Comparative study of the effect of sliver and selenium nanoparticles on bacterial and viral hepatic infection via modulating oxidative stress and DNA fragmentation

Background: Nanoparticles are recently playing a potential role in improving drug uptake and the and treatment of diseases. A variety of nanoparticles, such as selenium nanoparticles (SeNPs) and Silver nanoparticles (AgNPs) have been used as drug carriers in various ways for treatment of cancers and liver diseases. Our aim in this study is to investigate the ability of AgNPs and SeNPs to target and treat the viral and bacterial infection of liver in rats and cell lines. Methods: For assessment of antioxidant activity of silver nanoparticles, six adult male albino rats were included in this study, liver slices were taken and assigned to 6 groups. Markers of hepatic functions, oxidative stress and inammation in liver slices are carried out. While for assessment of antiviral activity of SeNPs, HBV-replicating human cell line HepG2 and normal human cell lines were used, hepatic and inammatory alterations are determined through quantitative polymerase chain reaction (PCR) and comet assay techniques. Results: The effect of Ag-NPs on interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) levels were reduced in different treated groups with Ag-NPs compared with the control and diseased groups. On the other hand, SeNPs revealed signicant alterations in the inammatory markers as well as DNA damage in the treated HBV- human cell line HepG2 compared to the diseased ones. Conclusion: Silver nanoparticles have the ability for producing various hepatic alterations and can inhibit the proliferation of hepatic stellate cells (HSCs) in a dose and size dependent manner. On the other hand, SeNPs showed excellent selectivity towards viral cells in the HepG2 cell lines. Both Ag-NPs and SeNPs might be a promising drug design for treating viral and bacterial liver diseases. Statistical analysis performed using one-way ANOVA followed by Tukey’s multiple comparison post hoc test; a : significant difference versus the control group.


Introduction
Nanoparticles have been widely investigated over the past decades, due to their special physical and chemical properties. The main properties of nanomaterials depend mainly on their morphology and particle size. AgNPs and SeNPs are the most commercialized nanoparticles used in medical researches for medical applications such as gene, antimicrobial agents and drug delivery carriers (1).
AgNPs have been shown to signi cantly inhibit the replication of HBV (2). Additionally, these AgNPs have been used to target speci c tissues and malignant cells in blood circulation through the combination of antibody-based targeting of ligands and material composition. These studies showed the potential effect of AgNPs for the cure of chronic liver disease including HBV infection and hepatic brosis (3).
Recently, traditional chemotherapy and anti-brotic treatments became ineffective in treatment of chronic liver diseases, due to the development of resistant drug tolerance. Thus, AgNPs may provide a safer therapeutic tool of treatment for targeting the hepatic stellate cells (HSCs), which are the main focus for treatment of liver cirrhosis and hepatic brosis (4).
SeNPs are regarded recently as new nanoparticles and have shown great interest due to their higher antioxidant, antiviral and anti-tumor activity, lower-toxicity to normal cells compared with other used nanoparticles (5). Taken into consideration, selenium (Se) is an essential element which has many important functions in the human body including protection of the cardiovascular and liver organs, regulation of secretion of hormones, and free radical scavenger. Recent studies have shown that SeNPs has potential therapeutic action for treatment of many diseases including in ammation, virus infection, and cancer (6). This new nanoparticle provides a promising tool for treatment of many liver diseases (7).
In this study, we will investigate the e ciency of different concentrations and particle sizes of AgNPs in treatment of liver bacterial infection in rats and the e ciency of SeNPs for targeting and treatment of HBV infected cell lines by evaluating their hepatic and in ammatory changes.

Preparation and synthesis of AgNPs
Two sizes of silver nanoparticles were purchased from (Egypt Center for nanotechnology), as small particle size (10 nm and 75 nm) and large particle size (250-300nm). The size, morphology and dispersion of the nanoparticles were characterized using a Tecnai™ G2 Twin Transmission Electron Microscope (FEI, Hillsboro, OR, USA) and dynamic light scattering (Compact Goniometer System 3; ALV-GmbH, Langen, Germany). Synthesis of silver nanoparticles was achieved by precipitation method using trisodium citrate (TSC) as reducing agent and capping one in the same time. AgNO3 solution (0.03M) was dissolved in 200 mL deionized water, heated to boiling, then 0.3 M TSC was added drop by drop with slowly stirring and heated until the color of solution become pale yellow. The nal solution was cooled at room temperature in isolated dark area to avoid lights (8).

Experimental animal design and conditions
Six adult male albino rats weighing from 200 to 250 gram were used. Rats were housed at MSA animal house under standard laboratory conditions and suitable temperature. They were housed in plastic cages, given standard normal diet and water. The work was done according to ethical committee guidelines in faculty of pharmacy and animal experimentations guideline at MSA University (code: PH8/EC8/2019F).
After reaching to target weight (250 gm), animals were sacri ced to take their livers.
The livers were immediately freezed then kept in fridge for 24 hours. Then, according to Olinga and Sshuppan (2016), a precision cut liver slices was carried out using Cryostat macrotome device in Animal health research institute, the thickness of the slice was optimized to 150 micrometers. The freshly liver slices (14 slice each 150 μm thickness) are transferred to six well plate under controlled physiological conditions. We used six well plate and distribute the slices evenly, every well contained two slices exactly.
After 24-hour incubation, plates were gently removed and the supernatant and tissue homogenate were all collected and then subjected for analysis. Same steps were repeated six times with each rat n=6.

Oxidative stress analysis:
GSH assay: Liver slices were kept frozen at −20 •C and tissue homogenate was made in glass homogenizer using 5% 5-suphosalicylic acid for (GSH). GSH level was assayed using the dithiobinitrobenzoic acid method (9).

Cytokine detection
The quantity of anti-in ammatory markers; IL-6 and TNF-α, in the liver slices homogenate -conditioned medium was quanti ed using an enzyme-linked immunosorbent assay (ELISA) kit (MyBiosource, Inc., San Diego., USA.) according to the manufacturer's instructions.

Histopathological Assessment
After xation of liver specimens in 10% formaldehyde in PBS, liver slices were then dehydrated, embedded into para n and sections were made at a thickness of 5μm. These sections were carefully stained with hematoxylin and eosin (H&E) for histopathology (10).

Preparation of selenium nanoparticles (Se-NPs)
Synthesis of Se-NPs have been done by precipitation -sono-chemical method using ascorbic acid and TSC as reducing-capping agent in the same time. Na 2 SeO 3 solution (0.1M) was dissolved in 120 ml of doubled deionized water until the solution become colorless using ultrasound instrument Hielscher 400UP S , Germany at amplitude of 74% for 1 minutes at temperature of 45°c. Then a mixture of ascorbic acid and TSC (0.2M to 0.1M for ascorbic acid and TSC, respectively.) are added drop by drop until the color of mixture become pale brown. The solution was cooled at room temperature in dark condition (11).

HBV infection and DNA analysis
HBV-replicating human HCC cell line HepG2. Were maintained in Dulbecco's Modi ed Eagle's Medium supplemented with 10% fetal bovine serum. Cell lines were cultured at 37°C in a humidi ed chamber supplemented with 5% CO2. One group left as control and 2 groups were infected by HBV-positive sera.
Cells were incubated with HBV-positive sera (10 9 particles/ml) for 14 h at 37°C, washed extensively with PBS and fresh medium was added. For PCR analysis, at 4 days post-infection cells were washed extensively in PBS, collected using a rubber policeman and lysed in a proteinase K lysis buffer for 6 h at 37°C, extracted with phenol-chloroform and precipitated with ethanol. The amount of DNA was determined by conducting semi-quantitative PCR analysis of HBsAg.

Determination of hepatic function tests and oxidative stress marker
The cell culture media from each of the 25 cm2 culture asks was collected, centrifuged at 3000×g for 10 min and stored at -70°C until assay. ALT and AST were assayed using (Biodiagnostics, CA, USA) while MDA was determined by thiobarbituric acid reactive substances (TBARS) method (12).

Cytokine detection
IL-2 was determined using IL-2 ELISA Kit (ab46032) and IL-8 was measured using IL-8 (CXCL8) ELISA Kit for cell culture supernatants by following the kit protocol while TNF-α was assayed by following the ELISA kit protocol (Cat: KIT10602) and TGF was measured using TGF -α Human (ab100647).

Quanti cation of HBV genomic DNA by Real time PCR
HBV genomic DNA extraction and sequencing analyses. HBV genomic DNA was extracted from the supernatants of co-cultured HepG2 cells using a Viral DNA Isolation Kit (Qiagen, germany) following the manufacturer's instructions. Brie y, cell supernatants were added to virus lysis buffer, and the lysates were loaded onto the spin column. After viral DNA was bound to the membrane, the column was washed and nally, the viral DNA was eluted. PCR was performed using HBV genomic DNA as template to amplify the X gene primer and probe sequences are as follows: forward primer HBV-F3, 5′-GGCCATCAGCGCATGC-3′, and reverse primer HBV-R3M3, 5′-C [5-NitIdl] GCTGCGAGCAAAACA-3′; and probe HBV-P3, 5′-R-CTCTGCCGATCCATACTGCGGAACTC-Q-3. The PCR conditions were: initial denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, and a nal extension was performed at 72°C for 10 min.

Comet Assay
The extent of DNA damage was accessed using the comet assay under alkaline conditions. Comet tail length was measured by uorescence microscopy and then analyzed using CaspLab Comet Assay X-ray Diffraction (XRD) Bruker D8 Discover device was used for XRD measurements. The X-ray source used was Cu Kα radiation with a current of 32 mA and voltage of 41 kV. The 2θ angles ranged from 20 to 70° for Se-NPs and from 35 to 90° for AgNPs with a scan speed of 0.3°/ min. The Raman spectra achieved by a Horiba lab RAM HR evolution spectrometer. The 532 nm edge laser line with Raman shift range from 100 to 1000 cm -1 for Se-NPs and AgNPs range from 20 to 200 cm -1 , grating (450-850 nm) and ND lter 10% to prevent oxidation of Se-NPs and AgNPs. Acquisition time was 15 secs, accumulations of 4 without spike lter and objective was X100.

Index class:
BET method (the Brunauer-Emmett-Teller isotherm) used to determine the speci c surface area by pore and surface area analyzer manufacturer by Quantachrome, model of NOVA touch LX2. Sample was degassed at 80°C for 3 h. under vacuum. DLS and zeta potential achieved by DLS and zeta potential analyzer (Malvern, UK).

Microscopic class:
Atomic Force Microscope (AFM) (5600LS, Agilent, USA) was used its 2D and 3D AFM images to determine surface topography of Se-NPs and AgNPs. Firstly, samples were prepared by subjecting samples to ultrasound waves for 15 minutes, a condition of 50 kHz, at an amplitude of 44% and 0.45 of a cycle (Up 400s manufacture by Hielscher, German). Finally, created a thin lm using Spain coater instrument model Laurell-650Sz at the condition of 700 rpm under vacuum. AFM images and data pro le have been done for 200 nm X 200 nm and its zoom 100 nm X 100 nm using contact mode, Al tap, 0.71 In/S speed, I. gain 2 and P. gain 4. Scanning electron microscopy (SEM) instrument have been done to study Se-NPs surface morphology. The SEM images was achieved by Jol 2000, Japan. Transmission electron microscopy (TEM) study was performed by added Se-NPs to deionized water and sonicating for 15 min using ultrasound prop with a 60 kHz, at an amplitude of 41% and 0.41 of a cycle (Up 400s, Hielscher, German). TEM experiments were achieved using (Jeol, JEM-2100 high-resolution, Japan).

Statistical Analysis
All data are expressed as mean ± SD. The difference between groups was statistically analyzed by GraphPad Prism 6 (La Jolla, CA, USA), using one-way ANOVA followed by Tukey's Kramer Multiple Comparison Test. P-value < 0.05 was considered as signi cant.

Microscopic class:
2D and 3D AFM images and data showed that colloidal Se-NPs nanoparticles samples have excellent homogenous surface topography. In addition, AFM images illustrated the rode shape (blue) with excellent caping of citrate (green) composed core-shell nanostructure with dramatic shape and structure change where most of literature synthesis spherical Se-NPs and the cap was not recognized as shown in Fig. (3). While for AgNPs as shown in Fig. (4), AFM images showed its spherical shape with very sharp edges and homogenous in size and shape. Scanning Electron Microscope (SEM) and Transmission Electronic Microscope (TEM) results for AgNPs were found in accordance with AFM results as show in Fig. (6). Colloidal Se-NPs nanoparticles have rode core-shell nanostructure with very sharp edge of both core and shell with width size about 30 nm and length about 200 nm. While AgNPs have spherical shape with size range from 15 to 25 nm. Sem image also determined the rode core-shell nanostructure of Se-NPs and spherical shape of AgNPs as shown in gure (5).

Index class:
BET surface area measured for Se-NPs and AgNPs was 25.8 m 2 /g and 70.1 m 2 /g respectively. The Nitrogen adsorption-desorption isotherm curve shows unity IV type isotherms with mesopoues as shown in gure (7) for Se-NPs and IV type for AgNPs as shown in gure (8). Zeta sizing and potential illustrated in table (1) and gures (7 B and 8 B), the results showed the very strong stability of Se-NPs and AgNPs in aqueous solution due to its high zeta potential value which con rm the colloidal properties of both of them. The DLS curve for Se-NPs illustrated the presence of two sharp peaks at size about of 50 nm and 200 nm which con rm the presence of two sizes; one for length and other for width of rode core-shell nanostructure as shown in gure (7C). While AgNPs curve showed one sharp peak at 15 nm as shown in gure (8 C).

AgNPs Improved Liver Function Tests
In group treated with Ags (0 to 100 nm), ALT level was ranged from (31: 44 U/L) with the mean equal to (37.5 U/L±2.5) showing signi cant increase compared to normal ones but signi cant decrease in comparison to LPS groups (P < 0.05). Also, in group of Ag with large particle size, ALT level was ranged from (35: 46 U/L) indicating signi cant increase in comparison to normal group but signi cantly decreased in comparison to LPS group (P < 0.05); while it shows no signi cant difference in comparison to group of Ag with small particle size. Similarly, AST level was ranged from (19: 22 U/L) with the mean equal to (20.3 U/L±0.6) showed signi cant decrease in comparison to LPS or NAC groups (P < 0.05). In NAC + AgL group, AST level showed (19.7 U/L±0.6) signi cant increase in comparison to normal while it causes a signi cant decrease in AST level in comparison to LPS or NAC group (P < 0.05) as shown in g.

Antioxidative and Anti-in ammatory Effects of AgNPs
The bacterial infection in LPS group resulted in a 2.6-fold decrease in the lipid peroxidation marker expressed as glutathione (GSH) compared with control liver slices indicating liver damage. Treatment with AgS increased GSH by around 40% and similarly In Ag L group, treatment caused 2-fold increase in GSH level compared to LPS group (Figure 9 C).
The anti-in ammatory properties of AgNPs in Liver slices were assessed by measuring the IL-6 and TNF-α levels. Treatment with both AgS and AgL resulted in the decrease of the anti-in ammatory IL-6 and the decrease of the pro-in ammatory TNF-α. Upon treatment with AgS, the IL-6 decreased by more than 1.6folds while AgL decreased it by 1.5-folds. On the other hand, AgS treated group decreased TNF-α by 21% compared to the diseased LPS group while AgL surpassed this to reach 32% decrease in the levels of TNF-α as shown in g. (10).

Histopathological Assessment
H&E staining revealed signi cant changes obtained in the histological examination between the control, LPS and treated groups with small and large particles of AgNPs. In the LPS group, the liver tissue shows activation of Kupffer cells and sporadic hepatocytes necrosis while both the AgS and the AgL groups showed slight activation of Kupffer cells and few necrosis effects of hepatocytes ( Figure 11).

SeNPs Improved Liver Function Tests and reduced oxidative stress marker
After treatment of HBV-hepG2 cell lines with SeNPs, both ALT (U/L) and AST (U/L) levels were increased by 1.7 and 2.5-folds while the MDA concentration decreased. SeNPs administration enhanced the liver function tests and decreased the level of both enzymes as well as decreasing the secretion of MDA by 1.2-fold ( Table 2).

Anti-in ammatory Effects of SeNPs in HBV-replicating human HCC cell line HepG2
SeNPs showed 1.3-fold and 1.5 decrease in both proin ammatory markers TNF-α and TGF levels in HepG2 cell lines after administering of SeNPs. These values were decreased signi cantly after treatment. Additionally, SeNPs decreased the level of IL-8 by 46 % and IL-2 by 43%. Treatment with SeNPs lowered the level of all proin ammatory markers signi cantly compared to the control group ( Figure 12).

Effects of SeNPs on DNA fragmentation
The effect of the administration of SeNPs on DNA fragmentation in HepG2 cell lines is illustrated in Fig.   13. A signi cant more than 8-fold increase in the tail length and 3.7-fold increase in tail DNA% (tDNA%) was shown in the HepG2 cell lines infected with HBV. Treatment with SeNPs signi cantly protected HepG2 cell lines from DNA damage as indicated by a decrease the tail's length% by 24% and the tDNA% by 40% compared to the diseased group.

Discussion
Recently, studies focused on the ability of nanoparticles in drug delivery and targeting, leading to treatment of various liver diseases (13,14). Consequently, this study investigated the effects of both AgNPs and SeNPs on liver tissue injury due to viral and bacterial causes. The study showed different mechanisms for the effect of AgNPs on liver tissue even at low concentration; either through the particle size of the AgNPs or through the down regulation of secretion of cytokines by hepatic cells, which may lead to HSC activation.
Another evidence was observed in the histological changes including, necrosis and hepatocellular degeneration that were dose dependent. Several studies con rmed that liver is the main target organ for the effect of AgNPs and showed that liver tissue after treatment with AgNPs exposure may be associated with reduction of oxidative stress (15,16). It is known that oxidative stress is a main mechanism by which silver nanoparticle affect treatment of injured liver tissues (17,18). In this study, markers of oxidative stress and in ammatory mediators were measured in the liver tissues. We observed a signi cant decrease in GSH level and a signi cant increase in TNF-and IL-6 in hepatocytes. Our study suggests that the ability of AgNPs to cause a signi cant decline in the in ammatory markers leads to reduce apoptosis through in ammatory and oxidative stress mechanisms.
Several previous studies have focused on the effect of nanoparticle size on cytotoxicity and cellular uptake (19). In this study, AgNPs of two different sizes classi ed as small particle size (10 nm and 75 nm) and large particle size (250-300nm) at different concentrations were included in the treatment of bacterial infected liver slices together with NAC. Activation of HSCs is signi cant in development of liver disease through the secretion of many cytokines. The results of the Elisa assay demonstrated that the anti-in ammatory effects exerted by AgNPs on HSCs are size-and dose-dependent. Larger AgNPs showed signi cant reduction in TNF-α than the smaller particles, while for the anti-in ammatory marker; IL-6, both small and large particles showed signi cant reduction in IL-6 compared to the LPS group. According to our study, the other mechanism by which AgNPs can treat the liver bacterial infection occurs through its effects on the up-regulation of antioxidants (GSH) and this was clearly obvious in the groups treated with small and large AgNPs compared to LPS group.
Moreover, AgNPs affected successfully the liver function tests, this was con rmed by the signi cant decrease of AST and ALT levels in groups treated with NAC together with small and large particles compared with LPS group and group treated with NAC only, which indicate a signi cant protective role of AgNPs. Therefore, the reducing effects of AgNPs on production of important cytokines as well as liver function tests which was also con rmed by other previous studies may suppress the progression of hepatic brosis; however, the detailed mechanisms require more studies (20). The size of AgNPs is highly signi cant in the cellular uptake by tissues, and thus affecting their bioactivity (21). the design of nanoparticles for medical applications should take into consideration the particle size to obtain the maximum effect.
There are several causes for chronic liver disease including; alcohol addiction, fatty liver, brosis, hepatitis B and C and cirrhosis (22). Recently SeNPs are considered a potential tool for treatment of cancers due to its chemical protective agent against toxic side effects of anticancer drugs (23). In the present study, the treatment of HBV-replicating human cell line HepG2 with SeNPs showed signi cant alterations in in ammatory mediators produced by injured liver tissue and DNA fragmentation.
Endocytosis has been proved as an important cellular uptake mechanism for nanoparticles. Many previous studies con rmed that selenium nanoparticles divide into smaller particles under the acidic conditions of the lysosomes, which helps the metabolism of SeNPs and the release of the loaded drugs (24).
Lipid peroxidation markers are products of oxidation and reduction process, including highly toxic compounds (25). These markers are considered to be a signi cant factor in cancer prevalence and recurrence. In addition, many studies have shown that alterations in lipid oxidation and in ammatory markers level in cells affect cell apoptosis and in turn could be used in treatment (26).
Our results agree with another study conducted by (27) who suggested that this injury is caused due to increase in the oxidative stress process in liver, and this was clearly obvious in our study which showed signi cant reduction in MDA levels in treated group with SeNPs compared to HepG2 cell lines. Moreover, our results were also con rmed by a recent study (24) which found that HBV causes in ammation and chronic liver injury which affects the damage of DNA, our current ndings showed signi cant decrease in the in ammatory markers; TNF-α, TGF, IL-6 and IL-2 in the HepG2 cell lines treated with SeNPs compared to HBV-HepG2 cell lines. Additionally, the increase in DNA damage in HBV cell lines which may be due to the release of free radicals including nitric oxide reactive species (NOS) and reactive oxygen species (ROS) which leads to liver injury (28). These ROS led to in ammatory responses, necrosis of liver cells and brogenesis (29).
Our current results of comet assay showed that the treatment of HBV-cell lines with SeNPs caused signi cant decrease in DNA damage compared to HBV-cell lines, these results were reported also by another study (30). Taken into consideration, previous studies have shown that SeNPs has a signi cant therapeutic action for several diseases such as in ammation, cancer, and viral infection (31,32), for this reason, this nano system is drawing great attention in the eld of therapy due to its higher stability, antiviral activity, anti-in ammatory activity and low toxicity compared to other nanoparticles.

Conclusion
In view of the present study, the e cacy of AgNPs was investigated by different biochemical approaches and it was particle dose and particle-dependent. The biochemical alterations in the in ammatory and oxidative stress markers may be an indication of the anti-in ammatory activity and the inhibitory activity of proliferation of injured cells caused by AgNPs in liver tissue. On the other hand, SeNPs played a potential role in reducing DNA damage as well as production of cytokines in HBV-cell lines. Taken together, both nanoparticles might be an innovative approach for treatment of viral and bacterial liver infection, however more studies are needed to support the use of AgNPs and SeNPs for human disease treatment and prevention.     Illustrate XRD pattern (A) and Raman spectra (B) of silver nanoparticles.