Attractive study of the antimicrobial, antiviral, and cytotoxic activity of novel synthesized silver chromite nanocomposites

Antibiotic resistance is a global problem. This is the reason why scientists search for alternative treatments. In this regard, seven novel silver chromite nanocomposites were synthesized and assayed to evaluate their antimicrobial, antiviral, and cytotoxic activity. Five bacterial species were used in this study: three Gram-positive (Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus) and two Gram-negative (Escherichia coli and Salmonella enterica). Three fungal species were also tested: Candida albicans, Aspergillus niger, and A. flavus. The MIC of the tested compounds was determined using the bifold serial dilution method. The tested compounds showed good antibacterial activity. Maximum antibacterial activity was attained in the case of 15 N [Cobalt Ferrite (0.3 CoFe2O4) + Silver chromite (0.7 Ag0.5Cr2.5O4)] against M. luteus. Concerning antifungal activity, C. albicans was the most susceptible fungal species. The maximum inhibition was recorded also in case of 15 N [Cobalt Ferrite (0.3 CoFe2O4) + Silver chromite (0.7 Ag0.5Cr2.5O4)]. The most promising antimicrobial compound 15 N [Cobalt Ferrite (0.3 CoFe2O4) + Silver chromite (0.7 Ag0.5Cr2.5O4)] was assayed for its antiviral and cytotoxic activity. The tested compound showed weak antiviral activity. The cytotoxic activity against Mammalian cells from African Green Monkey Kidney (Vero) cells was detected. The inhibitory effect against Hepatocellular carcinoma cells was detected using a MTT assay. The antimicrobial effect of the tested compounds depends on the tested microbial species. The tested compounds could be attractive and alternative antibacterial compounds that open a new path in chemotherapy.


Introduction
Antibiotics and antimicrobial compounds which can inhibit the growth of microbes or kill them are commonly applied in microbial diseases treatment in humans. The world production of different antimicrobial compounds is 100 -200 thousand tons [1,2]. The development of antimicrobial resistance to antibiotics led to a huge social and economic impact that causes a significant threat to our future [3]. Current treatments either are less effective or result in further acquired resistance [4]. Dangerous, antibiotic-resistant bacterial frequency increased over the past decades [5]. The emergence of bacterial resistance worldwide affects antibiotic efficacy [6]. Excessive uptake of antimicrobial compounds leads to the development of resistance to antibiotics which leads to the post-antibiotics era, where microorganisms developed multi-drug resistance [7].
Nanoparticle synthesis is a revolution that happened in all fields that attracted researchers from all branches Open Access BMC Chemistry *Correspondence: asmaa@sci.cu.edu.eg of science [8][9][10][11]. Nanoparticles existed on Earth and man life. It can be manufactured biologically, anthropogenically, and geologically in erosion, volcanoes, fires of forests, burning of charcoal, and industry [12]. Moreover, nanoparticles can be prepared by different methods such as the solid-state method and wet methods [13][14][15][16][17]. The easy, low cost, rapid, save time and give high yield method is the Flash auto-combustion technique in which all the investigated samples have been prepared with it [18][19][20][21][22]. Magnetic nanomaterials are important in many technological applications especially biomedical applications [23][24][25]. As reported, there is a relation between the saturation magnetization and the antimicrobial properties in which high saturation magnetization gives strong antibacterial activity [26]. The physical and magnetic properties of silver chromites nanoparticles were studied in detail in previous work [27]. Considering the mechanism of nanoparticles, nanoparticles of silver are strong candidates as antiviral compounds. Nanoparticles attack a wide range of microbes which is futile [28]. Developments in Nanomedicine lead to evaluation and understanding of the ability of silver nanoparticles (SNPs) to be good antibiotic alternatives [29]. Nanoparticles are used to target bacteria as an alternative to antibiotics [30]. Different types of interactions of nanoparticles with bacterial cells include reactive oxygen production, the release of cation, damage of biomolecules, depletion of ATP, and interaction with membrane [31].
Many methods are used to screen the in vitro antimicrobial activity, like disk-diffusion and broth methods [32].
Generally, the cytotoxicity of the nanoparticles increased with an increase in concentration [33]. Nanoparticles can cross biological barriers and access a wide range of tissues in the body, such as the brain [34]. The risk and hazard of nanoparticles are key to be used by humans. In vivo studies is the source of information regarding its effects on the physiology of organisms [35,36].
Silver chromite is applied in the preparation of important compounds that can be applied in many fields such as cancer diagnosis, biomedical applications, and antimicrobial activities [29,37]. The use of nanomaterials is a suitable way to overcome microbial resistance [3].
In this work novel synthesized silver chromite nanocomposites were evaluated for their antimicrobial, antiviral, and cytotoxic activity.

Synthesis of nanoparticles
Seven nanocomposites were prepared by flash method with initial ingredients metal nitrates such as silver nitrate, chromium nitrate, copper nitrate, lanthanum nitrate, cobalt nitrate, iron III nitrate, and urea mixed with a small amount of distilled water with a stoichiometric ratio. Then the mixture was heated at 250 °C. The ashes were produced then the powders were ground. Samples  ). Highresolution transmission electron microscopy was studied for the pure sample using the HRTEM model Tecnai G20, Netherlands. Moreover, the field emission scanning electron microscopy (FESEM) was studied for pure sample using SEM model quanta 250 FEG apparatus. Also, Atomic force microscopy (AFM) was studied for 8 N nanoparticles (0.3 CuFe 2 O 4 + 0.7Ag 0.5 Cr 2.5 O 4 ) using noncontact wet SPM 9600 Shimadzu.

Biological activity Materials
Fungi and bacteria were supplied by the Faculty of Science, Cairo University.

Isolation and identification of microorganisms
Fungal species were cultivated on a PDA medium; plates were incubated at 25 °C for 7 days. The identification was assured, according to Moubasher [38]. Bacteria were grown on nutrient agar plates. The identification was assured according to growing Bergey's Manual of Determinative Bacteriology [39].

Assay of antimicrobial activity
Sabouraud medium was inoculated with 10 6 colony forming units/ml (CFU/ ml) of microorganisms. Then, discs (6 mm in diameter) containing 100 µg/ml of the test compound were added to the agar surface. The Petri plates were incubated at 27 °C for 5 days. The diameters of inhibition growth zones were measured. Three fungal species: Aspergillus flavus, A. niger, and Candia albicans, were used in this study. Tioconazole (100 µg/ml) was used as a control [40]. Five bacterial species were assayed; three Gram-positive species (Staphylococcus aureus, Micrococcus luteus, and Bacillus subtilis) and two Gram-negative species (Salmonella enterica and Escherichia coli) were added at concentration 10 6 (CFU/ml) to Mueller-Hinton Agar before solidification. Plates were incubated at 35 °C for 2 days. Ampicillin (100 µg/ml) was used as a control [41].

Relative activity
The ratio between the activity of a sample of interest and the activity of the control sample.

Minimum Inhibitory Concentration
The MIC of the assayed compounds was determined [42]. The serial dilution method evaluated the inhibitory effect of the different compounds on bacterial growth. The studied concentrations were 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, and 0.9 µg/ml. The compounds were dissolved in dimethyl sulfoxide. Mueller-Hinton broth and Sabouraud liquid medium were used as culture media for bacteria and fungi, respectively. Test inoculums of 10 4 /CFU were applied.

Antiviral activity
It was done using the Regional Center for Mycology and Biotechnology (RCMB, Al-Azhar University, Cairo, Egypt). This assay was done by the MTT method [45]. Amantadine was used as a control. The viability of cells was determined [46]. The viral inhibition rate was: where A, B, and C are the absorbance of the tested compounds, the absorbance of the virus control, and the absorbance of the cell control, respectively.

Mammalian cell line
Vero cells (derived from the kidney of African green monkey) (ATCC, Manassas, VA, USA). Vero cells were propagated in Dulbecco's modified Eagle medium [47]. Viable cell yield was determined by a MTT [46]. The optical density was measured at 590 nm with the microplate reader (Sunrise, TECAN, Inc, USA). The viability percentage was calculated as [(ODt/ODc)] × 100%, where ODt is the optical density of the tested sample and ODc is the optical density of untreated cells.

Cell line propagation
The cells were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/ml gentamycin. The cells were maintained at 37 ºC. The optical density was measured at 590 nm with the microplate reader (Sunrise, TECAN, Inc, USA). The viability percentage was calculated as [(ODt/ ODc)] × 100%. IC50 is the concentration needed to cause toxic effects in 50% of intact cells (San Diego, CA. USA) [48].
Statistical analysis Data were analyzed using SPSS software version 22. According to Kolmogorov-Smirnova and Shapiro-Wilk tests, data was normally distributed within groups. Accordingly, parametric analysis was applied for the statistical analysis of data. ANOVA was utilized to study the effect of treatment on the studied parameters. Duncan's test was utilized to study the similarity among the studied groups. An Independent t-test was applied to estimate the statistical difference between the control and 15 N groups. Data were presented as mean (n = 3) ± standard deviation. Figure 1a shows the morphology of the pure sample (Ag 0.5 Cr 2.5 O 4 ) using High-Resolution Transmission Electron Microscopy to assure that the sample was in the nanoscale range. The nanoparticles showed an agglomeration due to no surfactant being added during the preparation method. Moreover, the smaller the particles, the easily agglomerated to each other. Figure 1b shows the histogram of the average particle size estimated from the HRTEM micrograph and shows that the nanosized of the histogram was 93.14 nm.

HRTEM analysis
The polydispersity index (P) was calculated using the following formula: The polydispersity index showed that the pure sample gave a 0.35 value.

Antibacterial activity
Seven nanoparticle compounds were assayed against five bacterial species: three Gram-positive (Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus) and two Gram-negative (Escherichia coli and Salmonella enterica).
Maximum inhibition zone diameter (29 mm) was attained in the case of compound 15 N against M. luteus. Other tested species recorded lower inhibition zone diameter. The minimum inhibition zone (11 mm) was shown in the case of compound 8 N against S. enterica (Table 1; Fig. 4).
The highest relative activity (53.57%) was achieved in the case of 17 N against S. enterica, followed by 10 N against E. coli (52.38%) and (50%) in the case of 15 N against S. enterica. Salmonella enterica was significantly inhibited by all tested nanoparticle compounds, while Bacillus subtilis was the most resistant bacterial species to the assayed compounds (Table 2).

Antifungal activity
The seven nanoparticle compounds were assayed for their antifungal activity against three fungal species: Candida albicans, Aspergillus flavus, and A. niger. Compounds showed significant antifungal activity against C. albicans, where 7-14 mm inhibition zone diameters were recorded. The maximum inhibition zone was attained by compound 2 N, while the minimum inhibition zone (7 mm) was recorded in the case of compound 10 N. Weak antifungal activity was recorded in the case of Aspergillus flavus and A. niger (Table 3; Fig. 5).
Concerning the relative activity, it was found that the highest relative activity (52%, 58%, and 44%) was achieved against C. albicans in the case of 1 N, 2 N., and 15 N, respectively (Table 4).

Minimum inhibitory concentration
The data shows that the least MIC (31.25 µg/ml) was obtained in the case of 10 N and 2 N against M. luteus; and compound 15 N against S. enterica and B. subtilis (Table 5; Fig. 6).

Antiviral assay
Based on the results of antimicrobial activity of the tested compounds, compound 15 N was chosen to evaluate its antiviral activity against the Hepatitis A virus. The tested compound showed weak antiviral activity compared to Amantadine (control). The inhibitory activity of the tested sample against the hepatitis A virus was detected under these experimental conditions with a 50% effective concentration (EC50) = 408.14 ± 21.62 µg/ml. The dose inhibited 50% (EC50) was estimated ( Table 6; Fig. 7).

Cytotoxicity test Against VERO cell line
The Cytotoxic activity of the synthesized nanoparticles was assayed against Mammalian cells from African Green Monkey Kidney (Vero) cells was detected under these experimental conditions with 50% cell cytotoxic concentration (CC50) = 280.4 ± 19.3 µg/ml. The results showed that the higher the sample concentration the greater the cytotoxicity. Concentrations less than 31.25 µg/ml of the test compound showed no cytotoxicity (Table 7; Fig. 8).

Cytotoxicity against hepatocellular carcinoma
Inhibitory activity against Hepatocellular carcinoma cells was detected using MTT assay under these experimental conditions with (IC50 = 26.7 ± 2.31 µg/ml) and this was for the compound (15 N). The results indicated that low concentrations of the tested compound (greater than 15.6 µg/ml) showed promising anticancer activity against the tested hepatocellular carcinoma cells (Table 8; Fig. 9).

Discussion
Antimicrobial resistance is one of the public health problems; it has a significant effect on the world. Therapeutic options to treat infectious diseases are limited due to antimicrobial resistance [49]. The misuse and overuse of antimicrobial compounds is a global phenomenon that increases the levels of antimicrobial compounds in the ecosystem and the rates of their spread [50]. The abuse   [51]. Nanoparticles have numerous properties, including antimicrobial activity against many microbes. The antimicrobial nanoparticle compounds can be used to overcome antibiotic resistance [52]. Antimicrobial nanoparticles such as silver nanoparticles have been utilized in biocides for over 120 years [53]. The technique used to prepare nanoparticles should be efficient, low cost, high yield, and produce very fine nanoparticles [54]. The XRD technique initially characterized the synthesized nanoparticles, which confirmed their crystalline nature. Moreover, the XRD analysis of Ag 0.5 Cr 2.5 O 4 was discussed in detail in our previous study [27] and showed the formation of a single-phase spinel structure of the nano sample with a crystallite size of 72.6 nm. The morphology was studied using HRTEM, FESEM, and AFM to confirm that the nanoparticles of the samples were in the nanoscale range. Also, the micrograph and the histogram in Fig. 1 assured that the pure sample (Ag 0.5 Cr 2.5 O 4 ) in the nanoscale range (93.14 nm) with a polydispersity index of 0.35 due to the aggregation of the nanoparticles. Moreover, the physical and magnetic properties are detailed in previous work [27].
Nanoparticles synthesized at low temperature have a small size which enables them to penetrate the cell wall and the cell membrane of the microbial cell to damage the internal organelles and molecules, causing inhibition of growth and even death [55]. The tested compounds Table 3 Antifungal activity of the synthesized nanoparticle compounds against Aspergillus flavus, Aspergillus niger, and Candida albicans Data are displayed as mean (n = 3) ± standard deviation According to Duncan's test, in the same column, means marked with the same superscript letters are insignificantly different (P > 0.05), whereas those marked with different ones are significantly different (P < 0.05). P < 0.000: represent significant effect

Aspergillus flavus Aspergillus niger Candida albicans
Tioconazole (control) 0.00 ± 0.00 A 0.00 ± 0.00 A 25.00 ± 1.00 D  bacteriostatic properties against E. coli at a concentration of nanoparticles of silver oxide of more than 0.01% [56]. The mode of antibacterial activity of metals includes disruption of enzyme function [57], reactive oxygen production [58], disruption of the membrane, prevention of absorption of essential elements [59], and genotoxic activity [60,61].
The tested compounds showed significant antifungal activity. C. albicans was the most susceptible fungal   ]. The dispersion of silver nanoparticles in a polymer matrix enhances antibacterial efficacy by the regulated release of Ag + cations, which may considerably limit infectious agent transmission [62][63][64][65].
The antiviral activity of the tested compound showed weak antiviral activity. This contrasted with Ting et al. 's conclusion that the antiviral impact of the as-prepared GO-AgNPs nanocomposites on virus replication was studied [66]. The findings suggested that exposure to GO-AgNPs nanocomposites was capable of suppressing PRRSV infection. Concerning the cytotoxicity test; it was found that the tested compounds showed low cytotoxicity against normal cells. When the concentration of silver oxide nanoparticles is less than 0.1 percent, the BS/ silver oxide NPs show no harmful impact on eukaryotic cell cultures. The use of the resultant silver oxide nanoparticle composite as a reusable dry disinfectant is justified by its low toxicity and bacteriostatic activity, which are comparable to those of the medicinal alloy nitinol [55]. However, the antitumor efficacy of the silver chromite nanoparticles studied was encouraging. The biological activity of the produced Ni-Zn chromites was evaluated using Hela cell lines [67] and gave promising results. The cytotoxic activity against Mammalian cells from African Green Monkey Kidney (Vero) cells was detected. The inhibitory activity against Hepatocellular carcinoma cells was detected using a MTT assay. The most promising antimicrobial compound 15 N [cobalt Fig. 7 The inhibitory activity of the tested sample against Hepatitis A Virus

Conclusion
The preparation of seven silver nanocomposites using flash auto combustion techniques was synthesized successfully. The morphology of the nanoparticles was investigated using high-resolution transmission electron microscopy (HRTEM), scanning electron microscopy (SEM), and atomic force microscopy to ensure that they were formed in nanosized. The tested compounds (especially 15 N due to the presence of cobalt ferrite and silver chromite) showed promising antibacterial and antifungal activity; at the same time the cytotoxicity was low, and they could be used as efficient therapeutic agents against multidrug-resistant microbes that cause human diseases.