Nonlinear spectroscopy of chromophore aggregates with entangled photon pulses

The response of the bacterial reaction center to entangled photons is compared with stochastic and chirped pulses. Nonlinear optical signals reveal how distributions of exciton states may be controlled by quantum light

Protein Corona Prevents TiO₂ Phototoxicity

<div><p>Background & Aim</p><p>TiO₂ nanoparticles have generally low toxicity in the <i>in vitro</i> systems although some toxicity is expected to originate in the TiO₂-associated photo-generated radical production, which can however be modulated by the radical trapping ability of the serum proteins. To explore the role of serum proteins in the phototoxicity of the TiO₂ nanoparticles we measure viability of the exposed cells depending on the nanoparticle and serum protein concentrations.</p><p>Methods & Results</p><p>Fluorescence and spin trapping EPR spectroscopy reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles’ surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes’ surface.</p><p>Conclusion</p><p>These results imply that TiO₂ nanoparticles should be applied with ligands such as proteins when phototoxic effects are not desired - for example in cosmetics industry. On the other hand, the nanoparticles should be used in serum free medium or any other ligand free medium, when phototoxic effects are desired – as for efficient photodynamic cancer therapy.</p></div

Measurements of hydroxyl radical formations by UV irradiation of the TiO₂-NTs.

<p>Spin trap DMPO was used to detect production of the hydroxyl radicals generated by UV irradiated TiO₂-NTs. TiO₂-NTs were mixed with DMPO spin traps and cell medium with 10% FBS or without FBS. The sample was irradiated for 5 min with UV light (wavelength of 356 nm) or left in dark (control), followed by EPR measurements immediately after the addition of the cell medium. In parallel experiments the dispersion of the TiO₂-NTs in the cell medium without or with the serum proteins (FBS) was put in the dark for one hour, than the spin trap DMPO was added and samples were irradiated with the UV light. (A) Representative EPR spectrum of a trapped hydroxyl radical in the presence of the FBS or (B) absence of the FBS. (C) The experimental EPR spectrum was simulated with hyperfine splitting constants: A_N = 1.49 G and A_H = 1.49 mT typical for OH radical. Simulation was done with EPRSim Wizard [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129577#pone.0129577.ref029" target="_blank">29</a>]. EPR spectrum intensity peak normalized to the experiment with highest intensity peak, of nanomaterial in the cell medium without the FBS and with the FBS.</p

Labelling of TiO₂-NTs with fluorophore Alexa via the two steps-reaction of the TiO₂-NTs surface modification.

<p>Firstly, 3-(2-aminoethylamino)propyltrimethoxysilane (AEAPMS) is attached to free–OH groups of the nanotubes’ surface (fTiO₂-NT). Secondly, the Alexa 488 SDP ester is covalently linked to the free amino groups of silane molecules (A-TiO₂-NT).</p

Toxicity and phototoxicity of TiO₂-NTs.

<p>MCF-7 cells were grown during the first day, exposed to 1000 μg/mL of nanotubes during the second day and incubated in dark or under UV irradiation (1.6 W/m², wavelength 356 nm, every 3 h for 15 min in a period of 24 h) during the third day. Control experiment were performed without the appropriate stressor (nanotubes during the second day or UV irradiation during the third day). On the fourth day: (A) optical density of samples was measured by MTS assay, (B) cells were trypsinized and counted manually using Trypan Blue, (C) the same number of MCF-7 cell were seeded in pools to grow for additional 14 days and colonies were counted. Survival factor (SF) was calculated as percentage of plating efficiency (PE) of the treated cells over PE of the untreated cells. Since normality test (Shapiro-Wilk) failed, we used Mann-Whitney U statistics instead. ** denotes the significant difference in the median values at P = <0.001.</p

Cell viability versus concentration of TiO₂-NTs in absence or presence of UV radiation.

<p>Absorbance of MTS was measured as described in Materials and methods section. (A) Each non-irradiated data set (red symbols) is compared to a sample irradiated with UV light (blue symbols) at given concentration of TiO₂-NTs. All Measurements are presented in a boxplot representing the distribution of the measured cell viability and described in Materials and methods. Differences in the cell viability between irradiated and non-irradiated cells are shown with the black arrows at each concentration of TiO₂-NTs. The differences in the median values between the two groups at 0 and 1000 μg/mL and the two groups at 1 and 50 μg/mL of nanotubes are considered to be significant at P = <0.001, and P = <0.05, respectively. (B) Differences of median values of MTS absorbance between irradiated and non-irradiated cells, shown with the black arrows within the frame A, are plotted with the open bars. Solid black line represents the prediction of the difference in cell viability, based on the effect of the UV irradiation and phototoxic effect due to irradiated nanotubes not covered by serum proteins. Both contributions are shown separately in the frame C. (C) Solid dark blue line shows the transmittance of UV light versus the concentration of the nanotubes obtained experimentally (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129577#pone.0129577.s002" target="_blank">S2 Information</a>. Optical properties of TiO₂-NTs dispersion.), while dashed red line represents the concentration of the free nanotubes calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129577#pone.0129577.s003" target="_blank">S3 Information</a>. Model of albumin binding to TiO2-NTs and best fit parameter values. (D) The relative amount of TiO₂-NTs covered by the serum proteins (red line) and the relative amount of serum proteins bound to TiO₂-NTs (yellow line) are shown as predicted by the model described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129577#pone.0129577.s003" target="_blank">S3 Information</a>. Model of albumin binding to TiO2-NTs and best fit parameter values.</p

Stability of the TiO₂-NTs in cell medium at different concentrations of FBS.

<p>(A) Rate of sedimentation measured through the absorbance at 400 nm on a UV-VIS spectrometer up to one day. (B) Relative proportion of the remaining TiO₂-NTs in dispersion after centrifugation at 15000 rpm (r = 6.2 cm) for 20 min. (C) Average hydrodynamic diameter of the remaining small aggregates and coated single nanotubes in dispersion of the TiO₂-NTs after centrifugation.</p

Characterization of the TiO₂-NTs and their photo-catalytic activity.

<p>(A) TEM image of the TiO₂-NTs. Inset in Fig 2A shows interlayer spacing of about 0.35 nm that agree well with [100] diffraction peak of anatase TiO₂. (B) X-ray powder diffraction of TiO₂-NTs, anatase peaks are marked with A. (C) OH• radical production of TiO₂-NTs (closed black circles) as a function of wavelength compared to the radical production by P25 from Degussa (open circles). At 365 nm production of radicals decreases to half of their maximal activity at lower wavelengths.</p