<i>In vitro</i> biomechanical properties, fluorescence imaging, surface-enhanced Raman spectroscopy, and photothermal therapy evaluation of luminescent functionalized CaMoO₄:Eu@Au hybrid nanorods on human lung adenocarcinoma epithelial cells

<p>Highly dispersible Eu³⁺-doped CaMoO₄@Au-nanorod hybrid nanoparticles (HNPs) exhibit optical properties, such as plasmon resonances in the near-infrared region at 790 nm and luminescence at 615 nm, offering multimodal capabilities: fluorescence imaging, surface-enhanced Raman spectroscopy (SERS) detection and photothermal therapy (PTT). HNPs were conjugated with a Raman reporter (4-mercaptobenzoic acid), showing a desired SERS signal (enhancement factor 5.0 × 10⁵). The HNPs have a heat conversion efficiency of 25.6%, and a hyperthermia temperature of 42°C could be achieved by adjusting either concentration of HNPs, or laser power, or irradiation time. HNPs were modified with antibody specific to cancer biomarker epidermal growth factor receptor, then applied to human lung cancer (A549) and mouse hepatocyte cells (AML12), and <i>in vitro</i> PTT effect was studied. In addition, the biomechanical properties of A549 cells were quantified using atomic force microscopy. This study shows the potential applications of these HNPs in fluorescence imaging, SERS detection, and PTT with good photostability and biocompatibility.</p

Representative AFM deflection mode images of HAECs treated with 10 µg/ml DEPs for different time (cells were fixed prior to observe in PBS).

<p>Column 1 shows images of cells treated with DEPs for 4 hours; column 2, 8 hours; column 3, 24 hours; column 4, 48 hours. Row 1 shows deflection mode images of single cells; rows 2 and 3 show images of membrane surface ultrastructures. Particles on cells representatively indicated by green arrows are DEP. Scanning size of row 1 is marked on respective image; and that of row 2 and row 3 (ultrastructures): 10 µm×10 µm. This group of images indicated that when treatment time increased from 4 hours to 48 hours, cell membrane damage appeared in a time-dependent manner.</p

Representative images of DEP (10 µg/ml) -treated HAECs (fixed cells) in PBS obtained by the coupled AF/FL microscope.

<p>The panel arrangement is similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036885#pone-0036885-g006" target="_blank">Figure 6</a>. Optical images were obtained using a 100× oil objective. The exposure time of DEPs is marked on respective optical images. It is seen that fluorescence intensity slightly decreased, although cytoskeletal structures can be seen after 48 hours of treatment. Interestingly, cellular mitosis was still progressing and a dividing cell nucleus can be seen (column 4), implying this low dosage did not completely inhibit cell activities, which coincided with assessment of cell viability. Additionally, DEPs attached on cell membrane surface are seen for all four experimental groups, as indicated by green arrows.</p

Representative observations of fixed HAECs using the coupled AF/FL microscope in PBS.

<p>Column 1 shows images of untreated (0 µg/ml) group; columns 2–4 are images of cells treated with different concentrations of DEPs for 4 hours. Row 1 shows bright-field images, row 2 is fluorescence images, and row 3 exhibits AFM images of the same cells. Optical images were obtained by the 60× OIL (or 100× OIL for 10 µg/ml group); scanning size of AFM images is marked on the respective images. The black dots representatively pointing by green arrows are DEPs attached on cell membrane.</p

Statistical analysis of adhesion force (<i>F</i>_ad, nN) and Young's Modulus (<i>E</i>, kPa) of live HAECs.

<p>Decreases of adhesion force implicates depression of cell membrane adhesion behavior in the presence of DEP, and down-regulation of Young's modulus suggests cells become softer in the context of cytoskeleton losing induced by DEP treatment. The data of histograms were obtained from multiple cells (N_cell = 12 for each group, and 26 datum points on each cell). Error bar: standard error (SE). (*, P<0.01).</p

Maps of cell mechanics including adhesion force (<i>F</i>_ad, nN) and Young's modulus (<i>E</i>, kPa) of live HAECs, which were measured on single cells in culture medium.

<p>These maps are only to exhibit heterogeneous property in cell mechanics and do not reflect alteration tendency of <i>F</i>_ad or <i>E</i> because of individuality of cell-cell. Column 1, untreated (0 µg/ml) cells; columns 2–4, images of cells treated with different concentrations of DEPs for 4 hours. Row 1 shows AFM deflection mode images; rows 2 and 3 are their corresponding maps of adhesion force and Young's modulus. The color bars showing at the right of maps display the value scale of <i>F</i>_ad and <i>E</i>. The scanning size of AFM images is marked on each image of row 1.</p

Representative fluorescence images of cell viability evaluation.

<p>HAECs cells were exposed to DEPs with three different concentrations: 10 µg/ml (row 1), 50 µg/ml (row 2), 100 µg/ml (row 3), and four treatment durations: 4 hours (column 1), 8 hours (column 2), 24 hours (column 3), and 48 hours (column 4), respectively. Cells are stained with Invitrogen LIVE/DEAD Viability/Cytotoxicity Assay Kit. Green fluorescence presents live cells, whereas red fluorescence shows dead or membrane-damaged cells. All images were obtained with 10× lens. These fluorescence images together revealed that DEPs impaired cell viability in a dosage- and a time-dependent manner. And the corresponding bright-field picture of each fluorescent image is shown as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036885#pone.0036885.s005" target="_blank">Information S5</a>, from which DEPs can be clearly seen.</p

Representative AFM deflection mode images of fixed HAECs obtained in PBS.

<p>Column 1, images of untreated cells (0 µg/ml group); columns 2–4, images of cells treated with different concentrations of DEPs for 4 hours. Row I, image of single cells, whose scanning size is marked on respective image; rows II and III, images of membrane surface ultrastructures, whose scanning size is 10 µm×10 µm. Particles (representatively shown by green arrows) on cells are DEPs. This group of images indicated that when DEP concentration increased from 0 µg/ml to 100 µg/ml, cytoskeletal structures became gradually degraded, suggesting that cell membrane damage appeared in a dosage-dependent manner. And poor resolution of ultra-structures of 100 µg/ml DEP treated cells is mainly resulted from influences of the large amount of DEPs on membrane surface.</p

Investigation of Free Fatty Acid Associated Recombinant Membrane Receptor Protein Expression in HEK293 Cells Using Raman Spectroscopy, Calcium Imaging, and Atomic Force Microscopy

G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293–GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293–GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca²⁺) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293–GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells