Multidimensional effects of biologically synthesized silver nanoparticles in Helicobacter pylori, Helicobacter felis, and human lung (L132) and lung carcinoma A549 cells
© Gurunathan et al.; licensee Springer. 2015
Received: 8 December 2014
Accepted: 10 January 2015
Published: 5 February 2015
Silver nanoparticles (AgNPs) are prominent group of nanomaterials and are recognized for their diverse applications in various health sectors. This study aimed to synthesize the AgNPs using the leaf extract of Artemisia princeps as a bio-reductant. Furthermore, we evaluated the multidimensional effect of the biologically synthesized AgNPs in Helicobacter pylori, Helicobacter felis, and human lung (L132) and lung carcinoma (A549) cells. UV-visible (UV–vis) spectroscopy confirmed the synthesis of AgNPs. X-ray diffraction (XRD) indicated that the AgNPs are specifically indexed to a crystal structure. The results from Fourier transform infrared spectroscopy (FTIR) indicate that biomolecules are involved in the synthesis and stabilization of AgNPs. Dynamic light scattering (DLS) studies showed the average size distribution of the particle between 10 and 40 nm, and transmission electron microscopy (TEM) confirmed that the AgNPs were significantly well separated and spherical with an average size of 20 nm. AgNPs caused dose-dependent decrease in cell viability and biofilm formation and increase in reactive oxygen species (ROS) generation and DNA fragmentation in H. pylori and H. felis. Furthermore, AgNPs induced mitochondrial-mediated apoptosis in A549 cells; conversely, AgNPs had no significant effects on L132 cells. The results from this study suggest that AgNPs could cause cell-specific apoptosis in mammalian cells. Our findings demonstrate that this environmentally friendly method for the synthesis of AgNPs and that the prepared AgNPs have multidimensional effects such as anti-bacterial and anti-biofilm activity against H. pylori and H. felis and also cytotoxic effects against human cancer cells. This report describes comprehensively the effects of AgNPs on bacteria and mammalian cells. We believe that biologically synthesized AgNPs will open a new avenue towards various biotechnological and biomedical applications in the near future.
Nanomaterials often have novel and size-related physico-chemical properties that differ significantly from their larger counterparts. Therefore, the growing interest in the field has driven the production of numerous nanomaterials with outstanding optical, magnetic, catalytic, and electrical properties [1,2]. Silver nanoparticles (AgNPs) have become increasingly popular and have been used in various applications such as antibiotic agents in textiles and wound dressings and in biomedical devices; furthermore, they are one of the most commonly used engineered nanoparticles in commercialized products [3,4]. Since AgNPs have widespread applications, academia and industry have paid more attention to the production of AgNPs than to their uses .
Among several methods, chemical methods provide an easy way to synthesize AgNPs in solution, and they are a commonly used method for the production of AgNPs . In contrast, physical methods appear to produce a low yield. Chemical methods, on the other hand, are more complex in that they require three main components, including metal precursors, reducing agents, and stabilizing/capping agents. Furthermore, chemical methods use various toxic materials including hydrazine, citrate, borohydride, or other organic compounds (e.g., reducing agents); all these agents can be toxic to living organisms including humans. Capping agents are playing an important role for the stabilization of nanoparticles, for example, capped AgNPs exhibit better antibacterial activity than uncapped AgNPs do [6,7]. Biological methods seem to be valuable for the preparation of AgNPs with controlled size and shape of the nanoparticles [8-13]. Given that conventional physical methods have low yields and chemical methods are toxic and consume a lot of energy, the development of environmentally friendly approaches has become the more preferred trend for the field of nanobiotechnology. Biologically prepared nanomaterials are extremely valuable because nanoparticles are easily soluble and stable . In addition, during the biological synthesis of AgNPs, the reducing agent and stabilizer are replaced by molecules produced by living organisms. These molecular compounds can be sourced from various living organisms such as bacteria, fungi, yeasts, algae, or plants . Biomolecules can be attached to various types of surfaces via diffusion, adsorption/absorption, covalent cross-linking, and affinity interaction .
Recently, numerous microorganisms have been reported to synthesize AgNPs, including bacteria like Pseudomonas stutzeri AG259 , Bacillus licheniformis , Brevibacterium casei , Escherichia coli , and Shewanella oneidensis  and fungi like Fusarium oxysporum , Trichoderma viride , and Ganoderma neo-japonicum . Extracellular synthesis of various types of nanoparticles was performed using plants, including geranium leaves  and lemongrass , via the reduction of aqueous AgNO3 and AuCl4, respectively. Previous studies suggest that leaf and other parts of plant extracts from various plants, such as Azadirachta indica , Aloe vera , Bryophyllum sp. , Gliricidia sepium, Alfalfa sprouts [27,28], aqueous stem extract of banana , and Allophylus cobbe , have also been explored for the synthesis of AgNPs. Compared to other reducing agents derived from microorganisms, the reduction of the Ag+ ions with the extracts of plants occurs quickly . Furthermore, biological methods seem to have less time required for complete reduction and be stable and readily available in solution at high densities . Similarly, shape and size, the rate of reduction of metal ions is faster, and more stable metal nanoparticles are formed using leaf extracts compared to using microorganisms [28,30].
The green juice of Artemisia princeps used to treat skin injuries and gastrointestinal disorders [31,32]. Yun et al.  have identified 16 water-soluble phenolic compounds in the leaf water extract of A. princeps, and its extract contains 192 volatile chemicals . Therefore, this plant extract can be used as a reducing and stabilizing agent for the synthesis of AgNPs.
Infections caused by multidrug-resistant bacteria lead to major public health issues, such as morbidity, mortality, cost of health care, and the need for implementation of infection control measures . Parsonnet et al.  reported that bacteria have been linked to cancer by the induction of chronic inflammation and the production of carcinogenic bacterial metabolites. A pertinent example of the inflammatory mechanism of carcinogenesis is the Helicobacter pylori infection. H. pylori are known to cause infection in the stomach and are found in about two thirds of the world’s population. H. pylori exist and are adherent to the epithelium of stomach. Non-pylori gastric Helicobacter organisms cause chronic gastritis and inflammation in humans . On the other hand, Fusobacterium nucleatum promotes colorectal carcinogenesis and intestinal tumorigenesis and modulates the tumor-immune microenvironment [37,38].
Recent surveys suggest that lung cancer accounts for 23% of all cancer-related mortality, outnumbering the total mortality of breast, colon, and prostate cancers combined [39,40]. To address the effect of AgNPs, several studies have reported the impact of AgNPs in various cell lines, such as BRL4A rat liver cells , PC-12 neuroendocrine cells , germ line stem cells , rat alveolar macrophages , and a human lung carcinoma cell line, A549 . Recent studies reported that biologically prepared AgNPs have been used for antibacterial and antifungal [46-48]. The results from previous studies suggest that the generation of reactive oxygen species (ROS) is an important and general mechanism of nanoparticle-mediated cytotoxicity through DNA damage, apoptosis, and necrosis [44,49-53]. Although various studies have addressed the effect of AgNPs in various cell lines, there has been no study on the multiple functions of biologically prepared AgNPs using A. princeps on bacteria causing carcinogenesis and human cancer cells. Therefore, this study was aimed to investigate the following objectives. Firstly, we aimed to develop an easy, consistent, cost-effective, and green approach to the synthesis of colloidal AgNPs using leaf extract of A. princeps. Secondly, we evaluated the antibacterial and anti-biofilm activity of AgNPs against H. pylori and non-pylori Helicobacter felis. Finally, we assessed the cell-specific cytotoxic effects of AgNPs in normal lung and lung cancer cells.
Bacterial strains and reagents
All culture media and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. The strains of H. pylori GS-13 and H. felis GS-14 used in the present study were obtained from our culture collection. All strains were maintained at −80°C in Brucella agar (BA) (Sigma, Cream Ridge, NJ, USA) supplemented with 2% fetal calf serum (FCS). Penicillin-streptomycin solution, trypsin-EDTA solution, RPMI 1640 medium, and 1% antibiotic-antimycotic solution were obtained from Life Technologies/Gibco (Grand Island, NY, USA). Silver nitrate, fetal bovine serum (FBS), and the in vitro toxicology assay kit were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Synthesis and characterization of AgNPs
A. princeps leaves were collected from plants growing in the Jeju Island, South Korea, and stored at 4°C until needed. The leaf extract was prepared according to method described earlier . Briefly, the filtered extract was used for the synthesis of AgNPs by adding 10 mL (1 mg/mL) to 100 mL of 1 mM AgNO3 in an aqueous solution at room temperature. The bio-reduction of the AgNO3 was monitored spectrophotometrically between 300 and 600 nm. The synthesized particles were characterized according to methods described previously . The size distribution of the dispersed particles was measured using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., Malvern, UK). X-ray diffraction (XRD) analyses were carried out on an X-ray diffractometer (Bruker D8 DISCOVER; Bruker AXS GmBH, Karlsruhe, Germany). The high-resolution XRD patterns were measured at 3 Kw with a Cu target using a scintillation counter (λ = 1.5406 °A) at 40 kV and 40 mA and were recorded in the range of 2θ = 5°–80°. Further characterization of changes in the surface and surface composition was performed by Fourier transform infrared spectroscopy (PerkinElmer Spectroscopy GX, PerkinElmer, Waltham, MA, USA). Transmission electron microscopy (TEM), using a JEM-1200EX microscope, was performed to determine the size and morphology of AgNPs. TEM images of AgNPs were obtained at an accelerating voltage of 300 kV.
Determination of minimum inhibitory concentrations of AgNPs and in vitro killing assay
Minimal inhibitory concentration (MIC) of H. pylori and H. felis was determined as described previously . To determine the MICs of AgNPs, H. pylori and H. felis were then exposed to different concentrations of AgNPs. Growth was monitored using a microtiter ELISA reader (EMax, Molecular Devices, Sunnyvale, CA, USA) by monitoring the absorbance at 600 nm. The MIC of AgNPs was defined as the lowest concentration that inhibited the visible growth of bacteria. The in vitro killing assay was performed as described previously .
Determination of biofilm activity using the tissue culture plate method
Inhibition of biofilm was determined as described earlier with suitable modifications [8,55]. Briefly, the cells were grown in Brucella broth supplemented with 2% FCS and individual wells of sterile, 96-well flat-bottom polystyrene tissue culture plates (TCPs) were filled with 180 μL of a single bacterial species (1 × 106/mL). The cell culture plates were then incubated with AgNPs for 24 h at 37°C. After incubation, the media were removed, and the wells were washed three times with 200 μL sterile distilled water to remove non-adherent bacteria. The crystal violet solutions in water were added for 45 min. The wells were then washed five times with 300 μL of sterile distilled water to remove excess stain. The absorbance of each well was measured at 595 nm using a microtiter ELISA reader. The percent inhibition of biofilm activity was calculated as described earlier [8,55].
Measurement of ROS generation in bacteria
ROS was determined according to the manufacturer’s instructions and according to previous publications [8,49,56]. All test strains were grown in BB. Cell suspensions were incubated with AgNPs at 37°C on a rotary shaker for 12 h. Aliquots were then removed and spun in a microfuge, and the absorption of the supernatant was measured at 450 nm.
Human lung cancer A549 cells and normal human lung L-132 cells were obtained from the Korean Cell Bank (Seoul, Korea) and cultured in RPMI 1640 medium supplemented with 10% FBS and 100 U/mL penicillin-streptomycin at 5% CO2 and 37°C. At 75% confluence, the cells were harvested using 0.25% trypsin and subcultured in 75-cm2 flasks, 6-well plates, or 96-well plates. Cells were allowed to attach the surface for 24 h before treatment. The medium was replaced three times per week, and the cells were passaged at subconfluency.
Cell viability and cytotoxicity assays
Cell viability was measured using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Briefly, A549 and L132 cells were plated onto 96-well flat-bottom culture plates with various concentrations of AgNPs. All cultures were incubated for 24 h at 37°C (5% CO2 in a humidified incubator). CCK-8 solution (10 μL) was added to each well, and the plate was incubated for another 2 h at 37°C. Absorbance was measured at 450 nm with a microplate reader (Multiskan FC; Thermo Fisher Scientific Inc., Waltham, MA, USA). Cytotoxicity was assessed using supernatants from the medium in lactate dehydrogenase (LDH) assays. An LDH Cytotoxicity Detection kit (Takara Bio Inc., Tokyo, Japan) was used according to the manufacturer’s protocol, and the absorbance was measured at 490 nm using a microplate reader.
ROS (H2-DCFH-DA) assay
Human lung normal L132 cells and A549 human lung epithelial adenocarcinoma cells were cultured in minimum essential medium (Hyclone Laboratories, Logan, UT, USA) containing 10 μM H2-DCFDA in a humidified incubator at 37°C for 30 min. Cells were washed in PBS (pH 7.4) and lysed in lysis buffer (25 mM HEPES [pH 7.4], 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, and 0.1 mM DTT supplemented with a protease inhibitor cocktail). Cells were cultured on coverslips in a 4-well plate. Cells were incubated in DMEM containing 10 μM H2-DCFDA at 37°C for 30 min. Cells were washed in PBS, mounted with Vectashield fluorescent medium, and viewed with a fluorescence microscope.
The change in mitochondrial transmembrane potential was evaluated using the cationic fluorescent indicator JC-1 (Molecular Probes, Eugene, OR, USA). J-aggregates of intact mitochondria were fluorescent red with an emission at 583 nm, and J-monomers in the cytoplasm were fluorescent green with emission at 525 nm and an excitation wavelength of 488 nm. A549 and L132 cells were incubated in RPMI containing 10 μM JC-1 at 37°C for 15 min, washed with PBS, and transferred to a clear 96-well plate. Cells were also cultured on cover slips, incubated in DMEM containing 10 μM JC-1 at 37°C for 15 min, and then washed with PBS. Finally, cells were mounted using Vectashield fluorescent medium and visualized with fluorescence microscopy.
All assays were carried out in triplicate and the experiments were repeated at least three times. The results are presented as means ± SD. All experimental data were compared using Student’s t-test. A p value less than 0.05 was considered statistically significant.
Results and discussion
Synthesis and characterization of AgNPs using leaf extract
XRD analysis of AgNPs
FTIR spectra of AgNPs
Dynamic light scattering analysis of AgNPs
Size and surface analysis of AgNPs by TEM
Determination of MIC of AgNPs against H. pylori and H. felis
In response to the overwhelming evidence linking H. pylori infection to human cancer, the International Agency for Research on Cancer listed H. pylori as a definite human oncogenic agent in 1994 [36,73-75]. Therefore, we were interested in finding out the MIC of AgNPs against both pylori and non-pylori strains, such as H. pylori and H. felis, respectively. The MIC of AgNPs was defined as the lowest concentration that completely inhibited visible growth of bacteria after incubation at 37°C for 24 h. In these studies, H. pylori and H. felis were used as a model bacteria for Gram negative to evaluate antibacterial activities of AgNPs. Both strains were incubated with the different concentration of AgNPs for 24 h in Brucella broth. The media without AgNPs were used as a control. The H. pylori and H. felis bacterial counts were significantly reduced by the treatment with AgNPs than control. The level of MIC of AgNPs was found to be 5.0 and 5.5 μg/mL to H. pylori and H. felis, respectively. The toxic effects of AgNPs depend on size, surface area, and surface functionalization which are major factors that influence bio-kinetics and toxicity in bacteria [76,77].
Dose-dependent antibacterial effects of AgNPs
Time-dependent antibacterial activity of AgNPs
Anti-biofilm activity of AgNPs against H. pylori
Effect of AgNPs on ROS production
ROS is a natural by-product of the metabolism of respiring organisms . Induction of ROS synthesis leads to the formation of highly reactive radicals that destroy the cells. The possible mechanisms of H. pylori and H. felis cell death are due to membrane damage by AgNPs which relates to metal depletion, that is, the formation of pits in the outer membrane and change in membrane permeability by the progressive release of lipopolysaccharide (LPS) molecules and membrane proteins . Previous studies proposed that the sites of interaction for AgNPs and membrane cells might be due to sulfur-containing proteins present in the bacterial membrane proteins [91-95]. Previous studies suggest that ROS may be a common mechanism of cell death induced by bactericidal antibiotics [96-99].
Effect of AgNPs on cell viability of L132 and A549 cells
Effect of AgNPs membrane leakage
AgNPs induce time-dependent ROS generation
AgNPs induce mitochondrial-mediated apoptosis
We have demonstrated an easy, simple, and environmentally friendly approach to the synthesis of AgNPs using the leaf extract of A. princeps as a reducing and stabilizing agent. In this method, highly crystalline, spherical-shaped AgNPs with an average size of 20 nm were prepared without using any harmful reducing or capping agents. The phyto-molecules of the A. princeps extract were not only responsible for the reduction of AgNO3 but also function as capping agents to the surfaces of the AgNPs. The novel AgNPs show multifunctional effects against bacteria and human cancer cells, yet were biocompatible with normal lung cells. This suggests that AgNPs could contribute to develop therapeutic molecules for anticancer and anti-angiogenic. Interestingly, this comprehensive report describes the effect of AgNPs in bacteria and human cell types. Our results highlight a common and possible mechanism of cell death in bacteria and human cancer cells that is due to the generation of ROS, eventually leading to cell death. We believe that biologically prepared AgNPs could open a new avenue for various biomedical applications, particularly infections and cancer.
This work was supported by the KU-Research Professor Program of Konkuk University. Dr Sangiliyandi Gurunathan was supported by a Konkuk University KU-Full-Time Professorship. This work was carried out by Woo Jang-Choon project (PJ007849) from the Rural Development Administration (RDA), Republic of Korea.
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