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.

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.

Growth Inhibition of Hepatocellular Carcinoma Huh7 Cells by Lactobacillus casei Extract

PURPOSE: Lactobacillus casei (L. casei) is known to exert anti-proliferation effects on many types of cancer cells. However, the effect of L. casei on liver cancer has not been reported. Accordingly, the aim of this study was to determine the anti-cancer effect of L. casei extract on Huh7 cells. MATERIALS AND METHODS: L. casei ATCC393 extract was prepared and purified. After the treatment of L. casei extract on Huh7 cells, cell viability, cell cycle arrest and cell death were analyzed by flow cytometry. The expression levels of tumor necrosis factor-alpha receptor 1 (TNFR1) and death receptor 3 (DR3) mRNA related with extrinsic apoptosis were assessed by reverse transcription polymerase chain reaction. Additionally, P21 and P27 cell cycle proteins as well as Caspase-3, -8, -9, phospho-Bad and Bcl-2 apoptosis proteins were analyzed by western blot analysis. To determine the effect of L. casei extract on cancer stem-like cells, we analyzed changes in side population fraction through flow cytometry. RESULTS: The cell viability of Huh7 cells treated with L. casei extract was decreased by 77%, potentially owing to increases in the rates of Huh7 cells arrested in the G2/M phase (3% increase) and that underwent apoptosis (6% increase). The expression levels of TNFR1 and DR3 mRNA, as well as P21 and P27 cell cycle proteins, were increased. Meanwhile, the expressions of caspase-8, -9, phospho-Bad and Bcl-2 proteins decreased. However, in the case of side population cells, no remarkable changes were observed. CONCLUSION: L. casei extract exerts a potent anti-tumor effect on the viability of liver cancer cells, although not on cancer stem-like cells.