Plasmonics on nanostructures for cell manipulation

A plasmon resonance based method for introducing foreign material into living cells is presented. By illuminating gold-coated or structured surfaces, near field enhancement is employed to selectively open the cell membrane using ultrashort laser pulses

Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine

Background: In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. Results: The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. Conclusion: This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.DFG/SFB/Transregio 37DFG/EXC/REBIRTHDFG/Transregio 37DFG/EXC/REBIRT

Why and how does shared language affect subsidiary knowledge inflows? A social identity perspective

We draw on social identity theory to conceptualize a moderated mediation model that examines the relationship between shared language among subsidiary and HQ managers, and subsidiaries’ knowledge inflows from HQ. Specifically, we study (1) whether this relationship is mediated by the extent to which subsidiary managers share HQ goals and vision, and the extent to which HR decisions are centralized; and (2) whether subsidiary type moderates these mediated relationships. Building on a sample of 817 subsidiaries in nine countries/regions, we find support for our model. Implications for research on HQ-subsidiary knowledge flows, social identity theory and international HRM are discussed

Investigation of biophysical mechanisms in gold nanoparticle mediated laser manipulation of cells using a multimodal holographic and fluorescence imaging setup.

Laser based cell manipulation has proven to be a versatile tool in biomedical applications. In this context, combining weakly focused laser pulses and nanostructures, e.g. gold nanoparticles, promises to be useful for high throughput cell manipulation, such as transfection and photothermal therapy. Interactions between laser pulses and gold nanoparticles are well understood. However, it is still necessary to study cell behavior in gold nanoparticle mediated laser manipulation. While parameters like cell viability or perforation efficiency are commonly addressed, the influence of the manipulation process on other essential cell parameters is not sufficiently investigated yet. Thus, we set out to study four relevant cell properties: cell volume and area, ion exchange and cytoskeleton structure after gold nanoparticle based laser manipulation. For this, we designed a multimodal imaging and manipulation setup. 200 nm gold nanoparticles were attached unspecifically to canine cells and irradiated by weakly focused 850 ps laser pulses. Volume and area change in the first minute post laser manipulation was monitored using digital holography. Calcium imaging and cells expressing a marker for filamentous actin (F-actin) served to analyze the ion exchange and the cytoskeleton, respectively. High radiant exposures led to cells exhibiting a tendency to shrink in volume and area, possibly due to outflow of cytoplasm. An intracellular raise in calcium was observed and accompanied by an intercellular calcium wave. This multimodal approach enabled for the first time a comprehensive analysis of the cell behavior in gold nanoparticle mediated cell manipulation. Additionally, this work can pave the way for a better understanding and the evaluation of new applications in the context of cell transfection or photothermal therapy

Gold nanoparticle mediated laser transfection for efficient siRNA mediated gene knock down.

Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types

Magnetic beads enhance adhesion of NIH 3T3 fibroblasts: A proof-of-principle in vitro study for implant-mediated long-term drug delivery to the inner ear

Introduction Long-term drug delivery to the inner ear may be achieved by functionalizing cochlear implant (CI) electrodes with cells providing neuroprotective factors. However, effective strategies in order to coat implant surfaces with cells need to be developed. Our vision is to make benefit of electromagnetic field attracting forces generated by CI electrodes to bind BDNF-secreting cells that are labelled with magnetic beads (MB) onto the electrode surfaces. Thus, the effect of MB-labelling on cell viability and BDNF production were investigated. Materials and Methods Murine NIH 3T3 fibroblasts-genetically modified to produce BDNF-were labelled with MB. Results Atomic force and bright field microscopy illustrated the internalization of MB by fibroblasts after 24 h of cultivation. Labelling cells with MB did not expose cytotoxic effects on fibroblasts and allowed adhesion on magnetic surfaces with sufficient BDNF release. Discussion Our data demonstrate a novel approach for mediating enhanced long-term adhesion of BDNF-secreting fibroblasts on model electrode surfaces for cell-based drug delivery applications in vitro and in vivo. This therapeutic strategy, once transferred to cells suitable for clinical application, may allow the biological modifications of CI surfaces with cells releasing neurotrophic or other factors of interest. © 2016 Aliuos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Potential causes of the calcium signaling during gold nanoparticle mediated laser manipulation.

<p>Two different pathways in the presence or absence of a calcium chelating agent like EGTA are depicted. The intercellular calcium wave occurs in the presence of EGTA because the cascade is possibly mediated by the IP₃ pathway or paracrine ATP signaling. ATP might also be released from the cell after perforation.</p

Schematic of the experimental setup.

<p>An epifluorescence microscope is modified to capture digital holography and fluorescence image data. Coherent illumination necessary for digital holography is realized by weakly focusing a cw Helium Neon Laser on the sample. The pulsed manipulation laser is coupled into the setup parallel to the HeNe-Laser using a dichroic mirror (DM) and focused onto the sample. For fluorescence excitation a mercury vapor lamp is employed. At the side port of the microscope the manipulation laser is attenuated using a notch filter (F1). A beam splitter directs 90% of the light to the fluorescence unit where it passes through a filter blocking the coherent illumination (F2) and an emission filter (F3). The remaining light travels to the digital holography module which is set up in off-axis Michelson configuration.</p

Cell phase volume change evaluated by holographic microscopy.

<p>a) Phase images show phase volume and area change after 40 ms irradiation time for two radiant exposures. See corresponding supporting information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124052#pone.0124052.s005" target="_blank">S1 Video</a>. Scale bars are 20 <i>μ</i>m. b) Shown here are box plots of the fit parameters for the relative phase volume change of cells over a time of 60 s. See supporting information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124052#pone.0124052.s007" target="_blank">S3 Video</a>. Whiskers for box plots depict one standard deviation. The upper box plot indicates that the total loss of phase volume increases with radiant exposure. The middle box plot presents the linear slope of the fit, which is an indicator of the slow process of cell phase volume change. It can be either positive or negative, corresponding to linear phase volume increase or decrease. In the bottom box plot, the exponential decay parameter is depicted. The percentage of cells, which show a combination of linear and exponential decay is indicated in the upper histogram panel. The graph below this shows the decay constants for those cells which exhibited an exponential volume decay after irradiation. The decay parameter is smallest on average for highest radiant exposure, suggesting fastest loss of volume. Data of at least 22 irradiated cells is shown.</p