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  1. 1. Poushpi Dwivedi, S.S. Narvi, R.P. Tewari / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 Vol. 2, Issue4, July-August 2012, pp.1490-1495Mythology Converges With Technology To Combat Biomaterials Associated Infections Poushpi Dwivedi*, S.S. Narvi**, R.P. Tewari*** *, ** (Chemistry Department, Motilal Nehru National Institute of Technology, Allahabad, UP, India *** (Applied Mechanics Department, Motilal Nehru National Institute of Technology, Allahabad, UP, India)ABSTRACT The integration of nanotechnology, chiefly in the nanomedicine landscape [1]. A varietybiotechnology, biology, physical science, of methods have been developed thus to achievechemistry, biomedical science and medicine; for control over the properties of nanoparticles andthe best scope, development and application in nanostructered materials strongly depended uponscience and technology is being studied for their dimensions and structure [2]. Furthermore, thedecades. But the intimation of the immense biosynthesis of nanoparticles has attracted worldpotentiality of mythology converging with wide attention because of the necessity to bringtechnology is scarce. For the first time, we report environmentally friendly, cost-effective and efficienthere the combo power of the mythological Peepal techniques. The biological reduction of metals bytree, Ficus religiosa, in convergence with plant extracts has been known since the early 1900s;nanotechnology to combat biomaterials associated however the reduction products were not studied.infections. Infections associated with biomaterials Only recently within the last three decades, thesuch as medical devices, implants and prosthetic synthesis of nanoparticles using plant materials, formaterials, are due to bacterial adhesion and the most part, is being properly investigated [3]. Thesubsequent biofilm formation at the implantation synthesis of silver nanoparticles from plant resourcessite. Although much research has been focused on is now an expanding research area as it eliminates thesterilization, aseptic procedures and on developing call for harsh or toxic reagents and minimizespolymers that resist biofilm formation; bacterial hazardous byproducts [4].infection still remains a major impediment in theutility of such biomaterials. The emergence of Thus in the field of nanoscience themultidrug resistant ‘super bugs’, like the ones synthesis of silver nanoparticles using plant-derivedrecently discovered, ‘NDM-l’ (New Delhi Metallo- materials is a simple yet effective method for thebeta-lactamase), have made this global problem production of nanoparticles having invaluablerather alarming. For reducing this incidence of applications in the field of biomedical science [5].biomaterials associated infections we have On-going research efforts in the biomedical arenadeveloped self-sterilizing silver/chitosan have brought forward the hypothesis that silver-bionanocomposite via biosynthesis for application containing material can mitigate the menace ofas coating over biomedical implants and devices. biomaterial associated nosocomial infection whichThis material has been characterized through has been confirmed by several in-vitro and in-vivovarious techniques and antimicrobial studies have experiments [6]. Silver nanoparticles having highalso been done. surface reactivity due to high surface to volume ratio, releases silver ions which is highly antimicrobialKeywords - nanotechnology, nanoparticles, with the ability to kill a very broad spectrum ofbionanocomposite, biomaterials, antimicrobial medically relevant bacteria (gram positive and gram negative) as well as fungi (molds and yeasts) [7].1. INTRODUCTION Ionic silver is also oligodynamic, which means that it Materials and material development are is antimicrobial at very low doses, as low as aboutfundamental to our very culture. With the system of 0.001- 0.05 ppm [8]. Although silver is a heavyascribing major epochs of our society to materials, metal, at the reference low concentration amounts, itsuch as the stone age, bronze age, iron age, steel age, is non toxic to human cells and much safe [9].silcon age; this is apparently and undoubtedly thenano age. In this nanoregime the use of Therefore we have designed silver/chitosannanotechnology and designing nanomaterials is bionanocomposite (Ag/CS BNC) havingbecoming increasingly popular in almost every field antimicrobial and self-sterilizing properties toand especially in the arena of biomedical engineering mitigate the menace of biomaterials associatedit holds a significant advantage over others. infections and which when leached out will cause Today silver nanocoomposites have got minimal harm to the human body. Chitosan used asmyriads of interesting and demanding applications the matrix can protect silver nanoparticles from 1490 | P a g e
  2. 2. Poushpi Dwivedi, S.S. Narvi, R.P. Tewari / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 Vol. 2, Issue4, July-August 2012, pp.uncontrolled oxidation and stabilize them from (AgNP pellet) was re-dispersed in distilled water.agglomeration. Chitosan is FDA approved, natural This procedure was repeated three times to isolate Agbiopolymer derived by deacetylation of chitin, a nanoparticles from proteins or other bio-organiccomponent commonly found in the exoskeleton of compounds present. The remnant pellet wascrab, shrimp and crawfish and is the second most dispersed in 15 ml of chitosan solution [2% (w/v) inabundant biopolymer after cellulose. It is composed 1% (v/v) acetic acid] and sonicated for 10 min.of poly [β-(1→4)-2-amino-2-deoxy-D- Finally silver/chitosan (Ag/CS) bionanocompositeglucopyranose] and has many advantageous features film (1’) was prepared by casting thelike biocompatibility, biodegradability, non-toxicity, bionanocomposite suspension on a glass platehydrophilicity, together with antimicrobial properties (solvent casting) and dried at room temperature [11].[10]. 2.5 Coating The mythological tree of eternal life, Peepal Coating of medical implant, such astree, Ficus religiosa, referred as, Lord Krishna, who stainless steel rod was done by pouring theis revered as the omnipotent universal savior, has bionanocomposite suspension material (1’) on itintervened for the biosynthesis of silver (solvent casting technique) and air dried. Facile andnanoparticles. This is certainly for the first time that less time consuming method of coating was adoptedthe divine intervention of this plant has been involved for preliminary and basic studies.for the formation of nanoparticles; for thedevelopment of bactericidal bionanocomposite 2.6 UV-visible absorbance spectroscopy studymaterial; for being applied as self-sterilizing coating The reduction of Ag+ to Ag0 was monitoredover biomedical implants, prosthesis and devices. by measuring the UV-Vis. spectrum of reactionMythology converges here with nanotechnology to mixture (having AgNO3 solution + plant extract) aftercombat biomaterials associated infections and 48 h when complete stabilization and no further colorconquer the race between bacterial adhesion and transformation was observed. The spectra of planttissue integration. extract alone and AgNO3 solution were also taken. The UV-visible spectra were recorded using UV-2. MATERIALS AND METHOD visible spectrophotometer (Shimadzu UV – 2450)2.1 Materials from 200 to 800 nm. Deionised water was used as Chitosan (degree of deacetylation: 79 %, blank.molecular mass: 500,000 g/mol) was purchased fromSea Foods (Cochin), India; AgNO3 of analytical 2.7 Nanoparticle size analysisgrade from Thomas Baker (Chemical) Pvt. Ltd. Analysis of the nanoparticle size was doneIndia; acetic acid glacial (extra pure) from Thomas with the help of (NANOTECH) particle size analyzerBaker (Chemical) Pvt. Ltd. India. Solutions were instrument.prepared using deionized water. Leaves werecollected freshly from the plant Ficus religiosa. 2.8 TEM observations Samples for transmission electron2.2 Preparation of plant extract microscopy (TEM) of the silver nanoparticles Leaves of Ficus religiosa were washed and synthesized were prepared by placing drops of theair dried. 21 g of clean leaves were cut into fine product solution onto carbon-coated copper grids andpieces and boiled in 100 ml of sterile distilled water allowing the solvent to evaporate. TEMin a 500 ml Erlenmeyer flask for 15 min at 100 ○C. measurements were performed on the (HR TEMThe crude plant extract was filtered using Whatman TECNAI 20 G2) instrument operated at anNo. 41 filter paper and stored in closed bottle at 4 ○C accelerating voltage of 200 kV.for further use. 2.9 SEM study2.3 Synthesis of silver nanoparticles Silver/chitosan bionanocomposite was 5 ml of the aqueous plant extract was added coated with a thin layer of graphite and examined in ato 100 ml of 1 mM silver nitrate (AgNO3) solution scanning electron microscope (SEM) (JEOL JXAand this was marked as (1). The reaction mixture was 8100).kept in closed bottle and incubated at RT forstabilization. 2.10 FTIR analysis FTIR spectrum was recorded over the range 2.4 Development of silver/chitosan of (500-4000) cm−1 with (with FTLA 2000 ABB).bionanocomposite After 48 h the reaction mixture was 2.11 Antimicrobial activity testcentrifuged at 9,000 rpm for 15 min and the residue 1491 | P a g e
  3. 3. Poushpi Dwivedi, S.S. Narvi, R.P. Tewari / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 Vol. 2, Issue4, July-August 2012, pp. The bionanocomposite (BNC) was assayed peak of silver nanoparticles due to its surfacefor antimicrobial activity against Pseudomonas plasmon resonance (SPR) shifts to longeraeruginosa (gram negative), and Staphylococcus wavelength with increasing particle size.aureus (gram positive). Disc diffusion method wasused to find out the standard zone of inhibition (ZOI) CS[12]. Antibacterial test was done against (1’) 1.0 1bionanocomposite film (disc shaped pieces 5 mm in Frdiameter); biomaterial like medical grade stainless AgNO3steel rod (30 x 12 mm) coated withbionanocomposite (1’) (SSR). Chitosan film (CS)was taken as positive control and uncoated absorbancebiomaterial (B) as negative control. The materials to 0.5be assayed were sterilized by UV radiation for 30min and placed on different cultured agar plates.Muller Hinton Agar was used as culture media andinoculated with 300 μl of bacterial organismcontaining broth. The plates containing the bacterialorganism and silver nanocomposite films were 0.0incubated at 37○C for 2 days. The plates were then 200 300 400 500 600 700 800 wavelength in nmexamined for evidence of zones of inhibition, whichappear as a clear area around the discs. The diameterof such zones of inhibition was measured using ameter ruler. Fig. 1. UV-Visible spectra of, nanoparticle suspension (1); plant extract (Fr); silver nitrate3. RESULTS AND DISCUSSION solution (AgNO3); chitosan (CS).3.1 UV-visible spectral studies The reaction mixture (1) demonstrated 3.2 Nanoparticle size determinationincrease in colour development with time. The Analysis of nanoparticles through particleprocess of colour development and the size analyzer emphasizes non uniform size of thetransformation from colourless to reddish brown was particles formed. Details of the determination arerapid, which indicated the formation of silver shown through the particle size distribution graph innanoparticles. The UV–visible absorption spectrum Fig. 2.recorded from the nanoparticle suspension of thereaction mixture, (1), after 48 h of reaction andcomplete stabilization with no further colortransformation, is shown in Fig. 1. In this case, asurface plasmon resonance (SPR) band absorptionpeak of reaction mixture (1) appears at 415 nm whichis characteristic of silver nanoparticles. Spectralanalysis after the development of the silver/chitosanbionanocomposites (1’), also exhibited similarabsorbance peak as its aqueous suspension of thenanoparticles. The spectrum of aqueous AgNO3 onlysolution was at ~ 220 nm, chitosan itself istransparent in the UV-visible region. The spectrum ofplant extract rises at ~ 500 nm without any maximaor minima. Fig. 2. Histogram of particle size distribution of nanoparticle suspension 1. According to Mie’s theory only a singleSPR band peak is expected in the absorption spectra 3.3 TEM observationsof spherical nanoparticles, whereas anisotropic The morphology of silver nanoparticlesnanostructures or aggregates of spherical synthesized by the intervention of the mythologicalnanoparticles could give rise to two or more SPR Peepal tree was observed by transmission electronbands depending upon the shape of the particles [1]. microscopy (TEM). Fig.3 shows TEM images ofIn the present investigation, the suspension showed a silver nanoparticles formed.single SPR band revealing spherical shape of silvernanoparticles. Strong absorbance between 420 – 480 TEM observations revealed that silvernm in UV Vis. spectral analysis signifies the nanoparticles formed are chiefly spherical but arepresence of silver nanoparticles. Also the absorption irregular in shape and non uniform in size. Most of 1492 | P a g e
  4. 4. Poushpi Dwivedi, S.S. Narvi, R.P. Tewari / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 Vol. 2, Issue4, July-August 2012, pp.the particles showed interparticle interactions, which Furthermore the silver/chitosanmay have been due to the peripheral complexation of bionanocomposite (1’), developed from thecapped biomolecules. The ring-like diffraction nanoparticle suspension was studied for itspattern indicated that the particles were crystalline in morphological characteristics through scanningnature. This finding was reflected in the electron microscopy (SEM). Images in Fig. 4 (a, b)approximately circular nature of the selected area show nanoparticles well dispersed and distributed inelectron diffraction (SAED) spots in Fig. 3. the chitosan biopolymer matrix with minimum aggregation. (a) (b) (c)Fig. 3. TEM micrographs; SAED pattern (top) andimages of silver nanoparticles of suspension 1, atdifferent magnifications.3.4 Characterization of the silver/chitosanbionanocomposite 1493 | P a g e
  5. 5. Poushpi Dwivedi, S.S. Narvi, R.P. Tewari / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 Vol. 2, Issue4, July-August 2012, pp. Table 1. Zone of inhibition (ZOI) in (mm) against selective bacterial strains. Name of BNC Stainless Uncoated Plant the micro- film steel rod bio- extract organism (1’) coated material (Fr) with disc (B) BNC 1’- (SSR) (d) P. aeruginosa 19 28 Nil NilFig. 4. SEM images; (a), (b), are micrographs ofbionanocomposite film 1’; (c) image showing S. aureus 23 32 Nil Niluncoated stainless steel rod; (d) micrograph of thestainless steel rod coated with bionanocomposite 1’. The interaction, between the lone pair ofelectrons present at the amine group of chitosan andthe partial positive charge developed at the surface ofthe silver nanoparticles due to electron drift,effectively stabilizes the silver nanoparticles andprevents them from agglomeration. Thebionanocomposite suspension was able to be coatedunifomly over the surface of medical grade implantsused inside the human body eg. stainless steel rod;together with imparting smooth surface modificationof the biomaterial; vividly elucidated through theSEM micrographs in Fig. 4 (c, d). In the FTIR spectra of the bionanocompositein Fig. 5, the peak at ~3500 cm-1 is more pronouncedcorresponding to the axial OH group of the chitosanmolecule. The bending vibrations between 1600 and1000 cm-1 intensifies indicating possible interactionbetween silver nanoparticles and amino group ofchitosan.Fig. 5. FTIR spectrum[Transmission/Wavenumber(cm-1)] of (a)silver/chitosan bionanocomposite film 1’.3.5 Antimicrobial activity test Silver/chitosan bionanocomposite film (1’),having the bioreduced silver and biomaterial coatedwith the bionanocomposite (SSR), were assayed forantimicrobial activity against Pseudomonasaeruginosa (gram negative) and Staphylococcusaureus (gram positive), which cause majority of thebiomedical implant related infections. Disc diffusionmethod was adapted to find out the standard zone of Fig. 6. Photographs taken after 48 h of incubation,inhibition (ZOI). Details of the result obtained are showing the antibacterial activity through zone ofshown in Fig. 6 and listed in Table 1. inhibition (ZOI); of silver/chitosan bionanocomposite 1494 | P a g e
  6. 6. Poushpi Dwivedi, S.S. Narvi, R.P. Tewari / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 Vol. 2, Issue4, July-August 2012, (1’); stainless steel rod (SSR) coated with 6. REFERENCESsilver/chitosan bionanocomposite (1’); chitosan film [1] K.L. Kelly, E. Coronado, L.L. Zhao and(CS); uncoated biomaterial (B); plant extract of Ficus G.C. Schatz, The optical properties of metalreligiosa (Fr); against Pseudomonas aeruginosa and nanoparticles: The influence of size, shape,Staphylococcus aureus. and dielectric environment, J Phys Chem B, 107, 2003, 668–677. It has been observed in the present study [2] R. Narayanan and M.A. El-Sayed, Effect ofthat the effect was well pronounced against gram colloidal nanocatalysis on the metallicpositive bacteria which lack the outer membrane but nanoparticle shape: the Suzuki reaction,has a peptidoglycan layer of about 30 nm thickness, Langmuir, 21, 2005, 2027–2033.and even against gram negative bacteria which [3] S.S. Shankar, A. Rai, A. Ahmad and M.J.contains only a thin peptidoglycan layer of 2-3 nm Sastry, Rapid synthesis of Au, Ag andbetween the cytoplasmic membrane and the tough bimetallic Au shell nanoparticles usingouter membrane. Neem, J Colloid Interf Sci, 275, 2004, 496- 502.4. CONCLUSION [4] K.N. Thakkar, S.S. Mhatre and R.Y. Parikh, Thus this first attempt to synthesize Biological synthesis of metallicsilver/chitosan bionanocomposite through herbal nanoparticles, Nanomedicine: Nanotechnolroute via the intervention of our divine Peepal tree, Biol Med, 6, 2010, 257–262.Ficus religiosa, shows significant potentiality to [5] E.A. Deitch, A.A. Marino, V. Malakanovmitigate the menace of medical implant associated and J.A. Albright, Silver nylon cloth: ininfections. Bio-reduction of silver ions with plant vitro and in vivo evaluation of antimicrobialextract provides a facile path for the production of activity, J Trauma, 27, 1987, 301–304.silver nanoparticles, avoiding the use of obnoxious [6] T. Gilchrist, D.M. Healy and C. Drake,reducing agents which persistently adhere to the Controlled silver-releasing polymers andsurface of the nanostructures, rendering them their potential for urinary tract infectionhazardous to be handled as well as to be applied. control, Biomaterials, 12, 1991, 76–78. This biodegradable, bactericidal, self [7] S. Saint, J.G. Elmore, S.D. Sullivan, S.S.sterilizing, biocompatible material holds sure shot Emerson and T.D. Koepsell, The efficacy ofpotency to ameliorate the face of existing silver alloy-coated urinary catheters inbiomaterials and provides a winning strategy to preventing urinary tract infection: a meta-vanquish biomaterials associated infections in the analysis, Am J Med, 105, 1998, 236–241.race between bacterial adhesion and tissue [8] T.J. Berger, J.A. Spadaro, S.E. Chapin andintegration. Advanced coating techniques can be R.O. Becher, Electrically generated silverapplied for coating of medical implants and surgical ions: quantitative effects on bacterial anddevices with this novel bionanocomposite material in mammalian cells, Antimicrob Agentsorder to impart smooth and uniform surface Chemother, 9, 1976, 357–358.modification. [9] R.L. Williams, P.J. Doherty, D.G. Vince, G.J. Grashoff and D.F. Williams, The5. ACKNOWLEDGEMENTS biocompatibility of silver, Crit Rev One of the authors PD sincerely Biocompat, 5, 1989, 221–223.acknowledges and pays gratitude to Prof. O.N. [10] L. Balau, G. Lisa, M.I. Popa, V. Tura and V.Srivastava, Physics Department, Banaras Hindu Melnig, Physico-Chemical properties ofUniversity, Varanasi, India, for the TEM chitosan films, Cent Eur J Chem, 2, 2004,micrographs. The author is also thankful to Prof. P.K. 638-647.Dutta, Dr. Ashutosh Pandey, Dr. Tamal Ghosh, Ishrar [11] S.K. Mazumder, Composites manufacturing,Ahmed, K.P. Shukla; MNNIT, Allahabad, India and materials, product and process engineeringDr. M.M. Dwivedi, NCEMP, University of (CRC Taylor & Francis, (ed.). ISBN 0-Allahabad, India; for their kind support during the 8493-0585-3, 2002).research work. [12] R. Cruickshank, 11th ed. Medical microbiology: a guide to diagnosis and control of infection (Edinburgh and London: E. & S. Livingston Ltd. 1968). 1495 | P a g e