Allen Lawrence Garner

Nonthermal Atmospheric Plasma Rapidly Disinfects Multidrug-Resistant Microbes by Inducing Cell Surface Damage

ABSTRACT Plasma, a unique state of matter with properties similar to those of ionized gas, is an effective biological disinfectant. However, the mechanism through which nonthermal or “cold” plasma inactivates microbes on surfaces is poorly understood, due in part to challenges associated with processing and analyzing live cells on surfaces rather than in aqueous solution. Here, we employ membrane adsorption techniques to visualize the cellular effects of plasma on representative clinical isolates of drug-resistant microbes. Through direct fluorescent imaging, we demonstrate that plasma rapidly inactivates planktonic cultures, with >5 log10 kill in 30 s by damaging the cell surface in a time-dependent manner, resulting in a loss of membrane integrity, leakage of intracellular components (nucleic acid, protein, ATP), and ultimately focal dissolution of the cell surface with longer exposure time. This occurred with similar kinetic rates among methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Candida albicans. We observed no correlative evidence that plasma induced widespread genomic damage or oxidative protein modification prior to the onset of membrane damage. Consistent with the notion that plasma is superficial, plasma-mediated sterilization was dramatically reduced when microbial cells were enveloped in aqueous buffer prior to treatment. These results support the use of nonthermal plasmas for disinfecting multidrug-resistant microbes in environmental settings and substantiate ongoing clinical applications for plasma devices.

Cell membrane thermal gradients induced by electromagnetic fields

While electromagnetic fields induce structural changes in cell membranes, particularly electroporation, much remains to be understood about membrane level temperature gradients. For instance, microwaves induce cell membrane temperature gradients (∇T) and bioeffects with little bulk temperature change. Recent calculations suggest that nanosecond pulsed electric fields (nsPEFs) may also induce such gradients that may additionally impact the electroporation threshold. Here, we analytically and numerically calculate the induced ∇T as a function of pulse duration and pulse repetition rate. We relate ∇T to the thermally induced cell membrane electric field (Em) by assuming the membrane behaves as a thermoelectric such that Em ∼ ∇T. Focusing initially on applying nsPEFs to a uniform membrane, we show that reducing pulse duration and increasing pulse repetition rate (or using higher frequency for alternating current (AC) fields) maximizes the magnitude and duration of ∇T and, concomitantly, Em. The maximum ∇T ini...

Platelet activation using electric pulse stimulation: growth factor profile and clinical implications.

BACKGROUND Autologous platelet gel therapy using platelet-rich plasma has emerged as a promising alternative for chronic wound healing, hemostasis, and wound infection control. A critical step for this therapeutic approach is platelet activation, typically performed using bovine thrombin (BT) and calcium chloride. However, exposure of humans to BT can stimulate antibody formation, potentially resulting in severe hemorrhagic or thrombotic complications. Electric pulse stimulation using nanosecond PEFs (pulse electric fields) is an alternative, nonbiochemical platelet activation method, thereby avoiding exposure to xenogeneic thrombin and associated risks. METHODS In this study, we identified specific requirements for a clinically relevant activator instrument by dynamically measuring current, voltage, and electric impedance for platelet-rich plasma samples. From these samples, we investigated the profile of growth factors released from human platelets with electric pulse stimulation versus BT, specifically platelet-derived growth factor, transforming growth factor &bgr;, and epidermal growth factor, using commercial enzyme-linked immunosorbent assay kits. RESULTS Electric pulse stimulation triggers growth factor release from platelet &agr;-granules at the same or higher level compared with BT. CONCLUSION Electric pulse stimulation is a fast, inexpensive, easy-to-use platelet activation method for autologous platelet gel therapy.

Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma

Background Activated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma. Aims To determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity. Methods PRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin. Results PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet-derived microparticles, platelets, and platelet aggregates whereas SMHEF pulses primarily resulted in platelet-derived microparticles. Microparticles and platelets in PRP activated with SMLEF bipolar pulses had significantly lower annexin V-positivity than those following SMHEF activation. In contrast, the % P-selectin positivity and surface P-selectin expression (MFI) for platelets and microparticles in SMLEF bipolar pulse activated PRP was significantly higher than that in SMHEF-activated PRP, but not significantly different from that produced by thrombin activation. Higher levels of EGF were observed following either SMLEF bipolar pulses or SMHEF pulses of PRP than after bovine thrombin activation while VEGF, PDGF, and PF4 levels were similar with all three activating conditions. Cell proliferation was significantly increased by releasates of both SMLEF bipolar pulse and SMHEF pulse activated PRP compared to plasma alone. Conclusions PEF activation of PRP at bipolar low vs. monopolar high field strength results in differential platelet-derived microparticle production and activation of platelet surface procoagulant markers while inducing similar release of growth factors and similar capacity to induce cell proliferation. Stimulation of PRP with SMLEF bipolar pulses is gentler than SMHEF pulses, resulting in less platelet microparticle generation but with overall activation levels similar to that obtained with thrombin. These results suggest that PEF provides the means to alter, in a controlled fashion, PRP properties thereby enabling evaluation of their effects on wound healing and clinical outcomes.

Extending membrane pore lifetime with AC fields: A modeling study

AC (sinusoidal) fields with frequencies from kilohertz to gigahertz have been used for gene delivery. To understand the impact of AC fields on electroporation dynamics, we couple a nondimensionalized Smoluchowski equation to an exact representation of the cell membrane voltage obtained solving the Laplace equation. The slope of the pore energy function, dφ/dr, with respect to pore radius is critical in predicting pore dynamics in AC fields because it can vary from positive, inducing pore shrinkage, to negative, driving pore growth. Specifically, the net sign of the integral of dφ/dr over time determines whether the average pore size grows (negative), shrinks (positive), or oscillates (zero) indefinitely about a steady-state radius, rss. A simple analytic relationship predicting the amplitude of the membrane voltage necessary for this behavior agrees well with simulation for frequencies from 500 kHz to 5 MHz for rss   10 nm), dφ/dr oscillates about a negative value, sugge...

Accounting for conducting inclusion permeability in the microwave regime in a modified generalized effective medium theory

We standardize the approach to predict composite permeability, μeff, to determine the reflection (R), transmission (T), and absorption (A) using the generalized effective medium theory. For stainless steel (SS) spheres and fibers, we calculate the inclusion permeability based on the effective susceptibility generated by eddy currents in a time-varying magnetic field. This gives reasonable agreement with measured μeff when used in the Bruggeman theory and agreement with R, T, and A within 10%. For SS flakes, we fit the imaginary component of μeff to a single-peak Lorentzian for each loading and use typical fitting parameters to predict μeff and, ultimately, R, T, and A within 5%. The fitting exponent t was constant for all frequencies for a given inclusion shape (4.7, 1.6, and 1.7 for spheres, fibers, and flakes, respectively) and Ax typically varied with inverse volume loading rather than percolation threshold. Interestingly, at percolation for the fibers, Ax approached a constant that approximately equaled the observed percolation threshold.

Predicting effective permittivity of composites containing conductive inclusions at microwave frequencies

We predict the effective dielectric properties at microwave frequencies of composites containing various volume loadings of high conductivity (stainless steel or iron) spheres or flakes by adapting the semi-empirical method developed by McLachlan. Rather than the typical approach of fixing A (A = 1/vc – 1, where vc is percolation threshold) for a given inclusion shape, we consider it as an unknown and fix one of the geometric exponents. We observe that A varies linearly with the inverse of volume loading with a higher slope for flakes than spheres. The exponent is consistently higher for spheres than flakes and for iron than stainless steel. We achieve good agreement between measured and calculated permittivity over a wide range of volume loadings, inclusion shapes, and materials from 3 to 20 GHz.

Design, characterization and experimental validation of a compact, flexible pulsed power architecture for ex vivo platelet activation

Electric pulses can induce various changes in cell dynamics and properties depending upon pulse parameters; however, pulsed power generators for in vitro and ex vivo applications may have little to no flexibility in changing the pulse duration, rise- and fall-times, or pulse shape. We outline a compact pulsed power architecture that operates from hundreds of nanoseconds (with the potential for modification to tens of nanoseconds) to tens of microseconds by modifying a Marx topology via controlling switch sequences and voltages into each capacitor stage. We demonstrate that this device can deliver pulses to both low conductivity buffers, like standard pulsed power supplies used for electroporation, and higher conductivity solutions, such as blood and platelet rich plasma. We further test the effectiveness of this pulse generator for biomedical applications by successfully activating platelets ex vivo with 400 ns and 600 ns electric pulses. This novel bioelectrics platform may provide researchers with unprecedented flexibility to explore a wide range of pulse parameters that may induce phenomena ranging from intracellular to plasma membrane manipulation.

Cathode spot motion in an oblique magnetic field

Improper control of cathode spot (CS) motion can lead to uneven cathode erosion and shorter life, so magnetic fields are often used to direct CS motion. Here, we incorporate an axial magnetic field component into a model for CS retrograde motion based on the difference between the plasma kinetic and self-magnetic pressures. We consider the motion of the positive space charge associated with retrograde motion to generate a current perpendicular to the axial magnetic field, introducing an additional component to CS motion. The predicted angle of CS motion agrees well with the experimental data and a prior model based on electron backflow to the cathode surface.