Ameliorating Effects of Green Synthesized Silver Nanoparticles on

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Ameliorating Effects of Green Synthesized Silver Nanoparticles on Glycated End Product Induced Reactive Oxygen Species Production and Cellular Toxicity in Osteogenic Saos‑2 Cells Sarbani Ashe, Debasis Nayak, Manisha Kumari, and Bismita Nayak* Immunology and Molecular Medicine Laboratory, Department of Life Science, National Institute of Technology Rourkela, Odisha 769008, India ABSTRACT: Advanced glycation end-products (AGEs) that result from nonenzymatic glycation are one of the major factors involved in diabetes and its secondary complications and diseases. This necessitates our urge to discover new compounds that may be used as potential AGEs inhibitors without affecting the normal structure and function of biomolecules. In the present study, we investigated the inhibitory effects of AgNP (silver nanoparticles) on AGEs formation as well as their inhibitory effects on glycation mediated cell toxicity via reactive oxygen species (ROS) production and DNA damage. The excitation−emission fluorescence spectroscopy was employed to investigate the interaction of AgNP during glycation. The values of conditional stability constant (log Ka = 4.44) derived from the Stern−Volmer equation indicate that AgNP have strong binding capacity for glycated protein. UV−vis, fluorescence, and Fourier transform infrared spectral data reveal complexation of AgNP with glycated bovine serum albumin, which significantly inhibits AGEs formation in a concentration-dependent manner. Cytotoxic evaluations suggest that simultaneous administration of AgNP and glycated product reduces cell death (42.82% ± 3.54) as compared to the glycated product alone. Similarly, ROS production in AgNP treated cells is significantly less compared to only glycated product treated cells. Although DNA damage studies show DNA damage in both GP and GP-AgNP treated cells, fluorescence activated cell sorting analysis demonstrates that glycated products induce cell death by necrosis, while AgNP cause cell death via apoptotic pathways. AgNP have a positive effect on restoring native protein structure deduced from spectral studies, and hence, inferences can be drawn that AgNP have ameliorating effects on glycated induced cytotoxicity observed in osteogenic Saos-2 cells. KEYWORDS: glycation products, silver nanoparticle, fluorescence, cytotoxic, ROS, apoptosis

1. INTRODUCTION Hyperglycemic conditions as in diabetes result in the nonenzymatic modification of proteins and other biomacromolecules (nucleic acids and lipids) by reducing sugars to form advanced glycation end products (AGEs).1 The process, known as Maillard reaction, initiates a progressive cascade of irreversible cross-link formation with long-lived proteins within the body causing structural and functional loss of the same. These glycation products and their array of intermediates are the known miscreants of diabetic complications2 (atherosclerosis, diabetic microangiopathy, diabetic retinopathy, diabetic nephropathy), neurodegenerative diseases, cancer, premature aging, hypertension, cardiovascular diseases, etc.3 As endogenous proinflammatory mediators, glycation products trigger cellular innate immune response and reactive oxygen species (ROS) generation in a positive feed-forward cycle that ultimately leads to apoptosis4,5 and reduced stem cell differentiation.6 Thus, increased level of glycation products and intermediates in blood predicates the onset of several diseases including diabetic complications7,8 and a higher risk of in-hospital mortality not only for diabetic individuals, but also for other pathological conditions9 prompting the frantic search © 2016 American Chemical Society

for novel inhibitors to prevent AGEs formation and to counter the nefarious effects of glycation in disease progression and pathogenesis. Certain compounds like aminoguanidine, aspirin, vitamin B6, vitamin C, taurine, quercetin, and anti-inflammatory drugs such as ibuprofen have been reported with antiglycating properties.10 Nanotechnology, with its multidisciplinary prospective in early detection, accurate diagnosis, and personalized treatment of cancer and other diseases is considered the future of molecular medicine.11 By encompassing broad fields like chemistry, engineering, biology, medicine, etc., nanotechnology has the potential to revolutionize disease diagnosis and treatment. This is because of the unprecedented small size of the nanoparticles (NPs), that is, 100−10 000-times smaller than human cells with interactive ability both in vivo and ex vivo.12 Nanoparticle production to meet industry demands is mainly by physical and chemical routes. Large-scale production by the above methods not only shoots up the production cost, but also Received: August 24, 2016 Accepted: October 17, 2016 Published: October 17, 2016 30005

DOI: 10.1021/acsami.6b10639 ACS Appl. Mater. Interfaces 2016, 8, 30005−30016

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ACS Applied Materials & Interfaces the byproducts formed are environmental hazards.13 This necessitated the development of biological route of nanoparticle synthesis also termed green synthesis.14 The vast natural diversity of bacteria, algae, fungi, and plants provides the enzymes for the synthesis of NPs. Nanoparticle synthesis using plant parts is minimalistic, simple, swift, economic, and occurs in a single step; hence, is the most favored method.15 The vast array of phytochemicals comprising flavonoids, terpenoids, alkaloids, carboxylic acids, quinones, aldehydes, ketones, and amides act as enzymes, cofactors, and reducing agents for biosynthesis of nanoparticles in the presence of metal salts.14 The biological applications of silver nanoparticles (AgNP) have seen an unparalleled increase due to their modifiable surface enhanced resonance properties.16 A singular chemical structure and dimensions lower than 100 nm permit exciting possible biomedical applications like drug delivery, tissue engineering, and hyperthermia cancer therapy. AgNP with their similarity to cellular components and small size enter living cells using cellular endocytic mechanisms and have been reported to exhibit antibiofilm,17 anticancer,18 antimicrobial,19,20 antiplasmodial,21 anti-inflammatory,22 and antioxidant activities.23 The inhibitory effect of AgNP on the structural alteration of H2A histone by 3-deoxyglucosone glycation was reported by Ashraf et al.24 Ashraf et al. have also reported the inhibitory effect of AgNP on AGEs formation using biophysical techniques.10 The current work aims to validate the detrimental effects of glycation on osteogenic cells and the antiglycating potential of AgNP. The diminishing effect of glycation on the structural changes in the native protein due to the presence of AgNP was corroborated by various biophysical methods (UV−vis spectroscopy, fluorescence spectroscopy, and infrared spectroscopy). The ability of AgNP to reduce the cytotoxic effects of glycation products in vitro was also studied.

characterized for size distribution, charge, morphology, and antibacterial activity. The methodology and characterization studies have previously been published.27 2.3. Biophysical Studies of the Effect of AgNP on GP Formation. 2.3.1. UV−visible and Fluorescence Spectroscopy. UV− visible absorption spectra of control and glycated BSA with or without AgNP were carried out in a UV−visible spectrophotometer [Lambda 35 (PerkinElmer, Waltham, MS, USA)] in the range of 300−400 nm. Fluorescence spectra were obtained with an LS55 Fluorimeter PerkinElmer at 25 ± 1 °C equipped with a 1.0 cm quartz cell emission in the wavelength range of 360−460 nm, respectively, with slit width set at 5 nm in spectrofluorometer. All the readings were performed in triplicate, and the data were reported as the average of these three readings. To calculate the percent inhibition of AGEs formation by various concentrations of AgNP, the glycated sample was used as a positive control. The percent inhibition was calculated using the following formula:

%GP = [1 − (fluorescence of test group − fluorescence of control group)] × 100 2.3.2. Estimation of Protein Bound Carbonyl Content. Carbonyl contents of control and GP-modified BSA with or without AgNP samples were determined as previously described, with slight modifications. Briefly, 15 μM control and GP-modified BSA with or without AgNP samples were dissolved in PBS buffer and incubated for 1 h at room temperature with intermittent vortexing. Then the samples were precipitated with 20% (v/v) TCA and centrifuged for 5 min at 10 000 rpm at 4 °C. The pellet obtained was then suspended in 1 mL of 6 M guanidium hydrochloride (pH 2.3) and incubated at 37 °C for 30 min. Carbonyl content was determined in the supernatant based on the absorbance at 370 nm against 6 M guanidium hydrochlorides (as blank) using the molar extinction coefficient of 22 000 M−1 cm−1. Protein carbonyl content was expressed as nmol/mg of protein. 2.3.3. Estimation of Fructosamine Content. Nitro blue tetrazolium (NBT) assay was performed to determine the fructosamine content in the control and GP-modified BSA with or without AgNP.28 Briefly, 200 μL of sample and 800 μL of NBT reagent (300 μM) were added and incubated at room temperature for 37 °C for 15−30 min. The absorbance was measured at 562 nm on a microplate reader (2030 Multilabel Processor VictorX3 PerkinElmer USA). The fructosamine content was estimated using the molar extinction coefficient of 12 640 M−1 cm−1 for monoformazan as devised by Ansari and Ali.29 2.3.4. Fourier Transformation Infrared (FTIR) Spectroscopy. FTIR spectroscopy was used to investigate the changes in structure in glycated protein with various concentrations of AgNP. Buffer subtracted transmission spectra were recorded with a nominal resolution of 4 cm−1 using Bruker ALPHA spectrophotometer (Ettlinger, Germany). The result obtained was analyzed through OPUS software. All the measurements were carried out at room temperature.30 2.4. Evaluation of Cytotoxic Effects of GP in the Presence of AgNP. 2.4.1. Cell Viability Assay (MTT Assay). The effect of GP and AgNP on cell viability of osteogenic Saos-2 cells was evaluated by the conventional MTT reduction assay.31,32 Briefly, after cell viability was determined by trypan blue staining, Saos-2 cells were seeded in 96-well plates at the density of 3 × 103 cells/well based on the doubling time in the presence of 200 μL/well DMEM supplemented with 10% FBS and 1% Penstrep and incubated at 37 °C under 5% CO2. After 24 h, the existing growth medium was replaced with 200 μL of experimental medium (1000-fold dilution of GP and GP-AgNP stock solutions) containing GP or AgNP (0.2 mM) and incubated for 24 h. To detect the cell proliferation, MTT working solution was prepared from a stock solution of 5 mg/mL in growth medium without FBS to the final concentration of 0.8 mg/mL. A sample of 100 μL of MTT solution was added to each well and incubated for 4 h at 37 °C. After 4 h of incubation, the supernatant was discarded, and 100 μL of DMSO solvent was added to each well under dark followed by an incubation of 15 min, and the optical density of the formazan product was read at

2. MATERIALS AND METHODS Bovine serum albumin (BSA), EDTA, 3-(4,5-dimethylthiazozyl)-2,5diphenyl tetrazolium bromide (MTT), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Trypan blue, 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA), agarose, crystal violet, nitroblue tetrazolium (NBT), propidium iodide (PI) and RNase, proteinase K, and ethidium bromide (EtBr) were all obtained from Hi-Media (Mumbai, India). D-Glucose, Penstrep (antibiotic solution), and rhodamine 123 were purchased from Sigma-Aldrich (Mumbai, India). Annexin V-FITC kit was obtained from Miltenyi Biotech, Germany. All other chemicals and reagents were purchased from commercial sources and were of analytical grade. Milli-Q water was used throughout the experiment. 2.1. Preparation of Glucose Derived Glycation Products. Glycation products (GP) were prepared following the protocol of refs 25 and 26 with slight modifications. Briefly, 10 mg/mL BSA in 1 M phosphate buffer (PBS; pH 7.4) was incubated with D-glucose (250 mM/L) in the presence of 1 mM EDTA and 1% PenStrep for 30 days at 37 °C. Pure BSA was incubated under the same conditions (in the absence of glucose) and was used as control. Autoclaved buffer and glasswares were used to inactivate proteases and remove contaminants. Prior to use, all solutions were sterile-filtered using Millipore Express 0.22-μ filter. For synthesis of GP in the presence of AgNP, varying concentrations (0.05−0.25 mM) of AgNP were added to the reaction mix and incubated. After incubation, glycated products and control BSA were extensively dialyzed against PBS at 4 °C, aliquoted, and stored at 4 °C. 2.2. Preparation of Silver Nanoparticle. The AgNP were prepared by mixing the aqueous petal extracts of Cucurbita maxima and 1 M silver nitrate solution at a temperature of 80 °C for 1 h on a hot plate magnetic stirrer. The prepared nanoparticles were later 30006


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ACS Applied Materials & Interfaces 595 nm in a microplate reader (2030 Multilabel Processor VictorX3 PerkinElmer USA) and the results expressed as the mean of three replicates as a percentage of control (taken as 100%). Statistical significance of difference among data of GP and GP-AgNP treated group was determined versus untreated cells using one-way analysis of variance (one-way ANOVA). P ≤ 0.05 was considered statistically significant. 2.4.2. Morphology Studies (SEM). Investigations of ultrastructural changes in the cells exposed to GP and AgNP were carried out by employing scanning electron microscopy (SEM, Jeol 6480LV JSM Microscope). Briefly, the cells upon trypsinization were suspended in a mixture of 0.7 mL of PBS and 0.3 mL of 2% paraformaldehyde and centrifuged at 100 rpm for 10 min. The supernatant was removed and to the pellet, 0.5 mL of 2% paraformaldehyde was added, and the mixture was incubated at 4 °C for 2 h. The fixative was removed by centrifuging at 1000 rpm for 10 min. The pelleted cells were dehydrated with ethanol (30%, 50%, 70%, 90%, and 100%). Finally, the cells were resuspended in 0.5 mL of 100% ethanol, and 20 μL of the sample was added onto a 1 mm glass slide and air-dried under the laminar hood. For SEM visualization, the slides were fixed on adequate support and gold coated using gold sputter module in a high vacuum evaporator. Observations were taken at different magnifications performed at 20 kV.33,34 The morphology of cells was also observed under inverted bright field microscope Olympus CKX41 10× magnification. 2.4.3. Clonogenic Assay. The effect of GP and AgNP on the clonogenic potential of Saos-2 cells was studied by colony formation assay.35,36 Briefly, 200 cells/well were seeded and after 6 h, and they were treated with GP or AgNP. After 7 days, colonies were stained with a mixture of 6.0% glutaraldehyde and 0.5% crystal violet in water. After 30 min of staining and cell fixation, colonies were washed with water. Air-dried colonies were calculated. Plating efficiency (PE) and the number of colonies that arise after treatment of cells, surviving fraction (SF), was measured by the following formulas:

ROS production (%) =

SF = (PE of treated cells/PE of untreated cells) × 100 The data were analyzed for statistical significance of difference among GP and GP-AgNP treated group versus untreated cells using one-way ANOVA with Tukey’s post hoc test for multiple comparisons. P ≤ 0.05 was considered statistically significant. 2.4.4. Respiratory Burst Activity and in Vitro Determination of Reactive Oxygen Species (ROS). Respiratory burst activity in Saos-2 cells was determined using nitroblue tetrazolium (NBT) reduction assay following the procedure by Munoz et al.37 Briefly, Saos-2 cells were seeded in 96-well culture plate, and following incubation, with both GP and AgNP, the cell lysates were incubated with 0.2% NBT solution for 1 h. The pellet obtained in each case was solubilized by adding 50% (v/v) acetic acid, and absorbance of NBT was measured at 595 nm in a microplate reader. The % superoxide production was calculated using the formula

Fsample − Fcontrol Fcontrol

Fcontrol

× 100

where Fsample is the OD of the sample and Fcontrol is the OD of the control. Further, the ROS produced in the external media were also quantified. H2O2-treated cells were considered as control. Intracellular ROS generation was also visualized under the fluorescent microscope (Olympus IX71) by DCFH-DA staining. The Saos-2 cells were analyzed by flow cytometry to ROS accumulation. The cells upon exposure to GP or AgNP were harvested, fixed in chilled ethanol, and rehydrated in PBS reaching a concentration of 3 × 106 cells/mL. A sample of 5 μL of DCFH-DA was added to the samples and incubated for 30 min in dark to analyze fluorescence intensity by flow cytometry (BD Accuri C6 Flow Cytometer). The NO produced within the cells was also determined. Griess reagent (1% sulphanilamide solution, 5% o-phosphoric acid, 0.1% of N-(1-naphthyl) ethylene diamine in distilled water) was added to the cell lysate and incubated in dark, at room temperature for 5−30 min. Reduced nitrite was measured by taking absorbance at 562 nm and the NO produced was defined as above. 2.4.5. Determination of Mitochondrial Membrane Potential. The effect of GP and AgNP on the membrane potential was assessed by using Rhodamine 123, which is sensitive to change in mitochondrial membrane potential. The cells upon exposure to GP or AgNP were labeled with 1 μM rhodamine 123 at 37 °C in DMEM for 1 h followed by washing to remove excess dye. Cells were visualized under fluorescent microscope (Olympus IX71). For flow cytometric analysis, treated cells were harvested, fixed with chilled ethanol, detached, and incubated with rhodamine 123 for 1 h in dark followed by washing.40 2.4.6. Effect of Glycation Products on DNA. The effect on DNA when the cells were incubated with GP and AgNP was studied by PI staining, comet assay, agarose electrophoresis, and flow cytometry. Briefly, Saos-2 cells were seeded in 12-well plates. The wells upon reaching >75% cell confluency were treated with GP and GP with AgNP and incubated for 12 h at 37 °C under 5% CO2. Thereafter, the cells were stained with 15 μL of PI for 30 min in dark at room temperature and visualized under fluorescent microscope (Olympus IX71). For DNA fragmentation assay, treated cells were harvested, and 5 × 105 cells from each well were transferred into 1.5 mL sterile microcentrifuge tubes. The samples were centrifuged at 2000 rpm for 5 min at 4 °C to obtain the cell pellet. The cell pellet was washed twice with PBS. The cell pellets were resuspended in 500 μL of TES buffer (100 mMTris; 0.8% SDS; 20 mM EDTA pH 8.0). Twenty microliters of RNase cocktail and 1 μL of Proteinase K were added to the cell suspension to remove RNA and protein. The cell suspension was incubated for 2 h with intermittent gentle tapping. After incubation, the samples were centrifuged at 12 000g for 10 min at 4 °C, and the supernatant was carefully removed into a fresh microcentrifuge tube. Forty microliters of the supernatant from each sample was loaded into 1% agarose ethidium bromide stained gel and run at 100 mA current until the loading dye had covered 2/3 of the gel. The agarose gel was visualized and documented by Gel documentation system to observe apoptotic or necrotic DNA. Further, the extent of DNA damage was also visualized by flow cytometry.41 Treated cells were harvested and centrifuged at 1000 rpm for 5 min at 4 °C. The supernatant was discarded, and then the cell pellet was washed with PBS. Afterward, the cells were resuspended in 100 μL of annexin V binding buffer (106 cells/mL) followed by 5 μL of annexin V-FITC. After 15 min incubation at room temperature in the dark, the cells were centrifuged at 1000 rpm for 5 min. Then the cell pellet was resuspended in 200 μL of annexin V binding buffer, and the cells were counter-stained with 5 μL of propidium iodide (PI) before analysis. The cells were analyzed using BD Accuri C6 Flow Cytometer with emission filters of 515−545 nm for FITC (green) and 600 nm for PI (red). A total event of 10 000 cells per sample was acquired. Comet assay was performed according to the protocol of Bausinger et al.42 Treated cells were harvested and mixed with 1% low melting

PE = (no. of colonies formed/no. of cells seeded) × 100

Superoxide produced (%) =

Fsample − Fcontrol

× 100

where Fsample is the OD of the sample and Fcontrol is the OD of the control. DCFH-DA is a nonpolar dye, which is converted into the polar derivative DCFH by cellular esterases upon cellular incorporation. Thus, it is a classic assay for evaluation of intracellular ROS level.38,36,39 Saos2 cells (3 × 103) were cultured in 96-well plates for 24 h with DMEM and 5% FBS, along with GP or AgNP. The medium was replaced with DMEM without FBS containing 10 μM DCFH-DA and incubated for 10 min at room temperature. Intracellular fluorescence was detected at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using microplate reader (2030 Multilabel Processor VictorX3 PerkinElmer USA) at 575 nm. The internal ROS activity was calculated using the formula: 30007


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Figure 1. Graph showing (a) UV spectra; (b) fluorescence spectra; and (c) % inhibition of glycation with AgNP.

Figure 2. Graph showing increased (a) carbonyl content; (b) fructosamine content; and (c) functional group changes in glycated, GP-AgNP, and unmodified BSA. point agarose in a tube. The cells in agarose were transferred to slides coated with normal melting point agarose. The agarose immobilized cells were lysed in alkaline solution, and the agarose trapped DNA was electrophoresed. DNA was stained with PI (2.5 μg/mL), and image acquisition was done by fluorescent microscope (Olympus IX71). Further, the change in the zeta potential of the DNA samples was also measured using Zeta sizer (ZS 90, Malvern Instruments Ltd., Malvern, UK).

3. RESULTS 3.1. Biophysical Studies of the Effect of AgNP on GP Formation. 3.1.1. UV−visible and Fluorescence Spectroscopy. The absorbance of each glycation product was scanned between the range of 300−400 nm. Glycated products showed characteristic peaks at around 340 nm. Figure 1, panel a shows the decreasing absorbance of the glucose-derived glycation 30008


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Figure 3. (a) Cell viability assay; (b) proliferative ability of cell upon treatment with GP and AgNP; (c) bright field images of treated and control cells; (d) SEM pictographs showing distorted morphology of treated cells in comparison to control untreated cells. Asterisks denote statistically significant differences (∗∗∗, P < 0.001; ∗∗, P < 0.01; and ∗, P < 0.05).

amino acid side chains in BSA. Carboxylation of lysine, arginine, threonine, and proline residues is a typical marker of protein oxidation. The decreasing carbonyl content with AgNP incubation was caused by the antiglycating activity of AgNP. The presence of carbonyl contents in vivo and in vitro is considered a biomarker of oxidative stress that predicts irreversible oxidative modifications in proteins during the glycation process. Similarly, fructosamine content also decreased when glycated products were incubated with AgNP as shown in Figure 2, panels a and b. The results show that the reactivity of both lysine and arginine residues of BSA with glucose gradually decreases with AgNP, which indicates that AgNP are capable of inhibiting the glycation reaction at the initial stage. 3.1.3. FTIR Spectroscopy. FTIR spectra of GP incubated with AgNP were analyzed in the range of 4000−500 cm−1 (Figure 2c). Change in the secondary structure was analyzed on Amide-I and Amide-II band shifts within the 1400−2000 cm−1 region. Amide I peak position occurs in the 1600−1700 cm−1 region, while the amide II band stretches occur from 1500− 1600 cm−1. Unmodified BSA showed spectral peaks at 1660.80 and 1547.0 cm−1 in the Amide I and II region, respectively. The slight peak shifts and increase in transmittance % in GP were due to the BSA reaction with glucose, which indicates that the altered secondary structure is due to the glycation reaction. In the presence of AgNP in GP solution, peak shifts showed slight changes relative to native BSA. Thus, FTIR analysis of the GP mixture with and without AgNP corroborates that AgNP play an important role in maintaining the secondary structure of BSA protein.

products in the presence of increased concentration of AgNP. Similar results were obtained with fluorescence studies at 360 and 460 nm where fluorescence maxima peaks tend to shifts to shorter wavelengths with increasing concentration of AgNP as shown in Figure 1, panel b signifying fluorescence quenching. Stern−Volmer analysis was employed to explain the fluorescence quenching behavior of AgNP using the relative fluorescence intensity (F0/F) as a function of quencher concentration [Q] using the following Stern−Volmer equation:

F0 = 1 + K sv[Q ] F F0 and F denote the fluorescence intensities of GP in the absence and presence of quencher (AgNP). Ksv denotes the Stern−Volmer constant. The quenching rate constant, binding constant, and Gibbs free energy were calculated using the following derivative of Stern−Volmer equation: ⎡F − F⎤ log⎢ 0 = log K + n log[Q ] ⎣ F ⎥⎦

The quenching rate constant and binding constant were found to be 4.445 and 2.352, respectively. ΔG value of −11.01 indicated spontaneous binding of AgNP with glycated products, thus signifying a favorable reaction. The % inhibition of glycation (Figure 1c) shows that 0.2 mM of AgNP is most effective in preventing structural changes in BSA at an initial stage; hence, for further experiments, 0.2 mM of AgNP concentration was used. 3.1.2. Estimation of Protein Bound Carbonyl Content and Fructosamine Content. Glycation leads to modification of 30009


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Figure 4. (a) Total ROS produced in %; (b) superoxide radical produced in %; (c) NO produced in %; (d) flow cytometric determination of increased ROS produced; (e) flow cytometric evaluation of decreased mitochondrial membrane potential.

3.2. GP Induces Cytotoxicity in Saos-2 Cells, and AgNP Attenuates the Cytotoxicity. 3.2.1. Cell Viability Assay (MTT Assay) And Morphology (SEM). To investigate the cytotoxic effects of GP administration on Saos-2 cells, the cells were incubated with GP, and cell viability was determined by MTT assay after 24 h. A considerable decline in the cell viability (68%) was observed when cells were incubated with GP (Figure 3). In contrast, when 0.2 mM AgNP were added along with GP at the same time, the cells showed around 50% cell viability (Figure 3a). Further, to screen the effects of both the treatment groups on Saos-2 cell morphology and density, we observed them under inverted phase-contrast microscope (Figure 3c). The morphological observation of untreated Saos2 cells showed the characteristic epithelial-like morphology. However, decreased cell density and irregular morphology were noticed upon incubation with GP. Preapoptotic features such as rounding of cells and formation of small apoptotic bodies were observed. However, a slight improvement in cellular morphology was visible when AgNP were added simultaneously. Similarly, a distorted morphology with membrane blebbing was observed for GP-treated cell under scanning electron microscope (Figure 3d). Although a distorted morphology can be seen in case of GP-AgNP treated cell, the membrane appeared to be smooth. 3.2.2. Clonogenic Assay. The colony forming assay was carried out to determine the long-term effect of GP and AgNP on the proliferative potential of Saos-2 cells. Our results show a reduction in clonogenic survival in Saos-2 cells upon treatment with GP as compared with the untreated cells. However, with simultaneous administration of AgNP, a significant increase in cell survival and proliferation could be seen (Figure 3b). 3.2.3. In Vitro ROS Activity. To elucidate whether GPinduced cytotoxicity and reduced cell proliferation in Saos-2 cells were aggravated by ROS intermediates, we initially performed a microtiter plate based spectroscopic assessment of total ROS, superoxide, and nitric oxide using NBT, DCHFDA, and Griess reagent assay after GP treatment in the presence and absence of AgNP. It is interesting to note that

ROS are produced in both GP treated and GP-AgNP treated cells but, as shown in Figure 4, the GP treated cells had higher superoxide, NO, and total ROS levels in comparison to both untreated control cells and GP-AgNP treated cells. This may be the reason for intensified cellular toxicity observed earlier. However, the presence of AgNP reduced the ROS levels, which corroborated the results from viability assays. Further, fluorescence imaging was done to confirm the intense ROS production in GP treated cells. Fluorescent pictographs in Figure 5, panel a show increasing fluorescent in treated groups signifying enhanced ROS production. Finally, we performed a flow cytometric analysis to determine the cell count that produces ROS with respect to untreated cells and GP-AgNP treated cells. 3.2.4. Determination of Mitochondrial Membrane Potential (MMP). To decipher the correlation between ROS and fall in MMP, we evaluated the mitochondrial membrane potential of GP and AgNP treated and untreated cells using rhodamine 123. Mitochondria are a decisive point for relay of both the extrinsic and intrinsic pathway apoptotic signals. Changes in the MMP directly corroborate with cell death; thus, a fall in MMP could explain the loss of cell viability. Initial determination of MMP intensity was done by observing cells under fluorescence microscope. Thus, untreated cells show a greater fluorescence as compared to treated cells. As shown in Figure 5, panel b, the rhodamine 123 positive cells are higher in number in the control group, and fluorescence decreases with treatment with GP. The fall in MMP as analyzed through flow cytometry also showed a similar pattern (Figure 4). Although the GP-AgNP treated cells also showed a decrease in MMP, the extent was less than only GP treated cells. The intact membrane of untreated cell showed a higher peak as compared to the GP and GP-AgNP treated cells. 3.2.5. Effect of Glycation Products on DNA. To determine the effect of GP on DNA damage, we performed nuclear staining with PI to confirm the induction of cell death with nuclear fragmentation. The GP treated cells showed a bright fragmented center sometimes more than one bright spots 30010


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Figure 5. (a) DCHF-DA staining showing increased ROS production; (b) rhodamine 123 staining showing disruptive mitochondrial membrane potential; (c) graphical representation of increased ROS production and decreased MMP in GP treated cells as compared to untreated cells and GPAgNP treated cells.

signifying fragmented nuclei (Figure 6a). Interestingly, GPAgNP treated cells also showed bright spots signifying DNA damage. Cell death may be necrotic, apoptotic, or a result of autophagy. To examine the correlation between ROS accumulation with apoptosis induction in Saos-2 cells, the percent of apoptotic population in both the treated group of cells was ascertained by flow cytometry using Annexin V-FITC. Figure 6, panel b shows that cells exposed to GP exhibited a concomitant increase in necrosis, whereas cells exposed with GP-AgNP showed apoptotic nature. Further, to investigate whether ROS generation aggravated DNA damage, cells exposed to GP were analyzed by comet assay. As shown in Figure 6, panel c, a relatively high level of DNA damage was observed in cells treated with GP. To validate the damaging effects, further, comet assay was performed. Minimal effects of DNA damage were seen in untreated cells, while both GP and GP-AgNP treated cell showed elongated tail. The tail lengths of GP treated cells were significantly higher in contrast to both untreated and GP-AgNP treated cells (Figure 6d). Additionally, DNA fragmentation assay confirmed the above findings, as

intense internucleosomal fragmentation in cells treated with GP was observed (Figure 6e). We have also tried to correlate DNA damage with the change in charge as measured by zeta potential. The DNA isolated from the untreated cell showed a higher zeta potential. The zeta potential drastically reduces by 78.06% and 61.30% in GP treated cell and GP-AgNP treated cell, respectively. The decrease in zeta potential may be due to nicks in the phosphate backbone of DNA molecule or due to loss of phosphate groups. Further, it is known that GP alters the structure of nucleic acids too.7 The zeta potential values of the DNA isolated from the various groups of cells are presented in Table 1. The zeta potential of the DNA from the untreated cell showed a higher negative value indicating the intact nucleic acid backbone. Treatment with GP or GP-AgNP disrupts the backbone; thus, there is a loss of phosphate groups leading to net loss of negative charge. 30011


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Figure 6. (a) Fluorescence micrographs showing damaged DNA by PI staining; (b) annexin V FITC pictographs showing live, necrotic, and apoptotic cells; (c) comet assay showing DNA damage; (d) graph showing tail length of damaged DNA obtained in the comet assay; (e) DNA fragmentation assay showing fragmented DNA in lanes A, B, and C corresponding to DNA isolated from GP treated cell and GP-AgNP treated cell and intact DNA in lane D; lame M is DNA ladder.

ment to complications such as oxidative stress and inflammation. The glycation products constitute an important pathogenic factor for various bone disorder and a potentially important therapeutic target for osteoporosis induced by multiple causes. However, a drug with anti-AGE activity involves a complex mechanism and thus poses a significant challenge. Various compounds such as aminoguanidine, pyridoxamine, carnosine, ALT-711 (Alagebrium), and phenyl thiazolium bromide have been reported to possess glycation inhibiting activity. Aminoguanidine, the first and the most effective compound to be identified, was abandoned due to major side effects (gastrointestinal disturbance, liver functioning abnormalities, renal neoplasms, anemia) during phase III clinical trials in diabetic patients.47−49 None of the other compounds has passed the clinical trials. Therefore, new antiglycating agents need to be identified that are safe with better efficacy. As discussed above, nanosized molecules with proven anticancer and antimicrobial clinical significance may possess potential antiglycation properties. Thus, we attempted to decipher the role of AgNP to inhibit AGE formation.

Table 1. Zeta Potential of the Various Cell Groups zeta potential (mV) untreated cells GP treated cells GP-AgNP treated cell

−11.1125 ± 1.27 −2.4375 ± 0.66 −4.3 ± 1.83

4. DISCUSSION Nanotechnology has extensively flourished into a multidisciplinary subject during the past decade with wide applicability in industry, renewable energy, environmental remediation, medical diagnostics, and therapeutics.43,44 Among the various nanoplatforms, AgNP have attained the utmost level of commercialization because of their characteristic physicochemical attributes, high electrical and thermal conductivity, surface-enhanced Raman scattering, stability, catalytic activity, nonlinear optical behavior, and broad spectrum antimicrobial activity.45 Recently, AgNP have developed into a tool for diagnosis and treatment of various malignancies.46 Hyperglycemic conditions influence the bone microenviron30012


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smooth round structures upon coaddition of AgNP observed in Figure 3, panels c(iii) and d(iii) implies formation of apoptotic bodies. AgNP induced apoptosis was confirmed by flow cytometric analysis using annexin V-FITC staining. Moreover, from our clonogenic assay results in Figure 3, panel b, it can be safely assumed that GP induces cell damage and AgNP carry out the cleansing of the dead cells by apoptosis. The factors released during this signal cell proliferation. The survival fraction of GP treated cells is only 27%, which doubles in case of AgNP coadministered cells. Thus, AgNP with modifications can be safely used as an antiglycating agent. Gold nanoparticle induced enhanced healing of diabetic wounds have previously been reported,59 although the gold particles were conjugated with antioxidants such as epigallocatechin gallate and a-lipoic acid. ROS are potent inducers of biomolecular damage with pathological significance. In addition, internucleosomal fragmentation of DNA is also considered to be an as a marker of DNA damage. The MMP of an intact normal cell is inversely proportional to the ROS level (Figure 5), where a lesser number of fluorescent peaks corresponding to ROS production (Figure 5a(i) is indirectly correlated to higher number of fluorescent peaks signifying higher MMP (Figure 5b(i)) and vice versa can be seen in GP treated and GP-AgNP treated cells. As compared to normal cells, there was a 66.66% drop in MMP in GP treated cells. AgNP addition restricted the MMP reduction by 82.14%. This corroborated with approximately 200% increase in ROS production in GP treated cells. Addition of AgNP with GP reduced ROS production to 150%. Thus, the rise in mitochondrial ROS load and fall in MMP clearly ascertain the loss of cellular integrity in GP treated cells, and simultaneous coadministration of AgNP reduced the dramatic increase in total ROS load and curbed the fall in MMP. Similarly, in case of Saos-2 cells treated with GP alone, the PI stained cells showed a greater number of brighter spots within the cells. Intense DNA smearing was also observed in the DNA fragmentation assay. However, a reduced number of brighter spots in PI stained cells and a less intense DNA smear was noticed in AgNP cotreated Saos-2 cells. To validate these observations, comet assay was done to check the status of DNA damage. Increased DNA damage was clearly observed in GP treated cells as compared with GP-AgNP treated cell. The images obtained were processed by computer-aided image analysis software (ImageJ) and the tail length, olive moment, and tail moment showed an increasing pattern of DNA damage. Next, to determine the nature of DNA damage, flow cytometric evaluation was performed using Annexin V-FITC/PI assay. While untreated cells were dual negative, GP treated cells were both Annexin V-FITC and PI positive signifying necrosis (Figure 6b). AgNP cotreatment also led to cell death, but the cells were Annexin V-FITC positive. Although AgNP cause cytotoxic cell death, the mode of action as observed from our results is apoptosis that not only removes the worn out cells, but also helps in wound healing and repair. The ability of AgNP to induce apoptosis has recently been reported to overcome tumors. The mechanism is unclear, but studies indicate that intracellular ROS overproduction could trigger DNA damage and cell apoptosis by activating of AKT, MAPKs, and p53 signaling pathways.58 On the other hand, GP induced cell death is by necrosis, which causes neighboring tissue damage and inflammation.60 This may be the reason for various diabetic complications (atherosclerosis, diabetic microangiopathy, diabetic retinopathy, diabetic nephropathy).6

This work validates the capacity of AgNP to modulate glycation in the initial stages. Preliminarily, we determined the binding of AgNP to glycated products in a concentrationdependent manner at 37 °C. As shown in Figure 1, with increasing concentration of AgNP, the fluorescence intensities of glycated products decreased gradually, which suggested that AgNP can interact with GP and quench the intrinsic fluorescence. To ascertain the interaction between AgNP and GP, the fluorescence quenching data were analyzed by the Stern−Volmer equation. A negative value of Gibbs free energy (ΔG0) −11.01 indicated a spontaneous reaction. Further, % inhibition of glycation was also calculated in the presence of different concentration of AgNP. When AgNP were added to the solution, the formation of GP decreased in a concentrationdependent manner. The % inhibition of glycation at the concentration of 0.2 mM AgNP onward showed a plateau trend. Hence, 0.2 mM AgNP were used for further experimentation. Next, we determined the effect of AgNP in reducing the fructosamine and protein carbonyl level in GP as compared to GP formed in the absence of AgNP during 28 days of incubation at 37 °C. The presence of AgNP reduced fructosamine and protein carbonyl content by 45.5% and 34.02%, respectively. This is in accordance with the works of Ashraf et al., the gum Arabic capped AgNP had reportedly inhibited methyl glyoxal induced glycation by 31.0 and 56.1% when used at a concentration of 0.09 and 0.18 mM, respectively.50 Similarly, 0.09 and 0.18 mM AgNP prepared from Aloe vera leaf extract inhibited AGE formation by 19.36% and 46.31%, respectively.10 Besides silver nanoparticles, gold nanoparticles have been reported to reduce the extent of nonenzymatic AGE formation.51−53 Silica-based cerium(III) chloride nanoparticles and selenium nanoparticles are also reported to inhibit the formation of AGE.54,55 However, the studies mostly focused on reducing glycation and its related structural and chemical changes and restoring the native structure of protein. There are no reports of in vitro cell based studies to validate the results within the cellular environment. Development of any therapeutic approach needs useful links regarding the mechanism of action of the compound and its efficacy. Moreover, the effects of GP on reducing cell viability are known, but its subsequent role in ROS production and DNA damage needs to be explored. Further, there are no reports on the role of AgNP in reducing cellular toxicity induced via GP. Hence, in the present study, along with evaluating the antiglycating potential of AgNP in physicochemical reaction, we have evaluated the same in vitro cell base studies in a first of a kind. As compared to control, both GP and GP-AgNP show reduced viability and proliferative ability. In comparison, to only GP treated cells, AgNP in conjunction with GP reduced cell death by 42.82%. In accordance, a 79.3% increase in proliferation was observed in cells cotreated with GP and AgNP with regard to only GP treated cells (Figure 3). However, the question of the efficacy of AgNP still remains, as in comparison to control GP-AgNP treated cells showed 50% viability. AgNP are known for their effective antibacterial properties and thus have been used in wound therapy.56,57 Wound healing occurs by cell replacement and regeneration of spent cells in response to injury or damage. The damaged cells self-destruct by apoptosis. Thus, it can be concluded that the cells undergo AgNP induced apoptosis58 to eliminate already dysfunctional, unwanted, and potentially dangerous cells from the surrounding to replace them with healthy neighbors. The formation of 30013


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(2) Yamagishi, S.; Takeuchi, M.; Inagaki, Y.; Nakamura, K.; Imaizumi, T. Role of Advanced Glycation End Products (AGEs) and Their Receptor (RAGE) in the Pathogenesis of Diabetic Microangiopathy. Int. J. Clin. Pharmacol. Res. 2002, 23 (4), 129−134. (3) Sena, L. A.; Chandel, N. S. Physiological Roles of Mitochondrial Reactive Oxygen Species. Mol. Cell 2012, 48, 158−166. (4) Zhu, W.; Tsang, S.; Browe, D. M.; Woo, A. Y. H.; Huang, Y.; Xu, C.; Liu, J.-F.; Lv, F.; Zhang, Y.; Xiao, R. Interaction of β1Adrenoceptor with RAGE Mediates Cardiomyopathy via CaMKII Signaling. JCI insight 2016, 1 (1), e84969. (5) Lu, Y. Q.; Lu, Y.; Li, H. J.; Cheng, X. B. Effect of Advanced Glycosylation End Products (AGEs) on Proliferation of Human Bone Marrow Mesenchymal Stem Cells (MSCs) in Vitro. In Vitro Cell. Dev. Biol.: Anim. 2012, 48 (9), 599−602. (6) Takeuchi, M.; Yamagishi, S. TAGE (Toxic AGEs) Hypothesis in Various Chronic Diseases. Med. Hypotheses 2004, 63 (3), 449−452. (7) Singh, V. P.; Bali, A.; Singh, N.; Jaggi, A. S. Advanced Glycation End Products and Diabetic Complications. Korean J. Physiol. Pharmacol. 2014, 18 (1), 1−14. (8) Guimarães, E. L. M.; Empsen, C.; Geerts, A.; van Grunsven, L. a. Advanced Glycation End Products Induce Production of Reactive Oxygen Species via the Activation of NADPH Oxidase in Murine Hepatic Stellate Cells. J. Hepatol. 2010, 52 (3), 389−397. (9) Ishihara, M.; Kojima, S.; Sakamoto, T.; Kimura, K.; Kosuge, M.; Asada, Y.; Tei, C.; Miyazaki, S.; Sonoda, M.; Tsuchihashi, K.; Yamagishi, M.; Shirai, M.; Hiraoka, H.; Honda, T.; Ogata, Y.; Ogawa, H. Comparison of Blood Glucose Values on Admission for Acute Myocardial Infarction in Patients with versus without Diabetes Mellitus. Am. J. Cardiol. 2009, 104 (6), 769−774. (10) Ashraf, J. M.; Ansari, M. A.; Khan, H. M.; Alzohairy, M. A.; Choi, I. Green Synthesis of Silver Nanoparticles and Characterization of Their Inhibitory Effects on AGEs Formation Using Biophysical Techniques. Sci. Rep. 2016, 6, 20414. (11) Cai, W.; Chen, X. Nanoplatforms for Targeted Molecular Imaging in Living Subjects. Small 2007, 3, 1840−1854. (12) Doane, T.; Burda, C. Nanoparticle Mediated Non-Covalent Drug Delivery. Adv. Drug Delivery Rev. 2013, 65, 607−621. (13) Sun, L.; Liu, A.; Tao, X.; Zhao, Y. A Green Method for Synthesis of Silver Nanodendries. J. Mater. Sci. 2011, 46, 839−845. (14) Mittal, A. K.; Chisti, Y.; Banerjee, U. C. Synthesis of Metallic Nanoparticles Using Plant Extracts. Biotechnol. Adv. 2013, 31, 346− 356. (15) Song, J. Y.; Kim, B. S. Rapid Biological Synthesis of Silver Nanoparticles Using Plant Leaf Extracts. Bioprocess Biosyst. Eng. 2009, 32 (1), 79−84. (16) Vasanth, K.; Ilango, K.; MohanKumar, R.; Agrawal, A.; Dubey, G. P. Anticancer Activity of Moringa Oleifera Mediated Silver Nanoparticles on Human Cervical Carcinoma Cells by Apoptosis Induction. Colloids Surf., B 2014, 117, 354−359. (17) Inbakandan, D.; Kumar, C.; Abraham, L. S.; Kirubagaran, R.; Venkatesan, R.; Khan, S. A. Silver Nanoparticles with Anti Microfouling Effect: A Study against Marine Biofilm Forming Bacteria. Colloids Surf., B 2013, 111, 636−643. (18) Nayak, D.; Ashe, S.; Rauta, P. R.; Kumari, M.; Nayak, B. Bark Extract Mediated Green Synthesis of Silver Nanoparticles: Evaluation of Antimicrobial Activity and Antiproliferative Response against Osteosarcoma. Mater. Sci. Eng., C 2016, 58, 44−52. (19) Lemire, J. a; Harrison, J. J.; Turner, R. J. Antimicrobial Activity of Metals: Mechanisms, Molecular Targets and Applications. Nat. Rev. Microbiol. 2013, 11 (6), 371−384. (20) Kora, A. J.; Sashidhar, R. B. Antibacterial Activity of Biogenic Silver Nanoparticles Synthesized with Gum Ghatti and Gum Olibanum: A Comparative Study. J. Antibiot. 2015, 68 (2), 88−97. (21) Ponarulselvam, S.; Panneerselvam, C.; Murugan, K.; Aarthi, N.; Kalimuthu, K.; Thangamani, S. Synthesis of Silver Nanoparticles Using Leaves of Catharanthus Roseus Linn. G. Don and Their Antiplasmodial Activities. Asian Pac. J. Trop. Biomed. 2012, 2 (7), 574−580. (22) David, L.; Moldovan, B.; Vulcu, A.; Olenic, L.; Perde-Schrepler, M.; Fischer-Fodor, E.; Florea, A.; Crisan, M.; Chiorean, I.; Clichici, S.;

The bone regeneration potential in diabetic individuals is also compromised.61 Repair of fractures and infected bone defects poses a significant challenge as diabetics are prone to frequent bacterial and fungal infections. In addition, bone forming ability and repair in case of fractures is drastically hampered in geriatric individuals.62 Despite the advances in therapeutics and bone tissue engineering, significant bone repair and regeneration is a challenge. Thus, administration of AgNP either as composites or embedded in scaffolds and matrices could be an ideal approach for the repair of bone in fractures and injuries because of its wider range of antibacterial activity18 as compared to traditional silver compounds, larger surface-tomass ratio, greater solubility, and chemical reactivity.63 The antiglycating ability of AgNP established through this work adds another dimension to its therapeutic use. Among many reports, researchers Banerjee and Das have stated that at very low concentration of protein, nanoparticles may come in contact with protein interfaces and cause unfolding facilitating greater coverage of surface area. However, with increase in protein concentration, they become less unfolded at the interface, thus retaining its native structure.64 This is in accordance with our observation (Figure 2c) where AgNP interact with protein at the metal/water interface restoring the native protein structure as reflected in our FTIR data. Although binding of proteins to planar surfaces induces significant changes to the secondary structure, the high curvature of NP can help proteins to retain their original structure.65,66 Thus, a great deal of optimizm exists regarding the potential impact of NP in restoring protein native structure. Thus, to conclude, we for the first time report a concentration dependent reduction in glycation in the presence of AgNP. By using the Stern−Volmer equation, the binding constant and Gibbs free energy were calculated showing favorable spontaneous binding of AgNP to glycated protein moiety. Although cotreatment with AgNP did not dramatically reduce cell death, a slight increase in cell viability was observed in comparison to GP treated cells. We also report that AgNP reduced the uncontrolled ROS production and DNA damage witnessed in cells treated with only GP. Further, we also report that GP induces necrotic cell death, while cotreatment with AgNP initiated apoptosis favoring tissue regeneration. Therefore, AgNP cotreatment in Saos-2 cells reduced the cytotoxic effects of GP by controlled orchestration of ROS production and apoptosis. Thus, administration of AgNP may play a role in attenuating the detrimental consequences of hyperglycemia manifested by diabetes-associated disorders.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +91-0661-2462682. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the National Institute of Technology Rourkela for providing the necessary financial support and infrastructure to carry out this work.

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REFERENCES

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