“ Sweet Chemistry ” : a Green Way for Obtaining Selenium Nanoparticles Active against Cancer Cells

We present an environment friendly synthesis of selenium nanoparticles and the study of their cytotoxic activity against uterine sarcoma cancer and fibroblasts cells. Amorphous selenium (a-SeNPs) and trigonal selenium (t-SeNPs) were synthesized using D-fructose as the reducing agent and characterized by high-resolution transmission electron microscopy (HRTEM), energydispersive X-ray spectroscopy (EDX), powder X-ray diffraction analysis (XRD), inductively coupled plasma optical emission spectrometry (ICP OES), dynamic light scattering (DLS) to obtain zeta potential values and cyclic voltammetry (CV). Particularly, a-SeNPs presented high toxicity toward the resistant cancer cell line MES-SA/Dx5 and its parental MES-SA line. However, they are not toxic against P4 fibroblast cells in comparative studies.


Introduction
Selenium is an essential element of significant importance not only for humans, but also for other forms of life. 1,2norganic seleno-compounds have strong antioxidant activity, as well as some pro-oxidant effects and, additionally, have great importance in nutrition and medicine. 3,46][7] For example, selenium supplementation showed effective reduction in the incidence of cancer in the cervix, lung and liver. 8[14] Nanoparticles have become increasingly prominent in the medical field in the last few decades, giving rise to a new area, nanomedicine.This new area is focused on developing nanomaterials with potential use in therapy and diagnosis of diseases, [15][16][17] including selenium nanoparticles (SeNPs). 180][21][22][23][24][25][26][27] In this sense, it is necessary to develop simple routes that focus on green chemistry and no harm to the environment.Research has demonstrated the use of microorganisms such as bacteria, to reduce selenite.][30] The use of plant extracts and biopolymers such as chitosan, konjac glucomannan, gum acacia, carboxymethyl cellulose, sodium alginate and glutathione, as reducing agents of selenite has also been an alternative, since they are highly biodegradable and non-toxic materials.][33] In this paper, we propose a method of synthesis of SeNPs that meet the principles of green chemistry, through a synthesis that requires little reaction time, non-toxic solvents, mild temperatures and uses a reducing agent that is biodegradable, cheap and not harmful to the environment.5][36] Glucose and fructose have aldehyde and hydroxyl groups at carbon 1, respectively, which make them effective reducing agents.Researchers used glucose as a reducing agent and stabilizer of SeNPs; however, in their synthesis they used high temperatures and toxic solvents. 35,36herefore, we chose D-fructose because it is a plentiful and cheap reducing sugar to act as a reducing agent for sodium selenite in clean and quick synthesis of SeNPs using water as solvent and at low temperature.The obtained nanoparticles were tested for cytotoxicity against normal cells (fibroblasts-P4) and uterine sarcomas cells (MES-SA and MES-SA/Dx5) in order to assess their potential use as an anticancer drug.

Experimental
Preparation and characterization of selenium nanoparticles (SeNPs) The SeNPs were prepared by the following procedure: an aqueous solution (1.0 mmol L -1 , 5 mL) of sodium selenite (Merck, Darmstadt, Germany) was slowly dripped in 10.0 mL of 1.0 mmol L -1 aqueous solution of D-fructose (Merck, Darmstadt, Germany).The reaction mixture was then stirred, under heat at 45 °C for 15 min, when red staining nanoparticles were obtained (amorphous selenium, a-SeNPs), turning black after 20 min (trigonal selenium, t-SeNPs).Each solution containing selenium nanoparticles (a-SeNPs and t-SeNPs) was centrifuged at 13,000 rpm for 10 minutes.The pellets obtained were then re-suspended in deionized water and centrifuged again under the same conditions.This process was repeated three times for the removal of organic impurities present in SeNPs.
The nanoparticles free of organic impurities were quantified regarding selenium using an optical emission spectrometer with inductively coupled plasma (ICP OES) of Spectro, model Arcos.The energy-dispersive X-ray spectroscopy (EDX) analyses were obtained on a Shimadzu, EDX-720 model.The corresponding powder X-ray diffraction analysis (XRD) pattern was collected using a diffractometer with Cu Ka operating at 30 kV, 15 mA, 5° to 2q = 90°, Rigaku, Miniflex model.The morphology and particle size were analyzed using a high-resolution transmission electron microscope (HRTEM) JEOL, JEM 2100 model.The preparation of the samples consisted in dispersing the nanoparticles in a coated copper grid with a thin film of carbon, after careful drying with a filter paper.The grid was observed by HRTEM, using an accelerating potential of 200 kV.Measurements of the zeta potential were carried out by dynamic light scattering (DLS) using a Malvern, Zetasizer Nano model.Electrochemical experiments were performed at 298 ± 1 K using potentiostat CH instruments, CH 660I model.A graphite bar (1 mm diameter) was used as working electrode, a platinum wire as auxiliary electrode and an Ag/AgCl (3 mol L -1 NaCl) reference electrode completing the three-electrode arrangement.Voltammetry of microparticles experiments were performed at microparticulate films of amorphous selenium (a-SeNPs) and trigonal selenium (t-SeNPs) prepared by abrasively transferring a few nanogram sample onto the surface of the graphite electrode.Aqueous 0.25 mol L -1 HAc/NaAc buffer solution at pH 4.75 was used as the electrolyte.

Cytotoxicity assays
For the in vitro studies of the cytotoxicity, the following cells were used: human foreskin fibroblasts isolated from tissue (cell P4), drug-sensitive human sarcoma cell line MES-SA and its multidrug-resistant (MDR) counterpart MES-SA/Dx5, as a model system for modulators' anti-MDR potency evaluation.P4 cells were maintained on Dulbecco's modified eagle medium (DMEM) containing 10% fetal calf serum, amphotericin B (0.63 µg mL -1 ) and penicillin (100 IU mL -1 ), streptomycin (100 µg mL -1 ), at 37 °C, in a humidity-controlled incubator at 90% relative humidity and 5% CO 2 .MES-SA cells were maintained on McCoy's 5A culture medium supplemented as above, and the MDR cell line MES-SA/Dx5 was cultivated at the same medium with doxorubicin 0.5 µM, at 37 °C, in a humidity-controlled incubator at 90% relative humidity and 5% CO 2 .After a few passages, cells were incubated at a concentration of 10 × 10 3 cell per wall for drug exposition of 24 h in 96-wall plates, with a final volume of 100 µL.After a period of 24 or 48 h, the cells were treated with concentrations 10 to 140 µM of the a-SeNPs.The mortality cell assay was performed in duplicate, 24 h after starting exposure to drugs.The tetrazolium reduction assay (MTT) was performed in triplicate, according to the literature method. 37Briefly, the medium was removed from the plates, the attached cells were washed with phosphate buffer solution and 200 µL of the MTT solution were added to replaced culture medium, at a concentration of 500 µg mL -1 .After an incubation period of 4 h at 37 o C, the MTT solution was removed, and the formazan crystals were solubilized in 100 µL of DMSO.Absorbance of this formazan solution was measured with a Tecan Infinite M200 microplate reader (Männendorf, Switzerland), at a wavelength of 570 nm.The cell viability rate was estimated from the number of intact cells relative to the total number of cells in the corresponding control, and it was expressed as the percentage of viable cells.The IC 50 was described as the concentration necessary to reach the mortality of 50% of cells.

Results and Discussion
Reduction of sodium selenite was clearly confirmed through observation with naked eye.A color change occurred, from colorless to a red solution within 15 min of reaction (Figure 1).This red coloring, which is characteristic of monoclinic colloidal selenium nanoparticles (a-SeNPs), is due to surface plasmon excitation. 38Within 20 min of reaction, black coloring nanoparticles were obtained, which is characteristic of selenium nanoparticles in the trigonal phase (t-SeNPs). 39gure 2a shows the XRD pattern of the sample obtained after 15 min of reaction, which has no sharp peak, confirming that the selenium nanoparticles synthesized under these conditions were in the amorphous phase (a-SeNPs). 24In the sample obtained after 20 min of reaction, peaks were observed (Figure 2b The HRTEM images corroborate the X-ray diffraction data, which show selenium nanoparticles obtained at 15 min of the reaction having spherical shape without aggregation of particles (Figures 3A and 3B).This is characteristic of the amorphous phase 30,31 (a-SeNPs) with the size ranging between 80-100 nm, and average particle size at 80 nm (Figures 3A and 3B).Figures 3C and 3D show that the shape of the nanoparticle depends on the reaction time.
After 20 min of reaction, the amorphous Se spheres (a-SeNPs) tend to form aggregates.Then, the particles start crystallization presenting rod-like shape, which can be characteristic of t-SeNPs. 30This behavior was observed in other studies in which conventional methods of synthesis of selenium nanoparticles were used. 24he EDX analysis shows qualitatively and quantitatively the elements that may be involved in the formation of nanoparticles. Figure 4 shows the high purity of the selenium nanoparticles synthesized.The two peaks at 11.2 and 12.1 keV, are related to the presence of selenium. 31he nanoparticle stability is very important for a number of applications, and can be determined through the zeta potential.For a-SeNPs synthesized in this work, the zeta potential was found to be -27.8± 0.5 mV.This result indicates the high stability of monodisperse nanoparticles. 32,41To the nanoparticles obtained after 20 min of reaction, t-SeNPs, the measured zeta potential was -18.2 ± 0.9 mV.This confirms the results obtained by the HRTEM image, which showed polydisperse particle aggregation, and indicates the poor stability of them.
Figure 5 compares the voltammetric responses of nanoparticulate deposits of a-SeNPs and t-SeNPs attached to graphite immersed into 0.25 mol L -1 HAc/NaAc aqueous buffer.Using square wave voltammetry as a detection mode, both oxidation and reduction signals can be seen in positive-going scans.Upon scanning the potential from -0.85 V vs. Ag/AgCl in the positive direction, the a-SeNPs displayed voltammetric signals at -0.80, -0.20 and a shoulder at +0.95 V. Remarkably, the t-SeNPs produced weaker shoulders at -0.80, -0.20 and +0.10 V intercalated between a sharp peak at -0.45 and a broad wave at +0.95 V.These responses can be interpreted in terms of the occurrence of two different electrochemical  "Sweet Chemistry": a Green Way for Obtaining Selenium Nanoparticles Active against Cancer Cells J. Braz.Chem.Soc.2024 pathways.First of all, the common signal at -0.80 V can be attributed to the reduction of Se to H 2 Se. 42The oxidation of SeNPs, however, occurs differently for a-and t-SeNPs.In this second case, a sharp signal at -0.45 V appears, corresponding to a typical stripping process 43 due to the oxidation of selenium(0) to selenium(II) species in solution.In contrast, the oxidation of a-SeNPs gives rise to a broad peak at -0.20 V which can be attributed to the selenium(0) oxidation to NP-associated selenium(II).][46] These features can be rationalized assuming that selenium(0) oxidation is quite sensitive to the crystalline structure, as observed in the electrochemistry of, for instance, gold NPs. 47,48In the case of t-SeNPs, which have a rigid structural arrangement, the electrochemical oxidation is dominated by the process at -0.45 V which yields mainly selenium(II) in solution.In contrast, the oxidation of a-SeNPs, where a more flexible coordinative environment for Se atoms exists, can result in a smoother oxidation (process at -0.20 V), producing selenium(II) species associated to the NP.In summary, the availability of electroactive sites cycling between the selenium(0) and selenium(II) states is the key difference between the a-and t-SeNPs.

Cytotoxicity data
The colloidal selenium nanoparticles a-SeNPs, obtained after 15 min of reaction, were evaluated for potential antitumor cytotoxicity against the cell line of uterine cancer MES-SA/Dx5 (doxorubicin-resistant mutants, with P-gp overexpression), and its parental cell line MES-SA.In addition, they were also tested for fibroblasts cell line P4.As shown in Figure 6A, the a-SeNPs were not able to affect P4 cells significantly, since the observed cell viability was 98 and 85% after 24 and 48 h of incubation, respectively, at a concentration of 140 µM.This data indicates that the selenium nanoparticles are non-toxic for this type of human healthy cell.In contrast, the results for cancer cells, shown in Figures 6B and 6C indicate that a-SeNPs present high toxicity with respect to the sarcoma cells.Cell viability decreases gradually with increasing concentration, and the activity is higher with increasing incubation time, for both cancer cell lines studied.IC 50 values were very similar, 110.01 ± 0.23 and 80.12 ± 0.41 µM, at 24 and 48 h, respectively, for MES-SA and 110.19 ± 0.12 and 81.38 ± 0.34 µM, at 24 and 48 h, respectively, for MES-SA/Dx5.The MES-SA/Dx5 cells usually overexpress P-glycoproteins, which act as a drug efflux pump.Consequently, these cells require higher doses of cytotoxic agent in comparison to the non-resistant cells. 49Therefore, these results show that the nanoparticles are equally toxic toward both the MDR (MES-SA/Dx5) and the non-resistant (MES-SA) cancer cell lines.This makes a-SeNPs a promising candidate to pharmaceutical agents to overcome resistance in cancer cells.
A previous study in the literature 50 showed that SeNPs coupled with adenosine triphosphate (ATP, 2.5-40 µM) inhibit the cellular viability of hepatocellular cancer strain (HepG2), but do not exhibit toxicity to normal human kidney cells (HK-2).In a more recent paper, 51 SeNPs were applied to HCT-8 cells (ileocecal adenocarcinoma), indicating that after 48 h of treatment only 27% of the cells survive, when 200 µM of SeNPs were used.Associated with irinotecan, SeNPs are able to inhibit the growth of this strain more efficiently compared to each irinotecan or SeNPs alone.Concerning normal cells (IEC6), the SeNPs were less toxic than irinotecan. 51Breast cancer cells (MCF-7 and MDA-MB-231) were also treated with SeNPs, and an IC 50 value of 25 µg mL -1 was obtained versus the MCF-7 line.For the MDA-MB-231 cell line, the inhibition was only 33% when the same maximum concentration was used, suggesting that SeNPs possess greater selectivity for early stage breast cancer compared to the metastatic breast cancer cells (MDA-MB-231). 33SeNPs (10-40 µM) synthesized using vitamin C also showed activity against the lines MDA-MB-231 and HeLa (human cervical carcinoma).The viability of MDA-MB-231 cells was decreased to 60% and of HeLa cells was reduced between 73 and 50%, in the presence of Se-NPs. 52This is the first time that MES-SA and MES-SA/Dx5 cell lines are tested in studies with selenium nanoparticles.The obtained results confirmed the overall trends reported in the literature.As here discussed, SeNPs have great potential in the treatment against cancer cells, due mainly to its low toxicity toward normal cells.In addition, these results are quite attractive in terms of selectivity, since SeNPs were capable of inducing the mortality of MDR tumor cells (MES-SA/Dox5), which overexpress the glycoprotein P-gp responsible for the efflux effect in tumor cells, at the same intensity as compared with the standard MES-SA cells (Table 1).

Conclusions
In this study, we described a "green" synthesis of SeNPs using D-fructose as a reducing agent, by a simple, economic and fast process.This is the first report of synthesis of Se nanoparticles where D-fructose has been used.Characterization techniques used indicate that at 15 min of reaction colloidal nanoparticles (a-SeNPs) were obtained, and in 20 minutes the process of transformation of a-SeNPs to t-SeNPs starts.HRTEM showed that a-SeNPs were obtained with 80 nm average particle size.Their purity was confirmed by EDX.Further, the a-SeNPs are highly stable with determined zeta potential of (-27.8 ± 0.5 mV).These data indicate that the electron transfer ability of the a-SeNPs and t-SeNPs nanoparticles is clearly different.Those results are in agreement with the observed cytotoxic data, since selenium nanoparticles induce pronounced death of cancer cells (MES-SA/Dx5 and MES-SA cells), but did not show noticeable antiproliferative effects on fibroblasts cells (P4) growth.These results indicate that SeNPs can be good candidates as new therapeutic drugs for uterine cancer treatment.
Figure2ashows the XRD pattern of the sample obtained after 15 min of reaction, which has no sharp peak, confirming that the selenium nanoparticles synthesized under these conditions were in the amorphous phase (a-SeNPs).24In the sample obtained after 20 min of reaction, peaks were observed (Figure2b) at 2q = 23, 29, 41, 43, 45, 51, 55, 61 and 65°, corresponding to the crystalline planes 100, 101, 110, 102, 111, 201, 112, 103.All the peaks could be indexed to the trigonal phase of selenium nanoparticles (t-SeNPs).40The lattice constants of a = 4.3662 Å and c = 4.9521 Å are consistent with the standard values for bulk Se with a = 4.3662 Å and c = 4.9536 Å, according to JCPDS file No. 73-0465.The HRTEM images corroborate the X-ray diffraction data, which show selenium nanoparticles obtained at 15 min of the reaction having spherical shape without aggregation of particles (Figures3A and 3B).This is characteristic of the amorphous phase30,31 (a-SeNPs) with the size ranging between 80-100 nm, and average particle size at 80 nm (Figures3A and 3B).Figures3C and 3Dshow that the shape of the nanoparticle depends on the reaction time.

Figure 1 .
Figure 1.Selenium nanoparticles formation: reaction as a function of time.

Figure 6 .
Figure 6.Effect of a-SeNPs synthesized by reduction with D-fructose on the viability of (A) P4 cell lines; (B) MES-SA cells and (C) MES-SA/Dx5.