Gary Friedman

Physical and biological mechanisms of direct plasma interaction with living tissue

Mechanisms of plasma interaction with living tissues and cells can be quite complex, owing to the complexity of both the plasma and the tissue. Thus, unification of all the mechanisms under one umbrella might not be possible. However, the authors are attempting to make first steps in this direction. In this paper, analysis of interaction of floating electrode dielectric barrier discharge (FE-DBD) with living tissues and cells is presented and biological and physical mechanisms are discussed. In physical mechanisms, charged species are identified as the major contributors to the desired effect and a mechanism of this interaction is proposed. Biological mechanisms are also addressed and a hypothesis of plasma selectivity and its effects is offered.

Effects of Non-Thermal Plasma on Mammalian Cells

Thermal plasmas and lasers have been widely used in medicine to cut, ablate and cauterize tissues through heating; in contrast, non-thermal plasma produces no heat, so its effects can be selective. In order to exploit the potential for clinical applications, including wound healing, sterilization, blood coagulation, and cancer treatment, a mechanistic understanding of the interaction of non-thermal plasma with living tissues is required. Using mammalian cells in culture, it is shown here that non-thermal plasma created by dielectric barrier discharge (DBD) has dose-dependent effects that range from increasing cell proliferation to inducing apoptosis. It is also shown that these effects are primarily due to formation of intracellular reactive oxygen species (ROS). We have utilized γ-H2AX to detect DNA damage induced by non-thermal plasma and found that it is initiated by production of active neutral species that most likely induce formation of organic peroxides in cell medium. Phosphorylation of H2AX following non-thermal plasma treatment is ATR dependent and ATM independent, suggesting that plasma treatment may lead to replication arrest or formation of single-stranded DNA breaks; however, plasma does not lead to formation of bulky adducts/thymine dimers.

High field gradient targeting of magnetic nanoparticle-loaded endothelial cells to the surfaces of steel stents

A cell delivery strategy was investigated that was hypothesized to enable magnetic targeting of endothelial cells to the steel surfaces of intraarterial stents because of the following mechanisms: (i) preloading cells with biodegradable polymeric superparamagnetic nanoparticles (MNPs), thereby rendering the cells magnetically responsive; and (ii) the induction of both magnetic field gradients around the wires of a steel stent and magnetic moments within MNPs because of a uniform external magnetic field, thereby targeting MNP-laden cells to the stent wires. In vitro studies demonstrated that MNP-loaded bovine aortic endothelial cells (BAECs) could be magnetically targeted to steel stent wires. In vivo MNP-loaded BAECs transduced with adenoviruses expressing luciferase (Luc) were targeted to stents deployed in rat carotid arteries in the presence of a uniform magnetic field with significantly greater Luc expression, detected by in vivo optical imaging, than nonmagnetic controls.

Nonthermal Dielectric-Barrier Discharge Plasma-Induced Inactivation Involves Oxidative DNA Damage and Membrane Lipid Peroxidation in Escherichia coli

ABSTRACT Oxidative stress leads to membrane lipid peroxidation, which yields products causing variable degrees of detrimental oxidative modifications in cells. Reactive oxygen species (ROS) are the key regulators in this process and induce lipid peroxidation in Escherichia coli. Application of nonthermal (cold) plasma is increasingly used for inactivation of surface contaminants. Recently, we reported a successful application of nonthermal plasma, using a floating-electrode dielectric-barrier discharge (FE-DBD) technique for rapid inactivation of bacterial contaminants in normal atmospheric air (S. G. Joshi et al., Am. J. Infect. Control 38:293-301, 2010). In the present report, we demonstrate that FE-DBD plasma-mediated inactivation involves membrane lipid peroxidation in E. coli. Dose-dependent ROS, such as singlet oxygen and hydrogen peroxide-like species generated during plasma-induced oxidative stress, were responsible for membrane lipid peroxidation, and ROS scavengers, such as α-tocopherol (vitamin E), were able to significantly inhibit the extent of lipid peroxidation and oxidative DNA damage. These findings indicate that this is a major mechanism involved in FE-DBD plasma-mediated inactivation of bacteria.

Mechanism of Blood Coagulation by Nonthermal Atmospheric Pressure Dielectric Barrier Discharge Plasma

Mechanisms of blood coagulation by direct contact of nonthermal atmospheric pressure dielectric barrier discharge (DBD) plasma are investigated. This paper shows that no significant changes occur in the pH or Ca2+ concentration of blood during discharge treatment. Thermal effects and electric field effects are also shown to be negligible. Investigating the hypothesis that the discharge treatment acts directly on blood protein factors involved in coagulation, we demonstrate aggregation of fibrinogen, an important coagulation factor, with no effect on albumin. We conclude that direct DBD treatment triggers selective natural mechanisms of blood coagulation.

Targeting stents with local delivery of paclitaxel-loaded magnetic nanoparticles using uniform fields

The use of stents for vascular disease has resulted in a paradigm shift with significant improvement in therapeutic outcomes. Polymer-coated drug-eluting stents (DES) have also significantly reduced the incidence of reobstruction post stenting, a disorder termed in-stent restenosis. However, the current DESs lack the capacity for adjustment of the drug dose and release kinetics to the disease status of the treated vessel. We hypothesized that these limitations can be addressed by a strategy combining magnetic targeting via a uniform field-induced magnetization effect and a biocompatible magnetic nanoparticle (MNP) formulation designed for efficient entrapment and delivery of paclitaxel (PTX). Magnetic treatment of cultured arterial smooth muscle cells with PTX-loaded MNPs caused significant cell growth inhibition, which was not observed under nonmagnetic conditions. In agreement with the results of mathematical modeling, significantly higher localization rates of locally delivered MNPs to stented arteries were achieved with uniform-field–controlled targeting compared to nonmagnetic controls in the rat carotid stenting model. The arterial tissue levels of stent-targeted MNPs remained 4- to 10-fold higher in magnetically treated animals vs. control over 5 days post delivery. The enhanced retention of MNPs at target sites due to the uniform field-induced magnetization effect resulted in a significant inhibition of in-stent restenosis with a relatively low dose of MNP-encapsulated PTX (7.5 μg PTX/stent). Thus, this study demonstrates the feasibility of site-specific drug delivery to implanted magnetizable stents by uniform field-controlled targeting of MNPs with efficacy for in-stent restenosis.

Control of methicillin-resistant Staphylococcus aureus in planktonic form and biofilms: A biocidal efficacy study of nonthermal dielectric-barrier discharge plasma

BACKGROUND Bacterial contamination of surfaces with methicillin-resistant Staphylococcus aureus (MRSA) is a serious problem in the hospital environment and is responsible for significant nosocomial infections. The pathogenic contaminants form biofilms, which are difficult to treat with routine biocides. Thus, a continuous search for novel disinfection methods is essential for effective infection control measures. This demonstration of a novel technique for the control of virulent pathogens in planktonic form as well as in established biofilms may provide a progressive alternative to standard methodology. METHODS We evaluated a novel technique of normal atmospheric nonthermal plasma known as floating-electrode dielectric-barrier discharge (FE-DBD) plasma against a control of planktonic and biofilm forms of Escherichia coli, S aureus, multidrug-resistant methicillin-resistant S aureus (MRSA) -95 (clinical isolate), -USA300, and -USA400, using widely accepted techniques such as colony count assay, LIVE/DEAD BacLight Bacterial Viability assay, and XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay. RESULTS Exposure of free living planktonic forms of E coli, S aureus, and MRSA were rapidly inactivated by DBD plasma. Approximately 10(7) bacterial cells were completely (100%) killed, whereas 10(8) and 10(9) were reduced by approximately 90% to 95% and 40% to 45%, respectively, in less than 60 seconds (7.8 J/cm(2)) and completely disinfected in < or =120 seconds. In established biofilms, the susceptibility of MRSA USA400 was comparable with USA300 but less susceptible than MRSA95 (clinical isolate), S aureus, and E coli (P < .05) to FE-DBD plasma, and plasma was able to kill MRSA more than 60% within 15 seconds (1.95 J/cm(2)). The killing responses were plasma exposure-time dependent, and cell density dependent. The plasma was able disinfect surfaces in a less than 120 seconds. CONCLUSION Application of DBD plasma can be a valuable decontamination technique for the removal of planktonic and biofilm-embedded bacteria such as MRSA -USA 300, -USA 400, methicillin-sensitive S aureus (MSSA), and E coli, the more common hospital contaminants. Of interest, E coli was more resistant than S aureus phenotypes.

An approach to targeted drug delivery based on uniform magnetic fields

The capability to deliver high effective dosages to specific sites in the human body has become the holy grail of drug delivery research. Drugs with proven effectiveness under in vitro investigation often reach a major roadblock under in vivo testing due to a lack of an effective delivery strategy. In addition, many clinical scenarios require delivery of agents that are therapeutic at the desired delivery point, but otherwise systemically toxic. We propose a method for targeted drug delivery by applying uniform magnetic fields to an injected superparamagnetic colloidal fluid carrying a drug. The experimental and theoretical models presented give insight into the use of magnetic microspheres for site-specific delivery of therapeutic agents and blood flow occlusion for embolotherapy.

Nanosecond-Pulsed Uniform Dielectric-Barrier Discharge

The authors report a new nanosecond-pulsed dielectric-barrier discharge (DBD) for sterilization and other medical applications. In the literature, several discharges have been reported, with pulse durations on the order of hundreds of nanoseconds. In this paper, a novel pulsed DBD has been developed, with only few tens of nanosecond pulsewidths working uniformly over large range of electrode gap distance in air under atmospheric pressure.

Magnetically driven plasmid DNA delivery with biodegradable polymeric nanoparticles

Targeting gene therapy remains a challenge. The use of magnetic force to achieve this was investigated in the present study. It was hypothesized that nanoparticles with both controllable particle size and magnetic properties would enable magnetically driven gene delivery. We investigated this hypothesis by creating a family of novel biodegradable poly‐meric superparamagnetic nanoparticle (MNP) formulations. Polylactide MNP were formulated using a modified emulsification‐solvent evaporation methodology with both the incorporation of oleate‐coated iron oxide and a polyethylenimine (PEI) oleate ion‐pair surface modification for DNA binding. MNP size could be controlled by varying the proportion of the tetrahydrofuran cosolvent. Magnetically driven MNP‐mediated gene transfer was studied using a green fluorescent protein reporter plasmid in cultured arterial smooth muscle cells and endothelial cells. MNP‐DNA internalization and trafficking were examined by confocal microscopy. Cell growth inhibition after MNP‐mediated adiponectin plasmid transfec‐tion was studied as an example of a therapeutic end point. MNP‐DNA complexes protected DNA from degradation and efficiently transfected quiescent cells under both low and high serum conditions after a 15 min exposure to a magnetic field (500 G). There was negligible transfection with MNP in the absence of a magnetic field. Larger sized MNP (375 nm diameter) exhibited higher transfection rates compared with 185 nm‐ and 240 nm‐sized MNP. Internalized larger sized MNP escaped lysosomal localization and released DNA in the perinuclear zone. Adiponec‐tin plasmid DNA delivery using MNP resulted in a dose‐dependent growth inhibition of cultured arterial smooth muscle cells. It is concluded that magnetically driven plasmid DNA delivery can be achieved using biodegradable MNP containing oleate‐coated magnetite and surface modified with PEI oleate ion‐pair complexes that enable DNA binding.—Chorny, M., Polyak, B., Alferiev, I. S., Walsh, K., Friedman, G., Levy, R. J. Magnetically driven plasmid DNA delivery with biodegradable polymeric nanoparticles. FASEB J. 21, 2510–2519 (2007)