The following instruments were used in this study. Water bath-type Ultrasonicator (EngoTech, Zurich, Switzerland). Ultraviolet (UV) visible (UV-vis) absorption measurements were performed with a double-beam Schimadzu-3100 spectrophotometer (Kyoto, Japan). Spectrofluorimetric measurements were performed using a Shimadzu Model RF-5000 spectrofluorometer (Kyoto, Japan). Far-UV circular dichroism (CD) spectra were obtained using a J-810 spectropolarimeter (JASCO, Tokyo, Japan). The ζ-potential and hydrodynamic diameter of the Cd-NCs were measured by dynamic light scattering (DLS) using a zeta sizer Nano ZS instrument (Malvern, Worcestershire, UK). For Fourier transform infrared (FT-IR) spectroscopy, samples were lyophilized, mixed with KBr (Sigma Chem. Co. St Louis, USA) to make pellets, and analyzed using a Perkin-Elmer spectrophotometer (Perkin-Elmer Inc., USA). The scanning electron microscopy (SEM) was done using a TESCAN VEGA//XMU and TESCAN’s Essence™ software (Kohoutovice, Czech Republic). The flame atomic absorption spectroscopy (FAAS, AA-7000, Shimadzu, Kyoto, Japan) was used to determine the Cd in samples. Sonicator-3000 (Misonix, Farmingdale, NY, USA) was used to homogenize tissue samples.

The Cd-NC was synthesized using a modified method described previously [9]. Briefly, the BSA solution (Sigma Chem. Co. St Louis, USA), 20 mg/mL in distilled water (DW), was gradually mixed with the same volume of cadmium chloride (CdCl₂, 5 mmol/L, Merck Co., Germany), on a stirrer. After 1 min, the pH was gradually increased to 13 by adding the NaOH (1 mol/L, Sigma Chem. Co. St Louis, USA). Then the temperature was increased to 45℃. After 7 h, the solution was sonicated for 1 h, with 5–10 min intervals, using a Bath Sonicator. After that, it was dialyzed using a dialysis bag [molecular weight cut off (MWCO): 12 kDa] against phosphate buffer saline (PBS). After 72 h, it was dried using a freeze dryer (Eyela, FDU-1200) and retained at –20℃ until use.

In the next step, HA [5 mg/mL in DW, sodium hyaluronate, molecular weight (MW) = 100–120 kDa was obtained from Lifecore Biomedical] was functionalized using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC, Sigma Chem. Co. St Louis, USA) and N-hydroxysuccinimide (NHS, Sigma Chem. Co. St Louis, USA), and were added to the Cd-NC solution, resulting in hyaloronized-Cd-NC (HA-Cd-NC) formation. Then, different methods, including UV-vis spectroscopy, fluorescence, DLS, FT-IR, CD, and SEM were used to characterize the formation of both Cd-NC and HA-Cd-NC.

Cro was extracted and purified from saffron (Crocus Sativus L.) according to the previously approved method [22]. Briefly, the experimental procedure for Cro loading on HA-Cd-NC was as follows. HA-Cd-NC powder (1 mg) was mixed with different amounts of Cro (0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, and 0.5 mg) and dissolved in PBS. The final volume was adjusted to 1 mL and then gently stirred in the dark for 24 h in a shaker incubator. After that, it was centrifuged at 15,000 g for 10 min. The supernatant was separated, and the pellet was mixed with 1 mL PBS and centrifuged. The procedure was repeated two times until a colorless supernatant was obtained. The supernatants of three times repeats were mixed (3 mL). The pellet containing Cro-HA-Cd-NC@BSA (Cro-NC) was freeze-dried and stored at –20℃ for further analysis and application. The percentages of the Cro encapsulation efficiency (EE) were estimated using Equation 1 [23].

EE (%) = W_Cro-Cd-NC/W_Cro × 100

Where W_Cro-Cd-NC is the amount of Cro loaded on this nanocluster and W_Cro is the total (initial) amount of Cro was added to HA-Cd-NC.

To evaluate the percentage of the EE of Cro, at each W:W ratio of Cro:NC, the absorbance of the supernatants obtained from the previous steps containing free Cro was read at 440 nm, and using a standard curve of Cro, the concentration of free Cro (unbound) was calculated and subtracted from the initial amount (W_Cro-cd-NC = W_Cro – W_{free Cro}). The remaining was the amount of bound Cro (loaded in the HA-Cd-NC), and using Equation 1, the EE percentage (EE%) of Cro was calculated.

The Cro releasing test was conducted under both physiological and acidic pH (7.4 and 5.3, respectively). For this purpose, the dried HA-Cd-NC containing Cro (0.5 mg) was weighed and dissolved in 2 mL PBS, pH = 7.4 or pH = 5.3, and mixed for 10 min, using a Vortex. The resulting clear solution was transferred into a 12 kDa dialysis tube, immersed in 10 mL PBS, pH = 7.4 or pH = 5.3, and placed in a shaker incubator at 37℃, in the dark. Then, at different time intervals, the absorbance of 2 mL buffer was read at 440 nm. This buffer was returned to the Becker to avoid the volume change. This process was completed in up to 24 h. Free Cro released from a similar dialysis tube was applied as a control.

Since Cd²⁺ is highly toxic and has been known as a potent oxidative stress inducer, its toxicity after incorporation into the NC should be investigated. For this purpose, the cytotoxicity studies were performed in dose- and time-dependent manners. At first, the genomic toxicity of Cd-NC was investigated by the comet assay on HeLa cells as a human cancer cell line other than breast cancer. The results were compared with the toxicity of free Cd ions in the form of CdCl₂ and KMnO₄. The cells with no treatment were also used as controls.

Comet assay or single-cell gel electrophoresis (SCGE) was performed according to the method described previously [24, 25]. Briefly, HeLa cells were cultured in appropriate numbers in 6-well flasks and Dulbecco’s Modified Eagle Medium (DMEM-F12), high glucose medium containing 2 mmol/L L-glutamine (Sigma Chem. Co. St Louis, USA), 10% FBS and 1% pen/strep (Life Technology, Paisley, UK). Then, they were treated with different concentrations of CdCl₂ (150 μmol/L, 200 μmol/L, and 300 μmol/L), Cd-NC (0.1 mg/mL, 0.5 mg/mL, and 1 mg/mL), and KMnO₄ (250 μmol/L). The control group received no more treatment. After 24 h and providing the appropriate number, the cells were trypsinized. Live cells were counted using trypan blue staining, and the images were analyzed using Image J software (version 1.49t, NIH approved) and a Comet analyzer (CAA-500).

In the second step, oxidative stress parameters such as malondialdehyde (MDA) and glutathione (GSH) concentrations and the specific activity of some antioxidant enzymes [GSH peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD)] were determined in the HeLa cells. MDA (lipid peroxidation colorimetric/fluorometric assay kit, K739, Biovision, USA) and GSH were measured by commercial kits (GSH colorimetric assay kit, K261, Biovision, USA) on cellular extracts of the cells in different groups. The GPX and CAT activities were measured by the GPX assay kit (K762-100 Biovision, USA) and CAT activity colorimetric/fluorometric assay kit (K773-100, Biovision, USA). The SOD activity was measured by the SOD assay kit (7500-100-K, Funakoshi, Japan). The protocols were performed according to the manufacturer’s kit. The protein concentration of all samples was determined using the Bradford method [26], and the specific activity of the enzymes was determined by dividing the enzyme unit per mg of protein.

The in vivo biodistribution of Cd-NC and the possibility of its toxicity were investigated in a murine model. For this purpose, twenty female BALB/c mice, eight weeks old were purchased from Pasture Institute, Karaj, Iran. Animals were kept under ad libitum access to water, an ordinary diet, and a 12 h/12 h light/dark cycle in the Tarbiat Modares University (TMU) Animal Laboratory. The animal care protocol and use were based on the guidelines of laboratory animals prepared by the TMU. After two weeks of acclimation, mice were entered into a 30-day study. Thus, they were randomly divided into four groups (5 in each) and named as follows:

(1)

D0: control group with no treatment. They received 100 μL of PBS through intra-peritoneal (i.p.) injection on 1st day of the study and then sacrificed on the 30th day.

The body weight of all groups was determined weekly. Before sacrificing, the samples of urine and feces were collected by the previously described method [27]. Then, the mice were weighed, and after anesthesia with ketamine/xylazine, their blood was collected via cardiac puncture. Major organs, including the liver, kidney, spleen, lung, and heart were removed, washed with PBS, and weighed. A section of each tissue was prepared and kept in neutral buffered formalin (4%) for hematoxylin and eosin (H & E) staining. The remaining was kept at –80℃ for further experiments. The body indices were calculated as the organ weight/total body weight ratio. Pathological examination with a digital microscope was done by a specialist in the field.

The blood and urine samples were prepared for Cd determination, with some modifications of the previously described method [28]. Briefly, blood or urine samples (50 μL) were mixed with 200 μL DW. Then, hydrochloric acid (10%, 500 μL, Merck Co., Germany) was added and the samples were analyzed using a FAAS.

The feces samples were defrosted, weighted, and digested in 70% nitric acid (Sigma Chem. Co. St Louis, USA). The solution was heated to evaporate the water, and then the remnant was homogenized in 4.5 mL DW. Then, 500 μL hydrochloric acid 10% was added. All samples were analyzed in duplicate, using a FAAS [29].

The tissue samples were weighted and homogenized in PBS using a Sonicator-3000. The resulting mixture was centrifuged at 3000 g for 10 min. The supernatant was used for determination of protein content using the Bradford method [26], as well as the CAT [30], SOD [31], and GSH s-transferase (GST) activities [32]. In addition, MDA and GSH content were measured using previously explained methods [33, 34]. All parameters were presented as values per mg protein of the tissue sample.

At this stage, the viability of four breast cancer cell lines (MDA-MB-468, MDA-MB-231, BT-474, and MCF-7) was investigated and compared in the presence of free Cro, Cd-NC, HA-Cd-NC, and Cro-NC using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-tetrazolium bromide (MTT) assay, as we described recently [21].

Data are expressed as the mean ± standard error of mean of at least three independent repeats. Statistical analysis was performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The data were analyzed using an unpaired t-test or one-way analysis of variance (ANOVA). Statistical significance for all tests was set at 95% confidential limits, P < 0.05.

The Cd-NC was synthesized and targeted toward the breast cancer cells by adding HA. The characteristic peaks of Cd-NC at pH = 13 were plotted using spectrophotometry (Figure 1A), CD (Figure 1B), and fluorescence intensity [FI, (Figure 1C)]. The FI of the complex was increased by increasing pH up to 13 (Figure 1C), which is the best situation for forming the Cd-NC. The plot obtained by Zeta Sizer is shown in Figure 1D, indicating the nano size of the Cd-NC. The SEM images (Figure 1E) indicates that the Cd-NC coated with a layer of gold. The images are shown at different magnifications, indicating the Cd-NC’s nano size.

The fluorescence microscopy images of the HeLa cells in the absence of any treatment (Figure 2A) and after 12 h incubation with Cd-NC (Figure 2B), KMnO₄ (Figure 2C), or CdCl₂ (Figure 2D) are shown in Figure 2. In this experiment, KMnO₄ was used as a positive control, and CdCl₂ was used to compare the effect of Cd in the atomic and ionic forms. In contrast to Figure 2B, Figure 2C and 2D show significant changes and tail formation, which are the characteristics of DNA damage in eukaryotic cells.

The comet, head, and tail areas (at 12 h, 24 h, and 48 h) are shown in Figure 2E–G, respectively, in different concentrations of Cd²⁺ or equivalent amounts of Cd-NC, and KMnO₄. The results indicate significant differences in the groups treated with Cd²⁺ or KMnO₄ compared to the control. For more clarity, the ratio of the head area/tail area in each condition was calculated and represented in Table 1. This table also clearly indicates differences between these ratios in different groups. There were no significant changes in these ratios between groups treated with the equivalent amounts of Cd-NC compared to the control group.

Changes in the comet, head, and tail lengths at 12 h, 24 h, and 48 h, respectively, under different conditions (Figure 2H–J). These figures and analysis of the data in Table 1 also indicate significant changes in the head-to-tail ratios in the presence of either Cd²⁺ or KMnO₄ compared with the control and Cd-NC treated cells. There were also no significant changes in these parameters due to Cd-NC treatment. Although there were no significant changes in the tail length after Cd-NC treatment of the cells, it was increased up to 5,890 µmol/L and 6,308 µmol/L (more than 10 times) after 48 h of 300 µmol/L of Cd and KMNO₄ treatment, respectively.

After separating the head and tail sections, the genomic DNA content was determined and analyzed in each part to estimate the degree of DNA damage in terms of DNA strand break. These parameters are represented by the DNA% at each segment. The DNA% at 12 h, 24 h, and 48 h and at different conditions are shown in Figure 2K–M. The results indicate a significant and time-dependent increase in the DNA% in the tail segments of the cells treated with Cd²⁺ compared to the control cells at 48 h. The DNA% in the tail was increased more than 50 times after 48 h treatment of the cells with higher Cd concentration or KMNO₄ compared with the Cd-NC.

The data presented in Figure 3A and 3B indicate that after 12 h of incubation, higher concentrations of Cd²⁺ caused a more significant decrease in the GSH and a more significant increase in the MDA concentrations, respectively. Significant decreases in the specific activities of the antioxidant enzymes (CAT, GPX, and SOD, respectively) were observed in the dose- and time-dependent manners due to the treatment of cells with either Cd²⁺ or KMnO₄ (Figure 3C–E). At the same time, the Cd-NC with similar loading concentrations of Cd²⁺ did not affect the specific activity of these enzymes.

As observed in Figure 3F and 3G, the observed changes in all parameters were continued up to 24 h and 48 h of treatments, indicating the time-dependent alterations. However, the Cd-NC had no significant effect on these parameters at any time or at equivalent concentrations.

The weekly changes in the body weight of all mouse groups during the experimental period are shown in Figure 4A. It indicates no significant changes in the body weight of animals in different groups. The tissue indexes (the ratio of tissue weight/ body weight) of the animal groups at the end of the experimental period are seen in Figures 4B (D1, D7, D30). These indicate no significant changes in the animal tissues after treatment with Cd-NC compared to the control group. It means that treatment of the mice with this Cd-NC did not cause any adverse effect on the growth and body weight of animals in the experiment period. In addition, no mortality, abnormal clinical signs, and behaviors were observed in the animals.

The biodistribution of Cd-NC into mice’s blood, urine, and feces, after 1 day, 7 days, and 30 days of administration are shown in Figure 4C–E. Considering the basal level of Cd in the blood of the control animals (Figure 4C), there were no significant changes after 1 day and 7 days of Cd-NC treatment, but the blood concentration of Cd significantly increased after 30 days. In animals’ urine and feces (Figure 4D and 4E), Cd increased significantly until day seven and then decreased. However, changes in the feces Cd concentration were more than in the urine.

The biodistribution of Cd in various tissues of different groups after sacrificing the animals at the end of the experiment are shown in Figure 4F. It indicates that the accumulation of Cd was more in the liver. The microscopic investigation of these tissues is shown in Figure 5, it indicates no significant pathologic changes in the Cd-NC-treated mice’s liver, kidney, spleen, lung, and heart tissues after 30 days (Figure 5A–E), unless there are some little changes in the liver tissue (Figure 5A).

The data in Figure 6A and 6B show the amount of GSH and MDA in the livers of the control and Cd-NC-treated mice in all groups. The specific activities of CAT and GST in the livers of the control and Cd-NC-treated mice are shown in Figures 6C and 6D. These data indicate no significant changes in these values between the control and Cd-NC-treated mice. However, a significant increase in SOD-specific activity is observed in the liver after 1 day and 7 days of Cd-NC administration (Figure 6E). Although an increasing trend was also seen after 30 days, it was not statistically significant.