The AgNPs were prepared based on the interaction between sodium nitroprusside and silver nitrate in an aqueous solution. After purification by centrifugation and drying of the nanoparticles, several physicochemical characterizations were performed in vitro using breast cancer cells (MCF-7) and non-malignant breast epithelial cells (MCF-10A) and in vivo using female mice (C57BL/6J) with murine mammary adenocarcinoma tumors (EO771).
Initially, to detect functional groups present in AgNPs Fourier Transformed Infrared Spectroscopy (FTIR) was used. Figure 1 (A) illustrates the vibrational spectra in the infrared region of AgNPs. The two characteristics of nitroprusside peaks attributed to the functional groups -C ≡ N (2,145 cm− 1) and –N = O (1,942 cm− 1) exhibited a displacement for shorter wavelength regions at 2,137 cm− 1 and 1,932 cm− 1, respectively. This shift suggests the replacement of sodium ions with silver ions after the formation of AgNPs [25].
The distinctive peaks for δ (FeNO) and δ (FeCN) were observed at 658 cm− 1 and 512 cm− 1, respectively, and this is in agreement with previously published literature [25].
The two peaks at 3,629 cm− 1 and 3,547 cm− 1 correspond to the characteristic peaks of water molecules present in the crystalline structure of sodium nitroprusside.
XRD analysis of the nanoparticles was performed to verify their crystallinity. Figure 1 (B) illustrates the XRD spectrum of AgNPs that indicates the crystallinity of the materials and presents a monoclinic unit cell. The observed diffraction peaks (2θ = 13.0, 13.8, 21.5, 25.2, 30.0, 33.4, and 36.2) indicate the crystalline nature of the prepared material, and this is consistent with previously published literature [25].
Using the Debye-Scherrer equation (Eq. 1) [45], it was possible to estimate the diameter of the AgNPs as 133 nm.
\(d=(k. {\lambda })/({\beta }.{cos}{\theta })\) (Eq. 1)
where:
d = is the diameter of the grain
k = is constant (0.9 for spherical particles)
λ = is the X-ray wavelength (1.5418 Å)
β = total width at half height
θ = is the Bragg angle
The nanoparticle diameter was consistent with that obtained by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis.
The surface morphology of the AgNPs was evaluated using SEM and TEM, and these analyses revealed the shape and size of the prepared AgNPs. Based on SEM and TEM analyses, Figs. 1 (C) and (E), respectively, indicate that the prepared material exhibited predominantly irregular shapes that were analogous to certain silver nanoparticles that were previously described in the literature [25] with particle sizes ranging from 103.3 to 226 nm.
Dispersive energy spectroscopy (EDX) was used to analyze the elemental composition of the AgNPs and the elemental composition of Ag, Fe, N, C, and O as illustrated in Fig. 1 (D).
The Ag+/ Fe3+ ratio was 4.21, and this was very close to the values reported in the literature [25]. The results obtained in this analysis indicate that the preparation of the nanoparticles occurred successfully, as the sodium ions are entirely replaced by silver ions in the ionic sphere of the nitroprusside complex to thus lead to the formation of AgNPs.
<Figure 1>
Nitric oxide exhibits broad biochemical activity, and therefore, it is essential to quantify its concentration in the material. Within this context, the total concentration of NO released in an aqueous solution of AgNPs (1 mg/mL) was determined according to NOmeter analysis to be 250.0 µmol L− 1. A 0.1 mol L− 1 solution of oxyhemoglobin was prepared to verify if nitric oxide was detected. After adding oxyhemoglobin, a current decay was observed. Specifically, all NO present was consumed, thus indicating that the species generated by the system was nitric oxide.
Atomic absorption spectroscopy (AAS) was performed to determine the internalization time of AgNPs. Thus, a study was performed using two concentrations of AgNPs (1.0 and 3.0 µg/mL) at two time points (1 and 3 h). MCF-7 and MCF-10A cells were plated onto 24 well adherent plates (6×104 cells per well) using a specific cell culture medium. After 48 h, treatment with AgNPs (1.0 and 3.0 µg/mL) was performed, and the plates were incubated for 1 h and 3 h at both concentrations.
After each treatment, the medium containing the AgNPs was carefully removed from the wells, and the cells were washed with PBS 1X buffer solution. After washing, 500 µL of isopropanol was added to each well for cell lysis, and the incorporated nanoparticles were released into the isopropanol. After cell lysis, the lysates were analyzed using the analytical method of atomic absorption spectroscopy using a graphite furnace to quantify silver within the medium.
After atomic absorption spectroscopy analysis, we observed that at both concentrations of nanoparticles (1.0 and 3.0 µg/mL of AgNPs) and at the two different treatment times (1:30 and 3:00 h), the concentrations of Ag obtained ranged from 12–14 ppb. Thus, to ensure effective internalization and based on its wide use in similar studies [46], a 3 hour incubation time for treatment with AgNPs was adopted for all studies.
To evaluate the morphological effects on the cell lines (MCF-10A and MCF-7) exposed to different concentrations (1.0 and 3.0 µg/mL) of AgNPs, a morphological analysis was performed using bright field microscopy at 10× magnification.
From the morphology images (Supplementary data: Fig. 1 [A] and [B]) it was possible to observe the cell morphology of the two cell lines (MCF-10A and MCF-7) in monolayer in the absence of nanoparticles (control) and after treatment with AgNPs (incubation for 3 h) at 0, 24, and 48 h after treatment withdrawal.
It is possible to verify that for the 1.0 µg/mL concentration, there was almost no change in the morphological structure of the cell lines after treatment with the lowest concentration of AgNPs. Initially, a slight decrease in cell quantity was observed at time 0 (shortly after the treatment was withdrawn) in all strains, and the cells proliferated again and returned to morphology similar to that of the control over time (24 and 48 h after withdrawal from treatment).
However, after treatment with AgNPs at the highest concentration (3.0 µg/mL), there was a considerable change in the cell morphological structure. After the treatment was withdrawn, the cell morphology was altered and numerous cells were observed in suspension (rounded shape). Over time, the percentage of cells in the suspension increased considerably in the tumorigenic line MCF-7.
Conversely, it is possible to observe in the micrographs of the MCF-10A healthy line that after 3 hours of treatment, they exhibit a change in morphology that is indicative of a reduction in the number of viable cells. However, at 24 and 48 h after the withdrawal of the treatment, the cells proliferated again, and this indicated that there was a cell recovery compared to observations after treatment with the highest concentration of AgNPs. This is similar to the results obtained from the colorimetric viability assays (MTT and Resazurin), thus suggesting that AgNPs are selective for non-malignant breast epithelial cells (MCF-10A).
To evaluate the cytotoxicity of AgNPs in the context of cellular biochemical processes, MTT and resazurin colorimetric assays were used to measure cellular metabolic activity to indirectly assess cell viability after treatment with nanoparticles. MTT is reduced by the activity of mitochondrial dehydrogenases in living cells [47], and after the complete reduction of MTT, the cells were lysed with 2-propanol. The absorbance of formazan dye was then measured.
Figure 2 (A [i] and B [ii]) illustrates cell viability graphs based on the colorimetric MTT assay, and Fig. 2 (A [i] and B [ii]) illustrates cell viability graphs based on the colorimetric resazurin assay for MCF-10A and MCF-7 cell lines, respectively. The data demonstrate cell viability after 3 h of treatment with two different concentrations of nanoparticles (1.0 and 3.0 mg/mL) and with readings acquired at 0 (right after treatment withdrawal), 24, and 48 h after treatment withdrawal.
Sensitivity studies have demonstrated a slight difference between MTT and resazurin when applied for in vitro studies. Based on the colorimetric assays evaluated in Fig. 2 (C), it was possible to observe a reduction in the metabolic rate as the concentration of AgNPs increased.
<Figure 2>
Treatment with the lowest concentration of nanoparticles (1.0 µg/mL) at time 0 resulted in decreased viability of the epithelial cell line MCF10-A; however, it was possible to observe a recovery over time with increased viability at 24 h and 48 h after treatment in this cell line. For MCF-7 cells, this minimum concentration (even after 48 h) was not able to induce significant changes in cytotoxicity. Thus, both cell lineages did not enter a cell death process at this low concentration over time. In contrast, treatment with 3.0 µg/mL AgNPs decreased viability for both cell lineages; however, a drastic decrease in cell viability was observed for the tumorigenic lineage (MCF-7), compared to that of the epithelial cell line (MCF-10A). These differences were statistically significant between the groups (Supplementary Fig. 1). Taken together, these results suggest AgNPs at 3.0 µg/mL could be used as a potential treatment to target mammary tumors, as this concentration leads to a potential decrease with three-fold higher mortality in the tumorigenic cell line MCF-7 compared to that in the epithelial healthy cell line MCF-10A.
To test the inhibitory effect of AgNPs on tumor growth in vivo, female mice (wild-type C57BL/6J) with mammary adenocarcinoma induced by orthotopic inoculation of luciferase-labeled EO771 cells were used as a model [48]. Tumors were induced in all groups, and the animals were divided into a control untreated group (n = 4) and treated groups with AgNP at 3.0 µg/mL (n = 4) and AgNP at 10.0 µg/mL (n = 4). Bioluminescent signals were monitored using an in vivo imaging system (IVIS) (Fig. 3 [A]). Figure 3 (B) indicated a higher intensity of the bioluminescent tumor signal in the control group compared to that in the treated groups (3.0 and 10.0 µg/mL). Interestingly, the effectiveness of AgNPs was maintained in the group treated with 3.0 µg/mL compared to the highest treatment concentration 10.0 µg/mL. In this context, it is established that NO concentration can inhibit or promote the progression of these tumors and is in some situations responsible for activating signal transduction pathways that lead to cell death. NO also appears to protect cells from apoptosis [49].
To confirm the effect of AgNP (3.0 µg/mL) on tumor growth inhibition, histological analysis (Fig. 3 [C]) was performed on the total resected tumor. Indeed, the 3.0 µg/mL -treated group exhibited a decreased tumor area according to percentage as shown in Fig. 3 (D). Histological analysis also revealed that the control untreated group exhibited necrosis in extensive areas compared to levels in the AgNP (3.0 µg/mL)-treated group with a statistically significant difference. The presence of necrosis in the histopathological context of the mammary gland has been classically recognized as a clinical adverse effect of tumors in advanced stages and is normally associated with rapid tumor growth, excessive angiogenesis, and increased tumor grade (Putti et al 2005). Therefore, this result demonstrates the inhibition of tumor progression in the AgNP (3.0 µg/mL)-treated group and the prevention of necrosis that is associated with advanced stages.
<Figure 3>
To address the toxic effect of AgNPs, hematological analyses were performed on mice from all groups. The hematological results (Fig. 4) revealed that the animals treated with AgNP at 10.0 µg/mL possessed a higher number of lymphocytes (1.6–7.6×103/uL) and presented with lymphocytosis. The animals in the control and AgNP (3.0 µg/mL) groups exhibited no abnormalities. The literature value for the normal range of mouse lymphocytes is 0.7–5.7×103/uL. Immune reactions or inflammatory diseases can cause lymphocytosis that is characterized by varying degrees of lymphocyte activation [50], thus suggesting an inflammation reaction was caused by the nanoparticles.
<Figure 4>
Additionally, for any studied group, there were no behavioral or clinical changes (diarrhea, hair loss, or decreased motor activity throughout the experiment). The weights of the animals were recorded at the beginning of the study, on the days of treatment, and immediately before euthanasia. There was no significant change in the body weight of animals in any group (Supplementary data: Fig. 2). These results provide evidence of the effectiveness of AgNP at 3.0 µg/mL without any systemic inflammatory condition or toxicity.