1,720,964 research outputs found
Perspectives on polymeric nanostructures for the therapeutic application of antimicrobial peptides
No full textAntimicrobial peptides (AMPs) are a class of promising anti-infective molecules but
their therapeutic application is opposed by their poor bioavailability, susceptibility to
protease degradation and potential toxicity. The advancement of nanoformulation
technologies offers encouraging perspectives for the development of novel
therapeutic strategies based on AMPs to treat antibiotic resistant microbial infections.
Additionally, the use of polymers endowed per-se with antibacterial properties,
stands out as an innovative approach for the development of a new generation of
drug delivery systems in which an enhanced antimicrobial action could be obtained by
the synergic combination of bioactive polymer matrices and drugs. Herein, the latest
AMPs drug delivery research is discussed
Preparation, physical-chemical and biological characterization of chitosan nanoparticles loaded with lysozyme
No full textA commercially available chitosan (CS) was employed in the formulation of nanoparticles loaded with lysozyme (LZ) as antimicrobial protein drug model. Due to the variability of commercially available batches of chitosans and to the strict dependence of their physical and biological properties to the molecular weight (Mw) and deacetylation degree (DD) of the material, the CS was fully characterized resulting in weight-average molecular weight of 108,120 g/mol and DD of 92%. LZ-loaded nanoparticles (LZ-NPs) of 150 nm diameter were prepared by inotropic gelation. The nanoparticles were effectively preserving the antibacterial activity of the loaded enzyme, which was slowly released over 3 weeks in vitro and remained active toward Staphylococcus epidermidis up to 5 days of incubation. Beyond the intrinsic antibacterial activity of CS and LZ, the LZ-NPs evidenced a sustained antibacterial activity that resulted in about 2 log reduction of the number of viable S. epidermidis compared to plain CS nanoparticles. Furthermore, the LZ-NPs showed a full in vitro cytocompatibility toward murine fibroblasts and, in addition to the potential antimicrobial applications of the developed system, the proposed study could serve as an optimal model for development of CS nanoparticles carrying antimicrobial peptides for biomedical applications
Composizione profumata e sistema per il rilascio controllato di un essenza e⁄o profumo
No full textComposizione profumata e sistema per il rilascio controllato di un essenza e⁄o profum
Levofloxacin-loaded star poly(ε-caprolactone) scaffolds by additive manufacturing
Full text linkThe employment of a tissue engineering scaffold able to release an antimicrobial agent with a controlled kinetics represents an effective tool for the treatment of infected tissue defects as well as for the prevention of scaffolds implantation-related infectious complications. This research activity was aimed at the development of additively manufactured star poly(ε-caprolactone) (*PCL) scaffolds loaded with levofloxacin, investigated as antimicrobial fluoroquinolone model. For this purpose a computer-aided wet-spinning technique allowing functionalizing the scaffold during the fabrication process was explored. Scaffolds with customized composition, microstructure and anatomical external shape were developed by optimizing the processing parameters. Morphological, thermal and mechanical characterization showed that drug loading did not compromise the fabrication process and the final performance of the scaffolds. The developed *PCL scaffolds showed a sustained in vitro release of the loaded antibiotic for 5 weeks. The proposed computer-aided wet-spinning technique appears well suited for the fabrication of anatomical scaffolds endowed with levofloxacin-releasing properties to be tested in vivo for the regeneration of long bone critical size defects in a rabbit model
Chitosan nanoparticles for the linear release of model cationic peptide
No full textThe present study is focused on the development of a model drug delivery system (DDS) based on Chitosan (CS) nanoparticles using Renin substrate I (RSI) as model agent. RSI shares the main chemical-physical features of several biologically active antimicrobial peptides (AMPs). AMPs have a great therapeutic potential that is hampered by their lability in the biological fluids and as such they are perfect candidates for DDS. The development studies of quality DDS loaded with AMPs would require highly sensitive and specific quantification assays. The use of RSI allowed for the fine-tuning and optimization of the formulation parameters to promote the hydrophobic interactions between CS and the cationic peptide, favour the loading of the active ingredient and enhance the release properties of the carrier
Antimicrobial peptides: promising molecules for the treatment of biofilm-associated infections.
No full textIntroduction: Biofilms are complex bacterial communities living attached to biotic or abiotic surfaces and embedded in a self-produced extracellular matrix. It is now clear that biofilm formation may account for a large majority (up to 80%) of infections treated by physicians in the developed world and contributes significantly to morbidity and mortality especially in hospital settings. The treatment of biofilm infections is particularly challenging, as cells in this mode of growth are intrinsically refractory to antimicrobial drugs. The difficulty of successfully treating biofilm associated infections and the increasing resistance of microbes to traditional treatments call for the discovery of compounds with novel mechanisms of action. In this regard, over the last years, antimicrobial peptides (AMPs) have attracted considerable interest and are taken in increasingly consideration as new antibiofilm drugs.
Materials and Methods: AMPs from humans or frog skin were tested for their ability to inhibit biofilm formation or to eradicate preformed biofilms. Antibiofilm activity was assessed by evaluating biofilm biomass (by crystal violet
staining) biofilm architecture (by confocal microscopy) or by counting biofilm-associated viable cells. Synergy studies between AMPs and other antimicrobial compounds were also performed.
Results: Different AMPs have shown ability to inhibit biofilm formation and/or eradicate preformed biofilm of clinical isolates or laboratory strains of Gram-positive and Gram-negative bacteria. Some peptides exerted their antibiofilm activity at concentrations equal or very close to their MIC values, indicating a classical bactericidal mechanism of antibiofilm action. In contrast, other peptides inhibited biofilm formation at sub-MIC values suggesting mechanisms of action specifically targeting the biofilm mode of growth. Encapsulation of a model peptide into chitosan nanoparticles demonstrated linear releasing kinetics of the loaded molecule and prolonged antibacterial activity in in vitro models.
Discussion and Conclusions: In recent years, there has been increased interest in the use of AMPs as antibiofilm molecules. Nevertheless, optimization of AMP-antibiofilm activity is a research area still at an early stage and intensive discovery and design of new peptides is highly desirable. In this regards, a new database recently developed by a collaborative work from different research institutions in Pisa, and fully dedicated on AMPs tested on microbial biofilms, will represent a valid tool for peptide optimization and will be briefly presented
Doxorubicin loaded polyurethanes nanoparticles
No full textA poly(ether-ester-urethane) block copolymer based on poly(ε-caprolactone) and polyethylene glycol (PEGCL) was employed for the production of Doxorubicin loaded polymeric nanoparticles to be used as a drug delivery system. Nanoparticles were obtained by means of the nanoprecipitation/solvent evaporation technique, possessing a spherical shape and an average diameter of about 80 nm. The optimization of the formulation process led to high formulation yield and appreciable drug content. One of the main findings arising from the present study is represented by the protective activity played by nanoparticles in terms of preventing doxorubicin degradation in physiological conditions; moreover the observed time-delayed release of the drug was associated to PEGCL chemical structure
Preparazione e caratterizzazione di nanoparticelle polimeriche biodegradabili caricate con farmaci antitumorali e/o core magnetici per la terapia, diagnosi e teranostica di patologie tumorali
No full textLa nanomedicina è una nuova disciplina scientifica che si propone di applicare le nanotecnologie in ambito biomedico, offrendo prospettive molto promettenti per la cura e la diagnosi di malattie ad altissimo impatto sanitario, quali le patologie tumorali e le malattie neurodegenerative. Tra le nanotecnologie emergenti, lo sviluppo di sistemi nanostrutturati progettati per il rilascio controllato e mirato di farmaci e principi attivi, offre notevoli benefici terapeutici. Recentemente l’impiego di sistemi nanostrutturati nell’ambito della medicina oncologica è focalizzato sullo sviluppo di sistemi “teragnostici”, in grado di effettuare contemporaneamente la diagnosi e la terapia delle patologie neoplastiche. Nanoparticelle magnetiche infatti, possono essere usate sia nella parte diagnostica dei sistemi teranostici, come strumenti per l’imaging, sia come componente terapeutica vera e propria, sfruttando l’ipertermia magnetica da esse generata come strumento per la soppressione delle cellule tumorali.
Il presente lavoro di tesi è stato incentrato sulla preparazione, caratterizzazione e valutazione biologica, di nanoparticelle (NPs) polimeriche sia di natura esclusivamente organica che contenenti un core ferromagnetico a base di Magnetite (NPs ibride) caricate con il chemioterapico Paclitaxel, al fine di ottenere un rilascio mirato e controllato dello stesso combinato all’azione ipertermica svolta dal core magnetico.
Il polimero impiegato per la formulazione dei sistemi nanoparticellari è un copolimero a blocchi poli(estere-etere-uretano) a base di [poli(-caprolattone)2000-co-poli(etilenglicol)2000] (PCL2000-PEG2000) sintetizzato nel laboratorio in cui il presente lavoro di tesi è svolto. Il copolimero in oggetto è stato anche biofunzionalizzato con acido folico [(PCL2000-PEG2000) – FA] al fine di ottenere sistemi nanoparticellari in grado di svolgere una terapia mirata verso le cellule tumorali che sovra esprimono il recettore per questa vitamina [Wu, 2006; Cavallaro, 2006]. Le nanoparticelle sono state preparate mediante nano-precipitazione, una tecnica che prevede la preparazione di una soluzione polimerica contenente il farmaco da somministrare in un solvente organico appropriato e la successiva precipitazione della stessa in un non solvente. La rapida desolvatazione delle catene polimeriche, che si verifica durante la nanoprecipitazione, determina la formazione di nanoparticelle con diametro generalmente inferiore ai 200 nm. Modulando opportunamente i parametri della formulazione è stato possibile preparare nanoparticelle a base di (PCL2000-PEG2000) e [(PCL2000-PEG2000) – FA] sia bianche, che caricate con Paclitaxel e/o con core magnetico di Magnetite (NPs ibride). Le nanoparticelle preparate sono state caratterizzate da un punto di vista dimensionale mediante Light Scattering Dinamico (DLS) mostrando un diametro medio compreso tra gli 85 ed i 165 nanometri, a seconda del tipo di formulazione nanoparticellare. È stata inoltre effettuata un’analisi del potenziale Zeta volta ad evidenziare l’effettiva esposizione dell’acido folico sulle nanoparticelle a base di [(PCL2000-PEG2000) – FA]; la variazione del valore del potenziale Z dai -9 mVolt delle NPs a base di (PCL2000-PEG2000) ai -19.5 mVolt delle NPs a base del polimero biofunzionalizzato con acido folico, suggerisce l’effettiva presenza dell’acido folico sulla superficie delle nanoparticelle. La morfologia delle nanoparticelle preparate è stata studiata mediante Microscopia a Forza Atomica (AFM) e l’indagine della stabilità dei diversi campioni nanoparticellari preparati, condotta per 15 giorni a 4 ºC, ha mostrato una buona stabilità degli stessi. Le valutazioni biologiche della citotossicità delle nanoparticelle preparate sono state effettuate utilizzando la linea di fibroblasti murini embrionali balb/3T3 Clone A31. La vitalità e la proliferazione cellulare sono state valutate dopo l’esposizione delle cellule a diverse concentrazioni di nanoparticelle impiegando il sale di tetrazolio WST-1. In generale i sistemi prodotti hanno mostrato un’elevata citocompatibilità, anche nel caso dei sistemi ibridi. Attualmente sono in corso prove volte a valutare la risposta infiammatoria indotta dalle nanoparticelle sulla linea di macrofagi murini RAW 264.7. L’analisi delle citochine pro-infiammatorie eventualmente prodotte sarà condotta mediante tecnologia luminex
Development of polymeric micro/nanoformulates for the controlled and/or targeted release of antimicrobial peptides
No full textWith the dramatic rise in antibiotic resistance, including untreatable infections of multi-resistant bacteria and microbial biofilm-related diseases, there is no doubt that new effective antimicrobials or novel delivery systems for antibiotic compounds are urgently needed. Antimicrobial peptides (AMPs) represent a promising class of novel antimicrobials active against a wide range of microorganisms; despite an excellent antibiotic activity, their therapeutic potential is currently limited by their poor bioavailability, potential for systemic toxicity and sensitivity to protease degradation. The encapsulation of AMPs in polymeric carriers could protect them from degradation and secure their transport and delivery to the specific site of action at a controlled and tunable rate, rendering them effective and powerful antimicrobial tools.
The present PhD project has been carried out in the interdisciplinary field of nanomedicine, with the aim of developing chitosan nanoparticles for the controlled delivery of the antimicrobial peptide Temporin-1b, which proved a strong antimicrobial activity against nosocomial multidrug-resistant Gram-positive bacteria.
Chitosan (CS) was selected for nanoparticles (NPs) development due to its biocompatibility and biodegradability, fundamental characteristics in various biomedical applications. Due to the variability of commercially available batches of CS and to the strict dependence of its physical-chemical and biological properties to its structural features, commercial CS was fully characterized for its molecular weight (108 kDa, Mw/Mn 2.4) and deacetylation degree (~92%), key parameters for the development of CS NPs.
Lysozyme (LZ) was encapsulated into chitosan nanoparticles as the first proteic model for cationic AMPs, as it is a relatively small protein (~14 kDa) with an optimal pH in the range of 6-9 and an isoelectric point near 9.2 (similarly to cationic AMPs), it exerts an antimicrobial activity against Gram-positive bacteria and it is considered to be the precursor of AMPs. LZ-loaded nanoparticles with good dimensional features (mean diameter 159 ± 24 nm) were successfully prepared by means of a mild ionic gelation technique. LZ loading in the NPs was up to 8% and the release kinetic studies showed that up to 20% of the loaded enzyme was slowly released over 3 weeks, in a controlled and sustained manner. The developed formulations showed a full in vitro cytocompatibility towards mammalian cells and LZ-loaded nanoparticles exhibited a good antimicrobial activity on Staphylococcus epidermidis, selected as a model Gram-positive pathogen.
Renin substrate I (RSI) was then employed as an improved fluorogenic peptidic model for cationic peptides. In fact, despite the lack of an antimicrobial activity, RSI shares similar features with cationic AMPs (e.g. Temporin-1b), such as its small dimensions (8 amino acids), positive charge (+2) and hydrophobicity (75% of hydrophobic residues). The formulation parameters set for LZ were applied to the encapsulation of different amounts of RSI into CS nanoparticles and eventually tuned and optimized for maximum RSI encapsulation efficacy and loading. RSI encapsulation efficacy was raised to almost 100% by adjusting the pH of the CS solution from 3 to 5, thus promoting the hydrophobic interaction between CS backbone and peptide. RSI release kinetic from the NPs in vitro was evaluated: after a first equilibration time, all the formulations displayed a progressive linear release of the peptide. Through a mathematical modeling evaluation of RSI release profiles, Fickian diffusion was proposed as the peptide release mechanism from CS nanoparticles in vitro, dependent on the amount of loaded RSI and on the NPs radius.
Finally, the optimized parameters set for the model peptide RSI were applied to the encapsulation of the AMP Temporin-1b (T-1b) into CS nanoparticles, thus reducing the waste of expensive AMPs during the initial research phases. T-1b-loaded CS nanoparticles with good dimensional features were prepared; T-1b EE% in the formulations was up to 75% and the release kinetic studies highlighted a linear release of the peptide from the NPs in the experimental conditions, confirming the results obtained from RSI-loaded CS NPs, selected as a model. Temporin-1b toxicity towards mammalian cells in vitro was compared to that of T-1b-loaded nanoparticles, proving the capability of the polymeric carrier to significantly reduce the peptide toxicity. Finally, the antibacterial activity of the developed nanoparticles was tested against S. epidermidis. T-1b-loaded NPs proved a long term antibacterial activity statistically superior to that of plain CS nanoparticles and plain T-1b, active in the initial incubation times.
The antimicrobial NPs developed in this study seem to match the requirements of the “ideal” antibacterial delivery system: the bactericidal activity exerted by the nanocarrier itself (CS NPs) ensures an initial burst activity, markedly reducing the starting inoculum; afterwards, the linear release of T-1b from CS NPs secures a further reduction of viable bacterial counts, preventing the regrowth of the residual cells and ensuring a long-lasting antibacterial activity.
The proposed AMPs-loaded CS nanoparticles could find their applications in different critical field of modern medicine, such as the treatment of multidrug-resistance systemic or topical infections and the antimicrobial pre-treatment or treatment of medical devices-related infections, thus having a huge impact on the progress of antimicrobial therapies
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