Ultrasound and microbubble induced release from intracellular compartments

Abstract

Background: Ultrasound and microbubbles (USMB) have been shown to enhance the intracellular uptake of molecules, generally thought to occur as a result of sonoporation. The underlying mechanism associated with USMB-enhanced intracellular uptake such as membrane disruption and endocytosis may also be associated with USMB-induced release of cellular materials to the extracellular milieu. This study investigates USMB effects on the molecular release from cells through membrane-disruption and exocytosis.

Results: USMB induced the release of 19% and 67% of GFP from the cytoplasm in viable and non-viable cells, respectively. Tfn release from early/recycling endosomes increased by 23% in viable cells upon USMB treatment. In addition, the MFI of LAMP-1 antibody increased by 50% in viable cells, suggesting USMB-stimulated lysosome exocytosis. In non-viable cells, labeling of LAMP-1 intracellular structures in the absence of cell permeabilization by detergents suggests that USMB-induced cell death correlates with lysosomal permeabilization.

Conclusions: In conclusion, USMB enhanced the molecular release from the cytoplasm, lysosomes, and early/recycling endosomes.

Keywords: Acoustic cavitation bioeffects; Cellular bioeffects of ultrasound; Endocytosis; Exocytosis; In vitro ultrasound bioeffects; Intracellular release; Intracellular uptake; Sonoporation; Ultrasound and microbubble.

Fig. 1

a In-suspension ultrasound and microbubble exposure setup. It consists of a waveform generator connected to a power amplifier that sends the signal to a 500 kHz ultrasound transducer that is focused on an acoustic window in the treatment chamber using a micro-positioning system. The sample chamber is placed on a magnetic stirrer. b Monolayer USMB exposure setup. It consists of a waveform generator connected to a power amplifier that sends the signal to a 500 kHz ultrasound transducer which is focused on an acoustic window where the sample is placed

Fig. 2

The percentage of viable cells in ultrasound and microbubble (USMB) treated and untreated control at 1.5, 11.5, and 21.5 min assessed using 7-Aminoactinomycin D (7-AAD). The number of samples is n = 12 for the untreated group and n = 14 for the USMB treated group. The error bars represent the standard deviation

Fig. 3

The mean fluorescent intensity (MFI) in untreated controls and ultrasound and microbubble (USMB) treated Alexa647-Transferrin (Tfn) loaded cells at 1.5, 11.5, and 21.5 min. The number of samples is n = 4 for all groups and the (*) indicates statistical significance (p < 0.05) using a Mann–Whitney U test. The error bars represent the standard deviation

Fig. 4

a The percentage of Green Fluorescent Protein positive (GFP+) cells measured in ultrasound and microbubble (USMB) treated and untreated groups at 1.5, 11.5, and 21.5 min. b The mean fluorescent intensity (MFI) in GFP+ cells in untreated and USMB treated cells at 1.5, 11.5, and 21.5 min. The (*) indicates that p < 0.05 using a Mann–Whitney U test. The number of samples is n = 4 for the untreated control and n = 6 for USMB treated group. The error bars represent standard deviation

Fig. 5

The mean fluorescent intensity (MFI) from Lysosomal Associated Membrane Protein-1. (LAMP-1) antibody binding measured in untreated control and ultrasound and microbubble (USMB) treated samples at 1.5, 11.5, and 21.5 min from the start of USMB. The (*) indicates a statistically significant difference (p < 0.05) using a Mann–Whitney U test. The number of samples n = 4 for both USMB treated and untreated groups. The error bars represent the standard deviation

Fig. 6

The relationship between the release from cytoplasm and lysosomes obtained by plotting the mean fluorescent intensities (MFI) of Green Fluorescent Protein (GFP) and Lysosomal Associated Membrane Protein-1 (LAMP-1) antibody. The MFI values for each marker were obtained from independent experiments and were normalized to the MFI values of the untreated control at t = 1.5 min for each marker. The error bars represent standard deviation

Fig. 7

The localization of Lysosomal Associated Membrane Protein-1 (LAMP-1) antibody in Green Fluorescent Protein (GFP)-transfected Retinal Pigmented Epithelial cells (RPE). a LAMP-1 antibody binding in untreated control (b) LAMP-1 antibody binding in ultrasound and microbubble (USMB) treated cells (c) GFP-clathrin in untreated control (d) GFP-clathrin in USMB-treated cells (e) Merged channels of GFP-clathrin (green) and LAMP-1 antibody (red) in untreated control (f) Merged channels of GFP-clathrin and LAMP-1 antibody in USMB-treated cells (n = 3). Scale bar = 50 μm

Fig. 8

A schematic diagram summarizing the hypothesized mechanisms of USMB induced/enhanced release from cytoplasm, lysosomes, and early/recycling endosomes through both membrane disruption and exocytosis. These mechanisms can occur simultaneously and can affect the release from more than one compartment at the same time (a) Membrane disruption by USMB induces diffusion of molecules from the cytoplasm and causes an increase in Ca²⁺ influx which triggers lysosomes fusion for membrane repair and can also be involved in USMB enhanced endocytosis. b USMB induced production of H₂O₂ can induce the permeabilization of lysosomal membranes and the release of lysosomal content and this oxidative stress can also be involved in the slower rate of recycling from early/recycling endosomes (c) USMB induced biomechanical stress can cause enhancing endocytosis and triggering a cellular response driving release from early/recycling endosomes