Validation of damage on vascular endothelial cells under ultrasound exposure according to adhered situation of bubbles

Abstract

We investigated the viability of vascular endothelial cells with the existence of lipid bubbles under ultrasound exposure. First, we estimated the various situations of bubbles on the cells including either adhesion, floating, or both of them using not only image analysis but also an experiment to retain the cells in flow. Then we examined the viability measurement of the cells using the ultrasound conditions with the frequency of 3 MHz, a maximum sound pressure of 400 kPa-pp, and a maximum irradiation time of 60 s. We found that the floating bubbles caused more damage on the cells rather than the adhered bubbles. Because insufficient adhesion of bubbles might cause damage by floating bubbles, we consider that the adhered bubbles were protective of cells against floating bubbles. However, excessive bubbles with a higher concentration than the saturation also might cause damage by destructing both of floating and adhered bubbles.

Experimental study of ultrasound retention of bubble-surrounded cells under various conditions of acoustic field and flow velocity

“…We confirmed that BSCs were retained by a single focal point acoustic field under flow condition and a multi-focal acoustic field with tempo-spatial division emission, [21][22][23][27][28][29] which forms the shaping of acoustic field, using a twodimensional array transducer. To apply this technique, we developed a cell delivery system in an internal organ.…”

“…We proposed a similar technique involving the formation of bubble-surrounded cells (BSCs), [9][10][11][12] in which lipid bubbles 13) are attached to the surface of cells by making use of the connection between antibodies and their receptors to enhance the controllability of the cells under exposure to ultrasound. We have reported methods for the active control of these cells [9][10][11][12] using acoustic radiation force, which included active induction, [14][15][16][17] trapping, [18][19][20][21][22][23] and the formation of aggregates [24][25][26][27] of microbubbles in artificial blood vessels.…”

Experimental conditions for efficient retention of vascular endothelial cells on channel wall using lipid bubbles and acoustic interference

“…By forming a bubble-surrounded cell (BSC), in which the microbubbles are attached to the surface of a core cell, the dynamics of the cells contained in BSCs are controlled under ultrasound exposure. We confirmed that not only T-cells [13][14][15][16][17] or Colon-26 cells 18,19) but also vascular endothelial cells 20,21) are retained on the inner wall of a flow channel under an acoustic radiation force. These studies suggest that propelling forces acting on the cells are enhanced by the traveling wave reflected on the surface of the cells due to discontinuity in the acoustic impedance.…”

“…The obtained LBs had an average diameter of 100 nm and were encapsulated with the phosphate buffer solution in a liposome. We then modified LBs by conjugating cyclic-RGD (cyclo Arg-Gly-Asp-D-Phe-Cys, cRGD) peptides, 20,21,[36][37][38] which covalently adhered to vascular endothelial cells via DSPE-PEG on the LB surfaces. DSPE-PEG3.4k-Mal and cRGD were conjugated for a 1 h incubation at room temperature, overnight at 4 °C using a rotary mixer.…”

“…2; (a) single source acoustic field at amplitudes of P 1 = 300 kPa-pp, and (b) interferential acoustic field at amplitudes of P 1 = P 2 = 200 kPa-pp, when sinusoidal ultrasound was applied with a central frequency of 3 MHz and θ = 60°. The distribution of sound pressure was measured using an acoustic intensity measurement system (Onda AIMS III) by translating the hydrophone (Onda HNR-1000) [13][14][15][16][17][18][19][20][21] with the spatial pitch of 0.1 mm in degassed water, where the tip diameter of the hydrophone was 1.0 mm. The width of the nodes in the sound pressure distribution was regarded as 0.5 mm, which agreed with the theoretical value of the interval between nodes.…”