Acoustic Tweezing Cytometry
Mechanosensitivity to extracellular mechanical signals is central to many developmental, physiological, and pathological processes, affecting cell functions including growth, migration, differentiation, and apoptosis. Understanding the molecular mechanisms underlying mechanotransduction process rely on tools capable of applying controlled mechanical forces to cells to elicit and assess cellular responses.
The goal of this research is to develop a novel ultrasound-based technology, acoustic tweezing cytometry (ATC), as a cell mechanics and mechanobiology tool. In this research, we will develop and demonstrate the utility of ATC for stem cell applications, specifically to enable novel advances in human pluripotent stem cell (hPSC) maintenance/differentiation and understanding of mechanobiology of hPSCs. Capable of replicating themselves while retaining the ability to give rise to any type of specialized cells, hPSCs provide promising sources for disease modeling, drug screenings, and future cell-based therapeutics to treat degenerative diseases such as diabetes mellitus and spinal cord injury.
See publication list for our recent progress in the project.
Advanced Ultrasound Techniques for Tissue Engineering
Mechanical forces are a key component of the cellular microenvironment, and are well established to have potent effects on cells and tissues. The passive mechanical properties of two-dimensional cell substrates and three-dimensional extracellular matrices have been shown to influence progenitor cell phenotype and can be used to direct cell function. In addition, active stimulation of cells and tissues using externally applied forces has been applied at both the cell and tissue level to induce a variety of responses. Mechanobiology is particularly relevant to musculoskeletal tissues, but there is a gap in our understanding of the physical properties of the tissue environment on length scales that cells sense. This project builds on preliminary work by the project team in applying advanced ultrasound techniques to studying the microscale physical properties of engineered musculoskeletal tissues composed of cell-seeded mineralizing hydrogels. It integrates spectral ultrasound imaging (SUSI), dual-mode ultrasound elastography (DUE), and ultrasound-induced compressive stimulation. SUSI is a technique that uses the backscattered radiofrequency spectrum to derive information about the composition of a sample. DUE applies acoustic radiation force to deform hydrogels and measure their mechanical properties. Focused ultrasound-induced compression also applies acoustic pressure to mechanically stimulate tissues. A key feature of ultrasound techniques is that they are noninvasive and therefore can be used to study developing tissues over time. In addition, imaging and deformation can be applied at sub-millimeter resolution. This project will combine these advanced ultrasound techniques to create a system that can comprehensively characterize and stimulate engineered musculoskeletal tissues at the microscale. The target application is to potentiate bone formation using mesenchymal stem cells (MSC) embedded in a 3D hydrogel matrix. This project will investigate musculoskeletal mechanobiology using an innovative new tool that could have important impact on regenerative medicine. The long term goal is a therapeutic intervention to potentiate bone formation in indications where accelerated healing would lead to improved outcomes, such as treatment of non-unions and recalcitrant spinal fusions.
See publication list for our recent progress in the project.
ADVANCED ULTRASOUND ABLATION THERAPY FOR ATRIAL FIBRILLATION
AF is the most common sustained cardiac arrhythmia, characterized by uncoordinated atrial activation with consequent deterioration of atrial mechanical function. It increases a patient’s risk of stroke and has a significant negative impact on quality of life. It is a significant public health problem incurring substantial health care costs. Current pharmacotherapy of AF is temporary, expensive, and has well-recognized limitations such as relatively low efficacy and often poorly tolerated systemic side effects. The surgical Cox-maze procedure remains the gold standard to treat AF with > 90% efficacy. However, the procedure has not been gained wide spread application because the operation, requiring the creation of complex set of surgical incisions and reconstruction of the atria, is technically challenging and associated with significant morbidity and mortality. Therefore less invasive, ablation techniques have been exploited to replicate the cut-and-sew maze procedure by replacing the incisions with lines of ablation. Radiofrequency (RF), microwave, ultrasound, and cryo-therapy have been exploited for AF ablation, but current ablation technologies are not optimal for epicardial ablation, mainly due to their limited ability to create desired set of linear transmural lesions with minimum collateral damage.
The ability of ultrasound (US) to penetrate soft tissue makes it a preferred energy source for epicardial AF ablation. With its focus readily placed at depth to reach subsurface sites, high intensity focused ultrasound (HIFU) has the potential to overcome the limitations of current technologies to achieve better ablation outcome. However, despite encouraging initial development, major problems remain that have hindered the development of HIFU AF ablation. This project seeks to address these very problems in order to develop successful HIFU ablation therapy for AF by the means of intraoperative or thoracoscopic operations.
Hsiao YS, Kumon RE, Deng CX. Characterization of Lesion Formation and Bubble Activities during High Intensity Focused Ultrasound Ablation using Temperature-Derived Parameters. Infrared Phys Technol. 2013 Sep 1;60:108-117.
Successful high-intensity focused ultrasound (HIFU) thermal tissue ablation relies on accurate information of the tissue temperature and tissue status. Often temperature measurements are used to predict and monitor the ablation process. In this study, we conducted HIFU ablation experiments with ex vivo porcine myocardium tissue specimens to identify changes in temperature associated with tissue coagulation and bubble/cavity formation. Using infrared (IR) thermography and synchronized bright-field imaging with HIFU applied near the tissue surface, parameters derived from the spatiotemporal evolution of temperature were correlated with HIFU-induced lesion formation and overheating, of which the latter typically results in cavity generation and/or tissue dehydration.
Hsiao YS, Wang X, Deng CX. Dual-wavelength photoacoustic technique for monitoring tissue status during thermal treatments. J Biomed Opt. 2013 Jun;18(6):067003.
Photoacoustic (PA) techniques have been exploited for monitoring thermal treatments. However, PA signals depend not only on tissue temperature but also on tissue optical properties which indicate tissue status (e.g., native or coagulated). The changes in temperature and tissue status often occur simultaneously during thermal treatments, so both effects cause changes to PA signals. A new dual-wavelength PA technique to monitor tissue status independent of temperature is performed. By dividing the PA signal intensities obtained at two wavelengths at the same temperature, a ratio, which only depends on tissue optical properties, is obtained.
Gudur MS, Kumon RE, Zhou Y, Deng CX. High-frequency rapid B-mode ultrasound imaging for real-time monitoring of lesion formation and gas body activity during high-intensity focused ultrasound ablation.IEEE Trans Ultrason Ferroelectr Freq Control. 2012 Aug;59(8):1687-99.
The goal of this study was to examine the ability of high-frame-rate, high-resolution imaging to monitor tissue necrosis and gas-body activities formed during high-intensity focused ultrasound (HIFU) application. Ex vivo porcine cardiac tissue specimens (n = 24) were treated with HIFU exposure (4.33 MHz, 77 to 130 Hz pulse repetition frequency (PRF), 25 to 50% duty cycle, 0.2 to 1 s, 2600 W/cm(2)). RF data from B-mode ultrasound imaging were obtained before, during, and after HIFU exposure at a frame rate ranging from 77 to 130 Hz using an ultrasound imaging system with a center frequency of 55 MHz. The time history of changes in the integrated backscatter (IBS), calibrated spectral parameters, and echo-decorrelation parameters of the RF data were assessed for lesion identification by comparison against gross sections.
Laughner JI, Sulkin MS, Wu Z, Deng CX, Efimov IR. Three potential mechanisms for failure of high intensity focused ultrasound ablation in cardiac tissue. Circ Arrhythm Electrophysiol. 2012 Apr;5(2):409-16.
High intensity focused ultrasound (HIFU) has been introduced for treatment of cardiac arrhythmias because it offers the ability to create rapid tissue modification in confined volumes without directly contacting the myocardium. In spite of the benefits of HIFU, a number of limitations have been reported, which hindered its clinical adoption. In this study, we used a multimodal approach to evaluate thermal and nonthermal effects of HIFU in cardiac ablation. We designed a computer controlled system capable of simultaneous fluorescence mapping and HIFU ablation. This study identified 3 potential mechanisms responsible for the failure of HIFU ablation in cardiac tissues. Both acoustic radiation force and acoustic cavitation, in conjunction with inconsistent thermal deposition, can increase the risk of lesion discontinuity and result in gap sizes that promote ablation failure.
Deng CX, Qu F, Nikolski VP, Zhou Y, Efimov IR. Fluorescent real-time monitoring of cardiac focal ablation with HIFU in vitro. Annals of Biomedical Engineering. 2005; 33: 1417-1424.
Studies of high intensity focused ultrasound (HIFU) for cardiac arrhythmia ablation therapy have been restricted to conventional approaches to assess only tissue necrosis after thermal damage. In this paper, we proposed and demonstrated the innovative strategy using spatio-temporal cardiac action potentials measurements as a more relevant and functional metric for monitoring HIFU cardiac ablation. This unique study related for the first time the cardiac electrophysiological changes with HIFU dose directly.
MECHANISMS OF ULTRASOUND MEDIATED INTRACELLULAR DRUG AND GENE DELIVERY
There exists a widely recognized need to develop methods to achieve targeted delivery of drugs and to improve the methods of gene delivery for gene-based therapy of numerous diseases. As ultrasound exposure is safe and non-invasive, and allows targeted application both temporarily and spatially, ultrasound mediated delivery has the potential to provide an advantageous strategy especially for in vivo clinical applications to overcome the limitations of safety concerns, possibly mutagenesis and immune responses associated with methods such as electroporation and viral transfection.
It has been demonstrated that ultrasound application results in enhanced intracellular uptake of chemotherapeutic compounds, genetic materials, nanoparticles, and fluorescent dextran molecules, which are normally not permeable through intact cell membrane. The hypothesis is that sonoporation, during which pores form in the cell membrane as the result of ultrasound exposure, allowing entry of extracellular molecules and substances into the cell before resealing. However, despite of the recent progress made in the field, the mechanisms of sonoporation are not completely understood and many problems and challenges remain to improve delivery efficiency and cell survival rate.
The long term goal of this research is to develop robust and reliable ultrasound strategy for intracellular delivery of desirable agents (e.g. drugs, genes, imaging markers) for biomedical applications such as targeted cancer treatment, molecular imaging, and gene therapy. In particular, we focused on investigating the mechanisms of sonoporation by studying the dynamic processes of sonoporation at both the single cell level and the cellular level based on a large number of statistical events.
Fan Z, Sun Y, Di Chen, Tay D, Chen W, Deng CX, Fu J. Acoustic tweezing cytometry for live-cell subcellular modulation of intracellular cytoskeleton contractility. Sci Rep. 2013 Jul 12;3:2176
Mechanical forces are critical to modulate cell spreading, contractility, gene expression, and even stem cell differentiation. Yet, existing tools that can apply controllable subcellular forces to a large number of single cells simultaneously are still limited. Here we report a novel ultrasound tweezing cytometry utilizing ultrasound pulses to actuate functionalized lipid microbubbles covalently attached to single live cells to exert mechanical forces in the pN – nN range. Ultrasonic excitation of microbubbles could elicit a rapid and sustained reactive intracellular cytoskeleton contractile force increase in different adherent mechanosensitive cells. Further, ultrasound-mediated intracellular cytoskeleton contractility enhancement was dose-dependent and required an intact actin cytoskeleton as well as RhoA/ROCK signaling. Our results demonstrated the great potential of ultrasound tweezing cytometry technique using functionalized microbubbles as an actuatable, biocompatible, and multifunctional agent for biomechanical stimulations of cells.
Fan Z, Liu H, Mayer M, Deng CX. Spatiotemporally controlled single cell sonoporation. Proc Natl Acad Sci U S A. 2012 Oct 9;109(41):16486-91.
This paper presents unique approaches to enable control and quantification of ultrasound-mediated cell membrane disruption, or sonoporation, at the single-cell level. Ultrasound excitation of microbubbles that were targeted to the plasma membrane of HEK-293 cells generated spatially and temporally controlled membrane disruption with high repeatability. Using whole-cell patch clamp recording combined with fluorescence microscopy, we obtained time-resolved measurements of single-cell sonoporation and quantified the size and resealing rate of pores. We measured the intracellular diffusion coefficient of cytoplasmic RNA/DNA from sonoporation-induced transport of an intercalating fluorescent dye into and within single cells. We achieved spatiotemporally controlled delivery with subcellular precision and calcium signaling in targeted cells by selective excitation of microbubbles. Finally, we utilized sonoporation to deliver calcein, a membrane-impermeant substrate of multidrug resistance protein-1 (MRP1), into HEK-MRP1 cells, which overexpress MRP1, and monitored the calcein efflux by MRP1. This approach made it possible to measure the efflux rate in individual cells and to compare it directly to the efflux rate in parental control cells that do not express MRP1.
Fan Z, Kumon RE, Park J, Deng CX. Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles. J Control Release. 2010 Feb 25;142(1):31-9.
Ultrasound application in the presence of microbubbles is a promising strategy for intracellular drug and gene delivery, but it may also trigger other cellular responses. This study investigates the relationship between the change of cell membrane permeability generated by ultrasound-driven microbubbles and the changes in intracellular calcium concentration ([Ca(2+)](i)). Cultured rat cardiomyoblast (H9c2) cells were exposed to a single ultrasound pulse (1MHz, 10-15cycles, 0.27MPa) in the presence of a Definity(TM) microbubble. Intracellular transport via sonoporation was assessed in real time using propidium iodide (PI), while [Ca(2+)](i) and dye loss from the cells were measured with preloaded fura-2. The ultrasound exposure generated fragmentation or shrinking of the microbubble. Only cells adjacent to the ultrasound-driven microbubble exhibited propidium iodide uptake with simultaneous [Ca(2+)](i) increase and fura-2 dye loss. The amount of PI uptake was correlated with the amount of fura-2 dye loss. Cells with delayed [Ca(2+)](i) transients from the time of ultrasound application had no uptake of PI. These results indicate the formation of non-specific pores in the cell membrane by ultrasound-stimulated microbubbles and the generation of calcium waves in surrounding cells without pores.
Zhou Y, Shi J, Cui J, Deng CX. Effects of extracellular calcium on cell membrane resealing in sonoporation. Journal of Controlled Release 2008; 126 (1):34-43.
Sonoporation has been exploited as a promising strategy for intracellular drug and gene delivery. The technique uses ultrasound to generate pores on the cell membrane to allow entry of extracellular agents into the cell. Resealing of these non-specific pores is a key factor determining both the uptake and post-ultrasound cell survival. This study examined the effects of extracellular Ca(2+) on membrane resealing in sonoporation, using Xenopus oocytes as a model system. The cells were exposed to tone burst ultrasound (1.06 MHz, duration 0.2 s, acoustic pressure 0.3 MPa) in the presence of 0.1% Definity at various extracellular [Ca(2+)] (0-3 mM). Sonoporation inception and resealing in a single cell were monitored in real time via the transmembrane current of the cell under voltage clamp. The time-resolved measurements of transmembrane current revealed the involvement of two or more Ca(2+) related processes with different rate constants and characteristics. Rapid resealing occurred immediately after ultrasound application followed by a much slower resealing process. Complete resealing required [Ca(2+)] above 0.54 mM. The cells resealed in 6-26 s at 1.8 mM Ca(2+), but took longer at lower concentrations, up to 58-170 s at 0.54 mM Ca(2+).
Kumon RE, Aehle M, Sabens D, Parikh P, Kourennyi D, Deng CX. Ultrasound-induced calcium oscillations and waves in chinese hamster ovary cells in the presence of microbubbles. Biophys J 2007; 93 (6): L29-31.
Demonstrating the novel usage of real-time fluorescent calcium (Ca2+) imaging for sonoporation study, we showed that ultrasound (US) can induce specific intracellular Ca2+ transients in targeted cells. The Ca2+ transients include intracellular Ca2+ increase/recovery and periodic oscillation. We also found that the Ca2+ events are spreading from cells to the neighboring cells forming the so-called Ca2+ waves involved in intercellular signal transduction. These unique findings could have important implications beyond sonoporation as Ca2+ is an important signaling ion involved in many cellular functions/processes.
Deng CX, Sieling F, Pan H, Cui J. Ultrasound induced cell-membrane porosity. Ultrasound Medicine and Biology 2004; 30:519-526.
This is the first of a series of papers from our laboratory reporting novel results obtained by innovatively applying voltage clamp techniques for real-time measurement of US-generated cell membrane poration (sonoporation) at the single cell level. We obtained measurements of the transient and sub-micron scale pores generated by US. Distinctly different from typical studies in the field, this work reveals new information that was not available before yet useful for US mediated intracellular drug and gene delivery.
QUANTITATIVE ENDOSCOPIC ULTRASOUND IMAGING
The goal of this project is to develop improved endoscopic ultrasound (EUS) imaging techniques for pancreatic cancer detection or other gastrointestinal (GI) applications. Working with practicing physicians and clinical EUS scanners, we have developed imaging processing algorithms and conducted pilot and validation studies with groups of enrolled patients. We are continuing the development and implementation of the techniques in the digital EUS systems and performing additional studies with a larger population of patients, in addition to working on systematic studies focusing on the fundamentals of US imaging of tissue microstructures.
Kumon RE, Pollack MJ, Faulx AL, Olowe K, Farooq FT, Chen VK, Zhou Y, Wong RCK, Isenberg GA, Sivak, MV, Chak A, Deng CX. In vivo characterization of pancreatic and lymph node tissue using endoscopic ultrasound spectrum analysis: Validation study. Gastrointestinal Endoscopy 2009; 71(1): 53-63
In this follow-up study, our purpose was to validate RF spectral analysis as a method to distinguish between (1) benign and malignant lymph nodes and (2) normal pancreas, chronic pancreatitis, and pancreatic cancer. Midband fit, slope, intercept, and correlation coefficient from a linear regression of the calibrated RF power spectra were determined. Discriminant analysis of mean pilot-study parameters was then performed to classify validation-study parameters. For benign versus malignant lymph nodes, midband fit and intercept provided classification with 67% accuracy and area under the receiver operating curve (AUC) of 0.86. For diseased versus normal pancreas, midband fit and correlation coefficient provided 93% accuracy and an AUC of 0.98. For pancreatic cancer versus chronic pancreatitis, the same parameters provided 77% accuracy and an AUC of 0.89. Results improved further when classification was performed with all data. This study confirms that mean spectral parameters provide a noninvasive method to quantitatively discriminate benign and malignant lymph nodes as well as normal and diseased pancreas.
Kumon RE, Olowe K, Farooq FT, Chen V, Zhou Y, Faulx A, Iseenberg GA, Sivak MV, Chak A, Deng CX. EUS spectrum analysis for in vivo tissue characterization in the pancreas and intra-abdominal and mediastinal lymph nodes: A pilot study. Gastrointestinal Endoscopy 2007; 66(6): 1096-1106.
EUS is limited by variability in the examiner’s subjective interpretation of B-scan images to differentiate among normal, inflammatory, and malignant tissue. By using information otherwise discarded by conventional EUS systems, quantitative spectral analysis of the raw radiofrequency (RF) signals underlying EUS images enables tissue to be characterized more objectively. In this paper, linear regression parameters of calibrated power spectra of the RF signals were tested to differentiate normal pancreas from chronic pancreatitis and from pancreatic cancer as well as benign from malignant-appearing lymph nodes. The mean intercept, slope, and midband fit of the spectra differed significantly among normal pancreas, adenocarcinoma, and chronic pancreatitis when all were compared with each other (p < 0.01). On direct comparison, mean midband fit for adenocarcinoma differed significantly from that for chronic pancreatitis (p < 0.05). For lymph nodes, mean midband fit and intercept differed significantly between benign- and malignant-appearing lymph nodes (p < 0.01 and p < 0.05, respectively).