The combination of microbubbles and ultrasound has emerged as a promising method for local drug delivery. Microbubbles can be locally activated by a targeted ultrasound beam, which can result in several bio-effects. For drug delivery, microbubble-assisted ultrasound is used to increase vascular- and plasma membrane permeability for facilitating drug extravasation and the cellular uptake of drugs in the treated region, respectively. In the case of drug-loaded microbubbles, these two mechanisms can be combined with local release of the drug following destruction of the microbubble. The use of microbubble-assisted ultrasound to deliver chemotherapeutic agents is also referred to as sonochemotherapy. In this review, the basic principles of sonochemotherapy are discussed, including aspects such as the type of (drug-loaded) microbubbles used, the routes of administration used in vivo, ultrasound devices and parameters, treatment schedules and safety issues. Finally, the clinical translation of sonochemotherapy is discussed, including the first clinical study using sonochemotherapy.

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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.

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PurposeThe combination of ultrasound and microbubbles can facilitate cellular uptake of (model) drugs via transient permeabilization of the cell membrane. By using fluorescent molecules, this process can be studied conveniently with confocal fluorescence microscopy. This study aimed to investigate the relation between cellular uptake and fluorescence intensity increase of intercalating model drugs.ProceduresSYTOX Green, an intercalating fluorescent dye that displays >500-fold fluorescence enhancement upon binding to nucleic acids, was used as a model drug for ultrasound-induced cellular uptake. SYTOX Green uptake was monitored in high spatiotemporal resolution to qualitatively assess the relation between uptake and fluorescence intensity in individual cells. In addition, the kinetics of fluorescence enhancement were studied as a function of experimental parameters, in particular, laser duty cycle (DC), SYTOX Green concentration and cell line.ResultsUltrasound-induced intracellular SYTOX Green uptake resulted in local fluorescence enhancement, spreading throughout the cell and ultimately accumulating in the nucleus during the 9-min acquisition. The temporal evolution of SYTOX Green fluorescence was substantially influenced by laser duty cycle: continuous laser (100 % DC) induced a 6.4-fold higher photobleaching compared to pulsed laser (3.3 % DC), thus overestimating the fluorescence kinetics. A positive correlation of fluorescence kinetics and SYTOX Green concentration was found, increasing from 0.6 × 10−3 to 2.2 × 10−3 s−1 for 1 and 20 μM, respectively. Finally, C6 cells displayed a 2.4-fold higher fluorescence rate constant than FaDu cells.ConclusionsThese data show that the temporal behavior of intracellular SYTOX Green fluorescence enhancement depends substantially on nuclear accumulation and not just on cellular uptake. In addition, it is strongly influenced by the experimental conditions, such as the laser duty cycle, SYTOX Green concentration, and cell line.Electronic supplementary materialThe online version of this article (doi:10.1007/s11307-016-1042-x) contains supplementary material, which is available to authorized users.

INTRODUCTION Daily antiretroviral treatment (ART) can be challenging for some people living with HIV (PLHIV). Longacting injectable regimens (LAR) allow for non-daily dosing. We explored unmet needs associated with daily ART dosing and examined PLHIV's preference for LAR in the Netherlands, a country that has made enormous strides in improving HIV care; and 11 other European countries. METHODS Data were from the second wave of the Positive Perspectives survey of PLHIV on ART conducted in 2019 (Europe-wide, n=969 including Netherlands, n=51). Within four domains of ART-related challenges (emotional, psychosocial/stigma, physical, adherence), we used multivariable logistic regression to examine the relationship between the extent of unmet needs (tertiles) and LAR preference (p<0.05). RESULTS In pooled Europe-wide analysis, within each domain of unmet need, LAR preference increased with an increasing number of challenges. By the extent of ART-related emotional challenges, LAR preference odds were 1.76 (95% CI: 1.45-2.13) among those with a 'moderate' burden, and 4.05 (95% CI: 3.26-5.03) among those with a 'high' burden, compared to those with a 'low' burden. For anticipated stigma, LAR preference odds were 1.50 (95% CI: 1.11-2.04) for moderate and 2.33 (95% CI: 1.68-3.21) for high versus low. For adherence barriers, LAR preference odds were 1.53 (95% CI: 1.14-2.04) and 2.06 (95% CI: 1.45-2.91) among those with moderate and high levels of adherence barriers, respectively, compared to low. LAR preference odds were 1.71 (95% CI: 1.25-2.34) higher among PLHIV with 2+ non-HIV comorbidities versus HIV only, and 1.57 (95% CI: 1.12-2.34) higher among those on 2+ co-medications versus on ART exclusively. Of Dutch participants, 58.8% (30/51) indicated LAR preference, and 32.2% ranked LAR as the single most important ART improvement. Regarding daily oral ART dosing, 11.8% (6/51) of Dutch participants felt that it limited their daily life; 3.9% (2/51) were stressed by it, and 35.3% (18/51) said that it reminded them of HIV. Furthermore, 23.5% (12/51) hid/disguised their HIV medication to prevent unwanted disclosure of their HIV status. Also, 13.7% (7/51) reported adherence anxiety, 37.2% (19/51) missed ART 1+ times during the past month, and 13.7% (7/51) had difficulty swallowing pills. CONCLUSIONS PLHIV's preference for LAR can be for a variety of reasons other than clinical indications. LAR preference was associated with different domains of unmet needs, including emotional, psychosocial, physical, and adherencerelated challenges.