Unveiling the role of novel biogenic functionalized CuFe hybrid nanocomposites in boosting anticancer, antimicrobial and biosorption activities

Latest Research

Biosynthesis and physicochemical characterization of NPs

The biosynthesis of functionalized CuNPs (FCNPs) was initially validated by change of the color of S. cyaneofuscatus EM3 culture from pale green to faint brown. On the other hand, S. cyaneofuscatus EM3 culture containing functionalized FeNPs (FFNPs) and FCFNCs exhibited dark brown to black color. Comparatively, no obvious change of color occurred in the control samples, implying successful bioconversion of metal precursors into their corresponding NPs. The characteristic features and structural verification of biologically prepared NPs were elaborately evidenced with the following analysis:

Structural characteristics

The crystalline structure and phase identification of FCNPs, FFNPs and FCFNCs were determined through X-ray diffraction (XRD) (Fig. 1a) and their Joint Committee on Powder Diffraction Standards (JCPDS) (Supplementary Fig. S1). As a general observation, the sharp and distinct peaks in XRD diffractograms revealed the crystalline nature of biosynthesized NPs with the lowest surface energy22. However, other biologically or even chemically synthesized NPs signified the production of imperfect crystals or amorphous phase9,23. The diffractogram of FCNPs showed characteristic peaks at 2θ = 30.9°, 39.42°, 44.82°, 53.53°, and 68.38° which correspond to (110), (111), (− 202), (020), and (200), Bragg’s reflection, respectively. These diffraction planes were matched with values of the monoclinic cubic phase of CuO (JCPDS file no. 00-005-0661)24,25. The peak at 2θ = 60.95° (220) indexed cubic structure of Cu2O-NPs (JCPDS file no. 00-005-0667) as highlighted by Zhao et al.26. In addition, the presence of diffraction peaks at 49.9° and 74.89° matched reflection from (200) and (220) planes of the pure face-centered cubic structure of Cu0 (JCPDS file no. 00-004-0836)24. Obviously, the main phase was CuO with low amounts of Cu2O phase in the examined sample of FCNPs. Notably, the oxidation states of Cu in the examined sample, including (0), (+ 1), and (+ 2), were very typical. The position of these peaks matched well the reports by Zhao et al.26 and Karimzadeh et al.27, suggesting that Cu/Cu2O/CuO composites were successfully synthesized in the same sample.

Figure 1
figure1

Characteristic features of the biosynthesized FCFNCs; XRD (a), EDX (b), FTIR (c), UV–Vis (d), TEM micrograph of cytoplasmic-localized FCFNCs synthesized by S. cyaneofuscatus EM3 during stationary phase (e), after extraction (f), Particle size distribution curve (g) and Zeta potential (h).

On the other hand, the crystalline structure of the as-prepared FFNPs exhibited Fe0/FeOOH/Fe2O3/Fe3O4 as shown in Fig. 1a. The formed hexagonal Fe2O3 and cubic Fe3O4 nanostructures within the designed nanocomposite were indexed by (JCPDS file no. 01-89-0596] and (JCPDS file no. 01-071-6336], respectively28,29. The peak at 35.61° might be a combination of Fe2O3 and Fe3O4. The peak at 45.02° and 74.8° matched the reflection from (110) and (220) planes confirming the formation of cubic Fe0 based on the (JCPDS file no. 01-071-4649)30. Diffraction peak at 55.9° was assigned to the reflection from (251) plane of β-FeOOH31. Apparently, the polymorphic phase with heterostructure nanoparticles was produced via S. cyaneofuscatus EM3. These results revealed the synthesis of various crystalline types of the copper and iron derivates within the nanocomposite.

Notably, the XRD pattern of FCFNCs confirmed the formation of a hybrid composite of Fe3O4/FeOOH/Cu2O/CuO/Cu with a predominance of Fe3O4 and CuO indicated by the existence of their distinctive peaks. Thus, such result proposed that S. cyaneofuscatus EM3 mediated the synthesis of FCNPs, FFNPs and FCFNCs in mixed phases of metallic and oxidized states. The detection of oxides in metallic phase NPs is a well-known phenomenon that could be ascribed to the surface oxidation of metallic NPs during synthesis, processing, drying and XRD-preparation steps. This finding agreed with other studies9,32. The absence or weakening of some distinctive peaks of Cu, FeOOH, and Cu2O in FCFNCs revealed the low content and poor nature of crystallinity17,33. More so, the presence of a small background hump at 2θ range 20°–30° could be assigned to bacterial organic biomolecules associated with the NPs, which was frequently observed in green protocols and also reflected the successful functionalization with metal/metal oxides. That was supported by FT-IR as described below6,23,32.

Compositional characteristics

The elemental distribution of iron and copper in the examined FCFNCs was proved through mapping and energy dispersive X-ray spectrometry (EDX). As shown in Supplementary Fig. S2, the elemental mapping of FCFNCs referred to the presence of Cu, Fe, and O entities in a similar and homogeneous distribution, which deducing binary hybrid structure material. However, Fig. 1b illustrated three major peaks of Cu, Fe and O in the FCFNCs sample with weight percentages of around 33.1%, 30.6% and 29.5%, respectively. Accordingly, the EDX profile determined the atomic proportion of Cu:Fe (1:0.92) in the FCFNCs which was in consonance with the inoculated molar ratio (1:1) of the precursors Cu(NO3)2 and Fe(NO3)3·9H2O. Concerning EDX profiling of FCNPs and FFNPs, intense signals of Cu, Fe and O were observed at surface energies corresponding to 6.4 keV, 8 keV, and 0.5 keV, respectively (Supplementary Fig. S3). Additionally, the signals related to C, P and S at 0.27, 2.0 and 2.3 keV, correspondingly were significantly detected in the EDX spectrum. That could be assigned to the X-ray emission from the proteinaceous and nuclear moieties associated with as-synthesized NPs. This combination could furnish NPs with stability by acting as a capping agent. Thus, such self-functionalization property provided by the biological method in one step with a reduction during the biosynthesis process is considered indigenously behaved and replaces the additional surface modification step, that is critically required for coating chemically and physically synthesized NPs2. The involvement of these elements with NPs was commonly reported particularly with green approaches34.

Functional characteristics

Fourier-transform infrared spectroscopy (FTIR) measurements provided presumptive insight into the surface chemistry of biosynthesized NPs by identifying the functional groups of microbial biomolecules conjugated with them, which caused their biosynthesis and stabilization. Figure 1c and Table 1 manifested the presence of common bands in all examined samples, which coincided with the results of12,35. Regarding the fingerprint region, the vibrational modes for FCNPs and FFNPs were vividly displayed at wavenumbers 547 cm−1 and 583 cm−1 which belong, respectively, to Cu–O and Fe–O vibration stretching. These results agreed with He et al.33 and Pandharipande and Makode35. In general, in view of internuclear vibrations, the metal oxide absorption peaks were observed at a lower field in range the 400–700 cm−1, which reflected the metallic nature of any examined sample7,12,36. However, small shift in the absorption peaks was observed in FCFNCs profile which may be due to the interaction between CuNPs and FeNPs and also the association of FCFNCs with carboxylic acids in the protein, which led to functionalization and dispersion of FCFNCs as implied by Ismail et al.6. Virtually, the association of several functional groups like C=O, C=C, C–O–C, PO43−, amine and amide with NPs seemed to be advantageous4,6,37.

Table 1 FTIR peaks assignments and their corresponding functional groups associated with FCNPs, FFNPs and FCFNCs.

Optical characteristics

The optical and electronic structure of FCNPs, FFNPs, and its combined hybrid system FCFNCs were scrutinized by UV–Vis spectroscopy. As depicted in Fig. 1d, a single surface plasmon resonance (SPR) band was observed at 280 nm and 419 nm for FCNPs and FFNPs, respectively. Similar results were found elsewhere2,4. Remarkably, the SPR band is considered a special and unique property for materials of metallic nature; nevertheless, it is sensitive to surface modifications and varies with solution chemistry and synthesis method4. On the other hand, a relative change in the absorbance maxima of the combined hybrid system FCFNCs appeared at 369 nm, which was an intermediate location between the SPR band of FCNPs and FFNPs. As asserted by Banik et al.9, UV–Vis spectroscopy is a valuable tool to signal the architecture attained by any binary or hybrid NP, as a randomly organized hybrid metal/metal oxides identified by a single SPR peak at an intermediate position between the wavelengths of the two metals. Likewise, Thakore et al.38 and Al-Asfar et al.39 recorded the difference in optical responses due to the variation of the structural features derived from hybrid mixing of Ag/Cu and Ag @ Fe, respectively.

Morphological characteristics

The morphology, interior structure and textural properties of as-prepared NPs were visualized by transmission electron microscopy (TEM). Figure 1e and Supplementary Fig. S4a and c illustrated the nanofactory S. cyaneofuscatus EM3 cell encompassing NPs; Fig. 1f and Supplementary Fig. S4b and d showed the extracted NPs. The TEM micrographs showed heterogeneity of the shapes and sizes of the FCNPs and FFNPs. The FCNPs synthesized intercellularly by S. cyaneofuscatus EM3 displayed needle- or rod-like morphology, more or less of similar size (12–36 nm) and homogeneous distribution without aggregation (Supplementary Fig. S4a). Whereas the extracted FCNPs appeared aggregated in nanoclusters and embedded in protein microbial matrix as illustrated by elongated dark NPs in the less dense light matrix (Supplementary Fig. S4b). Such microbial matrix provided stability to NPs and functionalized them as was previously indicated by EDX and FTIR. Regarding FFNPs, S. cyaneofuscatus EM3 formed numerous, tiny, quasi-spherical or roughly globular NPs with size oscillated between 2 and 7 nm and showed a slight tendency for agglomeration (Supplementary Fig. S4c). The extracted FFNPs presented well separated, well-defined, monodispersed spheres (Supplementary Fig. S4d). Such disparity among the morphologies of FCNPs and FFNPs and even FCFNCs was observed and reported earlier by Nadeem et al.40, who ascribed that to metal type, microbial chemistry, selective interaction mechanism and the overall physical conditions of the mixture.

However, FCFNCs exhibited undefined, irregular shapes and assembled in bulks or nanoclusters, appearing as electron opaque spots (Fig. 1e). Besides, a small percent of rods was also observed which could be attributed to Ostwald ripening mechanism41. Upon extraction, FCFNCs seemed mostly spheres of 37 ± 2.2 nm, not well-separated from each other and agglomerated occasionally into chains (Fig. 1f). Such chain like FCFNCs were previously found by Sepúlveda et al.16 and Shubair et al.42, who attributed this aspect to the higher surface energy of individual Fe-NPs (2.63 eV/atom) than Cu-NPs (1.37 eV/atom), the physicochemical characterization of the sample and the higher stability of NPs of such morphology. The aggregated behavior of FCFNCs could be attributed to the magnetic forces, electrostatic attractions among NPs, surface energy differences between Cu/Fe and the presence of H-bonding in the microbial bioactive molecules associated with the NPs1,32. Interestingly, the size and morphology of FCFNCs in our study coordinated with those prepared by Tabrizian32. Furthermore, the particle size distribution (PSD) curve of FCFNCs (Fig. 1g) indicated that 73.16% of FCFNCs particles were around 66.3 nm with a standard deviation of 14.02%, while the hydrodynamic size of FCNPs, FFNPs was assessed by 83.2 nm (63.5%) and 21 nm (57.4%) with a standard deviation of 17.62% and 19.36%, respectively (Supplementary Fig. S5a and b). Such difference in size between TEM and PSD could be assigned to the presence of water and other biomaterial molecules associated with the surface of NPs4.

Surface charge characteristics

The electrokinetic potential of the streptogenic NPs and their long-term colloidal stability were evaluated by ζ-potential. It recorded − 31.3 mV, − 36.4 mV and − 26.2 mV for FCNPs and FFNPs and FCFNCs, respectively (Supplementary Fig. S5c and d and Fig. 1h). The stability limit of particles was in the range ± 25 mV, i.e., particles with ζ-potential values more than + 25 mV or less than − 25 mV exhibit higher electrostatic interaction, which provided them stability with a little chance for flocculation and agglomeration4,43. Accordingly, both FCNPs and FFNPs displayed higher stability and better monodispersity than FCFNCs, which could be attributed to the electro-static repulsion force among the smaller adjacent NPs that led to Brownian motion; eventually retaining NPs suspension away from aggregation and disposition. Negatively charged surface of the NPs indicated their association with negatively charged biomolecules such as polyacids of protein moieties and the sugar-phosphate backbone of nucleic acid moieties. Notably, ζ-potential of as-prepared nanomaterials in this study showed better results than other related investigations12,38.

Biosynthesis mechanism

Based on the preceding experimental data, the following sequence of events could be proposed to explicate the mechanism of NPs synthesis via S. cyaneofuscatus EM3. The mechanism involved a cascade of stages that fall in the scope of oxidation–reduction (redox) reaction illustrated in Fig. 2. Initially, the positively charged ions of metal precursors interacted electrostatically with different negatively charged functional groups that characterize actinomycetes cell wall10. Through various uptake systems, the acquired metals were internalized into the cell either to mitigate their toxicity or to utilize them in bacterial metabolism. In this sense, it is worth mention that the critical roles of Cu and Fe as the most common redox-active metals that conjugate with proteins44,45. The bioreduction step began once the metals entered the cells. Thereafter, the intracellular compartmentalization occurred due to the zero-oxidation Cu/Fe nuclei produced by the process summarized in the following equations

$$ {text{H}}_{2} {text{O }} to {text{ H}}^{ cdot } + {text{ OH}}^{ cdot } $$

(1)

$$ {text{Cu}}^{2 + } + , 2{text{H}}^{ cdot } + {text{ NAD}}({text{P}}){text{H-reductase}} to {text{ Cu}}^{ circ } + , 2{text{H}}^{ + } $$

(2)

$$ {text{Cu}}^{ + } + {text{ H}}^{ cdot } + {text{NAD}}({text{P}}){text{H-reductase }} to {text{ Cu}}^{ circ } + {text{ H}}^{ + } $$

(3)

$$ {text{Fe}}^{3 + } + , 3{text{H}}^{ cdot } + {text{NAD}}({text{P}}){text{H-reductase }} to {text{ Fe}}^{ circ } , + , 3{text{H}}^{ + } $$

(4)

$$ {text{Fe}}^{2 + } + , 2{text{H}}^{ cdot } + {text{NAD}}({text{P}}){text{H-reductase }} to {text{ Fe}}^{ circ } , + , 2{text{H}}^{ + } $$

(5)

Figure 2
figure2

Schematic diagram representing the biosynthesis process of FCFNCs via S. cyaneofuscatus EM3 in presence of Cu(NO3)2 and Fe(NO3)3.

Remarkably, various microbial biomolecules could participate substantially in the reduction phase and chelating activity to transform metal ions into their NPs analogues, including NADH-dependent enzymes, such as cytochrome reductase, hydrogenases, nitrate reductase, dehydrogenases4, superoxide dismutase and catalase46. Actually, in the biological redox reaction, the enzymes shuttle electrons to the metal ions that were reduced to the NPs through sequential variation in their oxidation states. During aerobic growth, the metallic Cu and Fe were oxidized, both by oxygen that was available in aerobic incubation and by the oxidizing biomolecules produced by microbial cells43, which generated Cu2+, Fe3+ and OH that are responsible for the formation of Cu(OH)2, Fe(OH)2 and Fe(OH)3 on the metallic Cu and Fe, respectively (Eqs. 6, 7 and 8).

$$ {text{ Cu}}_{{({text{s}})}} + , 2;{text{OH}}^{ – } + {text{ Oxidase }} to {text{ Cu}};({text{OH}})_{{2({text{s}})}} + , 2{text{H}}_{2} {text{O}} $$

(6)

$$ {text{Fe}}_{{({text{s}})}} + 2;{text{OH}}^{ – } + {text{ Oxidase }} to {text{ Fe}};({text{OH}})_{{2({text{s}})}} + , 2{text{H}}_{2} {text{O}} $$

(7)

$$ {text{Fe}}_{{({text{s}})}} + 3;{text{OH}}^{ – } + {text{ Oxidase }} to {text{ Fe}};({text{OH}})_{{3({text{s}})}} + , 3{text{H}}_{2} {text{O}} $$

(8)

Such hydroxide intermediates were dissociated to CuO, FeOOH and Fe3O4 as manifested in Eqs. (9), (10) and (11) below

$$ {text{Cu}};({text{OH}})_{4}^{2 – } to {text{CuO}}_{{({text{s}})}} + , 2;{text{OH}}^{ – } + {text{ H}}_{2} {text{O}} $$

(9)

$$ {text{Fe}};({text{OH}})_{{3({text{s}})}} to {text{ FeOOH}}_{{({text{s}})}} + {text{ H}}_{2} {text{O}} $$

(10)

$$ 2;{text{Fe}}^{3 + } + {text{ Fe}}^{2 + } + , 8;{text{OH}}^{ – } to , 2;{text{Fe}};({text{OH}})_{3} {text{Fe}};({text{OH}})_{2} to {text{ Fe}}_{3} {text{O}}_{4} + 4;{text{H}}_{2} {text{O}} $$

(11)

Simultaneously, under continuous aerobic incubation and at different microbial growth phases, the development of other oxides (e.g., Cu2O and Fe2O3) was observed as inferred from XRD (Fig. 1a). The reactions were presented in Eqs. (12) and (13).

$$ 4;{text{Cu }} + {text{ O}}_{2} to , 2;{text{Cu}}_{2} {text{O}} $$

(12)

$$ 4;{text{Fe }} + , 3;{text{O}}_{2} to , 2;{text{Fe}}_{2} {text{O}}_{3} $$

(13)

However, both products could be further oxidized to CuO and Fe3O4, which are the most stable phases, according to the following equations

$$ 2;{text{Cu}}_{2} {text{O }} + {text{ O}}_{2} to , 4;{text{CuO}} $$

(14)

$$ 3;{text{Fe}}_{2} {text{O}}_{3} + , 1/2;{text{O}}_{2} to , 2;{text{Fe}}_{3} {text{O}}_{4} $$

(15)

Interestingly, in the biosynthesis of FCFNC, the simultaneous redox of both metals was probably followed by S. cyaneofuscatus EM3, which implied cellular utilization of both metals at the same time without ionic selectivity property. This gave rise to such nanocomposite architecture (CuO/Cu2O/Cu/Fe3O4/FeOOH). In fact, the assembly of any binary elements to different composite nanoforms is based on several parameters that include the precursors ratio, pH, energy barrier, atomic size, oxidation/reduction potentials, electronegativity, and electronic/magnetic effects as well9,16. For FCFNCs in the current study, despite there being a difference in reduction potential of Fe3+/Fe0 (E0 = − 0.44 V) and Cu2+/Cu0 (E0 = 0.34 V)16, the nanocomposite with Cu and Fe in equivalent proportion was generated, as revealed by EDX. That could be attributed to the influence of pH. During S. cyaneofuscatus EM3 growth phases, pH fluctuated between 6.7–8.0 as a consequence of nitrate utilization and its conversion into nitrite (NO2) and ammonia (NH3+) by the act of nitrate reductase enzyme. Under these circumstances, Fe was rapidly reduced at a higher rate than Cu, which favors acidic conditions for precipitation32,47; forming Fe3O4 without any existence of Fe and/or Fe2O3 in the hybrid system (Fig. 1a). Nevertheless, the slower rate of Cu reduction led to the predominance of CuO on the surface of Cu2O and Cu, which existed in relatively low concentration. Therefore, in this study, the mutual effect of both redox potential and pH managed FCFNC synthesis in such configuration. These deductions were commensurate well with the earlier findings32,47.

Applications of as-prepared NPs

Cytotoxicity and anticancer activity of as-prepared NPs

Cancer is the second leading cause of human death. It is estimated that about 9.6 million deaths were caused by cancer in 2018 across the world48. The current form of chemotherapy did not cause a significant reduction in mortality among cancer patients; thus, the new and safer approaches are critically needed. Nanotherapy is the rational alternative remedial approach. The initial studies used NPs as drug carriers in which anticancer drugs were entrapped. Thereafter, the intrinsic antitumor effect of NPs was discovered, and their use as a drug by themselves to conquer tumor development began49. The potential of metallic NPs, especially transition metals (e.g., copper and iron) in the role of tumor inhibitors due to their unique properties and low toxicity has been demonstrated.

As recorded formerly, Cu and Fe are functional elements for many cellular processes, yet, in high concentrations, they adversely affect50. Therefore, herein, we examined the impact of all biosynthesized NPs on the viability of normal human cells. The safe doses (EC100) of FCNPs, FFNPs and FCFNCs that maintained 100% viability of normal human lung fibroblast Wi-38 cells were 823.5 ± 6.2 μg/mL, 1258.6 ± 24.8 μg/mL and 1999.7 ± 21.5 μg/mL, respectively. The lowest IC50 level of IC50 ≤ 17.21 μg/mL that had the strongest potential anticancer effect on all studied human cancer cells was demonstrated by hybrid FCFNCs. Figure 3a presents high anticancer activity of FFNPs (IC50 30.10–35.36 μg/mL) against all tested human cancer cell lines in comparison to FCNPs (IC50 83.77–105.52 μg/mL). Further, IC50 values of metal oxide NPs in the treated cancer cells were lower than their EC100 values in the treated normal cells. This reflected the selective preference of these metal oxide NPs for cancer cells, which has been reported in a recent study51. Previous studies found that CuNPs synthesized by Bacillus cereus display a similar effect at IC50 ≥ 20 μg/mL on MCF-7, Caco-2 and HepG2 cells52, while CuNPs produced by Sargassum polycystum brown seaweed at IC50 = 61.25 μg/mL were effective on MCF-753. On the other hand, Namvar et al.54 recorded the effect of IC50 of 18.75 μg/mL and 23.83 μg/mL of iron oxide (IO) NPs synthesized by Sargassum muticum (seaweed) on MCF-7 and HepG2 cells, respectively.

Figure 3
figure3

MTT results of the tested NPs against human cancer cells. IC50 (μg/mL) values (a) with the morphological alterations of cancer cell lines after 72 h treatment with these tested NPs (b). All values were expressed as mean ± SEM. FCFNCs were compared with FCNPs and FFNPs. This comparison considers significantly different at *p < 0.05, **p < 0.005, ***p < 0.0005.

Moreover, the morphological changes of FCFNCs-treated cells, including the complete collapse of the normal spindle shape and cell shrinkage, proved the strongest anticancer effect of FCFNCs among all examined NPs (Fig. 3b). More importantly, the combination index (CI) of FCFNCs anticancer activity was estimated at 50% inhibition in order to characterize the type of interaction between FCNPs and FFNPs as synergism, additivity or antagonism (< 1, = 1 or > 1, respectively). Therefore, the powerful anticancer effect of the hybrid nanocomposite might be attributed to high synergism between CuNPs and FeNPs (CIs = 0.422 ± 0.014, 0.628 ± 0.045, 0.543 ± 0.059 and 0.526 ± 0.122 against Caco-2, HepG-2, MCF-7 and PC-3, respectively). However, to our knowledge, the detailed synergistic antitumor features of FCFNCs were not studied before. Hence, further analysis of apoptotic mechanism using flow cytometry, fluorescence microscopy, immunostaining Ki-67 and expression levels of tumor suppressor gene (p53) and caspase 3 were investigated here.

Apoptosis-induced anticancer effect of as-prepared NPs

In Fig. 4a and Supplementary Fig. S6, the apoptotic potential of FCNPs, FFNPs and FCFNCs was illustrated using flow cytometric analysis by higher percentages of annexin V-stained apoptotic cells than that of propidium iodide-stained necrotic cancer cells. The percentages of annexin V-stained apoptotic cell populations in FCNPs-treated cancer cell lines were < 33%. In comparison, 59.27–67.56% apoptosis was estimated in the case of FFNPs treated cancer cells. Remarkably, FCFNCs exhibited the highest apoptosis percentage (77.71–82.8%) for all tested human cancer cells. Remarkably, a synergistic apoptotic anticancer effect (CI 0.619 ± 0.008, 0.558 ± 0.003, 0.585 ± 0.006 and 0.592 ± 0.009) was recorded between CuNPs and FeNPs in FCFNCs-treated colon, liver, breast and prostate cancer cells, respectively. Furthermore, the apoptosis was clarified with acridine orange/ethidium bromide (AO/EB) staining and visualized using a fluorescence microscope (Fig. 4b). The NPs-treated cancer cells had fluorescence ranging from yellow to red in comparison with the control (untreated viable cancer cells) that showed green fluorescence. The low anticancer effect of FCNPs was confirmed by yellow fluorescence staining of the treated cancer cells. While the nuclei of FFNPs-treated cancer cells showed orange fluorescence, which indicated high apoptotic effect. Notably, the most potent anticancer effect of FCFNCs was illustrated by red fluorescence of the nuclei of the treated colon, liver, breast and prostate cancer cell lines.

Figure 4
figure4

Apoptotic cell population in FCNPs, FFNPs and FCFNCs-treated human cancer cells compared with the untreated cancer cell lines. The percentage of apoptosis in the control and treated cancer cells using flow cytometry after staining with annexin V/propidium iodide (a). Fluorescence images of AO/EB double staining with green, yellowish-orange, and red fluorescence indicate viable, early apoptotic and late apoptotic cells, respectively (magnification × 200) (b). All values were expressed as mean ± SEM. FCFNCs were compared with FCNPs and FFNPs. This comparison considers significantly different at *p < 0.05, **p < 0.005, ***p < 0.0005.

Prooxidant effect-mediated anticancer activity of as-prepared NPs

One promising approach for collapsing cancer cells is enhancing the lethal reactive oxygen species (ROS) generation55. So, the redox activity of the active NPs was detected in HepG-2 cells (the most sensitive cancer cell line to be the tested with NPs based on previously mentioned results) using the ROS-sensitive fluorescent probe (DCF). As shown in Fig. 5a-I, II, the incubation of HepG-2 cells with FCNPs, FFNPs and FCFNCs caused a dramatic increase in the ROS level by 47.305 ± 1.34%, 69.925 ± 1.62% and 81.29 ± 0.75%, respectively. Also, GSSG level increased in FCNPs, FFNPs and FCFNCs-treated HepG-2 cells (26.58 ± 3.4, 16.81 ± 1.54 and 43.24 ± 2.01 nmol/mL, respectively) compared to 7.39 ± 0.17 nmol/mL in the untreated HepG-2 cells (Fig. 5b). This indicated that FCFNCs-treated HepG-2 cells had the highest levels of ROS and GSSG. Moreover, a synergistic interaction (CI = 0.721 ± 0.025 and 0.500 ± 0.034) between the prooxidant effect of CuNPs and FeNPs was recorded for ROS and GSSG, respectively in hybrid FCFNPs-treated HepG-2 cells. Therefore, our hybrid nanocomposites of Cu and Fe which demonstrated the highest prooxidant effect, were the most powerful inducer of the apoptosis-dependent death in all studied human cancer cell lines. Interestingly, Plotek et al.56 and Jungwirth et al.57 highlighted that Cu and Fe exhibited prooxidant-dependent nuclease activity by enhancing the formation of ROS, particularly hydroxyl radical which is the main cause of DNA damage mediate cell death. Dixon and Stockwell55 referred to transition metals (particularly, iron) and ROS as the most important triggers for cell death mediation. The synergistic prooxidant effect dependent anticancer activity of FCFNCs might be attributed to copper (II) which potentiates the prooxidant effect of Fe (III) by reducing it to Fe (II) by ROS produced by Cu (II)-catalyzing the oxidation of glutathione. This is elucidated by higher GSSG level in the FCNPs-treated HepG-2 cells than FFNPs-treated HepG-2 cells as demonstrated by the current study. To sustain their uncontrolled proliferation, cancer cells promote the reduction of GSSG to maintain a highly reduced microenvironment which prevent the accumulation of lethal ROS55,58. In conclusion, the strongest synergistic anticancer effect of hybrid NPs may due to their highest synergistic prooxidant activity that aggravated oxidative damage and enhanced the multiplicative apoptosis-dependent death among the FCFNCs-treated cancer cells.

Figure 5
figure5

Prooxidant effect of FCNPs, FFNPs and FCFNCs in the treated HepG-2 cells relative to the untreated cancer cell lines. (aI, II) Flow cytometric charts of fluorescence oxidized form of dichlorofluorescin diacetate (indicator of reactive oxygen species “ROS”) with quantitative histogram for the percentage of intracellular ROS. (b) Cellular content of GSSG (nmol/mL) in FCNPs, FFNPs and FFNPs -treated HepG-2 relative to the untreated cells. All values were expressed as mean ± SEM. FCFNCs were compared with all other treatments with significance at *p < 0.05, **p < 0.005, ***p < 0.0005.

The gene expression levels of tumor suppressor gene (p53) and caspase 3 and immunohistochemical expression of proliferation marker (Ki-67) in the NPs-treated cancer cells

Notably, FFNPs were able to upregulate the expression of p53 and caspase 3 (apoptosis executors) by 4.7 and 3.3 folds, respectively, while FCNPs did not show any marked change in the expressions of caspase 3. A change was recorded only in the expression of p53 as an increase by 2.4 folds in the treated HepG-2 cells (Fig. 6a). However, the hybrid nanocomposite was able to increase the expression of p53 and caspase 3 about 5 folds in the treated HepG-2 cells. Accordingly, it was found that CIs of FCFNCs for up-regulation p53 and caspase 3 expressions were 0.801 ± 0.15 and 0.621 ± 0.011, respectively, indicating the synergistic interaction. Noteworthy mention that p53 is one of the oxidative stress response transcription factors59. Thus, only up on ROS activation by these transition metals does; the apoptosis induction occurs. Maqusood et al.60 illustrated the anticancer effect of IONPs on HepG2 and A549 (human lung adenocarcinoma) cells via inducing ROS as well as p53, caspase 3 and caspase 9 expressions that trigger apoptosis. Our findings agreed with Shafagh et al.61 who showed that the generation of ROS, up-regulation of p53 expression and unchanged caspase 3 expression in CuO NPs-treated K562 leukemia.

Figure 6
figure6

Impact of FCNPs, FFNPs and FCFNCs on apoptotic and proliferation markers in the treated HepG-2 cells relative to the untreated cancer cell lines. (a) Relative change in the gene expression of p53 and caspase 3 using QPCR. (b-I, II) Microscopic image for Ki-67 of the untreated and treated cancer cells (magnification × 400) with representative percentage of positive immunostaining cancer cells. All values were expressed as mean ± SEM. FCFNCs were compared with all other treatments with significance at *p < 0.05, **p < 0.005, ***p < 0.0005.

Further, FFNPs-treated cancer cells revealed 22.47% Ki-67 staining (a marker for cell proliferation), which was lower than that (52.94%) in FCNPs-treated cancer cells (Fig. 6bI, II). Obviously, FCFNCs-treated cancer cells demonstrated the lowest percentage (8.3%) of Ki-67 staining (Fig. 6bI, II) with a CI value equals to 0.222, which considered as a sign of synergism between CuNPs and FeNPs in biosynthesized hybrid nanocomposites. Consequently, the utilization of biosynthesized FCFNCs in nanotherapy deemed promising by the dint of the enhanced properties of such hybrid that enable halting tumor progression.

Antimicrobial activity

The commercial and traditional organic antimicrobial agents suffer from several shortcomings such as toxicity to human beings, environmental pollution and increasing the chance of the microbes developing multidrug-resistance. Consequently, the recent development of inorganic agents is critical, especially the metals NPs, which exhibiting customized features of antimicrobial functionalities, without contaminating the ambient environment and avoiding damage the tissue in in-vivo applications like wound-healing. There are multidisciplinary applications of such biocidal metal-NPs in food packaging, water disinfection, dental restorative, medical devices, bone prostheses, surgical instruments, smart textiles and skin care products62. Accordingly, the antimicrobial, antibiofilm, algicidal activity of FCFNCs were compared to their sole NPs and antibiotics, at concentrations (250, 500 and 750 µg mL−1).

The cell viability was assessed using the turbidity assay. The data presented in Table 2 indicated that the reduction in the microbial viability induced by all the evaluated nano-species occurred in a dose-dependent manner. Also, the FCFNCs exhibited superior suppression activity than that displayed by their sole counterparts. The statistical analysis of the data with one-way ANOVA showed that treatments with NPs were significantly effective (P ˂ 0.05). Further, the maximum inhibition percentages, 92.7 and 81.6% were observed against B. cereus and S. aureuse, respectively, at 500 µg mL−1 of FCFNCs. These results were similar to those reported by Kim et al.63, and comparable relatively to those obtained by Bakina et al.12. Notably, B. cereus was most susceptible to all examined antimicrobial agents with all concentrations, while P. aeruginosa showed comparatively greater resistance. Hence, the image data demonstrates the sensitivity order of microbial pathogens versus examined NPs, which could be summarized as B. cereus ˃ S. aureus ˃ E. faecalis ˃ E. coli ˃ S. typhi ˃ P. aeruginosa. Moreover, 48.0 ± 0.81 and 22.5 ± 0.57 of viability percentages were noticed respectively for C. albicans under the stress of hybrid nanocomposite at concentrations 250 and 500 µg mL−1. This reflects the significant capability of the NPs to penetrate and corrupt the robust glycoprotein-glucan-chitin structure of fungal cell wall2,4. Though tetracycline and nystatin (reference antibiotics) were more effective, FCFNCs may be on a par with them in the term of activity and thereby could compete economically, especially at higher concentration. In contrast, Akinsiku et al.41 recorded lower efficiency of AgNi bimetallic nanostructures in comparison with the conventional antibiotics, despite well-known toxicity of silver and nickel. Ultimately, all tested planktonic pathogens were almost completely inhibited by FCFNCs with a dose of 750 µg mL−1.

Table 2 The antagonistic efficiency of FCNPs, FFNPs, FCFNCs and antibiotics (tetracycline for bacteria and nystatin for yeast) at 250, 500 and 750 µg mL−1 against planktonic prokaryotic and eukaryotic pathogens.

Antibiofilm activities (synthesis and detachment)

A biofilm lifestyle adopted by planktonic cells that encapsulated themselves in a self-produced protective polymeric matrix, which thereafter adhered to biotic and abiotic surfaces whether in natural, clinical, or even industrial and food-processing environments43. By such type of lifestyle, the microbial cells are refractory to disinfectants, antibiotics and host’ immune systems, which lead to outbreaks and environmental/industrial fouling issues2. Hence, the application of NPs in biofilm eradication during biofilm formation or disintegration of the preformed one is an important solution. In the current study, noticeably progressive inactivation of biofilm formation that increased with increasing concentrations of all treatments was observed (Table 3). Analysis of variance showed that FCFNCs at 750 µg mL−1 caused significant inhibition (P ˂ 0.05) of biofilm growth in S. aureus (90.6 ± 1.3%) and P. aeruginosa (76.11 ± 1.17%). In comparison, Chatzimitakos and coworkers46 found that 100 mg/mL of CuFe nanohybrid corrupted the biofilm formation of E. coli and S. aureus by 12 and 49%, respectively, proofing the efficiency of our FCFNCs in minuscule amounts.

Table 3 Antibiofilm potency of FCNPs, FFNPs, FCFNCs and the antibiotic (tetracycline) at different concentrations in response to biofilm evolution and degradation.

Meanwhile, the biofilm that are already formed showed notoriously tolerance to all treatments. Nonetheless, treatment with 750 µg mL−1 of FCFNCs induced the highest significant effective detachment of S. aureus and P. aeruginosa bulk biofilms reached 58.8 ± 0.8 and 41.8 ± 0.8%, respectively. Such lessness in the antibiofilm efficiency exerted against preformed biofilm deemed intuitive and expected due to the difficulties arose from the contact and interaction between NPs and bacterial cells that are colonized in robust, low permeable and pervasive extracellular polymeric substance (EPS). Let alone higher resistance of stationary cells in the dormant zone of biofilms. As documented by Xiong62, the influence of antimicrobial agents is constrained against dormant microbial cells. Therefore, it is plausible to mention that the higher antibiofilm potency of NPs during growth and evolution of biofilm could be ascribed to the vulnerability of the cells at the planktonic or the early stages of colonization, the direct and better contact between NPs and cells before the formation of polymeric barrier and also the possibility of destruction quorum sensing system2. Out of these results, the efficacy of FCFNCs of this study in the abatement of biofilm formation and disintegration of its architecture encourage their application as an antibiofilm agent in medical devices and also in fouling prevention.

Algicidal activity

The exposure of C. vulgaris cells to biofabricated FCNPs and FFNPs and FCFNCs decreased the cell viability significantly in all examined concentrations as revealed statistically (Table 3). Remarkably, the maximum suppression percentage exerted by FCFNCs of 500 and 750 µg mL−1 concentrations were 42.36 ± 3.7 and 79.31 ± 0.88%, respectively. The NPs may be ranked as follows, in the order of toxicity strength against C. vulgaris, FCFNCs > FFNPs > FCNPs. This considerable algicidal efficacy of FCFNCs empowers their employing in water and wastewater treatment systems to control the environmental issues of algal blooms propagation such as eutrophication and biofouling.

Notably, in this study, FCNPs and FFNPs displayed a considerable significant toxicity. On contrary, Tabrizian32 found the chemically prepared iron oxide NPs (Fe3O4, Fe2O3) didn’t show any microbial toxicity. Moreover, Liu et al.64 recorded a two-fold amelioration in the secondary metabolites production of S. coelicolor-M145 by exposure to 10 mg/L of CuO NPs. Such discrepancy in the results in antimicrobial activities among different studies could be attributed to three major parameters. The first one is concerned with NPs, including particle characteristics (type, size, charge, structure, shape, crystal quality, surface area, stability, agglomeration, magnetization, etc.), particle dosage and synthesis method as well65. The second reason is the microbial cell concentration and microbial cell characteristics (physiologically and biochemically) which definitely differ not only among various microbial genera, but also among the related species40,47,66. The third parameter was the nature of the inactivation process which included the properties of application conditions and the medium in which NPs were applied, including, pH, contact time and organic/inorganic nutrients content of the medium as explained by Pinto et al.13 and Noman et al.36. Various combinations of all such factors could accelerate or delay the microbial inactivation process and vary its effectiveness.

Broadly, CuFe in its composite nanoform manifested new functionalities and remarkable magnitude of toxicity as compared with FCNPs and FFNPs. That could be assigned to the synergistic effect of two different metals rather than merely additive effect. Other similar studies boosted this point of view10,14,40,67. In addition, the driving force of metal- NPs antimicrobial property is the metal ions released from the NPs62,68. In this regard, when microbial cells are exposed to different metal ions released by the nanomaterial of a binary nanostructure, the microbe may be suddenly shocked and become unable to develop multiple gene mutations to resist such concurrent, multiple and condensed dose. This may have enabled FCFNCs to target microorganism at numerous sites simultaneously and exerting more than a microbicidal mode of action. Likewise, Noman et al.36 discussed similar finding.

Interestingly, iron- and copper-containing nanoparticles (Fe, Fe2O3, Fe3O4, Cu, CuO) mostly displayed their toxicity via sequential stages. We could envisage them as nanoknives initiate their action by cracking in the microbial cell wall4, diminishing membrane integrity and generating osmotic imbalance14. Meanwhile, microbial cells internalize the NPs by endocytotic mechanisms62. At this moment, the increased release of metal ions occurs as NPs dissolve intracellularly in a process called Trojan horse mechanism. This causes massive oxidative stress. Then, redox-active particles generate oxygen free radicals or reactive ROS, such as hydroxyl radicals OH, superoxide radicals (O2) and singlet oxygen (1O2), due to the conversion of H2O2 through Fenton and Haber–Weiss reactions68,69.

Actually, elevating the level of ROS in the cell induces programmed death via consecutive paths including, polysaccharides depolymerization, lipid peroxidation, disruption of [4Fe–4S] clusters/thiol-groups of proteins and prohibits DNA replication. Nevertheless, Xiong62, and Palza68, recommended that the toxicity of metal-NPs could be originated from the replacement of other metal ions in their active sites of biomolecules, in a process called ionic mimicry or molecular mimicry. Aside from that, the metal ions released from metal-NPs interact selectively and/or specifically with some atoms such as oxygen, nitrogen and sulfur in the microbial functional groups; forming complexes that lead to serious alterations in the biomolecules conformational structure which leads eventually to malfunction.

Dye detoxification

Azo and triphenylmethane dyes are the most common classes of commercially produced synthetic colorants. For many decades, both types have been utilized as antiseptic agents, fungicides, anthelminthics, ectoparasiticides in aquaculture and commercial fisheries. Also, both recorded as coloring agents and additives in the food processing industry70. As well as, they have been used in the biological laboratories as stains, indicators of acidic media and in amyloidosis diagnosing71. Remarkably, as effluents, they pose a serious threat to the public health and aquatic ecosystem72. Their recalcitrance to degradation hinders light penetration, influences the photochemical activities such as photosynthesis and respiration of aquatic flora/fauna, which subsequently lead to increased chemical oxygen demand (COD) and biological oxygen demand (BOD) and ultimately deteriorate the aquatic ecosystem. Besides, their accumulation in aquatic creatures like fish, mollusks or crustaceans could cause teratogenicity, carcinogenesis, genotoxicity, mutagenicity, hematotoxicity, neurotoxicity as well as multi-organ tissue injuries to human through the food chain73.

Therefore, the efficacy of FCNPs and FFNPs and FCFNCs was examined for decolorization and detoxification of Congo red (CR) and Malachite green (MG) as a model reaction for anionic and cationic synthetic dyes, correspondingly. Thereafter, the microtoxicity and cytotoxicity of NPs-treated solutions were assessed. To evaluate the appropriate contact time, about 1 g/L of biosorbents FCNPs, FFNPs and FCFNCs were agitated with (50 mg/L) CR and MG solutions at different time intervals (30–360 min). As illustrated in Fig. 7, the overall removal process was time-dependent as indicated by gradual fading in the solution color and concurrent decrease in the intensity of absorption band, with time, at 497 nm and 617 nm for CR and MG, respectively. Faster removal was observed in MG solution, reaching up to 61.5 ± 1.5% by FCFNCs within 90 min. After 120 min, there was obvious increment in the removal process reached 94.6 ± 1.1%; a slight difference was shown after 180 min by 99.35 ± 1.8% removal. Concerning CR, only 48.4 ± 2.1% removal was achieved in the first 90 min and reached 93.7 ± 0.5% at 180 min, which remained constant up to 360 min. Conclusively, we could state that 180 min was enough time to obtain a maximum removal of MG and CR. Thus, 180 min as a contact time was selected to explore the effect of different dye concentrations on the removal process.

Figure 7
figure7

Contact time effect on removal of MG (a) and CR (b) using FCNPs, FFNPs and FCFNCs. Dye concentration (50 mg/L), NPs dose 1 g/L, temperature (30 °C), pH 7 and agitation speed (150 rpm); (c), photographic image showing MG and CR removal by FCFNCs with obvious color fading.

The initial concentration of MG and CR was varied from 200 to 500 mg/L with constant adsorbents dose (1 g/L); the decolorization percentages are represented graphically in Supplementary Fig. S7. As shown, the removal efficiency of FCFNCs declined from 81.35 ± 0.9 to 57.88 ± 1.5% by increasing MG concentration from 200 to 500 mg/L. Whereas, CR decolorization percentage recorded 68.6 ± 0.7% and 21.49 ± 1.8% at both dosages. In general, the maximum removal percentages were observed at low MG and CR concentrations, which could be attributed to the abundance of free and unoccupied adsorbent sites available for easily interaction with dye molecules. However, the elimination percentages decreased with increasing dye concentrations due to the saturation of almost adsorbent sites and the unavailability of more sites to adsorb the remaining dye molecules. A similar finding was documented by Luo et al.74 and Tamimi et al.75. Notably, all tested NPs exhibited higher affinity in the removal of MG than CR indicated by a higher removal rate. That could be ascribed to the electrostatic interaction of MG cationic groups with negatively charged NPs. Contrariwise, the lower affinity of tested NPs to anionic dye could be due to coulombic repulsion76. Additionally, the number of functional groups, the presence of acid (SO3) and basic (CH3N) groups, geometry, size and the arrangement of dye molecules could probably influence the dye adsorption capacity of NPs77. Furthermore, as noticed previously, the synergistic effect provided by FCFNCs improved the adsorptive performance for both dyes in a more satisfactory manner than FCNPs and FFNPs.

An exploratory adsorption study by FTIR

To confirm the removal of MG and CR by NPs under investigation, FTIR study of adsorbed dye-NPs was performed. As shown in Supplementary Fig. S8a, b and c, pronounced peaks were observed at 438 cm−1, which corresponded to the binding of cationic ions of MG; the peaks at 1194 cm−1 and 1163 cm−1 for aromatic C–N stretching vibrations. The peaks at 1597 cm−1 indicated C=C stretching of the benzene rings. Also, the bands at 1447 cm−1 and 1458 cm−1 were attributed to scissoring vibration of –CH277,78.

On the other hand, the vibrational modes for CR-treated samples exhibited absorption peaks at 1551, 1641 and 1643 cm−1, which distinguished stretching vibration band of the azo chromophore (–N=N) for the red azo dyes as stated by Bartošová et al.72. In addition, the presence of vibration bands at 1113, 1127 and 1180 cm−1, which characterized asymmetric stretching vibration of S–O(SO3-H) group might imply successful adsorption of CR on the surfaces of FCNPs, FFNPs and FCFNCs (Supplementary Fig. S9a, b and c). Notably, in the remediation profiles of both dyes, new unique bands were noticed including bands at wavelengths 884, 872 and 877 cm−1 for p-disubstituted benzene ring vibrations; 748 and 938 cm−1 for (=C–H) of aromatic rings78. Furthermore, there were shifts in some peak’s positions, especially of Cu and Fe in FCNPs and FFNPs and FCFNCs, with slight alterations in their intensities. Such observations could have several explanations, including a sufficient interaction between functional groups of the dye’s molecules with NPs, cleavage of some functional groups of dyes (e.g. amine) and involvement of new functionalities in the adsorption of dyes on the surface of NPs via Van der Waals forces or weak electrostatic interaction73,78,79.

Toxicity analysis

The crude untreated and NPs-treated MG and CR solutions (200 mg/L and 500 mg L−1) were subjected to microbial toxicity assay versus different microbial classes such as Proteus mirabilis, Bacillus mojavensis, and Metschnikowia pulcherrima (Supplementary Fig. S10). Initially, the control cultures (on NB and Sabouraud dextrose media) exhibited considerable growth by 1.46 × 109, 9.85 × 108 and 1.52 × 109 CFU/mL for P. mirabilis, B. mojavensis, and M. pulcherrima, respectively. While, untreated crude MG solutions of 200 mg/L and 500 mg/L concentrations caused growth reduction of 60.62 ± 2.5% and 83.18 ± 1.7% in P. mirabilis; 49.39 ± 1.3% and 60.68 ± 1.3% in B. mojavensis; and 39.95 ± 1.1 and 62.61 ± 1.8% in M. pulcherrima, respectively. Regarding CR (200 mg/L and 500 mg/L) the results showed microbial growth suppression by (50.08 ± 2.1, 68.1 ± 1.3%), (42.16 ± 2.5, 60.93 ± 1.5%) and (56.74 ± 2.5, 71.73 ± 0.8%) for P. mirabilis, B. mojavensis, and M. pulcherrima, correspondingly. Through FCNPs, FFNPs and CuFe-FCFNCs treatments, an improvement of microbial growth was noticed and inhibition percentage reduced to the range of 9–12% and 20–26% for 200 mg/L and 500 mg/L of MG, respectively. In the case of CR-treated solutions containing 200 mg/L and 500 mg/L, the inhibition percentages recorded 15–20% and 28–30%, respectively.

For cytotoxicity assessment of MG dye, the viability of Wi-38 and Vero human cells diminished to 54.16–58.49% and 49.78–52.54% after incubation with 200 mg/L and 500 mg/mL of MG, respectively (Supplementary Fig. S11a and b). However, incubation of these cells with 1 g/L of FCFNCs-treated MG solutions (200 mg/L and 500 mg/L) elevated viability of Wi-38 and Vero cells (p > 0.05) to (81.96, 85.38%) and (80.49, 81.64%), respectively. Regarding CR dye, after exposure to 200 mg/mL, the viability of Wi-38 and Vero cells decreased to 54.61% and 58.98%, respectively. A 500 mg/mL of the dye lowered the viability of Wi-38 and Vero cells to 50.38% and 54.81%, respectively. As depicted in Supplementary Fig. S11c and d, FCFNCs significantly suppressed the CR cytotoxicity, which was confirmed by the highest viability of Wi-38 and Vero cells by 94.87–98.82% at 200 mg/mL and 84.52–88.01% at 500 mg/mL, respectively.

From our perspective, the mutual effect of combined NPs in FCFNCs, which imitated the antibiotic’s effect and whatever the mechanism of antimicrobial action, showed their potential for application in the adjuvant or combination therapy to defeat pathogens that exhibit multidrug-resistance capability. Besides, the safety level offered by FCFNCs could permit their utilization in agriculture, prevention of biofouling, food packaging, anti-corrosion for water pipes and in pollution remediation instead of rare and expensive noble metals such as Ag, Au, Pt and Pd. The anticancer activity exhibited by our hybrid nanocomposite promotes its application medically in preventing infections, wounds-healing and in contraceptive devices and bone prostheses. Furthermore, the powerful remediation capacity of FCFNCs observed with MG and CR encourages its recruitment in detoxification of effluents from the textile industry, before discharging into water bodies, which represent more than 15% of water pollution as mentioned by Ismail et al.70.

Products You May Like

Leave a Reply

Your email address will not be published.