In vitro methods to study bubble-cell interactions: Fundamentals and therapeutic applications

Abstract

Besides their use as contrast agents for ultrasound imaging, microbubbles are increasingly studied for a wide range of therapeutic applications. In particular, their ability to enhance the uptake of drugs through the permeabilization of tissues and cell membranes shows great promise. In order to fully understand the numerous paths by which bubbles can interact with cells and the even larger number of possible biological responses from the cells, thorough and extensive work is necessary. In this review, we consider the range of experimental techniques implemented in in vitro studies with the aim of elucidating these microbubble-cell interactions. First of all, the variety of cell types and cell models available are discussed, emphasizing the need for more and more complex models replicating in vivo conditions together with experimental challenges associated with this increased complexity. Second, the different types of stabilized microbubbles and more recently developed droplets and particles are presented, followed by their acoustic or optical excitation methods. Finally, the techniques exploited to study the microbubble-cell interactions are reviewed. These techniques operate over a wide range of timescales, or even off-line, revealing particular aspects or subsequent effects of these interactions. Therefore, knowledge obtained from several techniques must be combined to elucidate the underlying processes.

FIG. 1.

Diverse cell models can be chosen depending on the desired level of complexity and type of information. (A) Models involving the study of single cells for obtaining mechanistic information. Single cells can be adherent (a, reprinted with permission from Fan et al., J. Controlled Release 170, 401 (2013). Copyright 2013 Elsevier), trapped in microfluidic structures (b, reprinted with permission from Li et al., Lab Chip 13, 1144 (2013). Copyright 2013 The Royal Society of Chemistry) and mammalian or not (Xenopus oocyte in c, reprinted with permission from Zhou et al., J. Controlled Release 157, 103 (2012). Copyright 2012 Elsevier). (B) More complete models make use of cell population that can be arranged as a monolayer (a, reprinted with permission from Sridhar et al., PLoS One 9, e93618 (2014). Copyright 2014 Author(s), licensed under a Creative Commons Attribution 4.0 License.⁷⁹) or be in suspension (b, reprinted with permission from Tandiono et al., Lab Chip 12, 780 (2012). Copyright 2012 The Royal Society of Chemistry) in order to learn about the cell behavior in a collaborative context. (C) More complex bottom-up models are developed in which the cells are placed in a 3D environment, that is more similar to the in vivo situation. These models can be classified in 3 main categories: cell clusters (a), organs-on-a-chip with fully developed blood vessels (b, reprinted with permission from Moya et al., Tissue Eng., Part C 19, 730 (2013). Copyright 2013 Mary Ann Liebert, Inc. publishers), and biofilms (c, reprinted with permission from D. J. Stickler, Nat. Clin. Pract. Urol. 5, 598 (2008). Copyright 2008 Macmillan Publishers Ltd.⁸⁰), which are particularly used to optimize cleaning processes. (D) Some ex vivo models were also used to study bubble-cell interactions such as excised tissues (a, reprinted with permission from Chen et al., Appl. Phys. Lett. 101, 163704 (2012). Copyright 2012 AIP Publishing LLC) or a chicken egg embryo (b, reprinted with permission from Faez et al., Ultrasound Med. Biol. 38, 1608 (2015). Copyright 2015 Elsevier) to which regulatory restrictions do not apply.

FIG. 2.

Schematic representation of microbubbles precursors (A) and functionalized microbubbles (B). (A) Superheated, liquid perfluorocarbon nanodroplets are stabilized by a surfactant layer and can be vaporized with ultrasound or laser pulses (a); fluorescently labeled nanoparticles can generate microbubbles when exposed to laser light (b); and polymeric nanoparticles encapsulating a small gas core can be used as sustainable microbubble precursors (c). (B) Microbubbles can be functionalized by attaching targeting ligands (a); loading them with drug-containing nanoparticles (b) or make them suitable for multimodal imaging by covering them with plasmonic nanoparticles (c).

FIG. 3.

(A) High-speed fluorescence imaging of the uptake of propidium iodide (PI) used as model drug by a cell as a result of membrane poration by an oscillating microbubble. (B) Ultra high-speed recording (interframe time of 58 ns) of the interaction of a vaporizing superheated microdroplet with a cell upon ultrasound exposure at 5 MHz. (C) Combined high-speed fluorescence imaging and bright-field ultra high-speed imaging to visualize the sonoporation of cells by short-lived cavitation bubbles. The bubbles are created by laser activation of polymeric microcapsules.

FIG. 4.

(A) Confocal images of membrane perforation and resealing in microbubble-induced sonoporation (Reprinted with permission from Hu et al., Ultrasound Med. Biol. 39, 2393 (2013). Copyright 2013 Elsevier). Upon the application of an ultrasound pulse, a disruption of 5.3 m is created in the cell, exactly at the site where the microbubble was. The cell membrane is stained with an orange dye (CellMask Orange). After ultrasound exposure, the pore progressively reseals within a few minutes. (B) SEM images of cell membrane damage after ultrasound radiation (Reprinted with permission from Kudo et al., Biophys. J. 96, 4866 (2009). Copyright 2009 Elsevier). The high magnification views show perforations in the cell membrane surrounded by a rough surface.

FIG. 5.

Summary of the diverse techniques used for the study of bubble-cell interaction in relation with the accessible time scales and length scales. The left panel presents techniques that can be used simultaneously with the ultrasound exposure, while the right panel shows the techniques that cannot be applied during ultrasound exposure and thus require a 2-step experiment.