Dual Activity of Rose Bengal Functionalized to Albumin-Coated Lanthanide-Doped Upconverting Nanoparticles: Targeting and Photodynamic Therapy

A modified version of a desolvation method was used to render lanthanide-doped upconverting nanoparticles NaGdF₄:Yb³⁺/Er³⁺ (Ln-UCNPs) water-dispersible and biocompatible for photodynamic therapy. Bovine serum albumin (BSA) was used as surface coating with a direct conjugation to NaGdF₄:Yb³⁺/Er³⁺ nanoparticles forming a ∼2 nm thick shell. It was estimated that approximately 112 molecules of BSA were present and cross-linked per NaGdF₄:Yb³⁺/Er³⁺ nanoparticle. Analysis of the BSA structural behavior on the Ln-UCNP surfaces displayed up to 80% loss of α-helical content. Modification of the Ln-UCNPs with a BSA shell prevents luminescence quenching from solvent molecules (H₂O) with high energy vibrations that can interact with the excited states of the optically active ions Er³⁺ and Yb³⁺ via dipole–dipole interactions. Additionally, the photosensitizer rose bengal (RB) was conjugated to albumin on the surface of the Ln-UCNPs. Emission spectroscopy under 980 nm excitation was carried out, and an energy transfer efficiency of 63% was obtained. In vitro cell studies performed using human lung cancer cells (A549 cell line) showed that Ln-UCNPs coated with BSA were not taken by the cells. However, when RB was conjugated to BSA on the surface of the nanoparticles, cellular uptake was observed, and cytotoxicity was induced by the production of singlet oxygen under 980 nm irradiation

Examples for the determination of radial magnification errors.

<p>(A) Radial intensity profile measured in scans of the precision mask. Blue lines are experimental scans, and shaded areas indicate the regions expected to be illuminated on the basis of the known mask geometry. In this example, the increasing difference between the edges corresponds to a calculated radial magnification error of -3.1%. (B—D) Examples for differences between the experimentally measured positions of the light/dark transitions (blue circles, arbitrarily aligned for absolute mask position) and the known edge distances of the mask. The solid lines indicate the linear or polynomial fit. (B) Approximately linear magnification error with a slope corresponding to an error of -0.04%. Also indicated as thin lines are the confidence intervals of the linear regression. (C) A bimodal shift pattern of left and right edges, likely resulting from out-of-focus location of the mask, with radial magnification error of -1.7%. (D) A non-linear distortion leading to a radial magnification error of -0.53% in the <i>s</i>-values from the analysis of back-transformed data. The thin grey lines in C and D indicate the best linear fit through all data points.</p

Analysis of the rotor temperature.

<p>(A) Temperature values obtained in different instruments of the spinning rotor, as measured in the iButton at 1,000 rpm after temperature equilibration, while the set point for the console temperature is 20°C (indicated as dotted vertical line). The box-and-whisker plot indicates the central 50% of the data as solid line, with the median displayed as vertical line, and individual circles for data in the upper and lower 25% percentiles. The mean and standard deviation is 19.62°C ± 0.41°C. (B) Correlation between iButton temperature and measured BSA monomer <i>s</i>-values corrected for radial magnification, scan time, scan velocity, but not viscosity (symbols). In addition to the data from the present study as shown in (A) (circles), also shown are measurements from the pilot study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref027" target="_blank">27</a>] where the same experiments were carried out on instruments not included in the present study (stars). The dotted line describes the theoretically expected temperature-dependence considering solvent viscosity.</p

Corrected best-fit apparent monomer molecular mass from integration of the <i>c</i>(<i>s</i>) peak when scanned with the absorbance system (green) and the interference system (magenta).

<p>Only data with rmsd less than 0.01 OD or 0.01 fringes were included. The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.</p

Examples of transient changes in the console temperature reading during the SV experiment, as saved in the scan file data.

<p>For comparison, the maximum adiabatic cooling of -0.3°C would be expected after approximately 300 sec, recovering to the equilibrium temperature after approximately 1,200 s (see Fig 3 in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref033" target="_blank">33</a>]).</p

Histogram and box-and-whisker plot of <i>s</i>-values of the BSA monomer after different corrections: Raw experimental <i>s</i>-values (black, with grey histogram), scan time corrected <i>s</i>_<i>t</i>-values (blue), rotor temperature corrected <i>s</i>_<i>20T</i>-values (green), or radial magnification corrected <i>s</i>_<i>r</i>-values (cyan), and fully corrected <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values (red with red histogram).

<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as a vertical line.</p

Distributions of calculated BSA monomer signals for the different kits and the different optical systems.

<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as vertical line.</p

Correlations of the <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values of the BSA monomer with the difference of the best-fit meniscus from the mean meniscus value, separately for absorbance data sets (A) and interference data sets (B).

<p>The difference of the best-fit meniscus to the mean was calculated separately for each kit, to eliminate offsets due to different sample volumes in each kit, and then merged into groups for the optical systems. Data are shown as a histogram with frequency values indicated in the colorbar. The dotted lines show the theoretically expected dependence of the apparent <i>s</i>-value on errors in the absolute radial position.</p

Root-mean-square deviation of the best-fit <i>c</i>(<i>s</i>) model of the BSA sedimentation experiment when scanned with the absorbance system (green) and the interference system (magenta).

<p>The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.</p

Absence of a long-term trend in <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values of the BSA monomer with time of experiment for the three kits (blue, green, and magenta).

<p>Highlighted as bold solid line is the overall average, and the grey area indicates one standard deviation.</p