Mechanistic Insight into Sonoporation with Ultrasound-Stimulated Polymer Microbubbles.
ABSTRACT: Sonoporation is emerging as a feasible, non-viral gene delivery platform for the treatment of cardiovascular disease and cancer. Despite promising results, this approach remains less efficient than viral methods. The objective of this work is to help substantiate the merit of polymeric microbubble sonoporation as a non-viral, localized cell permeation and payload delivery strategy by taking a ground-up approach to elucidating the fundamental mechanisms at play. In this study, we apply simultaneous microscopy of polymeric microbubble sonoporation over its intrinsic biophysical timescales-with sub-microsecond resolution to examine microbubble cavitation and millisecond resolution over several minutes to examine local macromolecule uptake through enhanced endothelial cell membrane permeability-bridging over six orders of magnitude in time. We quantified microbubble behavior and resulting sonoporation thresholds at transmit frequencies of 0.5, 1 and 2 MHz, and determined that sonic cracking is a necessary but insufficient condition to induce sonoporation. Further, sonoporation propensity increases with the extent of sonic cracking, namely, from partial to complete gas escape from the polymeric encapsulation. For the subset that exhibited complete gas escape from sonic cracking, a proportional relationship between the maximum projected gas area and resulting macromolecule uptake was observed. These results have revealed one aspect of polymeric bubble activity on the microsecond time scale that is associated with eliciting sonoporation in adjacent endothelial cells, and contributes toward an understanding of the physical rationale for sonoporation with polymer-encapsulated microbubble contrast agents.
Project description:This study presents a unique approach to understanding the biophysical mechanisms of ultrasound-triggered cell membrane disruption (i.e., sonoporation). We report direct correlations between ultrasound-stimulated encapsulated microbubble oscillation physics and the resulting cellular membrane permeability by simultaneous microscopy of these two processes over their intrinsic physical timescales (microseconds for microbubble dynamics and seconds to minutes for local macromolecule uptake and cell membrane reorganization). We show that there exists a microbubble oscillation-induced shear-stress threshold, on the order of kilopascals, beyond which endothelial cellular membrane permeability increases. The shear-stress threshold exhibits an inverse square-root relation to the number of oscillation cycles and an approximately linear dependence on ultrasound frequency from 0.5 to 2 MHz. Further, via real-time 3D confocal microscopy measurements, our data provide evidence that a sonoporation event directly results in the immediate generation of membrane pores through both apical and basal cell membrane layers that reseal along their lateral area (resealing time of ?<2 min). Finally, we demonstrate the potential for sonoporation to indirectly initiate prolonged, intercellular gaps between adjacent, confluent cells (?>30-60 min). This real-time microscopic approach has provided insight into both the physical, cavitation-based mechanisms of sonoporation and the biophysical, cell-membrane-based mechanisms by which microbubble acoustic behaviors cause acute and sustained enhancement of cellular and vascular permeability.
Project description:Sonoporation refers to the use of ultrasound and acoustic cavitation to temporarily enhance the permeability of cellular membranes so as to enhance the delivery efficiency of therapeutic agents into cells. Microbubble-based ultrasound contrast agents are often used to facilitate these cavitation effects. This study used nanodroplets to significantly enhance the effectiveness of sonoporation relative to using conventional microbubbles. Significant enhancements were demonstrated both in vitro and in vivo by using gold nanorods encapsulated in nanodroplets for implementing plasmonic photothermal therapy. Combined excitation by ultrasound and laser radiation is used to trigger the gold nanodroplets to induce a liquid-to-gas phase change, which induces cavitation effects that are three-to-fivefold stronger than when using conventional microbubbles. Enhanced cavitation also leads to significant enhancement of the sonoporation effects. Our in vivo results show that nanodroplet-vaporization-assisted sonoporation can increase the treatment temperature by more than 10 °C above that achieved by microbubble-based sonoporation.
Project description:Ultrasound application in the presence of microbubbles has shown great potential for non-viral gene transfection via transient disruption of cell membrane (sonoporation). However, improvement of its efficiency has largely relied on empirical approaches without consistent and translatable results. The goal of this study is to develop a rational strategy based on new results obtained using novel experimental techniques and analysis to improve sonoporation gene transfection. In this study, we conducted experiments using targeted microbubbles that were attached to cell membrane to facilitate sonoporation. We quantified the dynamic activities of microbubbles exposed to pulsed ultrasound and the resulting sonoporation outcome, and identified distinct regimes of characteristic microbubble behaviors: stable cavitation, coalescence and translation, and inertial cavitation. We found that inertial cavitation generated the highest rate of membrane poration. By establishing direct correlation of ultrasound-induced bubble activities with intracellular uptake and pore size, we designed a ramped pulse exposure scheme for optimizing microbubble excitation to improve sonoporation gene transfection. We implemented a novel sonoporation gene transfection system using an aqueous two phase system (ATPS) for efficient use of reagents and high throughput operation. Using plasmids coding for the green fluorescence protein (GFP), we achieved a sonoporation transfection efficiency in rate aortic smooth muscle cells (RASMCs) of 6.9%±2.2% (n=9), comparable with lipofection (7.5%±0.8%, n=9). Our results reveal characteristic microbubble behaviors responsible for sonoporation and demonstrated a rational strategy to improve sonoporation gene transfection.
Project description:Microbubble-mediated sonoporation has shown its great potential in facilitating intracellular uptake of gene/drugs and other therapeutic agents that are otherwise difficult to enter cells. However, the biophysical mechanisms underlying microbubble-cell interactions remain unclear. Particularly, it is still a major challenge to get a comprehensive understanding of the impact of cell cycle phase on the cellular responses simultaneously occurring in cell membrane and cytoskeleton induced by microbubble sonoporation. Methods: Here, efficient synchronizations were performed to arrest human cervical epithelial carcinoma (HeLa) cells in individual cycle phases. The, topography and stiffness of synchronized cells were examined using atomic force microscopy. The variations in cell membrane permeabilization and cytoskeleton arrangement induced by sonoporation were analyzed simultaneously by a real-time fluorescence imaging system. Results: The results showed that G1-phase cells typically had the largest height and elastic modulus, while S-phase cells were generally the flattest and softest ones. Consequently, the S-Phase was found to be the preferred cycle for instantaneous sonoporation treatment, due to the greatest enhancement of membrane permeability and the fastest cytoskeleton disassembly at the early stage after sonoporation. Conclusion: The current findings may benefit ongoing efforts aiming to pursue rational utilization of microbubble-mediated sonoporation in cell cycle-targeted gene/drug delivery for cancer therapy.
Project description:Sonoporation is a targeted drug delivery technique that employs cavitation microbubbles to generate transient pores in the cell membrane, allowing foreign substances to enter cells by passing through the pores. Due to the broad size distribution of microbubbles, cavitation events appear to be a random process, making it difficult to achieve controllable and efficient sonoporation. In this work a technique is reported using a microfluidic device that enables in parallel modulation of membrane permeability by an oscillating microbubble array. Multirectangular channels of uniform size are created at the sidewall to generate an array of monodispersed microbubbles, which oscillate with almost the same amplitude and resonant frequency, ensuring homogeneous sonoporation with high efficacy. Stable harmonic and high harmonic signals emitted by individual oscillating microbubbles are detected by a laser Doppler vibrometer, which indicates stable cavitation occurred. Under the influence of the acoustic radiation forces induced by the oscillating microbubble, single cells can be trapped at an oscillating microbubble surface. The sonoporation of single cells is directly influenced by the individual oscillating microbubble. The parallel sonoporation of multiple cells is achieved with an efficiency of 96.6 ± 1.74% at an acoustic pressure as low as 41.7 kPa.
Project description:The concurrent assessment of principal sonoporation factors has been accomplished in a single systemic study. Microbubble sonodestruction dynamics and cavitation spectral characteristics, ultrasound scattering and attenuation, were examined in relation to the intracellular delivery of anticancer drug, bleomycin. Experiments were conducted on Chinese hamster ovary cells coadministered with Sonovue microbubbles. Detailed analysis of the scattering and attenuation temporal functions culminated in quantification of metrics, inertial cavitation dose and attenuation rate, suitable for cavitation control. The exponents, representing microbubble sonodestruction kinetics were exploited to derive dosimetric, microbubble sonodestruction rate. High intracorrelation between empirically-attained metrics defines the relations which indicate deep physical interdependencies within inherent phenomena. Subsequently each quantified metric was validated to be well-applicable to prognosticate the efficacy of bleomycin delivery and cell viability, as indicated by strong overall correlation (R2?>?0.85). Presented results draw valuable insights in sonoporation dosimetry and contribute towards the development of universal sonoporation dosimetry model. Both bleomycin delivery and cell viability reach their respective plateau levels by the time, required to attain total microbubble sonodestruction, which accord with scattering and attenuation decrease to background levels. This suggests a well-defined criterion, feasible through signal-registration, universally employable to set optimal duration of exposure for efficient sonoporation outcome.
Project description:Ultrasound-mediated gene delivery (sonoporation) is a minimally invasive, nonviral and clinically translatable method of gene therapy. This method offers a favorable safety profile over that of viral vectors and is less invasive as compared with other physical gene delivery approaches (e.g., electroporation). We have previously used sonoporation to overexpress transgenes in different skeletal tissues in order to induce tissue regeneration. Here, we provide a protocol that could easily be adapted to address various other targets of tissue regeneration or additional applications, such as cancer and neurodegenerative diseases. This protocol describes how to prepare, conduct and optimize ultrasound-mediated gene delivery in both a murine and a porcine animal model. The protocol includes the preparation of a microbubble-DNA mix and in vivo sonoporation under ultrasound imaging. Ultrasound-mediated gene delivery can be accomplished within 10 min. After DNA delivery, animals can be followed to monitor gene expression, protein secretion and other transgene-specific outcomes, including tissue regeneration. This procedure can be accomplished by a competent graduate student or technician with prior experience in ultrasound imaging or in performing in vivo procedures.
Project description:Ultrasound-driven microbubble activities have been exploited to transiently disrupt the cell membrane (sonoporation) for non-viral intracellular drug delivery and gene transfection both in vivo and in vitro. In this study, we investigated the dynamic behaviors of a population of microbubbles exposed to pulsed ultrasound and their impact on adherent cells in terms of intracellular delivery and cell viability. By systematically analyzing the bubble activities at time scales relevant to pulsed ultrasound exposure, we identified two quantification parameters that categorize the diverse bubble activities subjected to various ultrasound conditions into three characteristic behaviors: stable cavitation/aggregation (type I), growth/coalescence and translation (type II) and localized inertial cavitation/collapse (type III). Correlation of the bubble activities with sonoporation outcome suggested that type III behavior resulted in intracellular delivery, whereas type II behavior caused the death of a large number of cells. These results provide useful insights for rational selection of ultrasound parameters to optimize outcomes of sonoporation and other applications that exploit the use of ultrasound-driven bubble activities.
Project description:Microbubbles interact with ultrasound to induce transient microscopic pores in the cellular plasma membrane in a highly localized thermo-mechanical process called sonoporation. Theranostic applications of in vitro sonoporation include molecular delivery (e.g., transfection, drug loading and cell labeling), as well as molecular extraction for measuring intracellular biomarkers, such as proteins and mRNA. Prior research focusing mainly on the effects of acoustic forcing with polydisperse microbubbles has identified a "soft limit" of sonoporation efficiency at 50% when including dead and lysed cells. We show here that this limit can be exceeded with the judicious use of monodisperse microbubbles driven by a physiotherapy device (1.0 MHz, 2.0 W/cm(2), 10% duty cycle). We first examined the effects of microbubble size and found that small-diameter microbubbles (2 µm) deliver more instantaneous power than larger microbubbles (4 & 6 µm). However, owing to rapid fragmentation and a short half-life (0.7 s for 2 µm; 13.3 s for 6 µm), they also deliver less energy over the sonoporation time. This translates to a higher ratio of FITC-dextran (70 kDa) uptake to cell death/lysis (4:1 for 2 µm; 1:2 for 6 µm) in suspended HeLa cells after a single sonoporation. Sequential sonoporations (up to four) were consequently employed to increase molecular delivery. Peak uptake was found to be 66.1 ± 1.2% (n=3) after two sonoporations when properly accounting for cell lysis (7.0 ± 5.6%) and death (17.9 ± 2.0%), thus overcoming the previously reported soft limit. Substitution of TRITC-dextran (70 kDa) on the second sonoporation confirmed the effects were multiplicative. Overall, this study demonstrates the possibility of utilizing monodisperse small-diameter microbubbles as a means to achieve multiple low-energy sonoporation bursts for efficient in vitro cellular uptake and sequential molecular delivery.
Project description:Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustical, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency ultrasound (7.44MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5MHz) ultrasound pulse (duration 13.3 or 40?s) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d=0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106±0.032?m (n=70) for acoustic pressure of 1.5MPa (duration 13.3?s), and increased to 0.171±0.030?m (n=125) for acoustic pressure of 1.7MPa and to 0.182±0.052?m (n=112) for a pulse duration of 40?s (1.5MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level.