A dose-response study of a novel method of selective tissue modification of cellular structures in the skin with nanosecond pulsed electric fields.

BACKGROUND AND OBJECTIVES: This study describes the effects of nanosecond pulsed electric fields (nsPEF) on the epidermis and dermis of normal skin scheduled for excision in a subsequent abdominoplasty. NsPEF therapy applies nanosecond pulses of electrical energy to induce regulated cell death (RCD) in cellular structures, with negligible thermal effects. Prior pre-clinical studies using nsPEF technology have demonstrated the ability to stimulate a lasting immune response in animal tumor models, including melanoma. This first-in-human-use of nsPEF treatment in a controlled study to evaluate the dose-response effects on normal skin and subcutaneous structures is intended to establish a safe dose range of energies prior to use in clinical applications using nsPEF for non-thermal tissue modification. STUDY DESIGN/MATERIALS AND METHODS: Seven subjects with healthy tissue planned for abdominoplasty excision were enrolled. Five subjects were evaluated in a longitudinal, 60-day study of effects with doses of six nsPEF energy levels. A total of 30 squares of spot sizes 25mm2 or less within the planned excision area were treated and then evaluated at 1 day, 5 days, 15 days, 30 days, and 60 days prior to surgery. Photographs were taken over time of each treated area and assessed by three independent and blinded dermatologists for erythema, flaking and crusting using a 5-point scale (0 = low, 4 = high). Punch biopsies of surgically removed tissue were processed and evaluated for tissue changes using hematoxylin and eosin, trichome, caspase-3, microphthalmia transcription factor, and elastin stains and evaluated by a dermatopathologist. The skin of two subjects received additional treatments at 2 and 4 hours post-nsPEF and was evaluated in a similar manner. RESULTS: Most energy settings exhibited delayed epidermal loss followed by re-epithelization by day 15 and a normal course of healing. Histologic analysis identified the appearance of activated caspase-3 at two and four hours after nsPEF treatment, but not at later time points. At the 1-day time point, a nucleolysis effect was observed in epidermal cells, as evidenced by the lack of nuclear staining while the epidermal plasma membranes were still intact. Cellular structures within the treatment zone such as melanocytes, sebaceous glands, and hair follicles were damaged while acellular structures such as elastic fibers and collagen were largely unaffected except for TL6 which showed signs of dermal damage. Melanocytes reappeared at levels comparable with untreated controls within 1 month of nsPEF treatment. CONCLUSIONS: The selective effect of nsPEF treatment on cellular structures in the epidermal and dermal layers suggests that this non-thermal mechanism for targeting cellular structures does not affect the integrity of dermal tissue within a range of energy levels. The specificity of effects and a favorable healing response makes nsPEF ideal for treating cellular targets in the epidermal or dermal layers of the skin, including treatment of benign and malignant lesions. NsPEF skin treatments provide a promising, non-thermal method for treating skin conditions and removing epidermal lesions. © 2019 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Nanoscale dose deposition in cell structures under X-ray irradiation treatment assisted with nanoparticles: An analytical approach to the relative biological effectiveness.

In this study, an analytical model for the assessment of the modification of cell culture survival under ionizing radiation assisted with nanoparticles (NPs) is presented. The model starts from the radial dose deposition around a single NP, which is used to describe the dose deposition in a cell structure with embedded NPs and, in turn, to evaluate the number of lesions formed by ionizing radiation. The model is applied to the calculation of relative biological effectiveness values for cells exposed to 0.5mg/g of uniformly dispersed NPs with a radius of 10nm made of Fe, I, Gd, Hf, Pt and Au and irradiated with X-rays of energies 20keV higher than the element K-shell binding energy.

Effect of dose-rate and irradiation geometry on the biological response of normal cells and cancer cells under radiotherapeutic conditions.

Biological efficacy of radiation depends on its energy, dose, dose rate, and on the type of cell irradiated. Changes in the radiation-energy spectrum due to passage through absorbing and scattering media affect the variability of biological responses of the cells. We investigated the impact of photon-radiation dose rate on the biological response of both normal and cancer cells in culture exposed to radiation in various positions (relative to the axis of the radiation beam) and depth of the absorbing medium (water). Human cancer cells (A549 and HCT116) as well as normal human cells (BEAS-2B) were placed in a water phantom at different medium depths (3 cm, 15 cm) and exposed to 6-MV photon radiation delivered at a beam rate of either 100 or 600 MU/min (Monitor Units per minute). The applied dose was 5 Gy. Cells were exposed in the axis and four cm outside the radiation field. Radiation-induced genetic changes were estimated as frequency of micro-nucleated and apopototic-like cells, by use of a cytokinesis-block micronucleus test. A smaller dose rate induced more severe cytogenetic damage (formation of micro-nucleated and apoptotic cells) than a higher dose rate, both in normal and in cancer cells. More micro-nucleated and apoptotic cells were formed at larger depth than at smaller depth. This holds true for both the normal and the two types of cancer cell investigated. The extent of cytogenetic damage arising in cells placed outside the irradiation field is independent of positioning depth and dose rate. Exposure of cells to smaller dose rates and larger depths in water medium resulted in a better ratio of cytogenetic damage to cancer cells irradiated in the beam axis vs damage to normal cells exposed outside the radiation field.

Free energies of stable and metastable pores in lipid membranes under tension.

The free energy profile of pore formation in a lipid membrane, covering the entire range from a density fluctuation in an intact bilayer to a large tension-stabilized pore, has been calculated by molecular dynamics simulations with a coarse-grained lipid model. Several fixed elongations are used to obtain the Helmholtz free energy as a function of pore size for thermodynamically stable, metastable, and unstable pores, and the system-size dependence of these elongations is discussed. A link to the Gibbs free energy at constant tension, commonly known as the Litster model, is established by a Legendre transformation. The change of genus upon pore formation is exploited to estimate the saddle-splay modulus or Gaussian curvature modulus of the membrane leaflets. Details are provided of the simulation approach, which combines the potential of mean constraint force method with a reaction coordinate based on the local lipid density.

Feasibility of imaging living cells at subnanometer resolutions by ultrafast X-ray diffraction.

Detailed structural investigations on living cells are problematic because existing structural methods cannot reach high resolutions on non-reproducible objects. Illumination with an ultrashort and extremely bright X-ray pulse can outrun key damage processes over a very short period. This can be exploited to extend the diffraction signal to the highest possible resolution in flash diffraction experiments. Here we present an analysis of the interaction of a very intense and very short X-ray pulse with a living cell, using a non-equilibrium population kinetics plasma code with radiation transfer. Each element in the evolving plasma is modeled by numerous states to monitor changes in the atomic populations as a function of pulse length, wavelength, and fluence. The model treats photoionization, impact ionization, Auger decay, recombination, and inverse bremsstrahlung by solving rate equations in a self-consistent manner and describes hydrodynamic expansion through the ion sound speed. The results show that subnanometer resolutions could be reached on micron-sized cells in a diffraction-limited geometry at wavelengths between 0.75 and 1.5 nm and at fluences of 1011-1012 photons microm-2 in less than 10 fs. Subnanometer resolutions could also be achieved with harder X-rays at higher fluences. We discuss experimental and computational strategies to obtain depth information about the object in flash diffraction experiments.

Ring-like nucleoid does not play a key role in radioresistance of Deinococcus radiodurans.

The conclusion based on transmission electron microscopy, "the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance", has instigated lots of debates. In this study, according to the previous research of Pprl's crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. radiodurans R1 strain, pprf function-deficient mutant (YR1), and pprl function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence microscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells exhibit the tightly packed ring-like morphology, while the pprl function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprl completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprl partly function-complementary strain YR1002's nucleiods exhibit about 60% ring-like structure; (3) a Pprl C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensitive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance.

Effects of ionizing radiation on cellular structures, induced instability and carcinogenesis.

Ionizing radiation is perhaps the most extensively studied human carcinogen. There have been a number of epidemiological studies on human populations exposed to radiation for medical or occupational reasons, as a result of protracted environmental exposures due to radiation accidents, or after atomic bombings. As a result of these studies exposure to ionizing radiation has been unambiguously linked to cancer causation. While cancer induction is the primary concern and the most important somatic effect of exposure to ionizing radiation, potential health risks do not only involve neoplastic diseases but also somatic mutations that might contribute to birth defects and ocular maladies, and heritable mutations that might impact on disease risks in future generations. Consequantly it is important we understand the long-term health risks associated with exposure to ionizing radiation.