has provided insight into both the physical, cavitation-based mechanisms of sonoporation and the biophysical, cell-membrane-based mechanisms by which microbubble acoustic behaviors cause acute and sustained enhancement of cellular and vascular permeability.

A Thermoelectric Device for Coupling Fluid Temperature Regulation During Continuous Skin Sonoporation or Sonophoresis.

During skin sonoporation and sonophoresis, time-consuming duty cycles or fluid replacement is often required to mitigate coupling fluid temperature increases. This study demonstrates an alternative method for temperature regulation: a circulating, thermoelectric system. Porcine skin samples were sonoporated continuously for 10 min at one of three intensities (23.8, 34.2, 39.4 W/m2). A caffeine solution was then applied to the skin and left to diffuse for 20 h. During sonoporation, the system was able to maintain the temperature between 10 and 16°C regardless of the intensity. No increase in transdermal transport was achieved with an intensity of 23.8 W/m2. Intensities of 34.2 and 39.4 W/m2 resulted in 3.5-fold (p < 0.05) and 3.7-fold (p < 0.05) increases in mean transport, relative to a control case with no ultrasound. From these results, it is concluded that a significant transport increase can be achieved with a system that circulates and cools the coupling fluid during ultrasound application. Relative to the previous methods of temperature control (duty cycles and fluid replacement), use of this circulation system will lead to significant time savings in future experimental studies.

Numerical and Experimental Evaluation of High-Intensity Focused Ultrasound-Induced Lesions in Liver Tissue Ex Vivo.

OBJECTIVES: Recent advances in the field of acoustics and piezoelectric and ultrasound transducers have led to new approaches to the diagnosis and treatment of certain diseases. One method of treatment with ultrasonic waves is high-intensity focused ultrasound (HIFU) treatment, which is a thermal therapeutic method used to treat malignant tumors. Although a variety of treatment-planning strategies using ultrasonic waves have been investigated, little clinical success has been achieved. Computational modeling is a powerful tool for predicting device performance. METHODS: The heating induced by a concave transducer with operating powers of 85 and 135 W was studied, and the experimental results presented in this article verify its applicability. Numerical simulations of the nonlinear acoustic field were performed by using the Westervelt and Khokhlov-Zabolotskaya-Kuznetsov equations. Heat transfer was measured for the 2 operational powers, and the results were compared with ex vivo experimental results. In addition, thermal dose contours for both the simulation and experimental results were calculated to investigate the ablated area. RESULTS: Good agreement was found between the experimental and numerical results. The results show that the average temperature deviations calculated at the focal point were 12.8% and 4.3% for transducer powers of 85 and 135 W, respectively. CONCLUSIONS: This study provides guidance to HIFU practitioners in determining lesion size and identifying nonlinear effects that should be considered in HIFU procedures.

Systemic Evaluation of Vascular Dysfunction by High-Resolution Sonography in an Nω -Nitro-l-Arginine Methyl Ester Hydrochloride-Induced Mouse Model of Preeclampsia-Like Symptoms.

OBJECTIVES: The purpose of this study was to evaluate vascular function, including arterial resistance and endothelial function, by high-resolution sonography in an Nω -nitro-l-arginine methyl ester hydrochloride (l-NAME)-induced mouse model of preeclampsia-like symptoms. METHODS: Pregnant mice were subcutaneously injected with a saline solution (control; n = 10) or l-NAME (n = 10) between the 7th and 18th days of gestation. The resistive index and pulsatility index (RI and PI, indicators of arterial resistance) of the uteroplacental, umbilical, femoral, and common carotid arteries and the flow-mediated dilatation (index of endothelial function) of the femoral artery were measured by high-frequency sonography in both groups. RESULTS: We noted significant increases in the RI and PI of the uteroplacental and umbilical arteries and a decrease in the flow-mediated dilatation of the femoral artery in the l-NAME group compared with the control group. We also found that the RI and PI of the uteroplacental and umbilical arteries were negatively correlated with fetal weight and crown-rump length. The results of the multivariate analysis using a logistic regression model indicated that the flow-mediated dilatation at 120 seconds was an independent diagnostic criterion for the l-NAME-induced preeclampsia-like model. A receiver operating characteristic analysis showed that flow-mediated dilatation at 120 seconds had the greatest area under the curve of 0.934, with an optimal cutoff point of 11.1%, yielding sensitivity of 100% and specificity of 84.6%. CONCLUSIONS: The PI and RI of the fetomaternal vasculature can identify fetuses in "high-risk" pregnancies, and flow-mediated dilatation is a reliable indicator for predicting preeclampsia. Assessment of vascular function by high-resolution sonography provides a useful platform for preeclampsia-related basic research with high reproducibility.

[The effect of secondary Bjerknes force in sonoporation on cell by ultrasound-mediated microbubbles].

In sonoporation, the cell membrane is broken-up temporarily by ultrasound mediated microbubbles, which is promoting drug or gene into the cell. In current literatures, there are numerous studies of single microbubble dynamics in sonoporation. However till now, little studies have been focused on the sonoporation incidence caused by more than one microbubble. In this article, the dynamic model of two adjacent microbubbles in stable cavitation has been introduced. By the model, the forces including secondary Bjerknes force on cell membrane given by microbubbles and their effects on sonoporation have been numerically studied. According to the experimental parameters, we numerically studied (1) effects of the ultrasound and microbubble parameters on the secondary Bjerknes forces; (2) the forces exerted on cell membrane by microbubble, including the secondary Bjerknes force; (3) the sonoporation possibility caused by those forces produced by microbubble. In this article, the ultrasound and microbubbles' parameters range were found to produce sonoporation by two adjacent microbubbles. Furthermore, it is the first time to found that the microbubbles' parameters are more important than ultrasound parameters on sonoporation.

Clinical study of ultrasound and microbubbles for enhancing chemotherapeutic sensitivity of malignant tumors in digestive system.

OBJECTIVE: To explore the safety of ultrasound and microbubbles for enhancing the chemotherapeutic sensitivity of malignant tumors in the digestive system in a clinical trial, as well as its efficacy. METHODS: From October 2014 to June 2016, twelve patients volunteered to participate in this study. Eleven patients had hepatic metastases from tumors of the digestive system, and one patient had pancreatic carcinoma. According to the mechanical index (MI) in the ultrasound field, patients were classified into four groups with MIs of 0.4, 0.6, 0.8 and 1.0. Within half an hour after chemotherapy, patients underwent ultrasound scanning with ultrasound microbubbles (SonoVue) to enhance the efficacy of chemotherapy. All adverse reactions were recorded and were classified in 4 grades according to the Common Terminology Criteria for Adverse Events version 4.03 (CTCAE V4.03). Tumor responses were evaluated by the Response Evaluation Criteria in Solid Tumors version 1.1 criteria. All the patients were followed up until progression. RESULTS: All the adverse reactions recorded were level 1 or level 2. No local pain occurred in any of the patients. Among all the adverse reactions, fever might be related to the treatment with ultrasound combined with microbubbles. Six patients had stable disease (SD), and one patient had a partial response (PR) after the first cycle of treatment. At the end of follow-up, tumor progression was restricted to the original sites, and no new lesions had appeared. CONCLUSIONS: Our preliminary data showed the potential role of a combined treatment with ultrasound and microbubbles in enhancing the chemotherapeutic sensitivity of malignant tumors of the digestive system. This technique is safe when the MI is no greater than 1.0.

Ultrasound and microbubble induced release from intracellular compartments.

BACKGROUND: Ultrasound and microbubbles (USMB) have been shown to enhance the intracellular uptake of molecules, generally thought to occur as a result of sonoporation. The underlying mechanism associated with USMB-enhanced intracellular uptake such as membrane disruption and endocytosis may also be associated with USMB-induced release of cellular materials to the extracellular milieu. This study investigates USMB effects on the molecular release from cells through membrane-disruption and exocytosis. RESULTS: USMB induced the release of 19% and 67% of GFP from the cytoplasm in viable and non-viable cells, respectively. Tfn release from early/recycling endosomes increased by 23% in viable cells upon USMB treatment. In addition, the MFI of LAMP-1 antibody increased by 50% in viable cells, suggesting USMB-stimulated lysosome exocytosis. In non-viable cells, labeling of LAMP-1 intracellular structures in the absence of cell permeabilization by detergents suggests that USMB-induced cell death correlates with lysosomal permeabilization. CONCLUSIONS: In conclusion, USMB enhanced the molecular release from the cytoplasm, lysosomes, and early/recycling endosomes.

Experimental and simulation studies on focused ultrasound triggered drug delivery.

To improve drug delivery efficiency in cancer therapy, many researchers have recently concentrated on drug delivery systems that use anticancer drug loaded micro- or nanoparticles. In addition, induction methods, such as ultrasound, magnetic field, and infrared light, have been considered as active induction methods for drug delivery. Among these, focused ultrasound has been regarded as a promising candidate for the active induction method of drug delivery system because it can penetrate a deep site in soft tissue, and its energy can be focused on the targeted lesion. In this research, we employed focused ultrasound as an active induction method. For an anticancer drug loaded microparticles, we fabricated poly-lactic-co-glycolic acid docetaxel (PLGA-DTX) nanoparticle encapsulated alginate microbeads using the single-emulsion technique and the aeration method. To select the appropriate operating parameter for the focused ultrasound, we measured the pressure and temperature induced by the focused ultrasound at the focal area using a needle-type hydrophone and a digital thermal detector, respectively. Additionally, we conducted a simulation of focused ultrasound using COMSOL Multiphysics 4.3a. The experimental measurement results were compared with the simulation results. In addition, the drug release rates of the PLGA-DTX-encapsulated alginate microbeads induced by the focused ultrasound were tested. Through these experiments, we determined that the appropriate focused ultrasound parameter was peak pressure of 1 MPa, 10 cycle/burst, and burst period of 20 µSec. Finally, we performed the cell cytotoxicity and drug uptake test with focused ultrasound induction and found that the antitumor effect and drug uptake efficiency were significantly enhanced by the focused ultrasound induction. Thus, we confirmed that focused ultrasound can be an effective induction method for an anticancer drug delivery system.

Microbubbles-Assisted Ultrasound Triggers the Release of Extracellular Vesicles.

Microbubbles-assisted ultrasound (USMB) has shown promise in improving local drug delivery. The formation of transient membrane pores and endocytosis are reported to be enhanced by USMB, and they contribute to cellular drug uptake. Exocytosis also seems to be linked to endocytosis upon USMB treatment. Based on this rationale, we investigated whether USMB triggers exocytosis resulting in the release of extracellular vesicles (EVs). USMB was performed on a monolayer of head-and-neck cancer cells (FaDu) with clinically approved microbubbles and commonly used ultrasound parameters. At 2, 4, and 24 h, cells and EV-containing conditioned media from USMB and control conditions (untreated cells, cells treated with microbubbles and ultrasound only) were harvested. EVs were measured using flow cytometric immuno-magnetic bead capture assay, immunogold electron microscopy, and western blotting. After USMB, levels of CD9 exposing-EVs significantly increased at 2 and 4 h, whereas levels of CD63 exposing-EVs increased at 2 h. At 24 h, EV levels were comparable to control levels. EVs released after USMB displayed a heterogeneous size distribution profile (30-1200 nm). Typical EV markers CD9, CD63, and alix were enriched in EVs released from USMB-treated FaDu cells. In conclusion, USMB treatment triggers exocytosis leading to the release of EVs from FaDu cells.

Intracellular Delivery of Bleomycin by Combined Application of Electroporation and Sonoporation in Vitro.

In this study, we aimed to determine whether the combination of electroporation (EP) and ultrasound (US) waves (sonoporation) can result in an increased intracellular delivery of anticancer drug bleomycin. CHO cells were treated with electric pulses (1 or 8 high voltage pulses of 800 or 1200 V/cm, 100 µs or 1 low voltage pulse of 100 or 250 V/cm, 100 ms) and with 880 kHz US of 320 or 500 kPa peak negative pressure, 100 % duty cycle, applied for 2 s in the presence or absence of exogenously added contrast agent microbubbles. Various sequential or simultaneous combinations of EP and sonoporation were used. The results of the study showed that i) sequential treatment of cells by EP and sonoporation enhanced bleomycin electrosonotransfer at the reduced energy of electric field and US; ii) sequential combination of EP and sonoporation induced a summation effect which at some conditions was more prominent when the cells were treated first by EP and then by sonoporation; iii) the most efficient intracellular delivery of bleomycin was achieved by the simultaneous application of cell EP and sonoporation resulting in percentage of reversibly porated cells above the summation level; and iv) compared with sequential application of EP and sonoporation, simultaneous use of electric pulses and US increased cell viability in the absence of bleomycin.