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Sviluppo e caratterizzazione di scaffold a base di polisaccaridi con proprietà antimicrobiche per la rigenerazione tissutale
No full textI difetti ossei sono un problema clinico irrisolto. Per promuovere la rigenerazione ossea, l'ingegneria tissutale sviluppa scaffold per aiutare la guarigione dei tessuti. Gli caffold porosi tridimensionali composti da alginato e idrossiapatite aiutano la rigenerazione dell'osso danneggiato. Gli scaffold tuttavia dovrebbero essere dotati anche di proprietà antimicrobiche, ma evitando l'uso di antibiotici ordinari. La resistenza batterica agli antibiotici, infatti, rende inutili la maggior parte degli attuali antibiotici. La necessità di nuovi antimicrobici è evidente e i peptidi antimicrobici (AMP) attirano sempre più attenzione a causa delle loro proprietà antibatteriche. Tra gli AMP, quelli ricchi di prolina (PrAMP) sono interessanti perché combinano potenza antimicrobica ed elevata biocompatibilità.
Lo scopo di questo progetto era caricare un PrAMP ottimizzato su scaffold di alginato / idrossiapatite (Alg / HAp) per fornire loro un effetto antimicrobico mantenendo la loro capacità di promuovere la rigenerazione ossea.
Per potenziare e adattare l'attività antimicrobica dei PrAMP, il Bac7 (1-16) è stato modificato sostituendo alcuni residui e aggiungendo quindi una frazione lipidica al suo N-terminale. Solo il primo approccio ha avuto successo fornendo il B7-005, un nuovo PrAMP che mostra una migliore attività antimicrobica. Per contro, la lipidizzazione di B7-005 è stata dannosa. Gli scaffold Alg / HAp sono stati prodotti e caricati in modo efficiente con B7-005. Tuttavia, una volta adsorbito sullo scaffold, il peptide non poteva essere rilasciato a causa della forte interazione tra alginato e B7-005. I tentativi di modulare l'interazione furono inutili. Le strutture a base di alginato non erano compatibili con gli AMP cationici. Per preparare scaffold compatibili con B7-005, l'alginato è stato sostituito con agarosio. Sono stati preparati scaffold tridimensionali porosi di agarosio/idrossiapatite (Aga/HAp). Essi hanno mostrato porosità simile agli scaffold Alg / HAp. Tali strutture si sono rivelate quindi idonee per scopi di rigenerazione ossea. Gli scaffold di Aga/HAp sono stati caricati con B7-005 e hanno mostrato un rilascio efficace di peptide. Gli scaffold Alg/HAp erano compatibili anche con le cellule umane, che proliferavano su di esse in presenza e in assenza di B7-005. Gli scaffold Aga / HAp hanno mostrato un rigonfiamento limitato e una resistenza meccanica molto bassa. Tuttavia non hanno mostrato segni di degrado dopo 2 mesi. Infine, gli scaffold B7-005 Aga/HAp hanno inibito la crescita di patogeni batterici, sebbene le loro proprietà antimicrobiche possano essere migliorate.
L'agarosio è quindi più adatto dell'alginato ad essere utilizzato in combinazione con peptidi antimicrobici cationici. Tale polimero può sostituire l'alginato in un protocollo per produrre scaffold porosi tridimensionali, mantenendo le proprietà desiderabili degli scaffold Alg / HAp.Bone defects are an unsolved clinical problem. To promote the bone regeneration tissue engineering for develops scaffolds to assist the healing of tissues. Porous three-dimensional scaffolds composed by alginate and hydroxyapatite help the regeneration of damaged bone. Scaffolds should also be endowed with antimicrobial properties, but avoiding the use of ordinary antibiotics. The bacterial antibiotic resistance, in fact makes useless most of the current antibiotics. The need of new antimicrobials is evident, and antimicrobial peptides (AMPs) attract increasing attention because of their antibacterial properties. Among AMPs, the proline-rich AMPs (PrAMPs) are interesting because they combine antimicrobial potency with high biocompatibility.
Aim of this project was to load an optimized PrAMP on alginate/hydroxyapatite (Alg/HAp) scaffolds to provide them with antimicrobial effect retaining their capability to promote bone regeneration.
To boost and tailor the antimicrobial activity of PrAMPs, the Bac7(1-16) was modified replacing some residues and adding then a lipid moiety to its N-terminus. Only the first approach was successful providing the B7-005, a new PrAMPs displaying better antimicrobial activity. On the other hand, the lipidation of B7-005 was detrimental. Alg/HAp scaffolds were produced and efficiently loaded with B7-005. However, once adsorbed on the scaffold, the peptide could not be released due to the strong interaction between alginate and B7-005. The attempts to modulate the interaction were useless. Alginate-based structures were not compatible with cationic AMPs. To prepare scaffolds compatible with B7-005, the alginate was replaced with agarose. Agarose/Hydroxyapatite (Aga/HAp) porous three-dimensional scaffolds were prepared. They displayed porosity similar to Alg/HAp scaffold. Aga/HAp were then suitable for bone-regeneration purposes. Aga/HAp scaffolds were loaded with B7-005 and they displayed effective release of peptide. Alg/HAp scaffolds were also compatible with human cells, that proliferated on them in the presence and in the absence of B7-005. Aga/HAp scaffolds displayed limited swelling and very low mechanical resistance. However they showed no signs of degradation after to 2 months. As last, B7-005 Aga/HAp scaffold inhibited the growth of bacterial pathogens, although their antimicrobial properties may be improved.
The agarose is therefore more suitable than alginate to be used in combination with cationic antimicrobial peptides. Agarose can replace alginate in a protocol to produce three-dimensional porous scaffolds, maintaining desirable properties of Alg/HAp scaffolds
Non-Membrane Permeabilizing Modes of Action of Antimicrobial Peptides on Bacteria
No full textAntimicrobial peptides (AMPs) are a large class of innate immunity effectors with a remarkable capacity to inactivate microorganisms. Their ability to kill bacteria by membranolytic effects has been well established. However, a lot of evidence points to alternative, non-lytic modes of action for a number of AMPs, which operate through interactions with specific molecular targets. It has been reported that non-membrane-permeabilizing AMPs can bind to and inhibit DNA, RNA or protein synthesis processes, inactivate essential intracellular enzymes, or affect membrane septum formation and cell wall synthesis. This minireview summarizes recent findings on these alternative, non-lytic modes of antimicrobial action with an emphasis to the experimental approaches used to clarify each step of their intracellular action, i.e. the cell penetration mechanism, intracellular localization and molecular mechanisms of antibacterial action. Despite the fact that such data exists for a large number of peptides, our analysis indicates that only for a small number of AMPs sufficient data have been collected to support a mode of action with an authentic and substantial contribution by intracellular targeting. In most cases, peptides with non-lytic features have not been thoroughly analyzed, or only a single aspect of their mode of action has been taken into consideration and therefore their mechanism of action can only be hypothesized. A more detailed knowledge of this class of AMPs would be important in the design of novel antibacterial agents against unexploited targets, endowed with the capacity to penetrate into pathogen cells and kill them from within
Search for Shorter Portions of the Proline-Rich Antimicrobial Peptide Fragment Bac5(1–25) That Retain Antimicrobial Activity by Blocking Protein Synthesis
No full textThe spread of antibiotic-resistant pathogens has boosted the
search for new antimicrobial drugs. Proline-rich antimicrobial
peptides are promising lead compounds for the development
of next-generation antibiotics, given their very low cytotoxicity
and their good antimicrobial activity targeting the bacterial ribosome.
Bac5(1–25) is an N-terminal fragment of the bovine
proline-rich antimicrobial peptide Bac5, whose mode of action
has been recently described. In this work we tested a number
of Bac5(1–25) fragments, and we characterized their antimicrobial
activity against Escherichia coli, Acinetobacter baumannii,
Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enterica,
and Pseudomonas aeruginosa. We evaluated their cytotoxicity
toward human cells and their efficacy in inhibiting bacterial
protein synthesis. This allowed us to identify some shorter
fragments of Bac5(1–25) with a good balance between antibacterial
efficacy, protein synthesis inhibition, and ease/cost-effectiveness
of synthesis, suitable as lead compounds to develop
new antibacterials
Antibacterial Electrospun Polycaprolactone Membranes Coated with Polysaccharides and Silver Nanoparticles for Guided Bone and Tissue Regeneration
Full text linkElectrospun polycaprolactone (PCL) membranes have been widely explored in the literature as a solution for several applications in tissue engineering and regenerative medicine. PCL hydrophobicity and its lack of bioactivity drastically limit its use in the medical field. To overcome these drawbacks, many promising strategies have been developed and proposed in the literature. In order to increase the bioactivity of electrospun PCL membranes designed for guided bone and tissue regeneration purposes, in the present work, the membranes were functionalized with a coating of bioactive lactose-modified chitosan (CTL). Since CTL can be used for the synthesis and stabilization of silver nanoparticles, a coating of this compound was employed here to provide antibacterial properties to the membranes. Scanning electron microscopy imaging revealed that the electrospinning process adopted here allowed us to obtain membranes with homogeneous fibers and without defects. Also, PCL membranes retained their mechanical properties after several weeks of aging in simulated body fluid, representing a valid support for cell growth and tissue development. CTL adsorption on membranes was investigated by fluorescence microscopy using fluorescein-labeled CTL, resulting in a homogeneous and slow release over time. Inductively coupled plasma–mass spectrometry was used to analyze the release of silver, which was shown to be stably bonded to the CTL coating and to be slowly released over time. The CTL coating improved MG63 osteoblast adhesion and proliferation on membranes. On the other hand, the presence of silver nanoparticles discouraged biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus without being cytotoxic. Overall, the stability and the biological and antibacterial properties make these membranes a valid and versatile material for applications in guided tissue regeneration and in other biomedical fields like wound healing
Core-shell electrospun polycaprolactone nanofibers, loaded with rifampicin and coated with silver nanoparticles, for tissue engineering applications
Full text linkIn the field of tissue engineering, the use of core-shell fibers represents an advantageous approach to protect and finely tune the release of bioactive compounds with the aim to regulate their efficacy. In this work, core-shell electrospun polycaprolactone nanofiber-based membranes, loaded with rifampicin and coated with silver nanoparticles, were developed and characterized. The membranes are composed by randomly oriented nanofibers with a homogeneous diameter, as demonstrated by scanning electron microscopy (SEM). An air-plasma treatment was applied to increase the hydrophilicity of the membranes as confirmed by contact angle measurements. The rifampicin release from untreated and air-plasma treated membranes, evaluated by UV spectrophotometry, displayed a similar and constant over-time release profile, demonstrating that the air-plasma treatment does not degrade the rifampicin, loaded in the core region of the nanofibers. The presence and the distribution of silver nanoparticles on the nanofiber surface were investigated by SEM and Energy Dispersive Spectroscopy. Moreover, SEM imaging demonstrated that the produced membranes possess a good stability over time, in terms of structure maintenance. The developed membranes showed a good biocompatibility towards murine fibroblasts, human osteosarcoma cells and urotheliocytes, reveling the absence of cytotoxic effects. Moreover, doble-functionalized membranes inhibit the growth of E. coli and S. aureus. Thanks to the possibilities offered by the coaxial electrospinning, the membranes here proposed are promising for several tissue engineering applications
Novel synthesis of 1,2-diaza-1,3-dienes with potential biological activity from cinnamic acids and diazonium salts of anilines
Full text linkCinnamic acids are an important class of phenolic compounds, which have many beneficial effects on human health but are also interesting synthetic intermediates thanks to the presence of several reactive sites. While studying the reactivity of cinnamic acids with diazonium salts from aromatic amines, an unexpected reactivity has been discovered, leading to the formation of 1,2-diaza-1,3-dienes instead of traditional diazo-coupling products. The new compounds have been fully characterized by mono and bidimensional NMR spectroscopy and mass spectrometry. Preliminary studies on the biological activity of the compounds have been carried out testing both their antibacterial and antitumor activity, leading to promising results
Lipopolysaccharide phosphorylation by the WaaY kinase affects the susceptibility of Escherichia coli to the human antimicrobial peptide LL-37
No full textThe human cathelicidin LL-37 is a multifunctional host defense peptide with immunomodulatory and antimicrobial roles. It kills bacteria primarily by altering membrane barrier properties, although the exact sequence of events leading to cell lysis has not yet been completely elucidated. Random insertion mutagenesis allowed isolation of Escherichia coli mutants with altered susceptibility to LL-37, pointing to factors potentially relevant to its activity. Among these, inactivation of the waaY gene, encoding a kinase responsible for heptose II phosphorylation in the LPS inner core, leads to a phenotype with decreased susceptibility to LL-37, stemming from a reduced amount of peptide binding to the surface of the cells, and a diminished capacity to lyse membranes. This points to a specific role of the LPS inner core in guiding LL-37 to the surface of Gram-negative bacteria. Although electrostatic interactions are clearly relevant, the susceptibility of the waaY mutant to other cationic helical cathelicidins was unaffected, indicating that particular structural features or LL-37 play a role in this interaction
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Methods for elucidating the mechanism of action of proline-rich and other non-lytic antimicrobial peptides
No full textA distinct group of antimicrobial peptides kills bacteria by interfering with internal cellular functions and without concurrent lytic effects on cell membranes. Here we describe some methods to investigate the mechanisms of action of these antimicrobial peptides. They include assays to detect the possible temporal separation between membrane permeabilization and bacterial killing events, to assess the capacity of antimicrobial peptides to cross the bacterial membranes and reside in the cytoplasm, and later to inhibit vital cell functions such as DNA transcription and protein translation
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