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.
KeywordsCationic silver nanoparticles Polyhexamethylene biguanide Antimicrobial activity
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.
- Dubas ST, Wacharanad S, Potiyaraj P: Tunning of the antimicrobial activity of surgical sutures coated with silver nanoparticles. Colloids Surf A 2011, 380: 25–28. 10.1016/j.colsurfa.2011.01.037View ArticleGoogle Scholar
- Dubas ST, Pimpan V: Green synthesis of silver nanoparticles for ammonia sensing. Talanta 2008, 76: 29–33. 10.1016/j.talanta.2008.01.062View ArticleGoogle Scholar
- Chen J, Luo Y, Liang Y, Jiang J, Shen G, Yu R: Surface-enhanced Raman scattering for immunoassay based on the biocatalytic production of silver nanoparticles. Anal Sci 2009, 25: 347–352. 10.2116/analsci.25.347View ArticleGoogle Scholar
- Kim S, Kim HJ: Anti-bacterial performance of colloidal silver-treated laminate wood flooring. Int Biodeterior Biodegrad 2006, 57: 155–162. 10.1016/j.ibiod.2006.02.002View ArticleGoogle Scholar
- Morones J, Elechiguerra J, Camacho A, Holt K, Kouri J, Ram J, Yacaman M: The bactericidal effect of silver nanoparticles. Nanotechnology 2005, 16: 2346–2353. 10.1088/0957-4484/16/10/059View ArticleGoogle Scholar
- Kumar A, Vemula PK, Ajayan PM, John G: Silver-nanoparticle-embedded antimicrobial paints based on vegetable oil. Nat Mater 2008, 7: 236–241. 10.1038/nmat2099View ArticleGoogle Scholar
- Sondi I, Salopek-Sondi B: Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for gram-negative bacteria. J Colloid Interface Sci 2004, 275: 177–182. 10.1016/j.jcis.2004.02.012View ArticleGoogle Scholar
- Dibrov P, Dzioba J, Gosink KK, Häse CC: Chemiosmotic mechanism of antimicrobial activity of Ag+ in Vibrio cholerae . Antimicrob Agents Chemother 2002, 46: 2668–2670. 10.1128/AAC.46.8.2668-2670.2002View ArticleGoogle Scholar
- Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO: A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus . J Biomed Mater Res 2000, 52: 662–668. 10.1002/1097-4636(20001215)52:4<662::AID-JBM10>3.0.CO;2-3View ArticleGoogle Scholar
- Lok CN, Ho CM, Chen R, He QY, Yu WY, Sun H, Tam PKH, Chiu JF, Che CM: Silver nanoparticles: partial oxidation and antibacterial activities. J Biol Inorg Chem 2007, 12: 527–534. 10.1007/s00775-007-0208-zView ArticleGoogle Scholar
- Lansdown ABG: Silver in health care: antimicrobial effects and safety in use. Curr Probl Dermatol 2006, 33: 17–34.View ArticleGoogle Scholar
- Lok CN, Ho CM, Chen R, He QY, Yu WY, Sun H, Tam PKH, Chiu JF, Che CM: Proteomic analysis of the mode of antibacterial action of silver nanoparticles. J Proteome Res 2006, 5: 916–924. 10.1021/pr0504079View ArticleGoogle Scholar
- Yamanaka M, Hara K, Kudo J: Bactericidal actions of a silver ion solution on Escherichia coli , studied by energy-filtering transmission electron microscopy and proteomic analysis. Appl Environ Microbiol 2005, 71: 7589–7593. 10.1128/AEM.71.11.7589-7593.2005View ArticleGoogle Scholar
- Bragg P, Rainnie D: The effect of silver ions on the respiratory chain of Escherichia coli . Can J Microbiol 1974, 20: 883–889. 10.1139/m74-135View ArticleGoogle Scholar
- McDonnell G, Russell A: Antiseptics and disinfectants: activity, action and resistance. Clin Microbiol Rev 1999, 12: 147.Google Scholar
- Furno F, Morley KS, Wong B, Sharp BL, Arnold PL, Howdle SM, Bayston R, Brown PD, Winship PD, Reid HJ: Silver nanoparticles and polymeric medical devices: a new approach to prevention of infection? J Antimicrob Chemother 2004, 54: 1019–1024. 10.1093/jac/dkh478View ArticleGoogle Scholar
- Ohashi S, Saku S, Yamamoto K: Antibacterial activity of silver inorganic agent YDA filler. J Oral Rehabil 2004, 31: 364–367. 10.1111/j.1365-2842.2004.01200.xView ArticleGoogle Scholar
- Maneerung T, Tokura S, Rujiravanit R: Impregnation of silver nanoparticles into bacterial cellulose for antimicrobial wound dressing. Carbohydr Poly 2008, 72: 43–51. 10.1016/j.carbpol.2007.07.025View ArticleGoogle Scholar
- Durán N, Marcato PD, Alves OL, De Souza GIH, Esposito E: Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains. J Nanobiotechnology 2005, 3: 8–14. 10.1186/1477-3155-3-8View ArticleGoogle Scholar
- Son WK, Youk JH, Lee TS, Park WH: Preparation of antimicrobial ultrafine cellulose acetate fibers with silver nanoparticles. Macromol Rapid Commun 2004, 25: 1632–1637. 10.1002/marc.200400323View ArticleGoogle Scholar
- Wigginton NS, Titta A, Piccapietra F, Dobias J, Nesatyy VJ, Suter MJF, Bernier-Latmani R: Binding of silver nanoparticles to bacterial proteins depends on surface modifications and inhibits enzymatic activity. Environ Sci Technol 2010, 44: 2163–2168. 10.1021/es903187sView ArticleGoogle Scholar
- Dal Lago V, de Oliveira LF, de Almeida Gonçalves K, Kobarg J, Cardoso MB: Size-selective silver nanoparticles: future of biomedical devices with enhanced bactericidal properties. J Mater Chem 2011, 21: 12267–12273. 10.1039/c1jm12297eView ArticleGoogle Scholar
- Elechiguerra JL, Burt JL, Morones JR, Camacho-Bragado A, Gao X, Lara HH, Yacaman MJ: Interaction of silver nanoparticles with HIV-1. J Nanobiotechnology 2005, 3: 1–10. 10.1186/1477-3155-3-1View ArticleGoogle Scholar
- Pal S, Tak YK, Song JM: Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli . Appl Environ Microbiol 2007, 73: 1712–1720. 10.1128/AEM.02218-06View ArticleGoogle Scholar
- Zhang Y, Peng H, Huang W, Zhou Y, Yan D: Facile preparation and characterization of highly antimicrobial colloid Ag or Au nanoparticles. J Colloid Interface Sci 2008, 325: 371–376. 10.1016/j.jcis.2008.05.063View ArticleGoogle Scholar
- Schäfer B, Tentschert J, Luch A: Nanosilver in consumer products and human health: more information required! Environ Sci Technol 2011, 45: 7589–7590. 10.1021/es200804uView ArticleGoogle Scholar
- Eby DM, Schaeublin NM, Farrington KE, Hussain SM, Johnson GR: Lysozyme catalyzes the formation of antimicrobial silver nanoparticles. ACS Nano 2009, 3: 984–994. 10.1021/nn900079eView ArticleGoogle Scholar
- Rowhani T, Lagalante AF: A colorimetric assay for the determination of polyhexamethylene biguanide in pool and spa water using nickel-nioxime. Talanta 2007, 71: 964–970. 10.1016/j.talanta.2006.07.042View ArticleGoogle Scholar
- Hiti K, Walochnik J, Haller-Schober E, Faschinger C, Aspöck H: Viability of Acanthamoeba after exposure to a multipurpose disinfecting contact lens solution and two hydrogen peroxide systems. Brit J Ophthalmol 2002, 86: 144–146. 10.1136/bjo.86.2.144View ArticleGoogle Scholar
- Rosin M, Welk A, Bernhardt O, Ruhnau M, Pitten FA, Kocher T, Kramer A: Effect of a polyhexamethylene biguanide mouthrinse on bacterial counts and plaque. J Clin Periodontol 2001, 28: 1121–1126. 10.1034/j.1600-051X.2001.281206.xView ArticleGoogle Scholar
- Rosin M, Welk A, Kocher T, Majic-Todt A, Kramer A, Pitten F: The effect of a polyhexamethylene biguanide mouthrinse compared to an essential oil rinse and a chlorhexidine rinse on bacterial counts and 4-day plaque regrowth. J Clin Periodontol 2002, 29: 392. 10.1034/j.1600-051X.2002.290503.xView ArticleGoogle Scholar
- Messick CR, Pendland SL, Moshirfar M, Fiscella RG, Losnedahl KJ, Schriever CA, Schreckenberger PC: In-vitro activity of polyhexamethylene biguanide (PHMB) against fungal isolates associated with infective keratitis. J Antimicrob Chemother 1999, 44: 297–298. 10.1093/jac/44.2.297View ArticleGoogle Scholar
- Donoso R, Mura J, Lopez M: Acanthamoeba keratitis treated with propamidine and polyhexamethyl biguanide (PHMB). Rev Méd Chile 2002, 130: 396–401.View ArticleGoogle Scholar
- Gray TB, Gross KA, Cursons R, Shewan JF: Acanthamoeba keratitis: a sobering case and a promising new treatment. Aust N Z J Ophthalmol 1994, 22: 73–76. 10.1111/j.1442-9071.1994.tb01700.xView ArticleGoogle Scholar
- Narasimhan S, Madhavan HN: Development and application of an in vitro susceptibility test for Acanthamoeba species isolated from keratitis to polyhexamethylene biguanide and chlorhexidine. Cornea 2002, 21: 203–205. 10.1097/00003226-200203000-00016View ArticleGoogle Scholar
- Cazzaniga A, Serralta V, Davis S, Orr R, Eaglstein W, Mertz PM: The effect of an antimicrobial gauze dressing impregnated with 0.2-percent polyhexamethylene biguanide as a barrier to prevent Pseudomonas aeruginosa wound invasion. Wounds 2002, 14: 169–176.Google Scholar
- Payne J, Kudner D: A new durable antimicrobial finish for cotton textiles. Am Dyestuff Rep 1996, 85: 26–30.Google Scholar
- Allen MJ, White GF, Morby AP: The response of Escherichia coli to exposure to the biocide polyhexamethylene biguanide. Microbiology 2006, 152: 989–1000. 10.1099/mic.0.28643-0View ArticleGoogle Scholar
- Broxton P, Woodcock P, Gilbert P: A study of the antibacterial activity of some polyhexamethylene biguanides towards Escherichia coli ATCC 8739. J Appl Microbiol 1983, 54: 345–353. 10.1111/j.1365-2672.1983.tb02627.xGoogle Scholar
- Gilbert P, Pemberton D, Wilkinson DE: Barrier properties of the gram negative cell envelope towards high molecular weight polyhexamethylene biguanides. J Appl Microbiol 1990, 69: 585–592. 10.1111/j.1365-2672.1990.tb01552.xGoogle Scholar
- Broxton P, Woodcock P, Heatley F, Gilbert P: Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli . J Appl Microbiol 1984, 57: 115–124. 10.1111/j.1365-2672.1984.tb02363.xGoogle Scholar
- Ikeda T, Ledwith A, Bamford C, Hann R: Interaction of a polymeric biguanide biocide with phospholipid membranes. Biochim Biophys Acta Biomembr 1984, 769: 57–66. 10.1016/0005-2736(84)90009-9View ArticleGoogle Scholar
- Allen MJ, Morby AP, White GF: Cooperativity in the binding of the cationic biocide polyhexamethylene biguanide to nucleic acids. Biochem Biophys Res Commun 2004, 318: 397–404. 10.1016/j.bbrc.2004.04.043View ArticleGoogle Scholar
- El Badawy AM, Silva RG, Morris B, Scheckel KG, Suidan MT, Tolaymat TM: Surface charge-dependent toxicity of silver nanoparticles. Environ Sci Technol 2011, 45: 283–287. 10.1021/es1034188View ArticleGoogle Scholar
- Gogoi SK, Gopinath P, Paul A, Ramesh A, Ghosh SS, Chattopadhyay A: Green fluorescent protein-expressing Escherichia coli as a model system for investigating the antimicrobial activities of silver nanoparticles. Langmuir 2006, 22: 9322–9328. 10.1021/la060661vView ArticleGoogle Scholar
- Amato E, Diaz-Fernandez YA, Taglietti A, Pallavicini P, Pasotti L, Cucca L, Milanese C, Grisoli P, Dacarro C, Fernandez-Hechavarria JM: Synthesis, characterization and antibacterial activity against gram positive and gram negative bacteria of biomimetically coated silver nanoparticles. Langmuir 2011, 27: 9165–9173. 10.1021/la201200rView ArticleGoogle Scholar
- Dutta S, Shome A, Kar T, Das PK: Counterion-induced modulation in the antimicrobial activity and biocompatibility of amphiphilic hydrogelators: influence of in-situ -synthesized Ag − nanoparticle on the bactericidal property. Langmuir 2011, 27: 5000–5008. 10.1021/la104903zView ArticleGoogle Scholar
- Saxena A, Tripathi RM, Singh RP: Biological synthesis of silver nanoparticles by using onion ( Allium cepa ) extract and their antibacterial activity. Dig J Nanomater Bios 2010, 5: 427–432.Google Scholar
- Nabikhan A, Kandasamy K, Raj A, Alikunhi NM: Synthesis of antimicrobial silver nanoparticles by callus and leaf extracts from saltmarsh plant, Sesuvium portulacastrum L. Colloids Surf B 2010, 79: 488–493. 10.1016/j.colsurfb.2010.05.018View ArticleGoogle Scholar
- Jaidev L, Naraismha G: Fungal mediated biosynthesis of silver nanoparticles, characterization and antimicrobial activity. Colloids Surf B 2010, 81: 430–433. 10.1016/j.colsurfb.2010.07.033View ArticleGoogle Scholar
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