Sonoporation Efficiency Research Articles

The effect of flow rate on sonoporation within individual ex vivo mesenteric arteries

This study investigates the impact of vascular flow rate on the efficiency of sonoporation in viable mesenteric arteries ((391 ± 35) μm in intralumenal diameter), shedding light on the applications of ultrasound and microbubble-mediated (USMB) therapies. Isolated viable rat mesenteric arteries, which retained physiological functionality, exhibited reversible responses to vasoactive agents when mounted on a pressure myograph throughout the experiments. We then subjected these vessels to ultrasound (2.25 MHz) and microbubble treatment (Definity) under varied acoustic and flow conditions and assessed sonoporation through propidium iodide (PI) fluorescence microscopy. Results disclosed a strong correlation between microbubble flow rate, duty cycle, and sonoporation efficiency. Higher flow rates and duty cycles were associated with increased PI-positive cell counts, signifying more efficient cellular permeability. Post-treatment viability assays affirmed vessel integrity. These findings underscore the pivotal role of vascular flow rate in shaping therapeutic efficacy within individual vessels. The implications extend to refining USMB therapies in diverse disease scenarios, emphasizing the necessity for meticulous parameter selection to ensure both effectiveness and safety. Overall, the study furnishes valuable insights for enhancing the success and applicability of USMB-based therapeutic approaches in cardiovascular and oncological contexts.

Extracellular matrix stiffness affects microbubble-assisted endothelial permeabilization under flow

Cancer immunotherapy has faced challenges in the treatment of solid cancers due to the complex tumor microenvironment (TME), including physical barriers that prohibit immune cell infiltration. Ultrasound (US) stimulated microbubbles present a novel way to potentiate cancer immunotherapy in tumors by permeabilizing the tumor vasculature, however the effect of individual TME parameters on treatment efficacy has not yet been elucidated. Here, we focus on one biophysical parameter, showing that an increase in matrix stiffness increases US-assisted membrane permeabilization. Using a novel setup that allows for real-time visualization under flow, HUVECs seeded on polyacrylamide hydrogels of different stiffnesses (800 and 1600 Pa) showed an increase in sonoporation rates. Collagen models corroborate this trend at two different flow rates for a range of stiffnesses (5, 25, and 75 Pa): for both 5 ml/min and 30 ml/min, there is a relative increase in sonoporation from the stiffer substrate. For a given collagen substrate, there is a significant increase in sonoporation efficiency with increasing flow rate. These data can be used to fine-tune treatments according to different TMEs; further, our findings have applications in designing US parameters for targeted drug delivery and other clinical contexts that consider a variety of tissue stiffnesses.

Three-dimensional array of microbubbles sonoporation of cells in microfluidics.

Sonoporation is a popular membrane disruption technique widely applicable in various fields, including cell therapy, drug delivery, and biomanufacturing. In recent years, there has been significant progress in achieving controlled, high-viability, and high-efficiency cell sonoporation in microfluidics. If the microchannels are too small, especially when scaled down to the cellular level, it still remains a challenge to overcome microchannel clogging, and low throughput. Here, we presented a microfluidic device capable of modulating membrane permeability through oscillating three-dimensional array of microbubbles. Simulations were performed to analyze the effective range of action of the oscillating microbubbles to obtain the optimal microchannel size. Utilizing a high-precision light curing 3D printer to fabricate uniformly sized microstructures in a one-step on both the side walls and the top surface for the generation of microbubbles. These microbubbles oscillated with nearly identical amplitudes and frequencies, ensuring efficient and stable sonoporation within the system. Cells were captured and trapped on the bubble surface by the acoustic streaming and secondary acoustic radiation forces induced by the oscillating microbubbles. At a driving voltage of 30 Vpp, the sonoporation efficiency of cells reached 93.9% ± 2.4%.

Dependence of sonoporation efficiency on microbubble size: An in vitro monodisperse microbubble study

Sonoporation is the process where intracellular drug delivery is facilitated by ultrasound-driven microbubble oscillations. Several mechanisms have been proposed to relate microbubble dynamics to sonoporation including shear and normal stress. The present work aims to gain insight into the role of microbubble size on sonoporation and thereby into the relevant mechanism(s) of sonoporation. To this end, we measured the sonoporation efficiency while varying microbubble size using monodisperse microbubble suspensions. Sonoporation experiments were performed in vitro on cell monolayers using a single ultrasound pulse with a fixed frequency of 1 MHz while the acoustic pressure amplitude and pulse length were varied at 250, 500, and 750 kPa, and 10, 100, and 1000 cycles, respectively. Sonoporation efficiency was quantified using flow cytometry by measuring the FITC-dextran (4 kDa and 2 MDa) fluorescence intensity in 10,000 cells per experiment to average out inherent variations in the bioresponse. Using ultra-high-speed imaging at 10 million frames per second, we demonstrate that the bubble oscillation amplitude is nearly independent of the equilibrium bubble radius at acoustic pressure amplitudes that induce sonoporation (≥ 500 kPa). However, we show that sonoporation efficiency is strongly dependent on the equilibrium bubble size and that under all explored driving conditions most efficiently induced by bubbles with a radius of 4.7 μm. Polydisperse microbubbles with a typical ultrasound contrast agent size distribution perform almost an order of magnitude lower in terms of sonoporation efficiency than the 4.7-μm bubbles. We elucidate that for our system shear stress is highly unlikely the mechanism of action. By contrast, we show that sonoporation efficiency correlates well with an estimate of the bubble-induced normal stress.

Dependence of sonoporation efficiency on microbubble size: An in vitro monodisperse microbubble study

Sonoporation is the process where intracellular drug delivery is facilitated by ultrasound-driven microbubble oscillations. Several mechanisms have been proposed to relate microbubble dynamics to sonoporation including shear and normal stress. The present work aims to gain insight into the role of microbubble size on sonoporation by varying the microbubble size of monodisperse microbubble suspensions. Sonoporation experiments were performed in vitro on cell monolayers at an ultrasound frequency of 1 MHz at varying acoustic pressures (250–750 kPa) and pulse length (10, 100, 1000 cycles). Sonoporation efficiency was quantified using flow cytometry by measuring the FITC-dextran (4 kDa and 2 MDa) fluorescence intensity in 10,000 cells per experiment. We demonstrate that the bubble oscillation amplitude is nearly independent of the equilibrium bubble radius at acoustic pressure amplitudes that induce sonoporation (≥500 kPa). However, we show that sonoporation efficiency is strongly dependent on the equilibrium bubble size and most efficiently induced by off-resonance 4.7-μm radius bubbles. These 4.7-μm bubbles are an order of magnitude more efficient than the polydisperse bubbles. We discuss that for our system shear stress is highly unlikely the mechanism of action, while we show that sonoporation efficiency correlates well with an estimate of the bubble-induced normal stress.

Ultrasound-assisted membrane permeabilization of endothelial cells under flow conditions

Ultrasound-stimulated microbubbles have been shown a feasible approach for localized therapeutic delivery. As applications of this technique span many anatomical sites, so too do the local fluid dynamics experienced by the circulating microbubbles and the adjacent endothelial cells. Our objective was to assess the relative effectiveness of endothelial cell sonoporation as a function of flow conditions. Human umbilical vein (HUVECs) or human brain endothelial cells (HBECs) were cultured as a monolayer in flow chamber slides connected to a fluidic system and placedupon an acoustically-coupled microscope. A suspension of diluted lipid-encapsulated microbubbles and propidium iodide (PI), used as a sonoporation marker, was constantly perfused over the monolayer at either 5 or 30 ml/min. Cells were treated with 1 MHz ultrasound (PRI= 1 ms, 20 cycles, duration = 2 s), and the video-microscopy data were quantified offline to assess the number of PI-positive cells. Our results demonstrate a marked increase in sonoporation efficiency at 30 ml/min as compared to 5 ml/min in both endothelial cell lines under identical acoustic conditions (9.7-fold increase and 2.3-fold increase for HUVECs and HBECs respectively, p < 0.001). Our results suggest the local fluid flow environment plays a role in US-mediated endothelial perforation efficiency and can modulate treatment strategies.

Non-Cavitation Targeted Microbubble-Mediated Single-Cell Sonoporation.

Sonoporation employs ultrasound accompanied by microbubble (MB) cavitation to induce the reversible disruption of cell membranes and has been exploited as a promising intracellular macromolecular delivery strategy. Due to the damage to cells resulting from strong cavitation, it is difficult to balance efficient delivery and high survival rates. In this paper, a traveling surface acoustic wave (TSAW) device, consisting of a TSAW chip and a polydimethylsiloxane (PDMS) channel, was designed to explore single-cell sonoporation using targeted microbubbles (TMBs) in a non-cavitation regime. A TSAW was applied to precisely manipulate the movement of the TMBs attached to MDA-MB-231 cells, leading to sonoporation at a single-cell level. The impact of input voltage and the number of TMBs on cell sonoporation was investigated. In addition, the physical mechanisms of bubble cavitation or the acoustic radiation force (ARF) for cell sonoporation were analyzed. The TMBs excited by an ARF directly propelled cell membrane deformation, leading to reversible perforation in the cell membrane. When two TMBs adhered to the cell surface and the input voltage was 350 mVpp, the cell sonoporation efficiency went up to 83%.

Ultrasound-Mediated Drug Delivery: Sonoporation Mechanisms, Biophysics, and Critical Factors.

Sonoporation, or the use of ultrasound in the presence of cavitation nuclei to induce plasma membrane perforation, is well considered as an emerging physical approach to facilitate the delivery of drugs and genes to living cells. Nevertheless, this emerging drug delivery paradigm has not yet reached widespread clinical use, because the efficiency of sonoporation is often deemed to be mediocre due to the lack of detailed understanding of the pertinent scientific mechanisms. Here, we summarize the current observational evidence available on the notion of sonoporation, and we discuss the prevailing understanding of the physical and biological processes related to sonoporation. To facilitate systematic understanding, we also present how the extent of sonoporation is dependent on a multitude of factors related to acoustic excitation parameters (ultrasound frequency, pressure, cavitation dose, exposure time), microbubble parameters (size, concentration, bubble-to-cell distance, shell composition), and cellular properties (cell type, cell cycle, biochemical contents). By adopting a science-backed approach to the realization of sonoporation, ultrasound-mediated drug delivery can be more controllably achieved to viably enhance drug uptake into living cells with high sonoporation efficiency. This drug delivery approach, when coupled with concurrent advances in ultrasound imaging, has potential to become an effective therapeutic paradigm.

The effect of low frequency and low intensity ultrasound combined with microbubbles on the sonoporation efficiency of MDA-MB-231 cells.

BackgroundUltrasound can produce certain biophysical effects including thermal and non-thermal effects on cells. Sonoporation, the most widely studied non-thermal biological effect of ultrasound, is considered to be the basis for new therapeutic applications. Ultrasound irradiation can increase the permeability of cell membranes through sonoporous effect, which makes molecules like those of drugs, protein, and DNA that normally cannot pass through the cell membranes be able to enter cells. Considering the poor therapeutic effect and poor prognosis of triple negative breast cancer, we aimed to explore the experimental conditions and find the optimal parameters to improve the therapeutic efficacy of chemotherapeutic drugs for MDA-MB-231 cells.MethodsBy establishing an experimental and control group, our study investigated the effect of low frequency and low intensity ultrasound combined with microbubbles on MDA-MB-231 cell membrane permeability at different times. We conducted factorial cross-design and set 3 levels of ultrasound intensity: 230, 300, and 370 mW/cm2; 3 levels of irradiation time: 1, 2, and 3 minutes; and 6 levels of microbubble doses: 0, 0.2, 0.4, 0.6, 0.8, and 1 mL.ResultsResults show that ultrasound intensity, time of irradiation, and microbubbles concentration are not only related to but also have interactive effects on the sonoporation efficiency of MDA-MB-231 cells, with the rank order being sound intensity, irradiation time, and microbubble concentration. The average positive rates (%) of FD4 staining in sound intensities of 230, 300, and 370 mW/cm2 levels were 1.20±0.71, 13.80±5.86, and 10.71±4.36, respectively; and in irradiated times of 1, 2, and 3 min they were 7.54±5.95, 9.74±8.42, and 8.59±5.80, respectively. When the microbubbles increased according to the gradient of 0, 0.2, 0.4, 0.6, 0.8, and 1 mL, the positive rates (%) of FD4 staining were 7.32±5.89, 9.26±7.39, 8.31±5.67, 10.12±8.42, 8.67±7.23, and 7.72±6.24.ConclusionsIn our study, the optimal parameters of the sonoporous effect for MDA-MB-231 cells were 300 mW/cm2 of ultrasound intensity, 2 minutes of irradiation time, and 20% microbubbles concentration.

Investigating the Role of Lipid Transfer in Microbubble-Mediated Drug Delivery.

Sonoporation, the permeabilization of cell membranes following exposure to microbubbles and ultrasound, has considerable potential for therapeutic delivery. To date, engineering of microbubbles for these applications has focused primarily upon optimizing microbubble size and stability, or attachment of targeting species and/or drug molecules. In this work, it is demonstrated that the microbubble coating can also be tailored to directly influence cell permeabilization. Specifically, lipid exchange mechanisms between phospholipid microbubbles and cells can be exploited to significantly increase sonoporation efficiency in vitro. A theoretical analysis of the energy required for pore formation was carried out. From this, it was hypothesized that sonoporation could be promoted by the transfer of lipid molecules with appropriate carbon chain length and/or shape (cylindrical or conical). Spectral imaging with a hydration-sensitive membrane probe (C-Laurdan) was used to measure changes in the membrane lipid order of A-549 cancer cells following exposure to suspensions of different phospholipids. Two candidate lipids were identified, a short-chain-length phospholipid (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) and a medium-chain-length lysolipid (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 lyso-PC)). Microbubbles were prepared with matched concentrations, size distributions, and acoustic responses. Confocal microscopy was used to measure cell uptake of a model drug (propidium iodide) with and without ultrasound exposure (1 MHz, 250 kPa peak negative pressure, 1 kHz pulse repetition frequency, 10% duty cycle, 15 s exposure). Despite significantly decreasing the cell membrane lipid order, DLPC did not increase sonoporation. Microbubbles containing 16:0 lyso-PC, however, produced a ∼5-fold increase in sonoporation compared to control microbubbles. Importantly, the lyso-PC molecules were incorporated into the microbubble coating and did not affect cell permeability prior to ultrasound exposure. These findings indicate that microbubbles can be engineered to exploit lipid exchange between microbubble shells and cell membranes to enhance drug delivery, a new optimization route that may lead to enhanced therapeutic efficacy of ultrasound-mediated treatments.

Intracellular Signaling in Key Pathways Is Induced by Treatment with Ultrasound and Microbubbles in a Leukemia Cell Line, but Not in Healthy Peripheral Blood Mononuclear Cells.

Treatment with ultrasound and microbubbles (sonoporation) to enhance therapeutic efficacy in cancer therapy is rapidly expanding, but there is still very little consensus as to why it works. Despite the original assumption that pore formation in the cell membrane is responsible for increased uptake of drugs, the molecular mechanisms behind this phenomenon are largely unknown. We treated cancer cells (MOLM-13) and healthy peripheral blood mononuclear cells (PBMCs) with ultrasound at three acoustic intensities (74, 501, 2079 mW/cm2) ± microbubbles. We subsequently monitored the intracellular response of a number of key signaling pathways using flow cytometry or western blotting 5 min, 30 min and 2 h post-treatment. This was complemented by studies on uptake of a cell impermeable dye (calcein) and investigations of cell viability (cell count, Hoechst staining and colony forming assay). Ultrasound + microbubbles resulted in both early changes (p38 (Arcsinh ratio at high ultrasound + microbubbles: +0.5), ERK1/2 (+0.7), CREB (+1.3), STAT3 (+0.7) and AKT (+0.5)) and late changes (ribosomal protein S6 (Arcsinh ratio at low ultrasound: +0.6) and eIF2α in protein phosphorylation). Observed changes in protein phosphorylation corresponded to changes in sonoporation efficiency and in viability, predominantly in cancer cells. Sonoporation induced protein phosphorylation in healthy cells was pronounced (p38 (+0.03), ERK1/2 (−0.03), CREB (+0.0), STAT3 (−0.1) and AKT (+0.04) and S6 (+0.2)). This supports the hypothesis that sonoporation may enhance therapeutic efficacy of cancer treatment, without causing damage to healthy cells.

Control of Acoustic Cavitation for Efficient Sonoporation with Phase-Shift Nanoemulsions

Acoustic cavitation can be used to temporarily disrupt cell membranes for intracellular delivery of large biomolecules. Termed sonoporation, the ability of this technique for efficient intracellular delivery (i.e., >50% of initial cell population showing uptake) while maintaining cell viability (i.e., >50% of initial cell population viable) has proven to be very difficult. Here, we report that phase-shift nanoemulsions (PSNEs) function as inertial cavitation nuclei for improvement of sonoporation efficiency. The interplay between ultrasound frequency, resultant microbubble dynamics and sonoporation efficiency was investigated experimentally. Acoustic emissions from individual microbubbles nucleated from PSNEs were captured using a broadband passive cavitation detector during and after acoustic droplet vaporization with short pulses of ultrasound at 1, 2.5 and 5 MHz. Time domain features of the passive cavitation detector signals were analyzed to estimate the maximum size (Rmax) of the microbubbles using the Rayleigh collapse model. These results were then applied to sonoporation experiments to test if uptake efficiency is dependent on maximum microbubble size before inertial collapse. Results indicated that at the acoustic droplet vaporization threshold, Rmax was approximately 61.7 ± 5.2, 24.9 ± 2.8, and 12.4 ± 2.1 μm at 1, 2.5 and 5 MHz, respectively. Sonoporation efficiency increased at higher frequencies, with efficiencies of 39.5 ± 13.7%, 46.6 ± 3.28% and 66.8 ± 5.5% at 1, 2.5 and 5 MHz, respectively. Excessive cellular damage was seen at lower frequencies because of the erosive effects of highly energetic inertial cavitation. These results highlight the importance of acoustic cavitation control in determining the outcome of sonoporation experiments. In addition, PSNEs may serve as tailorable inertial cavitation nuclei for other therapeutic ultrasound applications.

Electrochemical generation of microbubbles by carbon nanotube interdigital electrodes to increase the permeability and material uptakes of cancer cells

Artificial cavitation as a prerequisite of sonoporation, plays an important role on the ultrasound (US) assisted drug delivery systems. In this study, we have proposed a new method of microbubble (MB) generation by local electrolysis of the medium. An integrated interdigital array of three-electrode system was designed and patterned on a nickel-coated quartz substrate and then, a short DC electrical pulse was applied that consequently resulted in distributed generation of microbubbles at the periphery of the electrodes. Growth of the carbon nanotube (CNT) nanostructures on the surface of the electrodes approximately increased the number of generated microbubbles up to 7-fold and decreased their average size from ∼20 µm for bare to ∼7 µm for CNT electrodes. After optimizing the three-electrode system, biocompatibility assays of the CNT electrodes stimulated by DC electrical micropulses were conducted. Finally, the effect of the proposed method on the sonoporation efficiency and drug uptake of breast cells were assessed using cell cycle and Annexin V/PI flow cytometry analysis. These results show the potential of electrochemical generation of MBs by CNT electrodes as an easy, available and promising technique for artificial cavitation and ultrasound assisted drug delivery.

Breast Cancer Cell Line Phenotype Affects Sonoporation Efficiency Under Optimal Ultrasound Microbubble Conditions.

BackgroundUltrasound/microbubble (USMB)-mediated sonoporation is a new strategy with minimal procedural invasiveness for targeted and site-specific drug delivery to tumors. The purpose of this study was to explore the effect of different breast cancer cell lines on sonoporation efficiency, and then to identify an optimal combination of USMB parameters to maximize the sonoporation efficiency for each tumor cell line.Material/MethodsThree drug-sensitive breast cell lines – MCF-7, MDA-MB-231, and MDA-MB-468 – and 1 multidrug resistance (MDR) cell line – MCF-7/ADR – were chosen. An orthogonal array experimental design approach based on 3 levels of 3 parameters (A: microbubble concentration, 10%, 20%, and 30%, B: sound intensity, 0.5, 1.0, and 1.5 W/cm2, C: irradiation time, 30, 60, and 90 s) was employed to optimize the sonoporation efficiency.ResultsThe optimal USMB parameter combinations for different cell lines were diverse. Under optimal parameter combinations, the maximum sonoporation efficiency differences between different breast tumor cell lines were statistically significant (MDA-MB-231: 46.70±5.79%, MDA-MB-468: 53.44±5.69%, MCF-7: 59.88±5.53%, MCF-7/ADR: 65.39±4.01%, P<0.05), so were between drug-sensitive cell line and MDR cell line (MCF-7: 59.88±5.53%, MCF-7/ADR: 65.39±4.01%, p=0.026).ConclusionsDifferent breast tumor cell lines have their own optimal sonoporation. Drug-resistant MCF-7/ADR cells had higher sonoporation efficiency than drug-sensitive MCF-7 cells. The molecular subtype of tumors should be considered when sonoporation is applied, and optimal parameter combination may have the potential to improve drug-delivery efficiency by increasing the sonoporation efficiency.

FRET-based method for evaluation of the efficiency of reversible and irreversible sonoporation.

It is widely known that not all of the treated cells survive after introduction of exogenous molecules via any physical method. Therefore, it is important to develop methods that would allow simultaneous evaluation of both molecular delivery efficiency and cell viability. This study presents Förster resonance energy transfer (FRET)-based method that allows molecular transfer and cell viability evaluation in a single measurement by employing two common fluorescent dyes, namely, ethidium bromide and trypan blue. The method has been validated using cell sonoporation. The FRET-based method allows the efficiency evaluation of both reversible and irreversible sonoporation in a single experiment. Therefore, this method could be used to reduce time, labor, and material cost while improving the accuracy of evaluations.

An evaluation of the sonoporation potential of low-boiling point phase-change ultrasound contrast agents in vitro

BackgroundPhase-change ultrasound contrast agents (PCCAs) offer a solution to the inherent limitations associated with using microbubbles for sonoporation; they are characterized by prolonged circulation lifetimes, and their nanometer-scale sizes may allow for passive accumulation in solid tumors. As a first step towards the goal of extravascular cell permeabilization, we aim to characterize the sonoporation potential of a low-boiling point formulation of PCCAs in vitro.MethodsParameters to induce acoustic droplet vaporization and subsequent microbubble cavitation were optimized in vitro using high-speed optical microscopy. Sonoporation of pancreatic cancer cells in suspension was then characterized at a range of pressures (125–600 kPa) and pulse lengths (5–50 cycles) using propidium iodide as an indicator molecule.ResultsWe achieved sonoporation efficiencies ranging from 8 ± 1% to 36 ± 4% (percent of viable cells), as evidenced by flow cytometry. Increasing sonoporation efficiency trended with increasing pulse length and peak negative pressure.ConclusionsWe conclude that PCCAs can be used to induce the sonoporation of cells in vitro, and our results warrant further investigation into the use of PCCAs as extravascular sonoporation agents in vivo.

Manipulation of microbubbles and targeted single cell sonoporation with by surface acoustic waves in a microfluidic device

A microfluidic device was developed to transport the aggregated microbubbles to a targeted cell at arbitrary location in liquid solution by introducing the phase-shift to a planar standing surface acoustic wave (SAW). The device consists of two perpendicular pairs of interdigital transducer (IDTs) and a polydimethyl-siloxane microchannel. By adjusting the relative phase between electric voltages applied to the independent IDTs, the microbubbles can be trapped, continuously moved or rotated due to the translation of pressure nodes. We further demonstrate that a SAW is capable of inducing microbubble cluster destruction at a desired location to achieve a single cell’s reparable sonoporation. By controlling the position of the microbubble cluster relative to the targeted cell precisely, the effective size of the collapsing microbubbles is measured to be less than 0.68 times the diameter of microbubble cluster. The sonoporation efficiency and the cell viability are 82.4%± 6.5% and 90%±8.7%, respectively, when the targeted cell is within the effective microbubble destruction region.

Influence of tumor cell lines derived from different tissue on sonoporation efficiency under ultrasound microbubble treatment

To reveal the effect of tumor cell lines derived from different tissue on sonoporation efficiency under ultrasound microbubble (USMB) treatment, and meanwhile to determine the optimum parameter combination for each tumor cell line. Human breast tumor (MCF-7), ovarian tumor (A2780), liver tumor (Bel7402) and thyroid tumor (ARO) were exposed to ultrasound in the presence of SonoVue MBs. The major parameters for the designed experiments including MB concentration (A1:10%, A2:20%, A3:30%), sound intensity (B1:0.5, B2:1.0, B3:1.5W/cm2), irradiation time (C1:30, C2:60, C3:90s). An orthogonal array experimental design based on three levels L9 (33) of the above three parameters was employed to optimize the sonoporation efficiency. MTT experiment was used to calculate cell survival rate. FD500 uptake assay and cytometry were performed to evaluate transference percentage. The optimum parameter combination for each tumor cell line was different (MCF-7: A3B1C1, A2780: A1B3C3, Bel7402: A2B3C2, ARO: A2B3C2). Under their own optimum parameter combination, four kinds of tumor cell line exhibited different optimum sonoporation efficiency (MCF-7: 55.05±5.29%; A2780: 45.62±7.35%; Bel7402: 39.37±4.11%; ARO: 53.37±3.94%). Multiple comparison with LSD-t test showed that the sonoporation efficiency between four kinds of cell line was statistically significant (P<0.05), with the exception of MCF-7 VS. ARO (P=0.487). Tumor cell lines derived from different tissue can impact the sonoporation efficiency, and optimizing the exposure parameters can safely and efficiently increase the cell membrane permeability.

1H15 In vitroソノポレーションにおける分子導入の実時間観察 : 超音波照射条件の分子導入タイミングへの影響

Understanding the time when molecular delivery occurs during sonoporation is important to improve the efficiency of successful sonoporation and fraction of viable cells. Here, we report how ultrasound conditions affect the time for sonoporation. We employed real-time observations of propidium iodide (PI) delivery to 3T3-Swiss albino cells under 5 seconds 1 MHz continuous ultrasound waves treatment with fluorescence (PI) and microbubbles SONAZOID^[○!R]. Sonoporation timing was evaluated by analyzing the time courses of the altering fluorescence intensity of the cells. Three ultrasound intensity conditions, 0.15, 0.20 and 0.30 W/cm^2, were examined. Fluorescence intensity increased for 0.20 and .30 W/cm^2-sonication, indicating PI delivery into the cells, while no apparent increase was observed for 0.15 W/cm^2-sonication. Fluorescence intensity clearly started to increase earlier for 0.30 W/cm^2 than for 0.20 W/cm^2-sonication. Sonoporation timing ranged from 2 to 6 seconds for 0.30 W/cm^2-sonication. The most delayed timing was obtained for cells that had the largest adhesion area. As a conclusion, different microbubble responses stimulated by various ultrasound conditions may cause different sonoporation timing.

Acoustic Estimation of Resonance Frequency and Sonodestruction of SonoVue Microbubbles

AbstractAcoustic properties of ultrasound (US) contrast agent microbubbles (MB) highly influence sonoporation efficiency and intracellular drug and gene delivery. In this study we propose an acoustic method to monitor passive and excited MBs in a real time. MB monitoring system consisted of two separate transducers. The first transducer delivered over an interval of 1 s US pulses (1 MHz, 1% duty cycle, 100 Hz repetition frequency) with stepwise increased peak negative pressure (PNP), while the second one continuously monitored acoustic response of SonoVue MBs. Pulse echo signals were processed according to the substitution method to calculate attenuation coefficient spectra and loss of amplitude. During US exposure at 50–100 kPa PNP we observed a temporal increase in loss of amplitude which coincided with the US delivery. Transient increase in loss of amplitude vanished at higher PNP values. At higher PNP values loss of amplitude decreased during the US exposure indicating MB sonodestruction. Analysis of transient attenuation spectra revealed that attenuation coefficient was maximal at 1.5 MHz frequency which is consistent with resonance frequency of SonoVue MB. The method allows evaluation of the of resonance frequency of MB, onset and kinetics of MB sonodestruction.