Development of exosome membrane materials-fused microbubbles for enhanced stability and efficient drug delivery of ultrasound contrast agent

Lipid-coated microbubbles are widely used as an ultrasound contrast agent, as well as drug delivery carriers. However, the two main limitations in ultrasound diagnosis and drug delivery using microbubbles are the short half-life in the blood system, and the difficulty of surface modification of microbubbles for active targeting. The exosome, a type of extracellular vesicle, has a preferentially targeting ability for its original cell. In this study, exosome-fused microbubbles (Exo-MBs) were developed by embedding the exosome membrane proteins into microbubbles. As a result, the stability of Exo-MBs is improved over the conventional microbubbles. On the same principle that under the exposure of ultrasound, microbubbles are cavitated and self-assembled into nano-sized particles, and Exo-MBs are self-assembled into exosome membrane proteins-embedded nanoparticles (Exo-NPs). The Exo-NPs showed favorable targeting properties to their original cells. A photosensitizer, chlorin e6, was loaded into Exo-MBs to evaluate therapeutic efficacy as a drug carrier. Much higher therapeutic efficacy of photodynamic therapy was confirmed, followed by cancer immunotherapy from immunogenic cell death. We have therefore developed a novel ultrasound image-guided drug delivery platform that overcomes the shortcomings of the conventional ultrasound contrast agent and is capable of simultaneous photodynamic therapy and cancer immunotherapy.

The effect of secondary Bjerknes force in sonoporation on cell by ultrasound-mediated microbubbles / 生物医学工程学杂志

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.

Microbubbles used for contrast enhanced ultrasound and theragnosis: a review of principles to applications

Ultrasound was developed several decades ago as a useful imaging modality, and it became the second most popular diagnostic tool due to its non-invasiveness, real-time capabilities, and safety. Additionally, ultrasound has been used as a therapeutic tool with several therapeutic agents and in nanomedicine. Ultrasound imaging is often used to diagnose many types of cancers, including breast, stomach, and thyroid cancers. In addition, ultrasound-mediated therapy is used in cases of joint inflammation, rheumatoid arthritis, and osteoarthritis. Microbubbles, when used as ultrasound contrast agents, can act as echo-enhancers and therapeutic agents, and they can play an essential role in ultrasound imaging and ultrasound-mediated therapy. Recently, various types of ultrasound contrast agents made of lipid, polymer, and protein shells have been used. Air, nitrogen, and perfluorocarbon are usually included in the core of the microbubbles to enhance ultrasound imaging, and therapeutic drugs are conjugated and loaded onto the surface or into the core of the microbubbles, depending on the purpose and properties of the substance. Many research groups have utilized ultrasound contrast agents to enhance the imaging signal in blood vessels or tissues and to overcome the blood–brain barrier or blood-retina barrier. These agents are also used to help treat diseases in various regions or systems of the body, such as the cardiovascular system, or as a cancer treatment. In addition, with the introduction of targeted moiety and multiple functional groups, ultrasound contrast agents are expected to have a potential future in ultrasound imaging and therapy. In this paper, we briefly review the principles of ultrasound and introduce the underlying theory, applications, limitations, and future perspectives of ultrasound contrast agents.

Advances in ultrasound-targeted microbubble-mediated gene therapy for liver fibrosis

Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthesis and reduced degradation of extracellular matrix (ECM) and is a common pathological sequela of chronic liver disease. Excessive deposition of ECM in the liver causes liver dysfunction, ascites, and eventually upper gastrointestinal bleeding as well as a series of complications. However, fibrosis can be reversed before developing into cirrhosis and has thus been the subject of extensive researches particularly at the gene level. Currently, therapeutic genes are imported into the damaged liver to delay or prevent the development of liver fibrosis by regulating the expression of exogenous genes. One technique of gene delivery uses ultrasound targeting of microbubbles combined with therapeutic genes where the time and intensity of the ultrasound can control the release process. Ultrasound irradiation of microbubbles in the vicinity of cells changes the permeability of the cell membrane by its cavitation effect and enhances gene transfection. In this paper, recent progress in the field is reviewed with emphasis on the following aspects: the types of ultrasound microbubbles, the construction of an ultrasound-mediated gene delivery system, the mechanism of ultrasound microbubble-mediated gene transfer and the application of ultrasound microbubbles in the treatment of liver fibrosis.

Therapeutic Effects of Microbubbles Added to Combined High-Intensity Focused Ultrasound and Chemotherapy in a Pancreatic Cancer Xenograft Model

OBJECTIVE: To investigate whether high-intensity focused ultrasound (HIFU) combined with microbubbles enhances the therapeutic effects of chemotherapy. MATERIALS AND METHODS: A pancreatic cancer xenograft model was established using BALB/c nude mice and luciferase-expressing human pancreatic cancer cells. Mice were randomly assigned to five groups according to treatment: control (n = 10), gemcitabine alone (GEM; n = 12), HIFU with microbubbles (HIFU + MB, n = 11), combined HIFU and gemcitabine (HIGEM; n = 12), and HIGEM + MB (n = 13). After three weekly treatments, apoptosis rates were evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay in two mice per group. Tumor volume and bioluminescence were monitored using high-resolution 3D ultrasound imaging and in vivo bioluminescence imaging for eight weeks in the remaining mice. RESULTS: The HIGEM + MB group showed significantly higher apoptosis rates than the other groups (p < 0.05) and exhibited the slowest tumor growth. From week 5, the tumor-volume-ratio relative to the baseline tumor volume was significantly lower in the HIGEM + MB group than in the control, GEM, and HIFU + MB groups (p < 0.05). Despite visible distinction, the HIGEM and HIGEM + MB groups showed no significant differences. CONCLUSION: High-intensity focused ultrasound combined with microbubbles enhances the therapeutic effects of gemcitabine chemotherapy in a pancreatic cancer xenograft model.

Sonoporation-Invigorating Sound in Dentistry: A Review.

Ultrasound (US), traditionally a diagnostic modality, and is emerging as a non-invasive therapy using local drug delivery and gene therapy. US exposure gene rates bio eff ects that result in shear stress, tissue heating, and cavitation eff ects, which are used in therapeutic applications. Sonoporation employs these eff ects to enhance delivery of large molecules such as DNA into the cells which is applied to muscle, head and neck tumor, in a cell disruption process called transformation and increases the permeability to bioactive materials, which is usually used in molecular biology and gene therapy. Nevertheless, it has recently become popular as a technique to enhance drug release from drug delivery systems. Th is review presents the main fi ndings in the fi eld of sonoporation, namely drug delivery, gene delivery and DNA transfer and its applications in dentistry.

Damaging the membrane of Ehrlich ascitic tumor cells with focused ultrasound / 中华物理医学与康复杂志

Objective To study the damage focused ultrasound inflicts on the membrane permeability of Ehrlich ascitic tumor (EAC) cells and the relationship between changes in membrane permeability and focused ultra-sound exposure time. Methods The relative survival rate of tumor cells was examined at various intensities and dif-ferent exposure times using focused 2.2 MHz ultrasound. The uhrastructure changes were evaluated with a scanning electron microscope after different exposures. Membrane permeability was investigated by incorporating fluorescein isothiocyanate dextran (FD5OO) , and membrane damage was evaluated by measuring lactate dehydrogenase (LDH) release. Results Morphological observation showed there were numerous microvilli on the surface of un-exposed cells. When the cells had been irradiated with focused ultrasound for 30 s there was only a slight effect on the shape of the cells and the number of microvilli was slightly reduced. When the cells were exposed to ultrasound for 60 s, the surface of many cells became relatively smooth with no obvious microvilli, and several small craters were seen on the surfaces of cells where the cytoplasm seemed to have extruded through the membrane. The cell membrane was seri-ously damaged by sonoporation. The loading of FD500 in the unexposed cells was only 0.21%. When the cells had been sonicated with focused ultrasound for 30 s or 60 s, the loading of FD500 increased to 11.46% and 18.50% re-spectively. The released LDH activities in the 30 s group and 60 s group were 2.94±0.02 and 3.28±0.04 U/L, respectively. The activities of LDH increaased as the focused ultrasound exposure time was prolonged. Conclusion Focused ultrasound may damage the cell membrane permeability of EAC cells, and the damage increases as the expo-sure time is prolonged from 30 s to 60 s.

Effects of different pulsed ultrasound parameters and culture conditions on cell viability and sonoporation on cell membrane / 中华物理医学与康复杂志

Objective To investigate different pulsed ultrasound (PUS) parameters and culture conditionsthat would affect cell viability and sonoporation on cell membrane of human cervical cancer cells (HeLa). MethodsHeLa cells were cultured in two different conditions ( in suspension or in monolayer). Cells were exposed to differentPUS intensity (0.4 W/cm2, 1.0 W/cm2, 1.6 W/cm2, 2.2 W/cm2), duty cycle (10%, 20%, 50%) and expo-sure time ( 1 min or 3 min). Cell viability was analyzed by flow cytometry. Using microscope and scanning electronmicroscopy (SEM) , the changes of shape and the sonoporation on cell membrane induced by PUS were observed.Results Low intensity and duty cycle did not exert a great impact on the cell viability. Cell injury was found to in-crease progressively with high intensity ( 1.6 W/cm2 , 2.2 W/cm2 ) and duty cycle ( 50% ) ( P < 0. 01 ) , and celldetachment was significantly accompanied by PUS exposure in adherent HeLa cells. Results of factorial design showedthat the culture conditions and the PUS parameters had significant interaction ( P < 0.01 ). SEM demonstrated insome detail the phenomenon of transient pores in the cell membrane under suitable PUS irradiation. The ideal sonopo-ration conditions that cell viability was above 80% and more membrane holes were noted to be at 1.0 W/cm2 expo-sure for 3 min with a duty cycle of 20% in cell suspension. Conclusion The optimized conditions of the PUS pa-rameters and the culture conditions could lower the cell injury and exert a great impact on the sonoporation. It couldproduce remarkable membrane pores on cells and enhance cell membrane permeability, which facilitate transportationof macromolecules into cells.