Design and development of new nanosystem based on self-assembly peptides for nanomedicine applications

Peptide-based materials (PBMs) generate by the aggregation of amphiphilic monomers represent a rapidly growing tool within materials science. They have been considered for several applications in different fields from electronic to nanomedicine. According to an appropriate design, amphiphilic peptides can spontaneously assemble in well-structured supramolecular materials as result of an intricate network of inter- and/or intra-molecular interactions between hydrophobic and hydrophilic portions. The interaction manner can strongly influence both morphology and properties of the final supramolecular materials. Aromaticity is the terms used to describe the particular molecular stability associated to a cyclic shaped and planar chemical entity with a ring of resonance bonds. The peculiar electronic structure of aromatics molecules arouses their interesting physicochemical properties. All the classes of intermolecular forces known as the aromatic interaction give raise from it. The knowledge and the study of aromatic interaction contaminated peptide based material field too. To this day, using self-assembly as instrument of bottom-up nano-plenning, it is a clear evidence that aromatic peptide sequences, containing phenylalanine (Phe), tyrosine (Tyr) or tryptophan (Trp), can be opportunely modulated to the aim of generate the required supramolecular architecture. The forefather of this new class of peptide monomers is the well-known diphenylalanine (FF) homopeptide. Through a combination of π-π staking and hydrogen-bonding, different method of preparation, specific pH values or solvent, FF is able to self-organize in different kinds of nano- and macro- morphologies (hollow nanotubes, fibers, vesicles, metastable hydrogels or organogels). By this evidence, many structural analogues of diphenylalanine were studied and peptide nanostructures containing the FF motif or more extended aromatic sequences have been investigated for their mechanical, electrochemical and optical properties, and more recently for some nanomedicine applications. Nevertheless, the majority of studies reported in literature are principally focused on clarifying the physicochemical aspects responsible for array stability in FF based nanostructures, whereas only few studies have been devoted in the investigation of FF aggregates for biomedical applications. This essentially because of the intrinsic low water solubility of these peptide sequences. According to these considerations, during the three years of the PhD project, novel poly-phenylalanine self-assembling conjugates were carefully designed, synthetized and fully characterized. The final peptide materials were evaluated for potential applications in bio-imaging field (with particular bearing to Magnetic Resonance Imaging and fluorescence imaging). It was also appreciate as the chemical modification of the aromatic framework with chelating agents, gadolinium (Gd) complexes, and polyethylene glycol (PEG) fragments with different length can affect the structural organization and the supramolecular behavior of the nanomaterial. The result produced on the hierarchical organization by the chemical replacement of the Phe with others aromatic amino acids (such as tryptophan, tyrosine and 2-naphthylalanine) was also investigated. The entirety of collected data during this PhD project permits to highlight the possible relationship existing between the chemical structure of the proposed building blocks, the final supramolecular nanostructure, and their functional features. In order to simplify the comprehension and the discussion of the results (Chapter III), they will be argued in three separate sections: • Section I: PBMs as supramolecular contrast agents for MRI. • Section II: PBMs as photoluminescent supramolecular probes. • Section III: PBMs obtained by punctual chemical modifications of homophenylalanine sequences. In Section I, an innovative class of supramolecular CAs for MRI, based on peptide self-assembly monomers, is described and analyzed. Different design strategies were applied to obtain and improve the aggregation phenomenon. The structural and relaxometric properties of each self-assembling system are discussed and mutually compared. The improved values of relaxivity and the exanimated capability to encapsulate the doxorubicin anticancer drug suggest a potential use of the proposed nanostructures as new theranostic platform. Photoluminescent (PL) phenomena in peptide-based materials are the subject of Section II. A class of novel PEGylated homo-phenylalanine was synthetized and, due to the high content of β-sheet, the final self-assembled systems show blue PL emission. A red-shift of the fluorescence was actualized by FRET phenomena between the nanostructure and a internalized NBD dye. Committing to the hydrogen bonding hypothesis, a relationship between observed PL and the number of interaction sites in nanostructures was developed. In Section III the effect of a punctual chemical modification on peptide primary sequence will be elucidated. Hetero- and homo-sequences were derived simply by the replacement of Phe residues with tyrosine and tryptophan ones. Characteristic gelification behavior was found for peptides containing Tyr. WAXS/SAXS studies and molecular dynamic simulations supported the structural analysis of the new peptide-based materials. The experimental protocols are totally collected in the dedicated Experimental section. The full characterization of synthetized peptides, conjugates and derivatives is reported in Appendix I, meanwhile additional information, Tables and Figures are gather together in Appendix II

Fmoc-Diphenylalanine Hydrogels: Optimization of Preparation Methods and Structural Insights

Hydrogels (HGs) are tri-dimensional materials with a non-Newtonian flow behaviour formed by networks able to encapsulate high amounts of water or other biological fluids. They can be prepared using both synthetic or natural polymers and their mechanical and functional properties may change according to the preparation method, the solvent, the pH, and to others experimental parameters. Recently, many short and ultra-short peptides have been investigated as building blocks for the formulation of biocompatible hydrogels suitable for different biomedical applications. Due to its simplicity and capability to gel in physiological conditions, Fmoc-FF dipeptide is one of the most studied peptide hydrogelators. Although its identification dates to 15 ago, its behaviour is currently studied because of the observation that the final material obtained is deeply dependent on the preparation method. To collect information about their formulation, here are reported some different strategies adopted until now for the Fmoc-FF HG preparation, noting the changes in the structural arrangement and behaviour in terms of stiffness, matrix porosity, and stability induced by the different formulation strategy on the final materia

Peptide-Based Soft Hydrogels Modified with Gadolinium Complexes as MRI Contrast Agents

Poly-aromatic peptide sequences are able to self-assemble into a variety of supramolecular aggregates such as fibers, hydrogels, and tree-like multi-branched nanostructures. Due to their biocompatible nature, these peptide nanostructures have been proposed for several applications in biology and nanomedicine (tissue engineering, drug delivery, bioimaging, and fabrication of biosensors). Here we report the synthesis, the structural characterization and the relaxometric behavior of two novel supramolecular diagnostic agents for magnetic resonance imaging (MRI) technique. These diagnostic agents are obtained for self-assembly of DTPA(Gd)-PEG8-(FY)3 or DOTA(Gd)-PEG8-(FY)3 peptide conjugates, in which the Gd-complexes are linked at the Nterminus of the PEG8-(FY)3 polymer peptide. This latter was previously found able to form selfsupporting and stable soft hydrogels at a concentration of 1.0% wt. Analogously, also DTPA(Gd)PEG8-(FY)3 and DOTA(Gd)-PEG8-(FY)3 exhibit the trend to gelificate at the same range of concentration. Moreover, the structural characterization points out that peptide (FY)3 moiety keeps its capability to arrange into β-sheet structures with an antiparallel orientation of the β-strands. The high relaxivity value of these nanostructures (~12 mM−1·s−1 at 20 MHz) and the very low in vitro cytotoxicity suggest their potential application as supramolecular diagnostic agents for MRI

A Survey on the characteristics of sausage made from Sicilian and Sardinian native pigs

Four different mixtures were prepared using meat from native pigs. Mixtures 1 and 2 were made in Sardinia using meat from pigs of Sarda breed, mixtures 3 and 4 in Sicily, using meat from the Nera Siciliana breed. The manufacturing were different not only for the typology of the meat but also for the meat cuts used and the ingredients. Batches 1 and 3 were made in non conditioned natural environments, while batches 2 and 4 in conditioned environments. Chemical-physical and microbiological analyses were carried out on the fresh mixture, after 7 days of ripening (end of drying in conditioned environment and at the end of seasoning (28 days). The thermoigrometric environmental parameters and the variation in the weight of the production were measured during ripening. Regarding the Lactic Acid Bacteria and the Micrococcaceae, microorganisms of normal maturation, a rapid development, except for batch 1, was observed for all batches at 7 days of ripening. In batches 2 and 4 an increase of Enterobacteriaceae in the drying phase and a their decrease in the seasoning phase were noticed. In batch 3 their inactivation resulted slow. Such occurrences were probably due to the hygienic conditions and non inhibiting technological parameters. The initial humidity was less than 60% with the exception of batch 3; the quantity of NaCl ranged between 2 and 2.8%. The amount of fat varied between 14 and 24% at the beginning of the ripening, then reached values ranging between of 20 and 38% at the end of the seasoning phase. All the batches showed a decrease of aw included between 0.97 (mixture) and 0.87 (final product); the highest value of aw recorded in test 1 was attributable to the lower T recorded both during the drying and seasoning phase. The pH reached the lowest values at the end of the 7 days and then increased, with the exception of batch 3, which decreased during the whole seasoning phase

Self-assembled aggregates based on cationic amphiphilic peptides: structural insight

Short and ultra-short peptides have recently emerged as suitable building blocks for the fabrication of self-assembled innovative materials. Peptide aggregation is strictly related to the amino acids composing the sequence and their capability to establish intermolecular interactions. Additional structural and functional properties can also be achieved by peptide derivatization (e.g. with polymeric moieties, alkyl chains or other organic molecules). For instance, peptide amphiphiles (PAs), containing one or more alkyl tails on the backbone, have a propensity to form highly ordered nanostructures like nanotapes, twisted helices, nanotubes and cylindrical nanostructures. Further lateral interactions among peptides can also promote hydrogelation. Here we report the synthesis and the aggregation behaviour of four PAs containing cationic tetra- or hexa-peptides (C19-VAGK, C19-K1, C19-K2 and C19-K3) derivatized with a nonadecanoic alkyl chain. In their acetylated (Ac-) or fluorenylated (Fmoc-) versions, these peptides previously demonstrated the ability to form biocompatible hydrogels potentially suitable as extracellular matrices for tissue engineering or diagnostic MRI applications. In the micromolar range, PAs self-assemble in aqueous solution into nanotapes, or small clusters, resulting in high biocompatibility on HaCat cells up to 72 hours of incubation. Moreover, C19-VAGK also forms a gel at a concentration of 5 wt%

Codice di simulazione e potenzialità predittive per la stagionatura di salami tradizionali

Riassunto Gli Autori discutono le ipotesi ed i passi necessari allo sviluppo di un modello matematico “a principi primi” di essiccazione del salame in corso di stagionatura. L’articolo compara i dati sperimentali ottenuti da svariate lavorazioni industriali e pilota con i dati ottenuti dal modello matematico. I risultati ottenuti indicano una soddisfacente corrispondenza tra i valori teorici del calo peso ed i dati sperimentali, con un errore percentuale dell’ordine del 10% e mai superiore al 25%. Risulta pertanto disponibile un codice di calcolo snello e user friendly in MS Visual Basic 6.0, con documentate potenzialità predittive in termini di perdita di peso in corso di lavorazione e finale, contenuto d’acqua e temperatura di lavorazione del salame. Abstract The assumptions and the steps needed to develop a "first principles" mathematical model during sausage maturation are discussed. This article compares the experimental data obtained from a variety of industrial and pilot-scale processes with the data obtained by the mathematical model. The results obtained show a satisfactory correspondence between the theoretical values of weight loss and the experimental data, with a percent error of the order of 10% and never higher than 25%. It therefore provides a manageable and user friendly calculation code in MS Visual Basic 6.0, with documented predictive potentials in terms of weight los

Profili di umidità e di cloruro di sodio durante l’essiccazione del Capocollo

E’ stato sviluppato un modello matematico basato sulle leggi di Fick della diffusione per affrontare in modo semplice le interazioni reciproche tra i profili di concentrazione di umidità e di NaCl durante le varie fasi di lavorazione del Capocollo. Le concentrazioni di sale e di umidità sono determinate sperimentalmente su cinque sezioni concentriche. Un codice agile e di rapida esecuzione implementa il modello matematico per mezzo del software FlexPDE ® students’ edition. I risultati ottenuti dal modello matematico nelle differenti sezioni radiali sono in accordo con i dati sperimentali