Anti-microbial and anti-cancer activities of Mn0.5Zn0.5DyxFe2-xO4 (x ≤ 0.1) nanoparticles

Abstract Combining two or more nanoparticles is a promising approach. Previously we have reported synthesis of nanoparticles Dysprosium (Dy) substituted with manganese (Mn) zinc (Zn) by using ultrasonication method. The five different nanoparticles (NPs) Mn0.5Zn0.5DyxFe2-xO4 (x ≤ 0.1) have been structurally and morphologically characterized but there is no report on the biological application of these NPs. In the present study, we have examined the anti-cancer, anti-bacterial, and anti-fungal activities of Mn0.5Zn0.5DyxFe2-xO4 (x ≤ 0.1) NPs. Human colorectal carcinoma cells (HCT-116) were tested with different concentrations of NPs by using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. In addition, the impact of NPs was also examined on normal cells such as human embryonic kidney cells, HEK-293. After 48 h of treatment, Mn0.5Zn0.5DyxFe2-xO4 NPs (x = 0.02, 0.04 and 0.06) showed no inhibitory action on cancer cell’s growth and proliferation, whereas Mn0.5Zn0.5DyxFe2-xO4 NPs (x = 0.08 and 0.1) showed profound inhibitory action on cancer cell’s growth and proliferation. However, the treatment of Mn0.5Zn0.5DyxFe2-xO4 NPs on the normal cells (HEK-293) did not show cytotoxic or inhibitory action on HEK-293 cells. The treatment of Mn0.5Zn0.5DyxFe2-xO4 NPs (x ≤ 0.1) also inhibited both the bacteria (Escherichia coli ATCC35218 and Staphylococcus aureus) with lowest MIC and MBC values of 4 and 8 mg/mL and fungus (Candida albicans) with MIC and MFC values of 4 and 8 mg/mL on treatment with x = 0.08 and 0. 1.

In our previous studies, we have shown that different kinds of MNPs showed potent anti-cancer and anti-bacterial activities [30][31][32]. Recently it has been found that a combination of two or more nanoparticles is effective for improved NPs delivery and treatments [33,34]. NPs can be synthesized by chemically and green technology [35,36]. Previously we have reported that synthesis of Dysprosium (Dy) substituted with Manganese (Mn), Zinc (Zn) NPs by ultrasonication method. The Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) have been structurally and morphologically characterized [33], but there is no report on the biological application of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1). Interestingly, MNPs have useful applications in cancer detection and treatment [28,33,[37][38][39]. We have examined the impacts of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) on HCT-116, HEK-293 cells, bacteria (Escherichia coli ATCC35218, and Staphylococcus aureus), and fungus (Candida albicans).

Nanoparticle preparation and characterizations
The Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) were prepared as per previously described method [33] by ultrasonic irradiation method. All products were structurally and morphologically analysed by an X-ray powder diffractometer (XRD), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) methods.

Minimal inhibitory/bactericidal concentration (MIC/MBC)
Anti-bacterial activity of synthesized Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) was examined by obtaining the MIC and MBC using Escherchia coli ATCC35218 as gram negative and Staphlyloccocus aureus ATCC29213, as gram positive bacteria. The nanomaterial ranging in the concentration of 0.5-16 mg/ mL, was suspended and later sonicated in LB (Luria Bertaini) to achieve the suspended broth-drug solution. . The test bacteria were grown overnight and brought to cell concentration of 2.5 Â 10 5 CFU/mL. The Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) and culture broth media suspension were incubated at temperature of 37 C for 24 h. Both bacteria without the addition of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) were taken as negative control. The value of MIC is recorded as the effective amount of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) that arrests 99% of microbial growth [40].

Minimum bactericidal concentration (MBC)
After the study of MIC, 25 mL of the incubated Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) and bacterial suspension with no turbidity seen, were inoculated on new sterile Mueller Hinton Agar (MHA) plates and kept for incubation at 37 C for 24 h. MBC value is taken as the effective amount of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) that kills 100% of microbial cell growth.

Antifungal activity
Candida albicans ATCC 14053 (yeast) was used for the antifungal activity of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1). Candida was aerobically grown in Sabauraud's broth (SDB) at 28 ± 2 C for 24 h. Subsequently, the cells were harvested and washed using phosphate buffer saline (PBS), and cell density was adjusted to approximately 10 7 CFU/mL. The Mn 0.5 Zn 0.5 Dy x Fe 2- x O 4 NPs (x 0.1) was sonicated and prepared using SDB in the concentration, ranging from 0.5 to 16 mg/mL.

Minimal inhibitory concentration (MIC)
The standard broth dilution method was used for anti-fungal activity by evaluating the MIC of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1). The suspension of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) and SDB was added with freshly prepared inoculum and subjected to incubation with aeration at 28 ± 2 C for 24 h. Subsequently, the MIC was recorded as the minimum concentration of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) were used, which had no growth visible (absence of turbidity) [40].

Minimal fungicidal concentration (MFC)
MFC was obtained by selecting the MIC, having no visible growth. Briefly, 10 mL from MIC was inoculated onto the freshly prepared SDA plates and further incubated at 28 ± 2 C for overnight. The plates were observed for MFC, which was recorded as the minimum concentration of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1), which completely killed the Candida cells or had CFUs less than three.

Morphological analysis of treated Candida
The morphological effect of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) on Candida cells was studied by SEM. As described in previous section, Candida was treated with synthesized NPs at the concentration recorded as its MIC value. The untreated Candida was also included in the experiment, as a control. On completion of incubation period, the treated cells were obtained by centrifugation. The cell pellet was washed multiple times with PBS and re-centrifuged. The cells were fixed with 2.5% glutaraldehyde for 4 h, at 4 C and fixed again, with 1% osmium tetroxide for 30 min. The fixed cells were washed multiple times using PBS. Further, the Candida cells were dehydrated for 10 min by using varying concentration of ethanol, and later again washed with PBS. The fixed Candida was placed onto aluminium stub and dried using a desiccator. Dried cells were gold coated and examined at 20 kV by SEM [40].
One way ANOVA followed by Tukey's test was used to compare mean concentration. p value less than .05 was considered to show significant difference

Anticancer assay
In vitro cell culture and testing of NPs Human colorectal or colon carcinoma cells (HCT-116) were taken to study the impact of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1). The normal cells, such as human embryonic kidney cells (HEK-293), were taken as control cells. The cell culture method and in vitro testing method of nanoparticles were followed as per previously published work [32,41]. Briefly, the cells were treated with various concentrations of (2.

Statistical analysis
The mean ± standard deviation (SD) from control and NPs group was calculated. All statistical analyses were completed with GraphPad Prism 6.0 (GraphPad Software). The difference between control and NPs groups by a one-way analysis of variance (ANOVA), p-values were calculated by Student's t-test.

Characterization
The structural and morphological characterizations of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) were analysed by an XRD, TEM and SEM methods which has been previously published study by Almessiere et al. [33]. As per XRD, the average size of the MNPs was in the range from 11 to 18 nm. Direct optical energy band gaps were in a small band range of 1.61-1.67 eV. All products displayed super-paramagnetic properties at room temperature [33]. The SEM analysis showed that NPs revealed a regular size and uniform distribution of cubic particles with aggregation and the average particles size was less than 20 nm and was found to increase in size with increasing the Dy content. Similarly, the TEM analysis showed that NPs were agglomeration of cubic nanoparticles because of the magnetic interaction among nanoparticles [33].  (Figure 1(b)). It could be seen that the antibacterial action is getting slightly enhanced with the addition of 'x' content, hence the significant MIC/MBC was achieved by x ¼ 0.08 and 0.10. The significant variation was obtained with the increasing concentration of x content. Moreover, gram negative bacterium was seen to be affected slightly better than the gram-positive bacterium. This slightly selective action towards the gram positive and gram-negative bacteria is probably due to the structural variation in the chemistry of cell wall between the two [28]. In several studies many amalgamations of metal nanomaterial like zinc, copper, manganese are recorded to have antibacterial impact, however, the antibacterial impact of the current combination of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) is rare in the reported literature, to the best of author's knowledge [42].

MIC And MFC
In the present study, C. albicans was used for the antifungal activity of synthesized Mn 0. 5 Figure 2). The lowest MIC and MFC values of 4 and 8 mg/mL were recorded with x ¼ 0.08 and 0.1. The antifungal activity of treated Candida culture was achieved as the manipulation of the element Dy(x ¼ n) in the nanomaterial. The significant variation was obtained with the increasing concentration of x content. The obtained results proved, that the antifungal activity was relatively increased with the elevated concentration of 'x' content and goes in agreement with some previous studies, which stated the same effect, on combination of several metallic NPs [40,30,43,44].

Morphological analysis by scanning electron microscope (SEM)
It is evident from SEM micrographs (Supplementary Figure  S1), that Candida treated with Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) were appearing as deformed and distorted as compared to the untreated cells, which appeared normal and smooth in shape (Figure 3(a)). The cell wall and cell membrane of treated Candida indicated the loss of the integrity of membrane which resulted in distortion and ultimately to cell death [45]. The best possible reason of this action could be the size and easy attachment of NPs to the cellular surfaces and targeting the ergosterol, which is a main sterol responsible for the integrity of fungal cell wall [46,47]. Among all the ratios of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x 0.1) (x ! 0.02), x ¼ 0.08 and 0.1 has been the most effective in causing the morphogenesis as well as the reduction in the number of cells (Supplementary Figure S1). Different studies have also suggested that the NPs induces the production of hydrogen peroxide, creates oxidative stress and lipid peroxidation, and deactivation of cellular enzymes, which leads to inhibition of microbial growth and morphogenesis [48].

Dna disintegration analysis
We have also examined the DNA of cancer cell post-treatment of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x ¼ 0.08 and 0.1) by DAPI staining. The Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x ¼ 0.08 and 0.1) treated DAPI-stained cells showed that NPs treatment caused an inhibitory action on colon cancer cells (Figure 3(B, C)) compared to control cells (Figure 1(A)). Colon cancer cells treated with Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x ¼ 0.08 and 0.1) with concentration (0.71 mg/mL) and (1.48 mg/mL) showed a decrease in DNA stained cells due to cytotoxicity (Figure 3(B and C)).
Our results showed that the treatment of Mn 0.5 Zn 0.5 Dy x Fe 2-x O 4 NPs (x ¼ 0.08 and x ¼ 0.1) possess potential application in colon cancer treatments. Previously reports supported that nanoparticles can significantly improve cancer treatment by selectively inhibiting growth and proliferation of the colon cancer cells [32,41,[52][53][54][55]

Conclusion
There is no report on the biological application of

Disclosure statement
There is no potential conflict of interest was reported by the author(s).
The study is supported by Imam Abdulrahman Bin Faisal University project application number IRMC-015-2019.