Ultrasound insonification of microbubbles can locally enhance drug delivery, but the microbubble–cell interaction remains poorly understood. Because intracellular calcium (Ca_i ²⁺) is a key cellular regulator, unraveling the Ca_i ²⁺ fluctuations caused by an oscillating microbubble provides crucial insight into the underlying bio-effects. Therefore, we developed an optical imaging system at nanometer and nanosecond resolution that can resolve Ca_i ²⁺ fluctuations and microbubble oscillations. Using this system, we clearly distinguished three Ca_i ²⁺ uptake profiles upon sonoporation of endothelial cells, which strongly correlated with the microbubble oscillation amplitude, severity of sonoporation and opening of cell–cell contacts. We found a narrow operating range for viable drug delivery without lethal cell damage. Moreover, adjacent cells were affected by a calcium wave propagating at 15 μm/s. With the unique optical system, we unraveled the microbubble oscillation behavior required for drug delivery and Ca_i ²⁺ fluctuations, providing new insight into the microbubble–cell interaction to aid clinical translation.

Ultrasound insonification of microbubbles can locally increase vascular permeability to enhance drug delivery. To control and optimize the therapeutic potential, we need to better understand the underlying biological mechanisms of the drug delivery pathways. The aim of this in vitro study was to elucidate the microbubble-endothelial cell interaction using the Brandaris 128 ultra-high-speed camera (up to 25 Mfps) coupled to a custom-built Nikon confocal microscope, to visualize both microbubble oscillation and the cellular response. Sonoporation and opening of cell-cell contacts by single α_Vβ₃-targeted microbubbles (n = 152) was monitored up to 4 min after ultrasound insonification (2 MHz, 100–400 kPa, 10 cycles). Sonoporation occurred when microbubble excursion amplitudes exceeded 0.7 μm. Quantification of the influx of the fluorescent model drug propidium iodide upon sonoporation showed that the size of the created pore increased for larger microbubble excursion amplitudes. Microbubble-mediated opening of cell-cell contacts occurred as a cellular response upon sonoporation and did not correlate with the microbubble excursion amplitude itself. The initial integrity of the cell-cell contacts affected the susceptibly to drug delivery, since cell-cell contacts opened more often when cells were only partially attached to their neighbors (48%) than when fully attached (14%). The drug delivery outcomes were independent of nonlinear microbubble behavior, microbubble location, and cell size. In conclusion, by studying the microbubble–cell interaction at nanosecond and nanometer resolution the relationship between drug delivery pathways and their underlying mechanisms was further unraveled. These novel insights will aid the development of safe and efficient microbubble-mediated drug delivery.

Controlling microbubble-mediated drug delivery requires the underlying biological and physical mechanisms to be unraveled. To image both microbubble oscillation upon ultrasound insonification and the resulting cellular response, we developed an optical imaging system that can achieve the necessary nanosecond temporal and nanometer spatial resolutions. We coupled the Brandaris 128 ultra-high-speed camera (up to 25 million frames per second) to a custom-built Nikon A1R+ confocal microscope. The unique capabilities of this combined system are demonstrated with three experiments showing microbubble oscillation leading to either endothelial drug delivery, bacterial biofilm disruption, or structural changes in the microbubble coating. In conclusion, using this state-of-the-art optical imaging system, microbubble-mediated drug delivery can be studied with high temporal resolution to resolve microbubble oscillation and high spatial resolution and detector sensitivity to discern cellular response. Combining these two imaging technologies will substantially advance our knowledge on microbubble behavior and its role in drug delivery.

Ultrasound contrast agents consist of gas-filled coated microbubbles that oscillate upon ultrasound insonification. Their characteristic oscillatory response provides contrast enhancement for imaging and has the potential to locally enhance drug delivery. Since microbubble response depends on the local acoustic pressure, an ultrasound compatible chamber is needed to study their behavior and the underlying drug delivery pathways. In this study, we determined the amplitude of the acoustic pressure in the CLINIcell, an optically transparent chamber suitable for cell culture. The pressure field was characterized based on microbubble response recorded using the Brandaris 128 ultra-high speed camera and an iterative processing method. The results were compared to a control experiment performed in an OptiCell, which is conventionally used in microbubble studies. Microbubbles in the CLINIcell responded in a controlled manner, comparable to those in the OptiCell. For frequencies from 1 to 4 MHz, the mean pressure amplitude was -5.4 dB with respect to the externally applied field. The predictable ultrasound pressure demonstrates the potential of the CLINIcell as an optical, ultrasound, and cell culture compatible device to study microbubble oscillation behavior and ultrasound-mediated drug delivery.

One of the main challenges for ultrasound molecular imaging is acoustically distinguishing nonbound microbubbles from those bound to their molecular target. In this in vitro study, we compared two types of in-house produced targeted lipid-coated microbubbles, either consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, C16:0 (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine, C18:0 (DSPC) as the main lipid, using the Brandaris 128 ultrahigh-speed camera to determine vibrational response differences between bound and nonbound biotinylated microbubbles. In contrast to previous studies that studied vibrational differences upon binding, we used a covalently bound model biomarker (i.e., streptavidin) rather than physisorption, to ensure binding of the biomarker to the membrane. The microbubbles were insonified at frequencies between 1 and 4 MHz at pressures of 50 and 150 kPa. This paper shows lower acoustic stability of bound microbubbles, of which DPPC-based microbubbles deflated most. For DPPC microbubbles with diameters between 2 and 4μm driven at 50 kPa, resonance frequencies of bound microbubbles were all higher than 1.8 MHz, whereas those of nonbound microbubbles were significantly lower. In addition, the relative radial excursions at resonance were also higher for bound DPPC microbubbles. These differences did not persist when the pressure was increased to 150 kPa, except for the acoustic stability which further decreased. No differences in resonance frequencies were observed between bound and nonbound DSPC microbubbles. Nonlinear responses in terms of emissions at the subharmonic and second harmonic frequencies were similar for bound and nonbound microbubbles at both pressures. In conclusion, we identified differences in vibrational responses of bound DPPC microbubbles with diameters between 2 and 4μm that distinguish them from nonbound ones.

When exposed to ultrasound, microbubbles (MBs) deflate [1]. This occurs due to exchange of the MB filling gas with the gas of its environment, thus implying changes in its gas composition. Moreover, recent studies showed that gas composition affects the subharmonic (SH) response of phospholipid-coated contrast agents [2] [3]. In this work we investigated the SH emission from single MBs and the impact of deflation upon insonification.