Development of multifunctional nanopipettes for controlled intracellular delivery and single-entity detection.

The intracellular delivery of biomolecules and nanoscale materials to individual cells has gained remarkable attention in recent years owing to its wide applications in drug delivery, clinical diagnostics, bio-imaging and single-cell analysis. It remains a challenge to control and measure the delivered amount in one cell. In this work, we developed a multifunctional nanopipette - containing both a nanopore and nanoelectrode (pyrolytic carbon) at the apex - as a facile, minimally invasive and effective platform for both controllable single-cell intracellular delivery and single-entity counting. While controlled by a micromanipulator, the baseline changes of the nanopore ionic current (I) and nanoelectrode open circuit potential (V) help to guide the nanopipette tip insertion and positioning processes. The delivery from the nanopore barrel can be facilely controlled by the applied nanopore bias. To optimize the intracellular single-entity detection during delivery, we studied the effects of the nanopipette tip geometry and solution salt concentration in controlled experiments. We have successfully delivered gold nanoparticles and biomolecules into the cell, as confirmed by the increased scattering and fluorescence signals, respectively. The delivered entities have also been detected at the single-entity level using either one or both transient I and V signals. We found that the sensitivity of the single-entity electrochemical measurement was greatly affected by the local environment of the cell and varied between cell lines.

Dynamic single-cell intracellular pH sensing using a SERS-active nanopipette.

Glass nanopipettes have shown promise for applications in single-cell manipulation, analysis, and imaging. In recent years, plasmonic nanopipettes have been developed to enable surface-enhanced Raman spectroscopy (SERS) measurements for single-cell analysis. In this work, we developed a SERS-active nanopipette that can be used to perform long-term and reliable intracellular analysis of single living cells with minimal damage, which is achieved by optimizing the nanopipette geometry and the surface density of the gold nanoparticle (AuNP) layer at the nanopipette tip. To demonstrate its ability in single-cell analysis, we used the nanopipette for intracellular pH sensing. Intracellular pH (pHi) is vital to cells as it influences cell function and behavior and pathological conditions. The pH sensitivity was realized by simply modifying the AuNP layer with the pH reporter molecule 4-mercaptobenzoic acid. With a response time of less than 5 seconds, the pH sensing range is from 6.0 to 8.0 and the maximum sensitivity is 0.2 pH units. We monitored the pHi change of individual HeLa and fibroblast cells, triggered by the extracellular pH (pHe) change. The HeLa cancer cells can better resist pHe change and adapt to the weak acidic environment. Plasmonic nanopipettes can be further developed to monitor other intracellular biomarkers.

Relationship in Pacemaker Neurons Between the Long-Term Correlations of Membrane Voltage Fluctuations and the Corresponding Duration of the Inter-Spike Interval.

Several studies of the behavior in the voltage and frequency fluctuations of the neural electrical activity have been performed. Here, we explored the particular association between behavior of the voltage fluctuations in the inter-spike segment (VFIS) and the inter-spike intervals (ISI) of F1 pacemaker neurons from H. aspersa, by disturbing the intracellular calcium handling with cadmium and caffeine. The scaling exponent α of the VFIS, as provided by detrended fluctuations analysis, in conjunction with the corresponding duration of ISI to estimate the determination coefficient R 2 (48-50 intervals per neuron, N = 5) were all evaluated. The time-varying scaling exponent α(t) of VFIS was also studied (20 segments per neuron, N = 11). The R 2 obtained in control conditions was 0.683 ([0.647 0.776] lower and upper quartiles), 0.405 [0.381 0.495] by using cadmium, and 0.151 [0.118 0.222] with caffeine (P < 0.05). A non-uniform scaling exponent α(t) showing a profile throughout the duration of the VFIS was further identified. A significant reduction of long-term correlations by cadmium was confirmed in the first part of this profile (P = 0.0001), but no significant reductions were detected by using caffeine. Our findings endorse that the behavior of the VFIS appears associated to the activation of different populations of ionic channels, which establish the neural membrane potential and are mediated by the intracellular calcium handling. Thus, we provide evidence to consider that the behavior of the VFIS, as determined by the scaling exponent α, conveys insights into mechanisms regulating the excitability of pacemaker neurons.

Improving the development of human engineered cardiac tissue by gold nanorods embedded extracellular matrix for long-term viability.

A myocardial infarction (MI), commonly called a heart attack, results in the death of cardiomyocytes (CMs) in the heart. Tissue engineering provides a promising strategy for the treatment of MI, but the maturation of human engineered cardiac tissue (hECT) still requires improvement. Conductive polymers and nanomaterials have been incorporated into the extracellular matrix to enhance the mechanical and electrical coupling between cardiac cells. Here we report a simple approach to incorporate gold nanorods (GNRs) into the fibrin hydrogel to form a GNR-fibrin matrix, which is used as the major component of the extracellular matrix for forming a 3D hECT construct suspended between two flexible posts. The hECTs made with GNR-fibrin hydrogel showed markers of maturation such as higher twitch force, synchronous beating activity, sarcomere maturation and alignment, t-tubule network development, and calcium handling improvement. Most importantly, the GNR-hECTs can survive over 9 months. We envision that the hECT with GNRs holds the potential to restore the functionality of the infarcted heart.

Understanding spatiotemporal mechanical behavior, viscoelasticity, and functions of stem cell-derived cardiomyocytes.

Understanding myocytes' spatiotemporal mechanical behavior and viscoelasticity is a long-standing challenge as it plays a critical role in regulating structural and functional homeostasis. To probe the time-dependent viscoelastic behaviors of cardiomyocytes with cross-linked polymer networks, we measure stem cell-derived cardiomyocyte's (hiPSC-CM) deformation, adhesion, and contractility using atomic force microscopy (AFM) nanoindentation, fluidic micropipette, and digital image correlation (DIC). Our results show a cytoplasm load of 7-14 nN, a de-adhesion force of 0.1-1 nN, and an adhesion force between two hiPSC-CMs of 50-100 nN with an interface energy of 0.45 pJ. Based on the load-displacement curve, we model its dynamic viscoelasticity and discover its intimate associations with physiological properties. Cell detaching and contractile modeling demonstrate cell-cell adhesion and beating related strains manifesting viscoelastic behavior, highlighting viscoelasticity plays the primary role in governing hiPSC-CM spatiotemporal mechanics and functions. Overall, this study provides valuable information about the mechanical properties, adhesion behaviors, and viscoelasticity of single hiPSC-CM, shedding light on mechanical-structure relationships and their dynamic responses to mechanical stimuli and spontaneous contraction.

Membrane Remodeling of Human-Engineered Cardiac Tissue by Chronic Electric Stimulation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show immature features, but these are improved by integration into 3D cardiac constructs. In addition, it has been demonstrated that physical manipulations such as electrical stimulation (ES) are highly effective in improving the maturation of human-engineered cardiac tissue (hECT) derived from hiPSC-CMs. Here, we continuously applied an ES in capacitive coupling configuration, which is below the pacing threshold, to millimeter-sized hECTs for 1-2 weeks. Meanwhile, the structural and functional developments of the hECTs were monitored and measured using an array of assays. Of particular note, a nanoscale imaging technique, scanning ion conductance microscopy (SICM), has been used to directly image membrane remodeling of CMs at different locations on the tissue surface. Periodic crest/valley patterns with a distance close to the sarcomere length appeared on the membrane of CMs near the edge of the tissue after ES, suggesting the enhanced transverse tubulation network. The SICM observation is also supported by the fluorescence images of the transverse tubulation network and α-actinin. Correspondingly, essential cardiac functions such as calcium handling and contraction force generation were improved. Our study provides evidence that chronic subthreshold ES can still improve the structural and functional developments of hECTs.

Single-Entity Approach to Investigate Surface Charge Enhancement in Magnetoelectric Nanoparticles Induced by AC Magnetic Field Stimulation.

Magneto-electric nanoparticles (MENPs), composed of a piezoelectric shell and a ferromagnetic core, exhibited enhanced cell uptake and controlled drug release due to the enhanced localized electric field (surface charge/potential) and the generation of acoustics, respectively, upon applying alternating current (AC) magnetic (B)-field stimulation. This research, for the first time, implements an electrochemical single-entity approach to probe AC B-field induced strain mediated surface potential enhancement on MENP surface. The surface potential changes at the single-NP level can be probed by the open circuit potential changes of the floating carbon nanoelectrode (CNE) during the MENP-CNE collision events. The results confirmed that the AC B-field (60 Oe) stimulation caused localized surface potential enhancement of MENP. This observation is associated with the presence of a piezoelectric shell, whereas magnetic nanoparticles were found unaffected under identical stimulation.

Scanning Ion Conductance Microscopy Study Reveals the Disruption of the Integrity of the Human Cell Membrane Structure by Oxidative DNA Damage.

Oxidative stress can damage organs, tissues, and cells through reactive oxygen species (ROS) by oxidizing DNA, proteins, and lipids, thereby resulting in diseases. However, the underlying molecular mechanisms remain to be elucidated. In this study, employing scanning ion conductance microscopy (SICM), we explored the early responses of human embryonic kidney (HEK293H) cells to oxidative DNA damage induced by potassium chromate (K2CrO4). We found that the short term (1-2 h) exposure to a low concentration (10 µM) of K2CrO4 damaged the lipid membrane of HEK293H cells, resulting in structural defects and depolarization of the cell membrane and reducing cellular secretion activity shortly after the treatment. We further demonstrated that the K2CrO4 treatment decreased the expression of the cytoskeleton protein, ß-actin, by inducing oxidative DNA damage in the exon 4 of the ß-actin gene. These results suggest that K2CrO4 caused oxidative DNA damage in cytoskeleton genes such as ß-actin and reduced their expression, thereby disrupting the organization of the cytoskeleton beneath the cell membrane and inducing cell membrane damages. Our study provides direct evidence that oxidative DNA damage disrupted human cell membrane integrity by deregulating cytoskeleton gene expression.

Extrusion 3D Printing of Porous Silicone Architectures for Engineering Human Cardiomyocyte-Infused Patches Mimicking Adult Heart Stiffness.

Cardiac patches, three-dimensional (3D) constructs of polymer scaffold and heart muscle cells, have received widespread attention for regenerative therapy to repair damaged heart tissue. The implanted patches should mimic the micromechanical environment of native myocardium for effective integration and optimum mechanical function. In this study, we engineered compliant silicone scaffolds infused with cardiomyocytes (CMs) differentiated from human-induced pluripotent stem cells. Porous scaffolds are fabricated by extrusion 3D printing of room-temperature-vulcanized (RTV) silicone rubber. The stiffness and strength of scaffolds are tailored by designing a polymer strand arrangement during 3D printing. Single-strand scaffold design is found to display a tensile Young's modulus of ∼280 kPa, which is optimum for supporting CMs without impairing their contractility. Uniform distribution of cells in the scaffold is observed, ascribed to 3D migration facilitated by interconnected porous architecture. The patches demonstrated synchronized contraction 10 days after seeding scaffolds with CMs. Indentation measurements reveal that the contracting cell-scaffold patches display local moduli varying from ∼270 to 530 kPa, which covers the upper spectrum of the stiffness range displayed by the human heart. This study demonstrates the effectiveness of a porous 3D scaffold composed of flexible silicone rubber for CMs percolation, supporting a contractile activity, and mimicking native heart stiffness.

Fractal-like correlations of the fluctuating inter-spike membrane potential of a Helix aspersa pacemaker neuron.

We analyzed the voltage fluctuations of the membrane potential manifested along the inter-spike segment of a pacemaker neuron. Time series of intracellular inter-spike voltage fluctuations were obtained in the current-clamp configuration from the F1 neuron of 12 Helix aspersa specimens. To assess the dynamic or stochastic nature of the voltage fluctuations these series were analyzed by Detrended Fluctuation Analysis (DFA), providing the scaling exponent α. The median α result obtained for the inter-spike segments was 0.971 ([0.963, 0.995] lower and upper quartiles). Our results indicate a critical-like dynamic behavior in the inter-spike membrane potential that, far from being random, shows long-term correlations probably linked to the dynamics of the mechanisms involved in the regulation of the membrane potential, thereby endorsing the occurrence of critical-like phenomena at a single-neuron level.