Analyst View Article Online PAPER View Journal Synchrotron-based infrared microspectroscopy This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: DOI: 10.1039/c9an00792j study on the radiosensitization effects of Gd Open Access Article. Published on 31 July 2019. Downloaded on 8/26/2019 11:35:19 AM. nanoparticles at megavoltage radiation energies Immaculada Martínez-Rovira, *a,b Olivier Seksek, c,d Josep Puxeu,e Joan Gómez,b Martin Kreuzer,a Tanja Dučić,a Maria Josep Ferreres,f Manel Artiguese and Ibraheem Yousef *a The outcome of radiotherapy can be further improved by combining radiotherapy with nanoparticles. Previous biological studies showed a significant amplification of the biological damage in cells charged with nanoparticles prior to radiotherapy treatments. The rationale has been based on the physical dose enhancement. However, this subject is still a matter of controversy and there are clear indications that biochemical effects may play a key role in the radiosensitization effects of nanoparticles. Within this context, the main goal of our study was to provide new insights into the radiosensitization effects of F98 glioma cells exposed to gadolinium nanoparticles combined with clinical megavoltage beams, and compare them with respect to kilovoltage radiotherapy (commonly used in combination with nano- particles). For this purpose, we used synchrotron-based Fourier transform infrared microspectroscopy (SR-FTIRM) to provide relevant information on the treatment-induced biochemical changes of the main cell biomolecules. Biochemical differences were evaluated after the treatments to assess cellular damage. Multivariate analysis revealed nanoparticle-dependent changes in megavoltage treated cells. The main spectral variations were related to conformational changes in the protein secondary structures, which might be induced by radiation damage and by changes or rearrangements in the nucleic acid structures due to the initiation of DNA repair mechanisms. We also observed significant changes in the phosphate I and II bands, which concerns DNA damage, while few changes were detected in the lipid region. Received 2nd May 2019, Spectroscopic data showed that these changes increased as a function of the dose. Finally, PCA analysis Accepted 10th July 2019 did not discriminate clearly between megavoltage and kilovoltage groups treated with nanoparticles, indi- DOI: 10.1039/c9an00792j cating that megavoltage radiosensitization effects might not differ significantly from those in kilovoltage rsc.li/analyst radiotherapy. 1 Introduction in vivo through the preferential absorption of X-rays by 1.9 nm Au NP.1 This pioneering experiment, along with the results The use of nanoparticles (NP) as tumour radiosensitizers was obtained in subsequent biological studies (see ref. 2–7, among proposed by Hainfeld and collaborators in 2004, when they others), boosted the use of NP as a promising strategy to first reported the control of a malignant tumour (EMT-6) increase the efficiency of radiotherapy. The basis for NP use is their selective accumulation within tumours due to the enhanced permeability and retention a ALBA-CELLS Synchrotron, MIRAS Beamline, Carrer de la Llum 2-26, (EPR) effect,8 leading to a higher cell penetration and less 09290 Cerdanyola del Vallès, Spain. E-mail: immamartinez@gmail.com, adverse effects with respect to conventional radiosensitizers.9 iyousef@cells.es b Ionizing Radiation Research Group (GRRI), Physics Department, Therefore, the goal of combining NP with radiotherapy is to Universitat Autònoma de Barcelona (UAB), Avinguda de l’Eix Central, improve tumour cell killing, and thus, the differential effect Edifici C. Campus de la UAB, 08193 Cerdanyola del Vallès, Spain between healthy and tumour tissues. An increase in the radio- c Imagerie et Modélisation en Neurobiologie et Cancérologie (IMNC), CNRS, sensitivity of tumour cells treated with NP has been observed Univ Paris Sud, Université Paris-Saclay, F-91400 Orsay, France d in vitro and in vivo, using kilovoltage and also megavoltage Université de Paris, IMNC, F-91400 Orsay, France e Hospital Universitari Sant Joan de Reus, Institut d’Investigació Sanitària Pere photon beams.10–12 Virgili, Avinguda del Dr. Josep Laporte 2, 43204 Reus, Spain The biological findings have been basically described in f Laboratori ASPCAT Tortosa, Avinguda Santa Teresa 2–12, 43590 Tortosa, Spain terms of physics since the presence of (high-Z) NP increases This journal is © The Royal Society of Chemistry 2019 Analyst View Article Online Paper Analyst the probability of photoelectric and Compton interactions 2 Materials and methods (and, thus, the generation of short-range secondary elec- trons).13 This results in a local dose enhancement in the 2.1 F98 cells and cell culture tumour. However, the radiosensitization observed in some The F98 rat glioma cell line (ATCC-CRL-2397) was purchased biological experiments is greater than the predicted increase from LGC Standards (Molsheim, France). This type of cell has in the physical dose,14–16 indicating that the biological a highly invasive growth pattern and is weakly immunogenic response may not be driven only by dose enhancement in syngeneic Fischer rats.26 ( physical) effects. Other mechanisms such as the production The cells were cultured in high glucose DMEM medium of reactive oxygen species (ROS) and oxidative stress, DNA (Gibco) supplemented with 10% fetal calf serum, 1% penicil- This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. damage induction, cell cycle effects and potential inter- lin–streptomycin (10 000 units per mL each), 2 mM ference with the bystander effects may play a major L-glutamine, 1 mM sodium pyruvate and 10 mM HEPES in an Open Access Article. Published on 31 July 2019. Downloaded on 8/26/2019 11:35:19 AM. role.6,14–19 These mechanisms might be amplified in combi- incubator at 37 °C. The cells were seeded in 6-well plates on nation with radiotherapy, and thus increase the anticancer the day prior to the irradiation; 2 mL of a 5 × 104 cells per mL efficacy of NP. In addition, different radiosensitizing effects suspension were placed in each well and incubated overnight are expected according to the NP size, type, chemical nature, in order to reach a 75% confluence rate on the day of concentration, intracellular localization, radiotherapy con- irradiation. figuration (energy) and cell line used.17 That is why the bio- logical mechanisms of nanoparticle radiosensitization still 2.2 Nanoparticles remain unknown. Gadolinium NP (AGuIX®; Activation and Guiding of Within this context, the objective of this study is to use syn- Irradiation by X-ray) were purchased from Nano-H (Lyon, chrotron-based Fourier transform infrared microspectroscopy France). These nanoparticles consist of a polysiloxane network (SR-FTIRM) to disentangle radiosensitization effects in the surrounded by around 10 gadolinium chelates (diethyl- F98 glioma rat cell line treated with GdNP (AGuIX®) combined enetriaminepentaacetic acid, DTPA) covalently grafted to the with radiotherapy. In addition to its radiosensitization pro- polysiloxane inorganic matrix. The nanoparticles present a dia- perties, the use of Gd and its chemical derivatives is particu- meter of 3.0 ± 1.0 nm.27 The effectiveness of the AGuIX® NP larly interesting since it is the most widely used contrast agent has been proved in previous biological studies with several for magnetic resonance imaging (MRI); this makes Gd a types of cell lines or animal models, and irradiation perfect tool for theragnosis ( providing imaging and therapy at conditions.28–30 the same time).20 Thanks to this dual capacity, the use of A nanoparticle stock solution of 100 mM was obtained by GdNP is increasing. solubilization in ultrapure Millipore (DirectQ 8) water and kept FTIRM is a vibrational technique for the biochemical at 4 °C. For cell treatment, 3 mL of 1 mM nanoparticle solution analysis of cells on a microscopic scale. FTIRM methods in fresh supplemented medium was added into the wells and have been used to study the cell cycle, apoptosis, differen- incubated for 6 hours at 37 °C. At such concentrations, NP are tiation, and proliferation of different cell lines and tissues not toxic.9,31 A recent study with the same type of NP and cell (see, for instance ref. 21–24). Analysis of the infrared spectra line showed that the uptake of GdNP was time-dependent and provides relevant information on the treatment-induced bio- reached a plateau after a 5-hour incubation time.19 chemical modifications of the main cell biomolecules (nucleic acids, proteins and lipids). In our previous work,25 2.3 Irradiation we studied the biochemical structure alterations of bio- Kilovoltage and megavoltage irradiations were performed at molecules inside cells induced by low energy X-ray the Hospital Universitari Sant Joan de Reus. Kilovoltage irradiations and GdNP using FTIRM. In particular, we irradiations were performed with a Gulmay D3300 kilovoltage observed important spectral signature alterations in the fin- X-ray therapy unit (Gulmay Ltd, Byfleet, UK) with an accelerat- gerprint (DNA and protein) and lipid regions related to ing potential voltage of 60 kV (HVL = 1.19 mm Al). Dose con- changes in the cellular function and cell death processes. formation was performed with a circular 4 cm-diameter cone The main goal of the present study is to use SR-FTIRM to at 30 cm from the source. The cells were irradiated at a dose gain new insights into the radiosensitization effects involved rate of 2.1 Gy min−1. Megavoltage irradiations were performed when using GdNP at megavoltage (MV) photon energies using a 6 MV photon beam from a Varian 2100 linear accelera- (normally used in clinics) with respect to kilovoltage (kV) tor (Varian Medical System, Palo Alto, USA). The dose rate at photons (commonly used in combination with NP). To the the cells was also 2.1 Gy min−1 at a source to surface distance best of our knowledge, this is the first study that uses syn- (SSD) of 95 cm and at 5 cm-depth (cell location). In both cases, chrotron-based FTIRM to evaluate NP-based radiotherapy several irradiation doses were delivered to the cells: 0 Gy (no approaches. The biochemical information provided by the irradiation), 5, 10 and 20 Gy. highly brilliant infrared light source produced at ALBA Synchrotron leads to a clear advantage in spectral quality at 2.4 SR-FTIRM sample preparation a cellular level and will allow better characterization of NP- SR-FTIRM sample preparation was performed following our induced damage in cells. previous protocol.25 In order to evaluate immediate treatment- Analyst This journal is © The Royal Society of Chemistry 2019 View Article Online Analyst Paper induced effects, the cells were fixed two hours after the Violin plots showing the probability density of the data for irradiation. For this purpose, the medium was removed different ratios (PhI/AII, PhII/AII, AII/AI, Lip/Cell, CH2/Cell, (having previously collected floating cells) and 100 μL of a and CH2/CH3) were generated for the different configurations. 0.05% trypsin–EDTA solution (Lonza) was added to each well These ratios have been reported to provide valuable infor- in order to detach the cells; the floating cells were added back mation on cell response after treatments.32,34 to the trypsinised cells to take into account the whole cell population that was treated. Then, 500 μL of supplemented DMEM medium was added and the cell suspension was centri- 3 Results and discussion fuged at 1500 rpm for 5 minutes at 4 °C. The cellular pellet This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. was rinsed with PBS, and re-centrifuged at 1500 rpm for Details of the SR-FTIRM analysis are presented in this section 5 minutes. The pellet was then re-suspended in 10% formalin for several irradiation modalities (megavoltage and kilovol- Open Access Article. Published on 31 July 2019. Downloaded on 8/26/2019 11:35:19 AM. neutral buffered solution (Sigma-Aldrich) and incubated for tage), for several doses (from 0 to 20 Gy), and in the presence 1 hour at room temperature. Then, the samples were centri- (+NP) and absence (−NP) of GdNP. Section 3.1 shows the fuged at 1500 rpm for 5 minutes, and the pellet was rinsed 3 effects of GdNP at 0 Gy (without irradiation), while megavol- times in Millipore water to wash out the residual phosphate tage radiotherapy results are presented in section 3.2. In ions. Dried cells, previously deposited on 0.5 mm-thick infra- section 3.3, megavoltage radiosensitization effects by GdNP are red transparent calcium fluoride (CaF2), were then analyzed by compared with respect to kilovoltage radiation energies. SR-FTIRM. 3.1 Radiosensitization effects of GdNP 2.5 SR-FTIRM measurements The effects of the nanoparticles at 0 Gy (without irradiation) SR-FTIRM measurements (transmission mode) were per- are evaluated in this section. Fig. 1 shows the score plots (left) formed at the MIRAS beamline of ALBA Synchrotron using a and the loading plots (right) in the presence (+NP) and Hyperion 3000 microscope coupled to a Vertex 70 spectrometer absence (−NP) of nanoparticles in the 3000–2800 & (Bruker, Germany). The FTIR microscope is supplied with a 1760–950 cm−1 spectral regions. In the PCA score plot, each liquid nitrogen-cooled mercury cadmium telluride 50 μm MCT single point represents a cell spectrum. The PCA plot shows detector. This microscope operates with a 36× Schwarzschild clusters of samples based on their similarity, while the loading magnification objective (NA = 0.65) coupled to a 36× magnifi- plot expresses the influence of the variables (wavenumbers) in cation condenser. All spectra were obtained using a single each principal component. masking aperture size of 7 μm × 7 μm, covering the full cell The PC1–PC2 score plot shows the separation in the two clus- size. Single point maps of individual spectra were collected in ters (−NP and +NP groups) with some overlapping. The group the 3800–900 cm−1 mid-infrared range at 4 cm−1 resolution separation is mostly along PC-1, which explains 41% of the var- with 256 co-added scans per spectrum. For each condition, iance. The loading plot indicates that the largest biochemical between 60 and 80 cells were randomly collected. A back- changes induced by the NP are associated with protein modifi- ground measurement was performed under the same acqui- cations in the region 1700–1500 cm−1. These results indicate sition parameters every 10 samples. changes in the protein conformational structures (amide I and amide II) from α-helix toward β-sheet.25,34,35 2.6 SR-FTIRM data treatment and statistical analysis Fig. 2 and 3 show the second derivative for the two controls Spectral features to assess the differences between the control (−NP and +NP) and the distribution of the relative intensities and treated cells were highlighted by Principal Component of several spectral bands (Lip/Cell, PhI/AII and PhII/AII) Analysis (PCA) using Unscrambler X (CAMO Software AS, described in section 2.6, respectively. We observe changes in Norway). PCA was performed on vector normalized second the phosphodiester vibrations of the DNA. In particular, a shift derivative spectra (11 smoothing points, Savitzky–Golay algor- in the PO2− asymmetric band (1238 cm−1) is detected in the ithm). Vector normalization and PCA were applied in the presence of NP. We also observe a decrease in the ratio of PhII/ 3000–2800 & 1760–950 cm−1 spectral regions, as performed in AII in the NP-treated cells. These findings are in agreement previous FTIRM studies.21,32 with the ones obtained in our previous work, which was Raw spectra were corrected in the 3100–950 cm−1 spectral carried out with an internal source of infrared radiation.25 region following the rubber band method (32 baseline points) Protein and DNA modifications could be due to the biologi- using OPUS 7.5 (Bruker Optik GmbH, Germany). Then, the cal interactions of NP within the cells. Previous studies have area of the following bands/regions was evaluated: methylene shown that NP can induce conformational changes in cell asymmetric stretching, 2945–2900 cm−1 (CH2); methyl asym- proteins.36–38 Moreover, Taupin et al. reported proliferation metric stretching, 2980–2945 cm−1 (CH3); lipid spectral region, arrest and cell cycle modifications when using similar NP to 3000–2835 cm−1 (Lip); amide I, 1710–1598 cm−1 (AI); amide II, those used in our work.19 Thus, these FTIRM spectra changes 1590–1483 cm−1 (AII); phosphate I, 1270–1186 cm−1 (PhI); and could also be related to initial modifications in the cells phosphate II, 1146–1004 cm−1 (PhII). The total area induced by the NP.39,40 (3000–2835 and 1760–950 cm−1 regions) was associated with No significant differences were observed in the lipid region the total cell biomass (Cell) following previous studies.32,33 in the PCA scores. However, the violin plots show a slight 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 31 July 2019. Downloaded on 8/26/2019 11:35:19 AM. Fig. 1 Effect of the nanoparticles at 0 Gy (without irradiation). The PCA scores (left) and loading plots (right) are presented. ‘+NP’ labels the pres- ence of nanoparticles and ‘−NP’ the absence of nanoparticles. increase in the total lipid content (Lip/Cell ratio) for the popu- lation of NP-treated cells. It is important to note that the cells were fixed two hours after the irradiation to assess direct treat- ment-induced effects and that important lipid modifications are typically detected at longer times after the treatment.19,25 3.2 Radiosensitization effects of GdNP at megavoltage radiation energies Fig. 4 and 5 show the overall changes in the averaged absor- bance spectra and the corresponding Savitzky–Golay second derivative for several investigated groups, respectively. PCA scores and loadings are shown in Fig. 6, which illustrates sample grouping for the control and cells treated with mega- voltage radiotherapy with/without GdNP for several doses Fig. 2 Effect of the nanoparticles at 0 Gy (without irradiation). The (5, 10 and 20 Gy) in the 3000–2800 & 1760–950 cm−1 spectral Savitzky–Golay second derivative of the averaged absorbance spectra is regions. After the treatments (MV and/or NP), several infrared shown for the two controls. ‘+NP’ labels the presence of nanoparticles and ‘−NP’ the absence of nanoparticles. Fig. 4 Averaged absorbance spectra for controls, megavoltage (MV) Fig. 3 Effect of the nanoparticles at 0 Gy (without irradiation). The violin and kilovoltage (kV) radiotherapy (10 Gy). ‘+NP’ labels the presence of plot shows the distribution of the relative intensities of several spectral band nanoparticles and ‘−NP’ the absence of nanoparticles. For clarity, the ratios (Lip/Cell, PhI/AII and PhII/AII) described in section 2.6. ‘+NP’ labels different averaged absorbance spectra have been shifted along the the presence of nanoparticles and ‘−NP’ the absence of nanoparticles. absorbance axis. Analyst This journal is © The Royal Society of Chemistry 2019 View Article Online Analyst Paper the DNA organization. The most clear changes appear in the vibrations of the phosphodiester groups; there are modifi- cations in the PO2− asymmetric band (1238 cm−1), especially in the case of NP-treated cells. These modifications might be associated with local conformational changes in the DNA,47,48 such as the formation of pyrimidine dimers or DNA–DNA and DNA–protein cross-links.48 There is also a shift in the PO2− symmetric stretching (around 1085−1) in the NP-treated cells. We also observed treatment-dependent absorbance variations This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. in the phosphate II band, as it can be seen in the (top right) violin plots of Fig. 7. Taken together, these features could Open Access Article. Published on 31 July 2019. Downloaded on 8/26/2019 11:35:19 AM. point to an increase in DNA strand breaks, base cleavage reac- tions and chromatin fragmentation34,45,48–51 for cells charged with nanoparticles prior to MV radiotherapy irradiations. An analysis of the PCA loading plots reveals less contri- Fig. 5 Savitzky–Golay second derivative of the averaged absorbance bution from the lipid region (3000–2800 cm−1). We observe a spectra for controls, megavoltage (MV) and kilovoltage (kV) radiotherapy slight increase in the total quantity of lipids for the treated (10 Gy). The insets show a zoom of the 1700–1600 and 1300–1000 cm−1 regions. ‘+NP’ labels the presence of nanoparticles and ‘−NP’ the absence cells in Fig. 7 (bottom right). This is due to a slight increase in of nanoparticles. the absorbance in the CH2 and CH3 stretching modes in the treated cells (especially in the NP-treated group), indicating some changes following lipid peroxidation or changed lipid bands (mostly located in the protein and DNA regions) showed metabolism due to initial cell death processes.52,53 We do not intensity changes or frequency shifts. observe a significant increase in the ratio of CH2/CH3, which is The PCA scores do not discriminate clearly among the three normally observed several hours after the treatment due to oxi- treated groups (+NP, MV −NP, and MV + NP) for 5 Gy. dative processes occurring in response to treatment and con- Discrimination between the treated and control (−NP) groups sistent with cell death.54,55 is mainly associated with PC-1 and, in particular, with changes Finally, it is important to note that there is a clear increase in the protein spectral region in the range 1700–1500 cm−1 in the variance of the biochemical content of the cell popu- (amide I and amide II). The separation increases in the pres- lation after both treatments (nanoparticle and radiation), as ence of nanoparticles, as the MV + NP groups are clearly separ- can be seen in Fig. 7, probably as a result of the initiation of ated from the other treated groups, especially for 10 Gy and several DNA repair processes or cell death.50,56 20 Gy. The separation of the PCA scores for treated cells increases as a function of the dose. The shift in amide I in the 3.3 Comparison of the megavoltage radiosensitization effects treated cells with respect to the control is clearly seen in the of GdNP with respect to kilovoltage radiation energies Savitzky–Golay second derivative plot (see the inset in Fig. 5). PCA scores and loadings are shown in Fig. 8 for cells treated This shift in amide I indicates a change in the overall con- with megavoltage and kilovoltage radiotherapy with/without formational state in the secondary structure of proteins. In par- GdNP for several doses (5, 10 and 20 Gy) in the 3000–2800 & ticular, it might be related to an increase in the amount of 1760–950 cm−1 spectral regions. β-sheet secondary structures, which is typically associated with PCA does not show significant differences between the MV unfolding and/or denaturation of proteins or apoptosis.35,39 and kV groups in the absence of NP. Moreover, in the presence Immediately after the radiotherapy treatments, this protein of nanoparticles, both the ‘kV + NP’ and ‘MV + NP’ groups are damage might be related to direct damage of the ionizing equally separated (along PC-1) from the ‘−NP’ groups. radiation,41–44 and specifically to changes in the protein sec- Discrimination of NP-treated groups is mainly associated with ondary structures due to changes or rearrangements in the PC-1 and, in particular, with changes in the protein spectral DNA structure.34,45 Moreover, in the violin plots of Fig. 7 region (amide I and amide II). As has been discussed in the (middle), we observed a slight increase in the absorbance previous section, these spectral variations may be related to intensity of proteins (amide II) for the MV-treated cells with conformational changes of the protein secondary structures respect to controls, as in previous studies.34 This might indi- due to radiation damage or due to initial changes or rearrange- cate that the DNA repairing processes had started since they ments in the nucleic acid structures. We do not observe signifi- normally involve a large quantity of enzymes which recognize cant changes in the content of proteins (amide II) between and correct physical damage in DNA.46 megavoltage and kilovoltage groups (see Fig. 7, middle), which In the second derivative plot (Fig. 5), we can observe signifi- has been previously related to DNA repair mechanisms.46,48 cant changes between the ‘MV + NP’ and ‘MV − NP’ groups in The results presented here indicate that similar damage the DNA signatures in the region 1330–950 cm−1. Several occurs in the F98 glioma cells for both types of irradiation (MV changes are found simultaneously (changes in the intensity and kV) since PCA analysis does not allow clear discrimination and shifts), which implies a number of different variations in between the two ‘+NP’ groups. However, in the Savitzky–Golay 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 31 July 2019. Downloaded on 8/26/2019 11:35:19 AM. Fig. 6 Radiosensitization effects of GdNP at megavoltage (MV) radiation energies for several doses (5, 10 and 20 Gy). The PCA scores (left) and loading plots (right) are presented. ‘+NP’ labels the presence of nanoparticles and ‘−NP’ the absence of nanoparticles. 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 31 July 2019. Downloaded on 8/26/2019 11:35:19 AM. Fig. 7 Violin plots showing the distribution of the relative intensities of several spectral bands (PhI/AII, PhII/AII, AII/AI, Lip/Cell, CH2/Cell and CH2/ CH3 scaled by a factor 5) for controls, megavoltage (MV) and kilovoltage (kV) radiotherapy. The specific intervals for each ratio are described in section 2.6.32 For clarity, one dose (10 Gy) is presented. ‘+NP’ labels the presence of nanoparticles and ‘−NP’ the absence of nanoparticles. second derivative plot (Fig. 5) and the DNA-related violin plots the experimental results, the observed enhancement is higher (Fig. 7, top), we can observe some differences in the phosphate than the overall physical dose increase.14 Our results, in agree- I and II infrared bands (1270–1186 cm−1 and 1146–1004 cm−1, ment with these observations, indicated that the biochemical respectively). On average, there is a decrease in the intensities component in the nanoparticle radiosensitization plays a of these bands in the ‘kV + NP’ group with respect to the ‘MV + major role. NP’ group, which may indicate some differences in DNA damage in both treatments. Our SR-FTIRM analysis, supported by multivariate analysis, 4 Conclusions shows clear radiosensitization effects for megavoltage radio- therapy, which do not differ significantly from the ones This synchrotron-based infrared microspectroscopy investi- observed in kilovoltage radiotherapy. Despite Monte Carlo gation showed infrared spectral modifications in F98 cells simulations at the nanoscale predicting that dose enhance- exposed to Gd nanoparticles combined with megavoltage (nor- ment should be much higher using kV energies (compared to mally used clinically) and kilovoltage radiotherapy (commonly MV beams),57 previous biological studies have observed signifi- used in combination with nanoparticles). Biochemical differ- cant radiosensitization in studies where no dose enhancement ences were evaluated two hours after the irradiation to assess was expected.14 Butterworth et al. has reported that for most of cellular damage. 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 31 July 2019. Downloaded on 8/26/2019 11:35:19 AM. Fig. 8 Radiosensitization effects of GdNP at megavoltage (MV) and kilovoltage (kV) radiation energies for several doses (5, 10 and 20 Gy). The PCA scores (left) and loading plots (right) are presented. ‘+NP’ labels the presence of nanoparticles and ‘−NP’ the absence of nanoparticles. Analyst This journal is © The Royal Society of Chemistry 2019 View Article Online Analyst Paper PCA results showed NP-dependent changes in megavoltage 2 J. Hainfeld, F. Dilmanian, D. Slatkin and H. Smilowitz, treated cells. The main spectral variations were related to con- J. Pharm. Pharmacol., 2008, 60, 977–985. formational changes of the protein secondary structures 3 N. Chattopadhyay, Z. Cai, J. Pignol, B. Keller, E. Lechtman, (amide I and amide II), which were probably related to radi- R. Bendayan and R. Reilly, Mol. Pharm., 2010, 7, 2194–2206. ation damage and to changes or rearrangements in the nucleic 4 S. Jain, D. Hirst and J. O’Sullivan, Br. J. Radiol., 2009, 85, acid structures due to the initiation of DNA repair mecha- 101–113. nisms. We also observed significant changes in the phosphate 5 D. Joh, L. Sun, M. Stangl, A. Al Zaki, S. Murty, I and II bands (1270–1186 cm−1 and 1146–1004 cm−1, respect- P. Santoiemma, J. Davis, B. Baumann, M. Alonso-Basanta, ively), while minimal changes were observed in the lipids D. Bhang, G. Kao, A. Tsourkas and J. Dorsey, PLoS One, This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. region. Spectroscopic data showed that these changes 2013, 8, e62425. increased as a function of the dose, especially at 10 and 20 Gy. 6 Y.-W. Jiang, G. Gao, H.-R. Jia, X. Xhang, J. Zhao, N. Ma, Open Access Article. Published on 31 July 2019. Downloaded on 8/26/2019 11:35:19 AM. PCA analysis did not discriminate clearly between megavol- J.-B. Liu, P. Liu and F.-G. Wu, ACS Biomater. Sci. Eng., 2019, tage and kilovoltage groups in the presence of nanoparticles, 5, 1569–1579. indicating that GdNP radiosensitization effects in megavoltage 7 N. Ma, Y.-W. Jian, X. Zhang, H. Wu, J. N. Myers, P. Liu, radiotherapy do not differ significantly from those in kilovol- H. Jin, N. Gu, N. He, F.-G. Wu and Z. Chen, ACS Appl. tage radiotherapy. We observed some differences between the Mater. Interfaces, 2016, 8, 28480–28494. two types of radiation in the average intensities of the phos- 8 Y. Matsumura and H. Maeda, Cancer Res., 1986, 46, 6387– phate I and II bands, which might indicate higher DNA 6392. damage in kilovoltage irradiation. However, few differences 9 E. Porcel, S. Liehn, H. Remita, N. Usami, K. Kobayashi, were observed in the protein region. Y. Furusawa, C. Le Sech and S. Lacombe, Nanotechnology, Finally, it is important to note that it is difficult to draw 2010, 21, 085103. overall conclusions for other types of nanoparticles or cell 10 R. I. Berbeco, W. Ngwa and G. M. Makrigiorgos, lines, since radiosensitization effects depend on the nano- Int. J. Radiat. Oncol., Biol., Phys., 2011, 81, 270–276. particle size and type, surface functionalization, concentration 11 S. Jain, J. A. Coulter, A. R. Hounsell, K. T. Butterworth, and intracellular localization, cell type and irradiation con- S. J. McMahon, W. B. Hyland, M. F. Muir, G. R. Dickson, figuration. Thus, further work is required to shed more light K. M. Prise, F. J. Currell, J. M. O’Sullivan and D. G. Hirst, on the mechanisms involved in the radiosensitization effects Int. J. Radiat. Oncol., Biol., Phys., 2011, 79, 531–539. of nanoparticles. Comprehensive characterization studies of 12 S. J. McMahon, W. B. Hyland, M. F. Muir, different cellular responses to GdNP are required for the J. A. Coulter, S. Jain, K. T. Butterworth, G. Schettino, further development of this promising technique. G. R. Dickson, A. R. Hounsell, J. M. O’Sullivan, K. M. Prise, D. G. Hirst and F. J. Currell, Radiother. Oncol., 2011, 100, 412–416. Conflicts of interest 13 I. Martínez-Rovira and Y. Prezado, Med. Phys., 2011, 38, 4430–4439. There are no conflicts of interest to declare. 14 K. Butterworth, S. McMahon, F. Currel and K. Prise, Nanoscale, 2012, 4, 4830–4838. 15 S. Rosa, C. Connolly, G. Schettino, K. T. Butterworth and Acknowledgements K. M. Prise, Cancer Nanotechnol., 2017, 8, 1–25. The authors acknowledge the granted beamtime at CELLS-ALBA 16 I. Martínez-Rovira and Y. Prezado, Med. Phys., 2015, 42, Synchrotron, and at the Hospital Universitari Sant Joan de Reus 6703–6710. (Institut d’Investigació Sanitària Pere Virgili). 17 N. Ma, F.-G. Wu, X. Zhang, Y.-W. Jiang, H.-R. Jia, This project has received funding from the European H.-Y. Wang, Y.-H. Li, P. Liu, N. Gu and Z. Chen, ACS Appl. Union’s Horizon 2020 Research and Innovation Programme Mater. Interfaces, 2017, 9, 13037–13048. under the Marie Sklodowska-Curie grant agreement number 18 D. Choudhury, P. Xavier, K. Chaudhari, R. John, 748889, as well as from the Secretaria d’Universitats i Recerca A. Dasgupta, T. Pradeep and G. Chakrabarti, Nanoscale, del Departament d’Economia i Coneixement de la Generalitat 2013, 5, 4476–4489. de Catalunya. 19 F. Taupin, M. Flaender, R. Delorme, T. Brochard, Finally, we also acknowledge Pere, Esther, Guillermina, J.-F. Mayol, J. Arnaud, P. Perriat, L. Sancey, F. Lux, R. Barth, Maria Cinta and Marta from ASPCAT Laboratory (Tortosa) for M. Carriere, J.-L. Ravanat and H. Elleaume, Phys. Med. their support. Biol., 2015, 60, 4449–4464. 20 F. Lux, L. Sancey, A. Bianchi, Y. Cremillieux, S. Roux and O. Tillement, Nanomedicine, 2015, 10, 1801–1815. References 21 M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, 1 J. F. Hainfeld, D. N. Slatkin and H. M. Smilowitz, Phys. K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, Med. Biol., 2004, 49, 309–315. B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, This journal is © The Royal Society of Chemistry 2019 Analyst View Article Online Paper Analyst M. J. Walsh, B. R. Wood, P. Gardner and F. L. Martin, Nat. 37 S. R. Saptarshi, A. Duschl and A. L. Lopata, J. Nanobiotechnol., Protoc., 2014, 9, 1771–1791. 2013, 11, 1–12. 22 M. Diem, P. Griffiths and J. Chalmers, Vibrational spec- 38 D. Sahoo, P. Bhattacharya, H. K. Patra, P. Mandal and troscopy for medical diagnosis, John Wiley & Sons, New York, S. Chakravorti, J. Nanopart. Res., 2011, 13, 6755–6760. 2008. 39 H. Holman, M. Martin, E. Blakely, K. Bjornstad and 23 I. Yousef, J. Breard, N. SidAhmed-Adrar, A. Maamer-Azzabi, W. McKinney, Biopolymers, 2000, 57, 329–335. C. Marchal, P. Dumas and F. Le Naour, Analyst, 2011, 136, 40 S. Boydston-White, M. Romeo, T. Chernenko, A. Regina, 5162–5168. M. Miljkovic and M. Diem, Biochim. Biophys. Acta, 2006, 24 L. Buriankova, Z. Nadova, D. Jancura, M. Refregiers, 1758, 908–914. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. I. Yousef and M. J. P. Miskovsky, Laser Phys. Lett., 2010, 7, 41 J. Reisz, N. Bansal, J. Qian, W. Zhao and C. Furdui, 613. Antioxid. Redox Signaling, 2014, 21, 260–292. Open Access Article. Published on 31 July 2019. Downloaded on 8/26/2019 11:35:19 AM. 25 I. Yousef, O. Seksek, Y. Prezado, J. Sulé-Suso and 42 H. Fricke, Nature, 1952, 169, 965–966. I. Martínez-Rovira, Analyst, 2016, 141, 2238–2249. 43 M. J. Daly, DNA Repair, 2012, 11, 12–21. 26 R. Barth and B. Kaur, J. Neurooncol., 2009, 94, 299–312. 44 A. Singh and H. Singh, Prog. Biophys. Mol. Biol., 1982, 39, 27 R. Di Corato, F. Gazeau, C. Le Visage, D. Fayol, P. Levitz, 69–107. F. Lux, N. Letourneur, D. Luciani, O. Tillement and 45 N. Gault, O. Rigaud, J. Poncy and J. Lefaix, Int. J. Radiat. C. Wilhelm, ACS Nano, 2013, 7, 7500–7512. Biol., 2005, 81, 767–779. 28 L. Sancey, F. Lux, S. Kotb, S. Roux, S. Dufort, A. Bianchi, 46 A. Sancar and G. B. Sancar, Annu. Rev. Biochem., 1988, 57, Y. Cremillieux, P. Fries, J. Coll, C. Rodriguez-Lafrasse, 29–67. M. Janier, M. Dutreix, M. Barberi-Heyob, F. Boschetti, 47 D. Whelan, K. Bambery, P. Heraud, M. Tobin, M. Diem, F. Denat, C. Louis, E. Porcel, S. Lacombe, G. Le Duc, D. McNaughton and B. Wood, Nucleic Acids Res., 2011, 39, E. Deutsch, J. Perfettini, A. Detappe, C. Verry, R. Berbeco, 5439–5448. K. Butterworth, S. McMahon, K. Prise, P. Perriat and 48 E. Lipiec, K. Bambery, P. Heraud, C. Hirschmugl, J. Lekki, O. Tillement, Br. J. Radiol., 2014, 87, 20140134. W. Kwiatek, M. J. Tobin, C. Vogel, D. Whelan and B. Wood, 29 M. Luchette, H. Korideck, M. Makrigiorgos, O. Tillement J. Mol. Struct., 2014, 1073, 134–141. and R. Berbeco, Nanomedicine, 2014, 10, 1751–1755. 49 D. Dovbeshko, N. Gridina, E. Kruglova and O. Pashchuk, 30 L. Sancey, S. Kotb, C. Truillet, F. Appaix, A. Marais, Talanta, 2000, 53, 233–246. E. Thomas, B. van der Sanden, J. Klein, B. Laurent, 50 A. Meade, C. Clarke, H. Byrne and F. Lyng, Radiat. Res., M. Cottier, R. Antoine, P. Dugourd, G. Panczer, F. Lux, 2010, 173, 225–237. P. Perriat, V. Motto-Ros and O. Tillement, ACS Nano, 2015, 51 N. Gault, O. Rigaud, J. Poncy and J. Lefaix, Radiat. Res., 9, 2477–2488. 2007, 167, 551–562. 31 P. Mowat, A. Mignot, W. Rima, F. Lux, O. Tillement, 52 B. Vileno, S. Jeney, A. Sienkiewicz, P. Marcoux, L. Miller C. Roulin, M. Dutreix, D. Bechet, S. Huger, L. Humbert, and L. Forro, Biophys. Chem., 2010, 152, 164–169. M. Barberi-Heyob, M. Aloy, E. Armandy, C. Rodriguez- 53 E. Albi, S. Cataldi, G. Rossi and M. Viola, Arch. Biochem. Lafrasse, G. Le Duc, S. Roux and P. Perriat, J. Nanosci. Biophys., 2008, 478, 52–58. Nanotechnol., 2011, 11, 7833–7839. 54 G. Zhang, R. Zhu, Y. Xu, Y. Yan and Y. Dai, Acta. Bot. Sin., 32 G. Birarda, D. E. Bedolla, E. Mitri, S. Pacor, G. Grenci and 2004, 46, 711–718. L. Vaccari, Analyst, 2014, 139, 3097. 55 G. Blankenberg, P. Katsikis, R. Storrs, C. Beaulieu, 33 A. Kretlow, Q. Wang, M. Beekes, D. Naumann and D. Spielman, J. Chen, L. Naumovski and J. Tait, Blood, L. M. Miller, Biochim. Biophys. Acta, Mol. Basis Dis., 2008, 1997, 89, 3778–3786. 1782, 559–565. 56 K. M. Prise, G. Schettino, M. Folkard and K. D. Held, 34 N. Gault and J. Lefaix, Radiat. Res., 2003, 160, 238–250. Lancet Oncol., 2005, 6, 520–528. 35 A. Barth, Biochim. Biophys. Acta, 2007, 1767, 1073–1101. 57 J. C. Roeske, L. Nunez, M. Hoggarth, E. Labay and 36 N. Volkova, O. Pavlovich, O. Fesenko, O. Budnyk, S. Kovalchuk R. R. Weichselbaum, Annu. Rev. Biochem., 2007, 6, 395– and A. Goltsev, J. Nanomater., 2017, 16934757, 1–9. 401. Analyst This journal is © The Royal Society of Chemistry 2019