Preparation of PANI/CuPc/PDMS Composite Elastomer with High Dielectric Constant and Low Modulus Assisted by Electric Fields.

Dielectric elastomer is a kind of electronic electroactive polymer, which plays an important role in the application of soft robots and flexible electronics. In this study, an all-organic polyaniline/copper phthalocyanine/silicone rubber (PANI/CuPc/PDMS) dielectric composite with superior comprehensive properties was prepared by manipulating the arrangement of filler in a polymer matrix assisted by electric fields. Both CuPc particles and PANI particles can form network structures in the PDMS matrix by self-assembly under electric fields, which can enhance the dielectric properties of the composites at low filler content. The dielectric constant of the assembled PANI/CuPc/PDMS composites can reach up to 140 at 100 Hz when the content of CuPc and PANI particles is 4 wt% and 2.5 wt%, respectively. Moreover, the elastic modulus of the composites remains below 2 MPa, which is important for electro-deforming. The strain of assembled PANI/CuPc/PDMS three-phase composites at low electric field strength (2 kV/mm) can increase up to five times the composites with randomly dispersed particles, which makes this composite have potential application in the field of soft robots and flexible electronics.

Considering Joule heating in coupled electroporation and electrodeformation modeling of glioblastoma cells.

Cells exposed to a pulsed electric field undergo electroporation(EP) and electrodeformation(ED) under electric field stress, and a coupled model of EP and ED of glioblastoma(GBM) taking into account Joule heating is proposed. The model geometry is extracted from real cell boundaries, and the effects of Joule heating-induced temperature rise on the EP and ED processes are considered. The results show that the temperature rise will increase the cell's local conductivity, leading to a decrease in the transmembrane potential(TMP). The temperature rise also causes a decrease in the dynamic Young's modulus of the cell membrane, making the cell less resistant to deformation. In addition, GBM cells are more susceptible to EP in the middle portion of the cell and ED in the three tentacle portions under pulsed electric fields, and the cells undergo significant positional shifts. The ED of the nucleus is similar to spherical cells, but the degree of ED is smaller.

Methods to mechanically perturb and characterize GUV-based minimal cell models.

Cells shield organelles and the cytosol via an active boundary predominantly made of phospholipids and membrane proteins, yet allowing communication between the intracellular and extracellular environment. Micron-sized liposome compartments commonly known as giant unilamellar vesicles (GUVs) are used to model the cell membrane and encapsulate biological materials and processes in a cell-like confinement. In the field of bottom-up synthetic biology, many have utilized GUVs as substrates to study various biological processes such as protein-lipid interactions, cytoskeletal assembly, and dynamics of protein synthesis. Like cells, it is ideal that GUVs are also mechanically durable and able to stay intact when the inner and outer environment changes. As a result, studies have demonstrated approaches to tune the mechanical properties of GUVs by modulating membrane composition and lumenal material property. In this context, there have been many different methods developed to test the mechanical properties of GUVs. In this review, we will survey various perturbation techniques employed to mechanically characterize GUVs.

Thin-layer approximation for the multi-physics and multiscale simulation of cell membrane electrodeformation.

Multi-physics simulation techniques provide a platform that is used to gain insights into complex biological problems with multiple length scales such as cell electrodeformation (ED) and electropermeabilization (EP). However, owing to the large degrees of freedom required to compute the electromechanical properties at very different length scales (membrane thickness, cell size, and customized tissue scaffold) finite element (FE) simulations can be computationally very expensive. Here, we report on a general method of analysis by which we can systematically simulate multiscale ED under direct-current electric fields. In the context of electromechanical continuum behavior, the key novelty of our work is the introduction of a specific Dirichlet boundary condition, i.e. thin-layer approximation (TLA), to represent the capacitive elastic cell membrane. To test the robustness of this newly proposed procedure, Maxwell stress tensor (MST) and cell displacement arising from ED forces obtained with the TLA are compared with a model using a physical thickness of the cell membrane. Furthermore, we present our results in terms of benchmark points for vesicle deformation induced by an electric field excitation and we confirm our approximate results are relevant to predict the aspect ratio characterizing the ellipsoidal deformation of an initially spherical vesicle.

Recent advancements to measure membrane mechanical and transport properties.

The natural vesicles, microscopic spherical structures defined by a single or many lipid bilayer membranes, not only entrap but are also dispersed in the aqueous environment. The space division between inner and outer compartments is also the basic characteristics of cell membranes playing several essential functions in all living organisms. Thus, vesicles are a simple model system for studying various cellular properties. In the last few decades, synthetic vesicles (or liposomes) have gained substantial popularity from many academia as model membranes and from many pharmaceutical industries as targeted and controlled drug delivery systems. The manufacturing of vesicles with desired characteristics that can entrap and release the drugs as required is one of the major challenges in this research area. To this end, a better understanding of the mechanical and transport properties of vesicles is essential to gain deeper insight into the fundamental biological mechanisms of vesicle formation and cellular uptake. The requirement has brought the modifications in membrane composition (with cholesterol, charged lipid, proteins, peptides, polymers, etc.) and solution conditions (with salts, pH, buffers, etc.). This article mainly focuses on the different techniques developed for studying the mechanical and transport properties of natural/synthetic vesicles. In particular, I thoroughly review the properties such as bending and stretching elastic moduli, lysis tension, and permeability of vesicle membranes.

Electromechanical characterization of biomimetic membranes using electrodeformation of vesicles.

We describe a facile method to simultaneously measure the bending rigidity and capacitance of biomimetic lipid bilayers. Our approach utilizes the ellipsoidal deformation of quasi-spherical giant unilamellar vesicles induced by a uniform AC electric field. Vesicle shape depends on the electric field frequency and amplitude. Membrane bending rigidity can be obtained from the variation of the vesicle elongation on either field amplitude at fixed frequency or frequency at fixed field amplitude. Membrane capacitance is determined from the frequency at which the vesicle shape changes from prolate to oblate ellipsoid as the frequency is increased at a given field amplitude.

Preparation and Characterization of CCTO/PDMS Dielectric Elastomers with High Dielectric Constant and Low Dielectric Loss.

Dielectric elastomer (DE) is a type of electric field type electroactive polymer material that can produce greater deformation under the action of an electric field and has a faster recovery speed. It has the advantages of high energy density, large strain, low quality, and commercialization, and has become the most widely concerned and researched electroactive polymer material. In this study, copper calcium titanate (CCTO) particles with a large dielectric constant were selected as the filling phase, and a silicone rubber (PDMS) with better biocompatibility and lower elastic modulus was used as the matrix to prepare CCTO/PDMS, which is a new type of dielectric elastomer material. The structure of the dielectric elastomer is analyzed, and its mechanical properties, dielectric properties, and driving deformation are tested. Then, KH550, KH560, and KH570 modified CCTO is used in order to improve the dispersibility of CCTO in PDMS, and modified particles with the best dispersion effect are selected to prepare dielectric elastomer materials. In addition, mechanical properties, dielectric properties, and driving deformation are tested and compared with the dielectric elastomer material before modification. The results show that as the content of CCTO increases, the dielectric constant and elastic modulus of the dielectric elastomer also increase, and the dielectric loss remains basically unchanged at a frequency of 100 Hz. When the filling amount reaches 20 wt%, the dielectric constant of the CCTO/PDMS dielectric elastomer reaches 5.8 (100 Hz), an increase of 120%, while the dielectric loss at this time is only 0.0038 and the elastic modulus is only 0.54 MPa. When the filling amount is 5 wt%, the dielectric elastomer has the largest driving deformation amount, reaching 33.8%. Three silane coupling agents have been successfully grafted onto the surface of CCTO particles, and the KH560 modified CCTO has the best dispersibility in the PDMS matrix. Based on this, a modified CCTO/PDMS dielectric elastomer was prepared. The results show that the improvement of dispersibility improves the dielectric constant. Compared with the unmodified PDMS, when the filling content is 20 wt%, the dielectric constant reaches 6.5 (100 Hz). Compared with PDMS, it has increased by 150%. However, the improvement of dispersion has a greater increase in the elastic modulus, resulting in a decrease in its strain parameters compared with CCTO/PDMS dielectric elastomers, and the electromechanical conversion efficiency has not been significantly improved. When the filling amount of modified CCTO particles is 5 wt%, the dielectric elastomer has the largest driving deformation, reaching 27.4%.

Proximity-induced electrodeformation and membrane capacitance coupling between cells.

Membrane capacitance and transmembrane potential are sensitive to the proximity of neighboring biological cells which eventually induces anisotropic perturbation of the local electric field distribution in a cell assembly and/or a tissue. The development of robust and reliable multiphysics approaches is essential to solve the challenge of analyzing proximity-induced capacitance coupling (CC) between cells. In this study, we ask to what extent this CC is a minor perturbation on the individual cells or whether it can fundamentally affect bio-electromechanical cues. A key component of our continuum electromechanical analysis is the consideration of elastic models of cells under steady state electric field excitation to characterize electrodeformation (ED). Analyzing the difference between the ED force for a pair of cells and its counterpart for a single reference cell allows us to determine a separation distance-orientation angle diagram providing evidence of a separation distance beyond which the electrostatic interactions between a pair of biological cells become inconsequential for the ED. An attenuation-amplification transition of ED force in this diagram suggests that anisotropy induced by the orientation angle of the cell pair relative to the applied electric field direction has a significant influence on ED and CC. We furthermore observe that the shape of this diagram changes when extracellular conductivity is varied. The results obtained are then contrasted with the corresponding diagrams of similar cell configurations under an oscillating electric field excitation below and above the α-dispersion frequency. This investigation may provide new opportunities for further assessment of electromechanical properties of engineered tissues.

Active probing of the mechanical properties of biological and synthetic vesicles.

BACKGROUND: The interest in mechanics of synthetic and biological vesicles has been continuously growing during the last decades. Liposomes serve as model systems for investigating fundamental membrane processes and properties. More recently, extracellular vesicles (EVs) have been investigated mechanically as well. EVs are widely studied in fundamental and applied sciences, but their material properties remained elusive until recently. Elucidating the mechanical properties of vesicles is essential to unveil the mechanisms behind a variety of biological processes, e.g. budding, vesiculation and cellular uptake mechanisms. SCOPE OF REVIEW: The importance of mechanobiology for studies of vesicles and membranes is discussed, as well as the different available techniques to probe their mechanical properties. In particular, the mechanics of vesicles and membranes as obtained by nanoindentation, micropipette aspiration, optical tweezers, electrodeformation and electroporation experiments is addressed. MAJOR CONCLUSIONS: EVs and liposomes possess an astonishing rich, diverse behavior. To better understand their properties, and for optimization of their applications in nanotechnology, an improved understanding of their mechanical properties is needed. Depending on the size of the vesicles and the specific scientific question, different techniques can be chosen for their mechanical characterization. GENERAL SIGNIFICANCE: Understanding the mechanical properties of vesicles is necessary to gain deeper insight in the fundamental biological mechanisms involved in vesicle generation and cellular uptake. This furthermore facilitates technological applications such as using vesicles as targeted drug delivery vehicles. Liposome studies provide insight into fundamental membrane processes and properties, whereas the role and functioning of EVs in biology and medicine are increasingly elucidated.

Mechanical characterization of vesicles and cells: A review.

Vesicles perform many essential functions in all living organisms. They respond like a transducer to mechanical stress in converting the applied force into mechanical and biological responses. At the same time, both biochemical and biophysical signals influence the vesicular response in bearing mechanical loads. In recent years, liposomes, artificial lipid vesicles, have gained substantial attention from the pharmaceutical industry as a prospective drug carrier which can also serve as an artificial cell-mimetic system. The ability of these vesicles to enter through pores of even smaller size makes them ideal candidates for therapeutic agents to reach the infected sites effectively. Engineering of vesicles with desired mechanical properties that can encapsulate drugs and release as required is the prime challenge in this field. This requirement has led to the modifications of the composition of the bilayer membrane by adding cholesterol, sphingomyelin, etc. In this article, we review the manufacturing and characterization techniques of various artificial/synthetic vesicles. We particularly focus on the electric field-driven characterization techniques to determine different properties of vesicle and its membranes, such as bending rigidity, viscosity, capacitance, conductance, etc., which are indicators of their content and mobility. Similarities and differences between artificial vesicles, natural vesicles, and cells are highlighted throughout the manuscript since most of these artificial vesicles are intended for cell mimetic functions.

Effects of electrically-induced constant tension on giant unilamellar vesicles using irreversible electroporation.

Stretching in membranes of cells and vesicles plays important roles in various physiological and physicochemical phenomena. Irreversible electroporation (IRE) is the irreversible permeabilization of the membrane through the application of a series of electrical field pulses of micro- to millisecond duration. IRE induces lateral tension due to stretching in the membranes of giant unilamellar vesicles (GUVs). However, the effects of electrically induced (i.e., IRE) constant tension in the membranes of GUVs have not been investigated yet in detail. To explore the effects of electrically induced tension on GUVs, firstly a microcontroller-based IRE technique is developed which produces electric field pulses (332 V/cm) with pulse width 200 µs. Then the electrodeformation, electrofusion and membrane rupture of GUVs are investigated at various constant tensions in which the membranes of GUVs are composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). Stochastic electropore formation is observed in the membranes at an electrically induced constant tension in which the probability of pore formation is increased with the increase of tension from 2.5 to 7.0 mN/m. The results of pore formation at different electrically-induced constant tensions are in agreement with those reported for mechanically-induced constant tension. The decrease in the energy barrier of the pre-pore state due to the increase of electrically-induced tension is the main factor increasing the probability of electropore formation. These investigations help to provide an understanding of the complex behavior of cells/vesicles in electric field pulses and can form the basis for practical applications in biomedical technology.

Electrophoretic transport and dynamic deformation of bio-vesicles.

Study of the deformation dynamics of cells and other sub-micron vesicles, such as virus and neurotransmitter vesicles are necessary to understand their functional properties. This mechanical characterization can be done by submerging the vesicle in a fluid medium and deforming it with a controlled electric field, which is known as electrodeformation. Electrodeformation of biological and artificial lipid vesicles is directly influenced by the vesicle and surrounding media properties and geometric factors. The problem is compounded when the vesicle is naturally charged, which creates electrophoretic forcing on the vesicle membrane. We studied the electrodeformation and transport of charged vesicles immersed in a fluid media under the influence of a DC electric field. The electric field and fluid-solid interactions are modeled using a hybrid immersed interface-immersed boundary technique. Model results are verified with experimental observations for electric field driven translocation of a virus through a nanopore sensor. Our modeling results show interesting changes in deformation behavior with changing electrical properties of the vesicle and the surrounding media. Vesicle movement due to electrophoresis can also be characterized by the change in local conductivity, which can serve as a potential sensing mechanism for electrodeformation experiments in solid-state nanopore setups.

Electrodeformation of Vesicles Suspended in a Liquid Medium.

Deformation of flexible vesicles suspended in a fluid medium due to an applied electric field can provide valuable insight into deformation dynamics at a very small scale. In an electric field, the response of the vesicle membrane is strongly influenced by the conductivity of surrounding fluid, vesicle size and shape, and the magnitude of applied field. We studied the electrodeformation of vesicles immersed in a fluid media under a DC electric field. An immersed interface method is used to solve the electric field over the domain with conductive or non-conductive vesicles while an immersed boundary method is employed to solve fluid flow, fluid-solid interaction, membrane mechanics and vesicle deformation. Initial force analysis on the membrane surface reveals almost linear influence of vesicle size, but the vesicle size does not affect the long-term deformation which is consistent with experimental evidence. Highly nonlinear effect of the applied field as well as the conductivity ratios inside and outside of the vesicle are observed. Results also point towards an early linear deformation regime followed by an equilibrium stage for the membranes. Modeling results suggest that electrodeforming vesicles can create unique external flows for different conductivity ratios. Moreover, significant influence of the initial aspect ratio of the vesicle on the force distribution is observed across a range of conductivity ratios.

The Effect of Nanosecond, High-Voltage Electric Pulses on the Shape and Permeability of Polymersome GUVs.

Polymersomes, vesicles composed of block copolymers, are promising candidates as membrane alternatives and functional containers, e.g., as potential carriers for functional molecules because of their stability and tunable membrane properties. In the scope of possible use for membrane protein delivery to cells by electrofusion, we investigated the cytotoxicity of such polymersomes as well as the effects of nanosecond electric pulses with variable repetition rate on the shape and permeability of polymersomes in buffers with different conductivities. The polymersomes did not show cytotoxic effects to CHO and B16-F1 cells in vitro in concentrations up to 250 µg/mL (for 48 h) or 1.35 mg/mL (for 60 min), which renders them suitable for interacting with living cells. We observed a significant effect of the pulse repetition rate on electrodeformation of the polymersomes. The electrodeformation was most pronounced in low conductivity buffer, which is favorable for performing electrofusion with cells. However, despite more pronounced deformation at higher pulse repetition rate, the electroporation performance of polymersomes was unaffected and remained in similar ranges both at 10 Hz and 10 kHz. This phenomenon is possibly due to the higher stability and rigidity of polymer vesicles, compared to liposomes, and can serve as an advantage (or disadvantage) depending on the aim in employing polymersomes such as stable membrane alternative architectures or drug vehicles.

Electro-Deformation of Fused Cells in a Microfluidic Array Device.

We present a new method of analyzing the deformability of fused cells in a microfluidic array device. Electrical stresses-generated by applying voltages (4⁻20 V) across discrete co-planar microelectrodes along the side walls of a microfluidic channel-have been used to electro-deform fused and unfused stem cells. Under an electro-deformation force induced by applying an alternating current (AC) signal, we observed significant electro-deformation phenomena. The experimental results show that the fused stem cells were stiffer than the unfused stem cells at a relatively low voltage (<16 V). However, at a relatively high voltage, the fused stem cells were more easily deformed than were the unfused stem cells. In addition, the electro-deformation process is modeled based on the Maxwell stress tensor and structural mechanics of cells. The theoretical results show that a positive correlation is found between the deformation of the cell and the applied voltage, which is consistent with the experimental results. Combined with a numerical analysis and experimental study, the results showed that the significant difference of the deformation ratio of the fused and unfused cells is not due to their size difference. This demonstrates that some other properties of cell membranes (such as the membrane structure) were also changed in the electrofusion process, in addition to the size modification of that process.

Dielectrophoresis-Mediated Electrodeformation as a Means of Determining Individual Platelet Stiffness.

Platelets, essential for hemostasis, are easily activated via biochemical and mechanical stimuli. Cell stiffness is a vital parameter modulating the mechano-transduction of exogenous mechanical stimuli. While methods exist to measure cell stiffness, no ready method exists for measuring platelet stiffness that is both minimally-contacting, imparting minimal exogenous force and non-activating. We developed a minimal-contact methodology capable of trapping and measuring the stiffness of individual platelets utilizing dielectrophoresis (DEP)-mediated electrodeformation. Parametric studies demonstrate a non-uniform electric field in the MHz frequency range (0.2-20 MHz) is required for generating effective DEP forces on platelets, suspended in isotonic buffer with conductivity ~100-200 µS/cm. A nano-Newton DEP force (0.125-4.5 nN) was demonstrated to be essential for platelet electrodeformation, which could be generated with an electric field with strength of 1.5-9 V/µm. Young's moduli of platelets were calculated using a Maxwell stress tensor model and stress-deformation relationship. Platelet stiffness was determined to be in the range of 3.5 ± 1.4 and 8.5 ± 1.5 kPa for resting and 0.4% paraformaldehyde-treated cells, respectively. The developed methodology fills a gap in approaches of measuring individual platelet stiffness, free of inadvertent platelet activation, which will facilitate further studies of mechanisms involved in mechanically-mediated platelet activation.

Recent developments in the field of bending rigidity measurements on membranes.

This review gives a brief overview of experimental approaches used to assess the bending rigidity of membranes. Emphasis is placed on techniques based on the use of giant unilamellar vesicles. We summarize the effect on the bending rigidity of membranes as a function of membrane composition, presence of various inclusions in the bilayer and molecules and ions in the bathing solutions. Examples for the impact of temperature, cholesterol, some peptides and proteins, sugars and salts are provided and the literature data are discussed critically. Future directions, open questions and possible developments in this research field are also included.

Frequency-dependent electrodeformation of giant phospholipid vesicles in AC electric field.

A model of vesicle electrodeformation is described which obtains a parametrized vesicle shape by minimizing the sum of the membrane bending energy and the energy due to the electric field. Both the vesicle membrane and the aqueous media inside and outside the vesicle are treated as leaky dielectrics, and the vesicle itself is modeled as a nearly spherical shape enclosed within a thin membrane. It is demonstrated (a) that the model achieves a good quantitative agreement with the experimentally determined prolate-to-oblate transition frequencies in the kilohertz range and (b) that the model can explain a phase diagram of shapes of giant phospholipid vesicles with respect to two parameters: the frequency of the applied alternating current electric field and the ratio of the electrical conductivities of the aqueous media inside and outside the vesicle, explored in a recent paper (S. Aranda et al., Biophys J 95:L19-L21, 2008). A possible use of the frequency-dependent shape transitions of phospholipid vesicles in conductometry of microliter samples is discussed.