Biosynthesis and characterization of Cd NPs
Cd NPs were synthesized based on our previous procedures (Shakibaie et al. 2021). Reduction of aqueous Cd (NO3)2 ions by A. persica aqueous extract was observed by the opacification of the yellowish color of the mixture. It has been proposed that metal ions are reduced by secondary metabolites, including flavonoids, alkaloids, polyphenols, polysaccharides, as well as proteins, amino acids, enzymes, and vitamins present in the plant parts. One proposed mechanism of NPs synthesis is the reduction of metal ions by secondary metabolites, such as flavonoids, alkaloids, polyphenols, polysaccharides, proteins, enzymes, and vitamins present in the plant parts (Sahoo et al. 2021; Shakibaie et al. 2021). A similar endorsement was made during the rapid reduction of Cd chloride using aqueous marigold and rose flower petal extract. Most of the produced Cd NPs were spherical (Hajra et al. 2016). The SEM micrograph of Cd NPs synthesized using A. Persica showed Cd NPs with some aggregation between the NPs (Fig. 1a). Hexagonal Cd NPs were characterized by a single peak in particle size distribution between 11.2–18.6 nm, with the most frequent size of 14.8 nm (Fig. 1b). Similar hexagonal CdS NPs (produced by biological or chemical techniques) have been reported previously (Martinez-Alonso et al. 2014; Srinivasa Goud et al. 2016; Kamble et al. 2020). In the study of Martinez et al. (Martinez-Alonso et al. 2014), the hexagonal CdS NPs obtained by microwave heating method were 9.2–11.7 nm in diameter. Additionally, Hexagonal CdS NPs with an average size of 9 nm were biologically synthesized (Srinivasa Goud et al. 2016). Kamble et al. (Kamble et al. 2020) applied the hot-injection method to prepare hexagonal-type CdS NPs (18.3–37.5 nm). Azizi et al. (Azizi et al. 2018) have found spherical Cd NPs with a diameter of 5 nm synthesized by the chemical reduction method. In another major study, Alsaggaf et al. (Alsaggaf et al. 2020) found that CdS NPs produced by Aspergillus niger had spherical morphology with an average size of 5 nm. Malarkodi et al. (Malarkodi et al. 2014) reported that biogenic CdS NPs were produced with Klebsiella pneumoniae and had a spherical shape ranging from 10 nm to 25 nm in size. Different applied synthesis methods may contribute to the variation in the shape and size of Cd-containing NPs. Nevertheless, more researches are needed to investigate the factors affecting the size and shape of Cd NPs.
Cytotoxicity of Cd NPs
To determine the effects of Cd NPs, Cd (NO3)2, and cisplatin on the HepG2 and normal HUVEC cells, the MTT assay was carried out. As shown in Fig. 2 and Fig. 3, the tested compounds exhibited dose-dependent effects on the mentioned cell lines after 24 h and 48 h of incubation. The half-maximal inhibitory concentration value (IC50) of biosynthesized Cd NPs, Cd (NO3)2, and cisplatin was 586.8 ± 1.3 µg mL− 1, 16.83 ± 0.3 µg mL− 1, and 64.2 ± 0.4 µg mL− 1, respectively after 24 h of treatment on HepG2 cells (Fig. 2a). The calculated IC50 values of HepG2 cells after 48 h of treatment with Cd NPs, Cd (NO3)2, and cisplatin were 416.9 ± 1.7 µg mL− 1, 7.5 ± 0.04 µg mL− 1, and 15.4 ± 0.3 µg mL− 1, respectively (Fig. 2b). After 24 h and 48 h of incubation, Cd NPs showed less cytotoxicity than Cd (NO3)2 or cisplatin against HepG2 cells at concentrations between 5 µ mL− 1 to 640 µ mL− 1(p < 0.05). The Cd (NO3)2 was significantly more toxic than cisplatin when exposed to concentration ranges of 20 µg mL− 1 to 320 µg mL− 1 (p < 0.05), and there was no significant difference between the viability of HepG2 cells treated with 5µ mL− 1, 10 µ mL− 1 of Cd (NO3)2 and cisplatin after 24 h of treatment (p > 0.05). After 48 h of incubation, the Cd (NO3)2 displayed significantly higher toxicity effects on this cell line (5 µg mL− 1 to 640 µg mL− 1), compared with Cd NPs (p < 0.05).
In HUVEC cells, the measured 24 h IC50 value of Cd NPs, Cd (NO3)2, and cisplatin was 284.4 ± 2 µg mL− 1, 12.3 ± 0.1 µg mL− 1, and 26.2 ± 0.1 µg mL− 1, respectively (Fig. 3a). The viability of HUVEC cells following 48 h treatment with Cd NPs, Cd (NO3)2, and cisplatin is shown in Fig. 3b, with the calculated IC50 values of 142.2 ± 0.2 µg mL− 1, 3.7 ± 0.05 µg mL− 1, and 5.7 ± 0.2 µg mL− 1, respectively. Cd NPs showed lower toxicity than Cd (NO3)2 and cisplatin after 24 h or 48 h of exposure with all concentrations (p < 0.05). Compared with cisplatin, Cd (NO3)2 was more toxic at all concentrations (p < 0.05).
As shown in Fig. 2 and Fig. 3, the cell viability of HepG2 and HUVEC cells treated with 5 µg mL− 1 and 10 µg mL− 1 of Cd NPs did not differ significantly from the control group (0 µg mL− 1) after 24 and 48 h of treatment (p > 0.05). Substantially higher toxicity was observed at concentrations above 10 µg mL− 1 (p < 0.05). The obtained results of this study exhibited that the cytotoxicity of Cd NPs and Cd (NO3)2 against HepG2 and HUVEC cells was dose and time-dependent (Fig. 2 and Fig. 3).
In a study by Shikabaie et al. (Shakibaie et al. 2021), Cd NPs and Cd (NO3)2 were shown to induce a concentration-dependent response in 3T3, MCF-7, A549, U87, and HT-29 cell lines with the IC50 values of 10.6 µg mL− 1, 1.8 µg mL− 1, 47.2 µg mL− 1, 62.9 µg mL− 1, and 133.5 µg mL− 1, respectively. The viability of human erythroleukemia cells (K562 cells) and human embryonic kidney cells (HEK293T cells) was decreased in a dose- and time-dependent manner after treatment with CdTe quantum dots (QDs) at concentrations ranging from 3 µM to 187.5 µM (Su et al. 2009). Similarly, Zhang et al. (Zhang et al. 2007) investigated the size and time-dependent cytotoxicity of Cd telluride NPs (CdTe) (2–6 nm) in HepG2 cells.
The cytotoxicity effect of other Cd-containing NPs, like spherical CdTe-QDs (14 nm), caused intrinsic and extrinsic apoptosis in HepG2 cells after 6 h, 12 h, and 24 h treatment (0.001-100 µg mL-1). This hepatocellular toxicity was time and dose-dependent (Nguyen et al. 2013b). After 2 h, 4 h, 6 h, and 24 h of treatment with these spherical CdTe-QDs, macrophages (J774A.1) and HT-29 cells showed a dose- and time-dependent reduction in metabolic activity (Nguyen et al. 2013a). The toxicity of CdTe-QDs may result from oxidative stress induction through GSH and CAT activity depletion and reactive oxygen species (ROS) formation. Other Cd-containing NPs like albumin-coated Cd NPs (Cd [email protected], 88 nm) exhibited 57 times higher toxicity than Cd NPs (spherical shape, 5 nm) against MDA-MB-231 (Azizi et al. 2018). Alsaggaf et al. (Alsaggaf et al. 2020) reported that CdS NPs capped with A. niger proteins exhibited cytotoxicity against A549, MCF7, PC3, and cell lines with measured IC50 values of 149 µg mL− 1, 190 µg mL− 1, and 246 µg mL− 1, respectively. The biogenic CdS NPs (40–80 nm), prepared using a biological route with Escherichia coli sulphate reductase enzyme, showed IC50 values of 92.2 mM and 33.4 mM against Mus musculus skin melanoma and human epidermoid carcinoma cell lines, respectively (Shivashankarappa and Sanjay 2020). It seems that the chemical composition of Cd nanostructures, along with their synthesis routes and sizes, plays an essential role in the cytotoxicity of these Cd-containing NPs. Our study showed that Cd NPs had lower toxicity on HepG2 and HUVEC cell lines than Cd (NO3)2. Previously, several studies demonstrated decreasing cytotoxicity of biogenic metal(oid)-containing NPs compared with their ions (Forootanfar et al. 2014; Mohanty et al. 2014; Forootanfar et al. 2015; Ameri et al. 2020). In a similar study, Forootanfar et al. (Forootanfar et al. 2014) found that Se NPs produced by Bacillus sp. were less cytotoxic on the MCF-7 cell line compared with selenium dioxide.
Another study revealed that tellurium nanorods (22 nm diameter,185 nm length) display higher IC50 values than potassium tellurium against A549, MCF-7, HepG2, and HT1080 cell lines (Forootanfar et al. 2015). In the study of Mohanty et al. (Mohanty et al. 2014), biosynthesized tellurium nanorods (TeNRs) did not exhibit cytotoxicity to human bronchial epithelial cells (BEAS-2B) and murine macrophages (RAW264.7). The obtained results of our recently published investigation have demonstrated a significantly higher IC50 value for Cd NPs than Cd (NO3)2 against A549, HT-29, MCF-7, 3T3, and U87 cell lines (Shakibaie et al. 2021). It can be conferred from the literature review that Cd nanostructures induce cell death via mechanisms involved ROS production either through the formation of electron-hole pairs to transfer electrons to oxygen or by directly damaging the antioxidant system through the release of Cd2+ ions (Cho et al. 2007; Nguyen et al. 2013b; Alsaggaf et al. 2020; Shivashankarappa and Sanjay 2020). Ultimately, further research is necessary to determine the exact cytotoxicity mechanism of our Cd NPs.
The hemolytic properties of NPs can be measured to determine their interaction with blood components (Singh et al. 2020). A positive control (100% hemolysis) is the amount of hemoglobin released from the RBCs when water is added. As depicted in Fig. 4, at the highest concentration (640 µg mL− 1), the hemolysis rate for Cd NPS and Cd (NO3)2 was 0.88 ± 0.5% and 3.37 ± 0.3%, respectively. A similar result was observed in the study of Shivaji et al. (Shivaji et al. 2018), which demonstrated that the CdS quantum dots (2–5 nm) exhibited 1.83 ± 0.2% of hemolysis rate at the highest tested concentration (60 µg mL− 1). According to the previous reports, up to 5% of hemolysis is permissible for biomaterials and considered non-hemolytic (Muzquiz-Ramos et al. 2015; Shanthi et al. 2017). Cd NPs and Cd (NO3)2 exhibited no significant hemolysis at the tested concentrations (P < 0.05). In contrast, Shivashankarappa et al. (Shivashankarappa and Sanjay 2020) reported that biogenic CdS NPs (40–80 nm) showed 33.5% hemolysis activity at 0.2 mM concentration which was noted that accumulation in cell membranes leads to toxic effects on RBCs by positively charged CdS NPs
Antibacterial effect of Cd NPs
Cd NPs and Cd (NO3)2 were studied at different concentrations for their antimicrobial and anti-biofilm properties. A microplate serial dilution method was employed to study the MICs of Cd NPs and Cd (NO3)2 against various pathogenic bacteria. The calculated MIC values for Cd NPs against three clinically pathogenic strains of S. aureus, P. mirabilis, and P. aeruginosa strains, were more than 2560 µg mL− 1. Moreover, the measured MICs of Cd (NO3)2 against S. aureus, P. mirabilis, and P. aeruginosa pathogens were 1280 µg mL− 1, 1280 µg mL− 1, and 2560 µg mL− 1, respectively. Meanwhile, MICs of ciprofloxacin were 0.312 µg mL− 1, 0.312 µg mL− 1, and 0.625 µg mL− 1 against the S. aureus, P. mirabilis, and P. aeruginosa, respectively.
Previously, numerous metal(oid) based NPs such as ZnO, Cu2O, and Pd have been implicated as antimicrobial agents (Suresh et al. 2018; Bezza et al. 2020; Nasrollahzadeh et al. 2020). Cd-containing nanostructures have also been found to possess antimicrobial properties (Rajeshkumar et al. 2014; Abd et al. 2016; Haq Bhat and Yi 2019; Alsaggaf et al. 2020; Shivashankarappa and Sanjay 2020). Alsaggaf et al. (Alsaggaf et al. 2020) studied the antimicrobial activity of cubic CdS NPs and showed that these NPs were active against S.aureus, E. coli, Pseudomonas vulgaris, and Bacillus subtilis. Furthermore, Cd oxide (CdO) NPs synthesized by the chemical method have proven to inhibit the growth of S. aureus, P. aeruginosa, K. pneumoniae and, A. baumannii (Abd et al. 2016). Bhat et al. (Haq Bhat and Yi 2019) reported the efficacy of green synthesized CdS NPs against S. aureus and E. coli., with more sensitivity to gram-positive S. aureus. It has been proposed that the inhibition action of CdS NPs on bacteria is related to the interaction between the NPs and thiol groups present in key bacterial respiratory enzymes (Haq Bhat and Yi 2019). Similarly, Rajeshkumar et al. (Rajeshkumar et al. 2014) reported that spherical shaped CdS NPs exhibited the maximum growth inhibition zone against the clinical strains of E. coli, Vibrio sp., Serratia nematodiphila, Klebsiella planticola, Aspergillus niger, and Aspergillus flavus. Furthermore, they indicated that CdS NPs with a positive charge interact with proteins of the microorganism's cell membrane to disrupt them. Sekar et al. (Sekar et al. 2019) reported that CdS NPs were highly active against S. aureus and E.coli.
Unlike the previously mentioned antimicrobial activities of Cd-containing NPs, our Cd NPs biosynthesized in this study did not show antibacterial potency against the S. aureus, P. mirabilis, and P. aeruginosa strains at the tested concentrations (0-2560 µg mL− 1). The chemical structure of Cd-containing NPs seems to impact their antimicrobial activity significantly. Furthermore, our clinical strains of S. aureus, P. mirabilis, and P. aeruginosa have been previously exhibited high ability in biofilm formation, which might be a critical factor in microbial resistance against antimicrobial agents (Shakibaie et al. 2019b).
The antibacterial efficacy of commercial antibiotics alone and in combination with biosynthesized Cd NPs (100 µg/disk) and sub-MIC value of Cd (NO3)2 (100 µg/disk) was evaluated by disc diffusion method against MRSA strain. Meropenem, levofloxacin, streptomycin, vancomycin, gentamicin, tetracycline, and kanamycin were the only antibiotics that showed antibacterial activity against MRSA. At the same time, blank discs containing Cd NPs and Cd (NO3)2 did not exhibit antibacterial activity (Fig. 5). The presence of Cd NPs (100 µg/disk) significantly increased the antibacterial effect of disks containing antibiotics that inhibit the protein synthesis (gentamicin, tetracycline, and streptomycin) and vancomycin (a cell wall synthesis inhibitor) in MRSA (p < 0.05). Meanwhile, the presence of Cd (NO3)2 considerably increased the antibacterial activity of both gentamicin and streptomycin discs against MRSA (p < 0.05) (Fig. 5). The Cd (NO3)2 increased the growth inhibition zone of cefalexin (an inhibitor of cell wall synthesis), which did not show an inhibitory effect on MRSA alone (p < 0.05). The antibacterial effects of levofloxacin on MRSA were also significantly reduced by exposure to Cd NPs as well as Cd (NO3)2 (p < 0.05). It is noteworthy that meropenem (a cell wall synthesis inhibitor) and kanamycin (a protein synthesis inhibitor) discs did not have an inhibitory effect on MRSA alone. As a result of combining these antibiotics with Cd NPs or Cd (NO3)2 (100 µg/disk), the MRSA growth inhibition zone was significantly increased.
Previously Shakibaie et al. (Shakibaie et al. 2019b) reported that spherical shaped bismuth NPs (40–120 nm) enhanced the antibacterial effect of antibiotics that inhibit the protein and cell wall synthesis which had no inhibitory effect on MRSA alone. A synergistic effect of silver NPs (Ag NPs) combined with kanamycin (a protein synthesis inhibitor) against S. aureus, E. coli, S. Typhimurium was observed in a study by Vazquez-Munoz et al. (Vazquez-Munoz et al. 2019). Metallic NPs have been combined with conventional antibiotics to increase antibiotic effectiveness against pathogenic bacteria (Thati et al. 2010; Nazari et al. 2014; Bankier et al. 2019). Previously it has been reported that chemically synthesized CdO NPs (32–41 nm) increased the antibacterial activity of ciprofloxacin against K. pneumoniae (Abd et al. 2016). It has been reported that the sub-inhibitory concentration of ZnO NPs (20–45 nm) increased the antibacterial activities of erythromycin, methicillin, penicillin, ampicillin, amoxicillin, streptomycin amikacin, clindamycin, oxacillin, gentamicin, cloxacillin, cefotaxime, ceftazidime, vancomycin, cephalexin, and tetracycline against S. aureus (Thati et al. 2010).
Anti-biofilm activity of Cd NPs
The inhibitory effect of Cd NPs as well as Cd (NO3)2 on the biofilm formation of S. aureus, P. mirabilis, and P. aeruginosa is illustrated in Fig. 6. As mentioned previously, the sub-inhibitory concentrations of Cd NPs and Cd (NO3)2, (0-640 µg mL− 1) were selected for screening the formation of biofilms by planktonic cells. According to Fig. 6a, the biofilm formation of S. aureus decreased to 75.3 ± 9% and 23 ± 4% in the presence of Cd NPs, and Cd (NO3)2 (640 µg mL− 1), respectively which this reduction was significant compared to the control group (0 µg mL− 1) (P < 0.05). The biofilm formation by P. mirabilis was significantly reduced in the presence of Cd NPs, and Cd (NO3)2 (640 µg mL− 1) and reached 68.3 ± 2% and 74.3 ± 4%, respectively (Fig. 6b) (P < 0.05). P. aeruginosa's biofilm formation was reduced to 73.5 ± 8% and 95.4 ± 4% when exposed to Cd NPs and Cd (NO3)2 (640 µg mL− 1), respectively (Fig. 6c). While the reduction in the biofilm formation of P. aeruginosa in the presence of Cd (NO3)2 was not significant (P > 0.05).
According to the present study, at concentrations above 5 mg mL− 1, Cd (NO3)2 exhibited significantly higher anti-biofilm activity than Cd NPS against S. aureus (P < 0.05). The biofilm reduction of P. mirabilis in the presence of Cd NPs was not notably higher than Cd (NO3)2 (p > 0.05). At a concentration above 160 µg ml− 1, biogenic Cd NPs inhibited the growth of P. aeruginosa biofilms significantly more than Cd (NO3)2 (p < 0.05). Our result complies with Shakibaie et al. (2019b), who reported a notably higher anti-biofilm activity of bismuth NPs on P. aeruginosa rather than P. mirabilis and S. aureus. In a study by Dhanabalan et al. (2015), hydrophilic CdS NPs in a microemulsion medium significantly reduced the biofilm formation of gram-negative E. coli. As demonstrated in Fig. 6, The anti-biofilm effect of Cd (NO3)2 on S. aureus was significantly higher than P.mirabilis and P. aeruginosa (P < 0.05). The antibiofilm activity of Cd NPs on P.mirabilis was higher than S. aureus and P. aeruginosa, but this was not a significant effect (p > 0.05). The discovery of potent anti-biofilm compounds is a challenging endeavor for biofilm eradication. Using NPs is an approach to prevent the formation of biofilms and destroy those that have formed (Shakibaie et al. 2019b). Together, our results suggest that Cd NPs may have beneficial properties as an antibiofilm agent against clinical strains. This study did not discuss the mechanisms relating to eradicating biofilms by Cd NPs; thus, further investigation is required.