Antitumor activity and selectivity of a unique nanostructured polyoxoniobate

Antitumor activity and selectivity of a unique nanostructured polyoxoniobate | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Antitumor activity and selectivity of a unique nanostructured polyoxoniobate Luiz C. A. Oliveira, Poliane Chagas, Ana Pacheli Heitmann, José B. Gabriel, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4033921/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In vitro and in vivo experiments were carried out to evaluate the activity and antitumor selectivity of new niobium oligomers. A polyoxoniobate (PONb) and its chemical structure linked to the methylene blue dye (PONb-MB) showed promising results for some types of tumor cells. Molecular structures were determined for PONb and PONb-MB based on spectroscopic analyses with density functional theory calculations. Both caused a significant cell viability reduction in tumor cells (HeLa) with high effectiveness of 10.0% at 10 mg L-1, because of the reactivity oxygen species (ROS) generated on its chemical structure. Experimental quantitative analyzes demonstrated high selectivity for tumor cells (apoptosis 3x higher compared to MRC-5 healthy cells). Lower toxicities for healthy cells (e.g., MRC-5 and L929) confirm the selectivity of the synthesized compounds. It is noteworthy that PONb-MB and PONb-MB are physiologically soluble compounds and can be suitable for intravenous treatments. Ex vivo biodistribution studies in mice revealed a very low accumulation in organs such as the spleen, heart, liver, lungs, and kidneys. Additionally, almost no toxicity was observed in the mice post-injection, which was confirmed by biochemical analyzes. Biological sciences/Cancer Physical sciences/Chemistry Physical sciences/Materials science Physical sciences/Nanoscience and technology Polyoxoniobate ROS Cancer treatment Cervical adenocarcinoma Epidermoid carcinoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION Polyoxometalates (POMs) are anionic metal oxo clusters usually composed of d 0 species of the Groups VI and V (e.g., Mo, W, V, Ta, and Nb)[1,2]. They are oligomeric aggregates of early transition metal cations linked by oxide anions that are formed by self-assembly processes [1,2]. The POMs have high-water solubility, low toxicity for humans, and their redox and photochemical capacities can present diversified usages, mainly in catalysis and medicine [3,4]. Mo-based POMs have been applied for antitumor activity while those based on V are known for their antiviral effect[1]. Although POMs based on W, Mo, and V have been deeply investigated in the last decades, the chemistry of Nb-based POM (called polyoxoniobates, PONb) is less explored [1]. Nevertheless, it must be emphasized that PONb has shown distinctive properties such as high negative charge, base surface oxygen atoms, and a wide range of cluster sizes that are suitable for several applications, for example inhibiting tumor development. Particularly, antitumor activities of PONb can be associated with the apoptosis process and oxidation of cellular components[3,5–7]. Its bio-application and understanding its behavior in a biological environment is one great challenge. Furthermore, it is also important to investigate how size, shape, and composition can influence its toxicity profile. In general, the experimental determination of molecular structures is not an easy task. From this point of view, molecular modeling techniques based on computational methods of quantum chemistry appear as an alternative to calculating structural, electronic, and spectroscopic properties. Thus, it is possible to establish the structure-reactivity-activity relationship in the design of new drugs for various applications in medicinal chemistry [8–11]. Particularly, we have recently prepared a PONb complex by proposing a new class of materials to be used as anticancer agents [7]. Experimental and theoretical analyzes demonstrated that the Nb complex species presented reactivity and stability when docked into the DNA crystallographic structure. Herein we report the synthesis of unique PONb species with emphasis on its application to cancer treatment. Two strains of carcinogenic cells were used to demonstrate the versatility of PONb for inhibiting tumor cells, with quite low toxicity to healthy ones. Furthermore, ex vivo biodistribution and toxicological studies with healthy mice revealed negligible damage, showing the potential application of PONb as a drug candidate for tumor treatment with particular attention to the achievements of several human cell carcinomas in vitro . 2. MATERIAL AND METHODS 2.1- Preparation of polyoxoniobates. For the synthesis of niobium polyoxoniobates, 46.2 mmol of the NH 4 [NbO(C 2 O 4 ) 2 (H 2 O)](H 2 O)n salt (provided by CBMM) was dissolved in 100 mL of distilled water at 90 °C. Then, an NH 4 OH solution (5 mol L −1 ) was slowly added to the salt solution for precipitation of the niobium oxyhydroxide. The suspension was aged under magnetic stirring for 12 h and the obtained solid was filtered. Afterward, 8 mL of H 2 O 2 (30 %, v/v) and 100 mL of distilled water were added to 300 mg of niobium oxyhydroxide. After 12 h, the solid was centrifuged and the liquid supernatant contained the leached species of peroxoniobate complex (PONb). The PONb yellow solution was used to prepare the PONb-MB after reaction with a methylene blue solution (MB). For that, 10 mL of a 50 mg L -1 solution of the MB dye was added to 10 mL of the PONb. The cationic dye strongly binds to the structure of the PONb by electrostatic attraction, forming the PONb-MB compound, of blue coloration. 2.2 - Characterization of the compounds. The Nb concentration in the polyoxoniobate solutions was determined by Inductively Coupled Plasma Mass Spectrometry, ICP-MS Agilent 7700, using an analytical curve for the Nb element. The Polyoxoniobate solution (PONb) and the solution after the methylene blue dye addition (PONb-MB) were characterized by UV-Vis spectroscopy (Shimadzu - 2600/2700) and Zeta potential measurements (Zetasizer Nano ZS). The analytical procedure was conducted in a Zeta-sizer Nanoseries Zs apparatus (Malvern Instruments, Malvern, UK) after the adequate dilution of the samples in ultra-pure MilliQ® water. The results were expressed as mean ± standard deviation for at least three different batches of each sample. The Polyoxoniobate solution was placed in contact with the 5-GMP molecule (Sodium guanosine 5-monophosphate, which simulates the DNA) to analyze the possible interaction between the 5-GMP molecule and the PONb compound. This interaction was verified by NMR of 31 P (Bruker spectrometer 200 MHz), using 1024 scans and phosphoric acid as standard. The spectrum of the 5-GMP pure molecule was obtained using 20 mg of 5-GMP dissolved in 600 µL of D 2 O. Afterward, a 5-GMP and PONb mixture was prepared and irradiated for 7 min using a light wavelength of 365 nm. Then, 400 µL of D 2 O was added to 200 µL of the irradiated sample, and the mixture was taken to the spectrometer. The reactive oxygen species (ROS) were characterized by the EPR spin trapping technique using PBN (N-tert-butyl-α-phenylnitrone) and TEMP (2,2,6,6-tetramethyl-4-piperidone) as spin trapping molecules. The PBN is known to react with radicals and produces very stable spin adducts[12], while the TEMP reacts with singlet oxygen producing a nitroxide radical called TEMPO [13]. One milliliter of PBN (85 mM) or TEMP solution (1M) was added to 1 mL of PONb, PONb-MB, and MB aqueous solutions. These mixtures were placed in a 5 mL beaker and magnetically stirred for 20 min. Aliquots of the samples were extracted every 5 min using a capillary glass tube and the data were acquired using a X-band Magnettech miniscope 400 spectrometer. The tests were performed at room temperature and illumination. Moreover, Raman data were collected on a Bruker Senterra spectrometer with a 532 nm laser operating at 20 mW. The structure of the materials was further investigated by X ray powder diffraction and PDF patterns. The XRPD patterns were collected using a STADI-P diffractometer (Stoe®, Darmstadt, Germany) with MoKα1 radiation (λ = 0.7093 Å), operating in transmission geometry. The measurements for the application of the low-energy PDF method were performed using 0.5-mm diameter special glass capillaries nº 14 (Hilgenberg®, Malsfeld, Germany), which reduces the container contribution to the measurement. The data were measured from 2.0 to 136.4° (2q). An empty capillary was measured with the same conditions as the filled capillaries to remove the instrumental contributions of the measurements. A Q-range from 0.1 to 16.4 Å -1 was considered for the S(Q) (total structure-function) that was Fourier transformed to obtain the PDF pattern. S(Q) and PDF patterns were obtained using the PDFGetX3 software[14]. In this software, all non-structural contributions are determined through an ad hoc approach, then they are parameterized, and the parameters are used to obtain the structure function S(Q). The structure functions for lyophilized PONb and Nb 2 O 5 were presents in Figure S5a and the Fourier Transform of these results yielded the PDF pattern for the samples, shown in Figure S5b. The main distances up to 10 Å were calculated using the Bond_Str software [15] based on the crystallographic information framework file (CIF file) for the structure of Nb 2 O 5 obtained in the Inorganic Crystal Structure Database®, under the 51176 reference code. The calculated distances for individual atomic pairs were used to compare the distances observed in the PDF patterns. 2.3- In vitro comparative tests using HeLa (tumoral) and MRC-5 (healthy) cells. The assays were performed using the cell lines HeLa (ATCC®-CCL-2 ™), a tumor cell derived from human cervical adenocarcinoma, and MRC-5 (ATCC®-CCL-171 ™), a healthy cell line derived from human lung fibroblast. The cells were cultured in a CO 2 incubator (5% CO 2 Cole Parmer) with a humid atmosphere at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/amphotericin B (complete DMEM medium). Upon reaching 80% confluency, cells were harvested using sterile PBS to wash the plate and sterile 1% Trypsin solution to detach the cells to be collected in the falcon tube. The cells were then counted with the aid of a Neubauer chamber. The cell viability assay with sulforhodamine B (SRB) was performed according to the literature [16]. For this assay, the cells were seeded in 96-well culture plates (1 x 10 4 /well) and incubated at 37°C/5% CO 2 . After 24 h, the cells were treated with PONb and PONb-MB at concentrations of 1, 5, 10, 50, and 100 ppm. The group defined as cell control received only full DMEM medium and was maintained under the same conditions as the treated groups. After 24 h, the cells were fixed through protein precipitation with 10% trichloroacetic acid at 4°C (100 µL/well) for 1 h. After five washing steps, the cells were stained with 0.04 % SRB (100 µL/well) at room temperature for 1 h. Afterward, the plate was washed with 1% acetic acid four times to remove the unbound stain. The plate was air-dried at room temperature and the bound protein stain of each well was solubilized with 100 µL of 10 mM Tris base [tris(hydroxymethyl)aminomethane]. The optical density was measured by spectrophotometry on a UV-Visible Microplate Reader (Molecular Devices) at 510 nm. The survival fraction was calculated as a percentage of the control (Absorbance in control = 100% survival). The experiments were carried out in triplicate. The ability of the material to damage the cell membrane and promote cell death signaling was assessed by fluorescence microscopy imaging. For the cell dead assay, the cells were seeded in 96-well culture plates (1 x 10 4 /well) and incubated at 37°C/ 5% CO 2 . After 24 h, the cells were treated with PONb and PONb-MB at a 10 mg/L concentration. The group defined as control cells received only a complete medium and were maintained under the same conditions as the treated group. After 24 h of incubation at 37°C, the cells were supplemented with 1 μM calcein-MB (Life Technologies) and 2 μM propidium iodide (Life Technologies) in DMEM culture medium. The images were collected with 4x objective lens on an inverted fluorescence microscope Olympus IX70 (Olympus America, Melville, NY). The percentage of apoptosis was calculated from the ratio between the number of dead and living cells, which were counted with the aid of ImageJ software. Each treatment was performed in triplicate, three different images were counted, and the means were used for statistical analysis. Reactive oxygen species (ROS) are the result of oxygen reduction during aerobic respiration by various enzymatic systems within the cell. The assay detects the formation of intracellular ROS (especially superoxide and hydroxyl radicals) in live cells employing the fluorometric method. For this assay, the cells were seeded in 96-well culture plates (1 x 10 4 /well) and incubated at 37°C/ 5% CO 2 . After 24 h, 100 µL of ROS Detection Reagent was added to each well. The plate was incubated for another 1 h and then the cells were treated with PONb and PONb-MB in PBS medium at the concentration of 10 mg/L. The group defined as control cells received only medium and were maintained under the same conditions as the treated group. The Multi-Mode Reader Cytation 5 Cell Imaging (Bio tek) was used to measure the fluorescence intensity and to collect the images with a 4x objective lens. The detection of ROS was performed using an excitation wavelength of 490 nm and collecting the fluorescence emission of 525 nm. Each treatment was performed in triplicate, three different images were counted, and the means were used for statistical analysis. 2.4- In vitro tests using L929 (healthy) cells: cell viability with MTT assay. Healthy cell lines L929 (connective tissue fibroblasts) were cultured; Manassas, VA, USA in Dulbecco's Modified Eagle's Medium, supplemented with 10% FBS (Fetal Bovine Serum) and 1% Amphotericin B-Streptomycin (100 IU mL -1 ). Cells were maintained under the following conditions: 37°C and 5% CO 2 , with medium exchange every two days. After reaching confluence, the cells were seeded in 96 well plates in concentrations around 1 x 10 5 cells/well for the tests. The tests were performed after the cells reached 70% confluence in which different concentrations of PONb and PONb-MB were added in each well: 3.0; 5.0; 8.0; 10.0; 12.0; 15.0 and 20.0 mg mL -1 , in six replicates. An aqueous solution of methylene blue dye (MB) at 12.5 μg mL -1 was used as a positive control. The solutions were sterilized by filtration (0.22 μm membrane). The contact time (incubation) with the PONb, PONb-MB, and MB solutions was 24 and 48 h at 37°C and 5% CO 2 . Then, the reagent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT was added to the plates. After 4 h of incubation at 37°C for the formazan crystals formation, a sodium dodecyl sulfate (SDS) detergent solution was added. After 14 h, the UV-Vis Spectrophotometer readings were taken at a wavelength of 570 nm. The respective negative controls (containing only untreated cells) and blank controls (no cells and no treatment, containing all solutions involved in the test) were analyzed on all plaques. The effects on cell viability were expressed as a percentage of the viability detected for the treated cells versus the viability of the cells incubated with culture medium alone. Data are expressed as the mean ± standard deviation (n = 6). 2.5- In vivo experiments. The study was carried out in compliance with the ARRIVE guidelines. Third Swiss CF1 female mice (6-8 weeks old, 24-28 g) were obtained from the Faculty of Pharmacy of the Federal University of Minas Gerais (UFMG). The animals had free access to water and food and were kept under controlled environmental conditions. All animal procedures were approved by the Institutional Animal Care and Use Committee (CEUA/UFMG), under protocol 118/2019. Mice were randomly separated into groups of 6 animals for the in vivo studies. The number of mice was defined similarly to other papers from our group [17,18]. For the blood clearance experiments, aliquots of 100 μL of a tenfold diluted PONb-MB solution were injected intravenously into the healthy Swiss mice (n = 6, 6-8 weeks old, 20-24 g). An incision was made in the tail of the animals and blood was collected pre-weighed tubes at 15, 30, 60, 120, 240, 480, 1440, and 2880 min after administration. The mice were anesthetized with a mixture of xylazine (15 mg/kg) and ketamine (80 mg/kg). Blood was digested using HCl and HNO 3 solution, using a ratio of tenfold solution diluent to blood. In the biodistribution assays, aliquots of 100 μL of a tenfold diluted PONb_MB solution were injected intravenously into healthy Swiss mice (n = 6, 6-8 weeks old, 20-28 g). At the post-injection times of 30, 60, and 240 min, organs such as spleen, heart, liver, lungs, kidneys, and blood were removed and weighed [19]. The organs were macerated using tissue macerator, and the supernatant was used for the determination of the niobium content present in each organ. For the analysis by ICP-MS, the samples went through acid digestion, using distilled nitric acid (Merck), 65 % (w/v). For sample preparation, 1 mL of nitric acid was added to each 1 mL of the supernatant resulting from the digestion of each organ. The mixture was left under constant magnetic stirring at 50°C for two hours and then centrifuged using a QUIMIS centrifuge, model 0222E24. The supernatants were transferred to polyethylene tubes, completed to 10.0 mL, and analyzed by ICP-MS. For the biochemical analyses, aliquots of 100 μL of a tenfold diluted PONb_MB solution were injected intravenously into healthy Swiss mice (n = 6, 6-8 weeks of age, 20-28 g). After 24 h from the injection, blood was collected using an anticoagulant (0.18% w/v EDTA). The blood was centrifuged at 5000 rpm for 10 min to obtain the plasma used to perform the biochemical analyses. The biochemical tests were performed using commercial kits from Labtest® (Lagoa Santa, Brazil) through Bioplus BIO-2000 semiautomatic analyzer equipment (São Paulo, Brazil). Heart (CK-MB), liver (AST, aspartate aminotransferase, and ALT, alanine aminotransferase), and kidney (urea and creatinine) functions were tested. It is worth mentioning that all the reagents used in in vitro and in vivo experiments have > 95% purity by HPLC analysis. The methods presented in the work were carried out in accordance with relevant guidelines and regulations. 3. COMPUTATIONAL DETAILS All calculations were performed with the Gaussian 09 package [20] employing the density functional theory (DFT) methodology[21] with the B3LYP functional [22] using the 6-31G(d,p) basis set [23] for O, H, N, S, and C atoms and the LANL2DZ [24]effective core potential (ECP) for Nb(V) ion. B3LYP/LanL2Dz/6-31G(d,p) level of theory used in the present study has been widely used in the literature [25,26] and has demonstrated good agreement against experimental data for Nb compounds. Therefore, we point out that this level of theory can contribute to experimental analysis in elucidating the structural properties of the system. Firstly, the monomers corresponding to the structures of PONb and MB (Fig. S1, Supporting Information) were fully optimized in the gas phase and their geometries (Table S1) were used to build the structure of the PONb-MB system. The monomers (PONb and MB) and the PONb–MB system were characterized by harmonic frequency analysis indicating the optimized geometries as true minima. Finally, electronic excitations (UV-Vis spectra) were calculated using the B97-D functional [27] by TD-DFT formalism [28] and the molecular orbital analysis was computed, both using the Polarizable Continuum Model (PCM) [29] to describe solvent effects - water solvent (dielectric constant, ε=78.3553). 4. RESULTS AND DISCUSSION 4.1- Characterization of the polyoxoniobates. The Polyoxoniobate solutions were characterized by UV-Vis (blue lines in Figures 1a and b). The spectrum of the pure PONb shows an intense band in the UV region, centered at about 270 nm, typical of peroxide groups (ROS) that are linked to Nb atoms in the PONb structure (Figure 1a). However, the MB dye addition to PONb generates a new species (PONb-MB) that absorbs radiation in the visible light region of the spectrum, with maximums at 574, 694, and 775 nm, besides the high-intensity UV peak centered at 243 nm (Figure 1b). It is known that the MB dye has absorption bands at 612 and 665 nm,[7,30] which are not observed in the spectrum of PONb-MB (Figure 1b). This result indicates the formation of a new species of polyoxoniobate rather than a sole mixture of the two compounds of PONb and MB dye. It is also important to point out that the ROS observed (Bonded peroxide group) is not a radical species, and for this reason, the niobium compound might not be toxic to healthy cells. The theoretical spectra in the UV-Vis region (red lines in Figures 1a and b) show that the PONb species has an intense band at 287 nm, i.e ., 6.3 % shifted to the low-energy region compared to the experimental band (Figure 1a). After the formation of the PONb-MB system (Figure 1b), two absorption bands are observed: ( i ) an intense band at 274 nm assigned to an intramolecular transition between HOMO-2 (Figure 1d) and LUMO+5 (Figure S2a) orbitals and ( ii ) a weak and broad one in the 550 nm region assigned to a transition between HOMO-2 (Figure 1d) and LUMO (Figure 1) that can be attributed to the strong intermolecular interaction between the PONb and MB species. More details on the TD-DFT calculations observed in Figures 1c and d are found in the Supporting Information (Figure S3 a and b). Thermodynamic parameters of the PONb-MB interaction were calculated according to Equations S1 to S3. All calculated parameters are shown in Table S2. The interaction between PONb and MB molecules forms a strongly bound compound with energy stabilization of around -55.0 kcal mol -1 . TΔS term is negative and, according to Equation S3, it contributes repulsively to of the PONb-MB system. According to Equation S2, the value is negative enough to produce a large negative (-63.4 kcal mol -1 ), even though the thermal correction to enthalpy is negative (-8.4 kcal mol -1 ). Thus, these results suggest that the formation of the PONb-MB system is thermodynamically favorable to equal to -37.1 kcal mol -1 . Figure 1. UV-Vis absorption spectra (Experimental and simulation using TD-DFT calculation) for PONb (a) and PONb-MB system (b), fully optimized DFT structures of PONb monomer (c), and PONb-MB system (d). Moreover, to estimate the negative charge of PONb and PONb-MB molecules, the solutions were characterized by Zeta potential measurements. The results demonstrated a high negative value of the PONb cluster (-50.6 mV). These negative charges possibly allowed the binding of MB cationic dye to generate a new stable species of PONb-MB, in a good agreement with the theoretical calculations. This hypothesis was further attested by Zeta potential measurements of PONb-MB that presented a lower value (-36.5 mV) than the pure PONb, due to the electrostatic interactions between the cationic dye and the negative charge oxygen species in the PONb structure. In addition, the PONb species have an average size of 73(3) nm and the new polyoxoniobate (PONb-MB) presented a lower average size of 61(4) nm after reaction with MB. The prepared polyoxoniobates (PONb and PONb-MB) were characterized via DFT calculations, which indicate the formation of the structures shown in Figure S2. The optimized geometry of PONb (Figure S2a) has short- and medium-range hydrogen bonds in the range of 1.48 - 1.87 Å between the hydrogen atoms of peroxide ligands and neighboring oxygenated ligands. The bond lengths Nb(V)–O are in the range of 1.8 - 2.10 Å (), 2.14 - 2.22 Å () and 1.75 - 1.80 Å () and (). Similar results for these bond types were found by Grimme et al. [27] for the crystalline structure of the peroxopolioxoniobate ion salt [As 2 Nb 4 (O 2 ) 4 O 14 ] 6− and by Si et al. [26] also through an electronic DFT study based on the salt crystal of the peroxohexaniobate ion salt [H 3 Nb 6 O 19 ] 5- . The latter theoretical analysis used B3LYP as in the present study. The optimized geometry of the PONb-MB system (Figure S2c) demonstrated no significant changes in the Nb(V)–O bond lengths, but there are some dihedral angle rearrangements involving the terminal oxygenated groups. Figure S2c shows that the PONb—S-MB interaction appears to be electronically stronger (shorter intermolecular distance, » 2.11 Å) than the PONb—N-MB interaction (» 2.47 Å). Since each MB structure is cationic (charge +1) and the niobate model is anionic (charge -2), we can point out that both modes of interaction can be favorable and form a neutral PONb-MB system. The EPR analyzes were carried out to characterize the reactive oxygen species, which are the active groups in anticancer activity. No paramagnetic signal was observed in EPR spectra (not shown) for PBN experiments (Figure 2 a), i.e., the studied compounds cannot produce radicals in such experimental conditions. On the other hand, TEMPO was detected only for PONb-MB and MB. As presented in Figures 2 b and c, the singlet oxygen production initiated at five minutes and, after that, evolved until the end of the test (fifteen minutes). Moreover, the time-dependent signal was four times more intense for PONb-MB compared to MB, which indicates that the electrostatic compound produced from the PONb and MB reaction catalyzes the conversion of molecular oxygen from the triplet to the singlet state. These results show that the species formed by the reaction between PONb and MB dye to form PONb-MB has the potential to generate reactive species of the singlet oxygen type, indicating high potential for use as anticancer. In addition, even though the PONb species did not present this type of oxygenated species, the peroxo groups (observed in experimental characterizations and theoretical calculations) can present positive effects in anticancer tests [5,7]. Studies via Raman spectroscopy corroborated to identify the peroxo groups in the PONb species. In fact, the Raman spectra (Figure 2d) showed an intense band in the lower frequencies for niobium compounds. Such a band is usually associated with the Nb-O lattice band [19,31,32]. In the theoretical Raman spectrum, this band was observed in the range of 680-800 cm -1 (see the bands highlighted in Figure S4d and Table S3). Furthermore, the PONb shows a band around 880 cm -1 that is characteristic of the vibrational mode of peroxo species [33]. However, in the PONb-MB system, this signal was partially superimposed by MB dye bands. Our DFT calculations showed that the stretching modes of the peroxo groups in the PONb model are observed in the range of 1230-1400 cm -1 (see the bands highlighted in Figure S4d and Table S3). For the peroxo groups involved in the intermolecular interactions between PONb and MB, these bands are displaced to 740-770 cm -1 (see the bands highlighted in Figure S4f and Table S3). Experimentally, it was observed that the PONb-MB presents all the vibrational modes of MB, but they are more intense, and their frequencies slightly deviated (see Figure 2e), which again indicates that the PONb-MB is a new compound, not just a simple mixture of PONb and MB dye [34–36]. The differences found between theoretical and experimental frequencies for the analyzed vibrational modes are related to the theoretical unimolecular model proposed. The experimental solid sample has PONb-PONb interactions that can affect the absorption spectrum of the analyzed groups and that were not considered in our theoretical model. Nevertheless, the B3LYP calculations of the vibrational modes showed good agreement with the experimental data. Details of the theoretical assignments of the IR and Raman spectra (Figure S4 and Table S3) are found in the Supporting Information. Figure 2. Time-dependent EPR spectra of TEMPO for PONb (a), MB (b), and PONb-MB (c). Raman spectra of PONb and MB dye (d), PONb-MB, and MB dye (e). Differences in vibrational modes between PONb-MB and MB are in the range of 680 - 880 cm -1 . 4.2- Cell viability associated with PONb and PONb-MB in HeLa (tumoral), MRC-5 and L929 (healthy) cell lines. Figure 3 displays the cytotoxic effects of PONb (a) and PONb-MB (b) on HeLa and MRC-5 cells (treated for 24 h), evaluated by the MTT assay. There was a significant reduction in cell viability for polyoxoniobate compounds at concentrations above 1 mg L -1 , in the two tested strains. For PONb, this decrease is more significant and the viability for tumor cells is less than 4 % from the concentration of 5 mg L -1 (Figure 3a). The viability of the healthy cell, incubated at the same concentration, is about 40% indicating certain tumor selectivity in the PONb treatment. This cytotoxic profile can be verified also for PONb-MB, although to a lesser extent (Figure 3b). At the concentration of 5 mg L -1 , the cell viability is 15 % for tumor cells and 39 % for healthy cells. On the other hand, the compounds used in this study are still poorly understood regarding their biocompatibility, therefore, assays with different cell lines are required to evaluate cytocompatibility. In order to confirm the low toxicity for healthy cells, which is a highly desirable property for a drug candidate, thectionn of PONb and PONb-MB on the cell viability of L929 healthy cells was also investigated (Figure 3). The PONb (Figure 3c) and PONb-MB (Figure 3d) solutions had no significant cytotoxic effect at concentrations lower than 10 mg L -1 , after 24 and 48 h of incubation. These results further demonstrate that the two polyoxoniobate molecules have no cytotoxic effect on L929 healthy cells (fibroblasts). Fluorescence microscopy images of the cells after 24 h of incubation with the prepared materials are shown in Figure 3e. Detection of propidium iodide (DNA-specific red fluorescence) shows dead cells while calcein-MB makes the cell membrane of living cells visible (green fluorescence). A larger number of dead cells are observed in the images of the tumor cells incubated with the materials, mainly in the PONb group (Figure 3e). The percentage of apoptosis calculated after counting the cells is shown in Figure 3f. For the two studied lines, the control group had a death rate of less than 1 %. Concerning the groups incubated with PONb and PONb-MB (10 mg/L), the apoptosis was statistically comparable to tumor cells. Nevertheless, it should be emphasized that an apoptosis percentage of approximately 3x higher was obtained in tumor cells compared to healthy cells, for both PONb and PONb-MB (Figure 3f). This result confirms the effect observed in the cell viability assay, indicating a greater action of the prepared polyoxoniobates on tumor cell lineage. The ability to cause damage to tumor cells with significant selectivity makes both compounds promising materials for cancer therapy. Figure 3. Cell viability in HeLa (tumor) and MRC-5 (healthy) in the presence of PONb (a) and PONb-MB (b). Cell viability in L929 healthy cells with PONb (c) and PONb-MB (d) and solutions at different concentrations and incubation times. Fluorescence microscopy images of the cells after 24 h of incubation with PONb and PONb-MB (e) and cell apoptosis (f). The action mechanism of the polyoxoniobates selectivity related to tumor cell was investigated. For that, studies involving the formation of intracellular reactive oxygen species (ROS) were performed. Representative fluorescence microscopic images of the cells after 4 h of incubation are presented in Figure 4. The cells with higher ROS species are shown as red dots (Figure 4a). As such, the redder dots, the greater the ROS generation. It is known from the literature that when cells are subjected to ROS, these species can affect the vascular system of the tumor cell causing its death [37]. The percentages of dead and live cells were determined by counting (Figure 4b). In both cell lines, ROS species increased for PONb and PONb-MB groups compared to control cells. Moreover, the death of tumor cells showed a statistically significant increase (about 50 %). This result indicates that the capacity to promote higher cell death signaling in tumor cells is associated with a greater generation of ROS species. Habtetsion et al.[38], showed that adoptive immunotherapy deeply altered tumor metabolism, resulting in glutathione depletion and accumulation of ROS in tumor cells. Raza et al.[39] reported that a certain level of ROS is required by cancer cells, which can lead to cytotoxicity in them. In the present work, the new Nb-containing molecules are rich in labile oxygen species which might be interesting as a cancer therapy strategy. Figure 4. (a) Fluorescence microscopic images of the cells after 4 h of incubation and (b) the percentages of dead and live cells determined by counting. The results indicate that the formed niobium species have anti-cancer action through two different mechanisms: ( i ) the PONb species did not present radical species or the formation of singlet oxygen. Thus, the peroxide-type bonds (or peroxo group) present in the molecule (Figure 5a) must be directly responsible for the effect observed in the cell viability tests. An indication that the peroxo group in the PONb forms an interaction with the tumor cells decreasing their cell viability can be observed by the 31 P NMR spectra of the PONb in contact with the 5-GMP molecule (used to simulate the possible interaction between the active species and the DNA). Figure 5b shows the 31 P NMR spectrum for the 5-GMP molecule. It is observed that it has a single P signal with a chemical shift of 5.95 ppm, corresponding to the P of the phosphate group. However, in Figure 5c, it is shown the 31 P NMR spectrum of the molecule in contact with PONb. The presence of another signal was observed at a smaller chemical shift, 5.26 ppm, possibly corresponding to the P interacting with the peroxo group. The presence of this new group affects the chemical environment of the P (phosphate group) from the 5-GMP molecule, because the P atom that is interacting with the peroxo group is more shielded. After all, this group is more electronegative, which causes the appearance of a signal with less chemical shift [40]. The PONb-MB species showed a capacity for generating singlet oxygen (Figure 5d) probably due to the action of ambient light promoting the electronic transition for the conduction band of the species formed by the reaction of PONb with MB dye. The innovative results presented in this work indicate that ROS are formed and have their generation modulated when the dye is chemically bound to the polyoxoniobate species[39]. We believe that these results open a new line of possibilities since the dyes of various natures can present even better results. In addition, inorganic cations may also interact with the negative charges of the polyoxoniobate generating more versatile anticancer candidates. In this context, our new achievements might contribute to the development of new drugs with antitumoral and antiviral proposals. Figure 5. (a) Representative scheme of the peroxide-type bond (or peroxo group) and how it acts on tumor cells, (b) 31 P NMR spectrum for the 5-GMP molecule, (c) 31 P NMR spectrum showing the interaction between PONb and 5-GMP and, (d) the representative scheme of singlet oxygen generation promoted by the action of light in the compound PONb-MB. 4.3- In vivo tests for biosafety assessment . The promising results obtained for the polyoxoniobates for tumor cells motivated further investigation through in vivo tests with mice. Among several questions that can be raised, one can point out the concern of pharmaceutical accumulation, i.e ., the accumulation of the polyoxoniobates in mice’s organs. The results showed a biphasic clearance profile, indicated by the red curve in Figure 6a, with a fast half-life of 7.7 min and a slow half-life of 9.1 min. The biodistribution profile of the possible drug candidate was determined by the Nb loading in mice’s organs (Figure 6b). Some uptake was observed in kidneys and liver, which may represent the contribution of these organs in the elimination of the Nb compound. Noteworthy is the higher uptake in the heart at 60 min and 240 min post-injection, which indicates the affinity of PoNB-MB towards this organ and suggests potential cardiotoxicity. Figure 6. In vivo studies after PoNB-MB intravenous administration into healthy Swiss mice. Blood clearance assays (a) and the biodistribution profile post-injection of the PONb-MB determined by the Nb loading in the mice’s organs (b). Thus, to better analyze and prove the safety profile of PONb-MB, a toxicological assay was performed (Table 1). The data suggest that the treatment with PONb-MB may not cause tissue damage. The CKMB value indicates no cardiac tissue damage, even with the higher uptake presented in the biodistribution study. Therefore, the values found for all the tests are statistically like those of the control groups, the occurrence of acute toxicity not being indicating. Table 1. Biochemical parameters of healthy Swiss mice after intravenous administration of PONb-MB 1 h post-injection (n = 6). Control PONb-MB CKMB (U/L) 0.32 ± 0.13 0.26 ± 0.12 AST (U/L) 184.43 ± 55.98 178.26 ± 65.96 ALT (U/L) 22.17 ± 4.56 23.75 ± 4.39 Creatinine (mg/dL) 0.61 ± 0.05 0.56 ± 0.06 Urea (mg/dL) 47.54 ± 13.26 60.18 ± 14.95 5. CONCLUSIONS In this study, we describe the synthesis of unique polyoxoniobate compounds obtained through a new synthetic route from species of niobium oxide treated with hydrogen peroxide solution (PONb). The addition of methylene blue dye (MB) to PONb solution generated new oligomer species (PONb-MB). Theoretical analyses and thermodynamic properties contributed to propose a new molecular structure for the polyoxoniobate compounds. Both PONb and PONb-MB present reactive oxygen species (ROS), which resulted in high activity and selectivity as an antitumoral drug candidate. The results indicate that the proposed polyoxoniobate molecule can be useful in the case of selective cytotoxic effect on tumor cells. It is noteworthy that the viability of healthy cells was not strongly affected by the presence of both compounds, which may indicate that these Nb compounds can be widely and safely used as new possible drugs. Additionally, in vitro safety outcomes are corroborated by in vivo assays, which showed minimal uptake in the major organs with no sight of toxicity in the renal, liver, and heart biochemical analyses. In summary, all these findings open new perspectives for this class of compounds to be a promising alternative for cancer treatment with particular attention as antitumor and quite low toxicity activities. Further investigations on therapeutic efficacy and new developments of delivery systems are certainly necessary for future clinical tests. One can also envision that these new polyoxoniobate molecules can be tested in conjunction with other conventional approaches such as chemotherapies to treat cancer cells, instead of simply replacing other traditional drugs since it has demonstrated low toxicity for healthy cells that eventually surround the tumors. Declarations ACKNOWLEDGEMENTS The authors are immensely grateful for the financial support of CAPES, CNPq, and FAPEMIG. Furthermore, to the Companhia Brasileira de Metalurgia e Mineração (CBMM) and Nanonib for the donation of niobium compounds. Leonardo A. De Souza thanks the FAPERJ for the financial support (E-26/211.911/2021). AUTHOR CONTRIBUTIONS STATEMENT Luiz C. A. Oliveira: Term, Supervision, Conceptualization, Project administration, Funding acquisition, Resources. Cinthia C. Oliveira: Conceptualization, Methodology, Writing - Original Draft, Writing - Review & Editing, Visualization. Poliane Chagas : Investigation, Data Curation. Ana P. Heitmann: Investigation, Data Curation . José B. Gabriela: Investigation, Data Curation. Jadson C. Belchior: Formal analysis, Writing - Original Draft. Leonardo A. De Souza: Investigation, Formal analysis, Writing - Original Draft. Tiago H. Ferreira: Investigation, Methodology, Writing - Original Draft. Sued E. M. Miranda: Investigation, Methodology . André L. B. Barros: Methodology, Writing - Original Draft. Cynthia L. M. Pereira: Writing - Original Draft, Visualization. Vinícius D. N. Bezzon: Methodology, Writing - Original Draft. Fábio F. Ferreira: Methodology, Writing - Original Draft Data availability The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. Source data are provided with this paper. References Rhule, J. T.; Hill, C. L.; Judd, D. A.; Schinazi, R. F. Polyoxometalates in Medicine. Chem. Rev 98 , 327–357 (1998). Nyman, M. Polyoxoniobate Chemistry in the 21 St Century. Dalt. Trans 40 , 8049–8058 (2011). Zhang, Y. et al. Four polyoxonibate-based inorganic-organic hybrids assembly from bicapped heteropolyoxonibate with effective antitumor activity. Cryst. Growth Des. 14 , 110–116 (2014). Wang, S., Sun, W., Hu, Q., Yan, H. & Zeng, Y. 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Silverstein, R. M.; Webster, F. X.; Kiemle, D. J.; Bryce, D. L. The Spectrometric Identification Of Organic Compounds . (Wiley, 2014). Additional Declarations No competing interests reported. Supplementary Files SupInfPaperPolyoxoniobateScientificReports.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4033921","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":284775726,"identity":"77ce3b8f-7f86-42e2-9966-22b2451b2ad8","order_by":0,"name":"Luiz C. A. Oliveira","email":"","orcid":"","institution":"Universidade Federal de Minas Gerais","correspondingAuthor":false,"prefix":"","firstName":"Luiz","middleName":"C. 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Raman spectra of PONb and MB dye (d), PONb-MB, and MB dye (e). Differences in vibrational modes between PONb-MB and MB are in the range of 680 - 880 cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4033921/v1/5bba60512407364da82b7ab7.jpg"},{"id":53880527,"identity":"7bac578b-18d2-4880-ad18-5aac529e0627","added_by":"auto","created_at":"2024-04-01 17:42:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1431567,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability in HeLa (tumor) and MRC-5 (healthy) in the presence of PONb (a) and PONb-MB (b). Cell viability in L929 healthy cells with PONb (c) and PONb-MB (d) and solutions at different concentrations and incubation times. Fluorescence microscopy images of the cells after 24 h of incubation with PONb and PONb-MB (e) and cell apoptosis (f).\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4033921/v1/93333ba43cee4fa2d15085a2.jpg"},{"id":53880526,"identity":"35d67385-511d-479d-b347-f30f5c994c1f","added_by":"auto","created_at":"2024-04-01 17:42:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1205407,"visible":true,"origin":"","legend":"\u003cp\u003e(a)\u003cstrong\u003e \u003c/strong\u003eFluorescence microscopic images of the cells after 4 h of incubation and (b) the percentages of dead and live cells determined by counting.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4033921/v1/fa62c6915bbd2bac2f936004.jpg"},{"id":53880529,"identity":"e61636f4-f60c-4713-8d0e-daba77820075","added_by":"auto","created_at":"2024-04-01 17:42:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":164380,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Representative scheme of the peroxide-type bond (or peroxo group) and how it acts on tumor cells, (b) \u003csup\u003e31\u003c/sup\u003eP NMR spectrum for the 5-GMP molecule, (c) \u003csup\u003e31\u003c/sup\u003eP NMR spectrum showing the interaction between PONb and 5-GMP and, (d) the representative scheme of singlet oxygen generation promoted by the action of light in the compound PONb-MB.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4033921/v1/fd3afa8a410279be932897d0.jpg"},{"id":53880530,"identity":"fa039e19-bd7f-434f-a543-253179cb0587","added_by":"auto","created_at":"2024-04-01 17:42:58","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":90573,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo studies after PoNB-MB intravenous administration into healthy Swiss mice. Blood clearance assays (a) and the biodistribution profile post-injection of the PONb-MB determined by the Nb loading in the mice’s organs (b).\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4033921/v1/42c1d6579507b541266512ae.jpg"},{"id":64754130,"identity":"4e1d8b2f-370b-4b83-8ff9-5cabe140518b","added_by":"auto","created_at":"2024-09-18 11:23:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4442585,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4033921/v1/76f16891-4b11-4482-8a44-610c92bacf3d.pdf"},{"id":53880531,"identity":"e111fb4c-2046-4057-9200-83aa54bf96bb","added_by":"auto","created_at":"2024-04-01 17:42:58","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4021432,"visible":true,"origin":"","legend":"","description":"","filename":"SupInfPaperPolyoxoniobateScientificReports.docx","url":"https://assets-eu.researchsquare.com/files/rs-4033921/v1/c306c6ed4cac0473004cfdde.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antitumor activity and selectivity of a unique nanostructured polyoxoniobate","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003ePolyoxometalates (POMs) are anionic metal oxo clusters usually composed of d\u003csup\u003e0\u003c/sup\u003e species of the Groups VI and V (e.g., Mo, W, V, Ta, and Nb)[1,2]. They are oligomeric aggregates of early transition metal cations linked by oxide anions that are formed by self-assembly processes [1,2]. The POMs have high-water solubility, low toxicity for humans, and their redox and photochemical capacities can present diversified usages, mainly in catalysis and medicine [3,4]. Mo-based POMs have been applied for antitumor activity while those based on V are known for their antiviral effect[1]. Although POMs based on W, Mo, and V have been deeply investigated in the last decades, the chemistry of Nb-based POM (called polyoxoniobates, PONb) is less explored [1]. Nevertheless, it must be emphasized that PONb has shown distinctive properties such as high negative charge, base surface oxygen atoms, and a wide range of cluster sizes that are suitable for several applications, for example inhibiting tumor development. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eParticularly, antitumor activities of PONb can be associated with the apoptosis process and oxidation of cellular components[3,5\u0026ndash;7]. Its bio-application and understanding its behavior in a biological environment is one great challenge. Furthermore, it is also important to investigate how size, shape, and composition can influence its toxicity profile. In general, the experimental determination of molecular structures is not an easy task. From this point of view, molecular modeling techniques based on computational methods of quantum chemistry appear as an alternative to calculating structural, electronic, and spectroscopic properties. Thus, it is possible to establish the structure-reactivity-activity relationship in the design of new drugs for various applications in medicinal chemistry [8\u0026ndash;11]. Particularly, we have recently prepared a PONb complex by proposing a new class of materials to be used as anticancer agents [7]. Experimental and theoretical analyzes demonstrated that the Nb complex species presented reactivity and stability when docked into the DNA crystallographic structure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHerein we report the synthesis of unique PONb species with emphasis on its application to cancer treatment. Two strains of carcinogenic cells were used to demonstrate the versatility of PONb for inhibiting tumor cells, with quite low toxicity to healthy ones. Furthermore, \u003cem\u003eex vivo\u003c/em\u003e biodistribution and toxicological studies with healthy mice revealed negligible damage, showing the potential application of PONb as a drug candidate for tumor treatment with particular attention to the achievements of several human cell carcinomas \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e"},{"header":"2.\tMATERIAL AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e2.1-\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePreparation of polyoxoniobates.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the synthesis of niobium polyoxoniobates, 46.2 mmol of the NH\u003csub\u003e4\u003c/sub\u003e[NbO(C\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e(H\u003csub\u003e2\u003c/sub\u003eO)](H\u003csub\u003e2\u003c/sub\u003eO)n salt (provided by CBMM) was dissolved in 100 mL of distilled water at 90 \u0026deg;C. Then, an NH\u003csub\u003e4\u003c/sub\u003eOH solution (5 mol L\u003csup\u003e\u0026minus;1\u003c/sup\u003e) was slowly added to the salt solution for precipitation of the niobium oxyhydroxide. The suspension was aged under magnetic stirring for 12 h and the obtained solid was filtered. Afterward, 8 mL of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (30 %, v/v) and 100 mL of distilled water were added to 300 mg of niobium oxyhydroxide. After 12 h, the solid was centrifuged and the liquid supernatant contained the leached species of peroxoniobate complex (PONb).\u0026nbsp;The PONb yellow solution was used to prepare the PONb-MB after reaction with a methylene blue solution (MB). For that, 10 mL of a 50 mg L\u003csup\u003e-1\u003c/sup\u003e solution of the MB dye was added to 10 mL of the PONb. The cationic dye strongly binds to the structure of the PONb by electrostatic attraction, forming the PONb-MB compound, of blue coloration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 -\u003c/strong\u003e \u003cstrong\u003eCharacterization of the compounds.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Nb concentration in the polyoxoniobate solutions was determined by Inductively Coupled Plasma Mass Spectrometry, ICP-MS Agilent 7700, using an analytical curve for the Nb element. The Polyoxoniobate solution (PONb) and the solution after the methylene blue dye addition (PONb-MB) were characterized by UV-Vis spectroscopy (Shimadzu - 2600/2700) and Zeta potential measurements (Zetasizer Nano ZS).\u0026nbsp;The analytical procedure was conducted in a Zeta-sizer Nanoseries Zs apparatus (Malvern Instruments, Malvern, UK) after the adequate dilution of the samples in ultra-pure MilliQ\u0026reg; water. The results were expressed as mean \u0026plusmn; standard deviation for at least three different batches of each sample.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Polyoxoniobate solution was placed in contact with the 5-GMP molecule (Sodium guanosine 5-monophosphate, which simulates the DNA) to analyze the possible interaction between the 5-GMP molecule and the PONb compound. This interaction was verified by NMR of \u003csup\u003e31\u003c/sup\u003eP (Bruker spectrometer 200 MHz), using 1024 scans and phosphoric acid as standard. The spectrum of the 5-GMP pure molecule was obtained using 20 mg of 5-GMP dissolved in 600 \u0026micro;L of D\u003csub\u003e2\u003c/sub\u003eO. Afterward, a 5-GMP and PONb mixture was prepared and irradiated for 7 min using a light wavelength of 365 nm. Then, 400 \u0026micro;L of D\u003csub\u003e2\u003c/sub\u003eO was added to 200 \u0026micro;L of the irradiated sample, and the mixture was taken to the spectrometer.\u003c/p\u003e\n\u003cp\u003eThe reactive oxygen species (ROS) were characterized by the EPR spin trapping technique using PBN (N-tert-butyl-\u0026alpha;-phenylnitrone) and TEMP (2,2,6,6-tetramethyl-4-piperidone) as spin trapping molecules. The PBN is known to react with radicals and produces very stable spin adducts[12], while the TEMP reacts with singlet oxygen producing a nitroxide radical called TEMPO [13]. One milliliter of PBN (85 mM) or TEMP solution (1M) was added to 1 mL of PONb, PONb-MB, and MB aqueous solutions. These mixtures were placed in a 5 mL beaker and magnetically stirred for 20 min. Aliquots of the samples were extracted every 5 min using a capillary glass tube and the data were acquired using a X-band Magnettech miniscope 400 spectrometer. The tests were performed at room temperature and illumination. Moreover, Raman data were collected on a Bruker Senterra spectrometer with a 532 nm laser operating at 20 mW.\u003c/p\u003e\n\u003cp\u003eThe structure of the materials was further investigated by X ray powder diffraction and PDF patterns. The XRPD patterns were collected using a STADI-P diffractometer (Stoe\u0026reg;, Darmstadt, Germany) with MoK\u0026alpha;1 radiation (\u0026lambda; = 0.7093 \u0026Aring;), operating in transmission geometry. The measurements for the application of the low-energy PDF method were performed using 0.5-mm diameter special glass capillaries n\u0026ordm; 14 (Hilgenberg\u0026reg;, Malsfeld, Germany), which reduces the container contribution to the measurement. The data were measured from 2.0 to 136.4\u0026deg; (2q). An empty capillary was measured with the same conditions as the filled capillaries to remove the instrumental contributions of the measurements. A Q-range from 0.1 to 16.4 \u0026Aring;\u003csup\u003e-1\u003c/sup\u003e was considered for the S(Q) (total structure-function) that was Fourier transformed to obtain the PDF pattern. S(Q) and PDF patterns were obtained using the PDFGetX3 software[14]. In this software, all non-structural contributions are determined through an \u003cem\u003ead hoc\u003c/em\u003e approach, then they are parameterized, and the parameters are used to obtain the structure function S(Q).\u0026nbsp;The structure functions for lyophilized PONb and Nb\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u0026nbsp;\u003c/sub\u003ewere presents in Figure S5a and the Fourier Transform of these results yielded the PDF pattern for the samples, shown in Figure S5b.\u003c/p\u003e\n\u003cp\u003eThe main distances up to 10 Å were calculated using the Bond_Str software [15] based on the crystallographic information framework file (CIF file) for the structure of Nb\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e obtained in the Inorganic Crystal Structure Database\u0026reg;, under the 51176 reference code. The calculated distances for individual atomic pairs were used to compare the distances observed in the PDF patterns.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3-\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eIn\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003cem\u003evitro\u003c/em\u003e comparative tests using HeLa (tumoral) and MRC-5 (healthy) cells.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe assays were performed using the cell lines HeLa (ATCC\u0026reg;-CCL-2 \u0026trade;), a tumor cell derived from human cervical adenocarcinoma, and MRC-5 (ATCC\u0026reg;-CCL-171 \u0026trade;), a healthy cell line derived from human lung fibroblast. The cells were cultured in a CO\u003csub\u003e2\u003c/sub\u003e incubator (5% CO\u003csub\u003e2\u003c/sub\u003e Cole Parmer) with a humid atmosphere at 37\u0026deg;C in Dulbecco\u0026apos;s modified Eagle\u0026apos;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/amphotericin B (complete DMEM medium). Upon reaching 80% confluency, cells were harvested using sterile PBS to wash the plate and sterile 1% Trypsin solution to detach the cells to be collected in the falcon tube. The cells were then counted with the aid of a Neubauer chamber.\u003c/p\u003e\n\u003cp\u003eThe cell viability assay with sulforhodamine B (SRB) was performed according to the literature [16]. For this assay, the cells were seeded in 96-well culture plates\u0026nbsp;\u003cbr\u003e(1 x 10\u003csup\u003e4\u003c/sup\u003e/well) and incubated at 37\u0026deg;C/5% CO\u003csub\u003e2\u003c/sub\u003e. After 24 h, the cells were treated with PONb and PONb-MB at concentrations of 1, 5, 10, 50, and 100 ppm. The group defined as cell control received only full DMEM medium and was maintained under the same conditions as the treated groups. After 24 h, the cells were fixed through protein precipitation with 10% trichloroacetic acid at 4\u0026deg;C (100 \u0026micro;L/well) for 1 h. After five washing steps, the cells were stained with 0.04 % SRB (100 \u0026micro;L/well) at room temperature for 1 h. Afterward, the plate was washed with 1% acetic acid four times to remove the unbound stain. The plate was air-dried at room temperature and the bound protein stain of each well was solubilized with 100 \u0026micro;L of 10 mM Tris base [tris(hydroxymethyl)aminomethane]. The optical density was measured by spectrophotometry on a UV-Visible Microplate Reader (Molecular Devices) at 510 nm. The survival fraction was calculated as a percentage of the control (Absorbance in control = 100% survival). The experiments were carried out in triplicate.\u003c/p\u003e\n\u003cp\u003eThe ability of the material to damage the cell membrane and promote cell death signaling was assessed by fluorescence microscopy imaging. For the cell dead assay, the cells were seeded in 96-well culture plates (1 x 10\u003csup\u003e4\u003c/sup\u003e/well) and incubated at 37\u0026deg;C/ 5% CO\u003csub\u003e2\u003c/sub\u003e. After 24 h, the cells were treated with PONb and PONb-MB at a 10 mg/L concentration. The group defined as control cells received only a complete medium and were maintained under the same conditions as the treated group. After 24 h of incubation at 37\u0026deg;C, the cells were supplemented with 1 \u0026mu;M calcein-MB (Life Technologies) and 2 \u0026mu;M propidium iodide (Life Technologies) in DMEM culture medium. The images were collected with 4x objective lens on an inverted fluorescence microscope Olympus IX70 (Olympus America, Melville, NY). The percentage of apoptosis was calculated from the ratio between the number of dead and living cells, which were counted with the aid of ImageJ software. Each treatment was performed in triplicate, three different images were counted, and the means were used for statistical analysis.\u003c/p\u003e\n\u003cp\u003eReactive oxygen species (ROS) are the result of oxygen reduction during aerobic respiration by various enzymatic systems within the cell. The assay detects the formation of intracellular ROS (especially superoxide and hydroxyl radicals) in live cells employing the fluorometric method. For this assay, the cells were seeded in 96-well culture plates (1 x 10\u003csup\u003e4\u003c/sup\u003e/well) and incubated at 37\u0026deg;C/ 5% CO\u003csub\u003e2\u003c/sub\u003e. After 24 h, 100 \u0026micro;L of ROS Detection Reagent was added to each well. The plate was incubated for another 1 h and then the cells were treated with PONb and PONb-MB in PBS medium at the concentration of 10 mg/L. The group defined as control cells received only medium and were maintained under the same conditions as the treated group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Multi-Mode Reader Cytation 5 Cell Imaging (Bio tek) was used to measure the fluorescence intensity and to collect the images with a 4x objective lens. The detection of ROS was performed using an excitation wavelength of 490 nm and collecting the fluorescence emission of 525 nm. Each treatment was performed in triplicate, three different images were counted, and the means were used for statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4- \u003cem\u003eIn\u003c/em\u003e \u003cem\u003evitro\u003c/em\u003e tests using L929 (healthy) cells: cell viability with MTT assay.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHealthy cell lines L929 (connective tissue fibroblasts) were cultured; Manassas, VA, USA in Dulbecco\u0026apos;s Modified Eagle\u0026apos;s Medium, supplemented with 10% FBS (Fetal Bovine Serum) and 1% Amphotericin B-Streptomycin (100 IU mL\u003csup\u003e-1\u003c/sup\u003e). Cells were maintained under the following conditions: 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e, with medium exchange every two days. After reaching confluence, the cells were seeded in 96 well plates in concentrations around 1 x 10\u003csup\u003e5\u003c/sup\u003e cells/well for the tests. The tests were performed after the cells reached 70% confluence in which different concentrations of PONb and PONb-MB were added in each well: 3.0; 5.0; 8.0; 10.0; 12.0; 15.0 and 20.0 mg mL\u003csup\u003e-1\u003c/sup\u003e, in six replicates. An aqueous solution of methylene blue dye (MB) at 12.5 \u0026mu;g mL\u003csup\u003e-1\u003c/sup\u003e was used as a positive control. The solutions were sterilized by filtration (0.22 \u0026mu;m membrane). The contact time (incubation) with the PONb, PONb-MB, and MB solutions was 24 and 48 h at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Then, the reagent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT was added to the plates. After 4 h of incubation at 37\u0026deg;C for the formazan crystals formation, a sodium dodecyl sulfate (SDS) detergent solution was added. After 14 h, the UV-Vis Spectrophotometer readings were taken at a wavelength of 570 nm. The respective negative controls (containing only untreated cells) and blank controls (no cells and no treatment, containing all solutions involved in the test) were analyzed on all plaques. The effects on cell viability were expressed as a percentage of the viability detected for the treated cells versus the viability of the cells incubated with culture medium alone. Data are expressed as the mean \u0026plusmn; standard deviation (n = 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5- \u003cem\u003eIn\u003c/em\u003e \u003cem\u003evivo\u003c/em\u003e experiments.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was carried out in compliance with the ARRIVE guidelines. Third\u0026nbsp;Swiss CF1 female mice (6-8 weeks old, 24-28 g) were obtained from the Faculty of Pharmacy of the Federal University of Minas Gerais (UFMG). The animals had free access to water and food and were kept under controlled environmental conditions. All animal procedures were approved by the Institutional Animal Care and Use Committee (CEUA/UFMG), under protocol 118/2019. Mice were randomly separated into groups of 6 animals for the \u003cem\u003ein vivo\u003c/em\u003e studies. The number of mice was defined similarly to other papers from our group [17,18].\u003c/p\u003e\n\u003cp\u003eFor the blood clearance experiments, aliquots of 100 \u0026mu;L of a tenfold diluted PONb-MB solution were injected intravenously into the healthy Swiss mice (n = 6, 6-8 weeks old, 20-24 g). An incision was made in the tail of the animals and blood was collected pre-weighed tubes at 15, 30, 60, 120, 240, 480, 1440, and 2880 min after administration. The mice were anesthetized with a mixture of xylazine (15 mg/kg) and ketamine (80 mg/kg). Blood was digested using HCl and HNO\u003csub\u003e3\u003c/sub\u003e solution, using a ratio of tenfold solution diluent to blood.\u003c/p\u003e\n\u003cp\u003eIn the biodistribution assays, aliquots of 100 \u0026mu;L of a tenfold diluted\u0026nbsp;PONb_MB\u0026nbsp;solution were injected intravenously into healthy Swiss mice (n = 6, 6-8 weeks old, 20-28 g). At the post-injection times of 30, 60, and 240 min, organs such as spleen, heart, liver, lungs, kidneys, and blood were removed and weighed [19]. The organs were macerated using tissue macerator, and the supernatant was used for the determination of the niobium content present in each organ. For the analysis by ICP-MS, the samples went through acid digestion, using distilled nitric acid (Merck), 65 % (w/v). For sample preparation, 1 mL of nitric acid was added to each 1 mL of the supernatant resulting from the digestion of each organ. The mixture was left under constant magnetic stirring at 50\u0026deg;C for two hours and then centrifuged using a QUIMIS centrifuge, model 0222E24. The supernatants were transferred to polyethylene tubes, completed to 10.0 mL, and analyzed by ICP-MS.\u003c/p\u003e\n\u003cp\u003eFor the biochemical analyses, aliquots of 100 \u0026mu;L of a tenfold diluted PONb_MB solution were injected intravenously into healthy Swiss mice (n = 6, 6-8 weeks of age, 20-28 g). After 24 h from the injection, blood was collected using an anticoagulant (0.18% w/v EDTA). The blood was centrifuged at 5000 rpm for 10 min to obtain the plasma used to perform the biochemical analyses. The biochemical tests were performed using commercial kits from Labtest\u0026reg; (Lagoa Santa, Brazil) through Bioplus BIO-2000 semiautomatic analyzer equipment (S\u0026atilde;o Paulo, Brazil). Heart (CK-MB), liver (AST, aspartate aminotransferase, and ALT, alanine aminotransferase), and kidney (urea and creatinine) functions were tested.\u003c/p\u003e\n\u003cp\u003eIt is worth mentioning that all the reagents used in \u003cem\u003ein vitro\u003c/em\u003e and\u0026nbsp;\u003cem\u003ein vivo\u003c/em\u003e experiments have\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u0026gt; 95% purity by HPLC analysis.\u003c/p\u003e\n\u003cp\u003eThe methods presented in the work were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e"},{"header":"3.\tCOMPUTATIONAL DETAILS","content":"\u003cp\u003eAll calculations were performed with the Gaussian 09 package [20] employing the density functional theory (DFT) methodology[21] with the B3LYP functional [22] using the 6-31G(d,p) basis set [23] for O, H, N, S, and C atoms and the LANL2DZ [24]effective core potential (ECP) for Nb(V) ion. B3LYP/LanL2Dz/6-31G(d,p) level of theory used in the present study has been widely used in the literature [25,26] and has demonstrated good agreement against experimental data for Nb compounds. Therefore, we point out that this level of theory can contribute to experimental analysis in elucidating the structural properties of the system.\u003c/p\u003e\n\u003cp\u003eFirstly, the monomers corresponding to the structures of PONb and MB (Fig. S1, Supporting Information) were fully optimized in the gas phase and their geometries (Table S1) were used to build the structure of the PONb-MB system. The monomers (PONb and MB) and the PONb\u0026ndash;MB system were characterized by harmonic frequency analysis indicating the optimized geometries as true minima. Finally, electronic excitations (UV-Vis spectra) were calculated using the B97-D functional [27]\u0026nbsp;by TD-DFT formalism [28] and the molecular orbital analysis was computed, both using the Polarizable Continuum Model (PCM) [29] to describe solvent effects - water solvent (dielectric constant,\u0026nbsp;\u0026epsilon;=78.3553).\u003c/p\u003e"},{"header":"4.\tRESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003e4.1- Characterization of the polyoxoniobates.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Polyoxoniobate solutions were characterized by UV-Vis (blue lines in Figures 1a and b). The spectrum of the pure PONb shows an intense band in the UV region, centered at about 270 nm, typical of peroxide groups (ROS) that are linked to Nb atoms in the PONb structure (Figure 1a). However, the MB dye addition to PONb generates a new species\u0026nbsp;\u003cbr\u003e\u0026nbsp;(PONb-MB) that absorbs radiation in the visible light region of the spectrum, with maximums at 574, 694, and 775 nm, besides the high-intensity UV peak centered at 243 nm (Figure 1b). It is known that the MB dye has absorption bands at 612 and 665 nm,[7,30] which are not observed in the spectrum of PONb-MB (Figure 1b). This result indicates the formation of a new species of polyoxoniobate rather than a sole mixture of the two compounds of PONb and MB dye. It is also important to point out that the ROS observed (Bonded peroxide group) is not a radical species, and for this reason, the niobium compound might not be toxic to healthy cells.\u003c/p\u003e\n\u003cp\u003eThe theoretical spectra in the UV-Vis region (red lines in Figures 1a and b) show that the PONb species has an intense band at 287 nm, \u003cem\u003ei.e\u003c/em\u003e., 6.3 % shifted to the low-energy region compared to the experimental band (Figure 1a). After the formation of the PONb-MB system (Figure 1b), two absorption bands are observed: (\u003cem\u003ei\u003c/em\u003e) an intense band at 274 nm assigned to an intramolecular transition between HOMO-2 (Figure 1d) and LUMO+5 (Figure S2a) orbitals and (\u003cem\u003eii\u003c/em\u003e) a weak and broad one in the 550 nm region assigned to a transition between HOMO-2 (Figure 1d) and LUMO (Figure 1) that can be attributed to the strong intermolecular interaction between the PONb and MB species. More details on the TD-DFT calculations observed in Figures 1c and d are found in the Supporting Information (Figure S3 a and b).\u003c/p\u003e\n\u003cp\u003eThermodynamic parameters of the PONb-MB interaction were calculated according to Equations S1 to S3. All calculated parameters are shown in Table S2. The interaction between PONb and MB molecules forms a strongly bound compound with energy stabilization of around -55.0 kcal mol\u003csup\u003e-1\u003c/sup\u003e. T\u0026Delta;S term is negative and, according to Equation S3, it contributes repulsively to \u0026nbsp;of the PONb-MB system. According to Equation S2, the \u0026nbsp;value is negative enough to produce a large negative \u0026nbsp;(-63.4 kcal mol\u003csup\u003e-1\u003c/sup\u003e), even though the thermal correction to enthalpy is negative (-8.4 kcal mol\u003csup\u003e-1\u003c/sup\u003e). Thus, these results suggest that the formation of the PONb-MB system is thermodynamically favorable to equal to\u0026nbsp;\u003cbr\u003e-37.1 kcal mol\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 1.\u0026nbsp;\u003c/strong\u003eUV-Vis absorption spectra (Experimental and simulation using TD-DFT calculation) for PONb (a) and PONb-MB system (b), fully optimized DFT structures of PONb monomer (c), and PONb-MB system (d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, to estimate the negative charge of PONb and PONb-MB molecules, the solutions were characterized by Zeta potential measurements. The results demonstrated a high negative value of the PONb cluster (-50.6 mV). These negative charges possibly allowed the binding of MB cationic dye to generate a new stable species of PONb-MB, in a good agreement with the theoretical calculations. This hypothesis was further attested by Zeta potential measurements of PONb-MB that presented a lower value (-36.5 mV) than the pure PONb, due to the electrostatic interactions between the cationic dye and the negative charge oxygen species in the PONb structure. In addition, the PONb species have an average size of 73(3) nm and the new polyoxoniobate (PONb-MB) presented a lower average size of 61(4) nm after reaction with MB.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe prepared polyoxoniobates (PONb and PONb-MB) were characterized via DFT\u0026nbsp;calculations, which indicate the formation of the structures shown in Figure S2. The\u0026nbsp;optimized geometry of PONb (Figure S2a) has short- and medium-range hydrogen bonds in the range of 1.48 - 1.87 \u0026Aring; between the hydrogen atoms of peroxide ligands and neighboring oxygenated ligands. The bond lengths Nb(V)\u0026ndash;O are in the range of\u0026nbsp;1.8 - 2.10 \u0026Aring; (), 2.14 - 2.22 \u0026Aring; () and 1.75 - 1.80 \u0026Aring; () and (). Similar results for these bond types were found by Grimme et al. [27] for the crystalline structure of the peroxopolioxoniobate ion salt [As\u003csub\u003e2\u003c/sub\u003eNb\u003csub\u003e4\u003c/sub\u003e(O\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e]\u003csup\u003e6\u0026minus;\u003c/sup\u003e and by Si et al. [26] also through an electronic DFT study based on the salt crystal of the peroxohexaniobate ion salt [H\u003csub\u003e3\u003c/sub\u003eNb\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e19\u003c/sub\u003e]\u003csup\u003e5-\u003c/sup\u003e. The latter theoretical analysis used B3LYP as in the present study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe optimized geometry of the PONb-MB system (Figure S2c) demonstrated no significant changes in the Nb(V)\u0026ndash;O bond lengths, but there are some dihedral angle rearrangements involving the terminal oxygenated groups. Figure S2c shows that the PONb\u0026mdash;S-MB interaction appears to be electronically stronger (shorter intermolecular distance, \u0026raquo; 2.11 \u0026Aring;) than the PONb\u0026mdash;N-MB interaction (\u0026raquo; 2.47 \u0026Aring;). Since each MB structure is cationic (charge +1) and the niobate model is anionic (charge -2), we can point out that both modes of interaction can be favorable and form a neutral PONb-MB system.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe EPR analyzes were carried out to characterize the reactive oxygen species, which are the active groups in anticancer activity. No paramagnetic signal was observed in EPR spectra (not shown) for PBN experiments (Figure 2 a), i.e., the studied compounds cannot produce radicals in such experimental conditions. On the other hand, TEMPO was detected only for PONb-MB and MB. As presented in Figures 2 b and c, the singlet oxygen production initiated at five minutes and, after that, evolved until the end of the test (fifteen minutes). Moreover, the time-dependent signal was four times more intense for PONb-MB compared to MB, which indicates that the electrostatic compound produced from the PONb and MB reaction catalyzes the conversion of molecular oxygen from the triplet to the singlet state. These results show that the species formed by the reaction between PONb and MB dye to form PONb-MB has the potential to generate reactive species of the singlet oxygen type, indicating high potential for use as anticancer. In addition, even though the PONb species did not present this type of oxygenated species, the peroxo groups (observed in experimental characterizations and theoretical calculations) can present positive effects in anticancer tests [5,7].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStudies via Raman spectroscopy corroborated to identify the peroxo groups in the PONb species. In fact, the Raman spectra (Figure 2d) showed an intense band in the lower frequencies for niobium compounds. Such a band is usually associated with the Nb-O lattice band [19,31,32]. In the theoretical Raman spectrum, this band was observed in the range of 680-800 cm\u003csup\u003e-1\u003c/sup\u003e (see the bands highlighted in Figure S4d and Table S3). Furthermore, the PONb shows a band around 880 cm\u003csup\u003e-1\u003c/sup\u003e that is characteristic of the vibrational mode of peroxo species [33]. However, in the PONb-MB system, this signal was partially superimposed by MB dye bands. Our DFT calculations showed that the stretching modes of the peroxo groups in the PONb model are observed in the range of 1230-1400 cm\u003csup\u003e-1\u003c/sup\u003e (see the bands highlighted in Figure S4d and Table S3). For the peroxo groups involved in the intermolecular interactions between PONb and MB, these bands are displaced to 740-770 cm\u003csup\u003e-1\u003c/sup\u003e (see the bands highlighted in Figure S4f and Table S3). Experimentally, it was observed that the PONb-MB presents all the vibrational modes of MB, but they are more intense, and their frequencies slightly deviated (see Figure 2e), which again indicates that the PONb-MB is a new compound, not just a simple mixture of PONb and MB dye [34\u0026ndash;36]. The differences found between theoretical and experimental frequencies for the analyzed vibrational modes are related to the theoretical unimolecular model proposed. The experimental solid sample has PONb-PONb interactions that can affect the absorption spectrum of the analyzed groups and that were not considered in our theoretical model. Nevertheless, the B3LYP calculations of the vibrational modes showed good agreement with the experimental data. Details of the theoretical assignments of the IR and Raman spectra (Figure S4 and Table S3) are found in the Supporting Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 2.\u0026nbsp;\u003c/strong\u003eTime-dependent EPR spectra of TEMPO for PONb (a), MB (b), and PONb-MB (c). Raman spectra of PONb and MB dye (d), PONb-MB, and MB dye (e). Differences in vibrational modes between PONb-MB and MB are in the range of 680 - 880 cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2- Cell viability associated with PONb and PONb-MB in HeLa (tumoral), MRC-5 and L929 (healthy) cell lines.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 3 displays the cytotoxic effects of PONb (a) and PONb-MB (b) on HeLa and MRC-5 cells (treated for 24 h), evaluated by the MTT assay. There was a significant reduction in cell viability for polyoxoniobate compounds at concentrations above 1\u0026nbsp;mg L\u003csup\u003e-1\u003c/sup\u003e, in the two tested strains. For PONb, this decrease is more significant and the viability for tumor cells is less than 4 % from the concentration of 5\u0026nbsp;mg L\u003csup\u003e-1\u003c/sup\u003e (Figure 3a). The viability of the healthy cell, incubated at the same concentration, is about 40% indicating certain tumor selectivity in the PONb treatment. This cytotoxic profile can be verified also for PONb-MB, although to a lesser extent (Figure 3b). At the concentration of 5 mg L\u003csup\u003e-1\u003c/sup\u003e, the cell viability is 15 % for tumor cells and 39 % for healthy cells. On the other hand, the compounds used in this study are still poorly understood regarding their biocompatibility, therefore, assays with different cell lines are required to evaluate cytocompatibility.\u0026nbsp;In order to confirm the low\u0026nbsp;toxicity for healthy cells,\u0026nbsp;which is a highly desirable property for a drug candidate, thectionn of\u0026nbsp;PONb\u0026nbsp;and\u0026nbsp;PONb-MB on the cell viability of L929 healthy cells was also investigated (Figure 3). The\u0026nbsp;PONb\u0026nbsp;(Figure 3c) and\u0026nbsp;PONb-MB (Figure 3d) solutions had no significant cytotoxic effect at concentrations lower than 10 mg L\u003csup\u003e-1\u003c/sup\u003e, after 24 and 48 h of incubation. These results further demonstrate that the two polyoxoniobate molecules have no cytotoxic effect on L929 healthy cells (fibroblasts).\u003c/p\u003e\n\u003cp\u003eFluorescence microscopy images of the cells after 24 h of incubation with the prepared materials are shown in Figure 3e. Detection of propidium iodide (DNA-specific red fluorescence) shows dead cells while calcein-MB makes the cell membrane of living cells visible (green fluorescence). A larger number of dead cells are observed in the images of the tumor cells incubated with the materials, mainly in the PONb group (Figure 3e). The percentage of apoptosis calculated after counting the cells is shown in Figure 3f. For the two studied lines, the control group had a death rate of less than 1 %. Concerning the groups incubated with PONb and PONb-MB (10 mg/L), the apoptosis was statistically comparable to tumor cells. Nevertheless, it should be emphasized that an apoptosis percentage of approximately 3x higher was obtained in tumor cells compared to healthy cells, for both PONb and PONb-MB (Figure 3f). This result confirms the effect observed in the cell viability assay, indicating a greater action of the prepared polyoxoniobates on tumor cell lineage. The ability to cause damage to tumor cells with significant selectivity makes both compounds promising materials for cancer therapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 3.\u0026nbsp;\u003c/strong\u003eCell viability in HeLa (tumor) and MRC-5 (healthy) in the presence of PONb (a) and PONb-MB (b). Cell viability in L929 healthy cells with PONb (c) and PONb-MB (d) and solutions at different concentrations and incubation times. Fluorescence microscopy images of the cells after 24 h of incubation with PONb and PONb-MB (e) and cell apoptosis (f).\u003c/p\u003e\n\u003cp\u003eThe action mechanism of the polyoxoniobates selectivity related to tumor cell was investigated. For that, studies involving the formation of intracellular reactive oxygen species (ROS) were performed. Representative fluorescence microscopic images of the cells after 4 h of incubation are presented in Figure 4. The cells with higher ROS species are shown as red dots (Figure 4a). As such, the redder dots, the greater the ROS generation. It is known from the literature that when cells are subjected to ROS, these species can affect the vascular system of the tumor cell causing its death [37]. The percentages of dead and live cells were determined by counting (Figure 4b). In both cell lines, ROS species increased for PONb and PONb-MB groups compared to control cells. Moreover, the death of tumor cells showed a statistically significant increase (about 50 %). This result indicates that the capacity to promote higher cell death signaling in tumor cells is associated with a greater generation of ROS species. Habtetsion et al.[38], showed that adoptive immunotherapy deeply altered tumor metabolism, resulting in glutathione depletion and accumulation of ROS in tumor cells. Raza et al.[39] reported that a certain level of ROS is required by cancer cells, which can lead to cytotoxicity in them. In the present work, the new Nb-containing molecules are rich in labile oxygen species which might be interesting as a cancer therapy strategy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 4.\u003c/strong\u003e (a)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFluorescence microscopic images of the cells after 4 h of incubation and (b) the percentages of dead and live cells determined by counting.\u003c/p\u003e\n\u003cp\u003eThe results indicate that the formed niobium species have anti-cancer action through two different mechanisms: (\u003cem\u003ei\u003c/em\u003e) the PONb species did not present radical species or the formation of singlet oxygen. Thus, the peroxide-type bonds (or peroxo group) present in the molecule (Figure 5a) must be directly responsible for the effect observed in the cell viability tests. An indication that the peroxo group in the PONb forms an interaction with the tumor cells decreasing their cell viability can be observed by the \u003csup\u003e31\u003c/sup\u003eP NMR spectra of the\u0026nbsp;PONb\u0026nbsp;in contact with the 5-GMP molecule (used to simulate the possible interaction between the active species and the DNA).\u003c/p\u003e\n\u003cp\u003eFigure 5b shows the \u003csup\u003e31\u003c/sup\u003eP NMR spectrum for the 5-GMP molecule. It is observed that it has a single P signal with a chemical shift of 5.95 ppm, corresponding to the P of the phosphate group. However, in Figure 5c, it is shown the \u003csup\u003e31\u003c/sup\u003eP NMR spectrum of the molecule in contact with PONb. The presence of another signal was observed at a smaller chemical shift, 5.26 ppm, possibly corresponding to the P interacting with the peroxo group. The presence of this new group affects the chemical environment of the P (phosphate group) from the 5-GMP molecule, because the P atom that is interacting with the peroxo group is more shielded. After all, this group is more electronegative, which causes the appearance of a signal with less chemical shift [40]. The PONb-MB species showed a capacity for generating singlet oxygen (Figure 5d) probably due to the action of ambient light promoting the electronic transition for the conduction band of the species formed by the reaction of PONb with MB dye.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe innovative results presented in this work indicate that ROS are formed and have their generation modulated when the dye is chemically bound to the polyoxoniobate species[39]. We believe that these results open a new line of possibilities since the dyes of various natures can present even better results. In addition, inorganic cations may also interact with the negative charges of the polyoxoniobate generating more versatile anticancer candidates. In this context, our new achievements might contribute to the development of new drugs with antitumoral and antiviral proposals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 5.\u003c/strong\u003e (a) Representative scheme of the peroxide-type bond (or peroxo group) and how it acts on tumor cells, (b) \u003csup\u003e31\u003c/sup\u003eP NMR spectrum for the 5-GMP molecule, (c) \u003csup\u003e31\u003c/sup\u003eP NMR spectrum showing the interaction between PONb and 5-GMP and, (d) the representative scheme of singlet oxygen generation promoted by the action of light in the compound PONb-MB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3- \u003cem\u003eIn vivo\u003c/em\u003e tests for biosafety assessment\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe promising results obtained for the polyoxoniobates for tumor cells motivated further investigation through \u003cem\u003ein vivo\u003c/em\u003e tests with mice. Among several questions that can be raised, one can point out the concern of pharmaceutical accumulation, \u003cem\u003ei.e\u003c/em\u003e., the accumulation of the polyoxoniobates in mice\u0026rsquo;s organs. The results showed a biphasic clearance profile, indicated by the red curve in Figure 6a, with a fast half-life of 7.7 min and a slow half-life of 9.1 min. The biodistribution profile of the possible drug candidate was determined by the Nb loading in mice\u0026rsquo;s organs (Figure 6b). Some uptake was observed in kidneys and liver, which may represent the contribution of these organs in the elimination of the Nb compound. Noteworthy is the higher uptake in the heart at 60 min and 240 min post-injection, which indicates the affinity of PoNB-MB towards this organ and suggests potential cardiotoxicity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 6.\u003c/strong\u003e In vivo studies after PoNB-MB intravenous administration into healthy Swiss mice. Blood clearance assays (a) and the biodistribution profile post-injection of the PONb-MB determined by the Nb loading in the mice\u0026rsquo;s organs (b).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThus, to better analyze and prove the safety profile of PONb-MB, a toxicological assay was performed (Table 1). The data suggest that the treatment with PONb-MB may not cause tissue damage. The CKMB value indicates no cardiac tissue damage, even with the higher uptake presented in the biodistribution study. Therefore, the values found for all the tests are statistically like those of the control groups, the occurrence of acute toxicity not being indicating.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eBiochemical parameters of healthy Swiss mice after intravenous administration of\u0026nbsp;\u003cbr\u003e\u0026nbsp;PONb-MB 1 h post-injection (n = 6).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.632653061224488%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.510204081632654%\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.857142857142854%\"\u003e\n \u003cp\u003ePONb-MB\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.632653061224488%\"\u003e\n \u003cp\u003eCKMB\u0026nbsp;(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.510204081632654%\"\u003e\n \u003cp\u003e0.32 \u0026plusmn; 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.857142857142854%\"\u003e\n \u003cp\u003e0.26 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.632653061224488%\"\u003e\n \u003cp\u003eAST (U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.510204081632654%\"\u003e\n \u003cp\u003e184.43 \u0026plusmn; 55.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.857142857142854%\"\u003e\n \u003cp\u003e178.26 \u0026plusmn; 65.96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.632653061224488%\"\u003e\n \u003cp\u003eALT (U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.510204081632654%\"\u003e\n \u003cp\u003e22.17 \u0026plusmn; 4.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.857142857142854%\"\u003e\n \u003cp\u003e23.75 \u0026plusmn; 4.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.632653061224488%\"\u003e\n \u003cp\u003eCreatinine (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.510204081632654%\"\u003e\n \u003cp\u003e0.61 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.857142857142854%\"\u003e\n \u003cp\u003e0.56 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.632653061224488%\"\u003e\n \u003cp\u003eUrea (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.510204081632654%\"\u003e\n \u003cp\u003e47.54 \u0026plusmn; 13.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.857142857142854%\"\u003e\n \u003cp\u003e60.18 \u0026plusmn; 14.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"5.\tCONCLUSIONS","content":"\u003cp\u003eIn this study, we describe the synthesis of unique polyoxoniobate compounds obtained through a new synthetic route from species of niobium oxide treated with hydrogen peroxide solution (PONb). The addition of methylene blue dye (MB) to PONb solution generated new oligomer species\u0026nbsp;\u003cbr\u003e\u0026nbsp;(PONb-MB).\u0026nbsp;Theoretical analyses and thermodynamic properties contributed to propose a new molecular structure for the polyoxoniobate compounds.\u0026nbsp;Both PONb and PONb-MB present reactive oxygen species (ROS), which resulted in high activity and selectivity as an antitumoral drug candidate.\u0026nbsp;The results indicate that the proposed polyoxoniobate molecule can be useful in the case of selective cytotoxic effect on tumor cells.\u003c/p\u003e\n\u003cp\u003eIt is noteworthy that the viability of healthy cells was not strongly affected by the presence of both compounds, which may indicate that these Nb compounds can be widely and safely used as new possible drugs. Additionally, \u003cem\u003ein vitro\u003c/em\u003e safety outcomes are corroborated by \u003cem\u003ein vivo\u003c/em\u003e assays, which showed minimal uptake in the major organs with no sight of toxicity in the renal, liver, and heart biochemical analyses. In summary, all these findings open new perspectives for this class of compounds to be a promising alternative for cancer treatment with particular attention as antitumor and quite low toxicity activities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurther investigations on therapeutic efficacy and new developments of delivery systems are certainly necessary for future clinical tests. One can also envision that these new polyoxoniobate molecules can be tested in conjunction with other conventional approaches such as chemotherapies to treat cancer cells, instead of simply replacing other traditional drugs since it has demonstrated low toxicity for healthy cells that eventually surround the tumors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are immensely grateful for the financial support of CAPES, CNPq, and FAPEMIG. Furthermore, to the Companhia Brasileira de Metalurgia e Minera\u0026ccedil;\u0026atilde;o (CBMM) and Nanonib for the donation of niobium compounds. Leonardo A. De Souza thanks the FAPERJ\u0026nbsp;for the financial support (E-26/211.911/2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLuiz C. A. Oliveira:\u0026nbsp;\u003c/strong\u003eTerm, Supervision, Conceptualization, Project administration, Funding acquisition, Resources.\u003cstrong\u003e\u0026nbsp;Cinthia C. Oliveira:\u0026nbsp;\u003c/strong\u003eConceptualization, Methodology, Writing - Original Draft, Writing - Review \u0026amp; Editing, Visualization. \u003cstrong\u003ePoliane Chagas\u003c/strong\u003e: Investigation, Data Curation.\u003cstrong\u003e\u0026nbsp;Ana P. Heitmann:\u0026nbsp;\u003c/strong\u003eInvestigation, Data Curation\u003cstrong\u003e. Jos\u0026eacute; B. Gabriela:\u0026nbsp;\u003c/strong\u003eInvestigation, Data Curation.\u003cstrong\u003e\u0026nbsp;Jadson C. Belchior:\u0026nbsp;\u003c/strong\u003eFormal analysis, Writing - Original Draft.\u003cstrong\u003e\u0026nbsp;Leonardo A. De Souza:\u0026nbsp;\u003c/strong\u003eInvestigation, Formal analysis, Writing - Original Draft.\u003cstrong\u003e\u0026nbsp;Tiago H. Ferreira:\u0026nbsp;\u003c/strong\u003eInvestigation, Methodology, Writing - Original Draft.\u003cstrong\u003e\u0026nbsp;Sued E. M. Miranda:\u0026nbsp;\u003c/strong\u003eInvestigation, Methodology\u003cstrong\u003e. Andr\u0026eacute; L. B. Barros:\u0026nbsp;\u003c/strong\u003eMethodology, Writing - Original Draft.\u003cstrong\u003e\u0026nbsp;Cynthia L. M. Pereira:\u0026nbsp;\u003c/strong\u003eWriting - Original Draft, Visualization.\u003cstrong\u003e\u0026nbsp;Vin\u0026iacute;cius D. N. Bezzon:\u0026nbsp;\u003c/strong\u003eMethodology, Writing - Original Draft.\u003cstrong\u003e\u0026nbsp;F\u0026aacute;bio F. Ferreira:\u0026nbsp;\u003c/strong\u003eMethodology, Writing - Original Draft\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. Source data are provided with this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRhule, J. T.; Hill, C. L.; Judd, D. A.; Schinazi, R. F. Polyoxometalates in Medicine. \u003cem\u003eChem. Rev\u003c/em\u003e \u003cstrong\u003e98\u003c/strong\u003e, 327\u0026ndash;357 (1998).\u003c/li\u003e\n\u003cli\u003eNyman, M. Polyoxoniobate Chemistry in the 21 St Century. \u003cem\u003eDalt. Trans\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 8049\u0026ndash;8058 (2011).\u003c/li\u003e\n\u003cli\u003eZhang, Y. \u003cem\u003eet al.\u003c/em\u003e Four polyoxonibate-based inorganic-organic hybrids assembly from bicapped heteropolyoxonibate with effective antitumor activity. \u003cem\u003eCryst. 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(Wiley, 2014).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Polyoxoniobate, ROS, Cancer treatment, Cervical adenocarcinoma, Epidermoid carcinoma","lastPublishedDoi":"10.21203/rs.3.rs-4033921/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4033921/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"In vitro and in vivo experiments were carried out to evaluate the activity and antitumor selectivity of new niobium oligomers. A polyoxoniobate (PONb) and its chemical structure linked to the methylene blue dye (PONb-MB) showed promising results for some types of tumor cells. Molecular structures were determined for PONb and PONb-MB based on spectroscopic analyses with density functional theory calculations. Both caused a significant cell viability reduction in tumor cells (HeLa) with high effectiveness of 10.0% at 10 mg L-1, because of the reactivity oxygen species (ROS) generated on its chemical structure. Experimental quantitative analyzes demonstrated high selectivity for tumor cells (apoptosis 3x higher compared to MRC-5 healthy cells). Lower toxicities for healthy cells (e.g., MRC-5 and L929) confirm the selectivity of the synthesized compounds. It is noteworthy that PONb-MB and PONb-MB are physiologically soluble compounds and can be suitable for intravenous treatments. Ex vivo biodistribution studies in mice revealed a very low accumulation in organs such as the spleen, heart, liver, lungs, and kidneys. Additionally, almost no toxicity was observed in the mice post-injection, which was confirmed by biochemical analyzes.","manuscriptTitle":"Antitumor activity and selectivity of a unique nanostructured polyoxoniobate","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-01 17:42:47","doi":"10.21203/rs.3.rs-4033921/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"33954b83-e8f7-4ffb-9e20-1e2fa7faebcf","owner":[],"postedDate":"April 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29979107,"name":"Biological sciences/Cancer"},{"id":29979108,"name":"Physical sciences/Chemistry"},{"id":29979109,"name":"Physical sciences/Materials science"},{"id":29979110,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2024-09-18T11:15:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-01 17:42:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4033921","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4033921","identity":"rs-4033921","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}