Ultrasound and Microbubble-Targeted Delivery of Macromolecules Is Regulated by Induction of Endocytosis and Pore Formation

Abstract

Abstract-Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4-and 70-kDa dextrans through the cytosol, and localization of 155-and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin-and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4-to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size. Key Words: ultrasound microbubble targeted delivery Ⅲ cell membrane pore Ⅲ endocytosis Ⅲ dextran Ⅲ endothelial cells C onventional delivery methods for drugs or genes, such as systemic administration via intravenous injection or oral administration, often do not suffice for therapeutic compounds such as peptides, silencing RNAs and genes. 1 A recent development in delivery systems for therapeutic compounds is the microbubble-ultrasound (US) interaction. 2,3 Before its use as a clinical modality, it is of utmost importance to obtain new physiological insights into the mechanisms of uptake by US and microbubble-exposed cells.Microbubbles were originally developed as US contrast agents and are administered intravenously to the systemic circulation to enhance scattering of blood in echocardiography. They consist of a gas core stabilized with an encapsulation, ranging from 1 to 10 m in diameter. 4 Nowadays, research focuses on the use of US and microbubbles for therapeutic applications. It has been demonstrated that USexposed microbubbles can achieve safe and efficient local delivery of a variety of drugs 5,6 and genes.

Ultrasound and microbubble mediated drug delivery: Acoustic pressure as determinant for uptake via membrane pores or endocytosis

“…Meijering et al [14] demonstrated that both pore formation and endocytosis were involved in the uptake of dextrans, in agreement with our results. Furthermore, they found a greater contribution of endocytosis for larger molecules.…”

“…lowering the temperature to 4°C or ATP depletion, are frequently used. These tools were also applied in studies to investigate the role of endocytosis in ultrasound mediated delivery [14,15,19,20]. Cells display various pathways of endocytic internalization, with macropinocytosis, clathrin-mediated and caveolae-mediated endocytosis being the best studied ones [27].…”

“…Moreover, several papers demonstrated the uptake of cell-impermeable molecules when exposing cells to ultrasound [11][12][13]. However, more recently, Meijering et al [14] stated that besides pore formation, enhanced endocytosis also contributes to ultrasound mediated delivery. Molecule size was observed to determine decisively which route of uptake occurs, with endocytosis playing a greater role for larger molecules.…”

Ultrasound assisted particle and cell manipulation on-chip

“…In addition to the well-recognised involvement of cavitation, recent studies have demonstrated an association between oscillating microbubbles and some physiological or cell regulated mechanisms of porosity, or, for example, endocytosis [124,125].…”

Identification and Characterization of miR-148a as a New Oncogenic and Prognostic microRNA in Glioblastoma

“…In addition to providing effective delivery, it eliminates or reduces systemic exposure to the therapy, and also permits use of smaller doses [246]. A MRI- has been shown extensively in-vivo [244,247], and FUS parameters can be tuned to provide reversible BBB sonoporation [248][249][250] to permit the delivery drugs such as Herceptin, which is a humanized anti-human epidermal growth factor receptor 2 (HER2/c-erbB2) monoclonal antibody and 1-3-bis(2-chloroethyl)-1-nitrosourea (carmustine) (BCNU), which is chemotherapeutic drug [251,252] to the brain. FUS-MB-mediated tumor transfection has also been reported, yielding reduced carcinoma growth via the delivery of herpes simplex virus thymidine kinase, ganciclovir [253,254], and shRNA [255,256], supporting potential usage of FUS and MBs for BPN/miR/anti-miR delivery.…”