Analyst PAPER Study of the intracellular nanoparticle-based Cite this: Analyst, 2020, 145, 2345 radiosensitization mechanisms in F98 glioma cells treated with charged particle therapy through synchrotron-based infrared microspectroscopy I. Martínez-Rovira, *a O. Seksek, b,c I. Dokic,d,e S. Brons, d A. Abdollahi d,e and I. Yousef a The use of nanoparticles (NP) as dose enhancers in radiotherapy (RT) is a growing research field. Recently, the use of NP has been extended to charged particle therapy in order to improve the performance in radioresistant tumors. However, the biological mechanisms underlying the synergistic effects involved in NP-RT approaches are not clearly understood. Here, we used the capabilities of synchrotron-based Fourier Transform Infrared Microspectroscopy (SR-FTIRM) as a bio-analytical tool to elucidate the NP- induced cellular damage at the molecular level and at a single-cell scale. F98 glioma cells doped with AuNP and GdNP were irradiated using several types of medical ion beams ( proton, helium, carbon and oxygen). Differences in cell composition were analyzed in the nucleic acids, protein and lipid spectral regions using multivariate methods (Principal Component Analysis, PCA). Several NP-induced cellular modifications were detected, such as conformational changes in secondary protein structures, intensity variations in the lipid CHx stretching bands, as well as complex DNA rearrangements following charged particle therapy irradiations. These spectral features seem to be correlated with the already shown enhancement both in the DNA damage response and in the reactive oxygen species (ROS) production by the NP, which causes cell damage in the form of protein, lipid, and/or DNA oxidations. Vibrational features Received 21st November 2019, were NP-dependent due to the NP heterogeneous radiosensitization capability. Our results provided new Accepted 16th January 2020 insights into the molecular changes in response to NP-based RT treatments using ion beams, and high- DOI: 10.1039/c9an02350j lighted the relevance of SR-FTIRM as a useful and precise technique for assessing cell response to inno- rsc.li/analyst vative radiotherapy approaches. 1 Introduction some radioresistant tumors (such as gliomas) continues being a challenge since tumor doses are constrained by the sur- Radiotherapy (RT) is the most important non-surgical modality rounding healthy tissue tolerances. for cancer treatment since about 50% of cancer patients The combination of high-atomic number (Z) nanoparticles receive RT during the course of their illness. Latest technologi- (NP) as radiosensitizers in RT was born in the last decade with cal advances in RT brought a substantial gain in the balance the main goal of enhancing the differential effect between the between the probability of tumor control and the risk of tumor and the surrounding healthy tissue.1 The size of the NP normal tissue complications. However, despite the use of typically ranges between 1 and 100 nm. Small nanoparticles state-of-the-art RT approaches, the clinical management of have a reduced tendency to accumulate in the body after treat- ment.2 NP leak preferentially into the tumor through active tar- a geting or passively, through enhanced permeability and reten- MIRAS beamline BL01, ALBA-CELLS Synchrotron, Carrer de la Llum 2-26, 08290 tion (EPR) effects.3 Cerdanyola del Vallès, Spain. E-mail: immamartinez@gmail.com b Université Paris-Saclay, CNRS/IN2P3, IJCLab, 91405 Orsay, France The therapeutic efficacy of RT in the presence of the NP has c Université de Paris, IJCLab, 91405 Orsay, France been proved in numerous studies using kilovoltage and mega- d Heidelberg Ion Beam Therapy Center (HIT), Department of Radiation Oncology, voltage photon beams, both in vitro and in vivo (see ref. 4–6, Heidelberg University Hospital, 69120 Heidelberg, Germany among others). The initial rationale behind these studies is e Clinical Cooperation Unite Translational Radiation Oncology, German Cancer the enhanced photoelectric cross section of high-Z NP in com- Consortium (DKTK) Core Center, National Center for Tumor Diseases (NCT), Heidelberg University Hospital (UKHD) and German Cancer Research Center (DKFZ), parison to water and soft tissue, specially at kilovoltage 69120 Heidelberg, Germany energies.7 This journal is © The Royal Society of Chemistry 2020 Analyst, 2020, 145, 2345–2356 | 2345 Paper Analyst However, despite extensive research on NP radiosensitiza- infrared synchrotron radiation delivered at the MIRAS beam- tion using photon beams, few experimental studies have been line of ALBA synchrotron allowed an infrared spectroscopic carried out in charged particle therapy. The pioneering study microanalysis at the single-cell level. of Usami and collaborators showed an enhancement in DNA Our previous FTIRM works provided new insights into the breaks at the molecular scale ( plasmid DNA as a probe) using radiosensitization effects of gold NP (AuNP) and gadolinium platinum-based complexes combined with helium and iron NP (GdNP) in photon RT.33–35 In the present study, radiosensi- irradiations.8 The efficacy of the same Pt complexes was later tization effects of 1.9 nm AuNP (AuroVist®) and 3 nm GdNP proved in vitro by the same group.9 An enhancement of radi- (AGuIX®) were assessed in F98 glioma cells using several ation effects was also demonstrated in proton therapy10–14 and charged particle beams ( proton, helium, carbon and oxygen). carbon therapy15–18 in several biological studies using different The potential of both NP was demonstrated in previous bio- types of high-Z NP. An overview of experimental studies on logical studies.36–39 However, to the best of our knowledge, radiosensitization effects of high-Z compounds in charged par- this is the first study that evaluates radiosensitization of NP in ticle therapy can be found in the review of Lacombe and charged particle therapy using SR-FTIRM. collaborators.19 The mechanisms involved in the NP-induced amplification of radiation effects in charged particle therapy is still a matter 2 Materials and methods of controversy since they seem to differ from the ones in stan- 2.1 F98 cell culture dard photon RT. Moreover, the radiosensitization effects involved might be distinct from the well-established pathway The F98 rat cell line (ATCC® CRL-2397, LGC Standards, of radiation-induced direct DNA damage.20 The amplified radi- Molsheim, France) used in this study is a glioblastoma cell type ation damage in the presence of NP was initially associated to displaying a tumor growth infiltrative pattern within brain tissue. the increase in the ionizations and electron emissions induced F98 cells were cultured in high glucose (4.5 g L−1) DMEM by the incident ions or the electrons in the secondary tracks growth medium supplemented with 10% Fetal Calf Serum, 1% on the NP, resulting in a local dose enhancement.15 Recent penicillin–streptomycin, 1% glutamine and 1% sodium pyru- studies showed a significant increase in the yield of low-energy vate at 37 °C with 5% CO2 and 95% humidity. Prior to exper- secondary electrons emitted due to collective electron exci- imentation, cells were plated in order to be kept in the expo- tations in the NP ( plasmons).21 Other authors proposed that nential growing phase at around 75–80% confluence. Two days NP could be activated by projectile induced X-rays emission prior to irradiation, 500 μL of a 4 × 105 cells per mL suspen- (PIXE), being the origin of intra-atomic cascades of electron sion were incubated in each well (48-well microplates) at 37 °C emissions.10 At the same time, these electrons enhance cell with 5% CO2 in order to reach a 75–80% confluence rate on damage through the increased production of reactive oxygen the irradiation day. species (ROS). Indeed, several studies have pointed out the 2.2 Gd and Au nanoparticles important role of hydroxyl radicals.9,13,14 Furthermore, emer- ging data supported that radiosensitization effects might be Before the irradiation protocol, cells were treated with AuNP or also driven by biological pathways, such as oxidative stress, GdNP following our previous protocols.33–35 Gold nano- DNA damage induction, cell cycle effects and potential inter- particles (1.9 nm AuNP, AuroVist®) were purchased from ference with cell communication.22–25 Nanoprobes (Yaphank, NY). Prior to treatment, 40 mg of AuNP Both radiation chemistry and biological mechanisms, as were rapidly dissolved in 0.2 mL PBS and filtered through a well as distinct cell uptake and cell sensitivity as a function of 0.2 μm centrifugal filter at 15 000g for 8 min. From this stock NP type (size, shape, coating, etc.), should be considered since solution kept at 4 °C, 500 μg mL−1 were applied on the cells pure physical dose enhancement cannot explain the amplifica- during 24 hours prior to irradiation. As for gadolinium nano- tion effect observed in biological studies.26 Therefore, there is particles (3 nm GdNP, AGuIX®), which were purchased from an urgent need to understand the mechanisms involved in NH TherAguix (Lyon, France), a 100 mM stock solution was these novel approaches to progress on the development of NP- obtained by solubilization with PBS and kept at 4 °C. From based therapeutics. this solution, 1 mM NP solution was applied on the cells Within this context, the objective of our study is to shed during 6 hours prior to irradiation. The biochemical effects some light on the NP-based radiosensitization mechanisms at induced by the NP prior to the irradiations were previously the molecular level in charged particle therapy through syn- assessed by means of SR-FTIRM and are reported in our pre- chrotron-based Fourier Transform Infrared Microspectroscopy vious studies.33–35 (SR-FTIRM). Infrared microspectroscopy has become a power- ful tool for examining composition and/or conformational 2.3 Cell irradiations changes in biomolecules, including proteins, lipids, carbo- Irradiations were carried out at the fixed horizontal beam hydrates and nucleic acids.27 FTIRM methods have been exten- experimental station of the Heidelberg Ion Beam Therapy sively used to study cell functionality, cell cycle, cell death and Center (HIT). Several beams were used for the irradiations: apoptosis, as well as cell and tissue responses to different proton ( p), helium (4He), carbon (12C) and oxygen (16O). The treatments (see ref. 27–32, among others). The brilliance of the well plates were vertically irradiated and placed at the center of 2346 | Analyst, 2020, 145, 2345–2356 This journal is © The Royal Society of Chemistry 2020 Analyst Paper a 10 mm-long spread out Bragg peak (SOPB) region centered at Principal Component Analysis (PCA) was performed using 80 mm-depth. The following physical (absorbed) doses were the Orange software (Bioinformatics Lab, University of applied (the energy range, E, and mean dose-averaged LET Ljubljana, Slovenia)44 in order to evaluate the variations in the values are detailed between brackets): spectral features. Prior to the PCA application, the data was • p: 4.5, 9, 18.2 Gy (E = 101.9–108.2 MeV u−1; LET = 5 keV); pre-processed by vector normalized second derivative spectra • 4He: 3.3, 6.7, 13.3 Gy (E = 101.6–107.9 MeV u−1; LET = 22 (Savitzky–Golay algorithm; 9 smoothing points; 3rd polynomial keV); order). Vector normalization and PCA were separately per- • 12C: 2.5, 5, 10 Gy (E = 194.9–206.9 MeV u−1; LET = 96 formed in the 3025–2800 (‘lipids’) and 1800–950 cm−1 (‘finger- keV); print’) spectral regions. • 16O: 1.7, 3.3, 6.7 Gy (E = 228.2–244.2 MeV u−1; LET = 140 A curve-fitting analysis was carried out on the amide I band keV); to evaluate the relative attribution of each component repre- in order to achieve effective biological doses of approximatively senting a type of secondary structure. The second derivative 5 GyRBE, 10 GyRBE and 20 GyRBE for each beam function was used to assess the minima and, thus, to define configuration. SOBP beam intensities and energies, (dose- the position and number of Gaussian function energy com- averaged) LET values, as well as physical doses were computed ponents needed to fit the curve of amide I. The Origin 7.0 soft- using the experimental planning program TRiP.40–42 The range ware (OriginLab Corporation, Northampton, USA) was used to of doses was selected according to previous radiobiological perform the curve-fitting method. A linear baseline was per- studies.42,43 formed in the amide I range prior to curve-fitting. In order to evaluate the area under the main spectral bands 2.4 SR-FTIRM sample preparation (see Table 1), the Rubber-band baseline correction method (32 24 hours after the irradiations, cells were specifically prepared baseline points) was applied to the raw spectra using for SR-FTIRM measurements following our previous Orange.44 Then, violin plots were generated to assess the prob- protocols.33–35 First, the medium was removed and 100 μL of ability density of the data for several spectral ratios that were 0.05% Trypsin-EDTA solution were added to each well and used as specific markers for indices of the biochemical incubated for 5 minutes. Then, 500 μL of supplemented changes. More details can be found in section 3. DMEM medium were added and the cell suspension was cen- trifuged at 1500 rpm for 5 minutes. After discarding the super- natant, the cellular pellet was suspended in PBS and centri- fuged again at 1500 rpm for 5 minutes. The final pellet was 3 Results and discussion then re-suspended in 10% formalin neutral buffered solution and incubated for 1 hour at room temperature. Then, samples SR-FTIRM analysis is presented for several beams (p, 4He, 12C, 16 were rinsed three times in deionized water. Finally, SR-FTIRM O) and doses in the presence (+NP) and absence (−NP) of nano- was performed on dried cells deposited onto CaF2 particles. SR-FTIRM details on the biochemical effects induced coverglasses. by the GdNP and AuNP are reported separately for the fingerprint (section 3.1.1) and lipids (section 3.1.2) spectral regions. 2.5 SR-FTIRM data acquisition and data analysis The Hyperion 3000 microscope coupled to Vertex 70 spectro- Table 1 Main band assignments in the SR-FTIRM spectra (ν = stretching meter (Bruker Optik GmbH, Germany) installed at the infrared vibration; δ = bending vibration; s = symmetric; as = asymmetric)45–49 beamline MIRAS of ALBA synchrotron light source was used to perform the SR-FTIRM measurements. The infrared spectra Main band assignment Specta region were collected using the transmission operation mode of the νsPO2−; deoxyribose/ribose ν C–O (phosphate II; PhII) 1146–1004 FTIR microscope. This microscope is equipped with a 50 μm νasPO2− (phosphate I; PhI) 1270–1186 liquid nitrogen cooled mercury cadmium telluride (MCT) δ N–H; ν C–N (proteins: Amide II, AII) 1590–1483 detector optimized for operation in a range covering the mid- ν CvO (proteins: Amide I, AI) 1710–1598 ν CvO ester (lipids) 1760–1730 infrared spectral region. The FTIRM system is operating with a νsCH2 (lipids) 2862–2842 36× Schwarzschild magnification objective (NA = 0.65) coupled νsCH3 (lipids) 2882–2862 to a 36× magnification condenser. νasCH2 (lipids) 2945–2900 νasCH3 (lipids) 2980–2945 Between 100 to 120 cells were randomly selected to undergo ν = C–H (lipids) 3018–3002 SR-FTIRM measurements from each sample condition. The SR-FTIRM spectra were acquired using a single masking aper- ture size of 7 μm × 7 μm. Single point measurements of indi- vidual cells were obtained in the mid-infrared range at 4 cm−1 3.1 SR-FTIRM results on the radiosensitization mechanisms spectral resolution with 128 co-added scans per spectrum. A of GdNP and AuNP in charged particle therapy background spectrum was collected every 5 measurements in PCA on the Savitzky–Golay second derivative data was order to eliminate the residual contamination (water vapor, employed as an unsupervised method to classify the data and CO2) of the room ambient. identify the main sources of variation in the SR-FTIRM spectra This journal is © The Royal Society of Chemistry 2020 Analyst, 2020, 145, 2345–2356 | 2347 Paper Analyst in the presence of nanoparticles (GdNP or AuNP). Fig. 1–4 PCA results performed on the Savitzky–Golay second deriva- depict the PCA scores in the fingerprint (1800–950 cm−1) and tive (Fig. 1–4, top) show differences between NP-treated (AuNP in the lipids (3025–2800 cm−1) spectral regions for p, 4He, and GdNP) groups and NoNP groups for all ion types, with 12 C and 16O irradiations, respectively. The corresponding some overlapping in some of the configurations. An analysis of loading plots are also shown, which represent the influence of the PCA loading plots reveals that most of the variance the variables (wavenumbers) in each principal component. accounting for the separation between groups is associated The first two principal components (PC-1 and PC-2) were used with protein modifications in the region 1700–1600 cm−1 to explain the differences among the samples. The averaged (amide I). The percentage of the total variance described by Savitzky–Golay second derivative is also presented in the pro- the first and second principal components is 60–80% and teins, DNA and lipids regions in Fig. 5. 30–10%, respectively, depending on the configuration. 3.1.1 Fingerprint region (1800–950 cm−1). Between Fig. 5 (top) shows the vector normalized Savitzky–Golay 1800–1400 cm−1, the absorption is dominated by the peptide second derivative of the averaged absorbance spectra in the bonds of cell peptides and proteins, including membrane pro- protein spectral region. The conformation changes in the teins. In particular, the amide I (1700–1600 cm−1) band is due amide I can be clearly seen in both NP treatments, especially to the CvO stretching vibration of amide groups of proteins, in the case of proton and helium irradiations. Spectra decon- weakly coupled with the in-plane NH-bending and the CN- volution of the amide I region was performed by means of stretching modes.50 The amide II band, located in the curve-fitting to precisely evaluate the differences among the 1600–1480 cm−1 region, is mainly due to the combination of secondary structures of proteins for the several NP treatments, NH-bending and CN-stretching vibrations.50 Both the amide I as it can be seen in Fig. 6. The deconvoluted spectra of the and II bands provide valuable information on the secondary amide I band showed a composite of six bands, which are structure of proteins.50 The region between 1350–950 cm−1 reported in Table 2.51,52 Changes in the band around consists in a complex bands pattern due to contributions from 1628 cm−1 were observed, which is assigned to the vibrational carbohydrates and phosphates associated with nucleic acids modes of β-sheet structural components.51,52 The β-sheet (DNA, RNA). The main bands at 1085 cm−1 and 1238 cm−1 are intensity increased upon both NP treatments with respect to assigned to the symmetric and asymmetric stretching the absence of NP. Previous works correlated an increase in vibrations of phosphodiester bonds in nucleic acids. Finally, the β-sheet secondary structure with the hallmark character- the band in the region 1760–1730 cm−1 is related to the carbo- istics of cell death,32,53 as well as to lipid-auto-oxidation pro- nyl ester group stretching in lipids and it will be discussed in ducts that deform the protein secondary structure.54 the lipids section (3.1.2). Therefore, the modifications encountered in the protein spec- Fig. 1 Proton beam irradiations. SR-FTIRM results on the radiosensitization mechanisms of GdNP and AuNP in charged particle therapy. The PCA scores and the loading plots are presented in the fingerprint (1800–950 cm−1, top) and lipids (3025–2800 cm−1, bottom) spectral regions in the absence (NoNP; blue) and in the presence of AuNP (red) and GdNP (green) for 5 GyRBE (left) and 20 GyRBE (right). 2348 | Analyst, 2020, 145, 2345–2356 This journal is © The Royal Society of Chemistry 2020 Analyst Paper Fig. 2 Helium beam irradiations. SR-FTIRM results on the radiosensitization mechanisms of GdNP and AuNP in charged particle therapy. The PCA scores and the loading plots are presented in the fingerprint (1800–950 cm−1, top) and lipids (3025–2800 cm−1, bottom) spectral regions in the absence (NoNP; blue) and in the presence of AuNP (red) and GdNP (green) for 5 GyRBE (left) and 20 GyRBE (right). Fig. 3 Carbon beam irradiations. SR-FTIRM results on the radiosensitization mechanisms of GdNP and AuNP in charged particle therapy. The PCA scores and the loading plots are presented in the fingerprint (1800–950 cm−1, top) and lipids (3025–2800 cm−1, bottom) spectral regions in the absence (NoNP; blue) and in the presence of AuNP (red) and GdNP (green) for 5 GyRBE (left) and 20 GyRBE (right). This journal is © The Royal Society of Chemistry 2020 Analyst, 2020, 145, 2345–2356 | 2349 Paper Analyst Fig. 4 Oxygen beam irradiations. SR-FTIRM results on the radiosensitization mechanisms of GdNP and AuNP in charged particle therapy. The PCA scores and the loading plots are presented in the fingerprint (1800–950 cm−1, top) and lipids (3025–2800 cm−1, bottom) spectral regions in the absence (NoNP; blue) and in the presence of AuNP (red) and GdNP (green) for 5 GyRBE (left) and 20 GyRBE (right). Fig. 5 All ions ( p, 4He, 12C, 16O; from left to right). SR-FTIRM results on the radiosensitization mechanisms of GdNP and AuNP in charged particle therapy. Savitzky–Golay second derivative of the averaged absorbance spectra in the absence (NoNP; blue) and in the presence of AuNP (red) and GdNP (green) in the proteins (1700–1350 cm−1, top), DNA (1350–950 cm−1; middle) and lipids (3025–2800 cm−1, bottom) spectral regions. 2350 | Analyst, 2020, 145, 2345–2356 This journal is © The Royal Society of Chemistry 2020 Analyst Paper Fig. 6 Curve-fitting analysis of the amide I band in the absence (NoNP; blue) and in the presence of AuNP (red) and GdNP (green) (4He). The base- line-corrected spectrum was fitted with six Gaussian band profiles by approximating the number and their positions using the minima of the second derivative function, which fitted the experimental curve (in black). Table 2 Spectra deconvolution of the amide I band. Assignment of the Fig. 7, we can clearly observe an increase in the amide I to amide six Gaussian band profiles encountered in the curve-fitting analysis51,52 II ratio, especially in the case of AuNP-treated cells. Following previous works,56 these results seem to indicate that cellular Band assignment Wavenumber protein modifications in the folding and localization have Side chain 1619 cm−1 occurred following an enhanced cell death response induced by β-Sheet 1628 cm−1 the presence of the NP. Other works correlated the changes in Random coil 1638 cm−1 α-Helix 1655 cm−1 the amide I and II absorbances with the DNA repairing processes β-Turn 1680 cm−1 as a result of the radiation damage produced.57 β anti-parallel sheet 1691 cm−1 In the DNA region, several spectral areas appeared to be modified upon NP treatment for all ion beams, as it can be seen in the vector normalized Savitzky–Golay second derivative tral region could be correlated to an increase in the ROS pro- of the averaged absorbance spectra (see Fig. 5, middle). The duction in the presence of NP, which might cause cell damage multiple changes in the absorbance and position (shift) of in the form of protein oxidation (as well as lipids and DNA oxi- various peaks probably indicate a number of different modifi- dations), leading to an enhancement in the cell death. We also cations of the DNA conformational changes and/or rearrange- observed a reduction in the random coil structure content of ments attributed to distortions of the DNA double helix in the proteins, also previously detected in dying cells.55 presence of NP. The ratio of amide I to amide II was also used to show con- The modifications and the downward shift in the νasPO2− formational changes of proteins, since it is directly related to (named phosphate I) band at 1238 cm−1 suggest that the the secondary structure of proteins.56 In the violin plots in nucleic acid phosphodiester groups suffered conformation Fig. 7 All ions ( p, 4He, 12C, 16O; from left to right); 20 GyRBE. SR-FTIRM results on the radiosensitization mechanisms of GdNP and AuNP in charged particle therapy. Distribution of the relative intensities of the amide I (AI) to amide II (AII) spectral band ratio in the absence (NoNP; blue) and in the presence of AuNP (red) and GdNP (green). See Table 1 for more information on the bands assignment. This journal is © The Royal Society of Chemistry 2020 Analyst, 2020, 145, 2345–2356 | 2351 Paper Analyst modifications in the DNA backbone, such as partial transitions damage.28,57,60,61 The intensities in the phosphate I and phos- between B-DNA to A-DNA structures due to nucleotide base phate II bands (with respect to the amide II band) correlate damage.46,57,58 We also observe changes in the symmetric inversely with cell death, as detailed in previous works.46,56 In phosphate stretching band of phosphodiester nucleic acids the violin plots of Fig. 8, AuNP-induced DNA damage is (1085 cm−1; phosphate II), which was correlated with the expressed by the decrease in the PhI and PhII bands, which degree of intermolecular interactions in the nucleic acids.28 might indicate DNA condensation.28,29,46,53,56,57 A reduction on A series of peaks related to ribose and deoxyribose –C–O the absorbance of the phosphate I band was also reported upon and –PO2 stretching vibrations are located in the oxidative stress.48 1170–995 cm−1 range. The second derivative plot shows The combination of all these spectral changes showed NP- changes in these infrared bands in NP-treated cells, specially induced DNA damage in different groups of the DNA mole- in the peaks around 1060 and 1020 cm−1, suggesting changes in cule, which seems to correspond to the course of cell death the deoxyribose/ribose structures. They were reported to be corre- processes. However, unraveling the specific NP-induced cell lated with non-repaired damage in the DNA structure.53 In par- processes is a complex task due to the multiple contributions ticular, the furanose C–O stretching at 1020 cm−1 is an important in this region of the spectral range. marker for base-pairing and base-stacking in RNA.59 This might Finally the distinct LET of the several beams could be the indicate base cleavage reactions, probably due to ROS damage or responsible of the differences observed in the NP-induced bio- DNA repairing processes.60 Previous articles related these chemical modifications between ions, which might be due to changes with modifications in the structure of the RNA that distinct radiation-induced lethal effects. Despite an increase in could induce the formation of altered proteins as a result of the the ROS production was reported in ion beams, an enhanced incorrect translation processes.59 We observed an increase in DNA-damage complexity and accumulation of non-repairable such modifications with the presence of both types of NP. DSB was observed in the case of high LET particles.42 This, The band near 965 cm−1 is assigned to the ν C–C/C–O combined with the presence of NP, might induce a difference stretching vibration involved in the deoxyribose and phosphate of lethal effect on the cells as a function of the LET. moiety of the DNA backbone.47 Modifications in this band 3.1.2 Lipids region (3025–2800 cm−1). The lipids region is were previously related to single-strand breaks (SSB), double- mainly dominated by the symmetric and asymmetric stretch- strand breaks (DSB), crosslinks and deoxyribose ing modes of methylene and methyl groups, mainly present in Fig. 8 All ions ( p, 4He, 12C, 16O; from left to right); 20 GyRBE. SR-FTIRM results on the radiosensitization mechanisms of GdNP and AuNP in charged particle therapy. Distribution of the relative intensities of the phosphate I (PhI) and phosphate II (PhII) bands with respect to the amide II (AII) in the absence (NoNP; blue) and in the presence of AuNP (red) and GdNP (green). See Table 1 for more information on the bands assignment. 2352 | Analyst, 2020, 145, 2345–2356 This journal is © The Royal Society of Chemistry 2020 Analyst Paper the hydrocarbon acyl chain length of lipids, and to a lesser peaks at 2874 cm−1 and 2959 cm−1 are associated to the CH3 extend in proteins. Specifically, the intensities of the bands symmetric and asymmetric stretching.62 The region around around 2852 cm−1 and 2922 cm−1 correspond to the CH2 sym- 3010 cm−1 is originated from unsaturated fatty acids. In metric and asymmetric stretching, respectively, while the addition to the bands in the 3025–2800 cm−1 spectral range, Fig. 9 All ions ( p, 4He, 12C, 16O; from left to right); 20 GyRBE. SR-FTIRM results on the radiosensitization mechanisms of GdNP and AuNP in charged particle therapy. Distribution of the relative intensities of the CH2/CH3, CH2 stretching ratio, CH/CH2 and CvO ester/lipids spectral band ratios in the absence (NoNP; blue) and in the presence of AuNP (red) and GdNP (green). See Table 1 for more information on the bands assignment. This journal is © The Royal Society of Chemistry 2020 Analyst, 2020, 145, 2345–2356 | 2353 Paper Analyst the band from 1760–1730 cm−1 is assigned to the ester CvO hydrogen-bonded states in phospholipids was reported to be a stretching of the phospholipids in the cellular membrane, and good indicator of lipid peroxidation.49 Fig. 9 shows the distri- it is not overlapped by contributions from proteins and DNA.45 bution of this band with respect to the area under the lipids The PC1–PC2 scores scatter plots in the lipids region region in order to normalize to cell thickness differences.49 We (Fig. 1–4, bottom) display well-separated clusters between NP- detected an increase of the carbonyl ester stretching band in treated groups (GdNP and AuNP) and NoNP groups, mostly the presence of both types of NP, particularly in the case of along PC-1, for most of the ion beams and doses. PCA AuNP-treated cells. The increase was associated to an increase suggested a more marked difference for AuNP as the PCA of oxidized lipids present in the membrane of dying cells or in scores showed a clearer separation from NoNP' groups. In all cells under oxidative stress due to lipid peroxidation cases, the first two principal components explain around 50% products.32,49 and 10–15% of the total variance, respectively. The corres- Our results showed that RT-NP treatments induced several ponding loading plots demonstrated that NP-treated groups, modifications in the lipids spectral range, which might involve and specially AuNP-treated cells, are mainly correlated with a wide range of biological processes. The modifications conformational changes in the CHx vibration bands. Despite observed regarding lipids biophysical properties and chemical very subtle shifts can be observed in some lipid bands, the state might be related to the oxidative stress produced by the position of the most intense bands is preserved in all configur- production of ROS from indirect effects of ionizing radiation ations (see Fig. 5). in the presence of NP, which has been reported to be the main The most important markers for conformational disorders responsible of the amplification of the radiation damage in the cell membrane are the changes in the intensity in the observed in some charged particle therapy studies combined lipids spectral area. In particular, the ratios between the areas with NP.8,19 Indeed, the induction of AuNP-related oxidative of several absorption bands associated to lipids (CH2/CH3, CH2 stress was previously reported using the same type of NP via stretching ratio, CH/CH2 and carbonyl ester to lipids) are pre- increased production of endogenous reactive oxygen species sented in Fig. 9 and detailed hereafter. They provide relevant and depletion of intracellular antioxidants.36,67 The lipid differ- information on the lipids biophysical properties and chemical ences in cellular response between AuNP and GdNP might be state modifications within the cell membrane, such as to lipids attributed to the distinct radiosensitization capability of both structure, chain length, oxidative stress, degree of unsaturation NP, which might lead to distinct cell damage/death pathways. and membrane peroxidation.63 These ratios provided indications on the biochemical effects of the NP treatment on the cells. • The asymmetric CH2/CH3 ratio is associated with the 4 Conclusions hydrocarbon acyl chain length of lipids (increases with chain length). Petibois et al. correlated a reduction of CH2 /CH3 with The objective of our SR-FTIRM study was to shed some light membrane phospholipid free radical attacks during oxidative on the (Au and Gd) NP-based radiosensitization mechanisms stress.48 In our study (see Fig. 9), a slight decrease was in F98 cells treated with charged particle therapy ( proton, observed in AuNP-treated cells with respect to NoNP for all ion helium, carbon and oxygen). Thanks to the very high signal-to- beams and doses. In the case of GdNP, no significant changes noise ratio of synchrotron-based FTIRM, modifications in the in the CH2/CH3 ratio were observed. infrared spectra were assessed at a single-cell level. • The CH2 symmetric to CH2 asymmetric stretching ratio Observation of PCA results evidenced the separation between (CH2 stretching ratio) is another way to assess cell membrane the NP-treated cells and the NoNP control cells, with some morphology alterations.29,64 In particular, a decrease in the CH2 overlapping in some configurations. PCA in the fingerprint stretching ratio was associated to an increase in the membrane ( proteins and DNA) and lipids regions enabled to distinguish rigidity as a consequence of the presence of membrane stabiliz- the biochemical features induced by the nanoparticles on the ing agents or lipid raft aggregates, which are formed in dying main biomolecules present in F98 glioma cells. cells.64 Fig. 9 depicts the CH2 stretching ratio. In the presence In the proteins spectral region, a deconvolution study on of AuNP, there is a decrease in the CH2 stretching ratio. the amide I revealed an increase in the β-sheet secondary • The ratio between unsaturated fatty acids structure in the presence of NP. This, along with examination (3018–3002 cm−1) to the symmetric CH2 stretching provides a of the amide I to amide II ratio, indicated cellular protein good index for unsaturation.45 In Fig. 9, we detect a slightly modifications in the folding and localization induced by the higher concentration of unsaturated bonds in the lipid chains NP. In the DNA region, we revealed a number of structural and after AuNP treatment with respect to NoNP. The level of unsa- conformational changes in the presence of NP, as well as evi- turation of the fatty acids in the membrane has a high impact dences of DNA fragmentation represented by the decrease in on the characteristics of the membrane. In particular, an the νs PO2− and νas PO2− bands. In the lipids spectral region, increase of unsaturated fatty acids leads to alterations in the several specific markers were used as indices of the changes membrane fluidity,29 which can highly affect functional pro- (CH2/CH3, CH2 stretching ratio, CH/CH2 and CvO ester to perties of the cell.65,66 Lipids), which provided relevant information on the changes • The distribution of the carbonyl ester group stretching induced by the NP in the lipid structure, chain length, oxi- band (1760–1730 cm−1) found in non-hydrogen bonded and dative stress, degree of unsaturation and membrane peroxi- 2354 | Analyst, 2020, 145, 2345–2356 This journal is © The Royal Society of Chemistry 2020 Analyst Paper dation. The modulation of the band intensities specific to 9 N. Usami, Y. Furusawa, K. Kobayashi, S. Lacombe, DNA, proteins and lipids might evidence an enhanced pro- A. Reynaud-Angelin, E. Sage, T.-D. Wu, A. Croisy, duction of ROS in the presence of NP, as well as an enhanced J.-L. Guerquin-Kern and C. Le Sech, Int. J. Radiat. Biol., DNA damage response, correlated with cell damage/death. 2008, 84, 603–611. These results are in line with the amplification of radiation 10 J.-K. Kim, S.-J. Seo, K.-H. Kim, T.-J. Kim, M. H. Chung, effects induced by NP in previous radiobiological studies in K. R. Kim and T. K. Yang, Nanotechnology, 2010, 21, charged particle therapy. Finally, vibrational features were NP- 425102. dependent since radiosensitization effects differed between 11 J.-K. Kim, S.-J. Seo, H.-T. Kim, K.-H. Kim, M.-H. Chung, different NP (Au/Gd) types. K.-R. Kim and S.-J. Ye, Phys. Med. Biol., 2012, 57, 8309– To conclude, this is the first work that provides new 8323. insights into the radiosensitization effects of nanoparticles in 12 J. C. Polf, L. F. Bronk, W. H. P. Driessen, R. Pasqualini and charged particle therapy through SR-FTIRM. However, moni- M. Gillin, Appl. Phys. Lett., 2011, 98, 193702. toring the overall biochemical changes in the cell upon NP- 13 S. Li, S. Penninckx, L. Karmani, A.-C. Heuskin, K. Watillon, based RT treatmens is a very complex task since it involves a R. Marega, J. Zola, V. Corvaglia, G. Genard, B. Gallez, wide range of biochemical processes. Therefore, the under- O. Feron, P. Martinive, D. Bonifazi, C. Michiels and lying biochemical processes implicated in such novel NP-RT S. Lucas, Nanotechnology, 2016, 27, 455101. approaches will need to be more thoroughly experimentally 14 T. Schlatholter, S. Lacombe, P. Eustache, E. Porcel, investigated. D. Salado, L. Stefancikova, O. Tillement, F. Lux, P. Mowat, A. K. Biegun, M.-J. van Goethem, H. Remita and S. Lacombe, Int. J. Nanomed., 2016, 11, 1549–1556. Conflicts of interest 15 E. Porcel, S. Liehn, H. Remita, N. Usami, K. Kobayashi, Y. Furusawa, C. Le Sech and S. Lacombe, Nanotechnology, There are no conflicts of interest to declare. 2010, 21, 085103. 16 H. Kaur, G. Pujari, M. Semwal, A. Sarma and D. K. Avasthi, Nucl. Instrum. Methods Phys. Res., Sect. B, 2013, 301, 7–11. Acknowledgements 17 E. Porcel, O. Tillement, F. Lux, P. Mowat, N. Usami, The authors acknowledge the granted beamtime at K. Kobayashi, Y. Furusawa, C. Le Sech, S. Li and ALBA-CELLS Synchrotron. This project was funded by the S. Lacombe, Nanomedicine, 2014, 10, 1601–1608. European Union Horizon 2020 Research and Innovation 18 Y. Liu, X. Liu, X. Jin, P. He, X. Zheng, F. Ye, W. Chen and Programme under the Marie Sklodowska-Curie grant agree- Q. Li, J. Nanosci. Nanotechnol., 2016, 16, 2365–2370. ment number 748889. A. Abdollahi acknowledges the support 19 S. Lacombe, E. Porcel and E. Scifoni, Cancer Nanotechnol., from the German Research Foundation (DFG, SFB 1389, Unite) 2017, 8, 9. intramural funds of the National Center for Tumor Diseases 20 M. Sotiropoulos, N. T. Henthorn, J. W. Warmenhoven, (NCT Biodose program). R. I. Mackay, K. J. Kirkby and M. J. Merchant, Nanoscale, 2017, 9, 18431. 21 A. V. Verkhovtsev, A. V. Korol and A. V. Solovyov, J. Phys. References Chem. C, 2015, 119, 11000–11013. 22 K. Butterworth, S. McMahon, F. Currel and K. Prise, 1 J. F. Hainfeld, D. N. Slatkin and H. M. Smilowitz, Phys. Nanoscale, 2012, 4, 4830–4838. Med. Biol., 2004, 49, 309–315. 23 S. Rosa, C. Connolly, G. Schettino, K. T. Butterworth and 2 S. Akhter, M. Z. Ahmad, F. J. Ahmad, G. Storm and K. M. Prise, Cancer Nanotechnol., 2017, 8, 1–25. R. J. Kok, Expert Opin. Drug Delivery, 2012, 9, 1225–1243. 24 D. Choudhury, P. Xavier, K. Chaudhari, R. John, 3 Y. Matsumura and H. Maeda, Cancer Res., 1986, 46, 6387– A. Dasgupta, T. Pradeep and G. Chakrabarti, Nanoscale, 6392. 2013, 5, 4476–4489. 4 D. Kwatra, A. Venugopal and S. Anant, Transl. Cancer Res., 25 F. Taupin, M. Flaender, R. Delorme, T. Brochard, 2013, 2, 330–342. J.-F. Mayol, J. Arnaud, P. Perriat, L. Sancey, F. Lux, R. Barth, 5 K. Haume, S. Rosa, S. Grellet, M. A. Smialek, M. Carriere, J.-L. Ravanat and H. Elleaume, Phys. Med. K. T. Butterworth, A. V. Solovyov, K. M. Prise, J. Golding Biol., 2015, 60, 4449–4464. and N. J. Mason, Cancer Nanotechnol., 2016, 7, 1–20. 26 I. Martínez-Rovira and Y. Prezado, Med. Phys., 2015, 42, 6 S. Her, D. A. Jaffray and C. Allen, Adv. Drug Delivery Rev., 6703–6710. 2017, 109, 84–101. 27 M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, 7 I. Martínez-Rovira and Y. Prezado, Med. Phys., 2011, 38, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, 4430–4439. K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, 8 N. Usami, Y. Furusawa, K. Kobayashi, H. Frohlich, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, S. Lacombe and C. Le Sech, Int. J. Radiat. Biol., 2005, 81, M. J. Walsh, B. R. Wood, P. Gardner and F. L. Martin, Nat. 512–522. Protoc., 2014, 9, 1771–1791. This journal is © The Royal Society of Chemistry 2020 Analyst, 2020, 145, 2345–2356 | 2355 Paper Analyst 28 N. Gault and J. Lefaix, Radiat. Res., 2003, 160, 238–250. 45 R. Gasper, J. Dewelle, R. Kiss, T. Mijatovic and 29 W. Mihoubi, E. Sahli, A. Gargouri and C. Amiel, PLoS One, E. Goormaghtigh, Biochim. Biophys. Acta, 2009, 1788, 1263– 2017, 12, e0180680. 1270. 30 D. Ami, T. Neri, A. Natalello, P. Mereghetti, S. M. Doglia, 46 G. Birarda, D. E. Bedolla, E. Mitri, S. Pacor, G. Grenci and M. Zanoni, M. Zuccotti, S. Garagna and C. A. Redi, Biochim. L. Vaccari, Analyst, 2014, 139, 3097. Biophys. Acta, Mol. Cell Res., 2008, 1783, 98–106. 47 C. Sandt, C. Nadaradjane, R. Richards, P. Dumas and 31 D. R. Whelan, K. Bambery, L. Puskar, D. McNaughton and V. See, Analyst, 2016, 141, 870. B. R. Wood, Analyst, 2013, 138, 3891–3899. 48 C. Petibois and G. Deleris, Analyst, 2004, 129, 912–916. 32 H. Holman, M. Martin, E. Blakely, K. Bjornstad and 49 B. Vileno, S. Jeney, A. Sienkiewicz, P. R. Marcoux, W. McKinney, Biopolymers, 2000, 57, 329–335. L. M. Miller and L. Forro, Biophys. Chem., 2010, 152, 164– 33 I. Yousef, O. Seksek, Y. Prezado, J. Sulé-Suso and 169. I. Martínez-Rovira, Analyst, 2016, 141, 2238–2249. 50 A. Barth, Biochim. Biophys. Acta, 2007, 1767, 1073–1101. 34 I. Martínez-Rovira, O. Seksek, J. Puxeu, J. Gómez, 51 H. Ghimire, M. Venkataramani, Z. Bian, Y. Liu and M. Kreuzer, T. Ducic, M. J. Ferreres, M. Artigues and A. G. U. Perera, Sci. Rep., 2017, 7, 16993. I. Yousef, Analyst, 2019, 144, 5511–5520. 52 C. Chadefaux, A. Le Ho, L. Bellot-Gurlet and I. Reiche, e- 35 I. Martínez-Rovira, O. Seksek and I. Yousef, Analyst, 2019, Preserv. Sci., 2009, 6, 129–137. 144, 6352–6364. 53 N. Gault, O. Rigaud, J. Poncy and J. Lefaix, Int. J. Radiat. 36 J. A. Coulter, S. Jain, K. T. Butterworth, L. E. Taggart, Biol., 2005, 81, 767–779. G. R. Kickson, S. J. McMahon, W. B. Hyland, M. F. Muir, 54 A. Lamberti, C. Sanges and P. Arcari, Spectroscopy, 2010, 24, C. Trainor, A. R. Hounsell, J. M. O’Sullivan, G. Schettino, 535–546. F. J. Currell, D. G. Hirst and K. M. Prise, Int. J. Nanomed., 55 U. Zelig, J. Kapelushnik, R. Moreh, S. Mordechai and 2012, 7, 2673–2685. I. Nathan, Biophys. J., 2009, 97, 2107–2114. 37 K. T. Butterworth, J. A. Coulter, S. Jain, J. Forker, 56 F. Gasparri and M. Muzio, Biochem. J., 2003, 369, 239– S. J. McMahon, G. Schettino, K. M. Prise, F. J. Currell and 248. D. G. Hirst, Nanotechnology, 2010, 21, 2673–2685. 57 E. Lipiec, K. Bambery, P. Heraud, C. Hirschmugl, J. Lekki, 38 L. Sancey, F. Lux, S. Kotb, S. Roux, S. Dufort, A. Bianchi, W. Kwiatek, M. Tobin, C. Vogel, D. Whelan and B. Wood, Y. Cremillieux, P. Fries, J. Coll, C. Rodriguez-Lafrasse, J. Mol. Struct., 2014, 1073, 134–141. M. Janier, M. Dutreix, M. Barberi-Heyob, F. Boschetti, 58 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 59 E. Lipiec, G. Birarda, J. Kowalska, J. Lekki, L. Vaccari, O. Tillement, Br. J. Radiol., 2014, 87, 20140134. A. Wiechec, B. Wood and W. Kwiatek, Radiat. Phys. Chem., 39 L. Sancey, S. Kotb, C. Truillet, F. Appaix, A. Marais, 2013, 93, 135–141. E. Thomas, B. van der Sanden, J. Klein, B. Laurent, 60 A. Meade, H. Byrne and F. Lyng, Mutat. Res., 2010, 704, M. Cottier, R. Antoine, P. Dugourd, G. Panczer, F. Lux, 108–114. P. Perriat, V. Motto-Ros and O. Tillement, ACS Nano, 2015, 61 N. Gault, O. Rigaud, J. Poncy and J. Lefaix, Radiat. Res., 9, 2477–2488. 2007, 167, 551–562. 40 M. Krämer, O. Jäkel, T. Haberer, G. Kraft, D. Achardt and 62 Z. Movasaghi, S. Rehman and I. Rehman, Appl. Spectrosc. U. Weber, Phys. Med. Biol., 2000, 45, 3299–3317. Rev., 2008, 43, 134–179. 41 M. Krämer and M. Scholz, Phys. Med. Biol., 2000, 45, 3319– 63 F. S. Ruggeri, C. Marcott, S. Dinarelli, G. Longo, 3330. M. Girasole, G. Dietler and T. Knowles, Int. J. Mol. Sci., 42 I. Dokic, A. Mairani, M. Niklas, F. Zimmermann, 2008, 19, 2582. N. Chaudhi, D. Krunic, T. Tessonier, A. Ferrari, K. Parodi, 64 S. van der Driesche, F. Iuliano, C. Haiden, D. Pucciarelli, O. Jakel, J. Debus, T. T. Haberer and A. Abdollahi, H. Breiteneder, S. Pastorekova, C. Hafner and Oncotarget, 2016, 7, 56676–56689. M. J. Vellekoop, Sens. Actuators, B, 2013, 179, 150–156. 43 S. Combs, J. Bohl, T. Elsasser, K.-J. Weber, D. Schulz- 65 S. Baritaki, P. Kanellou, S. Apostolakis, D. A. Spandidos Ertner, J. Debus and W. Weyrather, Int. J. Radiat. Biol., and B. Bonavida, Proc. Amer. Assoc. Cancer Res., 2006, 47, 2009, 85, 126–137. 1262. 44 J. Demšar, T. Curk, A. Erjavec, V. Gorup, T. Hočevar, 66 M. K. Mishra, D. Ghosh, R. Duseja and A. Basu, Neurochem. M. Milutinovič, M. Možina, M. Polajnar, M. Toplak, Int., 2009, 54, 464–470. A. Starič, M. Štajdohar, L. Umek, L. Žagar, J. Žbontar, 67 Y. Pan, A. Leifert, D. Ruau, S. Neuss, J. Bornemann, M. Žitnik and B. Zupan, J. Mach. Learn. Res., 2013, 14, G. Schmid, W. Brandau, U. Simon and W. Jahnen-Dechent, 2349–2353. Small, 2009, 5, 2067–2076. 2356 | Analyst, 2020, 145, 2345–2356 This journal is © The Royal Society of Chemistry 2020