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      Chitosan nanoparticles loaded with the antimicrobial peptide temporin B exert a long-term antibacterial activity in vitro against clinical isolates of Staphylococcus epidermidis

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          Abstract

          Nowadays, the alarming rise in multidrug-resistant microorganisms urgently demands for suitable alternatives to current antibiotics. In this regard, antimicrobial peptides (AMPs) have received growing interest due to their broad spectrum of activities, potent antimicrobial properties, unique mechanisms of action, and low tendency to induce resistance. However, their pharmaceutical development is hampered by potential toxicity, relatively low stability and manufacturing costs. In the present study, we tested the hypothesis that the encapsulation of the frog-skin derived AMP temporin B (TB) into chitosan nanoparticles (CS-NPs) could increase peptide’s antibacterial activity, while reducing its toxic potential. TB-loaded CS-NPs with good dimensional features were prepared, based on the ionotropic gelation between CS and sodium tripolyphosphate. The encapsulation efficiency of TB in the formulation was up to 75%. Release kinetic studies highlighted a linear release of the peptide from the nanocarrier, in the adopted experimental conditions. Interestingly, the encapsulation of TB in CS-NPs demonstrated to reduce significantly the peptide’s cytotoxicity against mammalian cells. Additionally, the nanocarrier evidenced a sustained antibacterial action against various strains of Staphylococcus epidermidis for at least 4 days, with up to 4-log reduction in the number of viable bacteria compared to plain CS-NPs at the end of the observational period. Of note, the antimicrobial evaluation tests demonstrated that while the intrinsic antimicrobial activity of CS ensured a “burst” effect, the gradual release of TB further reduced the viable bacterial count, preventing the regrowth of the residual cells and ensuring a long-lasting antibacterial effect. The developed nanocarrier is eligible for the administration of several AMPs of therapeutic interest with physical–chemical characteristics analog to those of TB.

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          Antimicrobial properties of chitosan and mode of action: a state of the art review.

          Owing to its high biodegradability, and nontoxicity and antimicrobial properties, chitosan is widely-used as an antimicrobial agent either alone or blended with other natural polymers. To broaden chitosan's antimicrobial applicability, comprehensive knowledge of its activity is necessary. The paper reviews the current trend of investigation on antimicrobial activities of chitosan and its mode of action. Chitosan-mediated inhibition is affected by several factors can be classified into four types as intrinsic, environmental, microorganism and physical state, according to their respective roles. In this review, different physical states are comparatively discussed. Mode of antimicrobial action is discussed in parts of the active compound (chitosan) and the target (microorganisms) collectively and independently in same complex. Finally, the general antimicrobial applications of chitosan and perspectives about future studies in this field are considered. Copyright © 2010 Elsevier B.V. All rights reserved.
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            Antibiotics and Bacterial Resistance in the 21st Century

            Dangerous, antibiotic resistant bacteria have been observed with increasing frequency over the past several decades. In this review the factors that have been linked to this phenomenon are addressed. Profiles of bacterial species that are deemed to be particularly concerning at the present time are illustrated. Factors including economic impact, intrinsic and acquired drug resistance, morbidity and mortality rates, and means of infection are taken into account. Synchronously with the waxing of bacterial resistance there has been waning antibiotic development. The approaches that scientists are employing in the pursuit of new antibacterial agents are briefly described. The standings of established antibiotic classes as well as potentially emerging classes are assessed with an emphasis on molecules that have been clinically approved or are in advanced stages of development. Historical perspectives, mechanisms of action and resistance, spectrum of activity, and preeminent members of each class are discussed.
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              Preparation and antibacterial activity of chitosan nanoparticles.

              Chitosan nanoparticles, such as those prepared in this study, may exhibit potential antibacterial activity as their unique character. The purpose of this study was to evaluate the in vitro antibacterial activity of chitosan nanoparticles and copper-loaded nanoparticles against various microorganisms. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. Copper ions were adsorbed onto the chitosan nanoparticles mainly by ion-exchange resins and surface chelation to form copper-loaded nanoparticles. The physicochemical properties of the nanoparticles were determined by size and zeta potential analysis, atomic force microscopy (AFM), FTIR analysis, and XRD pattern. The antibacterial activity of chitosan nanoparticles and copper-loaded nanoparticles against E. coli, S. choleraesuis, S. typhimurium, and S. aureus was evaluated by calculation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results show that chitosan nanoparticles and copper-loaded nanoparticles could inhibit the growth of various bacteria tested. Their MIC values were less than 0.25 microg/mL, and the MBC values of nanoparticles reached 1 microg/mL. AFM revealed that the exposure of S. choleraesuis to the chitosan nanoparticles led to the disruption of cell membranes and the leakage of cytoplasm.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                28 April 2015
                2015
                : 6
                : 372
                Affiliations
                [1] 1Department of Chemistry and Industrial Chemistry, University of Pisa Pisa, Italy
                [2] 2National Interuniversity Consortium of Materials Science and Technology, Florence Italy
                [3] 3Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa Pisa, Italy
                Author notes

                Edited by: Tzi Bun Ng, The Chinese University of Hong Kong, China

                Reviewed by: Noton Kumar Dutta, Johns Hopkins University, USA; Yixin Shi, Arizona State University, USA

                *Correspondence: Giovanna Batoni, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Via San Zeno 35/39, 56127 Pisa, Italy giovanna.batoni@ 123456med.unipi.it

                These authors have contributed equally to this work.

                This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2015.00372
                4412066
                25972852
                93dfdfed-c377-448a-bae1-4455996b6c99
                Copyright © 2015 Piras, Maisetta, Sandreschi, Gazzarri, Bartoli, Grassi, Esin, Chiellini and Batoni.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 23 January 2015
                : 11 April 2015
                Page count
                Figures: 4, Tables: 2, Equations: 0, References: 45, Pages: 10, Words: 0
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                antimicrobial peptides,temporin b,delivery system,chitosan,nanoparticles,staphylococcus epidermidis

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