- Email: [email protected]
The effects of bacteria-nanoparticles interface on the antibacterial activity of green synthesized silver nanoparticles
Accepted Manuscript The effects of bacteria-nanoparticles interface on the antibacterial activity of green synthesized silver nanoparticles Aftab Ahmad, Yun Wei, Fatima Syed, Kamran Tahir, Aziz Ur Rehman, Arifullah Khan, Sadeeq Ullah, Qipeng Yuan PII:
S0882-4010(16)30746-X
DOI:
10.1016/j.micpath.2016.11.030
Reference:
YMPAT 2021
To appear in:
Microbial Pathogenesis
Received Date: 6 November 2016 Revised Date:
27 November 2016
Accepted Date: 29 November 2016
Please cite this article as: Ahmad A, Wei Y, Syed F, Tahir K, Rehman AU, Khan A, Ullah S, Yuan Q, The effects of bacteria-nanoparticles interface on the antibacterial activity of green synthesized silver nanoparticles, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2016.11.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The effects of bacteria-nanoparticles interface on the antibacterial activity of green synthesized silver nanoparticles Aftab Ahmad a, Yun Wei a, Fatima Syed b, Kamran Tahir a, Aziz Ur Rehman a, Arifullah Khan a
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical
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a
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Sadeeq Ullah a and Qipeng Yuan a*
Technology, No. 15 East Road of North Third Ring, Chao Yang District, Beijing 100029, China Institute of Chemical Sciences, University of Peshawar (25120) Peshawar-Pakistan
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b
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To whom correspondence should be addressed
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Email: [email protected]
[email protected]
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Abstract Neutralization of bacterial cell surface potential using nanoscale materials is an effective strategy to alter membrane permeability, cytoplasmic leakage, and ultimate cell death. In the present
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study, an attempt was made to prepare biogenic silver nanoparticles using biomolecules from the aqueous rhizome extract of Coptis Chinensis. The biosynthesized silver nanoparticles were surface modified with chitosan biopolymer. The prepared silver nanoparticles and chitosan
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modified silver nanoparticles were cubic crystalline structures (XRD) with an average particle size of 15 and 20 nm respectively (TEM, DLS). The biosynthesized silver nanoparticles were
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surface stabilized by polyphenolic compounds (FTIR). Coptis Chinensis mediated silver nanoparticles displayed significant activity against E. coli and Bacillus subtilus with a zone of inhibition 12 ± 1.2 (MIC = 25 µg/mL) and 18 ± 1.6 mm (MIC = 12.50 µg/mL) respectively. The bactericidal efficacy of these nanoparticles was considerably increased upon surface
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modification with chitosan biopolymer. The chitosan modified biogenic silver nanoparticles exhibited promising activity against E. coli (MIC = 6.25 µg/mL) and Bacillus subtilus (MIC = 12.50 µg/mL). Our results indicated that the chitosan modified silver nanoparticles were
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promising agents in damaging bacterial membrane potential and induction of high level of intracellular reactive oxygen species (ROS). In addition, these nanoparticles were observed to
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induce the release of the high level of cytoplasmic materials especially protein and nucleic acids into the media. All these findings suggest that the chitosan functionalized silver nanoparticles are efficient agents in disrupting bacterial membrane and induction of ROS leading to cytoplasmic leakage and cell death. These findings further conclude that the bacterial-nanoparticles surface potential modulation is an effective strategy in enhancing the antibacterial potency of silver nanoparticles.
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Keyword: Coptis Chinensis, silver nanoparticles, Bacteria, membrane potential 1. Introduction The chemists and technologists around the globe follow the basic principles of green chemistry
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to develop less hazardous synthesis procedures. Among the modern line of technological advancements, nano-biotechnology occupies an important position to create and enhance the utility of nano-size materials in advanced biotechnology [1]. Nanoscale materials and devices
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have unique physiochemical characteristics that make them applicable in fields ranging from electronic to medicine [2, 3]. Among the metal nanoparticles, nano silver is widely used in a
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wide array of consumer products and clinical care, as silver ions possess strong toxicity against a broad spectrum of pathogenic microbes [4-7]. Silver nanoparticles arrest bacterial growth by disrupting its cell membrane, DNA damage, inhibiting vital metabolic enzymes and production of reactive oxygen species (ROS) that destroy cellular constituents [4, 5, 8, 9]. Microbial cells
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are sensitive to reactive oxygen species and any therapy that generates enough reactive oxygen species that could bypass the antioxidant defense mechanism of microbial cell will be a promising strategy to treat infection and the emerging microbial resistance [10]. Metal
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nanoparticles are considered as promising agents producing significant level of ROS that can overcome the microbial antioxidant defense system leading to cell damage [11] [12].
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Because of its multi-sites action, nano-silver could be a promising candidate to overcome the emerging microbial resistance. Bacterial resistance is a serious health problem worldwide and the search for new therapeutic agents with broad-spectrum antibacterial activity is needed. One of the effective strategies is to reduce and cap silver ions with therapeutic molecules from the medicinal plants. The plant kingdom is a versatile source of biologically active phytochemicals that could be successfully delivered in combination with nano-silver to combat the emerging
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bacterial resistance. Such a combination therapy would present a synergistic approach from the antimicrobial properties of silver and the bioactive-capped biomolecules. Furthermore, functionalization of biogenic silver nanoparticles with a biocompatible biopolymer
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such as chitosan would further enhance their biological activities. Chitosan is a natural carbohydrate polymer possessing a broad-spectrum antibacterial activity and strong biocompatibility. Chitosan-functionalized nanoparticles change the bacterial and nanoparticles
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interfaces which in turn enhance the biological activity of metal nanoparticles [13]. Chitosan also has the ability to damage bacterial biofilms by penetrating (due to its cationic nature) the
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negatively charged bacterial membrane [14, 15]. Thus, the surface modification of biogenic silver nanoparticles with chitosan molecules would enormously enhance their antimicrobial activity and biocompatibility.
Various routes that include physical, chemical and biological procedures have been used to
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prepare silver nanoparticles. However, physical and chemical procedures generally rely on the use of expensive and hazardous chemicals and are therefore not eco-friendly. The synthesis of metal nanoparticles using eco-friendly and biocompatible reagents could minimize the toxicity of
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the resulting nanomaterials and the environmental impact of the byproducts [16]. Silver nanoparticles with desirable features can be prepared by the green approach. Plants represent a
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renewable source of bioactive molecules that can be effectively used as reducing and capping agents for the synthesis of large-scale metal nanoparticles [6, 17-19]. In the present contribution, an eco-friendly and facile method was developed to synthesize silver nanoparticles. The aqueous rhizome extract of Coptis Chinensis was used to reduce and stabilize silver ions into silver nanoparticles. Furthermore, the prepared biogenic silver nanoparticles were surface functionalized with chitosan biopolymer. The antibacterial activity of these nanoparticles
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was evaluated against E. coli (Gram-negative) and Bacillus subtilus (Gram-positive bacteria). Biogenic silver nanoparticles have been widely reported as antimicrobial agents; however, the antibacterial activity of chitosan functionalized biogenic silver nanoparticles has not been
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explored in detail. 2. Materials and methods 2.1. Preparation of plant extract
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A known amount (10 g) of the dust free plant material was extracted with 100 mL Millipore water. The suspended plant material in water was heated at 60 °C for 30 min and then
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magnetically stirred for another 60 min at room temperature (24 °C). Finally, the resulting water extract was filtered (Whatman filter paper No.1) and the clear supernatant was stored at 4 °C for subsequent uses. 2.2. Synthesis of silver nanoparticles
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Preparation of the biosynthesized silver nanoparticles was carried out according to the method as described in our previous work [6]. Briefly, 50 mL of 3 mM silver nitrate solution was mixed with different concentrations of the plant extract. The reaction mixture was allowed to stir (350
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rpm) at room temperature (24 °C) and the change in color from light yellow to dark brown
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indicated the reduction of silver ions into silver nanoparticles. At the end of the process (6 h), the colloidal suspension of the prepared nanoparticles (AgNPs) was subjected to centrifugation (10,000 rpm, 4 °C) for 15 min and the resulted product was washed three times with distilled water to remove any unbound silver and plant extract. The purified nanoparticles were freeze dried under vacuum and stored at 4 °C for further use. 2.3. Surface modification of silver nanoparticles
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Chitosan, a biopolymer, was used to modify the surface of the prepared silver nanoparticles. For this purpose, we followed a previously reported protocol with slight modification [13]. Briefly, 30 mg of chitosan was completely dissolved in distilled water containing 1 % (v/v) acetic acid.
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After stirring for 10 min, 70 mg of the prepared silver nanoparticles was dissolved (ultrasonic treatment 10 min) in the above chitosan solution. The reaction mixture was kept on a magnetic stirrer (200 rpm) for 14 h (dark). Chitosan molecules were adsorbed on the surface of silver
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nanoparticles during this time period, which was evident from the color change from brownish to blackish. The resulting suspension was then centrifuged at 10,000 rpm for 15 min and the pellet
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obtained was washed with distilled water to remove the unbound chitosan molecules. The chitosan bound silver nanoparticles were dried and stored at room temperature for further study. 2.4. Characterization of silver nanoparticles
The Coptis Chinensis mediated silver nanoparticles (AgNPs) and chitosan modified silver
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nanoparticles (CN-AgNPs) were characterized by various analytical techniques. The formation of biogenic silver nanoparticles and the appearance of localized surface plasmon resonance peak (SPR) were detected by UV-visible spectroscopy (Spectrophotometer Shimadzu, 2450). X-ray
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diffraction (Powder X-ray-D8 advanced diffractometer, BRUKER) and EDX (JEOL-JEM 3010) studies were performed to determine the crystalline structure and elemental composition
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respectively. The size and morphology of the prepared nanoparticles were determined by transmission electron microscopy (FEI- Tecnai G2 20 transmission electron microscope) and dynamic light scattering (HORIBA Zetasizer SZ100) techniques. The surface potential of the prepared nanoparticles and the bacterial strains was determined by zeta potential analysis. FTIR (BRUKER 3000 Hyperion Microscope) spectroscopy was employed to determine the possible biomolecules that are involved in metal reduction and capping.
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2.5. Antibacterial activity The antibacterial activity of the prepared biogenic and chitosan modified silver nanoparticles was evaluated against E. coli (Gram negative) and Bacillus subtilis (Gram-positive bacteria). Agar
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well diffusion method was used to determine the antibacterial activity of the selected extracts. Nutrient agar was prepared, autoclaved and allowed to solidify at room temperature. The autoclaved agar plates were inoculated with bacterial cell suspensions. Wells (8 mm) were made
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in each agar plate using a sterile metallic borer. To each well, 80 µL (1 mg/ml) of the sample was loaded with sterile distilled water and streptomycin as negative and positive control respectively.
inhibition in mm. 2.6. Determination of MIC
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All the plates were incubated at 37 °C for 24 h and the results were expressed as a zone of
The minimum inhibitory concentration (MIC) of the prepared silver nanoparticles was
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determined by a serial 2-fold dilution of test samples. Different concentrations (200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 µg/mL) of the biogenic silver nanoparticles and chitosan modified silver nanoparticles were prepared in autoclaved distilled water. 10 µL of the bacterial strain was
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inoculated to each tube containing different concentrations (1 mL) of the prepared nanoparticles and incubated at 37 °C (250 rpm) for 24 h. The MIC was defined as the minimum concentration
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of the sample inhibiting the visible growth of the microorganism. 2.7. Determination of bacterial cell surface potential The integrity of bacteria cell membrane before and after treatment with different concentrations (MIC and 2 x MIC) of silver nanoparticles was analyzed by measuring the cell surface charge using zeta potential. The bacteria cells were harvested (8,000 rpm, 10 min) from the early stationary phase and resuspended in PBS (0.06 M, pH 7.4). These cells (1 × 105) were then
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treated with the biosynthesized and chitosan modified silver nanoparticles and incubated for 4 h at 37 °C (200 rpm). A sample without treatment was taken as control. The zeta potential values of the treated and untreated samples were measured using a zeta potential analyzer.
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2.8. Determination of cells constituent release
The membrane damage caused by silver nanoparticles was further confirmed by measuring the cell constituent release into the media [20]. The cells treated with different concentrations (MIC
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& 2 x MIC) of silver nanoparticles and chitosan modified silver nanoparticles were incubated at 37 °C for 4 h. After incubation, the cells were harvested by centrifugation (8,000 rpm, 10 min)
absorbance at 260 nm and 280 nm. 2.9. Study of bacteria morphology
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and the supernatant collected was analyzed for the cell constituent release by measuring the
The ultra-structure of the bacterial cells treated with the prepared silver nanoparticles was
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studied by scanning electron microscope. The bacterial cells (E. coli) in phosphate buffer saline (0.06 M, pH 7.2) were exposed to different concentrations of the biosynthesized and chitosan modified silver nanoparticles and incubated at 37 °C for 4 h (200 rpm). The sample without
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treatment was taken as control. After 4 h of incubation, the cells were harvested (8,000 rpm for 5 min) and re-suspended in the same buffer. Afterward, a drop from the bacterial suspension was
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placed on silica glass and then fixed with 2.5% glutaraldehyde for 18 h at 4 °C. Finally, the fixed cells were dried by washing with 30, 50 and 100% ethanol respectively. The as-prepared samples were analyzed by scanning electron microscope (JEOL-J-7800F). 3.10. Cytotoxicity of silver nanoparticles The cytotoxicity assay of the biosynthesized and chitosan modified silver nanoparticles was tested against J-774 cell line by MTT test [21]. The cells (1 x 105) were allowed to fix in a 96
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wells plate for 24 h, and then exposed to different concentrations (10-100 µg/mL) of the biosynthesized and chitosan functionalized silver nanoparticles. The treated samples were incubated at 37 °C (48 h) and then analyzed for cytotoxicity assay. 100 µL of the MTT solution
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in BPS (5 mg/mL) was added to each well and kept for 4 h. After 4 h of incubation, the formed purple formazan crystals were dissolved by the addition of 100 µL of DMSO (each well) and the absorbance was measured at 590 nm (Eliza MAT 2000, DRG Instruments, GmbH).
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3. Results and discussion 3.1. UV-visible spectroscopy
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The appearance of a localized surface plasmon resonance peak (LSPR) indicates the formation of metal nanoparticles. UV-visible spectroscopy was used to detect the characteristic LSPR pattern for silver nanoparticles. Fig. 1 presents the time-dependent evolution of UV-vis spectra of silver nanoparticles synthesized from the aqueous rhizome extract of Coptis Chinensis using 3 mM
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AgNO3 as a silver precursor. When 10 mL of the plant extract was mixed with 50 mL silver nitrate solution (3 mM), the appearance of a characteristic LSPR band around 428 nm indicates the formation of silver nanoparticles (Fig. 1A) [6]. The intensity of the SPR peaks was gradually
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increased with time which did not show any blue-red shift, revealing an increase in the density and a uniform size particles distribution of the biosynthesized silver nanoparticles [22].
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However, when the chitosan molecules were adsorbed on the surface of silver nanoparticles, the LSPR peak slightly shifted towards longer wavelength (Fig. 1 B), suggesting a possible increase in particles size [13]. The observed red shift in the plasmon position by 7 nm confirms the adsorption of chitosan molecules on the surface of biogenic silver nanoparticles [13], which was further confirmed by FTIR, TEM and dynamic light scattering technique. The Coptis Chinensismediated silver nanoparticles are negatively charged (zeta value = -31.5 mV) whereas
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the chitosan molecules carry positive charge under mild acidic condition (due to partial protonation of the amino groups). It is believed that an electrostatic interaction will occur between the oppositely charged moieties, and the chitosan biopolymer will be adsorbed onto the
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surface of negatively charged silver nanoparticles [23]. The LSPR pattern of metal nanoparticles is sensitive to particles size, shapes and dielectric value of the reaction media [24, 25]. Generally, the SPR peaks of silver nanoparticles red-shift towards longer wavelength with the increase in
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particles size and vice versa [26]. We also investigated the effect of varying amount (mL) of plant extract on the size of silver nanoparticles. Our results indicated that a red shift in the SPR
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pattern was observed at very low (5 mL) and high amount (20 mL) of the plant extract used, which is an indicative of the formation of large size particles (results not shown). An increase in particles size at high concentration of plant extract (beyond optimum level, 10 mL) may be attributed to an additional interaction among the surface adhered biomolecules and secondary
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reduction processes [27]. Similarly, a very low concentration (5 mL) of the plant extract may not provide the necessary capping agents to stabilize the particles and prevent any possible aggregation. With aggregation, the particles are getting larger in size and results in the red shift
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of the localized surface plasmon bands for the resulting nanoparticles [28, 29].
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Fig. 1. Time-dependent evolution of UV-visible spectra of (A) biogenic silver nanoparticles and (B) chitosan modified silver nanoparticles
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3.2. XRD and EDX studies X-ray diffraction (XRD) and Energy dispersive X-ray (EDX) techniques are used to determine the crystalline nature and elemental composition of the prepared nanoparticles. XRD spectra of
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the prepared silver nanoparticles indicated four diffraction bands at 2 θ = 38°, 44°, 64° and 77°, which are the characteristic Bragg's diffraction plans (111), (200), (220) and (310) of the face-
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centered cubic crystalline structure of silver (reference file JCPDS no. 04-0783). Our result is in line with those reported previously [30, 31]. EDX spectra of the prepared silver nanoparticles displayed a major band around 3 keV, indicating the presence of elemental silver as a major component of the silver nanoparticles [30]. In addition, some weak signals from C and O were also detected at 0.3 and 0.5 keV respectively. These signals may be originated from the corban
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supported copper grid used for sample loading and the capping biomolecules.
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3.3. Particles size and morphology
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Fig. 2. XRD (A) and EDX (B) pattern of biogenic silver nanoparticles
Transmission electron microscopy and dynamic light scattering (DLS) techniques were used to determine the particles size, morphology, and dispersion. Fig. 3 shows the TEM micrographs of
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biogenic and chitosan functionalized silver nanoparticles. It was observed that the prepared silver nanoparticles are almost spherical in shapes with an average particle size of 15 nm. The nanoparticles are well separated from each other, suggesting a significant degree of stabilization
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by the phytochemicals in the aqueous extract of Coptis Chinensis (Fig. 3 A). Similarly, Fig. 3 B represents the TEM images of the chitosan functionalized silver nanoparticles. It can be seen that
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the chitosan functionalized silver nanoparticles are also spherical in shapes with an average particle size of 15-25 nm. Furthermore, these nanoparticles are well dispersed with no sign of aggregation. The particles size and distribution was also determined by DLS measurements. DLS measurements indicated that the particles size of biogenic silver nanoparticles was in the range of 10-20 nm, however, maximum proportion of the particles was in the size range of 15 nm. Similarly, the particles distribution of chitosan functionalized silver nanoparticles was
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comparatively wider (15-30) than that calculated from TEM measurements (20 nm). The difference in particles size as determined by two techniques may be attributed to the fact that these two techniques operate by different principles and detection methods. In addition, DLS
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technique measures the hydrodynamic diameter of the particles which is strongly augmented by water molecules. and the resulting particles size is generally wider than that obtained from TEM
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analysis.
Fig. 3. TEM micrograph of (A) biogenic silver nanoparticles and (B) chitosan functionalized
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silver nanoparticles, insights show the particles size distribution 3.4. Zeta potential of the prepared nanoparticles
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The zeta potential is considered as an important parameter to determine the stability and dispersion of metal nanoparticles in a solution. The zeta potential of metal nanoparticles indicates the overall charge that a sample retains in a solution. Biogenic silver nanoparticles and chitosan functionalized silver nanoparticles displayed a zeta potential value of -30 and 21 mV respectively. The negative value of zeta potential indicates that silver nanoparticles are capped with negatively charged biomolecules. The electrostatic repulsive interaction between the
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negatively charged silver nanoparticles may prevent the possible aggregation of the prepared silver nanoparticles, which might be responsible for their long-term stability 21, 42 21. On the other hand, a positive zeta value (21 mV) for chitosan functionalized silver nanoparticles reveals the
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adsorption of this biopolymer onto these nanoparticles. The repulsive interaction among the positively charged particles will prevent any possible aggregation among the metal nanoparticles,
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thus providing long-term stability.
Fig. 4. Dynamic light scattering study. Zeta values of (A) biogenic silver nanoparticles and (B)
3.4. FTIR
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chitosan functionalized silver nanoparticles, (Z.V= zeta value)
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FTIR study was performed to analyze the possible functionalities that are involved in metal reduction to metal nanoparticles. IR spectra of Coptis Chinensis rhizome extract and biogenic silver nanoparticles is given in Fig. 5 A, B. Fig. 5 shows the FTIR spectra of phytosynthesized silver nanoparticles and chitosan functionalized silver nanoparticles. The aqueous extract of Coptis Chinensis revealed three major bands at wave number 3390, 1618 and 1077 cm-1 that exhibited some degree shift in the corresponding silver nanoparticles. These peaks may be attributed to -OH stretching vibration of phenolic compounds, carbonyl group stretching
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vibration and ester bond in polyphenolic compounds respectively [32-34]. IR spectra of chitosan displayed prominent peaks at wave number 3433, 1640 and 1080 cm-1 that also revealed some degree shift in the chitosan functionalized silver nanoparticles, indicating the possible
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involvement of these moieties in the synthesis of conjugated nanoparticles. The observed peaks at 3433, 1640, and 1080 cm-1 may be originated from the -OH stretching vibration, -NH vibration, and C=O asymmetric stretch in chitosan molecule [34, 35]. Comparative FTIR spectra
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(Fig. 5 B) of biogenic silver nanoparticles and chitosan functionalized biogenic silver nanoparticles indicate that the peaks intensities at 1640 and 1080 cm -1 are more prominent in the
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case of chitosan functionalized biogenic silver nanoparticles. The observed increase in the peak intensities may be originated from an additional interaction of chitosan in the conjugated silver nanoparticles. Furthermore, the IR peak at 1640 cm -1 is more broader and larger in the chitosan functionalized nanoparticles as compared to pure chitosan, indicating a possible interactions
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between the oxygen atoms on the surface of biogenic silver nanoparticles and the NH2 group of chitosan (hydrogen bond formation). In addition, the absorption band of -OH group (̴ 3400 cm -1) in chitosan and chitosan functionalized silver nanoparticles did not show any shift in position,
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thus leaving the partial positive charge on the hydrogen atom (in -δO‒Hδ+ bond) in chitosan on the surface of nanoparticles [34]. FTIR spectra thus clearly indicate that biogenic silver
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nanoparticles are stabilized by polyphenolic moieties in the plant extract while, in case of chitosan functionalized silver nanoparticles; both the chitosan and some functionalities from the plant extract have capped the conjugated nanoparticles.
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Fig. 5. FTIR spectra of Coptis Chinensis rhizome extract, chitosan, biogenic and chitosan
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functionalized silver nanoparticles 3.5. Antibacterial activity
The antibacterial activity of the biosynthesized and chitosan functionalized silver nanoparticles was tested against E. coli and Bacillus subtilis and the results are expressed as a zone of inhibition (Fig. 6). It was observed that biogenic silver nanoparticles exhibited significant activity against Bacillus subtilis with a zone of inhibition 18 ± 1.6 mm (Fig. 6). However, these nanoparticles were comparatively less active against the Gram-negative E. coli with a zone of
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inhibition 12 ± 1.2 mm. The low toxicity of biogenic silver nanoparticles against the E. coli may be attributed to the difference in the cell wall composition between the Gram positive and Gram negative bacteria. The cell wall of Gram-negative E. coli is covered with an outer lipid
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membrane (lipopolysaccharide) which is more negatively charged than B. subtilis. As is evident from the zeta value, the biogenic silver nanoparticles are also negatively charged and the electrostatic repulsion between the nanoparticles and E. Colli hinders particles attachment and
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penetration into the cells. On the other hand, chitosan functionalized silver nanoparticles were significantly active against both the Gram-negative E. coli (22 ± 1.3 mm) and gram-positive
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Bacillus subtilus (20 ± 1.6 mm), indicating that modulation of bacteria-nanoparticles interfaces strongly influence the antibacterial activity. The chitosan functionalized biogenic silver nanoparticles have positive surface potential mainly due to the free hydroxyl groups of chitosan, which interact with the water molecules via hydrogen bondings. The positively charged chitosan
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functionalized nanoparticle would have a better surface for bacterial attachment than negatively charged silver nanoparticles. The positively charged chitosan binds electrostatically with negatively charged lipopolysaccharide (LPS) of Gram-negative cell surface, and also with the
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negatively charged teichoic acid, present in Gram-positive cells, and such interaction may create stress, leading to enhancement of cell permeability, cytoplasmic leakage and cell death [36, 37].
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Extraction of lipotechoic acid from the cytoplasmic membrane of B. subtilis by chitosan would have a drastic effect on the membrane integrity and subsequent leakage of protein and other cellular machinery [38]. Thus, the negatively charged teichoic acid might behave as a molecular link for positive chitosan on the cell surface, letting it to destroy membrane function. However, the enhanced bactericidal efficacy against the gram negative bacteria may be attributed to its higher negative potential and cell wall composition as compared to gram positive bacteria. A
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previous study has also demonstrated that chitosan modified iron oxide nanoparticles exert a promising antibacterial action against both the Gram positive B. subtilis and Gram negative E. coli as compared to their negatively charged counterparts [13]. Their results revealed that surface
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modification of nanoparticles with chitosan molecule improved the nanoparticles adhesion and penetration into the cell.
The effectiveness of these nanoparticles was further confirmed by determining the minimum
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inhibitory concentrations against the model bacteria used. The biosynthesized silver nanoparticles displayed an MIC value of 25 and 12.5 µg/ml against the Gram-negative E. coli
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and Gram-positive Bacillus subtilis respectively. On the other hand, chitosan functionalized biogenic silver nanoparticles had an MIC value of 6.25 µg/mL against E. coli and 12.5 µg/mL against Bacillus subtilis (Table 1). Our findings indicate that chitosan functionalized silver nanoparticles are more effective in arresting bacterial growth, supporting the idea that
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modulation of nanoparticles-bacteria interface has a strong influence on the antibacterial activity of metal nanoparticles [13]. Furthermore, the chitosan functionalized biogenic silver nanoparticles were more effective in inhibiting bacterial growth for several days. When the
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bacterial grown agar plates were incubated for 5 days, bacterial growth was re-appeared on the preformed zone of inhibition of standard drug and biogenic silver nanoparticles (Fig. 6 C).
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However, the chitosan functionalized silver nanoparticles were highly reactive against the tested bacteria as is obvious from the clear zone of inhibition. This result indicates that chitosan modified biogenic silver nanoparticles could be an effective nanomaterial to treat the emerging microbial resistance. Silver nanoparticles inhibit microbial growth by different mechanisms such as interfering with microbial DNA replication, contact killing, and generation of reactive oxygen
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species [8, 39, 40]. Metal nanoparticles generate a significant level of reactive oxygen species
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which are majorly responsible for the destruction of microbial cells.
Fig. 6. Antibacterial activity of biogenic and chitosan functionalized silver nanoparticles against
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(A) E. coli and (B) B. subtilis, 1, 2 presents the antibacterial activities of biogenic and chitosan functionalized silver nanoparticles (C), shows the antibacterial activity for 5 day. Streptomycin
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was used as a standard drug (S)
Table 1. Antibacterial activities of biogenic and chitosan functionalized silver nanoparticles against the tested bacteria Bacteria
E. coli
Zone of inhibition (mm)
MIC (µg/ml)
Zone of inhibition (mm)
MIC (µg/ml)
(AgNPs)
(AgNPs)
(CN-AgNPs)
(CN-AgNPs)
12 ± 0.8
25.0 ± 2.6
22 ± 1.6
6.25 ± 0.6
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B. subtilis
18 ± 1.4
12.50 ± 0.2
20 ± 1.6
12.50 ± 0.8
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3.6. Determination of bacterial membrane potential The zeta potential values of the tested bacteria (E. coli and Bacillus subtilus) were determined in order to monitor the effect of the biosynthesized and chitosan modified silver nanoparticles on
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the membrane surface potential. The bacterial cell surface potential plays an important role in the maintenance of cell growth and other metabolic activities. It also provides a valuable information
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about the membrane integrity and other surface characteristics. The change in membrane potential alters its permeability, leakage of cellular constituents and subsequent cell death. The untreated E. coli and Bacillus subitlus had zeta potential values of -37.6 and -29.0 mV respectively (PBS, 0.06 M pH 7.4). The strong negative surface potential of E. coli is due to an outer lipid membrane in Gram-negative bacteria. When E. coli and Bacillus subtilus in PBS were
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treated with biogenic silver nanoparticles, the negative cell surface potential was decreased to 20 and -13.5 mV for E. coli and Bacillus subtilus, indicating a significant degree of membrane
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alteration by silver nanoparticles. The zeta potential values of E. coli and Bacillus were further decreased when these cells were treated with chitosan functionalized biogenic silver
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nanoparticles. A zeta potential value of -11.6 and -7.5 mV was observed for E. coli and Bacillus subtilus upon treatment with chitosan functionalized silver nanoparticles respectively. The enhanced effect of chitosan modified silver nanoparticles on the alteration of membrane potential may be related to a favorable electrostatic interaction of the positively charged particles with the bacterial cell surface. Our results suggest that alteration in the surface potential values after treatment with biogenic and chitosan functionalized silver nanoparticles could be attributed to bacterial membrane damage followed by the release of cellular constituents. Previous studies
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also report a similar alteration in the membrane potential upon treatment with metal and metal oxide nanoparticles [41, 42]. The findings of those studies demonstrated that chitosan modified metal oxide nanoparticles had relatively stronger interaction with bacterial cell membrane and
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higher toxicity than unmodified (negatively charged) metal nanoparticles.
The bacterial cell morphology and membrane damage after exposure to silver nanoparticles was further studied by scanning electron microscopy (SEM). An intense damage to the bacterial cell
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was observed in samples treated with biogenic silver nanoparticles and chitosan modified silver nanoparticles (Fig. 6). The bacterial cells were hollow, deformed and squeezed after exposure to
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these nanoparticles for 4 h, indicating a severe membrane damage followed by cell constituents release into the media.
3.7. Determination of cell constituents release
The membrane damage caused by the biosynthesized silver nanoparticles and chitosan modified
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silver nanoparticles was further confirmed by the determination of cell constituent release into the media. Membrane damage results in the loss of cellular components, most importantly proteins and nucleic acids, which can be determined by measuring the absorbance at 280 and 260
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nm [43] [20]. An increase in the absorbance at 280 and 260 nm was observed with the increasing concentrations of the prepared silver nanoparticles (Table 1, 2). These results suggest that silver
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nanoparticles destroy bacterial membrane by a dose dependent manner followed by the release of periplasmic proteins and nucleic acids into the media [43, 44]. The cell constituent release was further increased when the bacterial cells were treated with chitosan functionalized biogenic silver nanoparticles. The improved activity of chitosan functionalized silver nanoparticles could be attributed to a more favorable interaction with bacterial cell surface as compared to biogenic silver nanoparticles.
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Table. 2. Cell constituent release from the tested microbes upon treatment with biogenic and chitosan functionalized silver nanoparticles at OD280 nm
Bacillus subtilis
OD280
Conc. CN- AgNPs
0.0
0.06
0.0
MIC (25.0 µg/mL)
0.22
MIC (6.25 µg/mL)
0.36
2 × MIC
0.32
2 × MIC
0.44
0.0
0.08
0.0
0.06
MIC (12.5 µg/mL)
0.27
2 × MIC
0.36
OD 280
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E. coli
Conc. AgNPs
0.06
MIC (12.5 µg/mL)
0.30
2 × MIC
0.38
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Microorganisms
Table 3. Cell constituent release from the tested microbes upon treatment with biogenic and chitosan functionalized silver nanoparticles at OD260 nm OD260
Conc. CN- AgNPs
OD 260
0.006
0.0
0.006
MIC (25.0 µg/mL)
0.15
MIC (6.25 µg/mL)
0.24
2 × MIC
0.24
2 × MIC
0.36
0.0
0.008
0.0
0.008
MIC (12.5 µg/mL)
0.18
MIC (3.125 µg/mL)
0.20
2 × MIC
0.28
2 × MIC
0.32
Conc. AgNPs
E. coli
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Bacillus subtilis
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Microorganisms
3.8. Determination intracellular of reactive oxygen species The cytotoxic effect of silver nanoparticles is majorly attributed to the generation of intracellular reactive oxygen species (ROS) in the bacterial cell [45, 46]. The interaction of silver nanoparticles with microbial cells often generates various reactive oxygen species such as superoxide ions (O2‒°), hydrogen peroxide (H2O2) and hydroxyl ions (OH°), which are
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responsible for the induction of oxidative stress and damages to biomolecules such as protein and DNA. The generation of ROS in bacteria was investigated by 2,7-dichlorofluorescin-diacetate (H2-DCFDA) assay (Fig. 7). It has been reported that 2,7-dichlorofluorescin-diacetate is
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oxidized into dichlorofluoroscein in the presence of reactive species which emit green fluorescence upon excitation at 488 nm [41]. An increase in the intracellular fluorescence intensity was observed with the increasing concentrations of the prepared silver nanoparticles.
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The relative fluorescence intensity was high in the samples exposed to chitosan modified silver nanoparticles. These findings suggest that the intracellular ROS production by silver
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nanoparticles is dose and surface charge dependent, which induce oxidative stress followed by damage to bacterial cell membrane leading to cell death. Previous study reports that the production of ROS by metal ions may be correlated to an inhibitory action of these metals on the thiol-containing enzymes of respiratory system [47]. It may be concluded that a bacterial cell on
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contact with silver nanoparticles may internalize silver ions, which in turn inhibit respiratory enzymes, thus facilitate the generation of reactive oxygen species leading to cell damage. The active oxygen species interact and destroy different cellular components such as DNA, cell
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membrane and other vital enzymes leading to cell death [5, 48]. Nanoscale materials in the size range of 10-80 nm can penetrate into the cell, producing various reactive oxygen species, which
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in turn disrupt bacterial cell membrane [49]. Our findings suggest that the prepared silver nanoparticles interact with the bacterial cell surface and arresting its growth by membrane damage, induction of intracellular reactive oxygen species and leakage of cytoplasmic materials.
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Fig. 7. Fluorescence microscopic and scanning electron microscopic studies of control and
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treated samples of E. coli, (A-C) presents the ROS production in control, biogenic silver nanoparticles, and chitosan modified silver nanoparticles treated E. coli respectively, (D-E) shows the SEM images of control group (A), treated with biogenic silver nanoparticles and (E)
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chitosan modified silver nanoparticles respectively.
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3.9. Cytotoxicity of the prepared silver nanoparticles The biocompatibility of the prepared silver nanoparticles was tested against J-774 cell line. Dose dependent cytotoxicity result indicated that both the prepared silver nanoparticles displayed an excellent biocompatibility in the dose range of 5 to 40 µg/mL after 24 h incubation with the selected cell line. These nanoparticles exhibited moderate cytotoxicity (75% cells survival) above 50 µg/mL, which is higher than the minimum inhibitory concentration of these
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nanoparticles against the tested bacterial strains. Hence, our results show that the biosynthesized silver nanoparticles could be a safe and effective class of antimicrobial agents. Conclusion
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The findings of the present study conclude that well dispersed and stable silver nanoparticles can be prepared by a green and facile approach. The phytochemicals in the aqueous rhizome extract of Coptis Chinensis were used as a source of reducing and stabilizing agents. The biosynthesized
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silver nanoparticles were spherical in shapes (TEM) with an approximate particle size of 15 nm (DLS). The surface potential of these nanoparticles was modified with chitosan biopolymer and
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the modified silver nanoparticles displayed an excellent biological activity. The biosynthesized silver nanoparticles were highly potent in arresting bacterial growth in a dose-dependent manner. The bactericidal potency of these nanoparticles was significantly enhanced by coating with chitosan molecules. The production of a high level of intracellular reactive oxygen species and
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the release of cytoplasmic materials indicated that interaction of silver nanoparticles with bacterial cell surface caused membrane damaged followed by cell death. The measurement of cell surface potential and ultrastructural analysis (SEM) of E. coli cells further supported the
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findings that bacterial membrane damage by silver nanoparticles (contact killing) and intracellular ROS production could be the major players involved in the cellular death.
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Furthermore, the favorable biocompatibility of these nanoparticles could be a safe and effective class of antimicrobial agents for the treatment of various bacterial infections. Acknowledgment
The authors are thankful to the International Science &Technology Cooperation Program of China (Grant No.2013DFR90290) and China Scholarship Council (CSC No. 2014DFH974) for supporting the current project.
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Highlights Biological synthesis of silver nanoparticles
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Surface modification of AgNPs with a biocompatible biopolymer Modulation of bacteria-nanoparticles interface for enhanced activity
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Detail mechanism of antibacterial action
S0882-4010(16)30746-X
DOI:
10.1016/j.micpath.2016.11.030
Reference:
YMPAT 2021
To appear in:
Microbial Pathogenesis
Received Date: 6 November 2016 Revised Date:
27 November 2016
Accepted Date: 29 November 2016
Please cite this article as: Ahmad A, Wei Y, Syed F, Tahir K, Rehman AU, Khan A, Ullah S, Yuan Q, The effects of bacteria-nanoparticles interface on the antibacterial activity of green synthesized silver nanoparticles, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2016.11.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The effects of bacteria-nanoparticles interface on the antibacterial activity of green synthesized silver nanoparticles Aftab Ahmad a, Yun Wei a, Fatima Syed b, Kamran Tahir a, Aziz Ur Rehman a, Arifullah Khan a
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical
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a
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Sadeeq Ullah a and Qipeng Yuan a*
Technology, No. 15 East Road of North Third Ring, Chao Yang District, Beijing 100029, China Institute of Chemical Sciences, University of Peshawar (25120) Peshawar-Pakistan
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b
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To whom correspondence should be addressed
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Email: [email protected]
[email protected]
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Abstract Neutralization of bacterial cell surface potential using nanoscale materials is an effective strategy to alter membrane permeability, cytoplasmic leakage, and ultimate cell death. In the present
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study, an attempt was made to prepare biogenic silver nanoparticles using biomolecules from the aqueous rhizome extract of Coptis Chinensis. The biosynthesized silver nanoparticles were surface modified with chitosan biopolymer. The prepared silver nanoparticles and chitosan
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modified silver nanoparticles were cubic crystalline structures (XRD) with an average particle size of 15 and 20 nm respectively (TEM, DLS). The biosynthesized silver nanoparticles were
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surface stabilized by polyphenolic compounds (FTIR). Coptis Chinensis mediated silver nanoparticles displayed significant activity against E. coli and Bacillus subtilus with a zone of inhibition 12 ± 1.2 (MIC = 25 µg/mL) and 18 ± 1.6 mm (MIC = 12.50 µg/mL) respectively. The bactericidal efficacy of these nanoparticles was considerably increased upon surface
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modification with chitosan biopolymer. The chitosan modified biogenic silver nanoparticles exhibited promising activity against E. coli (MIC = 6.25 µg/mL) and Bacillus subtilus (MIC = 12.50 µg/mL). Our results indicated that the chitosan modified silver nanoparticles were
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promising agents in damaging bacterial membrane potential and induction of high level of intracellular reactive oxygen species (ROS). In addition, these nanoparticles were observed to
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induce the release of the high level of cytoplasmic materials especially protein and nucleic acids into the media. All these findings suggest that the chitosan functionalized silver nanoparticles are efficient agents in disrupting bacterial membrane and induction of ROS leading to cytoplasmic leakage and cell death. These findings further conclude that the bacterial-nanoparticles surface potential modulation is an effective strategy in enhancing the antibacterial potency of silver nanoparticles.
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Keyword: Coptis Chinensis, silver nanoparticles, Bacteria, membrane potential 1. Introduction The chemists and technologists around the globe follow the basic principles of green chemistry
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to develop less hazardous synthesis procedures. Among the modern line of technological advancements, nano-biotechnology occupies an important position to create and enhance the utility of nano-size materials in advanced biotechnology [1]. Nanoscale materials and devices
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have unique physiochemical characteristics that make them applicable in fields ranging from electronic to medicine [2, 3]. Among the metal nanoparticles, nano silver is widely used in a
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wide array of consumer products and clinical care, as silver ions possess strong toxicity against a broad spectrum of pathogenic microbes [4-7]. Silver nanoparticles arrest bacterial growth by disrupting its cell membrane, DNA damage, inhibiting vital metabolic enzymes and production of reactive oxygen species (ROS) that destroy cellular constituents [4, 5, 8, 9]. Microbial cells
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are sensitive to reactive oxygen species and any therapy that generates enough reactive oxygen species that could bypass the antioxidant defense mechanism of microbial cell will be a promising strategy to treat infection and the emerging microbial resistance [10]. Metal
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nanoparticles are considered as promising agents producing significant level of ROS that can overcome the microbial antioxidant defense system leading to cell damage [11] [12].
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Because of its multi-sites action, nano-silver could be a promising candidate to overcome the emerging microbial resistance. Bacterial resistance is a serious health problem worldwide and the search for new therapeutic agents with broad-spectrum antibacterial activity is needed. One of the effective strategies is to reduce and cap silver ions with therapeutic molecules from the medicinal plants. The plant kingdom is a versatile source of biologically active phytochemicals that could be successfully delivered in combination with nano-silver to combat the emerging
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bacterial resistance. Such a combination therapy would present a synergistic approach from the antimicrobial properties of silver and the bioactive-capped biomolecules. Furthermore, functionalization of biogenic silver nanoparticles with a biocompatible biopolymer
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such as chitosan would further enhance their biological activities. Chitosan is a natural carbohydrate polymer possessing a broad-spectrum antibacterial activity and strong biocompatibility. Chitosan-functionalized nanoparticles change the bacterial and nanoparticles
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interfaces which in turn enhance the biological activity of metal nanoparticles [13]. Chitosan also has the ability to damage bacterial biofilms by penetrating (due to its cationic nature) the
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negatively charged bacterial membrane [14, 15]. Thus, the surface modification of biogenic silver nanoparticles with chitosan molecules would enormously enhance their antimicrobial activity and biocompatibility.
Various routes that include physical, chemical and biological procedures have been used to
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prepare silver nanoparticles. However, physical and chemical procedures generally rely on the use of expensive and hazardous chemicals and are therefore not eco-friendly. The synthesis of metal nanoparticles using eco-friendly and biocompatible reagents could minimize the toxicity of
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the resulting nanomaterials and the environmental impact of the byproducts [16]. Silver nanoparticles with desirable features can be prepared by the green approach. Plants represent a
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renewable source of bioactive molecules that can be effectively used as reducing and capping agents for the synthesis of large-scale metal nanoparticles [6, 17-19]. In the present contribution, an eco-friendly and facile method was developed to synthesize silver nanoparticles. The aqueous rhizome extract of Coptis Chinensis was used to reduce and stabilize silver ions into silver nanoparticles. Furthermore, the prepared biogenic silver nanoparticles were surface functionalized with chitosan biopolymer. The antibacterial activity of these nanoparticles
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was evaluated against E. coli (Gram-negative) and Bacillus subtilus (Gram-positive bacteria). Biogenic silver nanoparticles have been widely reported as antimicrobial agents; however, the antibacterial activity of chitosan functionalized biogenic silver nanoparticles has not been
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explored in detail. 2. Materials and methods 2.1. Preparation of plant extract
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A known amount (10 g) of the dust free plant material was extracted with 100 mL Millipore water. The suspended plant material in water was heated at 60 °C for 30 min and then
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magnetically stirred for another 60 min at room temperature (24 °C). Finally, the resulting water extract was filtered (Whatman filter paper No.1) and the clear supernatant was stored at 4 °C for subsequent uses. 2.2. Synthesis of silver nanoparticles
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Preparation of the biosynthesized silver nanoparticles was carried out according to the method as described in our previous work [6]. Briefly, 50 mL of 3 mM silver nitrate solution was mixed with different concentrations of the plant extract. The reaction mixture was allowed to stir (350
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rpm) at room temperature (24 °C) and the change in color from light yellow to dark brown
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indicated the reduction of silver ions into silver nanoparticles. At the end of the process (6 h), the colloidal suspension of the prepared nanoparticles (AgNPs) was subjected to centrifugation (10,000 rpm, 4 °C) for 15 min and the resulted product was washed three times with distilled water to remove any unbound silver and plant extract. The purified nanoparticles were freeze dried under vacuum and stored at 4 °C for further use. 2.3. Surface modification of silver nanoparticles
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Chitosan, a biopolymer, was used to modify the surface of the prepared silver nanoparticles. For this purpose, we followed a previously reported protocol with slight modification [13]. Briefly, 30 mg of chitosan was completely dissolved in distilled water containing 1 % (v/v) acetic acid.
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After stirring for 10 min, 70 mg of the prepared silver nanoparticles was dissolved (ultrasonic treatment 10 min) in the above chitosan solution. The reaction mixture was kept on a magnetic stirrer (200 rpm) for 14 h (dark). Chitosan molecules were adsorbed on the surface of silver
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nanoparticles during this time period, which was evident from the color change from brownish to blackish. The resulting suspension was then centrifuged at 10,000 rpm for 15 min and the pellet
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obtained was washed with distilled water to remove the unbound chitosan molecules. The chitosan bound silver nanoparticles were dried and stored at room temperature for further study. 2.4. Characterization of silver nanoparticles
The Coptis Chinensis mediated silver nanoparticles (AgNPs) and chitosan modified silver
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nanoparticles (CN-AgNPs) were characterized by various analytical techniques. The formation of biogenic silver nanoparticles and the appearance of localized surface plasmon resonance peak (SPR) were detected by UV-visible spectroscopy (Spectrophotometer Shimadzu, 2450). X-ray
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diffraction (Powder X-ray-D8 advanced diffractometer, BRUKER) and EDX (JEOL-JEM 3010) studies were performed to determine the crystalline structure and elemental composition
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respectively. The size and morphology of the prepared nanoparticles were determined by transmission electron microscopy (FEI- Tecnai G2 20 transmission electron microscope) and dynamic light scattering (HORIBA Zetasizer SZ100) techniques. The surface potential of the prepared nanoparticles and the bacterial strains was determined by zeta potential analysis. FTIR (BRUKER 3000 Hyperion Microscope) spectroscopy was employed to determine the possible biomolecules that are involved in metal reduction and capping.
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2.5. Antibacterial activity The antibacterial activity of the prepared biogenic and chitosan modified silver nanoparticles was evaluated against E. coli (Gram negative) and Bacillus subtilis (Gram-positive bacteria). Agar
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well diffusion method was used to determine the antibacterial activity of the selected extracts. Nutrient agar was prepared, autoclaved and allowed to solidify at room temperature. The autoclaved agar plates were inoculated with bacterial cell suspensions. Wells (8 mm) were made
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in each agar plate using a sterile metallic borer. To each well, 80 µL (1 mg/ml) of the sample was loaded with sterile distilled water and streptomycin as negative and positive control respectively.
inhibition in mm. 2.6. Determination of MIC
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All the plates were incubated at 37 °C for 24 h and the results were expressed as a zone of
The minimum inhibitory concentration (MIC) of the prepared silver nanoparticles was
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determined by a serial 2-fold dilution of test samples. Different concentrations (200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 µg/mL) of the biogenic silver nanoparticles and chitosan modified silver nanoparticles were prepared in autoclaved distilled water. 10 µL of the bacterial strain was
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inoculated to each tube containing different concentrations (1 mL) of the prepared nanoparticles and incubated at 37 °C (250 rpm) for 24 h. The MIC was defined as the minimum concentration
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of the sample inhibiting the visible growth of the microorganism. 2.7. Determination of bacterial cell surface potential The integrity of bacteria cell membrane before and after treatment with different concentrations (MIC and 2 x MIC) of silver nanoparticles was analyzed by measuring the cell surface charge using zeta potential. The bacteria cells were harvested (8,000 rpm, 10 min) from the early stationary phase and resuspended in PBS (0.06 M, pH 7.4). These cells (1 × 105) were then
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treated with the biosynthesized and chitosan modified silver nanoparticles and incubated for 4 h at 37 °C (200 rpm). A sample without treatment was taken as control. The zeta potential values of the treated and untreated samples were measured using a zeta potential analyzer.
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2.8. Determination of cells constituent release
The membrane damage caused by silver nanoparticles was further confirmed by measuring the cell constituent release into the media [20]. The cells treated with different concentrations (MIC
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& 2 x MIC) of silver nanoparticles and chitosan modified silver nanoparticles were incubated at 37 °C for 4 h. After incubation, the cells were harvested by centrifugation (8,000 rpm, 10 min)
absorbance at 260 nm and 280 nm. 2.9. Study of bacteria morphology
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and the supernatant collected was analyzed for the cell constituent release by measuring the
The ultra-structure of the bacterial cells treated with the prepared silver nanoparticles was
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studied by scanning electron microscope. The bacterial cells (E. coli) in phosphate buffer saline (0.06 M, pH 7.2) were exposed to different concentrations of the biosynthesized and chitosan modified silver nanoparticles and incubated at 37 °C for 4 h (200 rpm). The sample without
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treatment was taken as control. After 4 h of incubation, the cells were harvested (8,000 rpm for 5 min) and re-suspended in the same buffer. Afterward, a drop from the bacterial suspension was
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placed on silica glass and then fixed with 2.5% glutaraldehyde for 18 h at 4 °C. Finally, the fixed cells were dried by washing with 30, 50 and 100% ethanol respectively. The as-prepared samples were analyzed by scanning electron microscope (JEOL-J-7800F). 3.10. Cytotoxicity of silver nanoparticles The cytotoxicity assay of the biosynthesized and chitosan modified silver nanoparticles was tested against J-774 cell line by MTT test [21]. The cells (1 x 105) were allowed to fix in a 96
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wells plate for 24 h, and then exposed to different concentrations (10-100 µg/mL) of the biosynthesized and chitosan functionalized silver nanoparticles. The treated samples were incubated at 37 °C (48 h) and then analyzed for cytotoxicity assay. 100 µL of the MTT solution
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in BPS (5 mg/mL) was added to each well and kept for 4 h. After 4 h of incubation, the formed purple formazan crystals were dissolved by the addition of 100 µL of DMSO (each well) and the absorbance was measured at 590 nm (Eliza MAT 2000, DRG Instruments, GmbH).
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3. Results and discussion 3.1. UV-visible spectroscopy
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The appearance of a localized surface plasmon resonance peak (LSPR) indicates the formation of metal nanoparticles. UV-visible spectroscopy was used to detect the characteristic LSPR pattern for silver nanoparticles. Fig. 1 presents the time-dependent evolution of UV-vis spectra of silver nanoparticles synthesized from the aqueous rhizome extract of Coptis Chinensis using 3 mM
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AgNO3 as a silver precursor. When 10 mL of the plant extract was mixed with 50 mL silver nitrate solution (3 mM), the appearance of a characteristic LSPR band around 428 nm indicates the formation of silver nanoparticles (Fig. 1A) [6]. The intensity of the SPR peaks was gradually
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increased with time which did not show any blue-red shift, revealing an increase in the density and a uniform size particles distribution of the biosynthesized silver nanoparticles [22].
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However, when the chitosan molecules were adsorbed on the surface of silver nanoparticles, the LSPR peak slightly shifted towards longer wavelength (Fig. 1 B), suggesting a possible increase in particles size [13]. The observed red shift in the plasmon position by 7 nm confirms the adsorption of chitosan molecules on the surface of biogenic silver nanoparticles [13], which was further confirmed by FTIR, TEM and dynamic light scattering technique. The Coptis Chinensismediated silver nanoparticles are negatively charged (zeta value = -31.5 mV) whereas
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the chitosan molecules carry positive charge under mild acidic condition (due to partial protonation of the amino groups). It is believed that an electrostatic interaction will occur between the oppositely charged moieties, and the chitosan biopolymer will be adsorbed onto the
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surface of negatively charged silver nanoparticles [23]. The LSPR pattern of metal nanoparticles is sensitive to particles size, shapes and dielectric value of the reaction media [24, 25]. Generally, the SPR peaks of silver nanoparticles red-shift towards longer wavelength with the increase in
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particles size and vice versa [26]. We also investigated the effect of varying amount (mL) of plant extract on the size of silver nanoparticles. Our results indicated that a red shift in the SPR
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pattern was observed at very low (5 mL) and high amount (20 mL) of the plant extract used, which is an indicative of the formation of large size particles (results not shown). An increase in particles size at high concentration of plant extract (beyond optimum level, 10 mL) may be attributed to an additional interaction among the surface adhered biomolecules and secondary
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reduction processes [27]. Similarly, a very low concentration (5 mL) of the plant extract may not provide the necessary capping agents to stabilize the particles and prevent any possible aggregation. With aggregation, the particles are getting larger in size and results in the red shift
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of the localized surface plasmon bands for the resulting nanoparticles [28, 29].
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Fig. 1. Time-dependent evolution of UV-visible spectra of (A) biogenic silver nanoparticles and (B) chitosan modified silver nanoparticles
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3.2. XRD and EDX studies X-ray diffraction (XRD) and Energy dispersive X-ray (EDX) techniques are used to determine the crystalline nature and elemental composition of the prepared nanoparticles. XRD spectra of
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the prepared silver nanoparticles indicated four diffraction bands at 2 θ = 38°, 44°, 64° and 77°, which are the characteristic Bragg's diffraction plans (111), (200), (220) and (310) of the face-
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centered cubic crystalline structure of silver (reference file JCPDS no. 04-0783). Our result is in line with those reported previously [30, 31]. EDX spectra of the prepared silver nanoparticles displayed a major band around 3 keV, indicating the presence of elemental silver as a major component of the silver nanoparticles [30]. In addition, some weak signals from C and O were also detected at 0.3 and 0.5 keV respectively. These signals may be originated from the corban
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supported copper grid used for sample loading and the capping biomolecules.
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3.3. Particles size and morphology
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Fig. 2. XRD (A) and EDX (B) pattern of biogenic silver nanoparticles
Transmission electron microscopy and dynamic light scattering (DLS) techniques were used to determine the particles size, morphology, and dispersion. Fig. 3 shows the TEM micrographs of
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biogenic and chitosan functionalized silver nanoparticles. It was observed that the prepared silver nanoparticles are almost spherical in shapes with an average particle size of 15 nm. The nanoparticles are well separated from each other, suggesting a significant degree of stabilization
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by the phytochemicals in the aqueous extract of Coptis Chinensis (Fig. 3 A). Similarly, Fig. 3 B represents the TEM images of the chitosan functionalized silver nanoparticles. It can be seen that
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the chitosan functionalized silver nanoparticles are also spherical in shapes with an average particle size of 15-25 nm. Furthermore, these nanoparticles are well dispersed with no sign of aggregation. The particles size and distribution was also determined by DLS measurements. DLS measurements indicated that the particles size of biogenic silver nanoparticles was in the range of 10-20 nm, however, maximum proportion of the particles was in the size range of 15 nm. Similarly, the particles distribution of chitosan functionalized silver nanoparticles was
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comparatively wider (15-30) than that calculated from TEM measurements (20 nm). The difference in particles size as determined by two techniques may be attributed to the fact that these two techniques operate by different principles and detection methods. In addition, DLS
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technique measures the hydrodynamic diameter of the particles which is strongly augmented by water molecules. and the resulting particles size is generally wider than that obtained from TEM
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analysis.
Fig. 3. TEM micrograph of (A) biogenic silver nanoparticles and (B) chitosan functionalized
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silver nanoparticles, insights show the particles size distribution 3.4. Zeta potential of the prepared nanoparticles
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The zeta potential is considered as an important parameter to determine the stability and dispersion of metal nanoparticles in a solution. The zeta potential of metal nanoparticles indicates the overall charge that a sample retains in a solution. Biogenic silver nanoparticles and chitosan functionalized silver nanoparticles displayed a zeta potential value of -30 and 21 mV respectively. The negative value of zeta potential indicates that silver nanoparticles are capped with negatively charged biomolecules. The electrostatic repulsive interaction between the
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negatively charged silver nanoparticles may prevent the possible aggregation of the prepared silver nanoparticles, which might be responsible for their long-term stability 21, 42 21. On the other hand, a positive zeta value (21 mV) for chitosan functionalized silver nanoparticles reveals the
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adsorption of this biopolymer onto these nanoparticles. The repulsive interaction among the positively charged particles will prevent any possible aggregation among the metal nanoparticles,
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thus providing long-term stability.
Fig. 4. Dynamic light scattering study. Zeta values of (A) biogenic silver nanoparticles and (B)
3.4. FTIR
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chitosan functionalized silver nanoparticles, (Z.V= zeta value)
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FTIR study was performed to analyze the possible functionalities that are involved in metal reduction to metal nanoparticles. IR spectra of Coptis Chinensis rhizome extract and biogenic silver nanoparticles is given in Fig. 5 A, B. Fig. 5 shows the FTIR spectra of phytosynthesized silver nanoparticles and chitosan functionalized silver nanoparticles. The aqueous extract of Coptis Chinensis revealed three major bands at wave number 3390, 1618 and 1077 cm-1 that exhibited some degree shift in the corresponding silver nanoparticles. These peaks may be attributed to -OH stretching vibration of phenolic compounds, carbonyl group stretching
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vibration and ester bond in polyphenolic compounds respectively [32-34]. IR spectra of chitosan displayed prominent peaks at wave number 3433, 1640 and 1080 cm-1 that also revealed some degree shift in the chitosan functionalized silver nanoparticles, indicating the possible
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involvement of these moieties in the synthesis of conjugated nanoparticles. The observed peaks at 3433, 1640, and 1080 cm-1 may be originated from the -OH stretching vibration, -NH vibration, and C=O asymmetric stretch in chitosan molecule [34, 35]. Comparative FTIR spectra
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(Fig. 5 B) of biogenic silver nanoparticles and chitosan functionalized biogenic silver nanoparticles indicate that the peaks intensities at 1640 and 1080 cm -1 are more prominent in the
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case of chitosan functionalized biogenic silver nanoparticles. The observed increase in the peak intensities may be originated from an additional interaction of chitosan in the conjugated silver nanoparticles. Furthermore, the IR peak at 1640 cm -1 is more broader and larger in the chitosan functionalized nanoparticles as compared to pure chitosan, indicating a possible interactions
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between the oxygen atoms on the surface of biogenic silver nanoparticles and the NH2 group of chitosan (hydrogen bond formation). In addition, the absorption band of -OH group (̴ 3400 cm -1) in chitosan and chitosan functionalized silver nanoparticles did not show any shift in position,
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thus leaving the partial positive charge on the hydrogen atom (in -δO‒Hδ+ bond) in chitosan on the surface of nanoparticles [34]. FTIR spectra thus clearly indicate that biogenic silver
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nanoparticles are stabilized by polyphenolic moieties in the plant extract while, in case of chitosan functionalized silver nanoparticles; both the chitosan and some functionalities from the plant extract have capped the conjugated nanoparticles.
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Fig. 5. FTIR spectra of Coptis Chinensis rhizome extract, chitosan, biogenic and chitosan
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functionalized silver nanoparticles 3.5. Antibacterial activity
The antibacterial activity of the biosynthesized and chitosan functionalized silver nanoparticles was tested against E. coli and Bacillus subtilis and the results are expressed as a zone of inhibition (Fig. 6). It was observed that biogenic silver nanoparticles exhibited significant activity against Bacillus subtilis with a zone of inhibition 18 ± 1.6 mm (Fig. 6). However, these nanoparticles were comparatively less active against the Gram-negative E. coli with a zone of
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inhibition 12 ± 1.2 mm. The low toxicity of biogenic silver nanoparticles against the E. coli may be attributed to the difference in the cell wall composition between the Gram positive and Gram negative bacteria. The cell wall of Gram-negative E. coli is covered with an outer lipid
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membrane (lipopolysaccharide) which is more negatively charged than B. subtilis. As is evident from the zeta value, the biogenic silver nanoparticles are also negatively charged and the electrostatic repulsion between the nanoparticles and E. Colli hinders particles attachment and
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penetration into the cells. On the other hand, chitosan functionalized silver nanoparticles were significantly active against both the Gram-negative E. coli (22 ± 1.3 mm) and gram-positive
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Bacillus subtilus (20 ± 1.6 mm), indicating that modulation of bacteria-nanoparticles interfaces strongly influence the antibacterial activity. The chitosan functionalized biogenic silver nanoparticles have positive surface potential mainly due to the free hydroxyl groups of chitosan, which interact with the water molecules via hydrogen bondings. The positively charged chitosan
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functionalized nanoparticle would have a better surface for bacterial attachment than negatively charged silver nanoparticles. The positively charged chitosan binds electrostatically with negatively charged lipopolysaccharide (LPS) of Gram-negative cell surface, and also with the
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negatively charged teichoic acid, present in Gram-positive cells, and such interaction may create stress, leading to enhancement of cell permeability, cytoplasmic leakage and cell death [36, 37].
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Extraction of lipotechoic acid from the cytoplasmic membrane of B. subtilis by chitosan would have a drastic effect on the membrane integrity and subsequent leakage of protein and other cellular machinery [38]. Thus, the negatively charged teichoic acid might behave as a molecular link for positive chitosan on the cell surface, letting it to destroy membrane function. However, the enhanced bactericidal efficacy against the gram negative bacteria may be attributed to its higher negative potential and cell wall composition as compared to gram positive bacteria. A
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previous study has also demonstrated that chitosan modified iron oxide nanoparticles exert a promising antibacterial action against both the Gram positive B. subtilis and Gram negative E. coli as compared to their negatively charged counterparts [13]. Their results revealed that surface
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modification of nanoparticles with chitosan molecule improved the nanoparticles adhesion and penetration into the cell.
The effectiveness of these nanoparticles was further confirmed by determining the minimum
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inhibitory concentrations against the model bacteria used. The biosynthesized silver nanoparticles displayed an MIC value of 25 and 12.5 µg/ml against the Gram-negative E. coli
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and Gram-positive Bacillus subtilis respectively. On the other hand, chitosan functionalized biogenic silver nanoparticles had an MIC value of 6.25 µg/mL against E. coli and 12.5 µg/mL against Bacillus subtilis (Table 1). Our findings indicate that chitosan functionalized silver nanoparticles are more effective in arresting bacterial growth, supporting the idea that
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modulation of nanoparticles-bacteria interface has a strong influence on the antibacterial activity of metal nanoparticles [13]. Furthermore, the chitosan functionalized biogenic silver nanoparticles were more effective in inhibiting bacterial growth for several days. When the
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bacterial grown agar plates were incubated for 5 days, bacterial growth was re-appeared on the preformed zone of inhibition of standard drug and biogenic silver nanoparticles (Fig. 6 C).
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However, the chitosan functionalized silver nanoparticles were highly reactive against the tested bacteria as is obvious from the clear zone of inhibition. This result indicates that chitosan modified biogenic silver nanoparticles could be an effective nanomaterial to treat the emerging microbial resistance. Silver nanoparticles inhibit microbial growth by different mechanisms such as interfering with microbial DNA replication, contact killing, and generation of reactive oxygen
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species [8, 39, 40]. Metal nanoparticles generate a significant level of reactive oxygen species
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which are majorly responsible for the destruction of microbial cells.
Fig. 6. Antibacterial activity of biogenic and chitosan functionalized silver nanoparticles against
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(A) E. coli and (B) B. subtilis, 1, 2 presents the antibacterial activities of biogenic and chitosan functionalized silver nanoparticles (C), shows the antibacterial activity for 5 day. Streptomycin
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was used as a standard drug (S)
Table 1. Antibacterial activities of biogenic and chitosan functionalized silver nanoparticles against the tested bacteria Bacteria
E. coli
Zone of inhibition (mm)
MIC (µg/ml)
Zone of inhibition (mm)
MIC (µg/ml)
(AgNPs)
(AgNPs)
(CN-AgNPs)
(CN-AgNPs)
12 ± 0.8
25.0 ± 2.6
22 ± 1.6
6.25 ± 0.6
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B. subtilis
18 ± 1.4
12.50 ± 0.2
20 ± 1.6
12.50 ± 0.8
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3.6. Determination of bacterial membrane potential The zeta potential values of the tested bacteria (E. coli and Bacillus subtilus) were determined in order to monitor the effect of the biosynthesized and chitosan modified silver nanoparticles on
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the membrane surface potential. The bacterial cell surface potential plays an important role in the maintenance of cell growth and other metabolic activities. It also provides a valuable information
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about the membrane integrity and other surface characteristics. The change in membrane potential alters its permeability, leakage of cellular constituents and subsequent cell death. The untreated E. coli and Bacillus subitlus had zeta potential values of -37.6 and -29.0 mV respectively (PBS, 0.06 M pH 7.4). The strong negative surface potential of E. coli is due to an outer lipid membrane in Gram-negative bacteria. When E. coli and Bacillus subtilus in PBS were
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treated with biogenic silver nanoparticles, the negative cell surface potential was decreased to 20 and -13.5 mV for E. coli and Bacillus subtilus, indicating a significant degree of membrane
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alteration by silver nanoparticles. The zeta potential values of E. coli and Bacillus were further decreased when these cells were treated with chitosan functionalized biogenic silver
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nanoparticles. A zeta potential value of -11.6 and -7.5 mV was observed for E. coli and Bacillus subtilus upon treatment with chitosan functionalized silver nanoparticles respectively. The enhanced effect of chitosan modified silver nanoparticles on the alteration of membrane potential may be related to a favorable electrostatic interaction of the positively charged particles with the bacterial cell surface. Our results suggest that alteration in the surface potential values after treatment with biogenic and chitosan functionalized silver nanoparticles could be attributed to bacterial membrane damage followed by the release of cellular constituents. Previous studies
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also report a similar alteration in the membrane potential upon treatment with metal and metal oxide nanoparticles [41, 42]. The findings of those studies demonstrated that chitosan modified metal oxide nanoparticles had relatively stronger interaction with bacterial cell membrane and
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higher toxicity than unmodified (negatively charged) metal nanoparticles.
The bacterial cell morphology and membrane damage after exposure to silver nanoparticles was further studied by scanning electron microscopy (SEM). An intense damage to the bacterial cell
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was observed in samples treated with biogenic silver nanoparticles and chitosan modified silver nanoparticles (Fig. 6). The bacterial cells were hollow, deformed and squeezed after exposure to
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these nanoparticles for 4 h, indicating a severe membrane damage followed by cell constituents release into the media.
3.7. Determination of cell constituents release
The membrane damage caused by the biosynthesized silver nanoparticles and chitosan modified
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silver nanoparticles was further confirmed by the determination of cell constituent release into the media. Membrane damage results in the loss of cellular components, most importantly proteins and nucleic acids, which can be determined by measuring the absorbance at 280 and 260
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nm [43] [20]. An increase in the absorbance at 280 and 260 nm was observed with the increasing concentrations of the prepared silver nanoparticles (Table 1, 2). These results suggest that silver
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nanoparticles destroy bacterial membrane by a dose dependent manner followed by the release of periplasmic proteins and nucleic acids into the media [43, 44]. The cell constituent release was further increased when the bacterial cells were treated with chitosan functionalized biogenic silver nanoparticles. The improved activity of chitosan functionalized silver nanoparticles could be attributed to a more favorable interaction with bacterial cell surface as compared to biogenic silver nanoparticles.
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Table. 2. Cell constituent release from the tested microbes upon treatment with biogenic and chitosan functionalized silver nanoparticles at OD280 nm
Bacillus subtilis
OD280
Conc. CN- AgNPs
0.0
0.06
0.0
MIC (25.0 µg/mL)
0.22
MIC (6.25 µg/mL)
0.36
2 × MIC
0.32
2 × MIC
0.44
0.0
0.08
0.0
0.06
MIC (12.5 µg/mL)
0.27
2 × MIC
0.36
OD 280
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E. coli
Conc. AgNPs
0.06
MIC (12.5 µg/mL)
0.30
2 × MIC
0.38
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Microorganisms
Table 3. Cell constituent release from the tested microbes upon treatment with biogenic and chitosan functionalized silver nanoparticles at OD260 nm OD260
Conc. CN- AgNPs
OD 260
0.006
0.0
0.006
MIC (25.0 µg/mL)
0.15
MIC (6.25 µg/mL)
0.24
2 × MIC
0.24
2 × MIC
0.36
0.0
0.008
0.0
0.008
MIC (12.5 µg/mL)
0.18
MIC (3.125 µg/mL)
0.20
2 × MIC
0.28
2 × MIC
0.32
Conc. AgNPs
E. coli
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Bacillus subtilis
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0.0
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Microorganisms
3.8. Determination intracellular of reactive oxygen species The cytotoxic effect of silver nanoparticles is majorly attributed to the generation of intracellular reactive oxygen species (ROS) in the bacterial cell [45, 46]. The interaction of silver nanoparticles with microbial cells often generates various reactive oxygen species such as superoxide ions (O2‒°), hydrogen peroxide (H2O2) and hydroxyl ions (OH°), which are
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responsible for the induction of oxidative stress and damages to biomolecules such as protein and DNA. The generation of ROS in bacteria was investigated by 2,7-dichlorofluorescin-diacetate (H2-DCFDA) assay (Fig. 7). It has been reported that 2,7-dichlorofluorescin-diacetate is
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oxidized into dichlorofluoroscein in the presence of reactive species which emit green fluorescence upon excitation at 488 nm [41]. An increase in the intracellular fluorescence intensity was observed with the increasing concentrations of the prepared silver nanoparticles.
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The relative fluorescence intensity was high in the samples exposed to chitosan modified silver nanoparticles. These findings suggest that the intracellular ROS production by silver
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nanoparticles is dose and surface charge dependent, which induce oxidative stress followed by damage to bacterial cell membrane leading to cell death. Previous study reports that the production of ROS by metal ions may be correlated to an inhibitory action of these metals on the thiol-containing enzymes of respiratory system [47]. It may be concluded that a bacterial cell on
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contact with silver nanoparticles may internalize silver ions, which in turn inhibit respiratory enzymes, thus facilitate the generation of reactive oxygen species leading to cell damage. The active oxygen species interact and destroy different cellular components such as DNA, cell
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membrane and other vital enzymes leading to cell death [5, 48]. Nanoscale materials in the size range of 10-80 nm can penetrate into the cell, producing various reactive oxygen species, which
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in turn disrupt bacterial cell membrane [49]. Our findings suggest that the prepared silver nanoparticles interact with the bacterial cell surface and arresting its growth by membrane damage, induction of intracellular reactive oxygen species and leakage of cytoplasmic materials.
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Fig. 7. Fluorescence microscopic and scanning electron microscopic studies of control and
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treated samples of E. coli, (A-C) presents the ROS production in control, biogenic silver nanoparticles, and chitosan modified silver nanoparticles treated E. coli respectively, (D-E) shows the SEM images of control group (A), treated with biogenic silver nanoparticles and (E)
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chitosan modified silver nanoparticles respectively.
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3.9. Cytotoxicity of the prepared silver nanoparticles The biocompatibility of the prepared silver nanoparticles was tested against J-774 cell line. Dose dependent cytotoxicity result indicated that both the prepared silver nanoparticles displayed an excellent biocompatibility in the dose range of 5 to 40 µg/mL after 24 h incubation with the selected cell line. These nanoparticles exhibited moderate cytotoxicity (75% cells survival) above 50 µg/mL, which is higher than the minimum inhibitory concentration of these
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nanoparticles against the tested bacterial strains. Hence, our results show that the biosynthesized silver nanoparticles could be a safe and effective class of antimicrobial agents. Conclusion
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The findings of the present study conclude that well dispersed and stable silver nanoparticles can be prepared by a green and facile approach. The phytochemicals in the aqueous rhizome extract of Coptis Chinensis were used as a source of reducing and stabilizing agents. The biosynthesized
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silver nanoparticles were spherical in shapes (TEM) with an approximate particle size of 15 nm (DLS). The surface potential of these nanoparticles was modified with chitosan biopolymer and
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the modified silver nanoparticles displayed an excellent biological activity. The biosynthesized silver nanoparticles were highly potent in arresting bacterial growth in a dose-dependent manner. The bactericidal potency of these nanoparticles was significantly enhanced by coating with chitosan molecules. The production of a high level of intracellular reactive oxygen species and
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the release of cytoplasmic materials indicated that interaction of silver nanoparticles with bacterial cell surface caused membrane damaged followed by cell death. The measurement of cell surface potential and ultrastructural analysis (SEM) of E. coli cells further supported the
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findings that bacterial membrane damage by silver nanoparticles (contact killing) and intracellular ROS production could be the major players involved in the cellular death.
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Furthermore, the favorable biocompatibility of these nanoparticles could be a safe and effective class of antimicrobial agents for the treatment of various bacterial infections. Acknowledgment
The authors are thankful to the International Science &Technology Cooperation Program of China (Grant No.2013DFR90290) and China Scholarship Council (CSC No. 2014DFH974) for supporting the current project.
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Highlights Biological synthesis of silver nanoparticles
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Surface modification of AgNPs with a biocompatible biopolymer Modulation of bacteria-nanoparticles interface for enhanced activity
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Detail mechanism of antibacterial action