NANOFORMULATION OF ANTIFUNGAL DRUG (FLUCONAZOLE) AND THEIR EVALUATION FOR IMPROVED ANTIFUNGAL ACTIVITY

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1 NANOFORMULATION OF ANTIFUNGAL DRUG (FLUCONAZOLE) AND THEIR EVALUATION FOR IMPROVED ANTIFUNGAL ACTIVITY PROJECT REFERENCE NO. : 37S0342 COLLEGE : M. S. RAMAIAH INSTITUTE OF TECHNOLOGY, BANGALORE BRANCH : BIOTECHNOLOGY GUIDE : MR. SAMRAT K STUDENTS : CHAITRA M S VARSHA R THOTA HARISH M B SHWETA MANDAVALLI Introduction: Since the discovery of Antibiotic by Alexander Fleming, it is used rampantly which has lead to antibiotic resistance in microorganisms especially infections provoked by opportunistic fungi such as Candida albicans and Trycophiton rubrum. The emergency of Fluconazole and also other antibiotic such as Ampotericin B resistance in these fungi has promoted research on new antifungal agents. Nanotechnology provides excellent platform to tackle this drawback, various particles in nanometer range can be used as antifungal agent as well as vehicle for delivery of active materials to the targeted site without effecting the activity of active material. Due to unique biological properties of silver NPs such as biocompatibility and anti-bacterial affinity they have been applied for various medical purposes such as implants, catheters, and healing of wounds (Pham Van Dong et. al., 2012) In recent years, encapsulation of antimicrobial/fungal drugs in nanoparticle systems has emerged as an innovative and promising alternative that enhances therapeutic effectiveness and minimizes undesirable side effects of the drugs. Here the current progress and challenges in synthesizing nanoparticle platforms for delivering various antimicrobial drugs are reviewed. We also call attention to the need to unite the shared interest between nanoengineers and microbiologists in developing nanotechnology for the treatment of microbial diseases (L. Zhang et al, 2010 and Kheybari S et al, 2010). Taken together, silver nanoparticles may be considered for combination therapy against pathogenic microorganism due to its potential synergistic effect 1

2 with important antibiotics. Several studies are done on synergistic effects of nanoparticles with drugs, many cases it has proven good results (Karthick Raja Namasivayam. S and Avimanyu, 2011). In some cases encapsulating agents are used for encapsulation of drug and nanoparticles, many different encapsulating agents are used and chitosan is widely used among that. Objectives: Synthesis of Silver nanoparticles by chemical reduction method and its characterization. To determine the Minimum Inhibitory Concentration of Fluconazole against Candida albicans and Trichophyton rubrum. Conjugation of the Silver nanoparticles with drug (Fluconazole) and encapsulating with Chitosan. To evaluate and estimate the Improved Antifungal activity of nanoformulation of silver nanoparticles against Candida albicans and Trichophyton rubrum. Methodology: Organisms Candida albicans and Trichophyton rubrum: were obtained from Microbial type culture collection (MTCC) Chandigarh, India as lyophilized form. (MTCC No and 7859). Activation of Candida albicans was carried out by the direction of MTCC using Yeast Extract Peptone Dextrose Agar medium (YEPD). Activated culture was maintained on YEPD agar slants whereas Trichophyton rubrum was activated using Potato Dextrose agar (PDA) and maintained on the same media. Synthesis and Characterization of Silver nanoparticles Synthesis of Ag NP: Silver nanoparticles were synthesized by chemical reduction of 0.1M silver nitrate with 0.1M tri sodium citrate as stabilizing agent with 0.1M sodium borohydride as reducing agent. Both tri-sodium citrate and sodium borohydride is added drop wise to the continuous stirring silver nitrate solution. Synthesis of silver nano particles was confirmed by the conversion of the reaction mixture into black color. Once the particles are synthesized they are centrifuged 2

3 repeatedly for 3 times to remove the unreacted components, then the particles were oven dried and collected. Characterization of the synthesized silver nanoparticles was carried out with determination of Plasmon absorption maxima with UV-Vis spectroscopy, particle morphology (i.e., shape and size) with Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FT-IR) and X-Ray Diffraction (XRD). analysis was done which helps to detect the functional groups, structure of a compound and purity of the sample in a given environment in terms of frequencies of radiation present in the nanoparticles Antifungal activity of Silver nanoparticles Antifungal activity of Candida albicans is done by Well diffusion Assay. Antifungal activity for Trichophyton rubrum was done by Food poisoning technique. Antifungal activity of Fluconazole Active pharmaceutical ingredient (API) of Fluconazole was purchased from SRL (Sisco Research Lab). Different concentration of Fluconazole was prepared in sterilized deionised water and the antifungal activity was evaluated against Candida albicans by well diffusion assay and Trichophyton rubrum by Food poison technique, after the respective incubation time period the zone of inhibition were noted down. Synthesis and Characterization of Chitosan stabilized Silver nanoparticles loaded with fluconazole Chitosan stabilized silver and copper nanoparticles loaded with fluconazole were prepared by separately mixing 0.5mg and 0.1 mg of silver and copper nanoparticles with 58micrograms and 30 micrograms of Fluconazole respectively in 20ml of 0.15% acetic acid with 0.2% chitosan solution and stirred continuously for 3 hours until the solution becomes turbid. The solution was then oven dried at 45 o C for overnight and chitosan stabilized silver and copper nanoparticles loaded with fluconazole were collected. Characterization of chitosan stabilized nanoparticled was done by FT-IR. Antifungal activity of Chitosan stabilized Silver nanoparticles loaded with fluconazole Different concentration of Chitosan stabilized Silver nanoparticles were evaluated against Candida albicans by Well diffusion assay and Trichophyton rubrum by Food poison technique and the improved Antifungal activity is calculated by the following formula. 3

4 Improved = Zone of inhibition of Nanoformulation zone of inhibition of antibiotic antifungal activity (%) Zone of inhibition of Nanoformulation The antifungal activity of Chitosan stabilized Silver nanoparticles loaded with drugs are done in triplicates and results are noted down (Fig ). Antifungal activity of free Chitosan Antifungal activity of Chitosan against Candida albicans and Trichophyton rubrum were evaluated. 3mg/ml concentration of chitosan solution was prepared. Antifungal activity against Candida albicans was evaluated by Well diffusion assay and for Trichophyton rubrum was evaluated by Food poison technique. Results and Conclusions: In this study, evaluation of improved antifungal activity of fluconazole with silver nanoparticles against Candida albicans and Trichophyton rubrum was studied. Silver nanoparticles and nanoconjugates were synthesized by chemical reduction method and synthesized nanoparticles were characterized by UV-vis spectroscopy, Scanning Electron Microscopy, Fourier Transform Infrared Spectroscopy (FT-IR) and Energy dispersive X-ray spectroscopy (EDX). The synthesized particles were loaded into the antibiotics and the nanoformulated antibiotics were further characterized and this nanoformulation showed several fold increase of antifungal activity against both the tested fungi. This might be because of slow release of Fluconazole and Nanoparticles from the chitosan (encapsulating agent). This study suggests possible utilization of nanoformulated antibiotics as novel therapeutic agents against pathogenic fungi. Scope for future work: Future work on toxicological studies of the nano formulations can lead to efficiently develop therapeutic agents against pathogenic fungi. References: Kheybari S., Samadi N., Hosseini S.V., Fazeli A., Fazeli M.R., Synthesis and antimicrobial effects of silver nanoparticles, DARU (2010) 18 (3) L. Zhang, D. Pornpattananangkul, C.-M.J. Hu and C.-M. Huang, Development of Nanoparticles for Antimicrobial Drug Delivery, Current Medicinal Chemistry (2010), 17,

5 Karthick Raja Namasivayam.S and Avimanyu. Silver nanoparticle synthesis from lecanicillium lecanii and evalutionary treatment on cotton fabrics by measuring their improved antibacterial activity with antibiotics against staphylococcus aureus and e. coli strains. int j pharm pharm sci (2011), vol 3, issue 4, Pham Van Dong, Chu Hoang Ha, Le Tran Binh and Jorn Kasbohm, Chemical synthesis and antibacterial activity of novel-shaped silver nanoparticles, International Nano Letters (2012),