nanomaterials Article Nanoceria as Safe Contrast Agents for X-ray CT Imaging Ana García 1 , Juan Antonio Cámara 2 , Ana María Boullosa 3,4 , Muriel F. Gustà 4,5 , Laura Mondragón 1,5 , Simó Schwartz, Jr. 3,4,6 , Eudald Casals 7 , Ibane Abasolo 3,4,6 , Neus G. Bastús 4,5 and Víctor Puntes 1,4,5,8, * 1 Design and Pharmacokinetics of Nanoparticles, CIBBIM-Nanomedicine, Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain; ana.garcia@vhir.org (A.G.); lmondragon@carrerasresearch.org (L.M.) 2 Preclinical Imaging Platform, Vall d’Hebron Institute of Research (VHIR), Universitat Autònoma de Barcelona (UAB), 08035 Barcelona, Spain; juanantonio.camaraserrano@ucsf.edu 3 Clinical Biochemistry, Drug Delivery & Targeting (CB-DDT), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain; ana.boullosa@vhir.org (A.M.B.); simo.schwartz@vhir.org (S.S.J.); ibane.abasolo@vhir.org (I.A.) 4 Networking Research Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 08034 Barcelona, Spain; muriel.freixanet@icn2.cat (M.F.G.); neus.bastus@icn2.cat (N.G.B.) 5 Institut Català de Nanociència i Nanotecnologia (ICN2), Consejo Superior de Investigaciones Científicas (CSIC), The Barcelona Institute of Science and Technology (BIST), 08036 Barcelona, Spain 6 Servei de Bioquímica, Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain 7 School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, China; wyuchemecm@126.com 8 Institut Català de Recerca i Estudis Avançats, (ICREA), P. Lluis Companys 23, 08010 Barcelona, Spain * Correspondence: victor.puntes@icn2.cat Abstract: Cerium oxide nanoparticles (CeO2 NPs) have exceptional catalytic properties, rendering them highly effective in removing excessive reactive oxygen species (ROS) from biological envi- ronments, which is crucial in safeguarding these environments against radiation-induced damage. Additionally, the Ce atom’s high Z number makes it an ideal candidate for utilisation as an X-ray imaging contrast agent. We herein show how the injection of albumin-stabilised 5 nm CeO2 NPs into mice revealed substantial enhancement in X-ray contrast, reaching up to a tenfold increase at Citation: García, A.; Cámara, J.A.; significantly lower concentrations than commercial or other proposed contrast agents. Remarkably, Boullosa, A.M.; Gustà, M.F.; these NPs exhibited prolonged residence time within the target organs. Thus, upon injection into the Mondragón, L.; Schwartz, S., Jr.; tail vein, they exhibited efficient uptake by the liver and spleen, with 85% of the injected dose (%ID) Casals, E.; Abasolo, I.; Bastús, N.G.; Puntes, V. Nanoceria as Safe Contrast recovered after 7 days. In the case of intratumoral administration, 99% ID of CeO2 NPs remained Agents for X-ray CT Imaging. within the tumour throughout the 7-day observation period, allowing for observation of disease Nanomaterials 2023, 13, 2208. https:// dynamics. Mass spectrometry (ICP-MS) elemental analysis confirmed X-ray CT imaging observations. doi.org/10.3390/nano13152208 Keywords: nanoceria; CeO2 nanoparticles; X-ray CT imaging; reactive oxygen species scavengers Academic Editor: Zaicheng Sun Received: 30 June 2023 Revised: 24 July 2023 Accepted: 27 July 2023 1. Introduction Published: 29 July 2023 The precise enhanced medical imaging powered by contrast agents has become a universal diagnosis tool, especially in the case of techniques employing X-rays, which show high spatial resolution but relatively low sensitivity [1]. Currently, 40% of all X-ray medical images use contrast agents to improve diagnosis [2]. Iodine compounds are Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. generally employed due to their biocompatibility and high Z number [3]; however, they This article is an open access article are not exempt from limitations: ions are removed rapidly from the bloodstream making distributed under the terms and the imaging time window very short, so high doses are needed. These ions are filtered conditions of the Creative Commons in the kidney, ultimately producing nephrotoxicity [4], the third more common cause of Attribution (CC BY) license (https:// hospital-acquired acute renal injury [5]. creativecommons.org/licenses/by/ Intense research has prompted for the last 20 years to develop new contrast agents 4.0/). that overcome these limitations and allow new imaging applications [6,7]. Among them, Nanomaterials 2023, 13, 2208. https://doi.org/10.3390/nano13152208 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2023, 13, 2208 2 of 13 high Z number nanoparticles (NPs) have been proposed since their altered biodistributions and longer residence times permit extending the available imaging window and reducing administration volumes [8], opening the opportunity to study disease dynamics [9]. Ini- tially, NPs are captured by the liver and the spleen, allowing advanced imaging in these organs [10]. Remarkably, NPs biodistribution is sensitive to disease. Thus, NPs are also prone to maximise their accumulation in lymph nodes [11], solid tumours [12,13], and inflammation areas [14,15] via enhanced permeability and retention (EPR) effects [12,15,16]. Consequently, NPs containing heavy atoms have been employed to image hepatocellular carcinoma in the liver [17] and tumours in the lymph nodes [18,19] or the brain [20], where NPs biodistribution depends on size, surface charge, surface structure and the presence of targeting moieties [21], allowing for site-directed imaging [8,10]. Thus, several NPs have been studied as an alternative to the iodinated molecules, including Gold [22], Bismute [23] or Ytterbium [24] based NPs at 100 mg/mL, 75 mg/mL and 70 mg/mL, respectively. They all show good contrast efficiency. They are non-toxic and biocompatible. Despite low doses being used during X-ray imaging, about 700 traditional images are taken for a CT 3D reconstruction which is not exempt from side effects, especially in children. Several studies have reported the induction of chromosome aberrations, changes in gene expression and a significant increase in γ-H2AX foci levels in patients, which is indicative of DNA damage [25,26]. X-ray radiation can induce DNA damage at the molecular level, together with the ionisation of water and the generation of a large amount of reactive oxygen species (ROS), which ultimately accounts for 40% to 60% of the produced damage [27,28]. Unfortunately, it has been reported that the use of contrast agents during CT scans increases the radiation dose and toxicity [29]. This becomes even more dramatic when metallic NPs can catalyse the generation of free radicals, further increasing indirect damage [30]. In the meantime, CeO2 NPs have been extensively studied due to their radiopro- tective properties, degrading radiation-induced toxic free radicals (ROS) into innocuous species, which has been translated into protection from indirect radiative damage [31–36]. CeO2 NPs have a high capacity to buffer electrons in redox environments [37,38], followed by the capture or release of oxygen. It means that CeO2 NPs act as a free-radical scav- enger of ROS molecules such as OH• and H2 O2 [39]. Despite that Ce has a higher atomic number, Z number (58), than I (53), its potential as a CT contrast agent has been rela- tively unexplored [40]. Interestingly, recent studies have focused on the potential use of gadolinium-doped CeO2 NPs as contrast agents for magnetic resonance imaging with high T1 relaxivity [41,42]. Our study demonstrates the significant benefits of CeO2 NPs as a contrast agent for CT imaging, primarily due to their high X-ray attenuation, excellent biocompatibility, and radioprotective properties. Importantly, these CeO2 NPs exhibit slow biodegradation into innocuous species and efficient excretion, further enhancing their safety profile [43,44]. This combination of properties positions CeO2 NPs as a highly competitive contrast agent, offering improved safety for CT imaging. 2. Results and Discussion Non-aggregated highly soluble CeO2 NPs at 10 mg Ce/mL have been synthesised with an average size of 5.1 ± 1.4 nm (Figure 1). A size of ~5 nm was chosen to maximise their catalytic activity [45]. The as-synthesised NPs were immediately conjugated to an excess of Murine Serum Albumin (MSA), preventing them from aggregation. Conjugation with MSA increases biocompatibility and solubility in biological media [46], preventing rapid renal clearance [47] and avoiding aggregation in the highly ionic physiological media [48,49]. The colloidal solution presents a UV-Vis absorption peak at 290 nm, indicative of Ce4+ , a ζ potential value of −28.2 ± 0.73 mV (conductivity 0.74 ± 0.02 mS/cm at pH 7.4) and a hydrodynamic diameter of 38.7 ± 1.9 nm (Figure S1). DLS analysis shows a monomodal peak stable over time, indicative of good colloidal stability and lack of large aggregates [50]. Nanomaterials 2023, 13, 2208 3 of 13 and a hydrodynamic diameter of 38.7 ± 1.9 nm (Figure S1). DLS analysis shows a mono- Nanomaterials 2023, 13, 2208 3 of 13 modal peak stable over time, indicative of good colloidal stability and lack of large aggre- gates [50]. A B C 5 1/nm D 200 nm 5 nm 2 nm Figure 1. 1. Characterisation Characterisationof ofCeO CeO2NPs conjugated with murine serum albumin (10 mg Ce/mL). (A) Figure 2 NPs conjugated with murine serum albumin (10 mg Ce/mL). TEM image taken at 150,000× magnification showing CeO2NPs of 5.1 ± 1.4 nm average size; (B) (A) TEM image taken at 150,000× magnification showing CeO2 NPs of 5.1 ± 1.4 nm average size; HRTEM image taken at 450,000× magnification, (C) representative diffraction pattern; and (D) (B) HRTEM image taken at 450,000× magnification, (C) representative diffraction pattern; and HRTEM of a single CeO2NPs. (D) HRTEM of a single CeO2 NPs. To evaluate To evaluatethe theuse useofofCeO CeONPs2NPs as contrast agents for safe X-ray CT imaging, 200 μL as contrast agents for safe X-ray CT imaging, 200 µL of 2 aofCeO a CeO 2NPs-MSA solution (10 mg Ce/mL vs. standard Iodine at 175 mg/mL concentra- 2 NPs-MSA solution (10 mg Ce/mL vs. standard Iodine at 175 mg/mL concentrations) tions) was was injected injected intravenously intravenously through through the into the tail vain tail vain into awakened awakened female female athymic athymic nude mice nude mice of 7 weeks of age bearing xenografted subcutaneous of 7 weeks of age bearing xenografted subcutaneous sarcoma tumours of A-673 human sarcoma tumours of A- 673 human cancer cells cancer cells in in the right reartheflank. right rear Five flank. animalsFive animals with tumourwith tumourabove volumes volumes700above mm3 700 mm 3 were selected for the assay four weeks post-tumour inoculation. After NP ad- were selected for the assay four weeks post-tumour inoculation. After NP administration, ministration,ofsupervision supervision the animals of and the animals and body body weight weight measurement measurement were performedwere performed every day, every day, which did not indicate any symptoms of toxicity. Clinical which did not indicate any symptoms of toxicity. Clinical observations included possible observations in- cluded possible changes in skin, eyes, mucous membranes, alterations changes in skin, eyes, mucous membranes, alterations in respiratory pattern, behaviour, in respiratory pat- tern, behaviour, posture, response posture, response to handling to handling or abnormal or abnormal movements. movements. Seven Seven days after NP’sdays after injection, NP’s injection, animals were sacrificed. Blood, primary tumours and several animals were sacrificed. Blood, primary tumours and several organs -liver, spleen, kidneys, organs -liver, spleen, lung, kidneys, heart lung,were and brain- heartexcised, and brain- wereand weighed excised, at −20 ◦ Cand kept weighed kept at −20 for ICP-MS °C for ICP- measurements. MS measurements. Obtained results (TableObtained 1) showresults how CeO(Table 2 1) show NPs-MSA how CeO exhibited 2NPs-MSA efficient exhibited uptake by effi- the liver cientspleen and uptake by the upon liver and injection intospleen the tailupon vein, injection with 85%ID intobeing the tail vein, with recovered after85%ID 7 days.being recovered after 7 days. Table 1. ICP-MS analysis of Ce mass in mice organs (7 days post-i.v. injection), the weight of organs, TableCe total 1. mass ICP-MS analysis in each of and organ Ce mass in mice organs%ID the corresponding (7 days post-i.v. (2040 ± 98 mginjection), Ce). the weight of organs, total Ce mass in each organ and the corresponding %ID (2040 ± 98 mg Ce). Organ % Injected Dose Ce (µg/Organ g) Total Ce (µg) Organ Weight (g) (%ID) Ce (μg/Organ g) Total Ce (μg) % Injected Dose (%ID) Spleen Weight (g)± 87 1746 0.42 741 ± 42.3 36.3 ± 2.1% Spleen 1746 ± 87Liver 0.42579 ± 29 741 1.67 ± 42.3 969 ± 49.136.3 ± 2.1% 48.1 ± 2.4% Liver 579 ± 29Kidney 1.673.2 ± 0.2 0.38 969 ± 49.1 1.22 ± 0.0648.1 ± 2.4%0.06% Lung 102 ± 5.1 0.16 16.7 ± 0.84 0.8 ± 0.04% Kidney 3.2 ± 0.2 Heart 0.38 2.3 ± 0.12 1.22 0.14 ± 0.06 0.32 ± 0.02 0.06% 0.02% Lung 102 ± 5.1 Brain 0.16 0.05 ± 0.003 16.7 0.41 ± 0.84 0.02 ± 0.0010.8 ± 0.04%<0.01% Heart Tumour 2.3 ± 0.12 0.60 ± 0.03 0.14 0.32 1.40 ± 0.02 0.84 ± 0.04 0.02% 0.04% Brain 0.05 ± 0.003 Plasma 0.41 4.41 ± 0.22 0.02 ±2.65 0.001 11.68± 0.63 <0.01% 6.01% Tumour 0.60 ±Total 0.03Ce (µg/g): 1.40 0.84 ± 0.04 1754 ± 92.4 0.04%91.4 ± 4.3% Plasma 4.41 ± 0.22 2.65 11.68± 0.63 6.01% Total Ce (μg/g): 1754a±Quantum X-ray CT images were collected using 92.4 FX micro-CT91.4 ± 4.3% A total of instrument. 512 projections were obtained in a 120 s/scan. The incident X-ray tube potential was set at 90 kVp and current at 200 µA. For the animals injected via the tail vein, X-ray CT images were taken before injection and at various representative post-administration times using a field of view (FOV) of 40 × 40 mm. The images were reconstructed with the Quantum FX software, based on Feldkamp’s method. Three-dimensional rendering videos of the intratumoral study were performed with Amide Image analysis [51], consisting of 3D X-ray CT images were collected using a Quantum FX micro-CT instrument. A total of 512 projections were obtained in a 120 s/scan. The incident X-ray tube potential was set at 90 kVp and current at 200 μA. For the animals injected via the tail vein, X-ray CT images were taken before injection and at various representative post-administration times using Nanomaterials 2023, 13, 2208 a field of view (FOV) of 40 × 40 mm. The images were reconstructed with the Quantum 4 of 13 FX software, based on Feldkamp’s method. Three-dimensional rendering videos of the intratumoral study were performed with Amide Image analysis [51], consisting of 3D ra- diodensity evaluation of different biological structures, including liver, spleen, kidney, radiodensity evaluation of different biological structures, including liver, spleen, kidney, sub-lumbar muscle and tumour tissue. Previous to each scan, animals were anaesthetised sub-lumbar muscle and tumour tissue. Previous to each scan, animals were anaesthetised with Isofluorane. Once the scan was finished, animals were brought back to their cages with Isofluorane. Once the scan was finished, animals were brought back to their cages for recovery. for recovery. Combining the serial CT views and the quantitative analysis of contrast density al- Combining the serial CT views and the quantitative analysis of contrast density lowed us to gain insights into the distribution and behaviour of CeO2NPs within the mice allowed us to gain insights into the distribution and behaviour of CeO2 NPs within the [52]. Figure 2 displays obtained results after the intravenous injection of CeO2NPs-MSA mice [52]. Figure 2 displays obtained results after the intravenous injection of CeO2 NPs- solution via the tail vein of mice. The mice were examined at various time points, ranging MSA solution via the tail vein of mice. The mice were examined at various time points, from 0 h (before injection) up to 7 days, allowing us to capture the temporal evolution of ranging from 0 h (before injection) up to 7 days, allowing us to capture the temporal the NPs within evolution theNPs of the mice. In thethe within serial CT coronal mice. views, In the serial CTwe identified coronal the we views, regions of inter- identified the est: regions of interest: the liver, spleen, kidneys, and tumour (Figure 2A). These regionswith the liver, spleen, kidneys, and tumour (Figure 2A). These regions were labelled were their initials labelled within their the first image initials to facilitate in the first imageclear identification. to facilitate The spleen was clear identification. Thespecifically spleen was highlighted with arrowheads, while white arrows denoted the liver. Monitoring specifically highlighted with arrowheads, while white arrows denoted the liver. Monitoring the con- trasting evolutionevolution the contrasting of the spleen of theover spleentime wastime over achieved throughthrough was achieved the observation of CT the observation axial views (Figure 2B). In these images, the white areas correspond to the of CT axial views (Figure 2B). In these images, the white areas correspond to the bone, bone, while the black regions represent air. By analysing the changes in contrast density, while the black regions represent air. By analysing the changes in contrast density, we we quantified the temporalthe quantified evolution temporalofevolution CT contrastof CT density contrastvalues density(HU) for the values (HU) investigated tissues for the investigated (Figure tissues (Figure 2C). Detailed information on 3D rendering and specific HU valuesfound 2C). Detailed information on 3D rendering and specific HU values can be can be infound FigureinS2 and Table Figure S2 andS1.Table S1. Figure2.2.InInvivo Figure vivoCT CTanalysis analysis of of intravenous intravenous injection injection via viatail tailvein veinofofCeO CeO2 NPs-MSA solution (200 µL, 2NPs-MSA solution (200 10 mg Ce/mL) in mice, at various representative times from 0 h (before injection) μL, 10 mg Ce/mL) in mice, at various representative times from 0 h (before injection) up uptoto7 7days. days. (A) Serial CT coronal views showing the main regions of interest, i.e., liver, spleen, kidneys, and tumour, are indicated with their initials in the first image. Arrowheads point at the spleen, and the white arrows indicate the liver. (B) Corresponding CT axial views show the contrasting evolution of the spleen over time. (Other white areas correspond to the bone, and black regions correspond to air). (C) Temporal evolution of CT contrast density values (HU) of the tissues under study. Numerical HU values are provided in Table S1. (A) Serial CT coronal views showing the main regions of interest, i.e., liver, spleen, kidneys, and tumour, are indicated with their initials in the first image. Arrowheads point at the spleen, and the white arrows indicate the liver. (B) Corresponding CT axial views show the contrasting evolution of the spleen over time. (Other white areas correspond to the bone, and black regions correspond to Nanomaterials 2023, 13, 2208 5 of 13 air). (C) Temporal evolution of CT contrast density values (HU) of the tissues under study. Numer- ical HU values are provided in Table S1. Figure Figure 33 presents presents aa zoomed zoomed image image ofof the the liver, liver, providing providing insights insights into into the the distribution distribution of the NPs within the liver parenchyma. The image reveals of the NPs within the liver parenchyma. The image reveals that the NPs that the NPs areare homogene- homoge- ously neously distributed within the liver tissue without significant accumulation around the distributed within the liver tissue without significant accumulation around the blood vessels. These blood vessels. These results resultsindicate indicatethat thatNPsNPspossess possess good good solubility solubility andand dispersion dispersion in in the the organ, organ, suggesting suggesting the absence the absence of anofimmune an immune defensive defensive response response against against the CeO the CeO 2NPs 2 NPs [53] [53] following following the non-immunogenic the non-immunogenic character character of these of these NPs NPs previously previously described described in theinliter- the literature [54,55]. In contrast, intravenous injection of the commercial iodine ature [54,55]. In contrast, intravenous injection of the commercial iodine contrast agent contrast agent Iopamidol Iopamidol®®-370-370leads leadsto toaa fast fast accumulation accumulationin in kidneys kidneys(5 (5 min min post-injection) post-injection) and and subse- subse- quent quick renal excretion, with almost no remaining signal at 24 h quent quick renal excretion, with almost no remaining signal at 24 h post-administration post-administration (Figure S3). Figure Figure 3. 3. In Invivo vivocoronal coronalCT CTimages imagesafter afterintravenous intravenous injection injection of of (A) (A) the thecommercial commercial iodine iodine contrast contrast agent Iopamidol ®-370 (150 μL; 175 mg I/mL) and (B) CeO2NPs-MSA solution (200 μL, 10 mg Ce/mL) agent Iopamidol® -370 (150 µL; 175 mg I/mL) and (B) CeO2 NPs-MSA solution (200 µL, 10 mg Ce/mL) at various representative times, from 2 min post-injection up to 24 h. at various representative times, from 2 min post-injection up to 24 h. To investigate the effects of intratumoral administration, we administered 70 µL of CeO2 NPs-MSA solution directly into the tumour. Figure 4 shows serial CT coronal view images of the mouse at 15 min, 24 h and 7 days post-injection. CT scans were performed with a FOV of 24 × 24 mm and 60 × 60 mm for the mouse injected intratumorally. A quantitative analysis of the 3D tomographic reconstruction is given in Table 2. The 3D rendering videos at the three representative times can be seen in Figure S4. A clear To investigate the effects of intratumoral administration, we administered 70 μL of CeO2NPs-MSA solution directly into the tumour. Figure 4 shows serial CT coronal view images of the mouse at 15 min, 24 h and 7 days post-injection. CT scans were performed Nanomaterials 2023, 13, 2208 with a FOV of 24 × 24 mm and 60 × 60 mm for the mouse injected intratumorally. A quan- 6 of 13 titative analysis of the 3D tomographic reconstruction is given in Table 2. The 3D render- ing videos at the three representative times can be seen in Figure S4. A clear enhancement of contrast signal enhancement was observed of contrast in the signal was tumour observed in 15 themin after15 tumour injection (747.2 min after HU compared injection (747.2 HU to 75.5 HU), remaining enhanced for the 7 days of measurement. compared to 75.5 HU), remaining enhanced for the 7 days of measurement. This is in contrast This with is in intratumoral injection of iodine compounds such as Iohexol, which disappears contrast with intratumoral injection of iodine compounds such as Iohexol, which disappears from the tumour from theintumour 4 h, while in 4the nanoparticulate h, while polymericpolymeric the nanoparticulate Iodine, Poly(iohexol), loses 50% of Iodine, Poly(iohexol), the loses contrast 50% also of the at 4 h [56]. contrast also at 4 h [56]. Figure 4. Figure 4. InInvivo vivoCTCT images after images intratumoral after injection intratumoral of 70of injection μL70ofµL CeOof2NPs-MSA solutionsolution CeO2 NPs-MSA (10 mg Ce/mL) in mice at various time intervals: 15 min, 24 h and 7 days. (A) Snapshot of the 3D rendering (10 mg Ce/mL) in mice at various time intervals: 15 min, 24 h and 7 days. (A) Snapshot of the videos (videos are provided in Figure S4). (B) Coronal view of a 2D CT projection. A zoomed image 3D rendering videos (videos are provided in Figure S4). (B) Coronal view of a 2D CT projection. A of the tumoral area is shown in the insight of each image. zoomed image of the tumoral area is shown in the insight of each image. Table 2. CT contrast analysis (HU and volume) of the tumoral region after intratumoral injection of Table 2. CT contrast CeO2NPs-MSA analysis solution (HU (70 μL, 10and mg volume) Ce/mL). of the tumoral region after intratumoral injection of CeO2 NPs-MSA solution (70 µL, 10 mg Ce/mL). Non-Contrasted Muscle Ce Contrast Agent CT Ce Contrast Agent Tumour Vol TumourNon-Contrasted Tissue 3 3 CT Value (HU.)Muscle Value (HU) Agent Ce Contrast Ce Contrast Vol (mm Agent ) Tumour (mmVol) CT ValueTumour (HU) Tissue CT Value (HU) CT Value (HU) Vol (mm3 ) 3 (mm ) 15 min 75.5 ± 3.9 75.4 ±CT 5.7Value (HU) 747.2 ± 44.9 50.2 1027.1 15 min 24 h 76.7 ± 4.175.5 ± 3.9 73.9 ± 2.675.4 ± 5.7 898.3747.2 ± 44.9 ± 35.9 50.2 42.3 1027.1 1157.6 24 h 76.7 ± 4.1 73.9 ± 2.6 898.3 ± 35.9 42.3 1157.6 7 days 78.1 ± 1.0 72.6 ± 1.2 969.9 ± 38.7 37.4 1525.1 7 days 78.1 ± 1.0 72.6 ± 1.2 969.9 ± 38.7 37.4 1525.1 Nanomaterials 2023, 13, 2208 7 of 13 Long residence times of the contrast agent allow for observing the structural evolu- tion of tumours. During the 7 days of observation, the tumour grew from 700 mm3 to 1500 mm3 . Interestingly, contrast levels of the adjacent muscle remained at similar values over time, which indicates that CeO2 NPs did not spread through nearby tissues [57]. ICP-MS analyses performed after animal sacrifice on day 7 confirmed the CT observa- tions (Table S2). No trace of Ce was detected in the mouse organs (liver, spleen, kidneys, lungs, heart and brain) or blood plasma other than in the tumour. Tumour analysis yields a total Ce mass of 706.6 ± 35 µg, which corresponds to 98.9 ± 4.7%ID (ID: 714 ± 36 µg Ce), indicating that CeO2 NPs-MSA remain inside the tumour for the 7 days. Analysing contrast volume and intensity as a function of time, we observe how the measured contrasted area gets compressed with increased contrast intensities as the tumour grows. This fact is attributed to the interstitial stress and fluid pressure within a growing tumour [58]. Mechanical stress is an important parameter that regulates tissue oxygenation, can- cer cell proliferation [59] and drug delivery [60] during the progression of solid tumours. Heterogeneous mechanical solid stresses developed during tumour growth compress blood vessels, dramatically reducing the supply of oxygen and access to drugs in inner regions, promoting the formation of hypoxic and necrotic regions in the tumour [58]. Thus, CeO2 NPs-MSA may be used as a long-lasting probe to analyse tumour evolution over time for disease progression prediction, especially in cases where there is a need to clearly distinguish between pseudoprogression and tumour progression, like in glioblastoma [61]. Accurately evaluating animal models mimicking human disease is vital when translating these studies into the clinic. Non-invasive imaging is a clinical standard and has enormous yet underexploited benefits for advanced medical imaging and research if one can repeat- edly evaluate host response (inflammation, tissue remodelling) and disease progression needed to gain better insight into the dynamics of the pathology and treatment effects [9], especially with CeO2 NPs which are radioprotective [31–33], can be made safe [62] and have been observed to slowly biodegrade [43,44]. CeO2 NPs-MSA provide good contrast levels in vivo even when injected at compara- tively low concentrations. Moreover, the well-known antioxidant properties of CeO2 NPs (Figure S5) suppose an additional motivation for their clinical use, as they may help to mitigate the detrimental effects of the ionising radiation used in CT scans, especially in children. 3. Conclusions In this study, non-aggregated colloidally stable CeO2 NPs conjugated with murine serum albumin (MSA) were synthesised and administered intravenously to mice with xenografted subcutaneous sarcoma tumours. The distribution of the NPs was monitored using X-ray CT imaging. The results showed that the NPs were homogeneously distributed within the liver tissue without significant accumulation around blood vessels, indicat- ing good solubility and dispersion. In contrast, the commercial iodine contrast agent Iopamidol® -370 showed fast accumulation in the kidneys and subsequent renal excretion. Furthermore, intratumoral administration of CeO2 NPs-MSA resulted in a clear en- hancement of contrast signal in the tumour, which remained enhanced for the entire 7-day observation period and the long residence times of the contrast agent allowed for observing structural changes in the tumour over time. Analysis of the tumour and other organs con- firmed the presence of Ce only in the tumour, indicating that the CeO2 NPs-MSA remained inside the tumour for the entire 7-day period. This prolonged retention within the tumour can provide valuable insights into tumour growth and dynamics. Overall, the findings of this study demonstrate the potential of CeO2 NPs-MSA as a contrast agent for safer CT scans, offering prolonged residence times, good solubility, and dispersion. Further research and development in this area could lead to the translation of CeO2 NPs-MSA into clinical applications for enhanced medical imaging and improved diagnosis of diseases. Nanomaterials 2023, 13, 2208 8 of 13 4. Materials and Methods 4.1. Materials Cerium (III) nitrate hexahydrate (Ce(NO3 )3 ·6H2 O), tetramethylammonium hydroxide (TMAOH; 1M), and mouse serum albumin (MSA) (research grade, sterile filtered) were purchased from Sigma–Aldrich (Saint Louis, MO, USA). All reagents were used as received. Milli-Q water was used in the preparation of all solutions. 4.2. CeO2 NPs Synthesis CeO2 NPs of around 5 nm were synthesised by the chemical precipitation of cerium (III) nitrate hexahydrate (Ce(NO3 )3 ·6H2 O) in a basic aqueous solution [48]. A total of 37.5 mM of cerium (III) nitrate hexahydrate was dissolved in 50 mL Milli-Q water at room temperature. A total of 50 mL of tetramethylammonium hydroxide (TMAOH) solution (44 mM) was added slowly at room temperature (RT) under vigorous stirring. The mix- ture was left under soft stirring for about 30 min. During the first minutes, the solution is colourless, then turns progressively brownish. NPs were purified by centrifugation (20,000× g, 45 min, RT), and the resultant reddish pellet was resuspended in 100mL aque- ous solution of 1 mM TMAOH. As determined by the Xylenol Orange test (see below), the final Ce concentration was 1.06 ± 0.05 mg/mL (2.6 × 1015 NPs/mL). Before concentration, as-synthesised CeO2 NPs were first conjugated with MSA. A total of 10 mg of MSA was dissolved in 1 mL of PB 50 mM and kept undisturbed (without stirring) in the fridge until its complete dissolution. Then, 10 mL of CeO2 NPs were added dropwise to the protein solution while applying mild stirring. The sample was kept in the fridge for 24 h to allow the complete adsorption of MSA molecules onto the NP surface. The colloidal solution was then concentrated through 5 cycles of centrifugal filtration (Molecular weight cut-off 30 kDa, Millipore Amico Ultra) at 2500× g for 5 min, yielding 1ml of CeO2 NPs-MSA at 10.2 ± 0.5 mg Ce/mL (75.8 mM Ce, 2.6 × 1016 NPs/mL), as determined by ICP-MS and Xylenol-orange test. Both the synthesis and concentration of NP were performed under sterile conditions. 4.3. Characterisation The resultant NPs were characterised using a combination of techniques. 4.3.1. UV-Vis Spectroscopy UV-Vis spectra were acquired with a Cary 60spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) in the 200–800 nm range using 1.5 mL plastic cuvettes. 4.3.2. Dynamic Light Scattering (DLS) and ζ-Potential Malvern ZetaSizer Nano ZS instrument operating with a light source wavelength of 532 nm and a fixed scattering angle of 173◦ was used to determine the colloidal stability of the samples, the hydrodynamic diameter and ζ-potential value. Three independent measurements were performed. 4.3.3. Transmission Electron Microscopy Samples were prepared by drop casting 10 µL of the sample onto carbon-coated copper grids and left to dry at room temperature. TEM images were acquired with a JEOL 1010 electron microscope (Jeol, Tokyo, Japan) operated at 80 kV accelerating voltage. HRTEM images were acquired with an FEI Tecnai G2 F20, operated at 200kV. NPs size distribution was determined using ImageJ software (National Institutes of Health, Bethesda, Rockville, MD, USA) by analysing more than 500 particles. 4.3.4. Ce3+ Concentration by Xylenol Orange Test The test performed is based on the protocol developed by Tonosaki et al. [58]; orange forms a complex with free Ce3+ ions in a ratio of 1:1, which shows an absorption maximum at 575 nm. Briefly, 10 µL of the sample was added to a vial containing 700 µL of Xylenol Nanomaterials 2023, 13, 2208 9 of 13 Orange 1 mM, 3000 µL of acetate buffer (pH = 6.0) and 1290 µL of MilliQ water. The content of Ce3+ in an unknown sample was determined through the calibration graph obtained previously for 0–40 µg/mL of Ce3+ . Here, the supernatant of the reaction was analysed. 4.4. In Vivo Assays Female athymic nude mice (Janvier, Le Genest Saint Isle Saint Berthevin, France and Envigo Crs. SA, Barcelona, Spain) were kept in pathogen-free conditions and used at 7 weeks of age. Animal care was handled following the Guide for the Care and Use of Laboratory Animals of the Vall d’Hebron University Hospital Animal Facility, and the experimental procedures were approved by the Animal Experimentation Ethical Committee at the institution. In vivo studies were performed by the ICTS “NANBIOSIS” at the CIBER- BBN’s in vivo Experimental Platform of the Functional Validation and Preclinical Research (FVPR) area (https://www.nanbiosis.es/equipments/, accessed on 26 July 2023) (Barcelona, Spain). A-673-Fluc cells (2 × 106 ) suspended in 200 µL of cell culture media were subcuta- neously inoculated in the right rear flank of each animal. Tumour growth was monitored twice a week by conventional calliper measurements (D × d2 /2, where D is the ma- jor diameter and d is the minor diameter). Five animals with tumour volumes above 700 mm3 were selected for the assay four weeks post-tumour inoculation. In two of them, 200 µL of CeO2 NPs solution was administered intravenously through the tail vein in awakened animals. For comparison, another two mice were injected intravenously with a commercial iodine contrast agent (Iopamidol® -370, 150 µL of 175 µg I/mL). The fifth animal was anaesthetised with 1.5% isofluorane (Forane, Baxter, Deerfield, IL, USA) and administered intratumorally with 70 µL of the exact solution of CeO2 NPs-MSA. After NP administration, supervision of the animals and body weight measurement were performed daily, indicating no symptoms of toxicity. Clinical observations included possible changes in skin, eyes, mucous membranes, alterations in respiratory pattern, behaviour, posture, response to handling, and abnormal movements. Seven days after NP injection, animals were sacrificed. Blood plasma, primary tumours and several organs (liver, spleen, kidneys, lung, heart, brain and the tumour) were excised, weighed and kept at −20 ◦ C for ICP-MS measurements. The procedures for animal handling, humanitarian endpoint and animal euthanasia described in this project have been approved by the VHIR’s Animal Experimentation Ethics Committee (internal reference 38/17). 4.5. ICP-MS Analysis Ce concentration in the CeO2 NPs-MSA solution, blood plasma, tumour and various organs were determined by Inductively Coupled Plasma-Mass Spectrometry (Agilent, 7500ce). Plasma samples were diluted in a solution of EDTA 0.05% (p/v) and NH3 0.5% (v/v). The tumour and organs were dissolved in HNO3 concentrated (Merck; p.a.) and heated in a microwave digestion oven (Milestone, Ultrawave). In parallel, a control sample was also digested. The resultant digestions were diluted with HNO3 1% (v/v) before being injected into the ICP-MS instrument. 4.6. CT Imaging X-ray CT images were collected using a Quantum FX micro-CT instrument (Perkin Elmer, Waltham, MA, USA) (see Supplementary Figure S6). A total of 512 projections were obtained in a 120 s/scan at a maximal resolution of 0.05mm, incident X-ray tube potential was set at 90 kVp and current at 200 µA. For the animals injected via tail vein, X-ray CT images were taken before injection and at various representative post-administration times (15 min, 30 min, 1 h, 2 h, 24 h and 7 days) using a field of view (FOV) of 40 × 40 mm. CT scans were performed at 15 min, 24 h and 7 days post-injection with a FOV of 24 × 24 mm and 60 × 60 mm for the mouse injected intratumorally. Reconstruction of the studies was performed with the Quantum FX software, based on Feldkamp’s Nanomaterials 2023, 13, 2208 10 of 13 method. Three-dimensional rendering videos of the intratumoral study were performed with Amide [45]. Imaging data were analysed at the Preclinical Imaging Platform from the Vall d’Hebron Research Institute. Image analysis consisted of 3D radiodensity evaluation of different biological structures, including liver, spleen, kidney, sub-lumbar muscle and tumour tissue. In each of these structures, at least four different regions of interest (ROIs) were selected, and the average density was obtained. CT attenuation values are given in the standard Hounsfield Units (HU) by calibration with water (HU = 0) and air (HU = −1000). The CT equipment is not normalised; therefore, the density value of the sub-lumbar muscle is used as a reference. Before each scan, animals were anaesthetised with Isofluorane (5% during the induc- tion phase, 2% during maintenance). Air flow was set to 0.8 L/min. Once the scan was finished, animals were brought back to their cages for recovery. All the procedures were performed following the institutional ethic committee. Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/nano13152208/s1, Figure S1. Characterization of CeO2NPs conjugated with murine serum albumin; Table S1: CT contrast values (H.U.) corresponding to Figure 2 of the main article; Figure S2. Examples of in vivo 3D rendering videos before injection of CeO2NPs and 2 h after intravenous injection; Figure S3. CT coronal images 5 min and 24 h after intravenous injection of CeO2NPs-MSA; Figure S4. In vivo 3D rendering videos and their corresponding snapshots were recorded at 15 min, 24h and 7 days post-intratumoral injection; Table S2. ICP-MS analysis of organs after intratumoral injection; Figure S5. CeO2NPs-albumin conjugated and naked CeO2NPs capacity to scavenge hydrogen peroxide. Figure S6. (Left) Mouse anesthetized before a CT scans. (Right) Mouse placed in the plate of the Quantum FX micro-CT instrument (Perkin Elmer, Waltham, MA). References [57,63] are cited in the supplementary materials. Author Contributions: A.G. and M.F.G. synthesised the NPs under the supervision of E.C. and assisted in vivo experiments that were carried out by J.A.C. and A.M.B. in collaboration with L.M. after design and supervision by I.A. V.P. wrote the manuscript assisted by S.S.J. and N.G.B. All authors have read and agreed to the published version of the manuscript. Funding: We acknowledge financial support from the Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU) (RTI2018-099965-B-I00, AEI/FEDER, UE) proyectos de I+D+i de programación conjunta internacional MCIN/AEI (CONCORD, PCI2019-103436) co-funded by the European Union and Generalitat de Catalunya (2017-SGR-1431). ICN2 is supported by the Severo Ochoa program from Spanish MINECO (SEV-2017-0706) and is funded by the CERCA Programme/Generalitat de Catalunya. Data Availability Statement: Data available on request from the authors. Conflicts of Interest: The authors declare no competing interest. References 1. Kalender, W.A. 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