biology Article Differential Radiosensitizing Effect of 50 nm Gold Nanoparticles in Two Cancer Cell Lines Miguel Ángel Pérez-Amor 1 , Leonardo Barrios 2 , Gemma Armengol 1 and Joan Francesc Barquinero 1, * 1 Unit of Biological Anthropology, Department of Animal Biology, Plant Biology and Ecology, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain 2 Unit of Cell Biology, Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain * Correspondence: francesc.barquinero@uab.cat; Tel.: +34-935811317 Simple Summary: Nanoparticle treatment on tumor cells is proposed for its potential radiosensitizing properties, increasing the radiation effect on tumor cells and reducing the adverse effects on healthy tissues. The present study evaluates, on two cell lines derived from colon and breast adenocarcinomas, the impact of irradiation in the presence of specifically targeted gold nanoparticles. Cells were irradiated in the absence and in the presence of non-functionalized or specifically functionalized gold nanoparticles. The results pointed out that actively targeting gold nanoparticles has a clear radiosensitizing effect in both cell lines. Abstract: Radiation therapy is widely used as an anti-neoplastic treatment despite the adverse effects it can cause in non-tumoral tissues. Radiosensitizing agents, which can increase the effect of radiation in tumor cells, such as gold nanoparticles (GNPs), have been described. To evaluate the radiosensitizing effect of 50 nm GNPs, we carried out a series of studies in two neoplastic cell lines, Caco2 (colon adenocarcinoma) and SKBR3 (breast adenocarcinoma), qualitatively evaluating Citation: Pérez-Amor, M.Á.; Barrios, the internalization of the particles, determining with immunofluorescence the number of γ-H2AX L.; Armengol, G.; Barquinero, J.F. foci after irradiation with ionizing radiation (3 Gy) and evaluating the viability rate of both cell lines Differential Radiosensitizing Effect of after treatment by means of an MTT assay. Nanoparticle internalization varied between cell lines, 50 nm Gold Nanoparticles in Two though they both showed higher internalization degrees for functionalized GNPs. The γ-H2AX foci Cancer Cell Lines. Biology 2022, 11, counts for the different times analyzed showed remarkable differences between cell lines, although 1193. https://doi.org/10.3390/ biology11081193 they were always significantly higher for functionalized GNPs in both lines. Regarding cell viability, in most cases a statistically significant decreasing tendency was observed when treated with GNPs, Academic Editors: Charles D. Rice especially those that were functionalized. Our results led us to conclude that, while 50 nm GNPs and Armando Varela-Ramirez induce a clear radiosensitizing effect, it is highly difficult to describe the magnitude of this effect as Received: 28 June 2022 universal because of the heterogeneity found between cell lines. Accepted: 8 August 2022 Published: 9 August 2022 Keywords: gold nanoparticles; functionalization; internalization; radiation therapy; γ-H2AX foci; radiosensitization Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Radiation therapy is one of the most common treatments when fighting neoplasms and almost 60% of patients receiving anti-neoplasic treatment undergo radiation therapy [1], Copyright: © 2022 by the authors. be it alone or concomitant with another type of treatment, i.e., chemotherapy or surgery. Licensee MDPI, Basel, Switzerland. Despite its effectiveness in cancer treatment, exposure to radiation therapy can cause This article is an open access article several adverse side effects, such as dermatitis, fatigue, depression and radiation recall [2]. distributed under the terms and With the aim of either diminishing the appearance of undesired side effects or in- conditions of the Creative Commons creasing the radiation effect in tumoral cells, radioprotective and radiosensitizing agents Attribution (CC BY) license (https:// can be used. A radioprotective agent reduces the damage produced in healthy cells by creativecommons.org/licenses/by/ limiting the action on DNA of reactive radicals such as reactive oxygen species (ROS), 4.0/). Biology 2022, 11, 1193. https://doi.org/10.3390/biology11081193 https://www.mdpi.com/journal/biology Biology 2022, 11, 1193 2 of 17 mainly generated by the interaction of ionizing radiations and water [3]. A pioneering study showed that the fraction of the DNA damage that can be protected from radiation by the use of radioprotectors accounts for about 70% [4]. Radioprotective agents such as amifostine are already being used clinically [5], while others such as resveratrol and CBLB502 are being tested with promising results [6]. On the contrary, one of the main mechanisms of action of radiosensitizing agents is to enhance the level of radiation-induced ROS to cause damage to the DNA of tumoral cells. As radiosensitizing agents, various agents such as hyperbaric oxygen [7], nicotinamide and carbogen [8] are widely described and applied. A third type of agent exists, radiation mitigators [8], but their action is to reduce the impact of the adverse effects after an irradiation. An important limitation of some radiosensitizing treatments can be attributed to the unspecific distribution of the radiosensitizer throughout the body. One strategy for overcoming this limitation is the use of micro- or nanoparticles as radiosensitizers because they can naturally accumulate in tumor tissues due to the enhanced permeability and retention effect resulting from the imperfect endothelium of blood vessels formed during tumoral angiogenesis [9]. To increase the particles’ accumulation in specific desired tissues, active targeting strategies have been developed, such as the functionalization of the particle surface to be recognized by cell membrane receptors [10] even in fluidic conditions [11]. The effect of nanoparticles as radiosensitizers has been extensively reviewed [12–16], and their importance as therapeutic agents has been steadily rising with time. It has been described that high atomic number (Z) materials absorb more energy per unit mass than water when irradiated with X-rays [16]. In the case of gold (Z = 79), it can be 100 times more effective at absorbing photon energy than water. This local absorption triggers the emission of low-energy electrons from gold with a potential increase in DNA damage. The increase in the effect of a dose when it is delivered in the presence of gold nanoparticles (GNPs) is called “sensitization enhancement ratio” (SER). So, if GNPs can accumulate in specific tissues, it opens the door to a differential enhancement in tumoral tissues [13], allowing for lower radiation doses to have the same effect as higher ones [17,18]. However, it has been observed that GNPs induce higher SER than could be expected according only to their physical conditions [16,19,20]. This means that several factors influence the radiosensitization effect of GNPs. In relation to energy deposition, low-energy X-rays produce a higher enhancement than high-energy X-rays [21,22] and, the higher the diameter of GNPs, the lower the deposition of energy and the emission of low-energy electrons [23]. On the other hand, the radiosensitizing effect of GNPs is, partially, triggered by an increase in the formation of reactive oxygen species (ROS) when compared with cells irradiated in the absence of GNPs. ROS can react with DNA, inducing double strand breaks and affecting the cell viability. Moreover, without irradiation GNPs also increase the oxidative stress of the cells by interfering in the activity of some antioxidant enzymes [24–27]. Because ROS have a limited lifespan, it seems reasonable that a greater radiosensitizing effect is expected if GNPs are inside the cell. A higher cell uptake of 50 nm diameter GNPs when compared to 14 nm or 74 nm GNPs has been described [22]. Considering all this information, to take advantage of the radiosensitizing effect of GNPs in radiotherapy, we think that an effective targeting system could be of great help. The aim of the present study was to evaluate the radiosensitizing effect of 50 nm spherical GNPs specifically functionalized to interact with cell membrane receptors (EpCAM and transferrin receptor) of two different cell lines, Caco2 (colon adenocarcinoma) and SKBR3 (breast adenocarcinoma), respectively. Caco2 cells have been described as highly radioresistant due to a low 53BP1 expression [28–30]. SKBR3 cells showed a survival fraction and a mean inactivation dose, after irradiation, close to the mean of eight breast cancer cell lines [31], but other studies described a lower intrinsic sensitivity to radiation due to the overexpression of HER2 [32,33]. Radiosensitizing effect was evaluated by means of DNA damage by analyzing radiation-induced γ-H2AX foci and cell viability. Nanoparticle internalization was also assessed using confocal microscopy. Biology 2022, 11, 1193 3 of 17 2. Materials and Methods 2.1. Cell Lines In the present study, two different cell lines were used: Caco2 derived from colon adenocarcinoma cells (ATCC, Manassas, VA, USA) and SKBR3 derived from breast ade- nocarcinoma cells (ATCC). Both cell lines were cultured with Eagle’s modified enriched medium (EMEM, ATCC) and McCoy’s 5A medium (ATCC), respectively, at 37 ◦ C and in 5% CO2 conditions. Doubling times were determined by counting stained cells with trypan blue (Sigma-Aldrich, Madrid, Spain) and visualizing by optical microscopy with the help of a hemocytometer (Sigma-Aldrich). Doubling times were 68 h for Caco2 cells and 63 h for SKBR3 cells. 2.2. Receptor Expression To ensure that both cell lines behaved as expected and expressed the specific receptors (EpCam and TfR), an immunofluorescence staining was carried out. The primary antibodies used were Rabbit Anti-EpCam (cat. ab71916, Abcam, Cambridge, UK) and Mouse Anti- TfR (cat. ab9179, Abcam), and the secondary antibodies were Goat Anti-Rabbit IgG H&L Alexa Fluor® 488 (cat. ab150077, Abcam) and Goat Anti-Mouse IgG H&L Alexa Fluor® 488 (cat. ab150113, Abcam). First, cells were centrifuged and rinsed with 1 × PBS, then 35.000 cells were centrifuged using a Cytospin centrifuge (Cytospin 3, Shandon, Thermo Scientific, Madrid, Spain) and fixed to a polysine slide by applying 2% paraformaldehyde onto the sample. After 10 min, the slides were rinsed in 1 × PBS, and then fixed cells were permeabilized using 1 × PBS/0.5% Triton-100 at 4 ◦ C. After 15 min, slides were again rinsed with 1 × PBS before applying a blocking solution (1 × PBS/0.1% Tween20/2% FCS) for 30 min. Then, cells were incubated with the primary antibody overnight in a humid chamber at 4 ◦ C. Once incubation was complete, the slides were washed thrice for 5 min in a 1 × PBS/0.1% Tween20 solution to eliminate a possible primary antibody excess, then the secondary antibody was applied, and the slides were incubated for 1 h in a humid chamber at room temperature. After that, the slides were washed in 1 × PBS/0.1% Tween20 solution as previously described. Finally, 5 µL of Vectashield Mounting Medium (Vector Laboratories, Barcelona, Spain) with 4,6-diamidino-2-phenylindole (DAPI) as counterstain at a 1.5 µg·mL−1 concentration was applied. 2.3. Cytogenetic Characterization To know the genomic stability of the cell lines used in the present study, first we analyzed the karyotypes of one hundred cells for each cell line. Fluorescence in situ hy- bridization techniques using pancentromeric and pantelomeric PNA probes were used to establish the modal karyotypes and to check for the presence of unstable chromosome aber- rations. For this purpose, exponentially growing cell cultures were treated with colcemid at a final concentration of 0.3 µg·mL−1 for 4 h. Then, cultures were centrifuged and treated with a hypotonic solution (KCl 0.30 M) for 10 min, then cells were fixed with Carnoy’s solu- tion (1 acetic acid:3 ethanol) and dropped onto slides. For the in situ hybridization, slides were firstly treated with pepsin/HCl (Sigma-Aldrich) (50 µg·mL−1 ) for 10 min at room temperature, washed twice with 1 × PBS for 5 min, fixed with formaldehyde 4%–PBS1×, and washed again with PBS (3 times, 5 min each). After that, slides were dehydrated in ethanol series (70%, 85%, and 100%, 2 min each) and air-dried. Then, hybridization with FITC-labeled pancentromeric and Cy3-labeled pantelomeric PNA probes (Applied Biosystems, Foster City, CA, USA) was performed. Shortly after, 10 µL of hybridizing solution with both PNA probes, 0.6 µM of the pancentromeric probe, and 0.4 µM of the pantelomeric probe were applied to the slides and covered with a coverslip. Cells and probes were denatured for 3 min at 80 ◦ C and hybridized for 2 h at room temperature using a Vysis HYBrite Hybridization System (Abbott, Abbott Park, IL, USA). After hybridization, slides were washed twice, 15 min each, in 70% formamide–PBS1× and three times, 5 min each, in Tris-buffered saline solution, 1 × TBS/5% Tween20 (both from Sigma-Aldrich). Slides were then dehydrated with ethanol series and dried out, and 20 µL of the previously Biology 2022, 11, 1193 4 of 17 described DAPI solution were applied as counterstain and slides were observed on an epifluorescence microscope (AxioImager.Z2, Zeiss, Oberkochen, Germany). 2.4. Nanoparticles and Internalization In the present study, three types of GNPs were used. Citric acid stabilized 50 nm GNPs (cat. BG-50, CD Bioparticles, Shirley, NY, USA), from now on, non-functionalized gold nanoparticles (NF-GNPs), commercially Tf functionalized gold nanoparticles (TfGNPs) (cat. GCT-50, CD Bioparticles) and anti-EpCam antibody functionalized gold particles (AntiEpCamGNPs) functionalized with a conjugation kit (cat. GCK-50, CD Bioparticles) ac- cording to the manufacturer’s protocol. To measure the ζ-potential, functionalized and non- functionalized GNPs were separately resuspended in water or in EMEM (anti-EpCamGNP) and McCoy’s 5A (TfGNP) culture mediums and sonicated for 5 min (Fisherbrand FB15047, Fisher Scientific, Germany) to achieve a monodispersed sample. The ζ-potential was then measured with a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Cell cultures of both cell lines were treated with 50 nm NF-GNPs. Moreover, Caco2 and SKBR3 cell cultures were treated with AntiEpCamGNPs and TfGNPs, respectively. In all cases, functionalized and non-functionalized GNPs, diluted in 1 × PBS, were added to the cell cultures at a final concentration of 7 × 105 nanoparticles·mL−1 . Nanoparticle internalization was evaluated 24 h after treatment of the cell cultures with a broadband confocal microscope (Leica TCS SP5, Leica Microsystems, Wetzlar, Germany) located at the Microscopy Service of the Universitat Autònoma de Barcelona. CellMask™ Deep Red (cat. C10046, Thermofisher, Waltham, MA, USA) was used to stain cell membranes and Hoechst 33342 (cat. H3570, Thermofisher) to stain cell nuclei. Staining was applied 5 min prior to observation. Due to their reflective nature, no additional staining was required to visualize GNPs. 2.5. Irradiation Cell cultures were irradiated with gamma-rays 24 h after incubation in the absence or in the presence of nanoparticles (NF-GNPs for both cell lines and TfGNPs for SKBR3 cells and AntiEpCamGNPs for Caco2 cells). Irradiations were carried out at 3 Gy with a 137 Cs irradiator (IBL437C, CIS Biointernational, GIF Yvette, France) located at the Technical Unit for Radiation Protection of the Universitat Autònoma de Barcelona. Dose rate was 5.02 Gy·min−1 and the energy peak was 662 keV. 2.6. γ-H2AX Foci Detection To evaluate kinetics of γ-H2AX foci, five different cultures for each cell line were used, one to determine the basal frequency and four at different postirradiation times: 30 min, 2, 4 and 24 h. At each time, three irradiations were carried out for each cell line, one without GNP treatment, another in the presence of NF-GNPs and another with functionalized GNPs (TfGNPs and AntiEpCamGNPs for SKBR3 and Caco2 cell lines, respectively). To detect γ-H2AX foci, an immunofluorescence staining was conducted following the same protocol described in the Section 2.2. In this case, using a specific primary mouse antibody for γ-H2AX (cat. ab26350, Abcam) and a goat anti-mouse Cy3 secondary antibody (cat. AP181C, Sigma-Aldrich). After this procedure, a couple of extra steps were taken to ensure proper microscopic analysis of radiation-induced foci. First, after the incubation with the secondary antibody, samples were again washed as described after the first incubation. Lastly, the slides were dehydrated with ethanol at 70%, 85% and 100% concentrations for 1 min each. Then, a DAPI counterstain was applied to the samples to allow single nucleus detection. γ-H2AX foci analyses were carried out using an automated scanning fluorescence microscope system (Metafer 4, Meta Systems, Altlussheim, Germany) and processed using the MetaCyte software, version 3.10.2, (Meta Systems, Medford, MA, USA) coupled to a motorized z-stage Zeiss Axio Imager.Z2 microscope (MetaSystems) The images were captured using a 63_PlanApo objective. The foci signals in the selected nuclei were captured using the SpOr filter (red channel). All the SpOr signals were acquired as a z-stack with a allow single nucleus detection. γ-H2AX foci analyses were carried out using an automated scanning fluorescence microscope system (Metafer 4, Meta Systems, Altlussheim, Germany) and processed us- ing the MetaCyte software, version 3.10.2, (Meta Systems, Medford, MA, USA) coupled to a motorized z-stage Zeiss Axio Imager.Z2 microscope (MetaSystems) The images were Biology 2022, 11, 1193 captured using a 63_PlanApo objective. The foci signals in the selected nuclei were5 of 17 cap- tured using the SpOr filter (red channel). All the SpOr signals were acquired as a z-stack with a total of 10 focal planes and a z-step size of 0.35 μm between planes. A unique clas- total sifierofwas 10 focal usedplanes to countandfoci a z-step size2000 in about of 0.35 nuclei between µm for planes. A unique each experimental classifier was condition. used to count foci in about 2000 nuclei for each experimental condition. 2.7. MTT Cell Viability Assay 2.7. MTT Cell Viability Assay Cells were seeded at 4 × 104 cells·mL−1 concentration on a 96-well plate and cultured Cells were seeded at 4 × 10 4 cells·mL−1 concentration on a 96-well plate and cul- for 48 h before being treated with NF-GNPs, functionalized GNPs (TfGNPs and An- tured for 48 h before tiEpCamGNPs beingand for SKBR3 treated Caco2with cellNF-GNPs, functionalized lines, respectively), or notGNPs (TfGNPs treated at all inand the AntiEpCamGNPs for SKBR3 and Caco2 cell lines, respectively), or not treated case of our control cells. Twenty-four hours after GNP treatment, the culture medium was at all in the case of our control cells. Twenty-four hours after GNP treatment, the culture changed and then cells were irradiated at a 3 Gy dose. To determine the impact of treat- medium was mentschanged on cell and then acells viability, were irradiated at a 3 Gy dose. To determine thebromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium impact of te- treatments trazolium reduction assay (MTT cell viability assay) was conducted following bromide on cell viability, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium standard tetrazolium procedures reduction 24 h after assay (MTT(cat. irradiation cell viability assay) was conducted M2128, Sigma-Aldrich). following Viability standard was calculated in procedures 24 h after irradiation (cat. M2128, Sigma-Aldrich). Viability was calculated in relation to that observed in non-treated cells. Three replicates were carried out for each relation to that observed in non-treated cells. Three replicates were carried out for each experimental condition. experimental condition. 2.8.Statistical 2.8. StatisticalAnalysis Analysis Statisticalanalysis Statistical analysiswas wasconducted conductedwith withIBMIBMSPSS SPSSStatistics Statistics24. 24.Normality Normalitywaswastested tested in all cases using a Kolmogorov–Smirnov test with the Lilliefors correction. Sincecases in all cases using a Kolmogorov–Smirnov test with the Lilliefors correction. Since all all showed cases a non-normal showed distribution, a non-normal the statistical distribution, test chosen the statistical to compare test chosen foci frequencies to compare foci fre- was the Mann–Whitney quencies U test. Viability was the Mann–Whitney U test.comparisons were carriedwere Viability comparisons out with ANOVA carried with out with Tukey’s multiple comparison test. p values lower than 0.05 were considered ANOVA with Tukey’s multiple comparison test. p values lower than 0.05 were considered statistically significant.significant. statistically 3.3.Results Results 3.1. 3.1.Receptor ReceptorExpression Expression As Asexpected, expected,both bothcell celllines linesexpressed expressedthethecell cellmembrane membranereceptors receptors(EpCam (EpCamforforCaco2 Caco2 and andTfR TfRfor forSKBR3) SKBR3)described describedin inthe theliterature literature(Figure (Figure1), 1),confirming confirmingthe thevalidity validityof ofour our choice choicefor fornanoparticle nanoparticlefunctionalization. functionalization. Figure1.1.Caco2 Figure Caco2cells cellsexpressing expressingthe theEpCam EpCam(A)(A)and andSKBR3 SKBR3cells cellsexpressing expressing the the TfR TfR (B) (B)receptors. receptors. In both cases, the nuclei are marked in blue (DAPI) and the cell membrane receptors are marked in In both cases, the nuclei are marked in blue (DAPI) and the cell membrane receptors are marked in green (Alexa Fluor® 488, Abcam). green (Alexa Fluor® 488, Abcam). 3.2. Cytogenetic Characterization The cytogenetic analysis by pancentromeric and pantelomeric fluorescence in situ hybridization (Figure 2) was carried out to know the modal karyotype of each cell line and if they show structural chromosome instability that could influence the further results with the foci analysis. The analyses were performed on 100 cells of each of the cell lines. Caco2 cells showed two modal karyotypes of 58 and 86 chromosomes whilst SKBR3 showed a modal karyotype of 76 chromosomes (Figure 3). Only one Caco2 cell showed a dicentric and if they show structural The cytogenetic chromosome analysis instability by pancentromeric andthat could influence pantelomeric the further fluorescence in situresults with the foci analysis. hybridization The (Figure 2) analyses was carried were out toperformed know the modalon 100karyotype cells of each of the of each cellcell linelines. Caco2 and if cells showed they show two modal structural karyotypes chromosome of 58 instability thatand could86influence chromosomes whilst the further resultsSKBR3 with the showed a foci modal analysis. The analyses karyotype were performed of 76 chromosomes on 100 (Figure 3).cells Only ofone eachCaco2 of the cell cell showed lines. a Caco2 cells showed two modal karyotypes of 58 and 86 chromosomes dicentric chromosome plus an acentric fragment, all the other chromosomes in all ana- whilst SKBR3 Biology 2022, 11, 1193 showed lyzed cellsa showed modal karyotype of 76 chromosomes one centromere (Figure signals. and two telomere 3). Only Inonethe Caco2 casecell showedall of SKBR3, a 6 the of 17 dicentric chromosome plus an acentric fragment, all the other chromosomes cells showed a stable dicentric chromosome, which is consistent with the data provided in all ana- bylyzed cells showed one the manufacturer. Nocentromere cell showedandanother two telomere signals. structural In the case of chromosome SKBR3, alldetected aberration the cells showed a stable dicentric chromosome, which is consistent with the data provided by pancentromeric chromosome plus and pantelomeric an acentric labeling. fragment, all the other chromosomes in all analyzed cells by the manufacturer. No cell showed another structural chromosome aberration detected Theseone showed results indicateand centromere that two instead of the signals. telomere variability Ininthe thecase number of chromosomes, of SKBR3, all the cells by pancentromeric and pantelomeric labeling. the cellThese showed lines are a stable stable regarding the formation of unstable structural chromosome results indicate that instead of the variability in the number of chromosomes, by dicentric chromosome, which is consistent with the data provided aber- rations. the the cell lines are stable regarding the formation of unstable structural chromosome aber- by manufacturer. No cell showed another structural chromosome aberration detected pancentromeric rations. and pantelomeric labeling. Figure Figure2.2.Pancentromeric–pantelomeric fluorescenceinin Pancentromeric–pantelomeric fluorescence situ situ hybridization-stained hybridization-stained metaphases metaphases of of Figure 2. Pancentromeric–pantelomeric fluorescence in situ hybridization-stained metaphases of Caco2 Caco2(A)(A)and andSKBR3 SKBR3 (B) (B) cells. Greensignals cells. Green signalscorrespond correspond to to centromeres, centromeres, labeled labeled withwith FITC;FITC; and and Caco2 red (A) and signals SKBR3 (B)labeled cells. GreenCy3. signals correspond to centromeres, labeled with FITC; and red red signals tototelomeres, telomeres, labeled with with Cy3. signals to telomeres, labeled with Cy3. Figure 3. Violin plot showing chromosome distribution of both cell lines. The mean number of chro- mosomes for Caco2 was 72, although two modal karyotypes of 58 and 86 chromosomes were ob- Violin Figure3.3.Violin Figure plot plot showing showing chromosome chromosome distribution distribution of both cell lines. The number mean number of chro-of served. A unique modal karyotype of 76 chromosomes forofSKBR3 both cell waslines. The mean observed. mosomes for Caco2 chromosomes was 72, for Caco2 wasalthough two modal 72, although karyotypes two modal of 58 of karyotypes and5886 chromosomes and werewere 86 chromosomes ob- served. A unique observed. modal A unique karyotype modal of 76 karyotype ofchromosomes for SKBR3 76 chromosomes waswas for SKBR3 observed. observed. These results indicate that instead of the variability in the number of chromosomes, the cell lines are stable regarding the formation of unstable structural chromosome aberrations. 3.3. Nanoparticle Internalization In Figure 4 are shown the ζ-potentials of non-functionalized (NF-GNP) and function- alized GNPs (TfGNP and AntiEpCamGNP), measured in water and in culture media. As can be seen, in water NF-GNPs showed the most electronegative ζ-potential, a difference that disappeared in the culture medium where the ζ-potentials were less electronegative than in water. 3.3. Nanoparticle Internalization In Figure 4 are shown the ζ-potentials of non-functionalized (NF-GNP) and function- alized GNPs (TfGNP and AntiEpCamGNP), measured in water and in culture media. As can be seen, in water NF-GNPs showed the most electronegative ζ-potential, a difference Biology 2022, 11, 1193 7 of 17 that disappeared in the culture medium where the ζ-potentials were less electronegative than in water. Figure 4. Figure 4. ζ-potential ζ-potentialofofnon-functionalized non-functionalized(NF-GNP) and (NF-GNP) functionalized and GNPs functionalized (TfGNP GNPs and and (TfGNP An- tiEpCamGNP), measured in water and in EMEM (AntiEpCamGNP) and McCoy’s 5A (TfGNP) cul- AntiEpCamGNP), measured in water and in EMEM (AntiEpCamGNP) and McCoy’s 5A (TfGNP) ture media. culture media. Aqualitative A qualitativeassessment assessmentofofGNP GNPinternalization internalization through through confocal confocal microscopy microscopy imag- imaging ing allowed us to detect certain differences between the cell lines (Figures allowed us to detect certain differences between the cell lines (Figures 5 and 6). As can 5 and 6). As can be seen, for both cell lines the internalization of functionalized GNPs be seen, for both cell lines the internalization of functionalized GNPs (TfGNP for SKBR3 (TfGNP for SKBR3 andAntiEpCamGNP and AntiEpCamGNPfor forCaco2 Caco2cells) cells)isismore morevisible visiblethan thanthat thatfor forNF-GNPs. NF-GNPs. Moreover, Moreover,itit seems that seems thatSKBR3 SKBR3cells cellsshowed showedmoremoreinternalized internalizedfunctionalized functionalized GNPs GNPs than thanCaco2 Caco2cells cells (Figure6). (Figure 6).Finally, Finally,GNP GNPaggregation aggregationwas wasclearly clearlyvisible visibleforforTfGNP TfGNPin inSKBR3 SKBR3cells. cells.Despite Despite Biology 2022, 11, x FOR PEER REVIEWthe different the different degrees degrees ofof internalization internalization between between cell cell lines, lines, both both showed showed aa common 8common of 18 trait, trait, that is, the inability of GNPs to enter the nucleus, thus remaining in that is, the inability of GNPs to enter the nucleus, thus remaining in the cytoplasm. the cytoplasm. Figure 5. Figure 5.Confocal Confocalmicroscopy microscopycaptures of Caco2 captures cells.cells. of Caco2 The images show cells The images showtreated cellswith NF-GNPs treated with NF-GNPs (left) and cells treated with anti-EpCam-functionalized GNPs (right). Nuclei (A) are marked blue (left) and cells treated with anti-EpCam-functionalized GNPs (right). Nuclei (Hoechst 33342), plasma membranes (C) are marked red (CellMask™ Deep Red). Nanoparticles (B) (A) are marked blue (Hoechst 33342), are visualized plasma as green dotsmembranes (C) are marked due to their reflection red (CellMask™ being artificially assigned aDeep green Red). color byNanoparticles the microscope’s (B) software. are visualized Merged as green images dots due(D), and their to their orthogonal reflection beingprojections of assigned artificially the z-stacka recon- green color by structions the (right andsoftware. microscope’s bottom), where GNPs Merged (white (D), images arrows) andcan be seen. their orthogonal projections of the z-stack reconstructions (right and bottom), where GNPs (white arrows) can be seen. Figure 5. Confocal microscopy captures of Caco2 cells. The images show cells treated with NF-GNPs (left) and cells treated with anti-EpCam-functionalized GNPs (right). Nuclei (A) are marked blue (Hoechst 33342), plasma membranes (C) are marked red (CellMask™ Deep Red). Nanoparticles (B) Biology 2022, 11, 1193 are visualized as green dots due to their reflection being artificially assigned a green color by the 8 of 17 microscope’s software. Merged images (D), and their orthogonal projections of the z-stack recon- structions (right and bottom), where GNPs (white arrows) can be seen. Figure 6. Confocal microscopy captures of SKBR3 cells. The images show cells treated with NF- Figure 6. Confocal microscopy captures of SKBR3 cells. The images show cells treated with NF- GNPs (left) and cells treated with Tf-functionalized GNPs (right). Nuclei (A) are marked blue GNPs (Hoechst(left) andcytoplasmatic 33342), cells treatedmembranes with Tf-functionalized (C) are marked GNPs (right). Deep red (CellMask™ Nuclei (A)Nanopar- Red). are marked blue (Hoechst 33342), ticles (B) are cytoplasmatic visualized membranes as green dots (C)reflection due to their are marked redartificially being (CellMask™ Deepa green assigned Red). color Nanoparticles by the (B) are microscope’s visualized as software. Merged green dots dueimages (D), to their and their being reflection orthogonal projections artificially of theaz-stack assigned green color by reconstructions (right and bottom), where GNPs (white arrows) can be seen. the microscope’s software. Merged images (D), and their orthogonal projections of the z-stack reconstructions 3.4. γ-H2AX Foci (right and bottom), Induction where GNPs (white arrows) can be seen. and Kinetics The cytogenetic characterization did not show structural chromosome aberrations, 3.4. γ-H2AX Foci Induction and Kinetics but due to the variability in the number of chromosomes, about 2000 cells were analyzed Thedifferent for each cytogenetic characterization postirradiation did not time and GNP show structural treatment. Microscopechromosome images of γ-H2AX aberrations, but foci in Caco2 and SKBR3 cells are shown in Figure 7. In Caco2 cells (Figure 8), the higheranalyzed due to the variability in the number of chromosomes, about 2000 cells were for each different frequencies of foci postirradiation were observed 30 time minand GNP after treatment. irradiation Microscope for all treatments:images 64.3 ± 0.7of γ-H2AX foci in Caco2 and SKBR3 cells are shown in Figure 7. In Caco2 cells (Figure 8), the higher (mean ± SEM) without GNPs, 54.2 ± 0.8 with NF-GNPs, and 70.6 ± 0.7 with An- tiEpCamGNPs. frequencies As postirradiation of foci were observedtime went 30 min on, irradiation after the frequencies of foci for all decreased64.3 treatments: to sim- ± 0.7 (mean ilar values to unirradiated cells. At any postirradiation time, the frequencies ± SEM) without GNPs, 54.2 ± 0.8 with NF-GNPs, and 70.6 ± 0.7 with AntiEpCamGNPs. Biology 2022, 11, x FOR PEER REVIEW of foci 9 ofin18cells As postirradiation time went on, the frequencies of foci decreased to similar values to unirradiated cells. At any postirradiation time, the frequencies of foci in cells irradiated in the presence irradiated of AntiEpCamGNPs in the presence were of AntiEpCamGNPs weresignificantly higher significantly higher thanthan for for the the other two other two treatments treatments (p (p < 0.001). < 0.001). Figure 7. Nuclei from Caco2 (A) and SKBR3 (B) cells, stained with DAPI (blue), containing γ-H2AX Figure 7. Nuclei from Caco2 (A) and SKBR3 (B) cells, stained with DAPI (blue), containing γ-H2AX foci immunostained with cyanine 3 (Cy3, red). For both cell lines, nuclei with high (left) and low foci immunostained (right) with The foci counts are shown. cyanine images3 correspond (Cy3, red).toFor bothprepared samples cell lines, nuclei 30 min with (high foci high count)(left) and low (right) and foci counts 4 h (low areafter foci count) shown. The images irradiation, treated correspond to samples prepared with anti-EpCam-functionalized GNPs30(Caco2) min (high or foci count) transferrin-functionalized GNPs (SKBR3). and 4 h (low foci count) after irradiation, treated with anti-EpCam-functionalized GNPs (Caco2) or transferrin-functionalized GNPs (SKBR3). Figure 7. Nuclei from Caco2 (A) and SKBR3 (B) cells, stained with DAPI (blue), containing γ-H2AX foci immunostained with cyanine 3 (Cy3, red). For both cell lines, nuclei with high (left) and low (right) foci counts are shown. The images correspond to samples prepared 30 min (high foci count) Biology 2022, 11, 1193 9 of 17 and 4 h (low foci count) after irradiation, treated with anti-EpCam-functionalized GNPs (Caco2) or transferrin-functionalized GNPs (SKBR3). Figure 8. Box plots of the number of foci per cell in Caco2 cells before (basal) and at different times Figure 8. Box plots of(30 themin, number of 24 2, 4 and foci h) per aftercell in Caco2 irradiation, in cells beforeof(basal) the absence GNPs and at different (Without GNP) or times in the presence (30 min, 2, 4 and 24 h)ofafter irradiation, inGNPs non-functionalized the absence (NF-GNP)of GNPs (Without GNP) or in the or anti-EpCam-functionalized GNPspresence of (AntiEpCamGNP). non-functionalized GNPs (NF-GNP) Horizontal or anti-EpCam-functionalized lines inside boxes indicate the median. GNPs (AntiEpCamGNP). Hor- izontal lines inside boxes indicate the median. For SKBR3 (Figure 9), the differences between treatments were even more noticeable. Cells irradiated in the absence of GNPs or in the presence of NF-GNPs showed a low foci For SKBR3 (Figure 9), the differences between treatments were even more noticeable. frequency until two hours after irradiation (0.3 ± 1.0 and 1.3 ± 1.0 at 2 h, respectively). Then, Cells irradiated in the absence of GNPs or in the presence of NF-GNPs showed a low foci an increase was observed at 4 h after irradiation (9.1 ± 0.5 and 17.4 ± 0.8, respectively), frequency until two hours by followed after irradiation a decrease (0.3 to basal ± 1.0 levels 24 and 1.3irradiation. h after ± 1.0 at 2However, h, respectively). for cells irradiated OR PEER REVIEW Then, an increase was in theobserved presence of atTfGNPs, 4 h after theirradiation frequency of(9.1 foci±was0.5 maximum and 17.4 30 ± 0.8, 10 of irradiation, min respec- after 18 tively), followed by36,1 ± 0.7, followed a decrease by a progressive to basal levels 24 decrease with postirradiation h after irradiation. However,time but fortocells values signif- icantly higher than the basal ones at 24 h after irradiation, irradiated in the presence of TfGNPs, the frequency of foci was maximum 30 min after 8.9 ± 0.8. At all postirradiation times, the frequency of foci was significantly higher for cells irradiated in the presence of irradiation, the presence36,1 ± 0.7, followed of TfGNPs (p < 0.01).byTable a progressive 1 shows the decrease with postirradiation sensitization enhancement time ratio but (SER)to TfGNPs (p < 0.01). Table 1 shows the sensitization enhancement ratio (SER) for foci. As can values significantly for foci. As can be seen,higher be seen, than cells the cellsirradiatedbasal irradiated in ones inthe at thepresence 24 presence h after irradiation, of functionalized of functionalized 8.9 ± 0.8. particles particles At all (either (either AntiEpCamGNPs postirradiation times, AntiEpCamGNPs or the TfGNPs)frequency showed of foci ratioswas significantly higher than one or TfGNPs) showed ratios higher than one in all cases. higher in all for cases.cells irradiated in Figure 9. Box plots of the number of foci per cell in SKBR3 cells before (basal) and at different times Figure 9. Box plots of(30 themin, number of24 2, 4 and foci per cell h) after in SKBR3 irradiation, cells in the before absence of (basal) and at GNP) GNPs (Without different or intimes the presence of (30 min, 2, 4 and 24 h)non-functionalized after irradiation,GNPs in the(NF-GNP) absence oroftransferrin-functionalized GNPs (Without GNP) or in the GNPs presence (TfGNP). of Horizontal lines non-functionalized GNPsinside(NF-GNP) boxes indicateor the transferrin-functionalized median. GNPs (TfGNP). Horizontal lines inside boxes indicate the median. Table 1. Sensitization enhancement ratio (SER) for foci frequencies and viability. Caco2 and SKBR3 cells were irradiated at 3Gy in the presence of functionalized GNPs (AntiEpCamGNP and TfGNP, respectively) and results were compared with those after irradiation in the absence of GNPs (w/o GNP) or in the presence of non-functionalized GNPs (NF-GNP). Biology 2022, 11, 1193 10 of 17 Table 1. Sensitization enhancement ratio (SER) for foci frequencies and viability. Caco2 and SKBR3 cells were irradiated at 3Gy in the presence of functionalized GNPs (AntiEpCamGNP and TfGNP, respectively) and results were compared with those after irradiation in the absence of GNPs (w/o GNP) or in the presence of non-functionalized GNPs (NF-GNP). AntiEpCamGNP vs. TfGNP vs. w/o GNP NF-GNP w/o GNP NF-GNP Postirradiation Time Foci SER Foci SER 30 min 1.10 1.30 - - 2h 1.46 1.55 - - 4h 1.20 1.18 2.05 1.07 24 h 1.87 1.18 4.98 22.57 Viability SER Viability SER 1.11 1.07 1.12 1.02 To know if the presence of GNPs could have any effect on the rate of foci disappearance with postirradiation time, foci decay with postirradiation time has been adjusted to a one-phase decay function, obtaining the constant of the decay rate and the foci half-life estimation for each experimental condition (Table 2). For Caco2 cells, the decay rate constant gradually decreases from cells irradiated without GNPs, to cells treated with non-functionalized GNPs and in cells treated with AntiEpCamGNPs. Moreover, the foci half-life was higher for cells irradiated in the presence of AntiEpCamGNPs. For the SKBR3 cells, because of the behavior in foci kinetics, this approach was only possible for cells irradiated in the presence of TfGNPs. Table 2. Constant of the decay rate (K) and foci half-life for cells irradiated at 3 Gy in the three experimental conditions, in the absence of GNPs (w/o GNPs), in the presence of non-functionalized GNPs (NF-GNP) and in the presence of specifically targeted GNPs (AntiEpCamGNP and TfGNP for Caco2 and SKBR3 cells, respectively). Irradiation Conditions K (h−1 ) Foci Half-Life (h) w/o GNPs 0.35 2.00 CaCo2 NFGNP 0.32 2.17 AntiEpCamGNP 0.28 2.44 w/o GNPs - - SKBR3 NFGNP - - TfGNP 0.30 2.31 3.5. MTT Cell Viability Assay Cell viability was measured by MTT analysis 24 h after irradiation (Figure 10). For both cell lines, without irradiation there were no significant differences in viability between cells grown without GNPs and cells grown with NF-GNPs or functionalized GNPs. However, for both cell lines the highest viability was observed for cells grown without GNPs. As expected, after irradiation the viability decreased significantly in all cases (p < 0.05). Moreover, for both cell lines the decrease in viability was more pronounced for cells irradiated in the presence of functionalized GNPs but was only significant for Caco2 cells (p < 0.05). After irradiation, SKBR3 cells always showed significantly lower viabilities than Caco2 cells (p < 0.05). The sensitization enhancement ratio for viability was also higher than one for functionalized particles (Table 1). GNPs. As expected, after irradiation the viability decreased significantly in all cases (p < 0.05). Moreover, for both cell lines the decrease in viability was more pronounced for cells irradiated in the presence of functionalized GNPs but was only significant for Caco2 cells (p < 0.05). After irradiation, SKBR3 cells always showed significantly lower viabilities than Biology 2022, 11, 1193 Caco2 cells (p < 0.05). The sensitization enhancement ratio for viability was also higher 11 of 17 than one for functionalized particles (Table 1). Figure10. Figure Viabilityrates 10.Viability ratesofofCaco2 Caco2(A)(A)and andSKBR3 SKBR3(B) (B)cells, cells,left, left,non-irradiated non-irradiatedand andright, right,24 24hhafter after irradiation.From irradiation. Fromleft lefttotoright, right,the theboxes boxesmark markthe theviability viabilityof ofcells cellsin inthe theabsence absenceofofGNPs GNPs(w/o (w/oGNP), GNP), ininthe thepresence presenceofofnon-functionalized non-functionalizedGNPs GNPs(NF-GNP) (NF-GNP)and andininthe thepresence presenceofoffunctionalized functionalizedGNPs GNPs (AntiEpCam-GNP (AntiEpCam-GNPand andTf-GNP, Tf-GNP, respectively). respectively). 4. Discussion The interest in improving radiotherapeutic treatments has led, in recent decades, to the development of new irradiation strategies focused on a more precise dose delivery to tumoral cells but also to radiosensitize tumoral tissues. One of these is the use of GNPs as a radiosensitizer agent, that was first evidenced experimentally in vivo by Hainfeld and collaborators [34]. In the present study, our aim was to evaluate the radiosensitizing effect of 50 nm GNPs specifically functionalized to be actively targeted to two tumoral cell lines. To measure the radiosensitizing effect of GNPs, different approaches such as viability and cytogenetic assays can be used. However, it should be noted that the sensitization enhancement ratio (SER) [16,35] can differ within the methodologies used, as had been pre- viously described when the relative biological effectiveness of X-rays of different energies was checked [36–47]. The clonogenic assay has been used to measure the radioinduced cell death or loss of reproductive potential, though it has limitations such as the need for cell differentiation and the intrinsic variations in survivability between different cell types [48,49]. On the other hand, the cytogenetic approaches to the study of radiosensitivity are well known [50], with chromosomal damage and translocations being markers to test for radiation toxicity [51]. In the present study, we have used two methodologies to evaluate the radiosensitizing effect of GNPs: γ-H2AX foci detection as a genetic damage indicator and MTT as a cell viability assay. γ-H2AX is the phosphorylated variant of histone H2AX, forming discrete accumulations (foci) around the double strand breaks (DSBs) induced by the exposure to ionizing radiation [52–54]. γ-H2AX foci detection is a widely used technique for ra- diosensitivity assessment [55] because γ-H2AX foci can be detected almost immediately after the irradiation exposure, while other biomarkers such as chromosome aberrations require the cell to achieve the mitosis phase, thus yielding results only when the DNA repair mechanisms have already acted. Moreover, γ-H2AX foci can also be detected at different postirradiation time points to obtain a view on the kinetics of foci appearance and repair. Regarding the MTT cell viability assay, it allows a quick and easy sampling and quantification by spectrophotometry [56]. In our study, working with two different tumoral cell lines has helped us to achieve a better knowledge of the nanoparticles’ radiosensitizing effect in different cell models. First, Biology 2022, 11, 1193 12 of 17 to know if the cells used were genetically unstable, we evaluated the chromosome stability of both cell lines, and although the chromosome number was variable in both cell lines, no cells with chromosomes lacking telomere signals were observed, indicating no structural chromosome instability and hence no influence in the posterior foci counts. The radiosensitizing effect of GNPs is attributed, in part, to the increase in ROS production with irradiation, but in the absence of radiation, GNPs increase the intracellular ROS level [24,25,57,58]. It is known that ROS have a limited lifespan, so the radiosensitizing effect of GNPs should be more effective if nanoparticles are internalized by the cell. Our confocal microscope observations indicate that all GNPs are taken up by the cells, but the images obtained seem to indicate that functionalized GNPs (TfGNPs and AntiEpCamGNPs) were more effectively internalized by SKBR3 and Caco2 cells, respectively. It is well known that the surface charges of nanoparticles influence their internalization by the cells, with highly charged nanoparticles (positively or negatively) being taken up more by cells than neutral ones. It has also been described that functionalization of polystyrene macroparticles to make them positively charged significantly increases their internalization by HeLa cells [59]. We observed that the ζ-potential of all GNPs (functionalized and non-functionalized) becomes less electronegative when the ζ-potential was measured in culture medium, unless in water. Moreover, the differences observed in water disappeared in cell culture medium. This could be due to a protein corona formation, with serum proteins, when particles are in culture medium. This modification has been suggested to be responsible for the decrease in particle uptake by the cells [60,61], but it has also been described that the impact can vary between cell types [62]. In any case, for an accurate quantitative assessment of GNP uptake by the cells, alternative methods such as those based on inductively coupled plasma mass spectrometry (ICP-MS) should be applied. In our study, we chose nanoparticles of 50 nm in diameter because previous studies indicated an increased cell uptake for GNPs of 50 nm in diameter in relation to 14 nm and 74 nm [63] and it has been correlated with a higher radiosensitization effect [22]. On the other hand, it seems that smaller nanoparticles allow the deposition of high doses in their vicinity than bigger nanoparticles because of the increase in the surface to volume ratio [23]. However, because different studies have been carried out with different nanoparticle sizes, it is difficult to establish a unique optimum size of nanoparticles to increase cell uptake. Another factor, different to nanoparticles properties, to consider in studies of GNP radiosensitizing effect, is the intrinsic uptake capacities and intrinsic radiosensitivity of the cell lines. Not all cell types show a similar nano- or microparticle uptake capac- ity. SKBR3 cells can phagocytose 3 µm polysilicon–chromium–gold microparticles [64], 3 µm polystyrene microparticles functionalized with polyethyleneimine [61] and 1 µm and 3 µm polystyrene Tf-functionalized microparticles [65] without affecting viability. Caco2 cells can uptake functionalized polystyrene nanoparticles of 50 nm in diameter, but not of 100 nm or more [66], and were also unable to uptake TiO2 nanoparticles [67]. Actively targeting using anti-EpCam antibodies is an effective method to target Caco2 cells with magnetic and gold nanoparticles functionalized with these antibodies [68,69]. Other stud- ies indicate that the capability of Caco2 cells to uptake nanoparticles depends on their differentiated status, with only the undifferentiated cells being able to phagocytose nanopar- ticles [70,71]. Considering these results, one objective of our work was to determine if actively targeted GNPs could increase the radiosensitizing effect. Regarding the effect of GNPs in SKBR3 and Caco2 cell cultures without irradiation, we observed that the presence of NF-GNPs or functionalized GNPs did not significantly change the basal frequency of γ-H2AX foci or the cell viability when compared to cells grown without GNPs. This does not mean that the presence of GNPs does not have any effect on cells, because a slight decrease in cell viability, though not statistically significant, was observed when GNPs (either non-functionalized or functionalized) were present in the cell culture. This agrees with studies describing an increase in ROS by the presence of GNPs without irradiation [24,25,57,58] that could have a slight effect on cell viability. Biology 2022, 11, 1193 13 of 17 After irradiation, the γ-H2AX foci frequency was evaluated at four different postirra- diation times (30 min, 2, 4 and 24 h). For Caco2 cells, significant increases in foci frequencies were observed for cells irradiated in the presence of AntiEpCamGNPs when compared with cells irradiated in the presence of NF-GNPs or in the absence of GNPs. For these cells, the sensitization enhancement ratios (SERs) for foci obtained for AntiEpCamGNPs when compared with NF-GNPs or with irradiation without GNPs were, in general, close to the values described in the literature (reviewed in [15,16]). Moreover, the residual DNA damage was higher when irradiation was carried out in the presence of GNPs, either func- tionalized or non-functionalized. For SKBR3, the γ-H2AX foci kinetics showed a particular behavior when compared to Caco2 cells. The maximum foci frequency in those cells treated without GNPs or with NF-GNPs was delayed to 4 h postirradiation, while for cells treated with TfGNPs the maximum frequency of foci was observed 30 min after irradiation. It is difficult to explain the delayed appearance of foci in cells irradiated without GNPs or in the presence of NF-GNPs. A delayed appearance of γ-H2AX foci in different cell lines has been described [72], usually being related to an ATM deficiency, though the exact mechanism remains unclear. Despite this particular behavior, for all postirradiation times the frequency of foci in cells irradiated in the presence of TfGNPs was significantly higher than those for cells irradiated with NF-GNPs or in the absence of GNPs. For SKBR3 cells, TfGNPs always showed an elevated radiosensitizing effect when compared with NF-GNPs or no GNPs, but in this case the SER values were too high because of the strange behavior of foci frequencies for irradiations with NF-GNPs or without GNPs. In addition, it has been described that GNPs can interfere with the DNA repair machinery [24]. In the present study, and with Caco2 cells, the half-life of foci was higher in those cells treated with GNPs, being the highest in those treated with functionalized GNPs, indicating that GNPs would have this dual effect on cells irradiated in their presence, an increase in the DNA damage production but also a reduction in the DNA repair efficiency. Anyway, the overall results indicate that actively targeting GNPs has a radiosensitizing effect in both cell lines. This result is also supported by cell viability analyses. The mean viability of Caco2 cells irradiated in the presence of AntiEpCamGNPs (79.9% of viable cells) was lower than for cells irradiated in the presence of NF-GNPs (85.3%) or in the absence of GNPs (88. 7%). For SKBR3 cells, the mean viabilities were 59% for TfGNPs, 60% for NF-GNPs and 66.1% for cells irradiated in the absence of GNPs. These results are similar to those reported with gamma-rays [21,22]. Finally, when foci frequencies of both cell lines are compared, in all experimental conditions lower frequencies were observed in SKBR3 cells. This could be related to an elevated resistance to oxidative stress, as described for cells from breast cancer patients [73]. However, instead of the low levels of foci for SKBR3 cells when compared with Caco2 cells, their viability is clearly lower, indicating that Caco2 is a very radioresistant cell line, probably because of its lower expression of 53BP1. Although high Z metallic nanoparticles are currently the most used for radiosen- sitizing, other non-metallic nanoparticles are showing promising results. In a study by Yamaguchi et al. [74], silicon oxide nanoparticles coated with polyamidoamine have yielded a significant radiosensitizing effect on SKBR3 cells, due to their overexpression of HER2. Moreover, nanoparticle functionalization with therapeutic molecules, specifically targeted to a cell type, can be a powerful tool to fight against cancer. Using GNPs, a 43-fold decrease in viability was observed when SKBR3 cells were irradiated in the presence of trastuzumab- functionalized GNPs when compared with the same cells irradiated in the presence of non-functionalized GNPs, indicating the importance of active targeting [75]. Despite the radiosensitizing effect of gold nanoparticles previously described [76], the results of the present study led us to question the universality of their application. Working with only two cell lines has already shown how much the radiosensitizing effect of GNPs can vary due to the cell lines’ intrinsic radiosensitivity but also by the capability to take up GNPs. Besides the differences between cells, the choice of targeting molecules and nanoparticle composition, size and shape are also key factors to consider in further studies. Biology 2022, 11, 1193 14 of 17 5. Conclusions Even with the differences in the results of the present study between the two cell lines, gold nanoparticles remain an interesting radiosensitizing agent and a good candidate for future in vivo application in the treatment of certain types of tumors. Active targeting of GNPs has demonstrated its effectiveness to increase the genetic damage and to decrease the cell viability. This GNP radiosensitizer effect should be explored in further studies to find more effective radiation therapies against different tumor types. Author Contributions: Conceptualization, M.Á.P.-A., L.B. and J.F.B.; methodology, M.Á.P.-A., L.B. and J.F.B.; formal analysis M.Á.P.-A.; data curation M.Á.P.-A., L.B. and J.F.B.; writing—original draft preparation, M.Á.P.-A., L.B. and J.F.B.; writing—review and editing, M.Á.P.-A., L.B., J.F.B. and G.A.; funding acquisition, J.F.B. and G.A. All authors have read and agreed to the published version of the manuscript. Authorship is limited to those who have contributed substantially to the work reported. Funding: This research was funded by the Spanish Consejo de Seguridad Nuclear (BOE-A-2019-311). G.A. and JF.B. are part of an SGR recognized by the Generalitat de Catalunya (2017SGR0255). Institutional Review Board Statement: Not applicable for studies not involving humans or animals. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: We wish to thank Jessica Martínez (Unit of Biological Anthropology, UAB), Martí de Cabo (Microscopy Service, UAB) and Aritz Lafuente (Institut Català de Nanociència i Nan- otecnologia (ICN2)) for their technical assistance, and Manuel Higueras (Department of Mathematics and Computation, Universidad de la Rioja, UR). Conflicts of Interest: The authors declare no conflict of interest. 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