Biogenic silver nanoparticles synthesized with rhamnogalacturonan gum: Antibacterial activity, cytotoxicity and its mode of action

2.1 Synthesis and characterization of silver nanoparticles

The method for silver nanoparticles preparation using gum kondagogu was standardized and earlier reported by us. The silver nanoparticles were synthesized by autoclaving 0.5% homogenous gum solution prepared from 38 μm sized gum powder, containing 1 mM silver nitrate at 121 °C and15 psi for 60 min (Kora et al., 2010). The UV–visible absorption spectrum (UV–vis) of the prepared nanoparticle solution was recorded using an Elico SL 196 spectrophotometer (Hyderabad, India), from 250 to 800 nm, against corresponding gum blank. The shape of the nanoparticles was obtained with a NT-MDT Solver PRO-EC atomic force microscopy (AFM) (Zelenograd, Russia) operating in tapping mode. A minimum of 100 particles were counted and the size distribution and average particle size was obtained using OriginPro 7.0 software. The X-ray diffraction (XRD) analysis was conducted with a Rigaku, Ultima IV diffractometer (Tokyo, Japan) using monochromatic Cu Kα radiation (λ = 1.5406 Å) running at 40 kV and 30 mA. The intensity data for the nanoparticle solution deposited on a glass slide was collected over a 2θ range of 35–70° with a scan rate of 1°/min. The zeta potential of the nanoparticle solution was assessed with a Malvern Zetasizer Nano ZS90 (Malvern, UK).

2.2 Antibacterial assays

The Gram-negative (Escherichia coli ATCC 25922, E. coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853) and Gram-positive (Staphylococcus aureus ATCC 25923) bacteria were used as model test strains for checking the antibacterial activity and mode of action of silver nanoparticles. The absorbance and fluorescence measurements were carried out with Biotek Synergy™ H1 plate reader (Winooski, Vermont, USA). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of silver nanoparticles against test bacterial strains was investigated with micro-broth dilution method (Hernandez-Sierra et al., 2008; Martınez-Castanon et al., 2008). The effect of silver nanoparticles on biofilm formation was monitored with a modified microtiter plate assay (Uhlich et al., 2006). The bacterial growth curve in nanoparticle supplemented media was monitored for 48 h (Sondi and Salopek-Sondi, 2004; Venkatpurwar and Pokharkar, 2011). The effect of silver nanoparticles on membrane damage was studied by quantifying the leaked cytoplasmic nucleic acids and proteins (Chen and Cooper, 2002; Liu et al., 2004; Tiwari et al., 2008). The outer membrane damage in Gram-negative bacteria induced by silver nanoparticles was monitored using a fluorescent probe, N-phenyl naphthylamine (NPN) permeabilization assay (Helander and Mattila-Sandholm, 2000; Liu et al., 2004). To find out the possible involvement of free radicals in the bactericidal activity of nanoparticles, the antioxidant N-acetylcysteine (NAC) was used (Kim et al., 2007). The generation of intracellular reactive oxygen species (ROS) generation in nanoparticle treated bacterial cells was determined using a common fluorescent dye dichlorodihydrofluorescein diacetate (H₂DCFDA), an intracellular ROS indicator (Choi and Hu, 2008). In order to elucidate the antibacterial mode of silver nanoparticles, scanning electron microscopy (SEM) technique was used (Sondi and Salopek-Sondi, 2004). For evaluating the cytotoxicity of prepared nanoparticles on mammalian cells, the 3-(4,5-Dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out with human cervical cancer cell line HeLa (Cao et al., 2010). The detailed information on various assays is given in Appendix A. All the experiments were carried out in triplicate and the data were presented as mean ± standard deviation (SD). The statistical significance was determined by Student’s t-test.

3.1 Characterization of silver nanoparticles

After autoclaving, the appearance of yellow color in the reaction mixture indicated the formation of silver nanoparticles by the gum, a dual functional reductant and stabilizer. In the UV–vis spectrum a single strong peak with a maximum at 408 nm was observed, which corresponds to the typical surface plasmon resonance of silver nanoparticles [Fig. 1(a)]. From the AFM image, it was found that the nanoparticles are spherical in shape, polydisperse and the average particle size was about 4.5 ± 3.1 nm [Fig. 1(b)]. The XRD pattern of the silver showed characteristic diffraction peaks at 38.2°, 44.2° and 64.4°, respectively, corresponding to (1 1 1), (2 0 0) and (2 2 0) planes of face centred cubic (fcc) crystal structure of metallic silver. In addition, the broadening of the diffraction peaks suggested the effect of nano-sized particles [Fig. 1(c)]. The highly stable nature of the produced colloids was also confirmed from the measured zeta potential value of −32.7 ± 3.8 mV [Fig. 1(d)].

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Figure 1 (a) UV–vis spectrum, Inset: color of the reaction mixture (a) before and (b) after autoclaving, (b) two dimensional AFM topographic image, Scan scale 10 × 10 μm; height scale 10 nm, (c) XRD pattern and (d) zeta potential distribution of silver nanoparticles synthesized from gum.

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3.2 Determination of MIC and MBC

The biocidal nature of silver nanoparticles is determined by the size and stability of the produced colloids (Choi and Hu, 2008; Martınez-Castanon et al., 2008; Morones et al., 2005; Siddhartha et al., 2007; Tiwari et al., 2008). The bactericidal action of 5 nm sized silver nanoparticles produced with gum kondagogu against Gram-negative and Gram-positive bacteria was studied in detailed employing diverse susceptibility assays. The growth inhibition effect of silver nanoparticles against bacteria was measured by MIC and MBC (Table 1). For the bacterial strains S. aureus 25923, P. aeruginosa 27853, E. coli 25922 and E. coli 35218, the MIC values of silver nanoparticles were 10.0, 5.0, 2.0 and 2.0 μg mL⁻¹, respectively. In a previous antibacterial study, MIC values of 8–32, 16 and 32 μg mL⁻¹ were reported for S. aureus, P. aeruginosa and E. coli strains, respectively. These nanoparticles of 13 nm size were prepared using culture supernatant of P. aeruginosa (Kumar and Mamidyala, 2011). For the respective S. aureus, P. aeruginosa and E. coli strains, the MIC values were lower by 3.2, 3.2 and 16-folds. Thus, the silver nanoparticles synthesized using plant gum was found to be a more potent bactericidal agent in terms of concentration. The highest value of MIC was noted for S. aureus 25923 27853 followed by P. aeruginosa. Most of the MBC values of silver nanoparticles were same as MIC except for P. aeruginosa and S. aureus 25923, in which higher MBC values were observed. The MBC values detected for S. aureus and E. coli strains are lower by 4 and 50-folds, respectively; when compared to PVP stabilized nanoparticles of 10 nm size synthesized by ultrasonic irradiation (Cho et al., 2005). The lowest values of MIC and MBC were observed for both the E. coli strains. From the results, it is clear that among the strains selected for testing, E. coli was more susceptible towards the bactericidal activity of biogenic silver nanoparticles used in this study. These findings on susceptibility of E. coli towards silver nanoparticles are in similar lines with earlier reported studies (Cho et al., 2005; Kim et al., 2007; Martınez-Castanon et al., 2008).

3.3 Antibiofilm activity

The inhibition of biofilm formation of test strains by silver nanoparticles was studied with static microtiter plate assay at a concentration of 2 μg mL⁻¹ (Table 2). The silver nanoparticles were able to impede the biofilm formation in E. coli strains 25922 and 35218 by 83.9% and 93.1%, respectively. In the case of S. aureus 25923 and P. aeruginosa 27853, the nanoparticles were able to prevent the biofilm formation on the plate surface by 66.3% and 73.7%, respectively. These results are in line with an earlier antibiofilm studies carried out using starch stabilized silver nanoparticles (Mohanty et al., 2012) and silver nanoparticles synthesized using Bacillus licheniformis (Kalishwaralal et al., 2010); against P. aeruginosa and S. aureus 25923 and P. aeruginosa and S. epidermidis, respectively.

Among the selected strains, S. aureus 25923 and P. aeruginosa 27853 were known to produce exopolysaccharides, which are needed for the adhesion of bacterial cells to the surface during biofilm formation (Mohanty et al., 2012). The ability to form biofilms is one of important factor involved in the establishment of the infection. Though P. aeruginosa does not cause infection in healthy individuals, it is emerging as an opportunistic pathogen in immunocompromised humans (May et al., 1991). Interestingly, the activity of silver nanoparticles against P. aeruginosa 27853 biofilm formation was nearly 2-fold as compared to the positive control ciprofloxacin. The observations found in this study are very significant as both povidone–iodine and chlorhexidine were shown to be ineffective against biofilms of P. aeruginosa (Stickler and Hewett, 1991). The results indicate that the biogenic silver nanoparticles synthesized from the plant gum demonstrate strong bactericidal activity as well as antibiofilm activity which can have implications in the treatment of drug resistant bacterial infections caused by biofilms and biofouling control.

3.4 Bacterial growth kinetics in the presence of nanosilver

The growth inhibition of bacteria was checked in nutrient broth supplemented with different concentrations of nanoparticles (1–10 μg mL⁻¹), for 48 h at 600 nm. The growth curves are depicted for the untreated and silver nanoparticle treated bacteria to indicate the time dependent changes in bacterial growth. The kinetics of the all the growth curves obey typical pattern; a lag phase, an exponential phase and a stationary phase for the untreated and silver nanoparticle treated bacteria (Fig. 2). The growth of bacterial strains was inhibited progressively with increase in concentration of nanoparticles. At the concentrations beyond 1 μg mL⁻¹, the lag phase was continued for 8 h for the strain E. coli 25922. After 24 h of incubation, the growth was resumed at 2, 3, 4 and 5 μg mL⁻¹ and a concentration of 10 μg mL⁻¹ completely inhibited the growth [Fig. 2(a)]. But in an earlier report with 10–15 nm sized silver nanoparticles, even a higher concentration of 25 μg mL⁻¹ enhanced the lag phase about 8 h only for E. coli 25922 (Siddhartha et al., 2007).

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Figure 2 Growth curve of bacteria in nutrient broth supplemented with different concentrations (1–10 μg mL⁻¹) of silver nanoparticles, (a) E. coli 25922, (b) E. coli 35218, (c) P. aeruginosa 27853 and (d) S. aureus 25923.

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In the case of another Gram-negative E. coli 35218 strain, all the concentration tested (1–10 μg mL⁻¹) prolonged the lag phase for 8 h. The growth was resumed for concentrations of 1–2 μg mL⁻¹ and 3–5 μg mL⁻¹ after 8 and 24 h, respectively and a concentration of 10 μg mL⁻¹ completely inhibited the cellular division [Fig. 2(b)]. In an earlier antibacterial characterization study carried out with silver nanoparticles of 16 nm size against another E. coli strain ATCC 15224, no bacterial growth was noted at 60 μg mL⁻¹ concentration (Raffi et al., 2008). But such phenomenon of complete inhibition was observed at 10 μg mL⁻¹ concentration itself with gum reduced silver nanoparticles against E. coli 35218. With the same strain of E. coli 35218 and polylysine capped silver nanoparticles of 7.2 nm size, a concentration of 50 μg mL⁻¹ only prolonged the lag phase for 5 h (Dror-Ehre et al., 2009). For P. aeruginosa 27853, the concentrations of 1–2 and 3–5 μg mL⁻¹ of nanoparticles extended the lag phase for 8 h and 24 h, respectively. At a concentration of 10 μg mL⁻¹, the growth was arrested completely [Fig. 2(c)].

In the case of Gram-positive S. aureus 25923, 3–10 μg mL⁻¹ of silver nanoparticles caused a lag phase for 8 h and beyond which growth was continued for 24 h and reached stationary phase at 48 h [Fig. 2(d)]. With the same strain of S. aureus 25923, the concentration of 100 μg mL⁻¹ elicited only a partial growth inhibition with silver nanoparticles 10–15 nm synthesized with glucose and hydrazine (Siddhartha et al., 2007). With time, the concentration of nanoparticles decreases as a result of coagulation and removal during the interaction of nanoparticles with intracellular substances of the destroyed cells (Sondi and Salopek-Sondi, 2004). As a consequence, the bacterial cells resume the growth after 8 h due to a reduction in the effective concentration of nanoparticles. The bacterial cultures without nanoparticles did not show any inhibition and reached the stationary phase at the end of 48 h. The enhanced growth inhibition activity of the nanoparticles used here can be attributed to superior stability of the nanoparticle solutions (Siddhartha et al., 2007). The data on growth curve indicates a faster inhibition in Gram-negative bacteria compared to Gram-positive bacteria. In earlier studies on time and dose dependent antibacterial potential of silver nanoparticles, similar observations were noted with E. coli and B. subtilis (Tiwari et al., 2008); and E. coli and S. aureus strains (Siddhartha et al., 2007; Venkatpurwar and Pokharkar, 2011). The results conclusively suggest that the inhibition of growth depends upon the type of bacterial strain as well as concentration and particle size of the nanoparticles.

3.5 Leakage of cytoplasmic contents

As reported in literature, the leakage of cytoplasmic contents is a characteristic feature for indicating the damage to the bacterial cytoplasmic membrane (Chen and Cooper, 2002). Due to the interaction of nanoparticles with cells, the cell membrane looses its integrity leading to the release of intracellular contents such as nucleic acids and nucleotides; and proteins by membrane disruption (Fig. 3). Thus, the occurrence of these substances signifies the membrane damage. With an increase in nanoparticle concentration, the amount of nucleic acids and proteins released from the cells increased in a dose dependent manner. The enhancement suggests the rapid reaction kinetics of the nanoparticles with bacterial cells. These results are in line with previous reports (Tiwari et al., 2008). At 5 μg mL⁻¹ concentration of nanoparticles, the highest release of nucleic acids and proteins was observed with E. coli 25922, followed by P. aeruginosa 27853, E. coli 35218 and S. aureus 25923. In comparison with other Gram-negative strains, the Gram-positive S. aureus 25923 was found to be more resistant towards the nanoparticle mediated membrane damage [Fig. 2]. These observations are similar to the earlier antibacterial studies carried out with E. coli DH5α and B. subtilis strains (Tiwari et al., 2008). Hence, the results indicated the release of intracellular components, an evidence for membrane damage.

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Figure 3 Release of (a) 260 nm and (b) 280 nm absorbing materials from the cell suspensions of bacteria treated with silver nanoparticle solutions. Values are mean ± SD for n = 3; ^∗∗p < 0.005, ^∗∗∗p < 0.001 and ^∗∗∗∗p < 0.0001 compared to negative control.

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3.6 Outer membrane damage

An intact outer membrane acts as permeability barrier and excludes hydrophobic substances such as NPN. But once damaged, it can allow the entry of hydrophobic agents such as NPN to the phospholipid layer of outer membrane, resulting in prominent characteristic fluorescence. This is mainly due to the destabilizing interaction of nanoparticles with membrane components (Helander and Mattila-Sandholm, 2000). The bacterial cell suspensions treated with silver nanoparticles exhibited outer membrane damage in a dose dependent manner (Fig. 4). These findings are in agreement with studies on bactericidal activity of chitosan against E. coli (Liu et al., 2004). From the data it is apparent that among the Gram-negative strains, E. coli 35218 was least susceptible to the outer membrane permeabilization activity of silver nanoparticles. Even with less sensitive E. coli 35218, the activity of the nanoparticles was higher at both the tested concentrations of 2 and 3 μg mL⁻¹, when compared to the positive control hydrogen peroxide (Fig. 4).

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Figure 4 Uptake of NPN in bacteria induced by treatment with silver nanoparticle solutions. Values are mean ± SD for n = 3; ^∗∗∗∗p < 0.0001 compared to negative control.

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3.7 Effect of antioxidant on the bactericidal activity of silver nanoparticles

For studying the involvement of ROS in the antibacterial effect of silver nanoparticles, NAC was used as an antioxidant. In the control petriplates with NAC alone at 10 mM concentration, the bacterial colonies were clearly seen with no growth inhibition. However, in the petriplates supplemented with 5 μg mL⁻¹ of nanoparticles, no bacterial colonies were observed due to complete inhibition of growth. While, the petriplates supplemented with both NAC and silver nanoparticles, the bacterial colonies were observed (Fig. 5). From these photographs, it is clearly evident that NAC acted as a free radical scavenger and protected the bacterial cells from the bactericidal activity of silver nanoparticles.

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Figure 5 Petri plates showing the effect of antioxidant NAC on the bactericidal activity of silver nanoparticles on E. coli 25922.

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The % survival of bacteria in the presence of NAC and silver nanoparticles is depicted in Fig. 5. For the Gram-positive S. aureus 25923 and Gram-negative E. coli 35218, NAC was able to protect the 100% and 89.3% of bacterial cells from the toxicity of the silver nanoparticles at 5 μg mL⁻¹. Hence, S. aureus 25923 and E. coli 35218 were resistant to the bactericidal activity of silver nanoparticles. But, NAC was able to protect only 77.9% and 37.1% of cells from silver nanoparticles, for P. aeruginosa 27853 and E. coli 25922, respectively (Fig. 6). It indicates that these strains were not able to recover completely from the bactericidal activity of silver nanoparticles, even with NAC supplementation. The effective bactericidal activity of biogenic nanoparticles on E. coli 25922 and P. aeruginosa 27853 could be due to an increase in the bioavailability of the silver nanoparticles by gum kondagogu. The gum kondagogu is known to improve the bioavailability of the drug and the same is reported for the controlled release of diclofenac sodium (Naidu et al., 2009).

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Figure 6 Bar graph showing the% of viable cells in the presence of antioxidant NAC and 5 μg mL⁻¹ of silver nanoparticles. Values are mean ± SD for n = 3; ^∗∗p < 0.005, ^∗∗∗p < 0.001 and ^∗∗∗∗p < 0.0001 compared to control.

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The experimental data of the present study suggests that probably ROS are involved in the bactericidal activity of silver nanoparticles and these scavengers prevented this effect by interacting with the ROS. This observation is in concurrence with earlier studies on antimicrobial effects of silver nanoparticles (Kim et al., 2007; Kora and Arunachalam, 2011) and ZnO and TiO₂ nanoparticles (Inoue et al., 2002). As suggested by the previous researchers, our findings support the generation of free radicals from the surface of silver nanoparticles and inhibition of bacterial growth.

3.8 Detection of intracellular ROS

The levels of ROS are used as a collective marker for superoxide anion, hydroxyl radical and hydrogen peroxide, which act as an indicator for cellular oxidative stress. Hence, the production of intracellular ROS was monitored with H₂DCFDA by studying the response of bacterial strains towards silver nanoparticles (Fig. 7). For the bacterial strains S. aureus 25923 and E. coli 25922, the generation of intracellular ROS is comparable with positive control 30 μM H₂O₂, at 2 μg mL⁻¹ of nanoparticles (Fig. 7). In the case of P. aeruginosa 27853 and E. coli 35218, the intracellular ROS was more than the positive control (Fig. 7). The tolerance of P. aeruginosa towards H₂O₂ can be attributed to the protective role of constitutively expressed catalase enzyme (Elkins et al., 1999). Among the selected strains, the ROS generation was highest with S. aureus 25923. Thus, the production of intracellular ROS during the interaction of silver nanoparticles with bacteria are consistent with the previous studies on the involvement of oxidative stress in the toxicity of silver (Choi and Hu, 2008), ZnO and TiO₂ nanoparticles (Kumar et al., 2011).

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Figure 7 Production of intracellular ROS in the bacterial cell suspensions treated with silver nanoparticles. Values are mean ± SD for n = 3; ^∗∗p < 0.005 and ^∗∗∗∗p < 0.0001 compared to negative control.

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3.9 Effect of nanoparticles on bacterial morphology

The effect of 5 μg mL⁻¹ concentration of silver nanoparticles on the cell surface morphology of the Gram-positive S. aureus 25923 and Gram-negative P. aeruginosa 27853 strains was investigated using SEM technique (Fig. 8). In the untreated samples, the spherical and rod shaped cells of S. aureus and P. aeruginosa were clearly seen. When compared to the controls, the micrographs of the treated samples reveal the attachment of nanoparticles to the surface of bacterial cells. And after treatment, structural changes in the bacteria morphology such as cell surface damage were clearly observed and resulted in the formation of cell debris by majority of the bacterial cells. Additionally, the existence of elemental silver in the membrane was also confirmed from the EDX spectra of the treated bacteria. These samples exhibit a strong absorption peak at 3 keV, which corresponds to the characteristic absorption of silver nanoparticles due to surface plasmon resonance (Fig. 9). These results are in concurrence with the earlier antibacterial studies carried out with silver nanoparticles (Cho et al., 2005; Sondi and Salopek-Sondi, 2004).

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Figure 8 SEM photographs of bacterial cells at 200 nm scale. Cells of S. aureus (a) before and (b) after treatment; and P. aeruginosa 27853 (c) before and (d) after treatment with 5 μg mL⁻¹ of silver nanoparticles.

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Figure 9 EDX spectra of bacteria, S. aureus (a) before and (b) after treatment; and P. aeruginosa 27853 (c) before and (d) after treatment with 5 μg mL⁻¹ of silver nanoparticles.

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3.10 Proposed mode of antibacterial action

The antibacterial activity of silver nanoparticles was visualized with susceptibility assays and quantified in terms of MIC, MBC and inhibition of growth kinetics. The antibiofilm activity was calculated via in vitro microtiter plate crystal violet assay. The membrane damage was measured through UV–vis absorption and NPN. The involvement of ROS in bactericidal activity of silver nanoparticles was demonstrated via antioxidant assay and the generation of intracellular ROS was detected via fluorescent dye H₂DCFDA. The damage to cell surface morphology by nanoparticles is shown in SEM (Fig. 10). Based on the results, we tend to speculate that the ROS may be generated from the surface of silver nanoparticles; interact with the cell wall and membrane, damage the cell membrane, increase the cell permeability and leak the intracellular contents such as proteins and nucleic acids by cell disruption. In turn, the interaction of bacteria with nanoparticles also causes structural and morphological changes and cell surface damage. The observations noted in our study substantiate the association of ROS and cell membrane damage in the antibacterial action of silver nanoparticles. These observations confirm the earlier findings on the antibacterial mechanism of silver nanoparticles (Cho et al., 2005; Kim et al., 2007; Morones et al., 2005; Raffi et al., 2008; Sondi and Salopek-Sondi, 2004). However, we believe that the mode of action of silver nanoparticles on bacteria is probably more complex than assumed, involving more than one mechanism and a series of events, finally leading to cell death.

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Figure 10 Proposed mode of antibacterial action of silver nanoparticles demonstrated with various susceptibility assays.

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3.11 Cytotoxicity evaluation

The biocompatibility of the nanoparticles was evaluated with HeLa cell line for finding out their applications in biomedical field. The MTT assay assesses the mitochondrial activity by measuring the ability of viable cells to reduce MTT into purple formazon product. The % viability of the cells was plotted against various nanoparticle concentrations at different time intervals. The concentrations of silver nanoparticles up to 2.5 μg mL⁻¹ were not cytotoxic. Beyond the concentration of 2.5 μg mL⁻¹, they were cytotoxic. After 72 h of incubation, the cell viability was dropped drastically to 54% and 18.2% at 5 μg mL⁻¹ and 10 μg mL⁻¹, respectively (Fig. 11). Most of the previously reported cytotoxic studies with silver nanoparticles were carried out for 24 h only (Mohanty et al., 2012; Valodkar et al., 2011). With silver nanoparticles of 5–10 nm (Miura and Shinohara, 2009) and 10 nm (Valodkar et al., 2011) size, the cytotoxicity was noted at 100 μg mL⁻¹ and 50 μg mL⁻¹, respectively at 24 h. In the case of silver nanoparticles produced with garlic clove extract, the studies were extended for 48 h (Ahamed et al., 2011). While in our study, we have investigated the anti-proliferation effect of 4.5 nm sized plant gum synthesized silver nanoparticles for 24–72 h. It can be inferred from these results that there is a dependency of cytotoxic concentration on particle size. These results are in tune with earlier studies carried out with cytotoxic activities of chitosan nanoparticles of different sizes (Qi et al., 2005). Based on the observations, it can be inferred that nanoparticles with enhanced toxicity may be employed as bactericidal agents, while benign biocompatible ones as drug delivery and labeling agents (Suresh et al., 2010).

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Figure 11 Eeffect of silver nanoparticles on the cell viability of HeLa cell line at various concentrations. Values are mean ± SD for n = 3; ^∗∗p < 0.005, ^∗∗∗p < 0.001 and ^∗∗∗∗p < 0.0001 compared to untreated cells.

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