Analyst View Article Online PAPER View Journal A synchrotron-based infrared microspectroscopy This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. study on the cellular response induced by gold Open Access Article. Published on 16 September 2019. Downloaded on 9/27/2019 4:25:36 PM. Cite this: DOI: 10.1039/c9an01109a nanoparticles combined with X-ray irradiations on F98 and U87-MG glioma cell lines I. Martínez-Rovira, *a O. Seksek b,c and I. Yousef a The inclusion of nanoparticles (NP) in radiotherapy has been shown to increase the damaging effect on tumor cells. However, the mechanisms of action of NP combined with radiotherapy, and the influence of NP parameters and cell type on their radiosensitization capability at molecular and cellular levels still remain unclear. Gold NP (AuNP) have become particularly popular due to their multiple advantages. Within this context, our research work aimed to study the biochemical radiosensitization capacity of F98 and U87-MG glioma cell lines to 1.9 nm AuNP combined with X-ray irradiation. For this purpose, synchro- tron-based infrared microspectroscopy (SR-FTIRM) was used as a powerful tool for biochemical compo- sition and treatment response assessment of cells at a single-cell level. SR-FTIRM data, supported by multivariate analysis, revealed clear AuNP-induced changes in the DNA, protein and lipid spectral regions. The AuNP-related biochemical alterations appear prior to the irradiation, which gave us a first indication on the AuNP radiosensitization action. Biochemical modifications induced by the AuNP in the presence of radiotherapy irradiations include enhanced conformational changes in the protein secondary structures, Received 17th June 2019, variations in the intensity and position in the phosphodiester bands, and changes in the CH2 and CH3 Accepted 28th August 2019 stretching modes. These changes are better manifested at 24 hours post-irradiation time. SR-FTIRM DOI: 10.1039/c9an01109a results showed a clear heterogeneity in the biochemical cell response, probably due to the distinct cell– rsc.li/analyst NP interactions and thus, to different DNA damage and cell death processes. 1. Introduction Among all types of NP, gold (Z = 79) nanoparticles (AuNP) are particularly attractive since they are inert, highly biocompa- The radiosensitization potential of nanoparticles (NP) has tible, relatively easy to synthesize and functionalise, and they been previously reported in many biological studies (in vitro have an increased cross section for photoelectric photon and in vivo) in the past years (see, for instance ref. 1–5). Typical absorption (specially for low X-ray energies).1,4,5,8,9 The latter radiosensitizers include metallic high-atomic number (Z) enhances the secondary electron production (short-range materials that can augment the effectiveness of radiotherapy photoelectrons and Auger electrons) and the local dose depo- by inducing dose enhancement effects specifically in the sition near the NP.10 At the same time, these electrons can tumor site. This leads to an increase in the therapeutic index. further increase the generation of reactive oxygen species The use of NP over the conventional contrast agents resulted (ROS) and electron emission from nearby NP. Besides the pure in a larger NP accumulation in the tumor due to the enhanced physical effects, the presence of NP itself can also increase the permeability and retention (EPR) effects.6 In addition, previous ROS production, and thus, the cellular damage.4,11 studies reported that toxicity is highly reduced when using However, the radiosensitization mechanisms at molecular very small NP (<5 nm).7 and cellular levels still remain unclear. Results from the pre- vious biological studies indicated that radiosensitization effects might be driven by biological mechanisms induced by a MIRAS Beamline BL01, ALBA-CELLS Synchrotron, Carrer de la Llum 2–26, 08290 the NP rather than by physical (dose enhancement) effects. Cerdanyola del Vallès, Spain. E-mail: immamartinez@gmail.com These mechanisms include oxidative stress, DNA damage b Laboratoire d’Imagerie et Modélisation en Neurobiologie et Cancérologie (IMNC), induction, cell cycle effects and potential interference with cell Centre National de la Recherche Scientifique (CNRS); Université Paris Sud, Université communication.12–16 This, along with the influence of many Paris-Saclay, Campus Universitaire, Bât. 440, 15 rue Georges Clemenceau, F-91400 Orsay, France other parameters like NP size, shape and coating, which at the c Université de Paris, IMNC, F-91400 Orsay, France same time influence the NP distribution inside the cells, as This journal is © The Royal Society of Chemistry 2019 Analyst View Article Online Paper Analyst well as the distinct cellular responses, largely complicates 2.2. Gold nanoparticles research on radiosensitization effects of NP.4 Gold NP (1.9 nm AuroVist™) were purchased from Fourier transform infrared microspectroscopy (FTIRM) can Nanoprobes (Yaphank, NY, USA). The potential of these NP shed some light on the mechanisms involved in the radiosen- has been proved in previous biological studies.1,4,25,26 sitization effects of NP. FTIRM is a non-destructive A stock solution was extemporaneously prepared by mixing vibrational technique for the analysis of biological samples.17 the AuNP (40 mg of gold) with 1 mL ultrapure water (Millipore IR active molecular bonds of the main cell biomolecules Direct Q8). After rapid dissolution, the solution was filtered change the dipole moment as a result of the vibration that through a 0.2 μm centrifugal filter at 15 000g for 8 minutes occurs when infrared light is absorbed. The mid infrared This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. and kept at 4 °C. For cell treatment, the AuNP solution was spectral region is the most important for studying biological Open Access Article. Published on 16 September 2019. Downloaded on 9/27/2019 4:25:36 PM. diluted in fresh supplemented cell medium at a final concen- specimens since it includes the so-called ‘fingerprint region’ tration of 12 μM (500 μg mL−1).25 The cells were then incu- and the ‘lipids region’, which provides nucleic acid, protein, bated at 37 °C and 5% CO2 for 24 hours.25 Before the lipid, carbohydrate composition and/or conformational irradiation protocol, the AuNP-containing medium was dis- changes. This information enables the assessment of carded and replaced by 3 mL of fresh medium. cell functionality, cell division, differentiation, growth and metabolism.17–21 2.3. X-ray irradiation Within this context, the objective of this study is to use syn- Kilovoltage irradiations were performed at the Curie Institute chrotron-based FTIRM (SR-FTIRM) to disentangle radiosensiti- (Orsay, France) with an X-ray unit (XRAD 320, Cegelec). The zation effects of gold NP (1.9 nm AuroVist™)22 in glioma cells. mean energy of this X-ray generator is 90 keV and the dose rate Despite the current treatment options, high grade glioma has is 1.1 Gy min−1. Each well plate was irradiated separately at 5, poor prognosis in humans and thus, patient outcome could 10 and 20 Gy, respectively, for both cell lines, with and without benefit from the enhanced therapeutic index of combined AuNP. Two non-irradiated wells (0 Gy, without and with AuNP) RT-NP modalities. Gaining new insights into the radiosensiti- were kept for controls. Just after irradiation, the medium was zation effects of AuNP in glioma cells is essential to progress replaced by fresh medium. on the development of NP-based therapeutics. In this work, we present SR-FTIRM data on F98 and U87-MG glioma cell lines 2.4. SR-FTIRM sample preparation to assess distinct cellular responses as a function of the cell type. The highly brilliant infrared source used in SR-FTIRM For SR-FTIRM measurements, samples had to undergo a provided high spatial resolution and high quality information specific preparation protocol, i.e. cell fixation and deposition required for the single-cell analysis of our work. To the best of on infrared transparent calcium fluoride (CaF2) cover our knowledge, this is the first study that evaluates cell radio- glasses.21,27 The cells were fixed at two post-irradiation times: sensitization to AuNP by using SR-FTIRM. after the irradiation (T0h) to evaluate immediate treatment- induced effects, and after 24 hours (T24h) to assess the cellular response from the treatment. For both fixation times (T0h and T24h), the medium was 2. Materials and methods removed and kept in order to collect floating cells. Then, 2.1. Cell culture: F98 and U87-MG 100 μL of 0.05% trypsin–EDTA solution was added into each The response of the two glioma cell lines, F98 (rat, ATCC® well and incubated for 5 min. After cell detachment, the CRL-2397) and U87-MG (human, ATCC® HTB-14), was studied. medium with floating cells was added back to the trypsinized The F98 glioma rat cell line simulates the behavior of glioblas- cells, and 500 μL of fresh supplemented medium was added. toma tumors due to its highly invasive pattern of growth The cell suspension was then centrifuged at 1500 rpm for within the brain and low immunogenicity; it is commonly 5 min at 4 °C. The cellular pellet was rinsed with PBS, and used to evaluate the effectiveness of several therapeutic resuspended in 10% formalin neutral buffered solution agents.23 The U87-MG human tumor model presents a non- (Sigma-Aldrich). After 1 hour at room temperature, the diffusely infiltrative growth pattern and it is one of the most samples were centrifuged at 1500 rpm for 5 min and the pellet widely used cell lines, especially for anti-angiogenic therapy was washed 3 times in Millipore water. The final cell suspen- studies.24 sions in water were kept at 4 °C until cytospinned down onto Both cell lines were purchased from LGC Standards 0.5 mm thick infrared transparent CaF2 slides using a (Molsheim, France). They were maintained and passaged in a Cytospin 4 centrifuge (ThermoFisher; 700 rpm for 5 min). high glucose (4.5 g L−1) Gibco™ DMEM medium (Life Dried cells were then analyzed by SR-FTIRM. Technologies SAS, Courtaboeuf, France) supplemented with 1 mM GlutaMAX™, 1 mM sodium pyruvate, 10% fetal calf 2.5. SR-FTIRM at ALBA Synchrotron serum and 1% penicillin-streptomycin (10 000 U each), at SR-FTIRM experiments were performed at the infrared beam- 37 °C and 5% CO2. On the day prior to the treatments, 105 line MIRAS of the ALBA-CELLS synchrotron light source using cells per well were seeded in 6-well plates and incubated the Hyperion 3000 microscope coupled to a Vertex 70 spectro- overnight. meter (Bruker Optik GmbH, Germany). The measurements Analyst This journal is © The Royal Society of Chemistry 2019 View Article Online Analyst Paper were performed in the transmission mode of operation of the • Phosphate I: 1270–1186 cm−1 (PhI). FTIR microscope. This microscope is operating with a 36× • Phosphate II: 1146–1004 cm−1 (PhII). Schwarzschild magnification objective (NA = 0.65) coupled to a The total area under the spectra (except the 36× magnification condenser. The microscope is furnished 2800–1800 cm−1 region) was associated with the total cell with a liquid nitrogen cooled 50 μm mercury cadmium tellur- biomass (Cell) following previous studies.28,29 Violin plots ide (MCT) detector. Around 70–100 cells were randomly col- showing the probability density of the data for PhI/AII, PhII/ lected from each sample condition. Single point maps of indi- AII, AI/AII and CH2/CH3 ratios were generated, since these vidual cells were collected in the 3800–900 cm−1 mid infrared ratios have been reported to provide valuable information on range at 4 cm−1 spectral resolution with 256 co-added scans cell response after treatments.28,30,31 This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. per spectrum. All spectra were obtained using a single Open Access Article. Published on 16 September 2019. Downloaded on 9/27/2019 4:25:36 PM. masking aperture size of 8 μm × 8 μm. Reference or back- 2.7. Microphotography ground spectra were collected under the same acquisition F98 and U87-MG cells were seeded in a 35 mm polystyrene parameters of the sample. In particular, measurement in an Petri dishes using the same NP concentration and NP incu- empty region on the CaF2 slides (without cells) was repeated bation time as for SR-FTIRM measurements. After treatment, every 10 cell measurements, in order to take into account the the cells were washed in fresh medium and microscopy image differences under the ambient conditions along the measure- acquisition was performed using a 10× Nikon water-immersion ments. The final cell absorbance spectrum was obtained by DIC objective placed on an Eclipse 80i Nikon microscope subtracting the reference spectrum from the raw cell spectra. (Scop Pro, Marolles-en-Hurepoix, France), which is equipped The subtraction was done directly (automatically) using OPUS with a Zyla 5.5 MPX Andor SCMOS cooled camera (Scop Pro, 7.5 software (Bruker Optik GmbH, Germany). France) and MetaMorph acquisition software (Molecular Devices, Sunnyvale, CA). 2.6. SR-FTIRM data analysis Differences in the spectral features were evaluated by Principal 2.8. Cell viability assay Component Analysis (PCA) using Unscrambler X (CAMO Metabolic activity was evaluated using the resazurin–resorufin Software AS, Norway). PCA was performed on vector normal- cytotoxicity assay.32,33 Briefly, after irradiation treatment, the ized second derivative spectra (Savitzky–Golay algorithm; 3rd cells were harvested in each well of the 6-well plate by using polynomial order). Vector normalization and PCA were separ- the usual trypsin-EDTA procedure. The cells were counted and ately applied in the 3100–2800 and 1800–1000 cm−1 spectral 100 μL of 105 cells per mL were seeded in 96-well microplates regions. and incubated for 24 hours at 37 °C. After 24 hours, the Raw spectra were corrected following the rubber band medium was discarded and replaced by fresh medium contain- method (32 baseline points) using OPUS 7.5 (Bruker Optik ing 30 μg mL−1 of resazurin (from a filtered 1.2 mg mL−1 stock GmbH, Germany) in order to assess the area under several solution in PBS). The cells were incubated for 3 hours at 37 °C, spectral bands reported hereafter and represented in Fig. 1. and the fluorescence intensity measurement of each well was • Methyl asymmetric stretching: 2980–2945 cm−1 (CH3). performed using a Fluoroskan Ascent FL microplate reader • Methylene asymmetric stretching: 2945–2900 cm−1 (CH2). (Thermo Fisher Scientific, Illkirch, France) at 530 and 590 nm • Amide I: 1710–1598 cm−1 (AI). for excitation and emission, respectively. The percentage of the • Amide II: 1590–1483 cm−1 (AII). metabolic activity of the cell populations was evaluated as the Fig. 1 F98 (left) and U87-MG (right) glioma cells: averaged absorbance spectra for controls and treated cells, in the presence (‘+NP’) and absence (‘−NP’) of nanoparticles at two post-irradiation fixation times (0 h and 24 h) irradiated with a dose of 20 Gy. The limits of the band/regions described in section 2.6 are shown. For clarity, the different averaged absorbance spectra have been shifted along the absorbance axis. This journal is © The Royal Society of Chemistry 2019 Analyst View Article Online Paper Analyst ratio of the fluorescence intensity after treatment to the Whether this process is active (endocytosis) or passive ( per- control fluorescence intensity. meation) could not be addressed by this microscopy experi- ment. However, it has been shown elsewhere and on different cell types that the majority of 1.9 nm particles are endo- 3. Results and discussion cytosed.25 While on F98 cells, the size of the dots was quite the same inside and outside of the cells, on U87-MG cells, the size Microphotography images for the qualitative assessment of of the black dots seems to indicate that the AuNP are aggre- intracellular NP localization are shown in section 3.1. Then, gated. This might occur due to the intrinsic differences the results of SR-FTIRM analysis and cell viability assays are between the endocytosis processes in both cell lines. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. presented for both cell lines (F98 and U87-MG) and several Open Access Article. Published on 16 September 2019. Downloaded on 9/27/2019 4:25:36 PM. irradiation doses (from 0 to 20 Gy) in the presence (+NP) and 3.2. Biochemical effects induced by the AuNP without absence (−NP) of AuNP and two post-irradiation fixation times irradiation (0 Gy) (T0h and T24h). SR-FTIRM details on the biochemical effects Fig. 3 and 4 show the PCA scores and the corresponding induced by the AuNP without (0 Gy) and with radiation are loading plots on second derivative spectra for both F98 and presented in sections 3.2 and 3.3, respectively. The results of U87-MG cell lines, respectively. PCA allows the reduction of cell viability assays are reported in section 3.4. the dimensionality of features in order to assess the simi- larities and differences between spectra. In the PCA score plot, 3.1. Microphotography for intracellular NP localization each single point represents a cell spectrum. Loading plots assessment express the influence of the variables that are responsible for Fig. 2 shows the bright field micrographs of F98 and U87-MG each principal component as a function of the wavenumber. cells, respectively, with and without AuNP. While both cell PCA analysis was performed separately in the fingerprint lines show different morphologies (F98 is elongated and U87- (1800–1000 cm−1) and lipid (3100–2800 cm−1) regions. MG is flatter and more spread on the cell support), nuclei can For the F98 glioma cell line, and prior to the radiotherapy be easily distinguished as well as cell boundaries. After AuNP irradiation (0 Gy), the PC1–PC2 plot in the fingerprint region treatment and washing with fresh medium, free NP or NP shows a clear separation between NP-treated groups and the aggregates can still be clearly seen in the cell surroundings control group (−NP), mostly along PC-1, which explains 73% (black dots). An unambiguous punctuated pattern inside the of the variance. PC-1 shows more variance for the NP-treated cell cytoplasm can also reproducibly be shown, especially in groups. Fingerprint modifications are already observed at T0h, perinuclear areas. This strongly suggests an internalization which gives us a first indication of the AuNP action. For the process. U87-MG cell line, there is also some separation along PC-1 in Fig. 2 Micrographs of two F98 cells (top) and U87-MG cells (bottom), without (‘−NP’, left) and with (‘+NP’, right) 24 hour incubation of 500 μg mL−1 AuNP. Bar = 10 μm. Arrows = AuNP ( plain: inside cell; dotted: free AuNP). Analyst This journal is © The Royal Society of Chemistry 2019 View Article Online Analyst Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 16 September 2019. Downloaded on 9/27/2019 4:25:36 PM. Fig. 3 F98 glioma cells: biochemical effects induced by the AuNP prior to irradiation (0 Gy). The PCA scores (top) and the corresponding loading plots (bottom) are presented in the fingerprint (1800–1000 cm−1; left) and lipid (3100–2800 cm−1; right) spectral regions in the presence (‘+NP’) and absence (‘−NP’) of nanoparticles at two post-irradiation fixation times (0 h and 24 h). the score plot between the control and NP-treated cells in the in Fig. 6 (top). At T24h, there is a slight decrease in the PhII/AII fingerprint region mainly at T0h. ratio in the case of U87-MG cells, which might be correlated to The loading plots in the fingerprint region indicate that the cell cycle changes and, particularly, to an increase in the sub largest biochemical changes induced by the NP are associated G1 cell population induced by these AuNP.25 Previous studies with protein modifications in the region of 1700–1500 cm−1 have reported similar changes in the DNA region of the infra- for both F98 and U87-MG cell lines. The vector normalized red spectra due to cell cycle modifications.36,37 Finally, it is Savitzky–Golay second derivative calculated in the averaged important to note that we clearly observe an increase in the absorbance spectra (Fig. 5) shows NP-induced changes in the variance of the DNA biochemical content in NP-treated cells protein conformational structures in the amide I and amide II with respect to controls (−NP) both at T0h and T24h and for bands, especially in the F98 glioma cell line. These bands are both cell lines, as a result of different NP–cell interaction characteristic of peptide bonds of cell peptides and proteins. mechanisms. More precisely, there is a more explicit β-type secondary struc- In the region of 3000–2800 cm−1, mostly representative of ture inside the NP-treated cells,34–36 which could be related to fatty acids encountered in the membranes, there is a separ- an initial apoptotic response of the cells, since previous ation between the scores of control cells and NP-treated cells studies have reported changes in the expression of pre-apopto- in F98 glioma line (Fig. 3). NP-treated cells are correlated tic proteins using the same type of NP.25 with peaks at 2848 cm−1 and 2915 cm−1, while control cells The separation of the NP-treated group at T24h along PC-2 are correlated with peaks at 2935 cm−1 and 2858 cm−1. This also has some contribution of the asymmetric phosphodiester corresponds to changes in the asymmetric and symmetric band of the DNA (1238 cm−1), particularly in the case of the vibrations of CH2 stretching groups.38 Changes in these F98 cell line. We observe a shift towards the low frequency peaks were previously related to the changes in the lipid region for NP-treated cells (see Fig. 5). This shift has been structure and chain length due to oxidative stress and mem- observed in our previous work using gadolinium NP.21,27 We brane peroxidation.38–41 The separation in the PCA scores also observe changes in the distribution of the relative intensi- between −NP and +NP groups is less clear in the case of U87- ties of several DNA spectral bands (PhI/AII, PhII/AII), as shown MG cells (Fig. 4). This journal is © The Royal Society of Chemistry 2019 Analyst View Article Online Paper Analyst This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 16 September 2019. Downloaded on 9/27/2019 4:25:36 PM. Fig. 4 U87-MG glioma cells: biochemical effects induced by the AuNP prior to irradiation (0 Gy). The PCA scores (top) and the corresponding loading plots (bottom) are presented in the fingerprint (1800–1000 cm−1; left) and lipid (3100–2800 cm−1; right) spectral regions in the presence (‘+NP’) and absence (‘−NP’) of nanoparticles at two post-irradiation fixation times (0 h and 24 h). Fig. 5 F98 (left) and U87-MG (right) glioma cells: biochemical effects induced by the AuNP prior to irradiation (0 Gy). The vector normalized Savitzky–Golay second derivative calculated in the averaged absorbance spectra is shown for the −NP and +NP controls at two post-irradiation fixation times (0 h and 24 h) in the fingerprint (1800–1000 cm−1) spectral region. The insets show a zoom of the 1700–1600 cm−1 and 1350–1000 cm−1 regions. The violin plots in Fig. 6 (bottom) show a clear increase in the treatment (T0h), which seems to be reduced at T24h. The induction CH2/CH3 ratio distribution for F98 cells (both at T0h and T24h), of AuNP-related oxidative stress has been previously reported using which might indicate evidences of oxidative stress.39,40 There are the same type of NP via increased production of endogenous reac- also indications of oxidative stress in the U87-MG cells after NP- tive oxygen species and depletion of intracellular antioxidants.25,42 Analyst This journal is © The Royal Society of Chemistry 2019 View Article Online Analyst Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 16 September 2019. Downloaded on 9/27/2019 4:25:36 PM. Fig. 6 F98 (left) and U87-MG (right) glioma cells: biochemical effects induced by the AuNP prior to irradiation (0 Gy). The violin plots are showing the distribution of the relative intensities of several spectral band ratios (PhI/AII, PII/AII and CH2/CH3; see section 2.6 for more information) for the −NP (blue) and +NP controls at two fixation times (0 h, in orange; 24 h in green). Our findings evidence a different biochemical response for 10 Gy and 20 Gy and in the case of T24h. PC-1 and PC-2 depending on the tumor cell model, which could be related to take into account around 70% and 10% of the variance, a different radiosensitization capability according to NP local- respectively, for all doses. The loading plots show that wave- ization, NP-cell interaction processes or degree of sensitivity to numbers around 1628 cm−1 are highly correlated to NP- oxidative damage or others.5,25,26 This could explain the differ- treated groups, while wavenumbers around 1660−1 are corre- ences in NP internalization relative to the cell type shown in lated to irradiated groups without AuNP. This correlation is the microscopy pictures (see Fig. 2) and might be related to better manifested at T24h. While the first is attributed to the the subsequent variations in the biochemical effects observed β-sheet conformation of the amide I band, the second one is by SR-FTIRM. assigned to the α-helix form.34 This indicates a shift towards lower wavenumbers in the amide for irradiated cells in the 3.3. SR-FTIRM study on the radiosensitization effects at presence of AuNP, as can be seen in Fig. 9 (left). Previous several radiation doses studies correlated this shift to an increase in disordered Fig. 7 and 8 show the PCA scores and the corresponding structures due to protein denaturation.43 In our case, the loading plots on second derivative spectra for both F98 and secondary protein structure changes inside the cells corre- U87-MG cell lines, respectively, and several configurations: lated with protein unfolding and/or denaturation might be doses (from 5 to 20 Gy), with/without NP (−NP/+NP) and post- related to cell death under treatment-induced stress.36,44 irradiation times (T0h/T24h). PCA scores were separated as a This stress could be enhanced by the increased generation function of the dose to assess the effect of AuNP for the same of ROS due to the presence of AuNP, which has been associ- irradiation conditions. The two fixation times are compared in ated with changes in the amide I and II bands.44–46 An ampli- the same PCA scores to assess cell response to the treatments fication in the apoptotic response of irradiated cells in the as a function of time. presence of AuNP is in accordance with our previous FTIRM In F98 glioma cells, the protein region of the fingerprint study using gadolinium NP.21,27 The protein modifications as spectral range (1700–1500 cm−1) is the main factor respon- a function of the treatment are clearly shown on the vector sible for PCA score separation. PC1–PC2 score plots display normalized Savitzky–Golay second derivative plot (Fig. 9, clearly separated clusters (−NP/NP and T0h/T24h), particularly left). This journal is © The Royal Society of Chemistry 2019 Analyst View Article Online Paper Analyst This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 16 September 2019. Downloaded on 9/27/2019 4:25:36 PM. Fig. 7 F98 glioma cells: radiosensitization effects of AuNP for several doses: 5 Gy (left), 10 Gy (center) and 20 Gy (right). The PCA scores and the corresponding loading plots are presented in the fingerprint (1800–1000 cm−1; top) and lipid (3100–2800 cm−1; bottom) spectral regions. Analyst This journal is © The Royal Society of Chemistry 2019 View Article Online Analyst Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 16 September 2019. Downloaded on 9/27/2019 4:25:36 PM. Fig. 8 U87-MG glioma cells: radiosensitization effects of AuNP for several doses 5 Gy (left), 10 Gy (center) and 20 Gy (right). The PCA scores and the corresponding loading plots are presented in the fingerprint (1800–1000 cm−1; top) and lipid (3100–2800 cm−1; bottom) spectral regions. This journal is © The Royal Society of Chemistry 2019 Analyst View Article Online Paper Analyst This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 16 September 2019. Downloaded on 9/27/2019 4:25:36 PM. Fig. 9 F98 (left) and U87-MG (right) glioma cells: radiosensitization effects of AuNP (20 Gy). The vector normalized Savitzky–Golay second deriva- tive calculated in the averaged absorbance spectra is shown for the −NP and +NP controls at two post-irradiation fixation times (0 h and 24 h) in the fingerprint (1800–1000 cm−1) spectral region. The insets show a zoom of the 1700–1600 cm−1 and 1350–1000 cm−1 regions. In the case of U87-MG cells, the effect is less clear since the treated groups at T24h. The differential biochemical response PC1–PC2 scores in the fingerprint region show more overlap- to AuNP in the DNA region is more visible in the case of F98 ping between ‘−NP’ and ‘+NP’ groups. For 20 Gy, we observe a than in the case of U87-MG cells. slight separation of the NP-treated group for both fixation For F98 glioma cells, the violin plots of Fig. 10 show a slight times. Loading plots mainly correlate this separation with sec- reduction in the PhII/AII ratio for NP-treated cells at T24h, ondary protein structure changes and modifications in the which has been previously correlated with chromatin fragmen- asymmetric phosphodiester bands. These changes, amplified tation and apoptotic DNA condensation in previous in the presence of NP, are probably related to an enhanced cell studies.28,30,49 Previous studies have reported similar vari- death response due to the combination of radiation damage ations as a result of radiation damage.31,44,47,49 Instead, U87- and AuNP-induced biochemical modifications. However, we MG cells exhibit a clear increase in the PhI/AII and PhII/AII observed a reduced biochemical response to AuNP with ratios at T24h in NP-treated cells. An increase in the phospho- respect to F98 cells in the presence of radiation, since second- diester bands was already observed in the work of Lipiec and ary structure protein changes are less clear in the Savitzky– collaborators for PC-3 (human prostate adenocarcinoma) cells Golay second derivative plot (Fig. 9, right). following proton irradiation.47 The increase in the intensity of Fig. 10 shows the distribution of the relative intensities of the DNA backbone was correlated with multiple DNA several spectral band ratios (PhI/AII, PhII/AII, AI/AII and CH2/ breakages.47,50 CH3) defined in section 2.6. For the F98 cell line, we observe a When comparing NP-treated cells in the lipid region decrease in the absorbance of the AI/AII ratio in NP-treated (3100–2800 cm−1), the score plots do not display clear separ- cells. Changes in the amide I and amide II absorbance were ated clusters, despite the fact that the NP-treated groups at previously related to DNA repair processes, which involves T24h seem to be slightly separated towards positive PC-1 in many enzymes to repair the damage produced by the ionizing both cell lines from the other groups (T0h and T24h without radiation.47,48 The changes in relative intensity between the AuNP). Fig. 10 shows some increase in CH2/CH3 ratio distri- areas in amide I and amide II bands have also been correlated bution (defined in section 2.6) at T24h, which is more marked with the biochemical changes of cellular proteins following for U87-MG cells. The higher absorbance in the CH2 asym- apoptosis, which affects the overall protein folding and localiz- metric stretching mode with respect to the CH3 asymmetric ation and thus, the infrared absorption of peptide bonds.30,31 stretching has been reported with cell death processes in pre- By looking into the DNA region (1350–1000 cm−1) in the vious studies.28 Indeed, a higher CH2/CH3 ratio has been vector normalized Savitzky–Golay second derivative plot determined for apoptotic cells by proton nuclear magnetic (Fig. 9), which is assigned to phosphodiester groups mostly resonance spectroscopy in previous studies.51–53 found in nucleic acids, we observe several changes in the Finally, it is important to remark that we observe a higher intensity and position (shifts) of several peaks. This probably intra-cellular variation in treated cells, demonstrating the het- implies a number of different variations in DNA organization erogeneity in the biochemical cell response among the same or local conformation as a result of radiation damage, which is cell population and also among cell lines. This is probably due amplified in the presence of AuNP. The investigation of the to different NP–cell interaction processes, and degree of sensi- loadings shows a slight variance emerging from the asym- tivity to oxidative/radiation damage or others. The radiosensiti- metric phosphodiester band of the DNA at 1238 cm−1 for NP- zation potential dependence as a function of the cell type has Analyst This journal is © The Royal Society of Chemistry 2019 View Article Online Analyst Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 16 September 2019. Downloaded on 9/27/2019 4:25:36 PM. Fig. 10 F98 (left) and U87-MG (right) glioma cells: radiosensitization effects of AuNP (20 Gy). The violin plots are showing the distribution of the relative intensities of several spectral band ratios (PhI/AII, PhII/AII, AI/AII and CH2/CH3; see section 2.6 for more information) for the ‘−NP’ (blue) and ‘+NP’ (orange) controls at two post-irradiation fixation times (0 h and 24 h). Fig. 11 Metabolic activity of F98 cells (left) and U87-MG cells (right) with and without NP for several doses of radiation. The control cell group was considered to have 100% of living cells. The values obtained with the different cell treatments were normalized using the values of the control cell group. Data are presented as the mean and standard error of the mean (SEM). Statistical significance in the analysis of the effect of the radiation dose, with or without NP, was determined using a standard two-tailed unpaired t-test. If any, non-significant differences (ns) with p ≥ 0.05 are indi- cated on the graphs. This journal is © The Royal Society of Chemistry 2019 Analyst View Article Online Paper Analyst also been reported in previous studies using the same type of bands (F98), changes in amide I/II relative intensity (F98), and NP.5,25,26,54 The distinct cellular responses of different cell relative amplitude changes in the CH2 and CH3 stretching lines are a key point for the study of radiosensitization effects modes (U87-MG), along with several DNA organization or local of NP. Within this context, SR-FTIRM can provide new insights conformation modifications (F98, U87-MG) due to the pres- into the molecular and cellular levels for understanding the ence of AuNP. These changes are better manifested at T24h and complex radiosensitization processes involving nanoparticles they are probably related to an initial cell death response of and radiotherapy, as shown in this work. the cells, which is amplified in the presence of AuNP. SR-FTIRM results showed the heterogeneity in the biochemical 3.4. Cell viability assay cell response among different cell lines (and among the same This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. cell population), probably due to the different NP–cell inter- Open Access Article. Published on 16 September 2019. Downloaded on 9/27/2019 4:25:36 PM. Fig. 11 shows the results of the resazurin–resorufin prolifer- ation assay performed on treated cells, with or without AuNP, action processes, degree of sensitivity to oxidative/radiation and with or without X-ray irradiations at 24 hours post- damage, etc., which implies different radiosensitization capa- irradiation time, to correlate with respect to the infrared data bilities as a function of the cell line. at T24h. Despite the fact that SR-FTIRM provides a deeper under- F98 glioma cells seem to be more sensitive to AuNP, already standing of the NP-induced molecular and cellular response at in the absence of radiation. Also, in the presence of radiation, a biochemical level, further biological studies are required to U87-MG cells seem to be less sensitive to AuNP compared to assess the role of different ( physical and biochemical) radio- F98 cells since the metabolic activity is not significantly sensitization mechanisms as a function of NP and cell type. different in the presence of AuNP. In F98 glioma cells, we can Comprehensive characterization studies of cellular responses observe significant changes for the lowest dose (5 Gy), since to AuNP are required for the further development of this prom- the F98 metabolic activity is decreased in the presence of ising technique towards potential clinical translation. AuNP. It is interesting to note that the macroscopical measure- ment of the metabolic activity through the proliferation assay does not allow the differentiation of the effect of AuNP on Conflicts of interest both cell lines in all configurations, whereas SR-FTIRM shows There are no conflicts to declare. more detailed events occurring at the molecular level. In this study, the choice of performing the proliferation test at 24 hours post-irradiation was to be able to correlate with the SR-FTIRM data and early biological events. Larger effects Acknowledgements might be expected at larger post-irradiation times, as reported The authors acknowledge the granted beamtime at in previous studies using the same type of NP.25 ALBA-CELLS Synchrotron. We also thank Dr S. Heinrich, Dr F. Pouzoulet (Institut Curie, France) and Dr F. Gaudin (Institut Paris-Sud d’Innovation Thérapeutique, France) for their techni- 4. Conclusions cal support. 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