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Nanobio Pharmaceutical Technology Applications and Perspectives

Nanobio Pharmaceutical Technology Applications and Perspectives

Editors Latha Subbiah Selvamani Palanisamy Subramanian Natesan

ELSEVIER A division of Reed Elsevier India Pvt. Ltd.

Nanobio Pharmaceutical Technology Applications and Perspectives ELSEVIER A division of Reed Elsevier India Pvt. Ltd. © 2014 International Conference on Innovations and Research Dimensions in Nanobio Pharmaceutical Technology All rights reserved. This book contains information obtained from authentic and highly regarded sources. The printed material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means electronic, mechanical, photocopying, recording, or otherwise without the prior written permission of the publisher and copyright holder. ISBN: 978-935-107-293-5 Published by Elsevier, a division of Reed Elsevier India Private Limited Registered Office: 305, Rohit House, Tolstoy Marg, New Delhi 110 001. Corporate Office: 14th Floor, Tower 10B, DLF Cyber City, Phase-II, Gurgaon 122 002, Haryana Printed and bound in India.

Organising Committee CHIEF PATRON Dr. M. Rajaram, Hon’ble Vice-Chancellor, Anna University, Chennai

PATRON Dr. S. Ganesan, Registrar, Anna University, Chennai

CHAIR Dr. T. Senthil Kumar, Dean, Anna University, BIT Campus, Tiruchirappalli

ADVISOR Dr. K. Ruckmani, Professor & Head, Dept. of Pharmaceutical Technology, Director, Centre for Excellence in Nanobio Translational REsearch (CENTRE), Anna University, BIT Campus, Tiruchirappalli

ORGANIZING SECRETARIES Dr. S. Latha & Dr. P. Selvamani, Assistant Professor, Dept. of Pharmaceutical Technology, Anna University, BIT campus, Tiruchirappalli

MEMBERS Dr. A. Puratchikody Dr. N. Subramanian Dr. E. Sanmugarpriya Dr. R. Vijaya Mr. A. Shanmugarathinam Mrs. A. Umamaheswari Mr. R. Suriyakanth Dr. P. SenthamilSelvan Mrs. K. Akilandeswari Dr. K. Kavitha Dr. S. Lakshmanaprabu Mrs. M. Vijayalakshmi

PROCEEDINGS EDITORIAL BOARD CHAIRPERSON Mr. A. Shanmugarathinam

PROCEEDINGS EDITORIAL BOARD MEMBERS Mrs. M. Arputha Bibiana Mr. C. Prabu Ms. P.S. Dhivya Mr. S. Rajkumar Mrs. M. Poornima Ms. S. Monisha Ms. C. SherlinaDaphny Ms. T. Supassri

ADVISORY BOARD MEMBERS INTERNATIONAL SILVIO DUTZ Technische UniversitätIlmenau, Germany JAGAT KANWAR Deakin University, Australia MANIKAM SIVAKUMAR The Nottingham University, Malaysia R. THIRUMURUGAN International Medical University,  Malaysia.  R. RAJAN International Medical University,  Malaysia.  L. MANIKANDAN Forma Therapeutics, Singapore RENGANATHAN ARUN, University of Zurich, Switerland ASHOK KUMAR ASIA Metropolitan University, Malaysia. R. MANAVALAN NATIONAL Annamalai University, Tamil Nadu, India T.K. PAL Bioequivalence Study Centre, West Bengal, India DHIRENDER BAGADHUR IIT, Mumbai P.JAISANKAR Indian Institute of Chemical Biology, West Bengal, India J.K. GUPTA Jadavpur University, West Bengal, India P. GAUTAM Anna University, Tamil Nadu, India M. KRISHNAN Bharathidasan University, Tamil Nadu, India P. RAJAGURU Anna University, Tamil Nadu, India G.P. MOHANTA Annamalai University, Tamil Nadu, India S. KAVIMANI Mother Teresa Post Graduate and Research Institute, Pondicherry, India C. DHANAPAL Annamalai University, Tamil Nadu, India M.V. RAO Bharathidasan University, Tamil Nadu, India

TANAJI T. TALELE St. John’s University, USA RAVI PALANIAPPAN Mercer University, Atlanta, USA JITKANG LIM Universiti Sains Malaysia, Malaysia KARINA POMBO GARCIA Helmholtz-Zentrum Dresden-Rossendorf, Germany SHEEBA DAVID International Medical University,  Malaysia.  DEEPAK BALAJI Thimiri Consulting Group, France NIRMESH JAIN University of Sydney, Australia VASUDEVAN MANI Universiti Teknologi MARA, Malaysia RAJIV DAHIYA Global College of Pharmacy, UP, India T.N.K. SURIYA PRAKASH Al-Shifa College of Pharmacy, Kerala, India P. GOPINATH IIT, Roorkee RANU DUTTA University of Allahabad, Uttar Pradesh, India L.K. GHOSH Jadavpur University, West Bengal, India B.S. LAKSHMI Anna University, Tamil Nadu, India G. MATHAN Bharathidasan University, Tamil Nadu, India R. SIVAKUMAR Grace College of Pharmacy, Kerala, India G. MARIAPPAN Kamla Nehru Institute of Management and Technology,Uttar Pradesh, India R. NETHAJI Devakiammal College of Pharmacy, Kerala, India R.S. JEYA PRAKASH Manipal College of Pharmaceutical Sciences, Karnataka, India M. SIVAKUMAR Anna University, Tamil Nadu, India

M.B. VISWANATHAN Bharathidasan University, Tamil Nadu, India T.K. RAVI Sri Ramakrishna Institute of Paramedical Sciences, Tamil Nadu, India J. NIRMAL Dr.HarisinghGour University, Madhya Pradesh, India D.C. SUNDARAVELAN Sri Ramakrishna Institute of Paramedical Sciences, Tamil Nadu, India

K. NEHRU Anna University, Tamil Nadu, India V. RAJESHKANNAN Bharathidasan University, Tamil Nadu, India K. ASOKKUMAR Sri Ramakrishna Institute of Paramedical Sciences, Tamil Nadu, India C. VIJAYARAGHAVAN PSG College of Pharmacy, Tamil Nadu, India

Contents SECTION-I NANOMATERIALS  1 In vitro Antimicrobial Efficacy of Zinc Doped Nano Hydroxyapatite Against Human Pathogens R. Baskar, G. Devanand Venkatasubbu and K. Umamaheswari   2 Synergistic Antibacterial Efficacy of Two Drugs in Addition With Biosynthesized AgNPs From an Allergenic Fungus Chitra. N, B. K. Nayak, and Anima Nanda   3 Synthesis and Characterization of PAMAM Dendrimer and its Application for Removal of Heavy Metals E. Gomathi, P. Rajesh Prasanna & P. Selvamani   4 Bio-Toxicity of Silver Nanoparticles Against Multidrug Resistant Pathogens and Human Epidermoid Larynx Carcinoma (HEp-2) Cells Muthukrishnan Lakshmipathy and Anima Nandaa

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  5 Design and Evaluation of Polyvinyl-Gum Ghatti Self-Assembled Nanoscale Particles | for Oral Delivery of Simvastatin 25 Bibek Laha, Leena Kumari, Arpana Kashyap and Sabyasachi Maiti   6 Green Synthesis and Characterization of Herbal Mediated Silver Nano Particles Using Gymnema Sylvestre (Linn.) 31 Kalakotla Shanker and Gottumukkala Krishna Mohan   7 Synthesis and Characterization of Green Silver Nanoparticles Mediated by Aegle marmelos (L.) Leaf Extract 38 Sukumar Dandapat, Manoj Kumar and M. P. Sinha   8  Invitro Cytotoxicity Studies of Nickel Oxide Nanoparticles on Cancer Cells Using MTT Assay 45 K. Perumal Raj, V. Thangaraj, Dr. A. P. Uthirakumar, P. Sivakarthik   9 Characterization Studies of Sunlight Induced Biosynthesis of Silver Nanoparticles Using Solanum melongena (Egg Plant) 50 V. Aravindhan, C. Senthil Kumar, H. Linda Jeeva Kumari and K. Ruckmani 10  Thermal, Anti-Fungal and Primary Electrochemical Studies of Palladium Nanoparticles 59 N. John Sushma, K. Mallikarjuna, D. Prathyusha, G. Narasimha, G. Swathi, B.V. Subba Reddy and B. Deva Prasad Raju 11 Optimization Studies on Bioinspired Green Synthesis of Silver Nanoparticles Using Clitoria Ternatea66 N. John Sushma, G. Swathi, D. Prathyusha and B. Deva Prasad Raju 12 Biosynthesis and Characterization of Silver and Gold Nanoparticles in Piper Nigrum L. (Piperaceae) 73 M. Manogaran and M. B. Viswanathan 13 Synthesis of Gsh & Fa Conjugated Chitosan Functionalized Gold Nanospheres: Physicochemical Properties and Applications in Cancer Therapy 84 Kalpana Haria, Hemamalini Vedagirib, and Premkumar Kumpati 14 Antimicrobial, Antioxidant and Angiogenic Potential of Silver Nanoparticles From Leaves of Murraya Paniculata (L.) Jack 91 Rama. P, Vignesh. A and K. Murugesan 15 Biosynthesis of Silver Nanoparticles Using Tephrosia Tinctoria for Antibacterial Activity Against Multidrug Resistant Pathogens 105 D.C. Aiswaryaa, K. Rajaram and P. Sureshkumar

xContents 16  Green Synthesis of Silver Nanoparticles From Leaf Extracts of Breynia Vitis-Idaea111 B. Anandaraj, T. P. Rajesh, P. Suresh Babu and C. Narendhar 17  Characterization of Solid State Forms of Pioglitazone 117 B. Poornima, K. V. S. R. G Prasad and K. Bharathi 18  Synthesis of Metaloxide Nano Composites for Solar Cells 125 S. Shanthi and Dr. M. Dharmendirakumar 19  Phytogenic Synthesis of Silver Nanoparticles and its Potential Foron Blue Dye Decolorisation 133 P. Balashanmugam, P. Arthi and P. T Kalaichelvan 20 Aristolochia Indica L. Mediated Synthesis of Nano-Silver Particles for its Antimicrobial Activity Against Human Pathogens 139 G. Sathishkumar, C. Rajkuberan, K. Ravindran and S. Sivaramakrishnan 21  Pharmacokinetics of Enrofloxacin Loaded Solid Lipid Nanoparticles Following Oral Administration in Emu (Dromaius Novaehollandiae) Birds 149 P. Senthil Kumar, A. Arivuchelvan, A. Jagadeeswaran, N. Subramanian, C. Senthil Kumar and P. Mekala 22 Antidiabetic Activity of Chromium (iii) Nanoparticle Against Streptozotocin-Induced Diabetis in Rats 157 Kanakalakshmi Annamalai, Anjali Mohan Nair and Shanthi kuppusamy 23 Formulation of Soap and Cream From Biosynthesised Silver Nanoparticles of Adathoda Vasica Nees. Leaf Extract 162 H. Linda Jeeva Kumari, S. Sonia, R. Krishnamoorthy, M. Sivakumar and K. Ruckmani 24 Invitro Cytotoxicity Assessment of Silver Nanoparticles Synthesized Using Aspergillus Terreus Against Breast Cancer Cell Line 173 M. D. Bala Kumaran and P. T. Kalaichelvan 25 Larvicidal and Antimicrobial Potency of Silver Nanoparticles Synthesized Using an Actinomycete, Saccharopolyspora Erythraea178 Prabha S. B. and K. Murugesan 26  Biomimetic Production and Cytotoxic Effect of Silver Nanoparticles From Fungi 187 S. Akila and Anima Nanda 27 Synthesis and Characterization of Andrographis Paniculata Loaded Nanoparticles for the Development of Antimicrobial Poly Cotton Fabrics 193 R. Rajendran, K. Hemalatha, A. Manikandan and R. Radhai SECTION-II NANO DRUG DELIVERY SYSTEMS   1  M  odified Release Multiparticulate Delivery System for Tramadol HCL Using Gelucire 43/01 as Rate Retarding Material Divya Priya. S, Sruthi. S, Hema. R, Rajasekar. K, Ganesan. V, Lakshamana Prabu. S, Puratchikody. A, Shanmugarathinam. A.   2 Extraction and Characterization (Both Physico-Chemical and Analytical) of Pectin Obtained From Dillenia Indica and Abelmoschus Esculentus and Compatibility Study With Glipizide for Application in Novel Drug Delivery Systems Sivasankar Mohanty, G. Krishna Mohan and M. Sunitha Reddy.   3  Preparation and Characterization of Leflunomide Loaded Nanosuspensions for Rheumatoid Arthritis S. Lakshmana Prabu, S.P. Sharavanan, A. Shanmugarathinam, K. Ruckmani and A. Bhuvaneswari   4  Diclofenac Sodium Nanosuspension: An Approach To Improve Anti-Inflammatory Therapy S. Lakshmana Prabu, S.P. Sharavanan, A. Shanmugarathinam, K. Ruckmani, S. Aravindan, A. Bhuvaneswari and V. Manikandan

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Contentsxi   5 Formulation and In Vitro - In Vivo Evaluation of Wedelolactone Loaded Nanosuspension for Hepatoprotective Activity in CCl4 Induced Significant Hepatic Damage and Oxidative Stress Model S. Brito Raj, P. Sucharitha, T. Murali1, S. Wasim Raja and K. Bhaskar Reddy   6 Enhanced Oral Bioavailability of Naringenin Using Polymeric Nanoparticles: Formulation, Characterization and in Vivo Studies P. Suseelaa, K. Premkumarb, S.D. Saraswathya   7  Design and Characterisation of Baclofen Sustain Release Tablet Using Hydrophilic Matrix R. Mohan Kumar, M. Balakumaran, M. Annapoorni, P. Selvamani, N. Subramanian and K. Ruckmani.   8 Preparation and Evaluation of Miconazole Nitrate Nanoemulsion Using Tween 20 As Surfactant for Effective Topical and Transdermal Delivery P. Jaya Sree and Dr. C. Thirumal Azhagan   9 Development and Characterization of Enrofloxacin SLNs and its Pharmacokinetics Following Oral Administration in Emu (Dromaius Novaehollandiae) Birds P. Senthil Kumar, A. Arivuchelvan, A. Jagadeeswaran, N. Subramanian, C. Senthil Kumar and P. Mekala 10  Redox Environment Cleavable Polymeric Nanoparticles for Drug Delivery M. Gover Antoniraj, C. Senthil Kumar, Angeline Tisha and K. Ruckmani 11  Preparation of Prednisolone Loaded Caco3 Microparticles for Sustained Release C. Prabu, S. Latha and P. Selvamani 12 Development and Evaluation of Artemisinin Magnetic Nanosponges for Targeting Breast Cancer S. P. Saravanan , P. Chandrasekar, K. Sanjai, Padma @ Rajam, R. Suriyakanth and N. Subramanian 13 Quercetin Loaded Chitosan/ Peg Nanoparticles: Formulation and In-Vitro Characterisation Saravanakumar P, Vinoth J, Chandrasekar P and Subramanian N 14 Cancer Targeting With Artesunate Magnetic Nanoparticles Encapsulated With Thermo-Responsive Polymers Vinoth. J, Sharavanan. S.P, Senthil Kumar. C, Dhinakaran. P and Subramanian. N 15 Formulation and In Vitro Evaluation of Controlled Release Tablets of Norfloxacin Using Natural Polymers V.Ganesan, S.R.Senthilkumar 16 Exploration of Quail’s Egg Lecithin in Development and Evaluation of Novel Celecoxib Loaded Lecithin Organogel S. Balaguru, Ramya Devi.D, B.N. Vedha Hari 17 Development of Response Surface Methodology for Tenofovir Disoproxil Fumarate Microparticles Using Solvent Evaporation Method Fathima A, Vedha Hari BN, Ramya Devi D 18 Formulation and Evaluation of Mouth Dissolving Film Leflunomide for Rheumatoid Arthritis S. LakshmanaPrabu, A. Shanmugarathinam, S.P. Saravanan, M. Elakkiya, V. Manikandan, A. Bhuvaneswari, S. Aravindan 19 Development and Sustainable Release Evaluation of Enrofloxacin Solid Lipid Nanopartilces P. Senthil Kumara, A. Arivuchelvanb, A. Jagadeeswaranb, N. Subramanianc, C. Senthil Kumard and P. Mekalab

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xiiContents 20 Formulation and Evaluation of Stavudine Loaded Niosomes and Quantitative Estimation By HPLC351 S. Latha, P. Selvamani, R. Bharathi , R. Benaseer Begam, T. Keerthana, M. Ponmalar, K. Hari Gopala Reddy 21  Optimization and Formulation of Drug Loaded Magnetic Microcapsules 358 C. Prabu, S. Latha, P. Selvamani 22 Formulation and Characterization of Carisoprodol Magnetic Nano-Emulsion for Rheumatoid Arthritis 367 A. Stephen Prawin Kumar, G. Phonnavan, G. VinothKumar, M. Neelumathi, P. Selvamani, S. Latha 23  Lipid Nanocarriers of Methotrexate in the Topical Treatment of Psoriasis 376 Vanaja.K, Shailesh Biradar, Narendra C, Shobha Rani RH and Paradkar AV. SECTION-III BIOTECHNOLOGY   1  Comparison of Cell Surface Glycosylation on Apoptotic Cells 387 Keerthivasan Ambigapathy and Deepak Balaji Thimiri Govinda Raj   2 Packed Bed Column Adsorption of Cr(VI) onto Acid Treated Codium Tomentosum Biomass396 P. Suresh Babu, S. Eswaramoorthi, T.P. Rajesh and B. Anandaraj   3 Biotransformation of Clozapine to Norclozapine an Active Metabolite by Cunninghamella Elegans 404 S. Varalaxmi and M. Vidyavathi.   4  Production of Electricity From Municipal Waste Water Using Microbial Fuel-Cells 411 J. Jayabarath, K. Gobika, R. Yuvashri and K. Jeyaprakesh   5 Production of Antimicrobial Secondary Metabolites From Marine Associated Fluorescent Pseudomonas418 J. Jayabarath, J. Jeny Joseline and S. Sivakavi,   6 Hla G 14Bp Indel Polymorphism Implicated in Genetic Predisposition to Asthma in Kodaikanal 426 Renuka S, Thenmozhi M and V.J. Kavitha   7  Production and Characterization of Biosurfactant From Hydrocarbon Contaminated Soil 431 S. Monisha, T. Supassri, M. Dhivya, D. Venkatesan, P. Selvamani and S. Latha   8  Optimization of Media for the Production of Keratinase Enzyme 440 B. Anandaraj, T.P. Rajesh, N. Ilavarasan and P. Saranya Devi   9 Enhancementofpost-Harvest and Shelf Life of Carica papaya Using Aloe Vera Gel Based Coatings 450 Nandakumar. A, Ebenezer Samuel King. J and Arulvel. R 10 Biological Control of Damping Off and Stem Rot of Tomato (Lycopersicon Esculentum Mill.) Using an Actinomycete, Saccharopolyspora Erythraea457 Prabha S.B and K. Murugesan 11  Exploration for Antimicrobial Proteins From Marine Bivalve 467 G. Vinoth Kumar, M. Arputha Bibiana, C. Sherlina Daphny, P. Selvamani and S. Latha SECTION-IV PHARMACEUTICAL TECHNOLOGY   1 Phytochemical Investigation and Invitro Anti Diabetic Activity of Myxopyrum serratulum A. W. Hill K. Kavitha, K. Sujatha, S. Manoharan and Sundara Ramaprabhu

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Contentsxiii 2 In Vitro Antioxidant Activities, Total Flavonoids and Total Phenolic Content of Ethanolic Extract From Whole Plant of Lactuca Runcinata (Dc). 482 Jeyaraman Amutha Iswarya Devi and Arumugam Kottai Muthu 3  GC-MS Characterization of Volatile Odorous Compounds In Allium Cepa488 N.C.J. Packia Lekshmi, S. Viveka, M.B. Viswanathan, G. Manivannan and T. Mini Shobi 4 Antimicrobial Activity Study of Flavonoids and Salicylic Acid Extracted From Tagetes Erecta Linn.495 R. Devika and Y. Justin Koilpillail 5 Therapeutic Potential of Pleurotus ostreatus: An Edible Mushroom in Human Cancer Cell Line (Mcf-7) and In Rat Mammary Carcinoma 500 K. Deepalakshmi and S. Mirunalini 6 A Single Chemical Entity to Balance Anti-Amyloid and Anti-Cholinesterase Capacity: In Silico Drug Design 508 Pavadai Parasuraman, Ramalingam Suresh and Manathusamy Gopalakrishnan 7 A Calcium Channel Blocker - Amlodipine Attenuates Acetic Acid Induced Ulcerative Colitis in Mice 514 Rajinikanth. B and Venkatachalam. V 8 Antioxidant Activity and Mosquitocidal Activity of Allium Sativum of Kodaikanal Hilly Areas 521 M. Razia, K. Lavanya and B. Sri Shaila Devi 9  Phytochemical and Antimicrobial Investigations In Pseudarthria Viscida (Fabaceae) 527 S. Tamil Selvi, M. B. Viswanathan, and M. Venkatesan 10 Extraction and Antimicrobial Evaluation of Buffer Extracts of Marine Invertebrate Donax Cuneatus532 M. Arputha bibiana, C. Sherlina Daphny, M. Umamageshwari, P. Selvamani, S. Latha 11 Evaluation of Anticholinesterase Activity of Cadaba Indica by TLC Bioautography Method and Isolation of Active Phytoconstituents By Bioassay Guided Fractionation 540 P.S. Dhivya, P. Selvamani, S. Latha, M. Omrohini and M. Sobiya 12 Energy Conservation Opportunities In Paper Plant Using Combined Heat and Power for Pharmaceutical Application 546 N. Stalin 13 Energy Management and Integration of Pem Fuel Cell With UAV for Pharmaceutical Application 555 N. Stalin 14  Pharmacognostical Evaluation of Duranta Repens Leaves 567 Emdad Hossain, Ranadheer Rai and Rishikant Tripathi 15 Screening of Aegle Marmelos for Antidiabetic Activity In Streptozotocin Induced Diabetic Rats 575 S. Jebaseelan, Dr. P. Ramasubramanian and Dr. Venkatesh 16  Anthelminthic Activity of Acalypha Indica Belongs to The Family Euphoriaceae582 S. Kalimuthu, V. Balasubramanian, R. Xavier Arulappa 17 Spectroscopic Studies of Plant Extract of Ixora Coccinea (L.f) Belongs to the Family Rubiaceae 590 V. Balasubramanian, S. Kalimuthu, N. Balakrishnan 18  Diuretic Activity of Ethanolic Extract of Aristolochia Indica Linn. In Rats. 606 R. Mohan Kumar, M. Balakumaran, P. Selvamani, N. Subramanian and K. Ruckmani

xivContents 19 Invitro Antioxidant and Hepatoprotective Activity of Ethanolic Extract of Sesbania Grandifolia Linn Leaves K. S. Sridevi Sangeetha and S. Umamaheswari 20  Synthesis and Characterization of Novel Amino Acid Prodrug of Rosiglitazone S. Vijayaraj, G. Kalyana Chakravarthi and R. Shanmugam 21  Assessment of Carcinogenicity Potential By In Vitro Methods: Towards Better Strategy Darshan T. Valani, S. Rajesh Sundar, Mukul R. Jain and Sonal S. Bakshi 22  Antimicrobial Activity of Asystasia Gangetica (L.) T. Anderson Flower Extracts R. Jayalakshmi, P. Selvamani, K. Ruckmani and N. Subramanian 23 Computational Graphical User Interface Tool Development for Personalized Drug Selectivity and Designed Pharmacology M. Poornima, P. Selvamani and S. Latha

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Preface Multidisciplinary knowledge and skills are required to bring an approved drug product for clinical use which includes identification of molecular structures, creation of active molecules and development of drug delivery vehicles for comprehensive therapies. Recent understanding of chemistry and complex biology has served as a tool for enormous development of biotechnology which plays an important role to eradicate disease, to maintain good health and to provide essential requirements for all the life forms. Nanotechnology is a revolutionary technology of the 21st century and found applications in all facets of life. Products of nanotechnology are expected to modernize current trends in drug delivery and molecular medicine which will have significant contributions on the future healthcare system. Under the realm of unimaginable breakthroughs occurring in bio and nanotechnologies with promising applications in the area of drug development and delivery generated great research thrust on “Applications and Perspectives of Nanobio Pharmaceutical Technology.” We made an attempt to bring down the expertise of renowned academic and industrial researchers here to provide a comprehensive treatise on this subject through the TEQIP II and DBT, New Delhi sponsored International Conference on Innovations and Future Research Dimensions on Nanobio Pharmaceutical Technology. It is organized by the Department of Pharmaceutical Technology of the Centre for Excellence in Nanobio Translational REsearch (CENTRE) of the Anna University in its Bharathidasan Institute of Technology Campus, Tiruchirappalli, Tamil Nadu, India. Among the research reports attracted by the conference, selected research reports in the form of full papers has been incorporated in this book under various sections namely, Nanomaterials, Nano Drug Delivery Systems, Biotechnology & Pharmaceutical Technology. We hope this compilation of recent research reports will offer newer opportunities for the realization of the promises of Nanobio Pharmaceutical Technology.

Latha Subbiah Selvamani Palanisamy Subramanian Natesan

Acknowledgments We thank the authorities of Anna University, for providing us with the permission and assistance through TEQIP II grants to produce this proceedings based on the International Conference on Innovations and Future Research Dimensions on Nanobio Pharmaceutical Technology. The editors acknowledge the assistance of their doctoral scholars for their editorial assistance of the received research reports. Specifically, we thank all the individuals who have contributed papers and those who took time from their busy schedules to review these papers and for their valuable comments and helped to shape this book. We are grateful to M/s. Bridge People Consultancy Services Pvt. Ltd., Chennai and Grammarly INC; that helped us to remove many errors of grammar, spelling and related errors in all the published research reports. Also, we are truly indebted to our family members for their encouragement, patience, and support during all of our professional endeavors. Finally, we appreciate the support extended by Mr. K.V.C. Satish Kumar, Knowledge Curve, Chennai and Elsevier for the fine production of this proceedings.

Latha Subbiah Selvamani Palanisamy Subramanian Natesan

SECTION-I NANOMATERIALS

In Vitro Antimicrobial Efficacy of Zinc Doped Nano Hydroxyapatite against Human Pathogens R. Baskar1, G. Devanand Venkatasubbu2 and K. Umamaheswari3 Department of Biotechnology, University of Madras, Guindy Campus, Chennai-600025, TN 2, 3 Centre for Biotechnology, Anna University, Chennai-600025, TN e-mail: [email protected], Mob: 09940326510

1

Abstract: Hydroxyapatite (HA) nano crystals were synthesized by wet chemical precipitation reaction and doped with Zinc (Zn) ions at 2 % and 5 % concentration. The morphology, crystallinity and Zn incorporation into HA nanocrystals were studied using High Resolution Transmission Electron Microscopy (HRTEM), X-ray Diffraction (XRD) and Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) respectively. The Minimum Inhibitory Concentration (MIC) of Zn-HA nanocrystals was determined against various human pathogens by micro broth dilution assay. The synthesized Zn-HA nanocrystals exhibited a uniform morphology with size of 10-20 nm and crystalline in nature. The weight percentage of Zn in 2% and 5 % Zn-HA nanocrystals were found to be 0.4990 and 1.3740 respectively. The Zn-HA nanocrystals showed inhibition against E. coli, P. aeruginosa, K. pneumonia, S. aureus and C. albicans at a concentration between 15 – 500 µg/mL. The results of the present study shows that 5 % Zn-HA nanocrystals shows good antimicrobial activity than 2 % Zn-HA nanocrystals. Keywords: Hydroxyapatite, Zinc, Antimicrobial activity, Nanocrystals

INTRODUCTION Nanoscale structures and materials have been explored in many biological applications because their novel properties and functions drastically differ from their bulk counterparts. In particular, their high volume/ surface ratio, surface tailorability, improved solubility and multifunctionality open many new possibilities for biomedical application [1]. Calcium phosphate nanoparticles have gained increasing interest in medicine because of their high biocompatibility and good biodegradability which is because calcium phosphate is the inorganic mineral of human bone and teeth [2]. Hydroxyapatite (HA), Ca10(PO4)6(OH)2, is one of the most stable forms of the calcium phosphates. The recent trend in biomaterials research is focused on overcoming the limitations of HA ceramics (low bioresorbability, surface area and bioreactivity) and on improving their biological properties by exploring the unique advantages of nanotechnology. The use of nano-HA in orthopedics is considered to be very promising, due to its dimensional similarity with the bone crystals [3]. HA coated implant is more susceptible to bacterial infection as the micro-structure surface which is beneficial for osseointegration, could also become a reservoir for bacterial colonization [4]. However proteins, amino acids, and other organic substances are adsorbed easily on HA, which in turn favors the adsorption and replication of the bacteria in HA [5]. In order to reduce the incidence of implant-associated infections, several biomaterial surface treatments have been proposed [6]. It has been reported that incorporation of trace ions such as Ag, Zn, Ti, and Cu into HA structures not only provides crystallinity, but also improves their antimicrobial property [7]. Among the various elements that can be substituted, zinc (Zn) seems to be a potential candidate [8]. Zn is an essential element for cells. However, its increase above certain threshold level has been shown to inhibit some of the bacterial enzymes,



Nanobio Pharmaceutical Technology

including NADH dehydrogenase and protective enzymes such as thiol peroxidase and glutathione reductase [9]. Since, Zn ions has both direct proliferative effect on bone tissues and excellent antimicrobial effects it would be the suitable candidate to dope with HA nanoparticles to improve its biological potentials. In this study, Zn-HA nanocrystals were synthesized using a simple wet chemical precipitation reaction using two different weight percentage concentrations of Zn ions. The antimicrobial efficacy of the synthesized nanocrystals against various human pathogens was tested quantitatively by determining Minimum Inhibitory Concentration (MIC).

MATERIALS AND METHODS Preparation of Zinc doped Hydroxyapatite Nanocrystals The nanocrystals of HA were prepared involving the following reaction by wet chemical precipitation method: 10Ca (OH)2 + 6 H3PO4 → Ca10(PO4)6(OH)2 + 18H2O Calcium hydroxide and Orthophosphoric acid were used as precursors for the preparation of HA nanocrystals. One liter of an aqueous suspension of 85% orthophosphoric acid (0.6 M) was slowly injected to one litre of an aqueous suspension of calcium hydroxide (1.0 M) with continuous stirring for 2 h at room temperature. The final pH of the solution was adjusted to 11. The solution was centrifuged and washed using deionized water and dried [10]. Zn ions were doped with HA nanocrystals by adding zinc nitrate to a solution with Zn concentration of 2 and five Wt. %.

Characterization of Zinc doped Hydroxyapatite Nanocrystals The synthesized Zn-HA nanocrystals were characterized for their crystalline phase using powder X-ray diffractometer (Seifert, JSO-DE BYEFLEX 2002, Germany).The average size and shape of the sample were determined by High Resolution Transmission Electron Microscope (JEOL 2000Fx-II, Tokyo, Japan). The samples were analyzed by ICP-OES spectroscopy (Perkin Elmer Optima 5300 DV) to determine the amount of Zn present in the 2 % and 5 % Zn-HA nanocrystals.

Antimicrobial Testing by Microbroth dilution Technique The antimicrobial activity of 2% and 5% Zn-HA nanocrystals were tested against five bacterial and one fungal strains including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus aureus and Candida albicans. Minimum Inhibitory Concentration (MIC) was determined as per Clinical and Laboratory Standards Institute (CLSI) guidelines. Muller-Hinton broth and RPMI 1640 was used as a medium. The Zn-HA nanocrystals were serially diluted and were inoculated with a suspension of standard inoculum whose density was adjusted to that of 0.5 McFarland standard and were incubated at 37°C for 24 h. MIC80 was recorded as the lowest concentration of the nanocrystals at which growth was inhibited by 80% in an ELISA reader (Bio-Rad, [11]).

RESULTS AND DISCUSSION The formation of single phase nanocrystals of Zn-HA was confirmed from the XRD pattern shown in Figure 1a and 1b. The spectrum matches with the JCPDS values (09-0432) and the major peaks indicate the crystalline form. The crystalline size of the pure HA crystals was calculated to be 52.00 nm using Scherrer formula. The peak in the XRD pattern of Zn-HA is identical to the XRD pattern of pure HA, and no other crystalline phase is detected. As Zn concentration increases, the XRD peak of the samples become broader indicating lower crystallinity due to the addition of Zn.

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Figure 1: XRD pattern of (a) 2% Zn-HA nanocrystals (b) 5% Zn-HA nanocrystals

The morphology of the 2 % and 5 % Zn-HA nanocrystals were shown in Fig. 2a and 2b. The nanocrystals were needle-like in shape with sharp edges, and its average sizes range from 10 to 20 nm. The nanoparticles are crystalline as seen from XRD. As the concentration of Zn increases, agglomeration of particles also increases.

Figure 2: HRTEM image of (a) 2% Zn-HA nanocrystals (b) 5% Zn-HA nanocrystals.

The total amount of Zn present in the 2 % and 5 % Zn-HA nanocrystals were calculated using ICP-OES spectroscopy and the weight percentage of Zn ions were tabulated in Table 1. The amount of Zn increases with the increase in the amount of Zn ions added in the mother liquid. Whereas the Zn content of the final samples were lower than those of a corresponding amount of starting material that implies that some of the Zn ions remain in the mother solution after precipitation. Table 1: ICP-OES values of (Weight %) of Zn in Zn-HA nanocrystals.

S. No 1. 2.

Sample 2% Zn-HA nanocrystals 5% Zn-HA nanocrystals

Zn Concentration (Weight %) 0.4990 1.3740

Antimicrobial property is one of the most considerable characters of any nanomaterials that are predominately applied in biomedical applications. The in-vitro antimicrobial efficacy of synthesized Zn-HA was assayed quantitatively using microbroth dilution method and the minimum inhibitory concentration 80 % (MIC80) was determined. The MIC values ranged between 15 µg/mL and 500 µg/mL for both the

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Nanobio Pharmaceutical Technology

bacterial and fungal strains tested. The concentration-dependent inhibition was observed. The MIC80 values of 2 % and 5 % Zn-HA nanocrystals were shown in Table 2. C. albicans and K. pneumoniae followed by S.aureus were more susceptible to both 2 % and 5 % Zn-HA nanocrystals whereas P. aeroginosa, and E. coli was less susceptible. The reports on antimicrobial activity of Zn-HA nanoparticles were limited, and the previous studies were based on the principle of diffusion. Thian et al. [8] reported the 6-log reduction in the numbers of S. aureus treated with ZnHA at the day six from the log reduction assay whereas Stanic et al. [7] reported that S. aureus were less susceptible than E. coli to the Zn-HAP. Our study shows that S. aureus was more susceptible than E. coli. To Zn-HA nanocrystals. The results of the present study on the antimicrobial activity of the Zn-HA nanocrystals correlate well with the earlier documented reports. The antimicrobial mechanism of Zn-HA nanocrystals may be due to the formation of bonds between the Zn ions and cell membrane proteins, thereby causing structural changes causing cell death. Table 2: Minimum Inhibitory Concentration (MIC) (µg/mL). S. No

Organism Tested

1.

Escherichia coli

2. 3. 4. 5.

Pseudomonas aeroginosa Klebsiella pneumoniae Staphylococcus aureus Candida albicans

MIC80 (µg/mL)

2% Zn-HA nanocrystals 453.0 500.0 250.0 343.7 66.4

5% Zn-HA nanocrystals 427.8 468.0 218.7 234.3 16.5

CONCLUSION The present study involves wet chemical precipitation method for the synthesis of Zn-HA nanocrystals due to its high reproducibility and simplicity. The nature of nanoparticles was evident from the results of XRD, HRTEM and ICP-OES. The synthesized particles were crystalline and needle-shaped, and the size ranged from 10-20 nm. The antimicrobial efficacy of Zn-HA nanocrystals was studied against human bacterial and fungal pathogens using Microbroth dilution assay. The result showed that 5 % Zn doped samples inhibit the growth at lower concentration than 2 % Zn doped samples against all the tested pathogens. Furthermore, in vivo studies on microbial toxicity and cytotoxicity of Zn-HA nanocrystals will give better understanding about the long-term performance and use of HA as effective ceramic for biomedical applications.

REFERENCE 1. Gao J and Xu B, Applications of nanomaterials inside cells, Nano Today 2008 Oct 29; 4:37-51. 2. Epple M, Ganesan K, Heumann R, Klesing J, Kovtun A, Neumann S and Sokolova V, Application of calcium phosphate nanoparticles in Biomedicine, Journal of Material Chemistry 2010; 20:18-23. 3. Roveri N and Iafisco M, Evolving application of biomimetic nanostructured Hydroxyapatite, Nanotechnology, Science and Applications 2010 Nov 8; 3(1):107–125. 4. Saidin S, Chevallier P, Abdul Kadir MR, Hermawan H and Mantovani D, Polydopamine as an intermediate layer for silver and hydroxyapatite immobilisation on metallic biomaterials surface, Materials Science and Engineering: C 2013 Dec 1; 33(8):4715-4724. 5. Rameshbabu N, Sampath Kumar TS, Prabhakar TG, Sastry VS, Murty KVGK and Prasad Rao K, Antibacterial nanosized silver substituted hydroxyapatite: Synthesis and characterization, Journal of Biomedical Material Research A 2007 Mar 1; 80:581–591.

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6. Chen W, Liu Y, Courtney HS, Bettenga M, Agrawal CM Bumgardner JD and Ong JL, In vitro antibacterial and biological properties of magnetron co-sputtered silver-containing hydroxyapatite coating, Biomaterials Jul 26 2006; 27:5512–5517. 7. Stanic V, Dimitrijevic S, Stankovic JA, Mitric M, Jokic B, Plecas IB and Raicevic S, Synthesis, characterization and antimicrobial activity of copper and zinc-doped hydroxyapatite nanopowders, Applied Surface Science 2010 Apr 1; 256:6083-6089. 8. Thian ES, Konishi T, Kawanobe Y, Lim PN, Choong C, Ho B and Aizawa M, Zinc-substituted hydroxyapatite: a biomaterial with enhanced bioactivity and antibacterial properties, Journal of Materials Science: Materials in Medicine 2013 Nov 16; 24:437–445. 9. Kumar A, Pandey AK, Singh SS, Shanker R and Dhawan A, Engineered ZnO and TiO2 nanoparticles induce oxidative stress and DNA damage leading to reduced viability of Escherichia coli, Free Radical Biology & Medicine 2011Aug 28; 51 :1872–1881. 10. Pataquiva Mateus AY, Ferraz MP and Monteiro FJ, Microspheres based on hydroxyapatite nanoparticles aggregates for bone regeneration, Key Engineering Materials 2007; 330-332:243-246. 11. Annadhasan M, SankarBabu VR, Naresh R, Umamaheswari K and Rajendran N, A sunlight-induced rapid synthesis of silver nanoparticles using sodium salt of N-cholyl amino acids and its antimicrobial applications, Colloids and Surfaces B: Biointerfaces 2012 Apr 8; 96: 14– 21.

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Synergistic Antibacterial Efficacy of Two Drugs in Addition with Biosynthesized AgNPs from an Allergenic Fungus Chitra N,1 B.K. Nayak* and Anima Nanda Department of Botany, K. M. Centre for P.G. Studies (Autonomous), (Govt. of Puducherry), Lawspet, Pondicherry-605008, India Department of Biomedical Engineering, Sathyabama University, Rajiv Gandhi Salai, Chennai - 600119, India *Corres. author e-mail: [email protected], Tel.: +91 9443653844

1

Abstract: Efficacy of silver nanoparticles in order to control the growth and epidemics of pathogenic bacteria has been known from time immemorial. In recent days, bacteria are making themselves resistant to varied antibiotics based on their genetic affinities. In the present study, a green, simple and effective approach was performed to synthesize potent silver nanoparticles (AgNPs) from an airborne allergenic fungus, Alternaria sp. using both reducing and stabilizing agent. The appearance of yellowish brown color in the conical flask suggested the formation of AgNPs. The supernatant of the fungus culture changed the solution into brownish color upon the completion of 10 minute reaction. The characterization of silver nanoparticles was confirmed by Uv-VIS spectrophotometer, Field emission scanning electron microscopy (FESEM) and XRD analysis. Size of the nanoparticles measured between 20 nm to 30 nm by FESEM. Silver nanoparticles showed good antimicrobial activity against the selected pathogens, but when the nanoparticles were tested against the test pathogens in combined with the two drugs viz., Amoxyclav/Clavulanic Acid (30 mcg) and Oxacillin (1 mcg); the efficacy of the drugs was increased at some extent by one fold in the case of Oxacillin. Keywords: AgNPs, Alternaria sp., FESEM, XRD, Uv-vis Spectrophotometer

INTRODUCTION Nanosized particles have attracted worldwide attention in recent times due to their promising interdisciplinary fields of science that offers valuable nanomaterials having wide application in a range of areas, including catalysis, optics, mechanics, magnetics, energetics, and biomedical sciences [1]. Compared to physical and chemical process, biological methods have an increasing interest because of the necessity to develop new clean, cost-effective and efficient synthesis techniques. Synthesis of nanoparticles by biological systems such as bacteria, fungi, yeast and several plant extracts have been investigated due to their ability to reduce metal ions [2]. Silver nanoparticles (AgNPs) have drawn special attention owing to its immense potential as an antimicrobial agent in biomedical and other health care applications. They are an attractive option because they are nontoxic to the human body at low concentrations and have broad spectrum antibacterial actions. Silver ions are very reactive and are known to bind to the vital cell components, inducing cell death [3]. Duran et al. [4] described the extracellular synthesis of silver nanoparticles from Fusarium oxysporum strain of fungus by the presence of the hydrogenase enzyme that had exceptional redox properties, acting as an electron shuttle for reduction of metal ions. Fungal strains have the ability to resist environmental stresses and have the capability of growing in the presence of high metal concentrations.

Nanobio Pharmaceutical Technology

The aims and objectives of the recent study is to biosynthesize silver nanoparticles by extracellular method from an aero-allergenic fungus, Alternaria sp. isolated from vegetable market in order to confirm the formation of silver nanoparticles by UV-vis spectroscopy followed by various microscopic characterization and to evaluate its efficacy as a bactericide in order to combat the growth of selected bacterial pathogens viz., Staphylococcus aureus, Bacillus cereus, Proteus vulgaris, E. coli and Vibrio cholerae.

MATERIALS AND METHODS Isolation of Alternaria sp. The airborne fungi were collected from indoors of a vegetable market by exposing Sabouraud Dextrose agar for 5 minutes on media plates based on gravitation method and the plates were incubated in BOD incubator at 25 ± 3°C for 3  7 days in the Microbiology research Laboratory, Sathyabama University, Chennai for their enumeration. Alternaria sp was isolated and identified from the mixed culture of airborne fungi [5,6] put on pure culture and stored in a refrigerator at 4°C for further studies.

Synthesis of Silver Nanoparticles Isolated Alternaria sp. fungus was subjected to biosynthesis of silver nanoparticles. Fungal biomass was grown aerobically in a specific liquid medium containing (g/L): KH2PO4 7.0; 2.0 K2HPO4 MgSO4. 7H2O 0.1; (NH4)2SO4 1.0; yeast extract 0.6; glucose 10.0 at 25 ± 3°C and incubated at 25°C in a shaker at 140 rpm for 72 hours. After incubation, the biomass was filtered using Whatman filter paper No.1 and extensively washed with distilled water to remove all residual media components. The resulting fresh and clean biomass was taken into the Erlenmeyer flasks, containing 100 ml of deionized Milli-Q water. The flask was again incubated at 25° C in a shaker at 140 rpm for 72 hours. The biomass was filtered again with Whatman filter paper No.1, and the cell free extract was used in the following experiment. 1mM AgNO3 was prepared, and 50 ml was added to the cell free extract and kept in a dark condition for 48 hrs.

Characterization of silver nanoparticles The solution in the flask was observed for color change and maximum absorbance was analyzed using UV-visible spectrophotometer. 1 ml of sample supernatant was taken after 24 hours and absorbance were measured by using UV-visible spectrophotometer (T-60, PG Instruments Ltd. Lutterworh, United Kingdom) between 300-600 nm. FESEM analysis was used to determine the surface morphology and particle size of the silver nanoparticles. The AgNPs samples were sonicated and later centrifuged at 15000 rpm for 20 minutes. Before the process for FESEM analysis, the samples were further sonicated to get the uniformity and better observation. Later the supernatant were discarded, and pellet was washed with the Milli-Q water for three to four times. Later on the sample were transferred into the Petri plate and dried for about two hours at 50° C, after that the sample were subjected to FESEM analysis. XRD analysis was used to determine the crystallinity, and metallic nature and face centered cubic structure of silver nanoparticles. For XRD analysis, the sample was prepared by centrifugation of the silver nanoparticle solution at 15000 rpm for 20 minutes. The supernatant was discarded, and the pellet was washed with Milli-Q water three to four times and then dried in petri plates. The powder form of the sample was subjected for XRD analysis at International Research Centre, Sathyabama University, Chennai, Tamilnadu, India.

Antibacterial study of AgNPs The silver nanoparticles were checked for its antibacterial activity by disc diffusion method [7]. The antimicrobial activity of the prepared silver nanoparticles from Alternaria sp was tested against the pathogenic bacteria such as Staphylococcus aureus, Bacillus cereus, Proteus vulgaris, Vibrio cholerae and Escherichia

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coli. The Amoxyclav (AMC-30mcg) and Oxacillin (OX-1mcg) were taken separately as control parallel to the AgNPs to find a comparative assessment of the antibiotic efficacy over the pathogenic bacteria. The combined effects of AgNPs with antibiotics were used to find out synergistic effect against above bacterial pathogens. The zone of inhibition was measured after overnight incubation at 37° C.

RESULTS AND DISCUSSION The fungal strain, Alternaria sp used in this study was isolated from indoor air of the vegetable market and used for the biosynthesis of silver nanoparticles. AgNPs were synthesized by the reaction of Ag+ ions from AgNO3 with the supernatant of Alternaria sp under dark conditions. After 48 h incubation, appearance of yellowish brown color in the conical flask indicated the formation of AgNPs [8]. The supernatant of the Alternaria sp culture changed the solution to a brownish color upon completion of the 24 h reaction with Ag+ (Fig.1)

Figure 1: Synthesis of silver nanoparticles from Alternaria sp.

The AgNPs were characterized by Uv-vis spectroscopy, which has proved to be very useful for the analysis of nanoparticles. As illustrated in Fig.2, Uv-Vis spectra, a strong surface plasmon resonance were centered at approximately 430 nm indicating the presence of silver nanoparticles. The exact mechanism for the synthesis of silver nanoparticles has not been clear yet, but it has been attributed that the presence of NADH dependent nitrate reductase enzyme in the fungal biomass is responsible for the reduction reaction. When the silver ions come in contact with the cell wall of the fungal biomass, the nitrate reductase secreted by the fungus causes the reduction of silver ions into silver nanoparticles [9].

Figure 2: UV–vis spectrum of silver nanoparticles synthesized from Alternaria sp.

Field emission scanning electron microscopy (FESEM) was used to understand the surface topology and the size of silver nanoparticles. Analysis of AgNPs by FESEM showed spherical shaped silver nanoparticles which were well dispersed within the diameter ranges of 34 nm and 47 nm (Fig.3).

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Nanobio Pharmaceutical Technology

Figure 3: FESEM analysis of silver nanoparticles synthesized from Alternaria sp.

These biologically synthesized silver nanoparticles were further characterized by X-ray diffraction (XRD) technique to determine the metallic nature of nanoparticles. The XRD pattern clearly showed that silver nanoparticles have been formed resulting in the diffraction peaks at 38, 45, 64 and 77 respectively confirming the metallic nature of nanoparticles and peak was specific for the silver nanoparticles (Fig.4). The results obtained were similar to the earlier studies made by the following workers [10, 11].

Figure 4: XRD analysis of silver nanoparticles synthesized from Alternaria sp.

The antimicrobial activity of synthesized silver nanoparticles was studied by disc diffusion method against different clinically isolated pathogens viz., Staphylococcus aureus, Bacillus cereus, Proteus vulgaris, Vibrio cholerae and Escherichia coli. Synthesized silver nanoparticles showed good antimicrobial activity against the selected pathogens except Proteus vulgaris. While the antibiotics, Amoxyclav and Oxacillin, on their own didn’t show any impressive result over the test pathogens, the combined formulations of AgNPs with antibiotics showed remarkable results. Each disc is impregnated with 25µg silver nanoparticle solution. Vibrio cholerae was found to be more susceptible followed by Bacillus cereus in the combined formulation of Oxacillin and AgNPs (Table 1).

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Nanobio Pharmaceutical Technology

Table 1: Zone of inhibition (mm), Antibacterial Activity of AgNPs against bacterial pathogens Pathogens

AgNPs

AMC

AMC+AgNPs

OX

OX+AgNPs

Staphylococcus aureus

14

10

15

07

15

Bacillus cereus

15

08

18

10

17

Proteus vulgaris

13

09

15

07

15

Escherichia coli

18

11

18

08

11

Vibrio cholerae

18

10

17

20

25

The antimicrobial studies showed that combination formulation of Oxacillin and AgNPs were significantly effective compared to Amoxyclav and AgNPs combination. The studies confirmed that the biologically synthesized AgNPs from Alternaria sp amplified the antibacterial property of commercial antibiotics when used in combination. Further investigation is required to study its effect in vivo cytotoxicity for accessing its biocompatibility before administrating as antimicrobial drug for animals and human beings.

CONCLUSION During our study, in vitro biosynthesis of silver nanoparticles was made by extracellular method from Alternaria sp. Synthesized silver nanoparticles showed good antimicrobial activity against the selected pathogens and its activity were further enhanced in combination with antibiotics. Hence it may be concluded that combined formulation of available drugs with silver nanoparticles would be an alternative approach in order to treat the multi drug resistant pathogenic bacteria and also to minimize the antibiotic doses to cure the dreaded diseases.

REFERENCE 1. Chauhan A, Zubair S, Tufail S, Sherwani A, Sajid M, Raman SC and Azam A, Fungus-mediated biological synthesis of gold nanoparticles: potential in the detection of liver cancer, Int J Nanomed, 2011; 6: 2305–19. 2. Ghosh S, Patil S, Ahire M, Kitture R, Gurav DD, Jabgunde AM, Kale S, Pardesi K, Shinde V, Bellare J, Dhavale DD and Chopade BA, Gnidia glauca flower extract mediated synthesis of gold nanoparticles and evaluation of its chemocatalytic potential, J Nanobiotech, 2012; 10: 1–9. 3. Kulkarni N and Muddapur U, Biosynthesis of Metal Nanoparticles: A Review, J Nanotech 2014, doi. org/10.1155/2014/510246. 4. Duran N, Marcato PD, Alves OL, De Souza GIH and Esposito E, Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains, J Nanobiotech, 2005; 3: 1–7. 5. Barnett HL and Hunter BB, Illustrated genera of imperfect fungi, 3rd Ed. Burgess Publishing Co. Minnepolis. Minnesota, 1972. 6. Onions AHS, Allsopp D and Eggins HOW, Smith’s introduction to industrial Mycology, London, Edward Arnold, 1986. 7. Bauer AW, Kirby WM, Sherris JC and Turck M, Antibiotic susceptibility testing by a standardized single disk method, Am J Clin Pathol. 1966; 45: 493–96.

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8. Majeed S, Nanda A and Thirunavukarasu K, Evaluation of antimicrobial activity of biologically synthesized silver nanoparticles from filamentous fungi, International Journal of PharmTech Research. 2014; 6: 1049–53. 9. Duran N, Marcapto PD, Duran M, Yadav A, Gade A and Rai M, Mechanistic aspects in the biogenic synthesis of extracellular metal Nanoparticles by peptides, bacteria, fungi and plants, Appl Microbiol Biotechnol, 2011; 90: 1609–24. 10. Kathiresan K, Manivannan S, Nabeal MA and Dhivya B, Characterization and antibacterial analysis of silver nanoparticles synthesized by a marine fungus, Penicillium fellutanum, isolated from coastal mangrove sediment, Colloids Surf B Biointerfaces 2009; 71: 133-7. 11. Ingle A, Rai M, Gade A and Bawaskar M, Fusarium Solani: A navel biological agent for extracellular synthesis of silver nanoparticles.

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Synthesis and Characterization of Pamam Dendrimer and its Application for Removal of Heavy Metals E. Gomathi1, P. Rajesh Prasanna2 and P. Selvamani3 Department of Petrochemical Technology, 2Department of Civil Engineering, 3Department of Pharmaceutical Technology, BIT Campus - Anna University, Tiruchirappalli – 620 024, Tamil Nadu, India e-mail: [email protected]

1

Abstract: The objective of the present study is to synthesize silica based polyamidoamine (PAMAM) dendrimer with ester and amino groups at the outer surface. The synthesis of dendrimer is usually carried out in two ways namely convergent technique and divergent technique. PAMAM is normally synthesized by divergent methods starting from ammonia or ethylenediamine initiator core reagents. Amine terminated PAMAM dendrimers exhibit a high affinity for adsorption of metal ions to their surface via co ordination to the amine or the acid functionality. The structures were characterized by FTIR, SEM and AAS. FTIR methods were employed to monitor amidation reaction in order to judge the optimum reaction time. The experiments showed that both ester- and amino-terminated dendrimer-like PAMAM grafted silica-gel exhibited better adsorption capabilities for base metal ions: Cr, Hg,. It was shown that the adsorption data of the composite could be fitted using the Langmuir equation with a maximum adsorption capacity. Keywords: Silica-gel, Polyamidoamine-typed hyper branched polymer Preparation, Adsorption, Metal Ions.

INTRODUCTION The pollution of water resources due to the disposal of heavy metals has been increasing worldwide concern. High levels of heavy metals can damage soil fertility and may affect the flora and fauna [1]. Industrial effluents discharged from various industries like textile, tannery, sugar processing, dye and distilleries contain higher amount of metals like chromium, copper, nickel, lead, zinc, mercury and cadmium and results in a series of well documented problems in living beings because the heavy metals cannot be completely degraded [2]. Hence it is necessary to treat the effluent to reduce the concentration of heavy metal contamination and different systems like mechanical systems, aquatic systems and terrestrial systems were primarily used [3]. There are four main methods of wastewater treatment: physical, chemical, physical-chemical and biological methods. Despite the large number of methods, most are either too expensive to be applied in small plants or inefficient [4]. Low initial investment, simple design, large quantity of wastewater treatment and ease of use are only some advantages that make sorption one of the most advantageous methods for treatment of waste water containing heavy metals [5] Dendrimers represent a novel class of three –dimensional, highly branched, globular macromolecules, which fall a category of dendritic polymers. [6]. There are generally two synthesizing methods to fabricate dendrimers [7] the divergent method and the convergent method. Both methods can effectively contribute to generation growth. PAMAM dendrimers are dendrimer polymers synthesized with ethylenediamine as a monomer. PAMAM dendrimers have a 3-D stereoscopic structure, with a particle diameter of approximately 2  20 nm. Due to its unique spherical structure, PAMAM dendrimers have some excellent features, such as large internal cavities, high solubility, low viscosity, and the diversification of structure design. The notable features of PAMAM are that both the surface -NH2 groups and internal cavities play important roles in the adsorption of contaminants [8].

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MATERIALS AND METHODS Reagents Reagents included Ethanol, Tetra Hydro furan, methanol, ethylene diamine, methyl acrylate, toluene was purchased from Loba Chemicals, and Amino Propyl Triethoxysilane (APES) was purchased from Synergy Scientific Services. All the other solvents and reagents were of analytical or high performance liquid chromatography grade.

Preparation of Ester and Amino Terminated PAMAM Dendrimers The general scheme of synthesizing ester and amino terminated PAMAM dendrimers is by the introduction of amino groups onto the silica gel surface followed by the Michael’s addition of methyl acrylate to amino groups on the silica gel surface and ends with the terminal ester group amidation by ethylene diamine, given as [8, 9, 10, 11].

Preparation of SiO2-G0 Under an N2 atmosphere the reaction was carried out with 50.0 g of silica-gel and 50 ml of APES were stirred at 700 C in the 150 ml of toluene solution for 6 hours in a round bottomed flask mounted on a magnetic stirrer provided with a heater. The rate of mixing was controlled by the RPM controller provided. Condensing system was used to prevent toluene loss due to vaporization, since it is a highly volatile compound. The product was then filtered off, packed in a thimble bag and then transferred to a Soxhlet extraction apparatus for reflux-extraction in toluene and ethanol for 10 hours, respectively. The product thus extracted was dried under vacuum at 500 C over 48 hours.

Preparation of SiO2-G0.5 A mixture of 40 g of SiO2-G0 and 31 ml of Methyl Acrylate (MA) were added to a 500 ml flask with 240 ml of methanol as solvent. The mixture was stirred at 500 C for 3 days to react sufficiently under nitrogen atmosphere condition, brought about by purging the round bottomed flask with nitrogen gas after the reactants are added followed by the continuous passage of nitrogen gas into the round bottomed flask, at the time of reaction. The solid product was then filtered off, packed in a thimble bag, and transferred to the Soxhlet extraction apparatus for reflux extraction in ethanol and tetrahydrofuran for 24 hours, respectively. After extracting, the product was dried under vacuum at 500C over 48 h and SiO2-G0.5 was obtained.

Preparation of SiO2-G1.0 The reaction was carried out under a nitrogen atmosphere, a suspension of 30 g of SiO2-G0.5 and 300 ml of EDA was stirred at room temperature about 250 C in a flask using 200 ml of methanol as solvent for 5 days. The product was filtered off, packed in a thimble bag and transferred to the Soxhlet extraction apparatus for reflux-extraction in ethanol and tetrahydrofuran for 24 hours, respectively and then was dried under vacuum at 500 C over 48 hours. The product SiO2-G1.0 was obtained.

Preparation of SiO2-G1.5 Under a nitrogen atmosphere, the reaction involved mixing of a suspension of 19.4 g of SiO2-G1.0 and 62 ml of Methyl acrylate in 120 ml of methanol solvent. The reactant mixture was stirred for 4 days at 500 C and then the product SiO2- G1.5 was filtered off, packed in a thimble bag and transferred to the Soxhlet extraction apparatus for reflux-extraction in ethanol and tetrahydrofuran for 24 hours, respectively and then was dried under vacuum at 500 C over 48 hours. The product SiO2-G1.5 was obtained.

Preparation of SiO2-G2.0 The reaction was carried out under a nitrogen atmosphere and 11.4 g of SiO2-G1.5, 150 ml of Ethylene Diamine were mixed with 70 ml of methanol as solvent. The mixture was stirred at 250 C for 7 days .The

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solid product was then filtered, packed in a thimble bag and transferred to the Soxhlet extraction apparatus for reflux-extraction in ethanol and tetrahydrofuran for 24 hours, respectively and then was dried under vacuum at 500C over 48 hours. The product SiO2-G2.0 was obtained.

Characterization of Ester and Amino Terminated PAMAM Dendrimer Fourier Transforms Infra Red Spectrophotometer (FTIR) FTIR was used to measure changes in the chemical structure of the silica gel based ester and amino group terminated PAMAM dendrimer on which all the functional groups are added in a step wise manner. Infrared spectra were recorded on a Nicolet MAGNA IR 550 (series II) spectrophotometer, using ATR attachment at room temperature. The spectrum was acquired at 400  4000 cm-1 wave numbers with a 4 cm-1 resolution using EZOMNIC 6.0 (Thermo Nicolat) software.

Scanning Electron Microscope (SEM) The shapes and the surface morphology of the samples were examined on a SEM, to make sure that the synthesized particles are of nano size.

RESULT AND DISCUSSION Characterization of the PAMAM Dendrimer Nanoadsorbants have been characterized by using a wide range of techniques. The characterization of nanoadsorbants is a difficult task owing to their complexity, variety of structures and components involved in these systems, as well as the limitations associated with each technique, but such knowledge is essential for their successful commercial exploitation.

Scanning Electron Microscope (SEM) Scanning Electron Microscopy analysis was carried out at 10 Kv. The results of the scanning electron microscopy can be observed in the Figures 1 and 2.The figures reveal that the particle appearances of the G1 and G2 dendrimers were very similar, thus demonstrating that the particles of the dendrimer samples have good mechanical stability, and they had not been destroyed during the whole reaction.

Figure 1: SEM image of G1 PAMAM Dendrimer

Figure 2: SEM image report of G2 PAMAM dendrimer

Fourier transforms infrared spectroscopy The surface attachment of the amino groups on the surface of the silica gel was characterized by FTIR spectrum for the G0 and G 0.5 of the PAMAM dendrimer (Fig. 3 and 4) Thirteen characterization peaks at 3378.84, 3164.05, 3612.23, 3718.89, 3942.27, 3812.66, 2677.37, 1408.30, 1078.38, 1602.21, 727.43, 626.35 and 487.43 were observed to be NH bending (Primary and Secondary amides), NH stretching vibrations (Primary amides and Secondary

16

Nanobio Pharmaceutical Technology amides), C-N stretching band, conforming primary amine, C-N Stretching vibrations indicating primary and secondary amides, OH stretching vibrations showing the vibrations of the SiOH group and the torisonal oscillation of NH3 group.

Figure 3: FTIR Spectrum of G0 PAMAM Dendrimer

Figure 4: FTIR Spectrum of G0.5 PAMAM Dendrimer

Figure 5: FTIR Spectrum of G1 PAMAM Dendrimer

Figure 6: FTIR Spectrum of G1.5 PAMAM Dendrimer

Figure 7: FTIR Spectrum of G2 PAMAM Dendrimer

Characterization of Synthetic Solutions Atomic Absorption Spectroscopy (AAS): Atomic Absorption Spectrophotometer was used to measure the concentration of metal ions were detected by means of AAS. The adsorption amount was calculated according to the equation:

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Nanobio Pharmaceutical Technology Q=

(Co − C )V W

Q- Adsorption amount (mmol/g); C0- Initial concentrations of metal ions (mmol/ml); C- Final concentrations of metal ions (mmol/ml); V- Volume (ml); W- Weight of SiO2-G0–SiO2-G2.0 (g).

Adsorption properties of SiO2-G0–SiO2-G2 Static adsorption experiment was employed to determine the adsorption capabilities of SiO-G0–SiO-G2.0 for different kinds of metal ions. A typical way was that: a dose of desired amount of the metal ions solution were added to a 50 ml Pyrex glass tubes and then the glass tubes were placed in a thermo statcum-shaking assembly. A known amount of SiO-G0–SiO-G2.0 (0.05 g) was charged, and the mixture solutions were mechanically shaken at room temperature for 24 h, then the solutions in the tubes were separated from the adsorbent and the concentration of metal ions were detected by means of atomic absorption spectrometry (AAS).

Adsorption of Mercury The feed taken for mercury adsorption’s studies was mercury synthetic solution. And the initial concentration was found to be 7.036 mg after treated with SiO2-G1.5 and SiO2-G2 was found to be 4.301mg/l and1.153mg/l respectively. Table 1: Shows decrease in concentration of Mercury Generation

Concentration of Mercury (mg/l)

Feed

7.036

SiO2-G1.5

4.031

SiO2-G2

1.153

Figure 8: Generation of Dendrimers Vs Concentration of Mercury

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Adsorption of Chromium Table:2

Generation Feed SiO2-G0.5 SiO2-G1 SiO2-G1.5 SiO2-G2

Concentration of Chromium (mg/l) 0.325 0.275 0.210 0.027 0.043

The Table. 2, shows the decrease in concentration of chromium, when dendrimer generation increases. Comparing all adsorption studies of SiO2-GO.5 to SiO2-G2, it is clear that, dendrimer’s capacity with respect to different metal ions are, mercury >> chromium.

Figure 9: Generation of Dendrimers Vs Concentration of Chromium

CONCLUSION Ester and amino terminated PAMAM dendrimer was synthesized by divergent method of synthesis and they were grafted successfully on the surface of the silica gel. The percentage of grafting of the ester and amino group terminated PAMAM dendrimer on the surface of the silica gel increased with the increase in the number of generations as given by the FTIR reports. SEM analysis report makes it is evident that the size range of the PAMAM dendrimer is around 1nm and the pore surface diameter decreased after the series of grafting reactions. All of the ester and amino terminated PAMAM dendrimer presented regularities in adsorption of metals like chromium and mercury. It was found that the absortion efficiency increased with the generation of dendrimers, further the time to absorb the metals decreases with respect to the generation. The adsorption of ester and the amino terminated products increased with the increase in the increase in the grafting percentage and the addition of the surface functional groups. From this it can be concluded that the amine and the ester groups are alone responsible for the easy and efficient adsorption of the metal ions, the amine terminated groups exhibiting higher adsorption due to its coordination and acid functionality.

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REFERENCES 1. Mohammad Reza Hadj Mohammadi, Mina Salary Removal of Cr from aqueous solution using Pine needles powder as an adsorbant, 6, 1-2, (2011). 2. R. Muhammad and S.S. Ashraf, Chem.Engg.J. 209, 520 (2012). 3. Jameel M. Dhabab, Removal of Fe(II), Cu(II), Zn(II), and Pb(II) ions from aqueous solutions by duckweed, Journal of Oceanography and Marine Science. 2(1), 17-22 ,(2011). 4. K. Xie, W. Zhao and X. He, Carbohydr.polym, 83, 1516 (2011). 5. V.V. Panic, Z.P. Madzarevic, T. Volkov-Husovic and S.J. Velickovic, Chem.Eng.J., 217, 192 (2013). 6. D.A. Tomalia, H. Baker, J.R. Dewald, M. Hall, G. Kallos, S. Martin, J. Roeck, J. Ryder and P. Smith, polym.J., 17, 117(1985). 7. B. Klajnert and M. Bryszewska, Acta, 37, 39 (2004). 8. Chih-Ming Chou and Hsing-Lung Lien., Dendrimer-conjugated magnetic nanoparticles for removal of zinc (II) from aqueous solutions., J Nanopart Res. 13, 2099-2107, (2011). 9. Yuzhong Niu, Haifeng Lu, Dengxu Wang, Yuanzhi Yue and Shengyu Feng, Synthesis of siloxane-based PAMAM dendrimers and luminescent properties of their lanthanide complexes, Elseiver Journal of Organometallic Chemistry, 696, 544-550,( 2011). 10. Rongjun Qu, Yuzhong Niu, Changmei Sun and Chunnuan Ji, Syntheses, characterization, and adsorption properties for metal ions of silica-gel functionalized by ester-and amino-terminated dendrimer-like polyamidoamine polymer, Microporous and Mesoporous Materials, 97, 58–65, (2006). 11. Dey R.K., Tanushree Patnaik, Singh V.K., Sanjay K. Swain and Claudio Airoldi, Attachment of linear poly (amido amine) to silica surface and evaluation of metal-binding behavior, Elsevier Applied Surface Science, 255, 8176–8182, (2009).

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Bio-Toxicity of Silver Nanoparticles Against Multidrug Resistant Pathogens and Human Epidermoid Larynx Carcinoma (HEp-2) Cells Muthukrishnan Lakshmipathy and Anima Nandaa* Faculty of Bio & Chemical Engineering, Sathyabama University, Rajiv Gandhi Road, Chennai, Tamil Nadu 600119, India. * e-mail: [email protected], Tel.: +914424503804, Fax: +914424502344

1

Abstract: The nanotechnological advancement in reducing the size of noble metals (Ag, Au, Ti) with enhanced electrochemical properties has been a breakthrough in materials science research. Biological means of synthesis especially microbe-mediated synthesis of silver nanoparticles (AgNPs) using the soil bacterium, Bacillus subtilis A1 and its bio-toxicity against multidrug resistant strains and DNA damage have been reported. The AgNPs showed maximum absorbance in the range 420 – 450 nm, crystal structure and spherical shaped particles of size ~30.45 nm. The AgNPs showed potential antibacterial activity against multidrug-resistant strains with MIC ≤ 32 μg/mL. Further study on the dose-dependent DNA damage using comet assay revealed cleaved DNA fragments in the form of a comet with olive tail moment 6.4% and 1.1 % respectively at 100 μg/mL and 10μg/mL respectively. This toxicological potential plays a key role in inducing apoptosis, a limiting factor in cell death that could be harnessed for improved biomedical application. Keywords: Bio-toxicity, Silver nanoparticles, HEp-2 cells, MIC, Comet assay.

INTRODUCTION Nanobiotechnology, an interdisciplinary field has opened new vistas converging technological insights of nano applied at bio-molecular level. These wide applications of nanomaterials remain inevitable as far as healthcare industry, and biomedical field is concerned. Besides an application, focus on reliable, ecofriendly, pharmaceutically effective synthesis protocol using biological machinery draws much attention [1]. Micro-organisms, the so-called nature’s treasure have been involved in nullifying the adverse effects of metal ions released in the environment since time immemorial. The hunt for such novel route has attracted scientists across the globe implying microbes for preparing nanoparticles such as silver, gold, titanium, etc. Silver, known for their broad spectrum antibacterial and antifungal activity has been employed in sensors, biomedical imaging, etc. [2]. However, its level of toxicity needs to be assessed. This study investigates on bacteria-mediated synthesis of silver nanoparticles using environmental isolate, and Bacillus subtilis A1 and its antibacterial effect (inhibitory concentration) against multidrug-resistant clinical isolates. Next, the DNA damage induced by silver nanoparticles was demonstrated using single cell gel electrophoresis on HEp-2 cells (Human epidermoid larynx carcinoma).



Nanobio Pharmaceutical Technology

MATERIALS AND METHODS Bacterial Strains The clinical strains of bacteria were procured from M/s. Sharp Laboratory, Chennai and the antibiotic sensitivity of the clinical isolates was determined using Kirby-Bauer-disc diffusion method on MuellerHinton agar (MHA). The zone of inhibition was measured prior to incubation and maintained at 4° C.

Synthesis & Characterization The silver nanoparticles were synthesized using an environmental isolate Bacillus subtilis A1 extract and characterized by UV-visible spectroscopy, X-ray diffraction, field emission scanning electron microscopy as reported in our previous study [3].

Antimicrobial Activity The antimicrobial activity of silver nanoparticles was performed using the redox Resazurin microtiter assay plate (REMA) method [4] and the minimum inhibitory concentration determined as per the CLSI norms [5]. Susceptibility to silver nanoparticles was tested (1mg/mL), and the isolates were considered susceptible if the MIC was < 8 µg/mL and resistant if the MIC was > 8 µg/mL. The cultures were maintained at an inoculum of 1.5 x 107 CFU/mL.

Single Cell Gel Electrophoresis (Comet Assay) The single cell gel electrophoresis has been performed as per Singh et al. [6] In brief, 1% of normal melting point agarose was prepared on frosted slides along with Hep-2 cells mixed with 0.5 % low melting point agarose. The suspension was pipetted onto pre-coated slides and kept immersed in chilled lysis buffer (pH 10) for 60 min. The slides were then placed in alkaline buffer (pH > 13) in an electrophoresis tank and left for 30 min. Electrophoresis was performed at 25 V for 25 min at 4° C. The slides were neutralized in 0.4 M Tris (pH 7.5) and stained with Ethidium bromide (25 µL in 50µg/ mL) and visualized under fluorescent microscope. DNA damage was quantified by tail moment, tail length and Olive tail moment (OTM).

RESULTS AND DISCUSSION In the present study, antimicrobial efficacy of biogenic silver nanoparticles toward multidrug-resistant clinical isolates and its DNA fragmentation (single strand breaks) potential were investigated. The metabolic products of the B. subtilis A1 behaved as reducing an agent in the synthesis and growth of silver nanoparticles as evidenced from a color change to brown showing characteristic absorbance for silver. Further, the crystalline nature and fcc symmetry of the particles proved its nano-size of ~30.45 nm. The particles showed spherical to roughly spherical in shape with 97 % (w) purity of silver as observed from FESEM and EDX spectrum [3]. The antimicrobial activity of silver nanoparticles was investigated because of the increase in the drug resistant clinical strains to fourth-generation antibiotics. The clinical strains used in this study showed partial to complete drug resistance pattern as in Table 1. The emergence of resistance to imipenem and ciprofloxacin is of particular concern as these antibiotics remain as the drug of last resort. For the isolates CI 1 to 5, the MIC of silver nanoparticles was in the range 4 – 32 µg/mL (Table 2). The isolate CI 5, Acinetobacter baumannii was found highly resistant to antibiotics whereas susceptible on exposure to AgNPs. It is noteworthy to mention that all the strains exposed to silver nanoparticles were found susceptible. The modus operandi by which the NPs paralyze the cell remains unexplored. It was hypothesized that upon treatment, DNA replication and ribosomal subunit protein expression gets interrupted leading to loss of ATP production [7]. Moreover, Ag+ has a greater affinity toward membrane-bound enzymes involved in the respiratory chain [8]. It may be anticipated that

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Nanobio Pharmaceutical Technology

the generation of ROS (reactive oxygen species) and the tendency of soft acid (silver) to react with soft bases present in sulphur containing proteins in the membrane serve as a binding site accounting for cell damage [9]. Table 1: Showing the antibiogram pattern of clinical isolates Clinical Isolates CI 1 (E. coli)

AMP

AK

AMX

CAZ

CI

CP

IMP

R

R

S

S

R

S

S

CI 2 (Ps. aeruginosa)

S

S

R

R

R

S

S

CI 3 (S. aureus)

R

R

R

S

S

S

S

CI 4 (Enterococcus faecalis)

R

R

R

I

R

I

R

CI 5 (Acinetobacter baumanniii)

R

R

R

R

R

R

I

CI - Clinical isolates; AMP - Ampicillin, AK - Amikacin, AMX - Amoxicillin, CAZ - Ceftazidime, CI - Ceftriaxone, CP - Ciprofloxacin, IMP - Imipenem, R - Resistant, S - Sensitive, I - Intermediate Table 2: Showing the minimum inhibitory concentration of the clinical isolates Test Bacterial strains

MIC range (µg/mL)

E. coli

4–8

Ps. aeruginosa

8 – 16

S. aureus

16 – 32

E. faecalis

8 – 16

Acinetobacter baumannii

16 – 32

Silver nanoparticles induce significant DNA damage in HEp-2 cells through apoptosis as evidenced from comet assay. Fig. 1 shows single strand breaks in DNA induced by the nanoparticles account for the oxidative damage in treated cells. The level of DNA damage upon pre-treatment could be appreciated from the % of DNA in tail, tail moment and olive tail moment whereas no change was observed in untreated cell. The increase in concentration of AgNPs (10 µg to 100 µg/mL) significantly increases the level of DNA damage during single cell gel electrophoresis, indicative of apoptotic cell death. The antiproliferative effect of AgNPs toward HEp-2 cells at 31.25 µg/mL was determined as its IC50 concentration [10], whereas enhanced internucleosomal DNA fragmentation was observed in a dose dependent manner. This is another evidence for the generation of ROS as a result of oxidative stress induced by NPs forecasting apoptotic cell death [11]. It may be anticipated that the loss of mitochondrial membrane potential (ΔΨm) was regarded as a limiting factor in the apoptotic pathway [12].

Figure 1: Comet assay showing the DNA damage induced by AgNPs on Hep-2 cells (A) Control and Cells treated with AgNPs at 10 µg/mL (B) and 100 µg/mL (C)

Table 3: Showing data on the percentage of DNA damage induced by AgNPs at various concentrations HEp-2 cells Control % Nanoparticles (10 µg/mL) % Nanoparticles (100 µg/mL) % Head DNA

97.5

92.5

77.5

Tail DNA

2.5

7.5

22.5

Tail Moment

0.1

0.67

8.1

Olive Tail Moment

0.6

1.1

6.4

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Nanobio Pharmaceutical Technology

CONCLUSION The present study validates the potential antibacterial and apoptotic influence of silver nanoparticles. The broad spectrum antibacterial efficacy toward drug-resistant clinical isolates proves its size dependent physicochemical properties in bringing about cell death. Moreover, DNA damage induced by AgNPs remains one of the limiting factors in surpassing the specificity exhibited by antibiotics. This study would open new vistas of using AgNPs with improved biomedical applications.

ACKNOWLEDGEMENT The authors acknowledge Department of Biotechnology (DBT), Government of India for the financial support. The authors are grateful to Hon’ Chancellor, Managing Directors and the Department of Biomedical Engineering for providing infrastructural facilities.

REFERENCES 1. Nanda A and Saravanan M, Biosynthesis of silver nanoparticles from Staphylococcus aureus and its antimicrobial activity against MRSA and MRSE, Nanomedicine: Nanotechnology, Biol Med. 2009; 5(4):452 456. 2. Huang T, Nallathamby PD, Gillet D and Xu XHN, Design and Synthesis of Single Nanoparticle Optical Biosensors for Imaging and Characterization of Single Receptor Molecules on Single Living Cells, Anal Chem 2007 Sep; 79(20):7708–7718. 3. Muthukrishnan L and Nanda A, Geno-toxic study of silver bio-nanoparticles toward Gram-positive and Gram-negative clinical isolates, J Pharm Res 2013 Jul; 6(7):725  729. 4. Juan-Carlos P, Anandi M, Mirtha C, Humberto G, Jean S and Francoise P, Resazurin Microtiter Assay Plate: Simple and Inexpensive Method for Detection of Drug Resistance in Mycobacterium tuberculosis, Antimicrob Agents Chemother 2002 Aug; 46(8):2720–2722. 5. NCCLS, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, Approved standard, 6th ed. NCCLS document M7  A6. NCCLS, Wayne, Pa; 2003. 6. Singh NP, McCoy MT, Tice RR and Schneider EL, A simple technique for quantification of low levels of DNA damage in individual cells, Exp Cell Res 1988 Mar; 175:184  191. 7. Yamanaka M, Hara K and 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 Nov; 71:7589–7593. 8. Bragg PD and Rainnie DJ, The effect of silver ions on the respiratory chains of Escherichia coli, Can J Microbiol 1974 June; 20(6):883–889. 9. McDonnell G and Russell AD, Antiseptics and disinfectants: activity, action and resistance, Clin Microbiol Rev. 1999 Jan; 12(1):147–179. 10. Lakshmipathy M and Nanda A, In vitro oncogenic influence of silver nanoparticles on carcinoma model, Proc Inter Conf Mathemat Sci 2014 Jul: 664–667. 11. Haruna S, Kuroi R, Kajiwara K, Hashimoto R, Matsugo S, Tokumaru S and Kojo S, Induction of apoptosis in HL-60 cells by photochemically generated hydroxyl radicals, Bioorg Med Chem Lett 2002 Feb; 12(4):675–676. 12. Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT & Thompson CB, Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell. 1997 Nov; 91(5): 627–637.

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Design and Evaluation of Polyvinyl-Gum Ghatti SelfAssembled Nanoscale Particles for Oral Delivery of Simvastatin Bibek Laha, Leena Kumari, Arpana Kashyap and Sabyasachi Maiti Department of Pharmaceutics, Gupta College of Technological Sciences, Ashram More, G.T Road, Asansol-713301, West Bengal, India e-mail: [email protected], Mob: +919474119931

Abstract: This work describes the synthesis of a novel gum ghatti-based amphiphilic copolymer and its ability to form nanostructured particles in water. Hydrophobic polyvinyl chain was conjugated in the backbone of hydrophilic gum ghatti by etherification reaction mechanism to impart amphiphilic character. Simvastatin, a poor water soluble LDL-cholesterol lowering drug was incorporated by solvent evaporation technique by varying drug: copolymer weight ratio (1:2, 1:4 and 1:6). Microscopic analysis of the aqueous copolymer dispersion revealed spherical morphological structures. Thus it was suggested that the copolymer selfassembled in water and formed nanomicellar structures. The size of nanoparticles were characterized by Malvern Zetasizer Nano ZS 90 apparatus and was found to be in the range of 710.2  802.5 nm. The copolymer dispersion exhibited negative zeta potential values (-20.6  25.3 mV) suggesting physical stability of the dispersion. Even after an observation period of 3 months, no signs of aggregation were evident either macro- or microscopically. Compared to saturation water solubility of the drug, about 50 fold increase in drug solubility was observed after copolymer micellization. The drug loading was found to decrease with increasing amount of the copolymer. Only a small amount of drug (