# Internalization ofStaphylococcus aureus in Lymphocytes Induces Oxidative Stress and DNA Fragmentation: Possible Ameliorative Role of Nanoconjugated Vancomycin

Authors: Subhankari Prasad Chakraborty; Santanu Kar Mahapatra; Sumanta Kumar Sahu; Sabyasachi Das; Satyajit Tripathy; Sandeep Dash; Panchanan Pramanik; Somenath Roy
Journal: Oxidative Medicine and Cellular Longevity
 (2011)

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## Abstract

Staphylococcus aureus is the most frequently isolated pathogen causing bloodstream infections, skin and soft tissue infections and pneumonia. Lymphocyte is an important immune cell. The aim of the present paper was to test the ameliorative role of nanoconjugated vancomycin against Vancomycin-sensitive Staphylococcus aureus (VSSA) and vancomycin-resistant Staphylococcus aureus (VRSA) infection-induced oxidative stress in lymphocytes. VSSA and VRSA infections were developed in Swiss mice by intraperitoneal injection of 5×106 CFU/mL bacterial solutions. Nanoconjugated vancomycin was adminstrated to VSSA- and VRSA-infected mice at its effective dose for 10 days. Vancomycin was adminstrated to VSSA- and VRSA-infected mice at a similar dose, respectively, for 10 days. Vancomycin and nanoconjugated vancomycin were adminstrated to normal mice at their effective doses for 10 days. The result of this study reveals that in vivo VSSA and VRSA infection significantly increases the level of lipid peroxidation, protein oxidation, oxidized glutathione level, nitrite generation, nitrite release, and DNA damage and decreases the level of reduced glutathione, antioxidant enzyme status, and glutathione-dependent enzymes as compared to control group, which were increased or decreased significantly near to normal in nanoconjugated vancomycin-treated group. These findings suggest the potential use and beneficial role of nanoconjugated vancomycin against VSSA and VRSA infection-induced oxidative stress in lymphocytes.


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## Body

## 1. Introduction

Staphylococcus aureusis a major human pathogen causing significant morbidity and mortality in both community- and hospital-acquired infections [1]. It causes a diverse array of infections ranging from relatively minor skin and wound infections to more serious and life-threatening diseases such as pneumonia, endocarditis, osteomyelitis, arthritis, and sepsis. Concerns over the emergence of multidrug-resistant strains have renewed interest in understanding the virulence mechanisms of this pathogen at the molecular level and in elucidating host defense elements that either provide protection or limit infection [2, 3]. Many staphylococcal infections which tend to become chronic (e.g., osteomyelitis and mastitis) are associated with multiple recurrences and do not resolve even in the presence of what seems to be an adequate humoral immune response [4]. S. aureushas been shown to be ingested by nonprofessional phagocytes, such as mouse fibroblasts, mouse renal cells, and bovine mammary epithelial cells [5, 6]. S. aureusalso has the ability to invade mouse and human osteoblast cell lines, as well as normal mouse and human osteoblasts [7, 8].Polymorphonuclear neutrophils (PMNs) have long been thought to provide significant host defense againstS. aureusinfection primarily because patients who are neutropenic or who have congenital or acquired defects in PMN function are more susceptible to infection with this pathogen [9]. The possibility that phagocytes, particularly PMN, could facilitate S. aureusinfection has been raised by other investigators [10–13].  In vivo studies suggested that the ability of S. aureusto exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureusinfection [14]. Till now no studies have assessed whether survival of S. aureusinside lymphocyte occurs in vivo and whether this can promote infection.Chitosan (CS), the deacetylated form of chitin, is a linear polysaccharide, composed of glucosamine and N-acetyl glucosamine linked in aβ linkage [15]. CS has been reported to possess immune stimulating properties such as increasing accumulation and activation of macrophages and polymorphonuclear, suppressing tumor growth, augmenting antibody responses and inducing production of cytokines [16]. Carboxy methyl chitosan (CMC) is synthesized from CS by carboxylation of the hydroxyl and amine groups [17]. In our previous laboratory report, we have synthesized carboxymethyl chitosan-2,2′-ethylenedioxy bis ethylamine-Folate (CMC-EDBE-FA) nanoparticle based on carboxy methyl chitosan tagged with folic acid by covalently linkage through 2,2′-(ethylenedioxy) bis-(ethylamine); vancomycin was loaded onto it called “nanoconjugated vancomycin” and observe its in vitro bactericidal activity against S. aureus [18]. In our recent laboratory report, we observed that CMC-EDBE-FA is nontoxic and it inhibits the nitric oxide-mediated Staphylococcus aureus pathogenesis in lymphocytes in a dose- and duration-dependent manner [19, 20]. The present study was aimed at testing the ameliorative role of nanoconjugated vancomycin against VSSA- and VRSA-induced oxidative stress in lymphocytes.


## 2. Results

### 2.1. Characterization of CMC-EDBE-FA

The peak assignment of CMC was as follows: 1741 cm−1 (–COOH), 1070–1136 cm−1 (–C–O), and 1624 and 1506 cm−1 (–NH3+). FA-EDBE showed the characteristic absorption bands at 1650 and 1550 cm−1 located in the zone related to the (–CONH–), corresponding, respectively, to the (C=O) stretching band and to the (–NH) bending vibration band. The presence of these two bands indicates that an amide bond has been formed between –COOH of folic acid and the –NH2 amine end group of EDBE. The more characteristics of these two bands have become more prominent and intense in CMC-EDBE-FA. This provides evidence for the formation of an extra amide bond during the attachment of folic acid. 1H NMR spectrum of CMC-EDBE-FA showed the peaks at about 1.9 ppm attributed to the methyl hydrogen of acetamido-2-deoxy-β-D-glucopyranosyl unit; the peaks at about 2.9–3.2 ppm attributed to methylene hydrogen atoms of EDBE and 3.5–4 ppm observed the glucopyranosyl hydrogen atoms. It was clear the proton peaks of 8.7, 7.6, 6.9, 6.4 ppm were observed in 1H NMR spectrum of CMC-EDBE-FA. No such peaks were observed in the same chemical shifts of 1H NMR spectrum for CMC. The appearance of these peaks confirms the successful conjugation of FA-EDBE with CMC. The size of CMC-EDBE-FA self-assembled nanoparticles in aqueous medium measured by dynamic laser light-scattering (DLS) ranged from 210±40 nm. The morphology of CMC-EDBE-FA self-aggregated nanoparticles was investigated by TEM. The nanoaggregate shows a spherical geometry and having a uniform size. At lower magnification, nanoparticles having an average size of about 50 nm were observed (Figures are not shown) [18].


### 2.2. Nitrite Generation by Lymphocytes and Release in Serum

Nitrate (NO) generation is an indicator of free radical generation. NO generation was significantly (P<0.05) increased in lymphocytes by 123.56% and 149.66%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) decreased by 48.16% and 47.19% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin decreased NO generation significantly (P<0.05) in VSSA-infected lymphocytes by 32.17%, but by 5.08% in VRSA-infected lymphocytes which is not significant. Vancomycin, at its most effective doses used in this experiment, did not induce significantly NO generation in normal lymphocytes; however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly decreased (P<0.05) NO generation by 27.74% and 50.33% in normal lymphocytes (Figure 1). NO release was significantly (P<0.05) increased in serum by 99.91% and 105.71%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) decreased by 42.11% and 42.09% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin decreased NO release significantly (P<0.05) in VSSA-infected lymphocytes by 30.73%, but by 3.22% in VRSA infected group which is not significant. Vancomycin, at its most effective doses used in this experiment, did not induce significantly NO release in control groups; however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly decreased (P<0.05) NO release by 20.67% and 24.87% in normal serum (Figure 3).Figure 1
Nitrite generation (NO) in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocytes. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


### 2.3. Myeloperoxidase Activity of Lymphocyte

Myeloperoxidase (MPO) is an important enzyme to produce hypochlorous acid (HOCl) in cellular system that leads to oxidative damage. MPO activity was significantly (P<0.05) increased in lymphocytes by 92.65% and 104.28%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) decreased by 43.19% and 48.29% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin decreased MPO activity significantly (P<0.05) in VSSA-infected lymphocytes by 38.24%, but by 5.37% in VRSA infected lymphocytes which is not significant. Vancomycin, at its most effective doses used in this experiment, did not induce significantly MPO activity in normal lymphocytes; however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly decreased (P<0.05) MPO activity by 23.6% and 33.87% in normal lymphocytes (Figure 2).Figure 2
Myeloperoxidase (MPO) activity in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocytes. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 3
Release of nitrite in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated serum. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


### 2.4. Lipid Peroxidation Level in Lymphocytes

Lipid peroxidation is an important determinant to access the cellular damage. Lipid peroxidation in terms of malondialdehyde (MDA) level was significantly (P<0.05) increased in lymphocytes by 187.23% and 228.5%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) decreased by 51.24% and 54.35% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin decreased MDA levels significantly (P<0.05) in VSSA-infected lymphocytes by 33.49%, but by 4.05% in VRSA-infected lymphocytes which is not significant. Vancomycin, at its most effective doses used in this experiment, did not induce significantly lipid peroxidation in normal lymphocytes; however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly decreased (P<0.05) MDA level by 22.13% and 28.38% in normal lymphocytes (Figure 4).Figure 4
Lipid peroxide (MDA) level in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


### 2.5. Protein Oxidation Level in Lymphocytes

Like lipid peroxidation, protein oxidation is also an important determinant to access the cellular damage. Protein oxidation in terms of protein carbonyl (PC) level was significantly (P<0.05) increased in lymphocytes by 129.37% and 164.57%, respectively, due to VSSA and VRSA infections as compared to control group, which was significantly (P<0.05) decreased by 43.71% and 47.76% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin decreased protein carbonyl level significantly (P<0.05) in VSSA-infected lymphocytes by 33.68%, but by 4.99% in VRSA-infected lymphocytes which is not significant. Vancomycin, at its most effective doses used in this experiment, did not induce significantly protein oxidation in normal lymphocytes; however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly decreased (P<0.05) PC level by 26.51% and 32.61% in normal lymphocytes (Figure 5).Figure 5
Protein Carbonyl (PC) level in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


### 2.6. Glutathione Level and Redox Ratio in Lymphocytes

Glutathione is an important antioxidant in cellular system. So to understand glutathione level, we have measured both reduced and oxidized forms of glutathione. Reduced glutathione (GSH) and the redox ratio (GSH/GSSG) were significantly (P<0.05) diminished in lymphocytes by 51.2%, 60.71%, 73.64%, and 80.52%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) increased by 68.2%, 94.12%, 173.91%, and 236.76% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin increased reduced glutathione and the redox ratio significantly (P<0.05) in VSSA-infected lymphocytes by 46.59% and 103.26%, but by 15.03% and 26.47% in VRSA infected lymphocytes which is not significant (Figures 6 and 8). Oxidized glutathione (GSSG) level was significantly (P<0.05) elevated in lymphocytes by 84.89% and 101.76%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) decreased by 38.62% and 42.48% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin decreased oxidized glutathione level significantly (P<0.05) in VSSA-infected lymphocytes by 27.87%, but by 9.07% in VRSA-infected lymphocytes which is not significant (Figure 7). Vancomycin did not alter any significant change in reduced glutathione level, oxidized glutathione level, and redox ratio in normal lymphocytes at its effective doses used in this experiment. Nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly increased (P<0.05) reduced glutathione level and redox ratio by 30.4%, 51.93%, 68.48%, and 118.05% and significantly decreased (P<0.05) oxidized glutathione level by 22.71% and 30.4% in normal lymphocytes (Figures 6–8).Figure 6
Reduced glutathione (GSH) level in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 7
Oxidized glutathione (GSSG) level in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 8
Redox ratio (GSH/GSSG) in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


### 2.7. Antioxidant Enzyme Status in Lymphocytes

The super oxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-s-transferase (GST) were measured to understand the antioxidant enzymes status of different experimental group of lymphocytes. SOD, CAT, GPx, GR, and GST activity were significantly (P<0.05) diminished in lymphocytes by 44.65%,  54.41%, 64.24%, 50.83%, 50.01%, and 51.12%, 65.85%, 66.63%, 56.47%, 53.55%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) increased by 67.37%, 83.28%, 136.99%, 75.93%, 71.27%, and 79.29%, 129.08%, 184.31%, 94.13%, 86.35% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin increased all these antioxidant enzymes activity significantly (P<0.05) in VSSA-infected lymphocytes by 48.3%, 44.37%, 71.96%, 44.74%, and 36.99%, but by 8.52%, 13.84%, 6.65%, 7.66%, and 9.11% in VRSA-infected lymphocytes which is not significant. Vancomycin, at its most effective doses used in this experiment, did not alter any significant change in these antioxidant enzyme activity, however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly increased (P<0.05) these antioxidant enzymes activity by 22.5%, 35.4%, 27.08%, 32.92%, 31.73% and 29.22%, 48.23%, 38.91%, 52.11%, 48.14% in normal lymphocytes (Figures 9–13).Figure 9
Superoxide dismutase (SOD) activity in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 10
Catalase (CAT) activity in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 11
Glutathione peroxidase (GPx) activity in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 12
Glutathione reductase (GR) activity in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 13
Glutathione-s-transferase (GST) activity in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


### 2.8. Detection of Lymphocyte Apoptosis by DNA Fragmentation

DNA fragmentation is an indicator of apoptotic cell death; hence the quantitative and qualitative DNA fragmentation in lymphocyte was evaluated in this study. Quantitative DNA fragmentation in all groups of lymphocyte was evaluated by diphenylamine (DPA) assay spectrophotometrically. VSSA and VRSA infection produced 461.58% and 521.52% fragmented DNA, whereas control group showed negligible fragmented DNA in lymphocyte that was associated significantly (P<0.05). Treatment of nanoconjugated vancomycin significantly (P<0.05) decreased DNA fragmentation by 68.84% and 69.55%, respectively. Treatment of vancomycin decreased DNA fragmentation significantly (P<0.05) in VSSA-infected lymphocytes by 47.3%, but by 8.05% in VRSA-infected lymphocytes which is not significant. Vancomycin, at its most effective doses used in this experiment, did not induce significantly DNA fragmentation in normal lymphocytes; however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly decreased (P<0.05) DNA fragmentation by 27.58% and 32.85% in normal lymphocytes (Figure 14). To confirm the data obtained from the spectrophotometric method, we have analyzed the DNA fragmentation by DNA laddering in 1.2% agarose gel electrophoresis. Agarose gel electrophoresis of DNA, isolated from lymphocytes of VSSA- and VRSA-infected lymphocytes, showed the presence of typical DNA ladder (hall mark of apoptosis), while the DNA of normal lymphocytes did not show any ladder. We have found the negligible smear of DNA in nanoconjugated vancomycin-treated group (Figure 15).Figure 14
Quantitative estimation of DNA fragmentation assay by diphenylamine (DPA) assay in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 15
DNA fragmentation study by agarose gel electrophoresis in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


## 2.1. Characterization of CMC-EDBE-FA

The peak assignment of CMC was as follows: 1741 cm−1 (–COOH), 1070–1136 cm−1 (–C–O), and 1624 and 1506 cm−1 (–NH3+). FA-EDBE showed the characteristic absorption bands at 1650 and 1550 cm−1 located in the zone related to the (–CONH–), corresponding, respectively, to the (C=O) stretching band and to the (–NH) bending vibration band. The presence of these two bands indicates that an amide bond has been formed between –COOH of folic acid and the –NH2 amine end group of EDBE. The more characteristics of these two bands have become more prominent and intense in CMC-EDBE-FA. This provides evidence for the formation of an extra amide bond during the attachment of folic acid. 1H NMR spectrum of CMC-EDBE-FA showed the peaks at about 1.9 ppm attributed to the methyl hydrogen of acetamido-2-deoxy-β-D-glucopyranosyl unit; the peaks at about 2.9–3.2 ppm attributed to methylene hydrogen atoms of EDBE and 3.5–4 ppm observed the glucopyranosyl hydrogen atoms. It was clear the proton peaks of 8.7, 7.6, 6.9, 6.4 ppm were observed in 1H NMR spectrum of CMC-EDBE-FA. No such peaks were observed in the same chemical shifts of 1H NMR spectrum for CMC. The appearance of these peaks confirms the successful conjugation of FA-EDBE with CMC. The size of CMC-EDBE-FA self-assembled nanoparticles in aqueous medium measured by dynamic laser light-scattering (DLS) ranged from 210±40 nm. The morphology of CMC-EDBE-FA self-aggregated nanoparticles was investigated by TEM. The nanoaggregate shows a spherical geometry and having a uniform size. At lower magnification, nanoparticles having an average size of about 50 nm were observed (Figures are not shown) [18].


## 2.2. Nitrite Generation by Lymphocytes and Release in Serum

Nitrate (NO) generation is an indicator of free radical generation. NO generation was significantly (P<0.05) increased in lymphocytes by 123.56% and 149.66%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) decreased by 48.16% and 47.19% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin decreased NO generation significantly (P<0.05) in VSSA-infected lymphocytes by 32.17%, but by 5.08% in VRSA-infected lymphocytes which is not significant. Vancomycin, at its most effective doses used in this experiment, did not induce significantly NO generation in normal lymphocytes; however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly decreased (P<0.05) NO generation by 27.74% and 50.33% in normal lymphocytes (Figure 1). NO release was significantly (P<0.05) increased in serum by 99.91% and 105.71%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) decreased by 42.11% and 42.09% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin decreased NO release significantly (P<0.05) in VSSA-infected lymphocytes by 30.73%, but by 3.22% in VRSA infected group which is not significant. Vancomycin, at its most effective doses used in this experiment, did not induce significantly NO release in control groups; however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly decreased (P<0.05) NO release by 20.67% and 24.87% in normal serum (Figure 3).Figure 1
Nitrite generation (NO) in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocytes. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


## 2.3. Myeloperoxidase Activity of Lymphocyte

Myeloperoxidase (MPO) is an important enzyme to produce hypochlorous acid (HOCl) in cellular system that leads to oxidative damage. MPO activity was significantly (P<0.05) increased in lymphocytes by 92.65% and 104.28%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) decreased by 43.19% and 48.29% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin decreased MPO activity significantly (P<0.05) in VSSA-infected lymphocytes by 38.24%, but by 5.37% in VRSA infected lymphocytes which is not significant. Vancomycin, at its most effective doses used in this experiment, did not induce significantly MPO activity in normal lymphocytes; however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly decreased (P<0.05) MPO activity by 23.6% and 33.87% in normal lymphocytes (Figure 2).Figure 2
Myeloperoxidase (MPO) activity in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocytes. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 3
Release of nitrite in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated serum. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


## 2.4. Lipid Peroxidation Level in Lymphocytes

Lipid peroxidation is an important determinant to access the cellular damage. Lipid peroxidation in terms of malondialdehyde (MDA) level was significantly (P<0.05) increased in lymphocytes by 187.23% and 228.5%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) decreased by 51.24% and 54.35% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin decreased MDA levels significantly (P<0.05) in VSSA-infected lymphocytes by 33.49%, but by 4.05% in VRSA-infected lymphocytes which is not significant. Vancomycin, at its most effective doses used in this experiment, did not induce significantly lipid peroxidation in normal lymphocytes; however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly decreased (P<0.05) MDA level by 22.13% and 28.38% in normal lymphocytes (Figure 4).Figure 4
Lipid peroxide (MDA) level in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


## 2.5. Protein Oxidation Level in Lymphocytes

Like lipid peroxidation, protein oxidation is also an important determinant to access the cellular damage. Protein oxidation in terms of protein carbonyl (PC) level was significantly (P<0.05) increased in lymphocytes by 129.37% and 164.57%, respectively, due to VSSA and VRSA infections as compared to control group, which was significantly (P<0.05) decreased by 43.71% and 47.76% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin decreased protein carbonyl level significantly (P<0.05) in VSSA-infected lymphocytes by 33.68%, but by 4.99% in VRSA-infected lymphocytes which is not significant. Vancomycin, at its most effective doses used in this experiment, did not induce significantly protein oxidation in normal lymphocytes; however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly decreased (P<0.05) PC level by 26.51% and 32.61% in normal lymphocytes (Figure 5).Figure 5
Protein Carbonyl (PC) level in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


## 2.6. Glutathione Level and Redox Ratio in Lymphocytes

Glutathione is an important antioxidant in cellular system. So to understand glutathione level, we have measured both reduced and oxidized forms of glutathione. Reduced glutathione (GSH) and the redox ratio (GSH/GSSG) were significantly (P<0.05) diminished in lymphocytes by 51.2%, 60.71%, 73.64%, and 80.52%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) increased by 68.2%, 94.12%, 173.91%, and 236.76% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin increased reduced glutathione and the redox ratio significantly (P<0.05) in VSSA-infected lymphocytes by 46.59% and 103.26%, but by 15.03% and 26.47% in VRSA infected lymphocytes which is not significant (Figures 6 and 8). Oxidized glutathione (GSSG) level was significantly (P<0.05) elevated in lymphocytes by 84.89% and 101.76%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) decreased by 38.62% and 42.48% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin decreased oxidized glutathione level significantly (P<0.05) in VSSA-infected lymphocytes by 27.87%, but by 9.07% in VRSA-infected lymphocytes which is not significant (Figure 7). Vancomycin did not alter any significant change in reduced glutathione level, oxidized glutathione level, and redox ratio in normal lymphocytes at its effective doses used in this experiment. Nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly increased (P<0.05) reduced glutathione level and redox ratio by 30.4%, 51.93%, 68.48%, and 118.05% and significantly decreased (P<0.05) oxidized glutathione level by 22.71% and 30.4% in normal lymphocytes (Figures 6–8).Figure 6
Reduced glutathione (GSH) level in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 7
Oxidized glutathione (GSSG) level in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 8
Redox ratio (GSH/GSSG) in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


## 2.7. Antioxidant Enzyme Status in Lymphocytes

The super oxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-s-transferase (GST) were measured to understand the antioxidant enzymes status of different experimental group of lymphocytes. SOD, CAT, GPx, GR, and GST activity were significantly (P<0.05) diminished in lymphocytes by 44.65%,  54.41%, 64.24%, 50.83%, 50.01%, and 51.12%, 65.85%, 66.63%, 56.47%, 53.55%, respectively, due to VSSA and VRSA infection as compared to control group, which was significantly (P<0.05) increased by 67.37%, 83.28%, 136.99%, 75.93%, 71.27%, and 79.29%, 129.08%, 184.31%, 94.13%, 86.35% due to treatment of nanoconjugated vancomycin. Treatment of vancomycin increased all these antioxidant enzymes activity significantly (P<0.05) in VSSA-infected lymphocytes by 48.3%, 44.37%, 71.96%, 44.74%, and 36.99%, but by 8.52%, 13.84%, 6.65%, 7.66%, and 9.11% in VRSA-infected lymphocytes which is not significant. Vancomycin, at its most effective doses used in this experiment, did not alter any significant change in these antioxidant enzyme activity, however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly increased (P<0.05) these antioxidant enzymes activity by 22.5%, 35.4%, 27.08%, 32.92%, 31.73% and 29.22%, 48.23%, 38.91%, 52.11%, 48.14% in normal lymphocytes (Figures 9–13).Figure 9
Superoxide dismutase (SOD) activity in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 10
Catalase (CAT) activity in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 11
Glutathione peroxidase (GPx) activity in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 12
Glutathione reductase (GR) activity in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 13
Glutathione-s-transferase (GST) activity in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


## 2.8. Detection of Lymphocyte Apoptosis by DNA Fragmentation

DNA fragmentation is an indicator of apoptotic cell death; hence the quantitative and qualitative DNA fragmentation in lymphocyte was evaluated in this study. Quantitative DNA fragmentation in all groups of lymphocyte was evaluated by diphenylamine (DPA) assay spectrophotometrically. VSSA and VRSA infection produced 461.58% and 521.52% fragmented DNA, whereas control group showed negligible fragmented DNA in lymphocyte that was associated significantly (P<0.05). Treatment of nanoconjugated vancomycin significantly (P<0.05) decreased DNA fragmentation by 68.84% and 69.55%, respectively. Treatment of vancomycin decreased DNA fragmentation significantly (P<0.05) in VSSA-infected lymphocytes by 47.3%, but by 8.05% in VRSA-infected lymphocytes which is not significant. Vancomycin, at its most effective doses used in this experiment, did not induce significantly DNA fragmentation in normal lymphocytes; however, nanoconjugated vancomycin, at its most effective doses used in this experiment, significantly decreased (P<0.05) DNA fragmentation by 27.58% and 32.85% in normal lymphocytes (Figure 14). To confirm the data obtained from the spectrophotometric method, we have analyzed the DNA fragmentation by DNA laddering in 1.2% agarose gel electrophoresis. Agarose gel electrophoresis of DNA, isolated from lymphocytes of VSSA- and VRSA-infected lymphocytes, showed the presence of typical DNA ladder (hall mark of apoptosis), while the DNA of normal lymphocytes did not show any ladder. We have found the negligible smear of DNA in nanoconjugated vancomycin-treated group (Figure 15).Figure 14
Quantitative estimation of DNA fragmentation assay by diphenylamine (DPA) assay in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.Figure 15
DNA fragmentation study by agarose gel electrophoresis in control, 100 mg/kg bw/day vancomycin, 500 mg/kg bw/day vancomycin, 100 mg/kg bw/day nanoconjugated vancomycin, 500 mg/kg bw/day nanoconjugated vancomycin, VSSA infection control, VSSA infection + 100 mg/kg bw/day vancomycin,  VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin, VRSA infection control, VRSA infection + 500 mg/kg bw/day vancomycin,  VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin for 10-day treated lymphocyte. Values are expressed as mean ± SEM,n=6. *indicates significant difference (P<0.05) compared to control group. #indicates significant difference (P<0.05) compared to infection control group.


## 3. Discussion

S. aureushas long been considered as an extracellular pathogen, which may occasionally survive and even multiply within phagocytes resulting in prolonged and recurrent infections [21]. It is one of the most successful human pathogen with the ability to colonize and infect both hospitalized patients with or without compromised host defenses and healthy immunologically competent people in the community [22]. However, S. aureusmay occasionally become intracellular, at least within monocytes, macrophages, and polymorphonuclear neutrophils (PMNs) when host defense mechanisms are activated [23]. Our previous lab report showed that lymphocyte is susceptible to S. aureusinfection through generation of nitric oxide, and the infection was successfully eliminated by treatment of nanoconjugated vancomycin in a dose- and duration-dependent manner [20]. In this context, our present study proves to be more relevant and will help further study in investigating the ameliorative role of nanoconjugated vancomycin against VSSA and VRSA induced oxidative stress in lymphocytes.CMC-EDBE-FA nanoparticles were prepared by the carboxylic group (–COOH) of folic acid and –COOH group of functionalized carboxymethyl chitosan connected through the end-amino groups hydrophilic spacer, 2,2′-(ethylenedioxy)-bis-ethylamine. It is well known that carboxymethyl chitosan is easily soluble in water but folic acid has less solubility in water. When carboxymethyl chitosan is connected by folic acid through a spacer, carboxymethyl chitosan may act as a hydrophilic part and folic acid as a hydrophobic part. It is evident from our study that, in vivo VSSA and VRSA infection in mice lymphocytes is associated with enhanced nitrate generation, nitrate release, MPO activity, MDA level, PC level, GSSG level, and decreased GSH level, and as well as decreased enzymatic antioxidant (SOD, CAT, GPx, GR, and GST) activity, which are ameliorated by treatment of nanoconjugated vancomycin (Figures1–13). Moreover, DNA damage assessed by DPA assay and agarose gel electrophoresis due to VSSA and VRSA infection was also observed in lymphocytes, which are protected by treatment of nanoconjugated vancomycin (Figures 14 and 15).In this study, a significant elevation of nitrate generation, MPO activity in lymphocyte, and nitrate release in serum was observed in VSSA- and VRSA-infected mice, whereas nitrate generation, MPO activity in normal lymphocytes, and nitrate release in normal serum was decreased in nanoconjugated vancomycin-treated group. Treatment of nanoconjugated vancomycin to VSSA- and-VRSA infected mice decreased NO generation, MPO activity significantly in lymphocytes, and nitrate release in serum (Figures1–3). Nitric oxide (NO) is a free radical synthesized by nitric oxide synthase (NOS). NOS is composed of two identical monomers with molecular weights ranging from 130 to 160 kDa [24]. Our previous study had shown that nitric oxide synthesis in lymphocytes and release in serum is high during VSSA and VRSA infection, which can be related to an alteration in oxidant-antioxidant potential [20]. Thus, higher level of nitrite release by VSSA and VRSA infection may be due to high production of free radicals, mainly NO. Nanoconjugated vancomycin plays the role of antioxidant to prevent the nitrate generation may be through the inhibition of inducible nitric oxide synthase (iNOS) expression [25]. Hypochlorous acid (HOCl) is generated in the presence of myeloperoxidase and initiates the deactivation of antiproteases and the activation of latent proteases and leads to the cellular damage [26]. In this study, nanoconjugated vancomycin inhibited the myeloperoxidase activity which was increased due to VSSA and VRSA infection, suggesting a protective role of nanoconjugated vancomycin (Figure 2). These results suggest that either the cellular antioxidants level reached in a higher concentration in lymphocyte exert antioxidant effects or scavenged the free radical produced by the myeloperoxidase [27]. Thus, in addition to the cellular antioxidant system, nanoconjugated vancomycin may indirectly protect lymphocyte from VSSA- and VRSA-infection-induced cellular changes. Thus, free radical depletion by the antioxidant agents seems to be beneficial for preventing the damage of lipid and protein.In this study, significant elevation of malondialdehyde (MDA) and protein carbonyl level was observed in lymphocyte of VSSA- and VRSA-infected mice; whereas MDA and PC levels in normal lymphocytes were decreased in nanoconjugated vancomycin-treated group. Treatment of nanoconjugated vancomycin to VSSA- and VRSA-infected mice decreased lipid peroxidation and protein oxidation significantly in lymphocytes (Figures4 and 5). It may be due to the generation of free radicals (mainly NO) which may react with protein in addition to lipids. Lipid peroxidation is known to disturb the integrity of cellular membranes; leading to the leakage of cytoplasmic enzymes [28]. Protein carbonyls formation has been indicated to be an earlier marker of protein oxidation. Oxidation of protein may be due to either excessive oxidation of proteins or decreased capacity to clean up oxidative-damaged proteins. Oxidative modification of proteins may lead to the structural alteration and functional inactivation of many enzyme proteins [29], as evidenced by the decreased activity of different antioxidant enzymes like SOD, CAT, GPx, GR, and GST.Reactive oxygen species (ROS) are generated during oxidative metabolism and can inflict damage on all classes of cellular macromolecules and eventually leading to cell death. An elevation in free radical formation can be accompanied by an immediate compensatory increase in the activities of the free radical scavenging enzymes [30]. Imbalance between the generation of reactive oxygen species (ROS) and the antioxidant system causes oxidative stress. Glutathione, an important cellular reductant, is involved in protection against free radicals, peroxides, and toxic compounds in cellular systems [31]. In the present study, the reduced glutathione level and redox ratio were significantly decreased in lymphocyte of VSSA- and VRSA-infected mice, whereas GSH level and redox ratio in normal lymphocytes were increased in nanoconjugated vancomycin-treated group. Treatment of nanoconjugated vancomycin to VSSA and VRSA infected mice increased the GSH level and redox ratio in lymphocytes (Figures 6–8). In this study, it was observed that oxidized glutathione level was increased in VSSA- and VRSA-infected lymphocytes, which was ameliorated due to nanoconjugated vancomycin treatment. GSSG level in normal lymphocytes was decreased in nanoconjugated vancomycin-treated group (Figure 7). The decreased GSH levels represent its increased utilization due to VSSA and VRSA infection. On the other hand, decreasing GSH level may be due to increasing level of lipid oxidation products which may be associated with less availability of NADPH required for the activity of glutathione reductase (GR) to transform GSSG to GSH [32] due to the increasing production of ROS in form of NO. In our present study, the increasing levels of GSSG and decreasing GR activity (Figure 12) due to VSSA and VRSA infection may support the explanation.Antioxidant enzymes are considered to be a primary defense that prevents biological macromolecules from oxidative damage. SOD rapidly dismutates superoxide anion (O2.-) to less dangerous H2O2, which is further degraded by CAT and GPx to water and oxygen [33]. The results of the present study showed a significant fall in SOD and CAT activities in lymphocyte of VSSA- and VRSA-infected group; where as SOD and CAT activities in normal lymphocytes were increased in nanoconjugated vancomycin-treated group. Treatment of nanoconjugated vancomycin to VSSA- and VRSA-infected mice significantly increased the SOD and CAT activity in lymphocytes (Figures 9 and 10). SOD, dismutateO2.- and the same in turn, is a potent inhibitor of CAT [34]. The depletion in SOD activity was may be due to dispose off the free radicals, produced due to VSSA and VRSA infection. Beside this, during infection, H2O2 produced by dismutation of superoxide anion may have been efficiently converted to O2 by CAT and the enzyme activities showed a marked reduction. The depletion of antioxidant enzyme activity was may be due to inactivation of the enzyme proteins by VSSA and VRSA infection-induced NO generation, depletion of the enzyme substrates, and/or downregulation of transcription and translation processes.GPx works nonspecifically to scavenge and decompose excess hydroperoxides including H2O2, which may be prevalent under oxidative stress [35–37]. Glutathione-S-transferase (GST) mainly detoxifies electrophilic compounds [38] and has a well-established role in protecting cells from mutagens and carcinogens as a free radical scavenger along with glutathione. In the present study, the significant decreasing of GSH level and GSH-dependent enzymes, that is, GPx, GR, and GST (Figures 11–13) in lymphocyte of VSSA and VRSA infection may be due to increased utilization to scavenge the free radical generation. The results of the present study showed a significant fall of GPx, GR, and GST activities in lymphocyte of VSSA- and VRSA-infected group, whereas GPx, GR, and GST activities in normal lymphocytes were increased in nanoconjugated vancomycin treated group. Treatment of nanoconjugated vancomycin to VSSA- and VRSA-infected mice significantly increased the GPx, GR, and GST activity in lymphocytes (Figures 11–13). In the present study, it was observed that MDA level (Figure 4) and DNA fragmentation (Figures 14 and 15) were significantly elevated in lymphocytes due to VSSA and VRSA infection. This elevated MDA level decreases GSH level (Figure 6) and SOD activity (Figure 9), which may be associated with DNA fragmentation in lymphocytes. In this study, it was observed that DNA fragmentation increased in VSSA- and VRSA-infected lymphocytes, which was brought back near to control due to nanoconjugated vancomycin treatment. DNA fragmentation in normal lymphocytes was decreased in nanoconjugated vancomycin-treated group (Figure 14).In conclusion, in the study described here, lymphocytes are susceptible toS. aureus infection through the increased production of nitric oxide which leads to decreased antioxidant status in cell, and nanoconjugated vancomycin protects the lymphocytes from such infection by decreasing free radical generation, lipid and protein damage, and also by increasing the antioxidant status. Hence, the nanoconjugated vancomycin can be used as a potent free radical scavenger antioxidative product and can be used as a potential therapeutic agent against staphylococcal infection.


## 4. Materials and Methods

### 4.1. Chemicals and Reagents

Sodium dodecyl sulfates (SDS), 2,4-dinitrophenyl hydrazine (DNPH), 5′,5′-dithio (bis)-2-nitrobenzoic acid (DTNB), standard reduced glutathione (GSH), glutathione reductase (GR), NADPH Na4, NADPH, oxidized glutathione (GSSG), agarose, folic acid (FA),Chitosan (medium molecular weight), dicyclohexyl carbodiimide (DCC), Trifluoroacetic acid, 2,2′-(ethylenedioxy)-bis-(ethylamine) (EDBE), di-tert-butyldicarbonate (BoC2O), N-hydroxysuccinimide (NHS) and 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide Hydrochloride (EDC), RNase A,  and Proteinase K were purchased from Sigma Chemical Co., USA. Sodium chloride (NaCl), sodium dodecyl sulfate, ethylene diamine tetra acetate (EDTA), tryptic soy broth, luria broth, mannitol salt agar, agar powder, sucrose, magnesium chloride (MgCl2), sodium azide (NaN3), phenol/chloroform/iso-amyl alcohol, ethidium bromide, and bromophenol blue were purchased from Himedia, India. Tris-HCl, Tris buffer, potassium dihydrogen phosphate (KH2PO4), di potassium hydrogen phosphate (K2HPO4), sodium hydroxide (NaOH), sodium acetate, ammonium acetate, alcohol, sulfanilamide, phosphoric acid, N-C-1 naphthyl ethylene diamine dihydrochloride, diphenylamine (DPA), glycerol, boric acid, O-phenylenediamine, and other chemicals were procured from Merck Ltd., SRL Pvt. Ltd., Mumbai, India. All other chemicals were from Merck Ltd., SRL Pvt., Ltd., Mumbai and were of the highest grade available.


### 4.2. Animals

Experiments were performed using Swiss male mice 6–8 weeks old, weighing 20–25 g. The animals were fed standard pellet diet and water was givenad libitum and housed in polypropylene cage (Tarson) in the departmental animal house with 12 h light : dark cycle, and the temperature of 25 ± 2°C. The animals were allowed to acclimatize for one week. The animals used did not show any sign of malignancy or other pathological processes. Animals were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India and approved by the ethical committee of Vidyasagar University.


### 4.3. Bacterial Strain

We used coagulase-positive vancomycin-sensitive and -resistantStaphylococcus aureusstrains that were isolated from human postoperative pus sample [39]. These bacterial strains were grown at 37°C for overnight in tryptic soy broth. The bacterial culture was centrifuged at 15,000 rpm for 15 minutes. The pellet was resuspended and washed with sterile phosphate buffer saline (PBS). Using a UV-spectrophotometer (Schimadzu, USA) at an absorbance of 620 nm, we adjusted the viable bacterial count to approximately 1.0×109 colony-forming units (CFU)/mL, which corresponded to an optical density of 1.6. The bacterial suspension was adjusted by serial dilution in phosphate buffer saline (PBS) to give a final concentration of approximately 5×106 in 100 μL of bacterial suspension [14].


### 4.4. Preparation of CMC-EDBE-FA Nanoparticle and Loading of Vancomycin

CMC-EDBE-FA nanoparticle was prepared, and vancomycin was loaded onto it according to our previous laboratory report [18].


### 4.5. Development of VSSA and VRSA Infection in Swiss Mice

VSSA and VRSA infection was developed in male Swiss mice by intraperitoneal (i.p.) injection of 100μL of bacterial suspension containing 5×106 CFU/mL according to our previous laboratory report [20].


### 4.6. Experimental Design

VSSA- and VRSA-infected mice were treated with nanoconjugated vancomycin for successive 10 days at a dose of 100 mg/kg bw/day and 500 mg/kg bw/day, respectively. The dose and duration of nanoconjugated vancomycin was selected from our previous laboratory report [20]. The following groups were considered for the experiment.Group I: Control.Group II: 100 mg/kg bw/day vancomycin.Group III: 500 mg/kg bw/day vancomycin.Group IV: 100 mg/kg bw/day nanoconjugated vancomycin.Group V: 500 mg/kg bw/day nanoconjugated vancomycin.Group VI: VSSA infection.Group VII: VSSA infection + 100 mg/kg bw/day vancomycin.Group VIII: VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin.Group IX: VRSA infection.Group X: VRSA infection + 500 mg/kg bw/day vancomycin.Group XI: VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin.After the termination of experiment, animals were sacrificed by an intraperitoneal injection of sodium pentobarbital (60–70 mg/kg body weight) [40], and blood (n=6/group) was used for preparation of serum and separation of lymphocyte for biochemical estimation of oxidative parameters.


### 4.7. Separation of Serum and Lymphocyte

Serum was obtained by centrifugation of blood samples taken without anticoagulant at 1500 g for 15 min and was kept at −86°C for the further estimation. Heparinized blood samples were used for the separation of Lymphocyte. Lymphocytes were isolated from blood using standard isolation techniques [41]. Blood samples were diluted with equal amount of PBS (pH 7.0) buffer and then layered very carefully on the density gradient (Histopaque 1077, Sigma Chemical Co.) in 1 : 2 ratio. It was centrifuged at 500 g for 20 min and the white milky layer of mononuclear cells that is, lymphocytes was carefully removed. The layer was washed twice with the same buffer and centrifuged at 3000 g for 10 min to get the required pellet of lymphocytes [42]. The pellets of lymphocytes were lysed in a hypotonic lysis buffer for 45 min at 37°C and kept at −86°C until biochemical estimations [42].


### 4.8. Biochemical Estimation

#### 4.8.1. Nitrite (NO) Production by Lymphocyte

After treatment schedule, 100μL of Griess reagent (containing 1 part of 1% sulfanilamide in 5% phosphoric acid, and 1 part of 0.1% of N-C-1 napthyl ethylene diamine dihydrochloride) was added to 100 μL of sample, incubated at room temperature for 10 minutes, readings were taken in a UV spectrophotometer at 550 nm and compared to a sodium nitrite standard curve (values ranging between 0.5 and 25 μM). The levels of NO were expressed as μmol/mg protein [43].


#### 4.8.2. Determination of Myeloperoxidase Activity in Lymphocyte

200μL of cell lysate was reacted with 200 μL substrate (containing H2O2 and OPD) in dark for 30 min. The blank was prepared with citrate phosphate buffer (pH 5.2) and substrate, in absence of cell-free supernatant. The reaction was stopped with addition of 100 μL 2 (N) sulfuric acid and reading was taken at 492 nm in a spectrophotometer [44]. The MPO activity was expressed in terms of μM/mg protein.


#### 4.8.3. Determination of Lipid Peroxidation (MDA)

Lipid peroxidation was estimated by the method of Kar Mahapatra et al., 2009 [44]. Briefly, the reaction mixture contained Tris-HCl buffer (50 mM, pH 7.4), tetra-butyl hydroperoxide (BHP) (500 μM in ethanol), and 1 mM FeSO4. After incubating the samples at 37°C for 90 min, the reaction was stopped by adding 0.2 mL of 8% sodium dodecyl sulfate (SDS) followed by 1.5 mL of 20% acetic acid (pH 3.5). The amount of malondialdehyde (MDA) formed during incubation was estimated by adding 1.5 mL of 0.8% TBA and further heating the mixture at 95°C for 45 min. After cooling, samples were centrifuged, and the TBA reactive substances (TBARS) were measured in supernatants at 532 nm by using 1.53×105 M−1 cm−1 as extinction coefficient. The levels of lipid peroxidation were expressed in terms of μmol/mg protein.


#### 4.8.4. Determination of Protein Carbonyls (PC) Contents in Lymphocyte

Protein oxidation was monitored by measuring protein carbonyl contents by derivatization with 2,4-dinitrophenyl hydrazine (DNPH) [44]. In general, cell lysate proteins in 50 mM potassium phosphate buffer, pH 7.4, were derivatized with DNPH (21% in 2 N HCl). Blank samples were mixed with 2 N HCl incubated at 1 h in the dark; protein was precipitated with 20% trichloro acetic acid (TCA). Underivatized proteins were washed with an ethanol : ethyl acetate mixture (1 : 1). Final pellets of protein were dissolved in 6N guanidine hydrochloride, and absorbance was measured at 370 nm. Protein carbonyls content was expressed in terms of μmol/mg protein.


#### 4.8.5. Determination of Reduced Glutathione (GSH)

Reduced glutathione estimation in cell lysate was performed by the method of Kar Mahapatra et al., 2009  [44]. The required amount of sample was mixed with 25% of TCA and centrifuged at 2,000 ×g for 15 min to settle the precipitated proteins. The supernatant was aspirated and diluted to 1 mL with 0.2 M sodium phosphate buffer (pH 8.0). Later, 2 mL of 0.6 mM DTNB was added. After 10 minutes, the optical density of the yellow-colored complex formed by the reaction of GSH and DTNB (Ellman’s reagent) was measured at 405 nm. A standard curve was obtained with standard reduced glutathione. The levels of GSH were expressed as μg of GSH/mg protein.


#### 4.8.6. Determination of Oxidized Glutathione (GSSG)

The oxidized glutathione level was measured after derivatization of GSH with 2-vinylpyidine according to the method of Kar Mahapatra et al., 2009 [44]. In brief, with 0.5 mL sample, 2 μL of 2-vinylpyidine was added and incubated for 1 hr at 37°C. Then the mixture was deproteinized with 4% sulfosalicylic acid and centrifuged at 1,000 ×g for 10 min to settle the precipitated proteins. The supernatant was aspirated, and GSSG level was estimated with the reaction of DTNB at 412 nm in spectrophotometer and calculated with standard GSSG curve. The levels of GSSG were expressed as μg of GSSG/mg protein.


#### 4.8.7. Activity of Super Oxide Dismutase (SOD)

Superoxide dismutase (SOD) activity was determined from its ability to inhibit the auto-oxidation of pyrogalol according to Gautam et al., 2010 [45]. The reaction mixture considered of 50 mM Tris (hydroxymethyl) amino methane (pH 8.2), 1 mM diethylenetriamine penta acetic acid, and 20–50 μL of sample. The reaction was initiated by addition of 0.2 mM pyrogalol, and the absorbance measured kinetically at 420 nm at 25°C for 3 min. SOD activity was expressed as unit/mg protein.


#### 4.8.8. Activity of Catalase (CAT)

Catalase activity was measured in the cell lysate by the method of Gautam et al., 2010 [45]. The final reaction volume of 3 mL contained 0.05 M Tris-buffer, 5 mM EDTA (pH 7.0), and 10 mM H2O2 (in 0.1 M potassium phosphate buffer, pH 7.0). About 50 μL of sample was added to the above mixture. The rate of change of absorbance per min at 240 nm was recorded. Catalase activity was calculated by using the molar extinction coefficient of 43.6 M−1 cm−1 for H2O2. The level of CAT was expressed in terms of μmol H2O2 consumed/min/mg protein.


#### 4.8.9. Activity of Glutathione Peroxidase (GPx)

Glutathione peroxidase (GPx) activity was measured by the method of Gautam et al., 2010 [45]. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM sodium azide, 0.2 mM NADPH, 1 U glutathione reductase, and 1 mM reduced glutathione. The sample, after its addition, was allowed to equilibrate for 5 min at 25°C. The reaction was initiated by adding 0.1 mL of 2.5 mM H2O2. Absorbance at 340 nm was recorded for 5 min. Values were expressed as n mol of NADPH oxidized to NADP by using the extinction coefficient of 6.2×103 M−1 cm−1 at 340 nm. The activity of GPx was expressed in terms of n mol NADPH consumed/min/mg protein.


#### 4.8.10. Activity of Glutathione Reductase (GR)

Glutathione reductase (GR) activity was measured by the method of Gautam et al., 2010 [45]. The tubes for enzyme assay were incubated at 37°C and contained 2.0 mL of 9 mM GSSG, 0.02 mL of 12 mM NADPH Na4, 2.68 mL of 1/15 M phosphate buffer (pH 6.6), and 0.1 mL of sample. The activity of this enzyme was determined by monitoring the decrease in absorbance at 340 nm. The activity of GR was expressed in terms of n mol NADPH consumed/min/mg protein.


#### 4.8.11. Activity of Glutathione-s-Transferase (GST)

Glutathione-s-transferase (GST) activity was measured by the method of Gautam et al., 2010 [45]. The tubes of enzyme assay were incubated at 25°C and contained 2.85 mL of 0.1 M potassium phosphate (pH 6.5) containing 1 mM of GSH, 0.05 mL of 60 mM 1-chloro-2,4-dinitrobengene, and 0.1 mL of sample. The activity of this enzyme was determined by monitoring the increase in absorbance at 340 nm. The activity of GST was expressed in terms of n mol NADPH consumed/min/mg protein.


#### 4.8.12. Quantitative Estimation of DNA Fragmentation Assay by Diphenylamine (DPA) Assay

The diphenylamine (DPA) reaction of lymphocytes was performed by the method of Peradones et al., 1993 [46]. Perchloric acid (0.5 M) was added to the sample containing uncut DNA (resuspended in 200 μL of hypotonic lysis buffer) and to the other half of the supernatant-containing DNA fragments. Then two volumes of a solution consisting of 0.088 M DPA, 98% (v/v) glacial acetic acid, 1.5% (v/v) sulphuric acid, and a 0.5% (v/v) concentration of 1.6% acetaldehyde solution were added. The samples were stored at 4°C for 48 h. The reaction was quantified spectrophotometrically at 575 nm. The percentage of fragmentation was calculated as the ratio of DNA in the supernatants to the total DNA.


#### 4.8.13. DNA Fragmentation Study by Agarose Gel Electrophoresis

DNA gel electrophoresis was performed as described in our previous lab report [47]. In brief, after the treatment schedule, the lymphocytes were resuspended in 270 μL pre-cooled lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 10 mM EDTA, pH 7.4) with 30 μL SDS (10%). RNase A (final concentration 100 μg/mL) was then added, and incubation was continued at 45°C for 45 min. Subsequently, proteinase K (final concentration 100 μg/mL) was added to the cell lysate, and incubation was continued at 50°C for overnight to complete digestion. DNA was isolated from the lysate using phenol/chloroform/iso-amyl alcohol. Then, DNA was precipitated with one volume of 10 M sterile ammonium acetate, and two volumes of absolute ethanol followed by centrifugation at 13000 ×g for 10 min at 4°C. The extracted DNA samples were washed with 70% ethanol and dissolved in 50 μL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6). Gel loading buffer (10 mM EDTA, 0.25% bromophenol blue, 30% glycerol) was then added to the DNA sample at 1 : 5 ratio and loaded onto a 1.2% agarose gel. The electrophoresis was carried out at 50 V for 90 min in TBE buffer (90 mM Tris-HCl, 2 mM EDTA, 90 mM boric acid, pH 8.0). 100 bp molecular weight markers (Genai, Bangalore, India) were used. After electrophoresis, DNA was visualized by soaking the gel in TBE buffer containing 1.5 μg/mL ethidium bromide in UV light, and the picture was captured in Bio-Rad gel documentation system.


#### 4.8.14. Protein Estimation

Protein was determined according to Lowry et al., 1951 using bovine serum albumin as standard [48].


### 4.9. Statistical Analysis

The data were expressed as mean ± standard error,n=6. Comparisons of the means of control, and experimental groups were made by two-way ANOVA test (using a statistical package, Origin 6.1, Northampton, MA 01060 USA) with multiple comparison t-tests; P<0.05 as a limit of significance.


## 4.1. Chemicals and Reagents

Sodium dodecyl sulfates (SDS), 2,4-dinitrophenyl hydrazine (DNPH), 5′,5′-dithio (bis)-2-nitrobenzoic acid (DTNB), standard reduced glutathione (GSH), glutathione reductase (GR), NADPH Na4, NADPH, oxidized glutathione (GSSG), agarose, folic acid (FA),Chitosan (medium molecular weight), dicyclohexyl carbodiimide (DCC), Trifluoroacetic acid, 2,2′-(ethylenedioxy)-bis-(ethylamine) (EDBE), di-tert-butyldicarbonate (BoC2O), N-hydroxysuccinimide (NHS) and 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide Hydrochloride (EDC), RNase A,  and Proteinase K were purchased from Sigma Chemical Co., USA. Sodium chloride (NaCl), sodium dodecyl sulfate, ethylene diamine tetra acetate (EDTA), tryptic soy broth, luria broth, mannitol salt agar, agar powder, sucrose, magnesium chloride (MgCl2), sodium azide (NaN3), phenol/chloroform/iso-amyl alcohol, ethidium bromide, and bromophenol blue were purchased from Himedia, India. Tris-HCl, Tris buffer, potassium dihydrogen phosphate (KH2PO4), di potassium hydrogen phosphate (K2HPO4), sodium hydroxide (NaOH), sodium acetate, ammonium acetate, alcohol, sulfanilamide, phosphoric acid, N-C-1 naphthyl ethylene diamine dihydrochloride, diphenylamine (DPA), glycerol, boric acid, O-phenylenediamine, and other chemicals were procured from Merck Ltd., SRL Pvt. Ltd., Mumbai, India. All other chemicals were from Merck Ltd., SRL Pvt., Ltd., Mumbai and were of the highest grade available.


## 4.2. Animals

Experiments were performed using Swiss male mice 6–8 weeks old, weighing 20–25 g. The animals were fed standard pellet diet and water was givenad libitum and housed in polypropylene cage (Tarson) in the departmental animal house with 12 h light : dark cycle, and the temperature of 25 ± 2°C. The animals were allowed to acclimatize for one week. The animals used did not show any sign of malignancy or other pathological processes. Animals were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India and approved by the ethical committee of Vidyasagar University.


## 4.3. Bacterial Strain

We used coagulase-positive vancomycin-sensitive and -resistantStaphylococcus aureusstrains that were isolated from human postoperative pus sample [39]. These bacterial strains were grown at 37°C for overnight in tryptic soy broth. The bacterial culture was centrifuged at 15,000 rpm for 15 minutes. The pellet was resuspended and washed with sterile phosphate buffer saline (PBS). Using a UV-spectrophotometer (Schimadzu, USA) at an absorbance of 620 nm, we adjusted the viable bacterial count to approximately 1.0×109 colony-forming units (CFU)/mL, which corresponded to an optical density of 1.6. The bacterial suspension was adjusted by serial dilution in phosphate buffer saline (PBS) to give a final concentration of approximately 5×106 in 100 μL of bacterial suspension [14].


## 4.4. Preparation of CMC-EDBE-FA Nanoparticle and Loading of Vancomycin

CMC-EDBE-FA nanoparticle was prepared, and vancomycin was loaded onto it according to our previous laboratory report [18].


## 4.5. Development of VSSA and VRSA Infection in Swiss Mice

VSSA and VRSA infection was developed in male Swiss mice by intraperitoneal (i.p.) injection of 100μL of bacterial suspension containing 5×106 CFU/mL according to our previous laboratory report [20].


## 4.6. Experimental Design

VSSA- and VRSA-infected mice were treated with nanoconjugated vancomycin for successive 10 days at a dose of 100 mg/kg bw/day and 500 mg/kg bw/day, respectively. The dose and duration of nanoconjugated vancomycin was selected from our previous laboratory report [20]. The following groups were considered for the experiment.Group I: Control.Group II: 100 mg/kg bw/day vancomycin.Group III: 500 mg/kg bw/day vancomycin.Group IV: 100 mg/kg bw/day nanoconjugated vancomycin.Group V: 500 mg/kg bw/day nanoconjugated vancomycin.Group VI: VSSA infection.Group VII: VSSA infection + 100 mg/kg bw/day vancomycin.Group VIII: VSSA infection + 100 mg/kg bw/day nanoconjugated vancomycin.Group IX: VRSA infection.Group X: VRSA infection + 500 mg/kg bw/day vancomycin.Group XI: VRSA infection + 500 mg/kg bw/day nanoconjugated vancomycin.After the termination of experiment, animals were sacrificed by an intraperitoneal injection of sodium pentobarbital (60–70 mg/kg body weight) [40], and blood (n=6/group) was used for preparation of serum and separation of lymphocyte for biochemical estimation of oxidative parameters.


## 4.7. Separation of Serum and Lymphocyte

Serum was obtained by centrifugation of blood samples taken without anticoagulant at 1500 g for 15 min and was kept at −86°C for the further estimation. Heparinized blood samples were used for the separation of Lymphocyte. Lymphocytes were isolated from blood using standard isolation techniques [41]. Blood samples were diluted with equal amount of PBS (pH 7.0) buffer and then layered very carefully on the density gradient (Histopaque 1077, Sigma Chemical Co.) in 1 : 2 ratio. It was centrifuged at 500 g for 20 min and the white milky layer of mononuclear cells that is, lymphocytes was carefully removed. The layer was washed twice with the same buffer and centrifuged at 3000 g for 10 min to get the required pellet of lymphocytes [42]. The pellets of lymphocytes were lysed in a hypotonic lysis buffer for 45 min at 37°C and kept at −86°C until biochemical estimations [42].


## 4.8. Biochemical Estimation

### 4.8.1. Nitrite (NO) Production by Lymphocyte

After treatment schedule, 100μL of Griess reagent (containing 1 part of 1% sulfanilamide in 5% phosphoric acid, and 1 part of 0.1% of N-C-1 napthyl ethylene diamine dihydrochloride) was added to 100 μL of sample, incubated at room temperature for 10 minutes, readings were taken in a UV spectrophotometer at 550 nm and compared to a sodium nitrite standard curve (values ranging between 0.5 and 25 μM). The levels of NO were expressed as μmol/mg protein [43].


### 4.8.2. Determination of Myeloperoxidase Activity in Lymphocyte

200μL of cell lysate was reacted with 200 μL substrate (containing H2O2 and OPD) in dark for 30 min. The blank was prepared with citrate phosphate buffer (pH 5.2) and substrate, in absence of cell-free supernatant. The reaction was stopped with addition of 100 μL 2 (N) sulfuric acid and reading was taken at 492 nm in a spectrophotometer [44]. The MPO activity was expressed in terms of μM/mg protein.


### 4.8.3. Determination of Lipid Peroxidation (MDA)

Lipid peroxidation was estimated by the method of Kar Mahapatra et al., 2009 [44]. Briefly, the reaction mixture contained Tris-HCl buffer (50 mM, pH 7.4), tetra-butyl hydroperoxide (BHP) (500 μM in ethanol), and 1 mM FeSO4. After incubating the samples at 37°C for 90 min, the reaction was stopped by adding 0.2 mL of 8% sodium dodecyl sulfate (SDS) followed by 1.5 mL of 20% acetic acid (pH 3.5). The amount of malondialdehyde (MDA) formed during incubation was estimated by adding 1.5 mL of 0.8% TBA and further heating the mixture at 95°C for 45 min. After cooling, samples were centrifuged, and the TBA reactive substances (TBARS) were measured in supernatants at 532 nm by using 1.53×105 M−1 cm−1 as extinction coefficient. The levels of lipid peroxidation were expressed in terms of μmol/mg protein.


### 4.8.4. Determination of Protein Carbonyls (PC) Contents in Lymphocyte

Protein oxidation was monitored by measuring protein carbonyl contents by derivatization with 2,4-dinitrophenyl hydrazine (DNPH) [44]. In general, cell lysate proteins in 50 mM potassium phosphate buffer, pH 7.4, were derivatized with DNPH (21% in 2 N HCl). Blank samples were mixed with 2 N HCl incubated at 1 h in the dark; protein was precipitated with 20% trichloro acetic acid (TCA). Underivatized proteins were washed with an ethanol : ethyl acetate mixture (1 : 1). Final pellets of protein were dissolved in 6N guanidine hydrochloride, and absorbance was measured at 370 nm. Protein carbonyls content was expressed in terms of μmol/mg protein.


### 4.8.5. Determination of Reduced Glutathione (GSH)

Reduced glutathione estimation in cell lysate was performed by the method of Kar Mahapatra et al., 2009  [44]. The required amount of sample was mixed with 25% of TCA and centrifuged at 2,000 ×g for 15 min to settle the precipitated proteins. The supernatant was aspirated and diluted to 1 mL with 0.2 M sodium phosphate buffer (pH 8.0). Later, 2 mL of 0.6 mM DTNB was added. After 10 minutes, the optical density of the yellow-colored complex formed by the reaction of GSH and DTNB (Ellman’s reagent) was measured at 405 nm. A standard curve was obtained with standard reduced glutathione. The levels of GSH were expressed as μg of GSH/mg protein.


### 4.8.6. Determination of Oxidized Glutathione (GSSG)

The oxidized glutathione level was measured after derivatization of GSH with 2-vinylpyidine according to the method of Kar Mahapatra et al., 2009 [44]. In brief, with 0.5 mL sample, 2 μL of 2-vinylpyidine was added and incubated for 1 hr at 37°C. Then the mixture was deproteinized with 4% sulfosalicylic acid and centrifuged at 1,000 ×g for 10 min to settle the precipitated proteins. The supernatant was aspirated, and GSSG level was estimated with the reaction of DTNB at 412 nm in spectrophotometer and calculated with standard GSSG curve. The levels of GSSG were expressed as μg of GSSG/mg protein.


### 4.8.7. Activity of Super Oxide Dismutase (SOD)

Superoxide dismutase (SOD) activity was determined from its ability to inhibit the auto-oxidation of pyrogalol according to Gautam et al., 2010 [45]. The reaction mixture considered of 50 mM Tris (hydroxymethyl) amino methane (pH 8.2), 1 mM diethylenetriamine penta acetic acid, and 20–50 μL of sample. The reaction was initiated by addition of 0.2 mM pyrogalol, and the absorbance measured kinetically at 420 nm at 25°C for 3 min. SOD activity was expressed as unit/mg protein.


### 4.8.8. Activity of Catalase (CAT)

Catalase activity was measured in the cell lysate by the method of Gautam et al., 2010 [45]. The final reaction volume of 3 mL contained 0.05 M Tris-buffer, 5 mM EDTA (pH 7.0), and 10 mM H2O2 (in 0.1 M potassium phosphate buffer, pH 7.0). About 50 μL of sample was added to the above mixture. The rate of change of absorbance per min at 240 nm was recorded. Catalase activity was calculated by using the molar extinction coefficient of 43.6 M−1 cm−1 for H2O2. The level of CAT was expressed in terms of μmol H2O2 consumed/min/mg protein.


### 4.8.9. Activity of Glutathione Peroxidase (GPx)

Glutathione peroxidase (GPx) activity was measured by the method of Gautam et al., 2010 [45]. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM sodium azide, 0.2 mM NADPH, 1 U glutathione reductase, and 1 mM reduced glutathione. The sample, after its addition, was allowed to equilibrate for 5 min at 25°C. The reaction was initiated by adding 0.1 mL of 2.5 mM H2O2. Absorbance at 340 nm was recorded for 5 min. Values were expressed as n mol of NADPH oxidized to NADP by using the extinction coefficient of 6.2×103 M−1 cm−1 at 340 nm. The activity of GPx was expressed in terms of n mol NADPH consumed/min/mg protein.


### 4.8.10. Activity of Glutathione Reductase (GR)

Glutathione reductase (GR) activity was measured by the method of Gautam et al., 2010 [45]. The tubes for enzyme assay were incubated at 37°C and contained 2.0 mL of 9 mM GSSG, 0.02 mL of 12 mM NADPH Na4, 2.68 mL of 1/15 M phosphate buffer (pH 6.6), and 0.1 mL of sample. The activity of this enzyme was determined by monitoring the decrease in absorbance at 340 nm. The activity of GR was expressed in terms of n mol NADPH consumed/min/mg protein.


### 4.8.11. Activity of Glutathione-s-Transferase (GST)

Glutathione-s-transferase (GST) activity was measured by the method of Gautam et al., 2010 [45]. The tubes of enzyme assay were incubated at 25°C and contained 2.85 mL of 0.1 M potassium phosphate (pH 6.5) containing 1 mM of GSH, 0.05 mL of 60 mM 1-chloro-2,4-dinitrobengene, and 0.1 mL of sample. The activity of this enzyme was determined by monitoring the increase in absorbance at 340 nm. The activity of GST was expressed in terms of n mol NADPH consumed/min/mg protein.


### 4.8.12. Quantitative Estimation of DNA Fragmentation Assay by Diphenylamine (DPA) Assay

The diphenylamine (DPA) reaction of lymphocytes was performed by the method of Peradones et al., 1993 [46]. Perchloric acid (0.5 M) was added to the sample containing uncut DNA (resuspended in 200 μL of hypotonic lysis buffer) and to the other half of the supernatant-containing DNA fragments. Then two volumes of a solution consisting of 0.088 M DPA, 98% (v/v) glacial acetic acid, 1.5% (v/v) sulphuric acid, and a 0.5% (v/v) concentration of 1.6% acetaldehyde solution were added. The samples were stored at 4°C for 48 h. The reaction was quantified spectrophotometrically at 575 nm. The percentage of fragmentation was calculated as the ratio of DNA in the supernatants to the total DNA.


### 4.8.13. DNA Fragmentation Study by Agarose Gel Electrophoresis

DNA gel electrophoresis was performed as described in our previous lab report [47]. In brief, after the treatment schedule, the lymphocytes were resuspended in 270 μL pre-cooled lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 10 mM EDTA, pH 7.4) with 30 μL SDS (10%). RNase A (final concentration 100 μg/mL) was then added, and incubation was continued at 45°C for 45 min. Subsequently, proteinase K (final concentration 100 μg/mL) was added to the cell lysate, and incubation was continued at 50°C for overnight to complete digestion. DNA was isolated from the lysate using phenol/chloroform/iso-amyl alcohol. Then, DNA was precipitated with one volume of 10 M sterile ammonium acetate, and two volumes of absolute ethanol followed by centrifugation at 13000 ×g for 10 min at 4°C. The extracted DNA samples were washed with 70% ethanol and dissolved in 50 μL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6). Gel loading buffer (10 mM EDTA, 0.25% bromophenol blue, 30% glycerol) was then added to the DNA sample at 1 : 5 ratio and loaded onto a 1.2% agarose gel. The electrophoresis was carried out at 50 V for 90 min in TBE buffer (90 mM Tris-HCl, 2 mM EDTA, 90 mM boric acid, pH 8.0). 100 bp molecular weight markers (Genai, Bangalore, India) were used. After electrophoresis, DNA was visualized by soaking the gel in TBE buffer containing 1.5 μg/mL ethidium bromide in UV light, and the picture was captured in Bio-Rad gel documentation system.


### 4.8.14. Protein Estimation

Protein was determined according to Lowry et al., 1951 using bovine serum albumin as standard [48].


## 4.8.1. Nitrite (NO) Production by Lymphocyte

After treatment schedule, 100μL of Griess reagent (containing 1 part of 1% sulfanilamide in 5% phosphoric acid, and 1 part of 0.1% of N-C-1 napthyl ethylene diamine dihydrochloride) was added to 100 μL of sample, incubated at room temperature for 10 minutes, readings were taken in a UV spectrophotometer at 550 nm and compared to a sodium nitrite standard curve (values ranging between 0.5 and 25 μM). The levels of NO were expressed as μmol/mg protein [43].


## 4.8.2. Determination of Myeloperoxidase Activity in Lymphocyte

200μL of cell lysate was reacted with 200 μL substrate (containing H2O2 and OPD) in dark for 30 min. The blank was prepared with citrate phosphate buffer (pH 5.2) and substrate, in absence of cell-free supernatant. The reaction was stopped with addition of 100 μL 2 (N) sulfuric acid and reading was taken at 492 nm in a spectrophotometer [44]. The MPO activity was expressed in terms of μM/mg protein.


## 4.8.3. Determination of Lipid Peroxidation (MDA)

Lipid peroxidation was estimated by the method of Kar Mahapatra et al., 2009 [44]. Briefly, the reaction mixture contained Tris-HCl buffer (50 mM, pH 7.4), tetra-butyl hydroperoxide (BHP) (500 μM in ethanol), and 1 mM FeSO4. After incubating the samples at 37°C for 90 min, the reaction was stopped by adding 0.2 mL of 8% sodium dodecyl sulfate (SDS) followed by 1.5 mL of 20% acetic acid (pH 3.5). The amount of malondialdehyde (MDA) formed during incubation was estimated by adding 1.5 mL of 0.8% TBA and further heating the mixture at 95°C for 45 min. After cooling, samples were centrifuged, and the TBA reactive substances (TBARS) were measured in supernatants at 532 nm by using 1.53×105 M−1 cm−1 as extinction coefficient. The levels of lipid peroxidation were expressed in terms of μmol/mg protein.


## 4.8.4. Determination of Protein Carbonyls (PC) Contents in Lymphocyte

Protein oxidation was monitored by measuring protein carbonyl contents by derivatization with 2,4-dinitrophenyl hydrazine (DNPH) [44]. In general, cell lysate proteins in 50 mM potassium phosphate buffer, pH 7.4, were derivatized with DNPH (21% in 2 N HCl). Blank samples were mixed with 2 N HCl incubated at 1 h in the dark; protein was precipitated with 20% trichloro acetic acid (TCA). Underivatized proteins were washed with an ethanol : ethyl acetate mixture (1 : 1). Final pellets of protein were dissolved in 6N guanidine hydrochloride, and absorbance was measured at 370 nm. Protein carbonyls content was expressed in terms of μmol/mg protein.


## 4.8.5. Determination of Reduced Glutathione (GSH)

Reduced glutathione estimation in cell lysate was performed by the method of Kar Mahapatra et al., 2009  [44]. The required amount of sample was mixed with 25% of TCA and centrifuged at 2,000 ×g for 15 min to settle the precipitated proteins. The supernatant was aspirated and diluted to 1 mL with 0.2 M sodium phosphate buffer (pH 8.0). Later, 2 mL of 0.6 mM DTNB was added. After 10 minutes, the optical density of the yellow-colored complex formed by the reaction of GSH and DTNB (Ellman’s reagent) was measured at 405 nm. A standard curve was obtained with standard reduced glutathione. The levels of GSH were expressed as μg of GSH/mg protein.


## 4.8.6. Determination of Oxidized Glutathione (GSSG)

The oxidized glutathione level was measured after derivatization of GSH with 2-vinylpyidine according to the method of Kar Mahapatra et al., 2009 [44]. In brief, with 0.5 mL sample, 2 μL of 2-vinylpyidine was added and incubated for 1 hr at 37°C. Then the mixture was deproteinized with 4% sulfosalicylic acid and centrifuged at 1,000 ×g for 10 min to settle the precipitated proteins. The supernatant was aspirated, and GSSG level was estimated with the reaction of DTNB at 412 nm in spectrophotometer and calculated with standard GSSG curve. The levels of GSSG were expressed as μg of GSSG/mg protein.


## 4.8.7. Activity of Super Oxide Dismutase (SOD)

Superoxide dismutase (SOD) activity was determined from its ability to inhibit the auto-oxidation of pyrogalol according to Gautam et al., 2010 [45]. The reaction mixture considered of 50 mM Tris (hydroxymethyl) amino methane (pH 8.2), 1 mM diethylenetriamine penta acetic acid, and 20–50 μL of sample. The reaction was initiated by addition of 0.2 mM pyrogalol, and the absorbance measured kinetically at 420 nm at 25°C for 3 min. SOD activity was expressed as unit/mg protein.


## 4.8.8. Activity of Catalase (CAT)

Catalase activity was measured in the cell lysate by the method of Gautam et al., 2010 [45]. The final reaction volume of 3 mL contained 0.05 M Tris-buffer, 5 mM EDTA (pH 7.0), and 10 mM H2O2 (in 0.1 M potassium phosphate buffer, pH 7.0). About 50 μL of sample was added to the above mixture. The rate of change of absorbance per min at 240 nm was recorded. Catalase activity was calculated by using the molar extinction coefficient of 43.6 M−1 cm−1 for H2O2. The level of CAT was expressed in terms of μmol H2O2 consumed/min/mg protein.


## 4.8.9. Activity of Glutathione Peroxidase (GPx)

Glutathione peroxidase (GPx) activity was measured by the method of Gautam et al., 2010 [45]. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM sodium azide, 0.2 mM NADPH, 1 U glutathione reductase, and 1 mM reduced glutathione. The sample, after its addition, was allowed to equilibrate for 5 min at 25°C. The reaction was initiated by adding 0.1 mL of 2.5 mM H2O2. Absorbance at 340 nm was recorded for 5 min. Values were expressed as n mol of NADPH oxidized to NADP by using the extinction coefficient of 6.2×103 M−1 cm−1 at 340 nm. The activity of GPx was expressed in terms of n mol NADPH consumed/min/mg protein.


## 4.8.10. Activity of Glutathione Reductase (GR)

Glutathione reductase (GR) activity was measured by the method of Gautam et al., 2010 [45]. The tubes for enzyme assay were incubated at 37°C and contained 2.0 mL of 9 mM GSSG, 0.02 mL of 12 mM NADPH Na4, 2.68 mL of 1/15 M phosphate buffer (pH 6.6), and 0.1 mL of sample. The activity of this enzyme was determined by monitoring the decrease in absorbance at 340 nm. The activity of GR was expressed in terms of n mol NADPH consumed/min/mg protein.


## 4.8.11. Activity of Glutathione-s-Transferase (GST)

Glutathione-s-transferase (GST) activity was measured by the method of Gautam et al., 2010 [45]. The tubes of enzyme assay were incubated at 25°C and contained 2.85 mL of 0.1 M potassium phosphate (pH 6.5) containing 1 mM of GSH, 0.05 mL of 60 mM 1-chloro-2,4-dinitrobengene, and 0.1 mL of sample. The activity of this enzyme was determined by monitoring the increase in absorbance at 340 nm. The activity of GST was expressed in terms of n mol NADPH consumed/min/mg protein.


## 4.8.12. Quantitative Estimation of DNA Fragmentation Assay by Diphenylamine (DPA) Assay

The diphenylamine (DPA) reaction of lymphocytes was performed by the method of Peradones et al., 1993 [46]. Perchloric acid (0.5 M) was added to the sample containing uncut DNA (resuspended in 200 μL of hypotonic lysis buffer) and to the other half of the supernatant-containing DNA fragments. Then two volumes of a solution consisting of 0.088 M DPA, 98% (v/v) glacial acetic acid, 1.5% (v/v) sulphuric acid, and a 0.5% (v/v) concentration of 1.6% acetaldehyde solution were added. The samples were stored at 4°C for 48 h. The reaction was quantified spectrophotometrically at 575 nm. The percentage of fragmentation was calculated as the ratio of DNA in the supernatants to the total DNA.


## 4.8.13. DNA Fragmentation Study by Agarose Gel Electrophoresis

DNA gel electrophoresis was performed as described in our previous lab report [47]. In brief, after the treatment schedule, the lymphocytes were resuspended in 270 μL pre-cooled lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 10 mM EDTA, pH 7.4) with 30 μL SDS (10%). RNase A (final concentration 100 μg/mL) was then added, and incubation was continued at 45°C for 45 min. Subsequently, proteinase K (final concentration 100 μg/mL) was added to the cell lysate, and incubation was continued at 50°C for overnight to complete digestion. DNA was isolated from the lysate using phenol/chloroform/iso-amyl alcohol. Then, DNA was precipitated with one volume of 10 M sterile ammonium acetate, and two volumes of absolute ethanol followed by centrifugation at 13000 ×g for 10 min at 4°C. The extracted DNA samples were washed with 70% ethanol and dissolved in 50 μL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6). Gel loading buffer (10 mM EDTA, 0.25% bromophenol blue, 30% glycerol) was then added to the DNA sample at 1 : 5 ratio and loaded onto a 1.2% agarose gel. The electrophoresis was carried out at 50 V for 90 min in TBE buffer (90 mM Tris-HCl, 2 mM EDTA, 90 mM boric acid, pH 8.0). 100 bp molecular weight markers (Genai, Bangalore, India) were used. After electrophoresis, DNA was visualized by soaking the gel in TBE buffer containing 1.5 μg/mL ethidium bromide in UV light, and the picture was captured in Bio-Rad gel documentation system.


## 4.8.14. Protein Estimation

Protein was determined according to Lowry et al., 1951 using bovine serum albumin as standard [48].


## 4.9. Statistical Analysis

The data were expressed as mean ± standard error,n=6. Comparisons of the means of control, and experimental groups were made by two-way ANOVA test (using a statistical package, Origin 6.1, Northampton, MA 01060 USA) with multiple comparison t-tests; P<0.05 as a limit of significance.



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*Source: 942123-2011-09-18.xml*