Polyhexamethylene biguanide functionalized cationic silver nanoparticles for enhanced antimicrobial activity
© Ashraf et al.; licensee Springer. 2012
Received: 21 March 2012
Accepted: 23 April 2012
Published: 24 May 2012
Polyhexamethylene biguanide (PHMB), a broad spectrum disinfectant against many pathogens, was used as a stabilizing ligand for the synthesis of fairly uniform silver nanoparticles. The particles formed were characterized using UV-visible spectroscopy, FTIR, dynamic light scattering, electrophoretic mobility, and TEM to measure their morphology and surface chemistry. PHMB-functionalized silver nanoparticles were then evaluated for their antimicrobial activity against a gram-negative bacterial strain, Escherichia coli. These silver nanoparticles were found to have about 100 times higher bacteriostatic and bactericidal activities, compared to the previous reports, due to the combined antibacterial effect of silver nanoparticles and PHMB. In addition to other applications, PHMB-functionalized silver nanoparticles would be extremely useful in textile industry due to the strong interaction of PHMB with cellulose fabrics.
Antimicrobial activity of silver nanoparticles (Ag NPs) is well documented, and they are currently being used in a variety of commercial antimicrobial products including textile products and paints etc. [1–6]. Although the mechanism of their antimicrobial activity is not completely understood yet, silver, whether in the form of ionic or metallic state, is known to destabilize and increase the permeability of bacterial membranes . It results in the collapse of the plasma membrane potential, disruption of ion transport processes, and the depletion of the levels of intracellular ATP [8–10]. Silver also inactivates essential respiratory enzymes and proteins responsible for RNA and DNA replication in bacteria [7, 11, 12]. Ag NPs can penetrate inside the microbial cells, damaging sulfur and phosphorus containing compounds such as DNA and proteins [9, 13–15]. Although Ag NPs form complexes with various amino acids to inhibit protein's function, their toxicity to the mammalian cells is very limited, thus, warranting their antimicrobial applications in medical and healthcare products [16–20].
PHMB (20% w/v) was purchased from Arch Chemicals (Norwalk, CT, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), except tryptone, agar, and yeast extract which were purchased from MP Biomedicals (Santa Ana, CA, USA), and were used as received without further purification. Pure strain of E. coli was provided by Invitrogen (Carlsbad, CA, USA). Luria Bertani (LB) medium, used for the growth study of E. coli, was prepared using 1% tryptone, 0.5% yeast extract, and 1% sodium chloride in water, and pH was adjusted to 7 using 0.5 M solution of sodium hydroxide and hydrochloric acid. Ultrapure water with a resistivity of 18.2 MΩ cm was used as the solvent in all preparations.
Synthesis of Ag NPs
In a typical experiment to prepare PHMB functionalized Ag NPs, a given volume of PHMB aqueous solution, starting from 0.5 to 200 μg/mL (ideally, 15 mL, 20 μg/mL), was added to an aqueous solution of AgNO3 (25 mL, 1 mM) under vigorous stirring. The reaction mixture was stirred vigorously for 2 h to let PHMB form a complex with silver. Freshly prepared aqueous solution of sodium borohydride (5 mL, 0.8 mg/mL) was then added quickly to PHMB-Ag solution, and the reaction was allowed to complete for 5 h. The yellow/brownish suspension of silver nanoparticles was then filter purified to remove excess PHMB and other impurities using centrifuge filters (Amicon® Ultra centrifugal filters by Millipore Corporation, Billerica, MA, USA ) having a molecular weight cutoff value of 30 KDa and then stored at room temperature for further analysis and use in subsequent experiments.
Antimicrobial activity of PHMB-functionalized Ag NPs
In order to study the effect of PHMB-stabilized Ag NPs on bacterial growth, E. coli strain was grown on an agar plate, and its fresh colony was transferred with the help of a sterilized loop into a shake flask which was then incubated in an incubator shaker for 12 h at 37°C at a shaking speed of 150 rpm under aerobic conditions. The growth of bacteria was measured by taking optical density (OD) of samples at 600 nm. When the OD of growth culture approached 1 at 600 nm, the bacterial culture was then transferred into new shake flasks at a density of 2.5% bacteria into each flask. Different concentrations of clean PHMB-coated Ag NPs were then added in triplicate starting from 0.075 to 0.15 μg/mL of Ag NPs (different tested concentrations of Ag NPs were 0.075, 0.09, 0.1, 0.12, 0.135, and 0.15 μg/mL) into shake flasks just before inoculating with E. coli. Same concentrations of Ag NPs were also added into the LB media for the purpose of control experiment to serve as a blank while measuring their OD at 600 nm. All these shake flasks containing E. coli strains and Ag NPs, along with controls, were placed in an incubator shaker for 24 h at 37°C at a shaking speed of 150 rpm. The growth of bacteria was monitored by measuring OD of samples at regular intervals at 600 nm. The bacterial growth curves were then plotted against time based on the data obtained by carefully monitoring the bacterial growth. Based on these growth curves, the effect of silver nanoparticles on specific growth rate and doubling time of E. coli was observed.
Transmission electron microscopy of Ag NPs was carried out using a high-resolution transmission electron microscope (JEOL, JEM-3010, JEOL Ltd., Akishima, Tokyo, Japan) operating at 300 kV. Nanoparticle specimens, for inspection by transmission electron microscopy, were prepared by slow evaporation of one drop of a dilute aqueous solution of the particles on a carbon-coated copper mesh grid. ImageJ software was used to calculate the particle size distribution from transmission electron micrographs. The particle size distribution and surface potential of nanoparticles were determined using Zetasizer Nano ZS (Malvern Instruments, Westborough, MA, USA). The surface chemistry of Ag NPs was determined using Bruker Alpha-P FTIR (Bruker Optik Gmbh, Ettlingen, Germany) with a diamond ATR attachment. UV-visible spectrum of Ag NPs suspension was recorded using an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies Inc., Santa Clara, CA, USA). The approximate concentration of Ag NPs was determined using fast sequential atomic absorption spectrometer (AA240FS) by Varian Inc., Palo Alto, CA, USA.
Results and discussion
Antimicrobial activity of Ag NPs is well known and is believed to be dependent on the size [21, 22], shape [5, 23, 24], surface bound ligands [25, 26], and their subsequent surface charge . Recently, it was reported that the surface charge of Ag NPs is the most important factor in this regard while studying the effect of neutral, positively, and negatively charged Ag NPs on the growth of gram-positive bacteria belonging to bacillus species . Negatively charged Ag NPs were found to be the most effective to retard the growth of these bacteria. In this study, we have prepared Ag NPs which are positively charged and, in addition, are functionalized with a known antimicrobial agent, PHMB, to enhance the antimicrobial properties of Ag NPs towards gram-negative bacteria.
After confirming the presence of PHMB on the surface of clean Ag NPs, we set out to determine their antimicrobial activity. Ag NPs are well known for their antimicrobial activity, and their bacteriostatic and bactericidal concentrations are usually found in the range of 5 and 50 μg/mL, respectively [5, 7, 25, 45–50]. In order to improve the antimicrobial activity and broaden its spectrum, we conjugated Ag NPs with an antimicrobial agent, PHMB. PHMB, if used alone, shows bactericidal activity beyond 10 μg/mL . The bacteriostatic and bactericidal activities of PHMB-functionalized Ag NPs were, however, improved to be 0.075 and 0.150 μg/mL, respectively.
Effect of concentrations of silver nanoparticles
Concentration of Ag NPs in E. coli culture (μg/mL)
Specific growth rateμ (h-1)
0.92 ± 0.03
0.75 ± 0.02
0.25 ± 0.01
2.8 ± 0.01
0.20 ± 0.01
3.4 ± 0.01
0.197 ± 0.01
3.52 ± 0.01
0.18 ± 0.01
4.31 ± 0.01
0.15 ± 0.01
4.6 ± 0.01
0.11 ± 0.01
6.3 ± 0.02
PHMB alone (3 μg/mL)
0.788 ± 0.03
0.879 ± 0.02
To summarize, a cationic biocide (PHMB) was used for the synthesis of fairly uniform silver nanoparticles. The cationic silver particles thus formed showed much higher antibacterial activity against E. coli than the previous reports. PHMB is routinely used in textile industry due to its antimicrobial activity and affinity to the cellulose fabrics. Therefore, these PHMB-coated silver nanoparticles with enhanced antimicrobial activity may have very useful applications in textile industry.
We gratefully acknowledge Higher Education Commission (HEC), Government of Pakistan, for the financial support to Sumaira Ashraf during her PhD studies. We are also thankful to ex-National Commission on Nanoscience and Technology (NCNST) and Ministry of Science and Technology (MoST), Government of Pakistan for the financial support to initiate nano-biotechnology research at NIBGE. IH thanks LUMS School of Science & Engineering (SSE), Lahore, Pakistan for start-up funds to initiate nanomaterial research at LUMS. We are also thankful to Professor Wolfgang Parak for his support to characterize nanoparticle samples.
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