Microbubble-mediated ultrasound (MB-US) can be used to realize sonoporation and, in turn, facilitate the transfection of leukocytes in the immune system. Nevertheless, the bio-effects that can be induced by MB-US exposure on leukocytes have not been adequately studied, particularly for different leukocyte lineage subsets with distinct cytological characteristics. Here, we describe how that same set of MB-US exposure conditions would induce heterogeneous bio-effects on the three main leukocyte subsets: lymphocytes, monocytes and granulocytes. MB-US exposure was delivered by applying 1-MHz pulsed ultrasound (0.50-MPa peak negative pressure, 10% duty cycle, 30-s exposure period) in the presence of microbubbles (1:1 cell-to-bubble ratio); sonoporated and non-viable leukocytes were respectively labeled using calcein and propidium iodide. Flow cytometry was then performed to classify leukocytes into their corresponding subsets and to analyze each subset's post-exposure viability, sonoporation rate, uptake characteristics and morphology. Results revealed that, when subjected to MB-US exposure, granulocytes experienced the highest loss of viability (64.0 ± 11.0%) and the lowest sonoporation rate (6.3 ± 2.5%), despite maintaining their size and granularity. In contrast, lymphocytes exhibited the lowest loss of viability (20.9 ± 7.0%), while monocytes had the highest sonoporation rate (24.1 ± 13.6%). For these two sonoporated leukocyte subsets, their cell size and granularity were found to be reduced. Also, they exhibited graded levels of calcein uptake, whereas sonoporated granulocytes achieved only mild calcein uptake. These heterogeneous bio-effects should be accounted for when using MB-US and sonoporation in immunomodulation applications.
Keywords: Granulocyte; Leukocyte; Lymphocyte; Microbubble-mediated ultrasound; Monocyte; Sonoporation.
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