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Polycationic carriers promise low cost and scalable gene therapy treatments, however inefficient intracellular unpacking of the genetic cargo has limited transfection efficiency. Charge-reversing polycations, which transition from cationic to neutral or negative charge, can offer targeted intracellular DNA release. We describe a new class of charge-reversing polycation which undergoes a cationic-to-neutral conversion by a reaction with cellular nucleophiles. The deionization reaction is relatively slow with primary amines, and much faster with thiols. In mammalian cells, the intracellular environment has elevated concentrations of amino acids (~10x) and the thiol glutathione (~1000x). We propose this allows for decationization of the polymeric carrier slowly in the extracellular space and then rapidly in the intracellular milleu for DNA release. We demonstrate that in a lipopolyplex formulation this leads to both improved transfection and reduced cytotoxicity when compared to a non-responsive polycationic control.

A functional vascular system is a prerequisite for bone repair as disturbed angiogenesis often causes non-union. Paracrine factors released from human bone marrow derived mesenchymal stromal cells (BMSCs) have angiogenic effects on endothelial cells. However, whether these paracrine factors participate in blood flow dynamics within bone capillaries remains poorly understood. Here, we used two different microfluidic designs to investigate critical steps during angiogenesis and found pronounced effects of endothelial cell proliferation as well as chemotactic and mechanotactic migration induced by BMSC conditioned medium (CM). The application of BMSC-CM in dynamic cultures demonstrates that bioactive factors in combination with fluidic flow-induced biomechanical signals significantly enhanced endothelial cell migration. Transcriptional analyses of endothelial cells demonstrate the induction of a unique gene expression profile related to tricarboxylic acid cycle and energy metabolism by the combination of BMSC-CM factors and shear stress, which opens an interesting avenue to explore during fracture healing. Our results stress the importance of in vivo - like microenvironments simultaneously including biochemical, biomechanical and oxygen levels when investigating key events during vessel repair. Statement of significance: Our results demonstrate the importance of recapitulating in vivo - like microenvironments when investigating key events during vessel repair. Endothelial cells exhibit enhanced angiogenesis characteristics when simultaneous exposing them to hMSC-CM, mechanical forces and biochemical signals simultaneously. The improved angiogenesis may not only result from the direct effect of growth factors, but also by reprogramming of endothelial cell metabolism. Moreover, with this model we demonstrated a synergistic impact of mechanical forces and biochemical factors on endothelial cell behavior and the expression of genes involved in the TCA cycle and energy metabolism, which opens an interesting new avenue to stimulate angiogenesis during fracture healing.

Cell spheroids are in vitro multicellular model systems that mimic the crowded micro-environment of biological tissues. Their mechanical characterization can provide valuable insights in how single-cell mechanics and cell-cell interactions control tissue mechanics and self-organization. However, most measurement techniques are limited to probing one spheroid at a time, require specialized equipment and are difficult to handle. Here, we developed a microfluidic chip that follows the concept of glass capillary micropipette aspiration in order to quantify the viscoelastic behavior of spheroids in an easy-to-handle, more high-throughput manner. Spheroids are loaded in parallel pockets via a gentle flow, after which spheroid tongues are aspirated into adjacent aspiration channels using hydrostatic pressure. After each experiment, the spheroids are easily removed from the chip by reversing the pressure and new spheroids can be injected. The presence of multiple pockets with a uniform aspiration pressure, combined with the ease to conduct successive experiments, allows for a high throughput of tens of spheroids per day. We demonstrate that the chip provides accurate deformation data when working at different aspiration pressures. Lastly, we measure the viscoelastic properties of spheroids made of different cell lines and show how these are consistent with previous studies using established experimental techniques. In summary, our chip provides a high-throughput way to measure the viscoelastic deformation behavior of cell spheroids, in order to mechanophenotype different tissue types and examine the link between cell-intrinsic properties and overall tissue behavior.

The surface topography of implantable devices is of crucial importance for guiding the cascade of events that starts from the initial contact of the cells with the surface and continues until the complete integration of the device in its immediate environment. There is, however, limited quantitative information available regarding the relationships between the different stages of such cascade(s) and how the design of surface topography influences them. We, therefore, used direct laser writing to 3D-print submicron pillars with precisely controlled dimensions and spatial arrangements to perform a systematic study of such relationships. Using single-cell force spectroscopy, we measured the adhesion force and the work of adhesion of the preosteoblast cells residing on the different types of surfaces. Not only the adhesion parameters (after 2-60 s) but also the formation of focal adhesions was strongly dependent on the geometry and arrangement of the pillars: sufficiently tall and dense pillars enhanced both adhesion parameters and the formation of focal adhesions. Our morphological study of the cells (after 24 h) showed that those enhancements were associated with a specific way of cell settlement onto the surface (i.e., "top state"). The cells interacting with tall and dense pillars were also characterized by numerous thick actin stress fibers in the perinuclear region and possibly high internal stresses. Furthermore, living cells with highly organized cytoskeletal networks exhibited greater values of the elastic modulus. The early responses of the cells predicted their late response including matrix mineralization: tall and dense submicron pillars significantly upregulated the expression of osteopontin after 21 days of culture under both osteogenic and nonosteogenic conditions. Our findings paint a detailed picture of at least one possible cascade of events that starts from initial cell adhesion and continues to subsequent cellular functions and eventual matrix mineralization. These observations could inform the future developments of instructive surfaces for medical devices based on physical surface cues and early markers.

An early step of metastasis requires a complex and coordinated migration of invasive tumor cells into the surrounding tumor microenvironment (TME), which contains extracellular matrix (ECM). It is being appreciated that 3D matrix-based microfluidic models have an advantage over conventional in vitro and animal models to study tumor progression events. Recent microfluidic models have enabled recapitulation of key mechanobiological features present within the TME to investigate collective cancer cell migration and invasion. Microfluidics also allows for functional interrogation and therapeutic manipulation of specific steps to study the dynamic aspects of tumor progression. In this review, we focus on recent developments in cancer cell migration and how microfluidic strategies have evolved to address the physiological complexities of the TME to visualize migration modes adapted by various tumor cells.

We examine the role of preshearing on the flow properties of polymer solutions containing essentially an acrylamide-based copolymer obtained from an emulsified polymer emulsion inverted by a surfactant. The polymer solutions were presheared using three methods: (1) a Buddeberg disperser, (2) an Ultra-Turrax disperser, and (3) pressure-driven flow through a capillary. Shearing the polymer solution was done under fast flow to induce high stretching of the polymer chains and thus promote the break-up of the longest ones (i.e., decrease in relaxation time and shear-thinning level). The unsheared and presheared polymer solutions were forced through sand packs to compare their corresponding flow resistances. We observed that the reduction in the viscosity and screen factor of the presheared polymer solutions is path independent regardless of the shearing device. We found a critical Weissenberg number (Wic ∼ 13) above which the viscosity of the polymer solutions started to decrease. The resistance factor for the polymer solutions presheared with the Ultra-Turrax disperser at an energy input of 31.3 and 290.7 MJ/m3 was nearly 3 and 7 times, respectively, lower than for the unsheared polymer solution, while the viscosity decreased only by 27 and 48%, respectively. The sand-pack experiments were successfully interpreted using a numerical model taking into account time-dependent retention. The model showed that the flow of the presheared polymer solutions through the sand packs was enhanced mainly due to the breaking of the longest polymer chains, which results in smaller mechanical entrapment. This preshearing of the water-soluble polymers can be used in multiple industrial applications, including chemical enhanced oil recovery and optimization of polymer processing.

The surface topography of engineered extracellular matrices is one of the most important physical cues regulating the phenotypic polarization of macrophages. However, not much is known about the ways through which submicron (i.e., 100–1000 nm) topographies modulate the polarization of macrophages. In the context of bone tissue regeneration, it is well established that this range of topographies stimulates the osteogenic differentiation of stem cells. Since the immune response affects the bone tissue regeneration process, the immunomodulatory consequences of submicron patterns should be studied prior to their clinical application. Here, we 3D printed submicron pillars (using two-photon polymerization technique) with different heights and interspacings to perform the first ever systematic study of such effects. Among the studied patterns, the highest degree of elongation was observed for the cells cultured on those with the tallest and densest pillars. After 3 days of culture with inflammatory stimuli (LPS/IFN-γ), sparsely decorated surfaces inhibited the expression of the pro-inflammatory cellular marker CCR7 as compared to day 1 and to the other patterns. Furthermore, sufficiently tall pillars polarized the M1 macrophages towards a pro-healing (M2) phenotype, as suggested by the expression of CD206 within the first 3 days. As some of the studied patterns are known to be osteogenic, the osteoimmunomodulatory capacity of the patterns should be further studied to optimize their bone tissue regeneration performance., The surface topography of implants can regulate the phenotypic polarization of macrophage. Using 2PP technique, we fabricated series of submicron pillars with controlled topological features and analyzed their roles on the macrophage polarization.

Electroporation has become a powerful tool for nonviral delivery of various biomolecules such as nucleic acids, proteins, and chemotherapeutic drugs to virtually any living cell by exposing the cell membrane to an intense pulsed electric field. Different multiphysics and multiscale models have been developed to describe the phenomenon of electroporation and predict molecular transport through the electroporated membrane. In this paper, we critically examine the existing mechanistic, single-cell models which allow spatially and temporally resolved numerical simulations of electroporation-induced transmembrane transport of small molecules by confronting them with different experimental measurements. Furthermore, we assess whether any of the proposed models is universal enough to describe the associated transmembrane transport in general for all the different pulse parameters and small molecules used in electroporation applications. We show that none of the tested models can be universally applied to the full range of experimental measurements. Even more importantly, we show that none of the models has been compared to sufficient amount of experimental data to confirm the model validity. Finally, we provide guidelines and recommendations on how to design and report experiments that can be used to validate an electroporation model and how to improve the development of mechanistic models.

Epithelial to mesenchymal transition (EMT) is crucial during embryonic development, tissue fibrosis, and cancer progression. Epithelial cells that display a cobblestone-like morphology can undergo a switch to mesenchymal-like phenotype, displaying an elongated spindle shape or a fibroblast-like morphology. EMT is characterized by timely and reversible alterations of molecular and cellular processes. The changes include loss of epithelial and gain of mesenchymal marker expression, loss of polarity, increased cell migratory and invasive properties. Epithelial cells can progress unevenly during this transition and attain hybrid E/M states or metastable EMT states, referred to as epithelial cell plasticity. To gain a deeper insight into the mechanism of EMT, understanding the dynamic aspects of this process is essential. One of the most prominent factors to induce EMT is the cytokine transforming growth factor-β (TGF-β). This chapter discusses molecular and cellular techniques to monitor TGF-β-induced signaling and EMT changes in normal and cancer cell lines. These methods include measuring the TGF-β-induced activation of its intracellular SMAD effectors proteins and changes in epithelial/mesenchymal marker expression and localization. Moreover, we describe assays of cell migration and dynamic reorganization of the actin cytoskeleton and stress filaments that are frequently part of the TGF-β-induced EMT cellular response.

We developed a localized single-cell electroporation chip to deliver exogenous biomolecules with high efficiency while maintaining high cell viability. In our microfluidic device, the cells are trapped in a microtrap array by flow, after which target molecules are supplied to the device and electrotransferred to the cells under electric pulses. The system provides the ability to monitor the electrotransfer of exogenous biomolecules in real time. We reveal through numerical simulations that localized electroporation is the mechanism of permeabilization in the microtrap array electroporation device. We demonstrate the simplicity and accuracy of this microtrap technology for electroporation by delivery of both small molecules using propidium iodide and large molecules using plasmid DNA for gene expression, illustrating the potential of this minimally invasive method to be widely used for precise intracellular delivery purposes (from bioprocess engineering to therapeutic applications).

The ability of exogenous DNA cargo to overcome the active and viscoelastic eukaryotic cytoplasm is a principal determinant for the gene delivery efficacy. During DNA electrotransfer, DNA forms complexes with the membrane (DNA cargo) which is transported through the cytoplasm through a combination of passive diffusion and active transport. However, this process is poorly understood limiting rational optimization of DNA cargo to be delivered to different cell types. We have investigated the intracellular transport of DNA cargo (of sizes 100 bp, 250 bp and 500 bp) delivered by electrotransfer to non-cancerous and cancerous mammalian cells. We demonstrate that intracellular DNA cargo transport is governed by coexisting ergodic and non ergodic anomalous diffusion for all the tested DNA sizes and cell types. The apparent diffusion coefficient of the electrotransferred DNA cargo in the cytoplasm decreases when the DNA size is increased from 100 bp to 500 bp. Interestingly, the electrotransferred DNA cargo (500 bp) transport is strongly dependent on the cell’s cancer state. Intracellular electrotransferred DNA cargo transport has a higher probability of superdiffusive transport and lower probability of caging in metastatic cells compared to malignant cells followed by benign cells.

Transient physical disruption of cell membranes by electric pulses (or electroporation) has significance in biomedical and biological applications requiring the delivery of exogenous (bio)molecules to living cells. We demonstrate that actin networks regulate the cell membrane permeability during electroporation. Disruption of actin networks increases the uptake of membrane-impermeable molecules such as propidium iodide during electroporation. Our experiments at different temperatures ranging from 11 °C to 37 °C show that molecular uptake during electroporation increases with temperature. Furthermore, by examining the temperature-dependent kinetics of propidium iodide uptake, we infer that the activation energy barrier of electroporation is lowered when the actin networks are disrupted. Our numerical calculations of transmembrane voltage show that the reduced activation energy barrier for the cells with disrupted actin is not a consequence of the changes in transmembrane voltage associated with changes in the cell shape due to the disruption of actin, indicating that this could be due to changes in membrane mechanical properties. Our results suggest that the current theoretical models of electroporation should be advanced further by including the contributions of the cytoskeletal networks on the cell membrane permeability during the delivery of exogenous materials.

Water-soluble polyacrylamides have often been used to modify flow response in various water-based technologies and industrial processes, including paints, water treatment, paper manufacturing, and chemical enhanced oil recovery. Polymers are susceptible to degradation at combined high salinity and elevated temperature conditions which limits their overall performance. Hybrid mixtures of hydrophobically modified polyacrylamide (HMPAM) with hydrophobically modified silica nanoparticles (NPs) emerged as a promising strategy for achieving enhanced stability and high viscosity in brines having a high total dissolved solids (TDS) content and high hardness at elevated temperatures (>20 wt% TDS, including >1.5 wt% divalent cations at T > 70 °C). The rheological response of the hybrids at various concentrations of HMPAM and NPs was examined to investigate the synergic effects. Hybridization of HMPAM with NPs led to a higher viscosity at high salinity and elevated temperature. The viscosity improvement was more pronounced when the concentration of HMPAM was in the semi-dilute regime and concentration of NPs was higher than a critical threshold where the viscosity increased roughly by a factor of 1.5. Here we present the mechanisms of improved viscosity behaviour. The rheological data suggest the role of NPs in the bridging between HMPAM molecules, which in turn increases the hydrodynamic radius and consequently the viscosity of the hybrids.

Delivery of naked DNA molecules into living cells via physical disruption of the membrane under electric pulses has potential biomedical applications ranging from gene electro-transfer, electro-chemotherapy, to gene therapy, yet the mechanisms involved in DNA transport remain vague. To investigate the mechanism of DNA translocation across the cell membrane, giant unilamellar vesicles (GUVs) were electroporated in the presence of DNA molecules keeping the size of the DNA molecules as a variable parameter. We experimentally determined the translocation efficiency for each size of the DNA molecule, to compare the results with the existing and conflicting theories of the translocation mechanism i.e. stochastic threading and bulk electrophoresis. We observed that the translocation efficiency is independent of DNA size (ranging from 25–20 000 bp, bp = base pairs), implying that DNA molecules translocate freely across the electro-pores in the lipid membrane in their native polymer conformation, as opposed to unravelling and threading through the electro-pore. Bulk electrophoretic mobility determines the relationship between translocation efficiency and the size of the DNA molecule. This research provides experimental evidence of the mechanistic understanding of DNA translocation across lipid membranes which is essential for devising efficient and predictable protocols for electric field mediated naked DNA delivery.

Transient permeabilisation of the cell membrane is a critical step to introduce drugs or DNA into living cells, yet challenging for both biological research and therapeutic applications. To achieve this, electroporation (or electropermeabilisation) has become a widely used method due to its simplicity to deliver almost any biomolecule to any cell type. Although this method demonstrates promise in the field of drug/gene delivery, the underlying physical mechanisms of the response of the heterogeneous cell membrane to strong electric pulses is still unknown. In this study, we have investigated the role of gel-phase lipids in the electroporation of binary giant unilamellar vesicles (GUVs), composed from DPPC (gel-phase) and DPhPC (fluid-phase) lipids (molar ratio 8:2 and 2:8). We have observed that the exposure to electric pulses leads to expel of fluid-phase lipids and concomitant decrease in GUV size, whereas the gel-phase domains become buckled. Based on experiments on pure fluid-phase and gel-phase GUVs, we have found that fluid-phase lipids can be expelled by electrical forces and the highly viscous gel-phase lipids cannot. Moreover, our analyses suggest that pore formation occurs primarily in fluid-phase domains and that the pore size is similar in all GUVs containing fluid-phase lipids, irrespective of the gel-phase percentage.

We report the results of molecular dynamics simulations of stress relaxation tests in athermal viscous soft sphere packings close to their unjamming transition. By systematically and simultaneously varying both the amplitude of the applied strain step and the pressure of the initial condition, we access both linear and nonlinear response regimes and control the distance to jamming. Stress relaxation in viscoelastic solids is characterized by a relaxation time τ∗ that separates short time scales, where viscous loss is substantial, from long time scales, where elastic storage dominates and the response is essentially quasistatic. We identify two distinct plateaus in the strain dependence of the relaxation time, one each in the linear and nonlinear regimes. The height of both plateaus scales as an inverse power law with the distance to jamming. By probing the time evolution of particle velocities during relaxation, we further identify a correlation between mechanical relaxation in the bulk and the degree of non-affinity in the particle velocities on the micro scale.

Molecular conformations of individual polymers during flow through porous media are directly observed by single-DNA imaging in microfluidics. As the Weissenberg number increases during flow (Wi > 1), we observe two types of elastic instabilities: (a) stationary dead-zone and (b) time-dependant dead-zone washing. When stretched polymer chains enter a dead-zone, they first re-coil and, once inside the dead-zone, they rotate and re-stretch again. The probability distribution of DNA chains under the stretched condition inside the dead-zone is found to be heterogeneous with a broad distribution.

Size of DNA molecules governs their interaction with the cell membrane during electroporation and their subsequent transport inside the cell. In order to investigate the effect of DNA size on DNA-membrane interaction during electroporation, cells are electro-pulsed with DNA molecules; 15 bp, 25 bp, 50 bp, 100 bp and 1000 bp (bp = base pairs). Within the experimental parameter space, DNA-membrane complexes or DNA aggregates are observed at the cell membrane for DNA molecules containing 25 or more base pairs. No aggregates are observed for DNA molecules containing 15 bp. For all DNA sizes, direct access to the cytoplasm is observed, however the amount translocated decays with the size. The observed dependency of DNA aggregate formation on the size of the DNA molecules is consistent with the Onsager's theory of condensation of anisotropic rod-like molecules.

We study the role of a biomimetic actin network during the application of electric pulses that induce electroporation or electropermeabilization, using giant unilamellar vesicles (GUVs) as a model system. The actin cortex, a subjacently attached interconnected network of actin filaments, regulates the shape and mechanical properties of the plasma membrane of mammalian cells, and is a major factor influencing the mechanical response of the cell to external physical cues. We demonstrate that the presence of an actin shell inhibits the formation of macropores in the electroporated GUVs. Additionally, experiments on the uptake of dye molecules after electroporation show that the actin network slows down the resealing process of the permeabilized membrane. We further analyze the stability of the actin network inside the GUVs exposed to high electric pulses. We find disruption of the actin layer that is likely due to the electrophoretic forces acting on the actin filaments during the permeabilization of the GUVs. Our findings on the GUVs containing a biomimetic network provide a step towards understanding the discrepancies between the electroporation mechanism of a living cell and its simplified model of the empty GUV.

Fluidic rectification refers to anisotropic flow resistance upon changing the flow direction. Polymeric solutions, in contrast to Newtonian fluids, can exhibit an anisotropic flow resistance in microfluidic devices by tuning the channel shape at low Reynolds number. Such a concept has not been investigated in an anisotropic porous medium. We have developed a fluidic rectifier based on an anisotropic porous medium consisting of a periodic array of triangular pillars that can operate at a low Reynolds number. Rectification is achieved, when the type of high Weissenberg number elastic instabilities changes with the flow direction. The flow resistance differs across the two directions of the anisotropic porous medium geometry. We have identified the type of elastic instabilities that appear in both forward and backward directions. Particularly, we found a qualitative relation between the dead-zone instability and the onset of fluidic rectification.

We study the role of a biomimetic actin cortex during the application of electric pulses that induce electroporation or electropermeabilization, using giant unilamellar vesicles (GUVs) as a model system. The actin cortex, a subjacently attached interconnected network of actin filaments, regulates the shape and mechanical properties of the plasma membrane of mammalian cells, and is a major factor influencing the mechanical response of the cell to external physical cues. We demonstrate that the presence of an actin shell inhibits the formation of macropores in the electroporated GUVs. Additionally, experiments on the uptake of dye molecules after electroporation show that the actin network slows down the resealing process of the permeabilized membrane. We further analyze the stability of the actin network inside the GUVs exposed to high electric pulses. We find disruption of the actin layer that is likely due to the electrophoretic forces acting on the actin filaments during the permeabilization of the GUVs. Our findings on the GUVs containing a biomimetic cortex provide a step towards understanding the discrepancies between the electroporation mechanism of a living cell and its simplified model of the empty GUV.

Here, we investigate separately the dependence of the mixing time on the size and velocity of micro-droplets moving through serpentine channels. We find that the mixing time scales linearly with droplet size. All experimental data collapse on a master-line, when the convective time scale is multiplied by the dimensionless droplet size.

Dielectrophoresis (DEP) is a versatile technique for the solution of difficult (bio-)particle separation tasks based on size and material. Particle motion by DEP requires a highly inhomogeneous electric field. Thus, the throughput of classical DEP devices is limited by restrictions on the channel size to achieve large enough gradients. Here, we investigate dielectrophoretic filtration, in which channel size and separation performance are decoupled because particles are trapped at induced field maxima in a porous separation matrix. By simulating microfluidic model porous media, we derive design rules for DEP filters and verify them using model particles (polystyrene) and biological cells (S. cerevisiae, yeast). Further, we bridge the throughput gap by separating yeast in an alumina sponge and show that the design rules are equally applicable in real porous media at high throughput. While maintaining almost 100% efficiency, we process up to 9 mL min-1, several orders of magnitude more than most state-of-the-art DEP applications. Our microfluidic approach provides new insight into trapping dynamics in porous media, which even can be applied in real sponges. These results pave the way toward high-throughput retention, which is capable of solving existing problems such as cell separation in liquid biopsy or precious metal recovery.

Recent shear experiments in well-entangled polymer solutions demonstrated that interfacial wall slip is the only source of shear rate loss and there is no evidence of shear banding in the micron scale gap. In this work, we experimentally elucidate how molecular parameters such as slip length, b, influence shear inhomogeneity of entangled polybutadiene (PBD) solutions during shear in a small gap H ∼ 50 μm. Simultaneous rheometric and velocimetric measurements are performed on two PBD solutions with the same level of entanglements (Z = 54) in two PBD solvents with molecular weights of 1.5 kg mol(-1) and 10 kg mol(-1) that possess different levels of shear inhomogeneity (2bmax/H = 17 and 240). For the PBD solution made with a low molecular weight PBD solvent of 1.5 kg mol(-1), wall slip is the dominant response within the accessible range of the shear rate, i.e., up to the nominal Weissenberg number (Wi) as high as 290. On the other hand, wall slip is minimized using a high molecular-weight PBD solvent of 10 kg mol(-1) so that bulk shear banding is observed to take place in the steady state for Wi100. Finally, these findings and previous results are in good agreement with our recently proposed phase diagram in the parameter space of apparent Wi versus 2bmax/H suggesting that shear banding develops across the micron scale gap when the imposed Wi exceeds 2bmax/H [Wang et al., Macromolecules, 2011, 44, 183].

The present review focuses on the effects of pulsed electric fields on lipid vesicles ranging from giant unilamellar vesicles (GUVs) to small unilamellar vesicles (SUVs), from both fundamental and applicative perspectives. Lipid vesicles are the most popular model membrane systems for studying biophysical and biological processes in living cells. Furthermore, as vesicles are made from biocompatible and biodegradable materials, they provide a strategy to create safe and functionalized drug delivery systems in health-care applications. Exposure of lipid vesicles to pulsed electric fields is a common physical method to transiently increase the permeability of the lipid membrane. This method, termed electroporation, has shown many advantages for delivering exogenous molecules including drugs and genetic material into vesicles and living cells. In addition, electroporation can be applied to induce fusion between vesicles and/or cells. First, we discuss in detail how research on cell-size GUVs as model cell systems has provided novel insight into the basic mechanisms of cell electroporation and associated phenomena. Afterwards, we continue with a thorough overview how electroporation and electrofusion have been used as versatile methods to manipulate vesicles of all sizes in different biomedical applications. We conclude by summarizing the open questions in the field of electroporation and possible future directions for vesicles in the biomedical field.

We study the necking and pinch-off dynamics of liquid droplets that contain a semidilute polymer solution of polyacrylamide close to overlap concentration by combining microfluidics and single DNA observation. Polymeric droplets are stretched by passing them through the stagnation point of a T-shaped microfluidic junction. In contrast with the sudden breakup of Newtonian droplets, a stable neck is formed between the separating ends of the droplet which delays the breakup process. Initially, polymeric filaments experience exponential thinning by forming a stable neck with extensional flow within the filament. Later, thin polymeric filaments develop a structure resembling a series of beads-on-a-string along their length and finally rupture during the final stages of the thinning process. To unravel the molecular picture behind these phenomena, we integrate a T-shaped microfluidic device with advanced fluorescence microscopy to visualize stained DNA molecules at the stagnation point within the necking region. We find that the individual polymer molecules suddenly stretch from their coiled conformation at the onset of necking. The extensional flow inside the neck is strong enough to deform and stretch polymer chains; however, the distribution of polymer conformations is broad, and it remains stationary in time during necking. Furthermore, we study the dynamics of single molecules during formation of beads-on-a-string structure. We observe that polymer chains gradually recoil inside beads while polymer chains between beads remain stretched to keep the connection between beads. The present work effectively extends single molecule experiments to free surface flows, which provides a unique opportunity for molecular-scale observation within the polymeric filament during necking and rupture.

In geometries capable of producing uniform shear stress across the gap such as cone–plate, it is a challenge to study the nonlinear rheology of well-entangled polymers without wall slip and edge fracture. The task to obtain meaningful steady-state information is particularly daunting. Significant insights are required in the design of experiments and choice of reliable apparatuses that could fulfill the objective to determine the constitutive behavior. In a recent published article, Hu J. Rheol. 54, 1307–1323 (2010) reported particle-imaging velocimetric measurements of two highly entangled polybutadiene solutions with a cocylinder geometry. One of the solutions was prepared and provided to Hu by Ravindranath from the University of Akron. Rheological and particle-tracking velocimetric studies have been carried out on the same sample in our laboratory. In this letter, we will compare Hu’s results with our own measurements, clarify a few misrepresentations, and discuss several important issues concerning sh...

One-dimensional nanostructures such as nanochannels (and nanotubes) are characterized by extremely small transverse size and resultant high degree of spatial confinement that endow them a unique set of properties. When patterned laterally, these nanostructures are widely used as critical transport devices for a variety of applications such as sensing, nanomanipulation, and information processing.[1–8] While numerous fabrication techniques have been developed, few can generate large and highly ordered arrays of both nanochannels and nanowires with no defects and low-cost. The most notable high-resolution lithographic techniques include electron beam lithography (EBL) and focused ion beam milling (FIB),[9–13] but they are associated with either low throughput or high-cost. Another lithographic technique, nanoimprint lithography (NIL), is of high throughput and relatively low-cost, but it requires the use of highly specialized equipment and molds prepared typically by EBL.[14– 17] Many inexpensive techniques have been developed, but they are inadequate in terms of high precision, low defect rate, or large area fabrication of both nanochannels/tubes and nanowires/strands.[7,18–25] Moreover, these nanostructures need to be connected to the micro/macroscale structures, such as reservoirs and channels, to form functional devices. This is not a trivial task and the lack of a low-cost solution to this problem significantly limits the applicability of many nanoconstructs.

We carry out a systematic investigation into steady-state shear behavior of six entangled solutions based on a superposition of continuous shear and small amplitude oscillatory shear (SAOS). During steady shear in the shear thinning regime, the superimposed SAOS frequency sweep measurements reveal characteristics of viscous liquids, e.g., terminal dynamics, on the experimental time scale of the reciprocal shear rate. The residual entanglement network retains the same level of elastic stiffness as the equilibrium system does. Consistent with the convective constraint release idea, chains in the network are forced to pass around each other as they must do so to undergo steady flow. When such a sample is examined at significantly short time scales, chains are unable to pass around and the signature of this residual entanglement is that the storage modulus is greater than the loss modulus at higher frequencies than the applied shear rate. The particle-tracking velocimetric observations confirm that whether shear banding is present or not does not affect the basic “terminal flow” character revealed by the superimposed SAOS.

We have studied stress overshoot behavior in startup shear of four monodisperse polymer melts with a range of chain entanglement from Z=24 to 160 entanglement points per chain. In the elastic deformation regime defined by γτR>1, where τR is the Rouse relaxation time, (i) the peak shear stress σmax scales with the time tmax at the peak to −1/2 power, in contrast to an exponent of −1/4 in the viscoelastic regime (for γτR

(Received 28 November 2007; final revision received 6 October 2008 Synopsis Entangled DNA solutions are ideal as a model system to examine nonlinear shear flow behavior. Even when the number of entanglements per chain, Z, is higher than 100, the solution is still soft enough with an elastic plateau modulus under 100 Pa and is thus amenable to experimental study by commercial rotational rheometry without ambiguity and uncertainty. We have investigated nonlinear flow behavior of three entangled DNA solutions with Z=24, 60, and 156, respectively, using a combination of particle-tracking velocimetric PTV and conventional rheometric measurements. We explore questions such as a whether shear banding also occurs in moderately entangled solutions, b whether creep results in development of nonlinear velocity profile, c whether shear banding produced in startup shear and creep persists at long times in steady state, and d whether these entangled solutions exhibit homogeneous shear at the upper end of the stress plateau region. We found that the first DNA solution Z=24 only shows transient weakly inhomogeneous shear and steady linear velocity profile. In the more entangled solutions Z=60 and 156, shear banding is observed in startup rate- and stress-controlled shear in the shear thinning regime. Shear homogeneity eventually returns at the upper end of the stress plateau shear

In this work we attempt to answer several questions concerning the flow characteristics of entangled polymer solutions in a sliding plate shearing cell. We explore (a) how the molecular weight distribution affects the velocity profile in simple shear, (b) whether the observed shear banding is consistent with a nonmonotonic constitutive model, (c) whether the flow response and velocity profiles are different in simple shear depending on the different modes of shear. Our results provide a comparison with recent reports on a polydisperse polymer sample [Tapadia and Wang, Phys. Rev. Lett. 96, 016001 (2006); Tapadia, et al., Phys. Rev. Lett. 96, 196001 (2006)] that revealed the first evidence for inhomogeneous shear during startup in cone-plate flow geometry of a rotational rheometer. Using a highly monodisperse sample, we observed the sample to partition into two fractions with different local shear rates instead of possessing a smooth spatial variation of the local shear rate as seen for the polydisperse sam...

Nanochannel electroporation (NEP) was applied to deliver precise dosages of myeloid cell leukemia-1 (Mcl-1)-specific siRNA and molecular beacons to two types of acute myeloid leukemia (AML) cells, FMS-like tyrosine kinase-3 wild-type (WT) and internal tandem duplications (ITD) type at the single-cell level. NEP, together with single-cell quantitative reverse transcription PCR, led to an observation showing nearly 20-folds more Mcl-1 siRNA than MCL1 mRNA were required to induce cell death for both cell lines and patient blasts, i.e. , ∼8,800 siRNAs for ∼500 ± 50 mRNAs in ITD cells and ∼6,000 siRNAs for ∼300 ± 50 mRNAs in WT cells. A time-lapse study revealed that >75% MCL1 mRNA was downregulated within 1 hour after delivery of a small amount of siRNA. However, additional siRNA was required to inhibit the newly transcribed mRNA for >12 hours until the cell lost its ability of self-protection recovery. A multidelivery strategy of low doses and short delivery interval, which require 77% less siRNA and has the potential of lower side effects and clinical cost, was as effective as a single high-dose siRNA delivery. Our method provides a viable analytical tool to investigate gene silencing at the single-cell level for oligonucleotide-based therapy.

A living cell interrogation platform based on nanochannel electroporation is demonstrated with analysis of RNAs in single cells. This minimally invasive process is based on individual cells and allows both multi-target analysis and stimulus-response analysis by sequential deliveries. The unique platform possesses a great potential to the comprehensive and lysis-free nucleic acid analysis on rare or hard-to-transfect cells.

This article describes a systematic investigation of a discontinuous interfacial stick-slip transition (SST) in simple shear of monodisperse entangled 1,4-polybutadiene (PBD) and polyisoprene (PIP) melts with different molecular weights and architecture, using a specially designed controlled-force shear rheometer. The magnitude of the transition is found to be determined by the level of chain entanglement. Specifically, the dependence of extrapolation length b on molecular weight as b∼Mw3.4 and of the melt viscosity as b∼η is consistent with the observations based on capillary rheometric studies [X. Yang et al., Rheol. Acta 37, 415–423 (1998)]. The interfacial nature of the flow behavior is explicitly demonstrated by a surface treatment of the shearing plates and dependence of the abrupt increase of the apparent shear rate on the gap distance as well as by particle tracking velocimetry. The critical stress for different molecular weights of PBD and PIP is about 0.2 and 0.1MPa, respectively, independent of...

Despite the relevance and importance of slip, a fundamental understanding of the underlying molecular mechanisms of wall slip in polymer flow is still missing. In this work, we investigate the slip behavior of an entangled DNA solution at a molecular scale using a confocal microscope coupled to a microfluidic device. From microscopic measurement, we obtain both the velocity profile and conformation of polymeric chains by visualizing DNA molecules during flow on various surfaces (ranging from weak to strong interactions with DNA molecules). In channel flow at a low Weissenberg number (Wi= 0.14), we observe a parabolic flow for an APTES-treated glass (with strong interaction with DNA) in the absence of slip, while a significant amount of slip has been observed for a regular glass (with a weak interaction with DNA). At higher flow rates (Wi > 1.0), strong slip appears during flow on APTES-treated surfaces. In this case, only immobile DNA molecules are stretched on the surface and other bulk chains remain coiled. This observation suggests that the flow induced chain stretching at the interface is the main mechanism of slip during flow on strong surfaces. Conversely, for slip flow on surfaces with weak interactions (such as unmodified or acrylate-modified glasses), polymeric chains are desorbed from the surface and a thin layer of water is present near the surface, which induces an effective slip during flow. By imaging DNA conformations during both channel and shear flows on different surfaces, we elucidate that either chain desorption or flow-induced stretching of adsorbed chains occurs depending on the surface condition. In general, we expect that these new insights into the slip phenomenon will be useful for studying the biological flow involving single DNA molecule experiments in micro/nanofluidic devices.

The use of electric pulses to deliver foreign DNA, RNA, proteins or other therapeutic agents into living cells is one of the most popular physical methods for gene delivery. The basic theory of this treatment is that strong electric field can destabilize membranes and induce formation of pores thus increasing membrane permeability. This phenomenon is often known as electroporation (EP), sometimes also referred to electropermeabilization or electric break down. Unfortunately, the fundamental science of the electroporation and transport remains poorly understood, due to the lack of well-defined electroporation conditions on single cells.In this work, we employ an optical tweezer and micro/nano-fluidics as convenient tools to unravel the physics and mechanism behind the electropermeablization process in different conditions. We have measured the minimum electric field required for permeabilization of individual single cells in different geometries from bulk to micro and nanoscale for different cell line. In addition, we have measured the minimum electric field required for permeabilization of individual suspended spherical cells as a function of cell diameter for three different cell lines. Key to this work is employing an optical tweezer to manipulate single suspended cells and position in different fluidic geometries (from bulk to nano-scale). Furthermore, we also investigate how DNA move and transport inside these fluidic geometries and deliver into cells. We discovered that delivering a voltage through a nanochannel can generate high electric field over a very small area on the cell membrane, allowing DNAs to be delivered instantly into the cell cytoplasm. In this talk, both experimental work and modeling will be discussed to address the electroporation process.

The delivery of biomolecules, including siRNAs (≈21 bp) and large plasmids (≈10 kbp), into living cells holds a great promise for therapeutic and research applications. Lipoplex nanoparticles are popular nanocarriers for gene delivery. In conventional transfection methods, the cellular uptake of lipoplex nanoparticels occurs through the endocytosis process. The entrapment of lipoplex nanoparticles into endocytic vesicle is a major barrier in achieving efficient gene silencing and expression. Here, a novel nanochannel electroporation (NEP) method is employed to facilitate the cellular uptake and release of siRNAs/DNAs from lipoplexes. First, it is demonstrated that in a NEP device, lipoplex nanoparticles can be injected directly into the cell cytoplasm within several seconds. Specifically, it is found that lipoplexes containing MCL-1 siRNA delivered by NEP can more efficiently down-regulate the expression of MCL-1 mRNA in A549 cancer cells than conventional transfection. Quantum dot-mediated Forster resonance energy transfer (QD-FRET) reveals that lipoplexes delivered via NEP can directly release siRNA in the cytoplasm without going through the endocytosis route, which unravels the responsible mechanism for efficient gene delivery. Furthermore, the advantage of combining NEP with lipoplex nanoparticles by the successful delivery of large plasmids (pCAG2LMKOSimO, 13 kbp) into CHO cells is demonstrated.

Thanks to direct observation and manipulation of DNA in micro/nanofluidic devices, we are now able to elucidate the relationship between the polymer microstructure and its rheological properties, as well as to design new singlemolecule platforms for biophysics and biomedicine. This allows exploration of many new mechanisms and phenomena, which were previously unachievable with conventional methods such as bulk rheometry tests. For instance, the field of polymer rheology is at a turning point to relate the complex molecular conformations to the nonlinear viscoelasticity of polymeric fluids (such as coil-stretch transition, shear thinning, and stress overshoot in startup shear). In addition, nanofluidic devices provided a starting point for manipulating single DNA molecules by applying basic principles of polymer physics, which is highly relevant to numerous processes in biosciences. In this article, we review recent progress regarding the flow and deformation of DNA in micro/nanofluidic systems from both fundamental and application perspectives. We particularly focus on advances in the understanding of polymer rheology and identify the emerging research trends and challenges, especially with respect to future applications of nanofluidics in the biomedical field.

In the research of cancer cell invasion and metastasis, recreation of physiologically relevant and faithful three-dimensional (3D) tumor models that recapitulate spatial architecture, spatiotemporal control of cell communication and signaling pathways, and integration of extracellular cues remains an open challenge. Here, a programmable multifunctional 3D cancer cell invasion microbuckets-hydrogel (Mb-H) platform is developed by integrating various function-variable microbuckets and extracellular matrix (ECM)-like hydrogels. Based on this Mb-H micro platform, the aggregation of multi-cancer cells is well controlled to form cancer cell spheroids, and the guiding relationship of single-cell migration and collective cell migration during the epithelial-mesenchymal transition (EMT) of cancer cell invasion are demonstrated. By programming and precisely assembling multiple functions in one system, the Mb-H platform with spatial-temporal controlled release of cytokine transforming growth factor beta (TGF-beta) and various functionalized Mb-H platforms with intelligent adjustment of cell-matrix interactions are engineered to coordinate the 3D invasive migration of cancer cell spheroids. This programmable and adaptable 3D cancer cell invasion micro platform takes a new step toward mimicking the dynamically changing (localized) tumor microenvironment and exhibits wide potential applications in cancer research, bio-fabrication, cell signaling, and drug screening.