Article pubs.acs.org/IECR
Green Synthesis of Protein Stabilized Silver Nanoparticles Using Pseudomonas fluorescens, a Marine Bacterium, and Its Biomedical Applications When Coated on Polycaprolactam Veluchamy Prabhawathi,† Ponnurengam Malliappan Sivakumar,† and Mukesh Doble* ABSTRACT: Green synthesis of protein stabilized silver nanoparticles (AgNPs) using the supernatant of a marine isolate, Pseudomonas fluorescens PMMD3 (P.fluorescens), and its biomedical applications and biocompatibility when coated on polycaprolactam is reported here. The AgNPs are spherical and are 1−10 nm in size. AgNP-coated polycaprolactam showed 89.7% and 92.4% reduction in colony forming units (CFUs), when compared to bare polymer, against S.aureus and E.coli, respectively. In the biofilm on AgNP composite, when compared to the bare polymer, there were carbohydrate reductions by factors of 3.5 and 6.0 in S.aureus and E.coli biofilm, respectively, and protein reductions by factors of 6.5 and 3.0 in S.aureus and E.coli biofilm, respectively. Reduction in the adhesion of S.aureus, C.albicans, A.niger, and F.proliferatum were observed using scanning electron microscopy (SEM) and fluorescence microscopy. The C.albicans biofilm is 15 and 6 μm thick on polycaprolactam and AgNP composite, respectively. We observed 84% and 80% proliferation of 3T3-L1 adipocyte cells and 85% and 97% of L929 fibroblast on the AgNP composite and bare polymer, indicating that this new material is suitable for biomaterial applications.
1. INTRODUCTION Among the noble metals, silver, at a minimal concentration, has been found to have wide biological applications, because of its excellent biocompatibility and antibacterial property.1 Worldwide silver production reached about 500 t in 2007.2 Because of biocompatibility, the silver nanoparticle is used in different types of consumer products including health and food supplements, food packaging and storage, refrigerators, washing machines etc. Biologically synthesized nanoparticles are widely used in the field of medicine.1 The drawback of most of the antimicrobials is that they do not act on a broad range of microorganisms including Grampositive and Gram-negative bacteria, fungi, and yeast.1 This limits the application of common antimicrobials against multiple strains. Since the surface of a polymeric implant is found to be contaminated with a wide variety of microbes that have the ability to mutate, there is a need for antimicrobials with a broad range of activity. Silver nanoparticles are one such antimicrobial. Silver-nanoparticle-impregnated polyaniline-coated polyurethane has been reported to exhibit antibacterial activity against several microorganisms.3 Organic antimicrobial compounds or antibiotics coated on polymeric surfaces may get leached from the surface. However, impregnating these molecules through a covalent bond may affect their antimicrobial activity. Hence, the use of nanocomposites in biomaterial seems to be a good strategy. Polycaprolactam is used as a biomaterial for medical, marine, and food applications. It consists of an amide bond, which lies in the same direction of natural polypeptides making it to resemble them. On contact with water (or body fluids), microbial growth along with biofilm deposition on this material leads to health and material loss. Researchers have imparted antimicrobial properties on this polycaprolactam surface by various methods. Veluchamy et al. immobilized protease on polycaprolactam surface and used it as an active food pack with enhanced antimicrobial property against Gram-positive and Gram-negative microbes.4 Polycaprolactam has been blended © 2012 American Chemical Society
with gelatin and chondroitin sulfate, using a phase precipitation method, and its invitro biocompatibility has been evaluated.5 This modified polycaprolactam did not show a degenerative effect on NIH3T3 cells, as determined by MTT and neutral red uptake (NRU) assays.5 The silver microcomposite from polycaprolactam exhibits a weak antimicrobial efficiency when the filler content is 8 wt %.6 Curdlan sulfate and heparin are physically entrapped on polycaprolactone, which reduces the thrombus formation to 39% and 28%, when compared to 80% on bare polymer.7 In the present study, the metabolites of Pseudomonas fluorescens PMMD3 are used in the synthesis of silver nanoparticles. This is a common Gram-negative rod shaped bacterium that is isolated from steel plates deployed in seawater.8 It is a nitrate-reducing bacterium that is capable of using nitrate instead of oxygen as the final electron acceptor during cellular respiration. This bacterium is also involved in the biosorption of metals. The biologically synthesized, protein-stabilized AgNP are coated on polycaprolactam and the antimicrobial, as well as antibiofilm property of this composite against bacteria, fungi, and yeast are studied. The cytotoxicity of this material is also tested by growing 3T3-L1 adipocytes and L929 fibroblasts on its surface. It is expected that biologically synthesized nanoparticles would be biocompatible.
2. METHODS 2.1. Bacteria and Chemicals. The strains used in this study namely, Staphylococcus aureus NCIM 5021 (S.aureus), Escherichia coli NCIM 293 (E.coli), Pseudomonas aeruginosa NCIM 5029 (P.aerugonosa), Salmonella typhimurium NCIM 2501 (S.typhi), Proteus vulgaris NCIM 2813 (P.vulgaris), Bacillus subtilis NCIM 2718 (B.subtilis), Vibrio natriegens PMMD6 (V.natriegens), Bacillus flexus MDLD1 (B.flexus), Aspergillus Received: Revised: Accepted: Published: 5230
December 15, 2011 March 7, 2012 March 18, 2012 March 19, 2012 dx.doi.org/10.1021/ie2029392 | Ind. Eng. Chem. Res. 2012, 51, 5230−5239
Industrial & Engineering Chemistry Research
Article
Instruments, Fullerton, CA, USA).11 The purified sample was further characterized by ultraviolet−visible (UV−vis) spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, and transmission electron microscopy (TEM). 2.5. Transmission Electron Microscopy (TEM) Analysis. Purified AgNPs were placed onto carbon-coated copper TEM grids and the measurements were performed on a JEOL Model 3010 instrument operated at an accelerating voltage of 120 kV. 2.6. Coating and Characterization of AgNPs on the Polymer Surface. 2.6.1. Polymer Preparation and Nanoparticle Coating. A polycaprolactam sheet 1 mm thick was cut into 1 cm × 1 cm and soaked in 70% ethanol for 24 h to avoid contamination. A swelling method was followed to incorporate AgNPs on the polycaprolactam surface. In this method, the polycaprolactam was swollen using acetone and then coated with AgNPs. This was allowed to dry in a desiccator. The polymer returns to its original form and morphology traps the nanoparticle. The nanoparticle-coated polymer is called a AgNP composite. 2.6.2. Fourier Transform Infrared (FTIR) Spectroscopic Analysis. The FTIR analyses of the AgNP solution, uncoated polymer, and AgNP composite were recorded in the frequency range of 400−4000 cm−1, using a Perkin−Elmer Model PE 1600 FTIR spectrometer. The analysis was done thrice with three different samples to verify the repeatability of the coating. 2.6.3. Scanning Electron Microscopy (SEM) Analysis. The surface of the polymers before and after adhesion of the bacteria and fungi were observed using a SEM system. After adhesion of the microorganism the surface was washed with distilled water and then fixed using 3% glutaraldehyde (in 0.1% phosphate buffer at a pH of 7.2) for an hour. The surface then was washed twice with phosphate buffer and once using distilled water and then dehydrated using various alcohol gradients (20%, 50%, 70%, and 90%) for 10 min. The samples were dried overnight in a desiccator. The films then were coated with gold at 30 mA for 1 min and viewed under an SEM microscope (JEOL, Model JSM 5600 LSV) at a magnification of 3000×. 2.6.4. Contact Angle. The contact angles of the polymer surfaces were measured based on the sessile drop technique,12 using a goniometer (Kruss, Germany). The sample was held on a glass slide and a drop of water (Millipore-grade) was placed on it, using a syringe. The image of the drop was captured and processed using DSA2 software (“Determination of Static and Dynamic Contact Angle”, SW4001). The left and right angles of the drop made on the polymer surface were calculated to an accuracy of ±0.1°. It was measured on three different locations on the polymer, and the average values were reported here. 2.6.5. XRF Analysis. Elemental composition of the polymer samples was analyzed using X-ray fluorescence (XRF) spectrometer (Bruker aXS, Model S4 Pioneer). X-rays are absorbed by the element present on the polymer, via the photoelectric effect, leading to photo ejection. The vacancies thus created are filled by electrons from a higher-energy state and X-rays are emitted to balance the energy difference between the electron states. The X-ray emitted is characteristic of the element from which it originated. These X-rays are directed to a detector. The energy of each X-ray and the number of X-rays at each energy are recorded. To determine the quantity of the unknown sample, the X-ray intensities (counts) at each energy are compared to values for known standards. The amount of AgNP present on the polymer was quantified using the template
niger NCIM 596 (A.niger), Candida albicans NCIM 3471 (C.albicans) and Fusarium proliferatum NCIM 1105 (F.prolif) were purchased from National Chemical Laboratory (NCL) (Pune, India). They were stored in glycerol stock at −20 °C and used when required. Polycaprolactam was purchased from Marine Industrial Polymers (Chennai, India). All the chemicals and solvents used in the experiments were obtained from Sigma (USA), and from SRL and HiMedia (Mumbai, India). 2.2. Molecular Identification of the Microbial Isolate Using 16S rDNA. Pseudomonas fluorescens was isolated from biofilm formed on metal coupons deployed near Ennore Port of Chennai Harbor, South India (latitude = 13°6′ N, longitude = 80°18′ E) for three months. The biomass from the fouled surface of the coupon was scrapped into a tank containing autoclaved seawater (seawater was collected from the same place). The microorganism was isolated by serial dilution and grown on Zobell marine agar, as well as Zobell marine broth (Himedia Laboratories, Mumbai 86, India) at a temperature of 30 °C. The isolate was sent to an external agency for 16S rDNA analysis (Genei (India) Ltd., Banglore) and was identified as Pseudomonas fluorescens PMMD3.8 2.3. Biological Synthesis of AgNPs. Bacterial strains used for the synthesis of AgNPs include S.aureus NCIM 5021, E.coli NCIM 293, P.aerugonosa NCIM 5029, S.typhi NCIM2501, P.vulgaris NCIM 2813, B.subtilis NCIM 2718, V.natriegens, P.fluorescens, and B.flexus. V.natriegens, B.flexus, and P.fluorescens were cultured in Zobell marine broth and rest of the strains was cultured in nutrient broth for 24 h at 30 °C and 180 rpm. Synthesis of AgNPs was carried out according to a previously described method.9,10 After 24 h, the cultured broth was centrifuged at 1537 g and the supernatant was used for the nanoparticle synthesis. Aqueous silver nitrate solution (1 mM) was separately added to reaction vessels containing supernatant of the bacterial strains (1% v/v) and the resulting mixtures were incubated in a dark place at 30 °C. The absorption spectrum of the samples was recorded on a UV spectrophotometer (Perkin− Elmer, Lambda 35, Shelton, CT, USA). The time taken by each strain to synthesize AgNP was determined. P.fluorescens required the shortest time to synthesize the nanoparticle and it was used for further studies (Table 1). Table 1. Different Bacterial Strains Used for the Synthesis of Silver Nanoparticles (AgNPs) and the Synthesis Time sample
bacterian strain
time needed for synthesis (h)
1 2 3 4 5 6 7 8 9
S.aureus E.coli P.aeruginosa S.typhi P.vulgaris B.subtilis V.natriegens B.flexus P.fluorescens
40 24 34 23 28 48 10 15 6
2.4. Purification of AgNPs. The above-synthesized AgNPs were washed five times by centrifugation at 1537 g for 10 min at 30 °C and redispersed in water to remove the remaining unconverted Ag ions. They were then transferred to a dialysis tube with a molecular weight cutoff of 10 000. Nanoparticles were resuspended in phosphate buffer (20 mM, pH 7.0) supplemented with sucrose, and purified using sucrose gradient centrifugation at 627 200 g, using a SW41 rotor (Beckman 5231
dx.doi.org/10.1021/ie2029392 | Ind. Eng. Chem. Res. 2012, 51, 5230−5239
Industrial & Engineering Chemistry Research
Article
200 μL of MTT (Thiazolyl Blue tetrazolium bromide) solution was added into each well and incubated for 4 h. After incubation, the supernatant was discarded and 150 μL of dimethyl sulfoxide (DMSO) was added to each well and covered with aluminum foil. This was left for 1 h in a shaking incubator at 50 rpm. The absorbance was measured at 550 nm (Perkin− Elmer, Model Lambda 35, Shelton, CT, USA). The cell proliferation on the polymeric surface was visually observed under a microscope (Leica Model DM5000, Germany). This was performed after 24 h incubation with 3T3-L1 adipocytes from mouse cell lines and then washing the polymers with PBS. 2.10. Statistical Analysis. Data reported here were expressed as mean ± standard error (SE) of three samples in each experiment. One-way analysis of variance (ANOVA) and two sample t-tests were performed using MiniTab Ver 14.0 (MiniTab, Inc., State College, PA, USA). A p-value of
