Sonoporation Effect Research Articles

In Vivo Sonoporation Effect Under the Presence of a Large Amount of Micro-Nano Bubbles in Swine Liver.

Sonoporation as a method of intracellular drug and gene delivery has not yet progressed to being used in vivo. The aim of this study was to prove the feasibility of sonoporation at a level practical for use in vivo by using a large amount of carbon dioxide micro-nano bubbles. The carbon dioxide micro-nano bubbles and 100 mg of cisplatin were intra-arterially injected to the swine livers, and ultrasound irradiation was performed from the surface of the liver under laparotomy during the intra-arterial injection. After the intra-arterial injection, ultrasound-irradiated and nonirradiated liver tissues were immediately excised. Tissue platinum concentration was measured using inductively coupled plasma mass spectrometry. Liver tissue platinum concentrations were compared between the irradiated tissue and nonirradiated tissue using the Wilcoxon signed rank test. The mean (SD) liver tissue platinum concentration was 6.260*103 (2.070) ng/g in the irradiated liver tissue and 3.280*103 (0.430) ng/g in the nonirradiated liver tissue, showing significantly higher concentrations in the irradiated tissue ( P = 0.004). In conclusion, increasing the tissue concentration of administered cisplatin in the livers of living swine through the effect of sonoporation was possible in the presence of a large amount of micro-nano bubbles.

The Evolution and Recent Trends in Acoustic Targeting of Encapsulated Drugs to Solid Tumors: Strategies beyond Sonoporation.

Despite recent advancements in ultrasound-mediated drug delivery and the remarkable success observed in pre-clinical studies, no delivery platform utilizing ultrasound contrast agents has yet received FDA approval. The sonoporation effect was a game-changing discovery with a promising future in clinical settings. Various clinical trials are underway to assess sonoporation's efficacy in treating solid tumors; however, there are disagreements on its applicability to the broader population due to long-term safety issues. In this review, we first discuss how acoustic targeting of drugs gained importance in cancer pharmaceutics. Then, we discuss ultrasound-targeting strategies that have been less explored yet hold a promising future. We aim to shed light on recent innovations in ultrasound-based drug delivery including newer designs of ultrasound-sensitive particles specifically tailored for pharmaceutical usage.

Phosphomimetic endothelial nitric oxide synthase-loaded microbubbles-mediated cardiac shock wave transfection therapy for angiogenesis in the ischemic myocardium

Abstract Background Cardiac shock wave therapy is a potential and effective remedy to promote revascularization in the ischemic myocardium of patients with refractory coronary heart disease (CHD). Although gene therapy holds great promise as a treatment for coronary heart disease, current clinical application is hampered by the development of safe and efficient systems for local gene delivery to a specific tissue or organ. Purpose In this study, we developed an experimental pulsed- focused electrohydraulic shock wave therapy system and investigated its effectiveness on myocardial angiogenesis in the hypercholesterolemic rabbit accompanied by phosphomimetic endothelial nitric oxide synthase-loaded microbubbles administration, wherein a high resolution diagnostic ultrasound is adjuncted with the therapy system, with a goal of increased safety. Methods Briefly, New Zealand white rabbits underwent 2% cholesterol rich diet injury for sixteen weeks. Histopathology results showed the formation of foam cells-rich soft atherosclerotic plaques with severe stenosis in all of the rabbits' coronary arteries. Then treatment group underwent extracorporeal electrohydraulic pulsed-focused shock waves (25kV, 0.5Hz, 150 pulses) accompanied by phosphomimetic endothelial nitric oxide synthase-loaded PESDA (Perfluorocarbon Exposed Sonicated Dextrose Albumin) microbubbles (100 ml/kg, 2.5x105 bubbles/ml) administration. Results Results from ultrasonography and histopathology showed a significant increase in the mean value for myocardial neovessels density in the treatment group compared with the control group (p<0.05). Conclusion Enhanced angiogenesis and sonoporation effect of electrohydraulic shock wave therapy, induced by inertial collapsed microbubbles, accompanied by phosphomimetic endothelial nitric oxide synthase-mediated shock wave transfection therapy, can cause to enhance the nitric oxide synthesis and VEGF, interleukin8 (IL8), stromal cell- derived factor 1 (SDF1) and matrix metalloproteinase 9 (MMP9) secretion in the hypercholesterolemic rabbits' coronary arteries, resulting in the differentiation and recruitment of endothelial progenitor cells (EPCs), promotion of revascularization in the ischemic myocardium and improvement of the cardiac function. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Mehrad Research Lab

Early stage atherosclerosis regression using combined sonotherapy and mitoxantrone-mediated sonoporation therapy

Abstract Background In atherosclerosis, local inflammation and associated macrophage activity can lead to foam cell-rich plaque formation, which results to plaque rupture and thrombosis, making inflammation an important therapeutic target in cardiovascular disease. The early stage of atherosclerosis is characterized by the accumulation of low-density lipoprotein (LDL) droplets, leading to the creation of foam cells. Foam cells are generated by uncontrolled uptake of modified LDL, especially oxidized LDL (oxLDL). Purpose In this study, we developed an experimental focused ultrasound-based combined sonotherapy and sonoporation therapy system, and investigated its effectiveness on macrophage-derived foam cells reduction, wherein a high resolution diagnostic ultrasound is adjuncted with combination therapy system, with a goal of increased safety. Methods Briefly, New Zealand white rabbits underwent primary balloon dilatation injury at the right common carotid artery followed by a 1.5% cholesterol-rich diet injury for three weeks. Histopathology results showed the early stage atherosclerosis formation in all of the rabbits' arteries. Then common carotid arteries of the treatment group at the lesion region, treated using extracorporeal low-level pulsed-focused ultrasound (P=11 W, F=1.1 MHz, DF=50%) sonotherapy accompanied by mitoxantrone-loaded PESDA (Perfluorocarbon-Exposed Sonicated Dextrose Albumin) microbubbles (100 ml/kg, 2–5x105 bubbles/ml)-mediated sonoporation therapy. Foam cells density were evaluated in the treatment group compared with the other groups using B-mode ultrasonography and histopathology. Results Results from B-mode ultrasonography and histopathology showed a significant reduction in the mean value for immune cells and foam cells density within the early stage atherosclerotic lesion in the treatment group compared with the other groups (p<0.05). Conclusion Enhanced sonoporation effect of ultrasound, induced by collapsed PESDA microbubbles accompanied by anti-inflammatory effect of sonotherapy and antineoplastic and cytotoxic effect of mitoxantrone can cause to reduce the immune cells and foam cells density within the lesion. These finding provide the basis for developing low-level pulsed-focused ultrasound-based combined sonotherapy and sonoporation therapy for a successful clinical application in the treatment of the early stage atherosclerosis. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Mehrad Research Lab

Nanobubbles: Fundamentals and recent drug delivery applications

The emerging branch in pharmaceutical sciences known as pharmaceutical nanotechnology presents new tools, opportunities and scope, which are expected to have significant applications, in disease diagnostics and therapeutics. There is a growing interest in nanobubble (NB) technology because of its wide range of potential applications in theranostics and targeted drug delivery including anticancer drug delivery, antibiotic delivery, gene delivery etc. Nanobubbles could offer several features in anticancer drug delivery, improving cellular uptake of chemotherapy drugs into cancer cell lines. Nanobubbles opened a new field of ultrasound imaging and were used as a diagnostic method. Generally, the delivery of drugs to organs and tissues is affected by two main ultrasound effects including the cavitation and sonoporation effects. This review describes the history of nanobubble, nomenclature, stability of nanobubble, physicochemical properties, characterization of nanobubble, method of preparation and applications. This article aims at highlighting the most recent and promising research trends including oxygen nanobubble, silica nanobubble and pluronicnanobubble and enlist the various drug delivery approaches involving the application of nanobubbles.

Antitumor Proliferation and Related Mechanism of Ultrasound Irradiation Combined with Safflower Yellow.

Ultrasound irradiation is now the best method for evaluating benign and malignant tumor nodules. Chemotherapy has always played an important role in the treatment of malignant tumors. With the large-scale application of chemotherapy drugs, the problem of multidrug resistance of tumors has become more and more prominent, which has become one of the difficulties in tumor chemotherapy. This study mainly explores the antitumor proliferation and related mechanisms of ultrasound irradiation combined with safflower yellow. The breast cancer cell line 4T1 derived from BALB/c mice was selected. BALB/c is an albino laboratory mouse, which, like many commonly used sublines, originated from Mus musculus. BALB/c mice have been bred for more than 200 generations in research institutions around the world and are widely used in animal experiments in immunology and physiology. When the cell proliferation reached 80%–90% of the bottom area of the culture flask, it was resuspended, passaged, frozen, and resuscitated according to experimental needs. The 4T1 breast cancer cell line was cultured by conventional methods. 4T1 breast cancer cells in the logarithmic proliferation phase were collected. After 0.25% was digested with pancreatin, it was washed twice with PBS to adjust the concentration to 1 × 107/mL. A 0.1 mL tumor cell suspension was subcutaneously inoculated on the edge of the mouse chest, thereby establishing a breast cancer model of BALB/c mice. After 6-15 days, the tumor volume grew rapidly and became larger. When the length of the tumor is 2.5 × 2.5, the modeling is successful. Ultrasound-targeted microbubble destruction technology, as a novel drug delivery method with high efficiency and low toxicity, can form transient pores (sonoporation effect) on the cell surface, widen the intercellular space, and increase the membrane permeability, and thus effectively. The transport of drugs, genes, proteins, etc., is promoted to target organs and tissues. Tumor-forming mice were randomly divided into the following four groups: control group, safflower yellow group, ultrasound irradiation group, and ultrasound irradiation combined with safflower yellow group. From the second day of inoculation to the end of the experiment, the body weight of the mice successfully inoculated with 4T1 cells was measured every day; from the 5th day, tumors in each group were calculated body volume and tumor inhibition rate (TIR) of each group. The combined treatment group has a higher tumor inhibition rate than the ultrasound irradiation group, and the difference is statistically significant (P < 0.05). Ultrasound irradiation combined with safflower yellow pigment can effectively inhibit tumor proliferation, maintain, or even improve the efficacy of chemotherapy, thereby improving the patient's tolerance to chemotherapy.

Sonoporation based on repeated vaporization of gold nanodroplets.

BackgroundGold nanodroplets (AuNDs) have been proposed as agents for photothermal therapy and photoacoustic imaging. Previously, we demonstrated that the sonoporation can be more effectively achieved with synchronized optical and acoustic droplet vaporization. By applying a laser pulse at the rarefactional phase of the ultrasound (US) pulse, the vaporization threshold can be reached at a considerably lower laser average power. However, a large loading quantity of the AuNDs may increase the risk of air embolism. The destruction of phase‐shifted AuNDs at the inertial cavitation stage leads to a reduced drug delivery performance. And it also causes instability of echogenicity during therapeutic monitoring.PurposeIn this study, we propose to further improve the sonoporation effectiveness with repeated vaporization. In other words, the AuNDs repeatedly undergo vaporization and recondensation so that sonoporation effects are accumulated over time at lower energy requirements. Previously, repeated vaporization has been demonstrated as an imaging contrast agent. In this study, we aim to adopt this repeated vaporization scheme for sonoporation.MethodsPerfluoropentane NDs with a shell made of human serum albumin were used as the US contrast agents. Laser pulses at 808 nm and US pulses of 1 MHz were delivered for triggering vaporization and inertial cavitation of NDs. We detected the vaporization and cavitation effects under different activation firings, US peak negative pressures (PNPs), and laser fluences using 5‐ and 10‐MHz focused US receivers. Numbers of calcein‐AM and propidium iodide signals uptake by BNL hepatocarcinoma cancer cells were used to evaluate the sonoporation and cell death rate of the cells.ResultsWe demonstrate that sonoporation can be realized based on repeatable vaporization instead of the commonly adopted inertial cavitation effects. In addition, it is found that the laser fluence and the acoustic pressure can be reduced. As an example, we demonstrate that the acoustic and optical energy for achieving a similar level of sonoporation rate can be as low as 0.44 MPa for the US PNP and 4.01 mJ/cm2 for the laser fluence, which are lower than those with our previous approach (0.53 MPa and 4.95 mJ/cm2, respectively).ConclusionWe demonstrated the feasibility of vaporization‐based sonoporation at a lower optical and acoustic energy. It is an advantageous method that can enhance drug delivery efficiency, therapeutic safety and potentially deliver an upgraded gene therapy strategy for improved theragnosis.

Ultrasound and Microbubbles Enhance Uptake of Doxorubicin in Murine Kidneys.

The use of ultrasound and microbubble-enhanced drug delivery, commonly referred to as sonoporation, has reached numerous clinical trials and has shown favourable results. Nevertheless, the microbubbles and acoustic path also pass through healthy tissues. To date, the majority of studies have focused on the impact to diseased tissues and rarely evaluated the impact on healthy and collateral tissue. The aim of this study was to test the effect and feasibility of low-intensity sonoporation on healthy kidneys in a mouse model. In our work here, we used a clinical diagnostic ultrasound system (GE Vivid E9) with a C1-5 ultrasound transducer combined with a software modification for 20-µs-long pulses to induce the ultrasound-guided drug delivery of doxorubicin (DOX) in mice kidneys in combination with SonoVue® and Sonazoid™ microbubbles. The acoustic output settings were within the commonly used diagnostic ranges. Sonoporation with SonoVue® resulted in a significant decrease in weight vs. DOX alone (p = 0.0004) in the first nine days, whilst all other comparisons were not significant. Ultrasound alone resulted in a 381% increase in DOX uptake vs. DOX alone (p = 0.0004), whilst SonoVue® (p = 0.0001) and Sonazoid™ (p < 0.0001) further increased the uptake nine days after treatment (419% and 493%, respectively). No long-standing damage was observed in the kidneys via histology. In future sonoporation and drug uptake studies, we therefore suggest including an “ultrasound alone” group to verify the actual contribution of the individual components of the procedure on the drug uptake and to perform collateral damage studies to ensure there is no negative impact of low-intensity sonoporation on healthy tissues.

Intermediate stage atherosclerosis regression using combined methotrexate- mediated ultrasound sonoporation therapy and gold nanoparticles- mediated X- rays- based Auger electron therapy

Abstract Background Atherosclerosis is a slow, progressive disease that may begin as early as childhood. Although the exact cause is unknown, atherosclerosis may start with damage or injury to the inner layer of an artery. Atherosclerosis is the main cause of stroke and myocardial infarction. In atherosclerosis, local inflammation and associated macrophage activity can lead to foam cell-rich lesion formation, making inflammation an important therapeutic target in cerebrovascular and cardiovascular diseases. Aims In this study, we developed an experimental pulsed low level focused ultrasound system, and investigated its effectiveness on intermediate stage atherosclerosis regression accompanied by intravenous lipid-based encapsulated methotrexate nanoparticles administration and lipid based encapsulated gold nanoparticles-mediated X-rays-based Auger electron therapy in the golden Syrian hamster common carotid artery, wherein diagnostic B-mode ultrasound is combined with ultrasound therapy system, with a goal of increased safety. Methods In this study, common carotid arteries of hamsters submitted to intermediate stage atherosclerosis using intravascular balloon injury and high-cholesterol diet injury. Then common carotid arteries of the treatment group (n=10) at the injured region, underwent pulsed low level focused ultrasound (F=1.3 MHz, P=24 W, PD=100 ms)-mediated sonoporation therapy accompanied by intravenous lipid-based encapsulated methotrexate nanoparticles (10ml/kg) administration and lipid based encapsulated gold nanoparticles-mediated X-rays (25 Gy)-based Auger electron therapy. Wall mean thickness and percentage of luminal cross-sectional area of stenosis were measured by ultrasonography and histopathology. Results Results from ultrasonography and histopathology showed a significant reduction in the mean value for wall mean thickness, percentage of luminal cross-sectional area of stenosis, lipid-laden cells and lipid droplets density at the stenotic region in the treatment group compared with the other groups (P&amp;lt;0.05) Conclusions Enhanced apoptotic and anti-proliferative effect of methotrexate, induced by enhanced inertial cavitation-based sonoporation effect of focused ultrasound, due to collapsed capsules accompanied by enhanced apoptotic effect of Auger electron therapy, can cause to regress of the intermediate stage atherosclerosis and significantly dilate the luminal cross-sectional area of stenosis. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Mehrad Research Lab

Neointimal hyperplasia regression using combined paclitaxel- mediated confocal dual pulse shock wave sonoporation therapy and catheter- based 90Y- mediated brachytherapy

Abstract Background and aims Intimal hyperplasia refers to proliferation and migration of vascular smooth muscle cells primarily in the tunica intima, resulting in arterial wall thickening and decreased arterial lumen size. Neointimal hyperplasia is the major cause of restenosis after percutaneous carotid interventions such as stenting or angioplasty. The aim of this study was to investigate the effect of combined shock wave enhanced sonoporation therapy and catheter-based 90Y-mediated β-brachytherapy on neointimal hyperplasia regression in an animal model, wherein diagnostic B-mode ultrasound is combined with therapy system, with a goal of increased safety. Methods Endothelial balloon catheter denudation of the abdominal aorta of golden Syrian hamsters was performed. Histopathologic evaluation confirmed neointimal hyperplasia formation in all of the hamsters' arteries. The treatment group underwent intravenous lipid-based encapsulated paclitaxel nanoparticles (10mg/kg)-mediated extracorporeal confocal dual pulse low-level focused electrohydraulic shock wave (V=15 kV, F=2 Hz, Impulses = 50 and V=10 kV, F=0.2 Hz, Impulses = 150) enhanced sonoporation therapy accompanied by catheter-based 90Y-mediated β-brachytherapy (90Y, 15 Gy), guided by simultaneous B-mode ultrasound imaging. Results B-mode ultrasound guided combined shock wave enhanced sonoporation therapy and β-brachytherapy was feasible and appeared safe for the targeting of stenosis in the aorta artery. Furthermore, pathological results showed a significant reduction in the mean value for smooth muscle hyperplasia cells density, lumen wall thickness and percentage of luminal cross- sectional area of stenosis in the treatment group compared with the other groups (p&amp;lt;0.05). Conclusions Enhanced toxicity effect of paclitaxel, induced by enhanced sonoporation effect of shock wave therapy, due to inertial cavitation effect of collapsed capsules and dual pulse system application accompanied by apoptotic effect of brachytherapy, can cause to neointimal hyperplasia regression. Combined shock wave enhanced sonoporation therapy and β-brachytherapy is significantly associated with reduced aorta artery stenosis in hamsters. The mechanism may relate to reduced smooth muscle hyperplasia cells and inflammation in the tunica intima. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Mehrad Research Lab

Ultrasound-triggered imaging and drug delivery using microbubble-self-aggregate complexes

Co-delivery of microbubbles (MBs) with anticancer drugs is a promising theranostic approach that can enhance both the ultrasound contrast and local extravasation of drugs with the sonoporation effect. The simultaneous administration of MBs and hydrophobic drugs, however, is still challenging due to the limitations in drug loading or undesirable stabilization of MBs. In this research, MB-self-aggregate complexes (MB-SAs) were newly fabricated for the encapsulation of hydrophobic drugs, and their theranostic properties are investigated in vitro and in vivo. Glycol chitosan self-aggregates (GC-SAs) loaded with hydrophobic drugs or dyes were chemically conjugated on the surface MBs. Their conjugation ratio was determined to be 73.9%, and GC-SAs on MBs did not affect the stability of MBs. GC-SA attached MBs (GC@MBs) were successfully visualized with low-intensity insonation and showed enhanced cellular uptake via the sonoporation effect. In vivo biodistribution of GC@MBs was examined with tumor-bearing mice, confirming that their accumulation at the tumor site increased by 1.85 times after ultrasound irradiation. The anticancer drug-loaded GC@MBs also exhibited 10% higher cytotoxicity under ultrasound flash. In conclusion, it was expected that GC@MBs could be used both as an ultrasound contrast agent and a drug carrier even with conventional ultrasonic devices.

Effect of high- dose atorvastatin- loaded pesda microbubbles- mediated b-mode ultrasound- guided extracorporeal pulsed low- level focused confocal dual frequency insonation on intermediate stage atherosclerosis

Background and Aims: Intermediate stage atherosclerosis is highlighted by infiltration of oxidized low-density lipoprotein LDL and foam cell formation, resulting in the thickening of arterial walls. In this study, we aimed to investigate the effect of sonoporation- based atorvastatin therapy on intermediate stage atherosclerosis regression, wherein diagnostic ultrasound imaging system is combined with the therapy system, with a goal of increased safety.

In-vivo Sonoporation Effect Under the Presence of a Large Amount of Micro-Nano Bubbles in Swine Liver

Sonoporation as a method of intracellular drug and gene delivery has not yet progressed to being used in vivo. This is likely because the cavitation effect is too small to reach a level practical for in vivo use. The aim of this study was to prove the feasibility of sonoporation at a level practical for use in vivo by injecting a large amount of carbon dioxide micro-nanobubbles into swine livers and applying ultrasound irradiation. Nine swine placed under general anesthesia were used. The carbon dioxide micro-nanobubbles and drug were injected through a catheter into the hepatic artery. The carbon dioxide micro-nanobubbles and 100 mg of cisplatin were intraarterially injected over a period of 10 minutes, and ultrasound irradiation was performed from the surface of the liver under laparotomy during the intraarterial injection. The swine were euthanized immediately after the intraarterial injection, after which ultrasound-irradiated and non-irradiated liver tissues were immediately excised. Cross-sections of the excised liver tissue were examined with the naked eye for any abnormalities. Increased cisplatin concentration was evaluated as increased platinum concentration. Tissue platinum concentration was measured using inductively coupled plasma mass spectrometry. Liver tissue platinum concentrations were compared between the irradiated tissue and non-irradiated tissue using the Wilcoxon signed-rank test. The mean (± SD) liver tissue platinum concentration was 6.260*103 ± 2.070 ng/g in the irradiated liver tissue and 3.280*103 ± 0.430 ng/g in the non-irradiated liver tissue, showing significantly higher concentrations in the irradiated tissue than in the non-irradiated tissue (p = 0.004). No abnormal macroscopic findings were seen in the liver tissue harvested from any of the specimens. In conclusion, increasing the tissue concentration of administered cisplatin in the livers of living swine through the effect of sonoporation was possible by performing ultrasound irradiation of these livers in the presence of a large amount of micro-nanobubbles. A sonoporation effect practical for use in vivo can be achieved if a large amount of micro-nanobubbles is present.

Ultrasound-Stimulated Microbubbles Inhibit Aggressive Phenotypes and Promotes Radiosensitivity of esophageal squamous cell carcinoma

ABSTRACT Ultrasound (US) is reported to improve the delivery efficiency of drugs loading onto large nanoparticles due to the sonoporation effect. Microbubbles (MBs) can be used as contrast agents of US expanding and contracting under low-amplitude US pressure waves. Ultrasound-stimulated microbubbles (USMBs) therapy is a promising option for the treatment of various cancers as a radiosensitizer. However, its role in esophageal squamous cell carcinoma (ESCC) remains unknown. In our study, human ESCC cell lines (KYSE-410, KYSE-1140) were treated with radiation solely, US alone, or radiation in combination with US or USMBs. The migration and invasion abilities of ESCC cells were examined by wound healing and Transwell assays. ESCC cell apoptosis was assessed using flow cytometry analysis and TUNEL assays. The levels of proteins associated with cell apoptosis and angiogenesis were measured by western blot analysis. A tube formation assay was performed to detect the ESCC cell angiogenesis. We found that USMBs at high levels most effectively most efficiently enhanced the effect of radiation, and significant changes in the viability (48%-51%), proliferation (1%), migration (63%-71%), invasion (52%) and cell apoptosis (31%-50%) of ESCC cells were observed compared with the control group in vitro. The ESCC angiogenesis was inhibited by US or radiation treatment and further inhibited by a combination of radiation and US or USMBs. USMBs at high levels most effectively enhanced the inhibitory effect of radiotherapy on ESCC cell apoptosis. Overall, USMBs enhanced the radiosensitivity of esophageal squamous cell carcinoma cells. Graphical abstract USMBs treatment enhanced the anti-tumor effect of radiation on ESCC cell proliferation, migration, invasion, angiogenesis and apoptosis in vitro. 1USMBs enhance the radiation-induced inhibition on ESCC cell growth 2USMBs promote the radiation effect on ESCC cell apoptosis 3USMBs enhance radiation-caused suppression on ESCC cell migration and invasion 4USMBs enhance the suppression of radiation on ESCC angiogenesis

Sonoporation: Underlying Mechanisms and Applications in Cellular Regulation

Abstract Ultrasound combined with microbubble-mediated sonoporation has been applied to enhance drug or gene intracellular delivery. Sonoporation leads to the formation of openings in the cell membrane, triggered by ultrasound-mediated oscillations and destruction of microbubbles. Multiple mechanisms are involved in the occurrence of sonoporation, including ultrasonic parameters, microbubbles size, and the distance of microbubbles to cells. Recent advances are beginning to extend applications through the assistance of contrast agents, which allow ultrasound to connect directly to cellular functions such as gene expression, cellular apoptosis, differentiation, and even epigenetic reprogramming. In this review, we summarize the current state of the art concerning microbubble–cell interactions and sonoporation effects leading to cellular functions.

Tumor Targeted Multifunctional Magnetic Nanobubbles for MR/US Dual Imaging and Focused Ultrasound Triggered Drug Delivery.

The development of multifunctional nanoplatforms that are safe and have multiple therapeutic functions integrated with dual- or multi-imaging modality is one of the most urgent medical requirements for active cancer therapy. In our study, we prepared multifunctional magnetic nanobubbles (MF-MNBs) by co-encapsulating superparamagnetic iron oxide nanoparticles (SPIONs) and doxorubicin into polylactideco–glycolide–polyethylene glycol–folate (PLGA-PEG-FA) polymer-based nanobubbles for tumor-targeted ultrasound (US)/magnetic resonance (MR) imaging and focused ultrasound (FUS)-triggered drug delivery. Hydrophobic SPIONs were successfully embedded into MF-MNBs by a typical double emulsion process. The MF-MNBs were highly dispersed with well-defined spherical morphology and an average diameter of 208.4 ± 12.58 nm. The potential of MF-MNB as a dual-modal contrast agent for US and MR imaging was investigated via in vitro study, and the MF-MNB exhibits promising US/MR contrast ability. Moreover, tumor targeting ability was further enhanced by folate conjugation and assessed through in vitro cell test. Furthermore, FUS, as a non-invasive and remote-control technique, was adopted to trigger the release of doxorubicin from MF-MNB and generate the sonoporation effect to enhance drug release and cellular uptake of MF-MNBs. The 4T1 cell viability was significantly decreased by FA ligand-receptor-mediated targeting and FUS sonication. In addition, the developed MF-MNB also exhibits enhanced accumulation in tumor site by FA ligand-receptor-mediated tumor targeting, in which the accumulation of MF-MNB was further enhanced by FUS sonication. Hence, we believe that the MF-MNB could be a promising drug nanocarrier for US/MR-guided anticancer drug delivery to improve cancer treatment efficacy.

Ultrasound-enhanced precision tumor theranostics using cell membrane-coated and pH-responsive nanoclusters assembled from ultrasmall iron oxide nanoparticles

Design of novel theranostic nanoplatforms integrating precision dynamic imaging modalities, tumor targeting ligands and therapeutic components with enhanced tumor accumulation still remains to be challenging. Here we report a facile strategy to prepare cell membrane (CM)-coated nanoclusters assembled from ultrasmall iron oxide nanoparticles (USIO NPs) with pH-responsiveness as a nanocarrier to load anti-cancer drug doxorubicin (DOX). Citric-stabilized USIO NPs with a size of 3.2 nm were synthesized, modified to have surface amine groups, and crosslinked to obtain pH-responsive nanoclusters (NCs). Then, the formed USIO NCs were loaded with DOX, and coated with cancer CMs to render them with resistance to macrophage cellular uptake and homologous cancer cell targeting specificity. Within the acidic tumor microenvironment, the pH-responsive USIO NCs/DOX@CM with dominant T2 magnetic resonance (MR) imaging performance can be dissociated to form single USIO NPs with T1 MR imaging ability, and simultaneously rapidly release DOX, thereby enabling dynamic T2/T1-weighted MR imaging and chemotherapy of tumors. Further, through the ultrasound-induced sonoporation effect, the dynamic MR imaging and tumor chemotherapy can be further enhanced. The designed USIO NCs/DOX@CM may be developed as a versatile theranostic platform for ultrasound-enhanced targeted precision theranostics of different cancer types.

Ultrasound-enhanced fluorescence imaging and chemotherapy of multidrug-resistant tumors using multifunctional dendrimer/carbon dot nanohybrids

Development of innovative nanomedicine enabling enhanced theranostics of multidrug-resistant (MDR) tumors remains to be challenging. Herein, we report the development of a newly designed multifunctional yellow-fluorescent carbon dot (y-CD)/dendrimer nanohybrids as a platform for ultrasound (US)-enhanced fluorescence imaging and chemotherapy of MDR tumors. Generation 5 (G5) poly(amidoamine) dendrimers covalently modified with efflux inhibitor of d-α-tocopheryl polyethylene glycol 1000 succinate (G5-TPGS) were complexed with one-step hydrothermally synthesized y-CDs via electrostatic interaction. The formed G5-TPGS@y-CDs complexes were then physically loaded with anticancer drug doxorubicin (DOX) to generate (G5-TPGS@y-CDs)-DOX complexes. The developed nanohybrids display a high drug loading efficiency (40.7%), strong y-CD-induced fluorescence emission, and tumor microenvironment pH-preferred DOX release profile. Attributing to the DOX/TPGS dual drug design, the (G5-TPGS@y-CDs)-DOX complexes can overcome the multidrug resistance (MDR) of cancer cells and effectively inhibit the growth of cancer cells and tumors. Furthermore, the introduction of US-targeted microbubble destruction technology was proven to render the complexes with enhanced intracellular uptake and anticancer efficacy in vitro and improved chemotherapeutic efficacy and fluorescence imaging of tumors in vivo due to the produced sonoporation effect. The developed multifunctional dendrimer/CD nanohybrids may represent an advanced design of nanomedicine for US-enhanced theranostics of different types of MDR tumors.

Overcoming anticancer resistance by photodynamic therapy-related efflux pump deactivation and ultrasound-mediated improved drug delivery efficiency

One of the major obstacles to successful chemotherapy is multi-drug resistance (MDR). A multi-drug resistant cancerous cell abnormally overexpresses membrane transporters that pump anticancer drugs out of the cell, resulting in low anticancer drug delivery efficiency. To overcome the limitation, many attempts have been performed to inhibit the abilities of efflux receptors chemically or genetically or to increase the delivery efficiency of anticancer drugs. However, the results have not yet been satisfactory. In this study, we developed nanoparticle-microbubble complexes (DOX-NPs/Ce6-MBs) by conjugating doxorubicin loaded human serum albumin nanoparticles (DOX-NPs) onto the surface of Chlorin e6 encapsulated microbubbles (Ce6-MBs) in order to maximize anticancer efficiency by overcoming MDR. Under the ultrasound irradiation, DOX-NPs and Ce6 encapsulating self-assembled liposomes or micelles were effectively delivered into the cells due to the sonoporation effect caused by the microbubble cavitation. At the same time, reactive oxygen (ROS) generated from intracellularly delivered Ce6 by laser irradiation arrested the activity of ABCG2 efflux receptor overexpressed in doxorubicin-resistant breast cancer cells (MCF-7/ADR), resulting in increased the chemotherapy efficacy. In addition, the total number of side population cells that exhibit the properties of cancer stem-like cells were also reduced by the combination of photodynamic therapy and chemotherapy. In conclusion, DOX-NPs/Ce6-MBs will provide a platform for simultaneously overcoming MDR and increasing drug delivery and therefore, treatment efficiency, under ultrasound irradiation.

Phosphorus dendrimer-based copper(II) complexes enable ultrasound-enhanced tumor theranostics

Abstract Design of simple-component theranostic agents with enhanced tumor accumulation represents a novel strategy in the area of precision nanomedicine for effective tumor theranostics. Herein, we report an innovative theranostic strategy based on ultrasound-enhanced tumor accumulation of a theranostic agent of phosphorus dendrimer-copper(II) complexes (1G3-Cu). The employed 1G3-Cu complexes represent a drug-free nanotheranostic agent with a simple composition possessing an r1 relaxivity of 0.7024 mM−1 s−1 for T1-weighted magnetic resonance (MR) imaging and an IC50 of 1.24 μM to inhibit the growth of a pancreatic cancer cell line. We show that cancer cells can be inhibited by the 1G3-Cu complexes through the activation of the apoptotic process with the upregulation of Bax, P53 and PTEN, downregulation of Bcl-2, and decrease of intracellular ATP. With the assistance of ultrasound targeted microbubble destruction (UTMD) technology that can increase cell membrane permeability temporarily via sonoporation effect, the 1G3-Cu complexes enable enhanced tumor MR imaging and chemotherapy due to their improved tumor accumulation. With the proven minimal toxicity to normal tissues at imaging and therapeutic doses, the prepared 1G3-Cu complexes may be potentially employed for UTMD-enhanced theranostics of different types of cancer.