Nanomaterials have attracted attention in several research fields, such as chemical catalysis, medicine, and engineering. Among the broad range of nanostructured materials that have been developed in recent years, self-assembling short-peptides are attractive minimalistic bioactive motifs and low-cost building blocks for supramolecular nanostructured hydrogels. They are inherently biocompatible and biodegradable functional nanomaterials, useful to the creation of novel therapeutic paradigms in nanomedicine. This PhD thesis studied the self-assembly of heterochiral tripeptides, where the presence of both D- and L-amino acids at specific positions along the sequences is evaluated as a useful tool to obtain supramolecular nanostructured hydrogels at physiological conditions. These sequences typically contain two phenylalanine residues for self-assembly, and thus far those reported to form supramolecular hydrogels were composed of hydrophobic amino acids devoid of functional groups in the side chain. However, the presence of a sulfur atom in the peptide sequence could give interesting added properties, such as the possibility to undergo redox cycles, metal coordination, or sulfur-based reversible chemistry. In this PhD thesis, new self-assembling heterochiral tripeptides that contain two phenylalanine residues (to drive the process) and either methionine or cysteine are described in Chapter 3 and Chapter 4, respectively. Peptides were synthesized via a standard Fmoc-based solid-phase peptide synthesis and then purified by reverse-phase HPLC. ESI–MS, 1H-NMR, and 13C-NMR have been carried out to have a spectroscopic characterisation of the molecules. After having obtained nanostructured hydrogels from peptide self-assembly, supramolecular behaviour of these materials has been characterised performing rheology analysis, circular dichroism analysis (CD), Fourier-transform infrared spectroscopy (FT-IR) and Thioflavin T fluorescence assay. Moreover, nanomorphology of the hydrogels, which are mainly composed of nanofibers, has been assessed by means of transmission electron microscopy (TEM). Single-crystal X-ray diffraction (XRD) studies on self-assembling peptides showed the ability of these peptides to form water channels, thanks to a clear segregation of the hydrophilic part (peptide backbone) and the hydrophobic part (side chains) of the tripeptides. Their biocompatibility was tested in fibroblast cell culture in vitro. After 72 hours, the cells presented a good viability and the hydrogels did not lead to cytotoxicity. The possibility to disassemble such nanostructured hydrogels, using an external stimulus to create new smart materials, has been discussed in Chapter 5. In particular, thermoreversibility has been investigated as a useful tool to disassemble a hydrogel by heating it up. The re-assembly has been easily achieved by cooling down the system. Moreover, sulfur-based chemistry has been exploited to induce selective disassembly of a hydrogel deriving from a cysteine containing peptide, by means of reaction with bromomaleimide. A photo-triggered desulfurization protocol in water has been reported to irreversibly disassemble a nanostructured hydrogel for its final disposal after use, by converting an assembling peptide into a non-assembling one. The reaction was followed by 1H-NMR, 31P-NMR, LC-MS, and Raman analyses. The use of light as trigger allowed spatial patterning by using a photomask.

Negli ultimi anni si è assistito ad un crescente sviluppo di materiali nanostrutturati in diversi campi applicativi, tra cui catalisi chimica, medicina e ingegneria. Tra le numerose tipologie di nanomateriali identificati, piccoli peptidi capaci di auto-assemblarsi in idrogel nanostrutturati hanno ottenuto un peculiare interesse in particolar modo nel campo della nanomedicina, grazie alle loro proprietà uniche, quali biocompatibilità e biodegradabilità. In questa tesi di dottorato, tripeptidi eterochirali, contenenti, cioè, sia amminoacidi D che L nella stessa sequenza, sono stati studiati per le loro capacità di auto-organizzarsi in condizioni fisiologiche in idrogel nanostrutturati. I peptidi con questo design riportati finora sono generalmente costituiti da amminoacidi idrofobici e aromatici con lo scopo di favorirne l’autoassemblaggio; tuttavia, la presenza di gruppi funzionali nelle catene laterali degli amminoacidi, potrebbe fornire nuove prospettive applicative. Ad esempio, la presenza dello zolfo potrebbe essere sfruttata per integrare nuove proprietà dinamiche nel nanomateriale, come la possibilità di coordinare metalli o indurre reazioni di ossido-riduzione. In questo studio, dunque, sono stati individuati nuovi tripeptidi eterochirali in grado di auto-assemblare in idrogel. Le sequenze peptidiche sono costituite da due residui di fenilalanina e un terzo residuo amminoacidico contenente l’atomo di zolfo in catena laterale, ossia la metionina (Capitolo 3) o la cisteina (Capitolo 4). Il lavoro è iniziato con la sintesi in fase solida dei tripeptidi, che sono stati poi purificati mediante HPLC a fase inversa. L’identità e la purezza dei composti sintetizzati sono state verificate tramite studi di massa, 1H-NMR e 13C-NMR. L’organizzazione supramolecolare di materiali ottenuti è stata poi analizzata e caratterizzata mediante l’utilizzo di diverse tecniche, tra cui analisi di reologia, dicroismo circolare (CD), spettroscopia infrarossa e saggi di fluorescenza amiloide. Studi di microscopia a trasmissione elettronica hanno inoltre permesso di analizzare la nano-morfologia dei materiali ottenuti, che sono risultati essere costituiti principalmente da fibre di diametro dell’ordine dei nanometri. Le analisi di diffrazione ai raggi X sul singolo cristallo hanno inoltre mostrato come le molecole peptidiche sono in grado di organizzarsi in maniera altamente ordinata a formare interessanti strutture canalari, in cui la catena peptidica identifica la cavità idrofilica del canale, completamente separata dall’ ambiente idrofobico esterno, in cui le catene amminoacidiche laterali sono immerse. Inoltre, saggi di tossicità cellulare in vitro effettuati su fibroblasti hanno evidenziato una buona biocompatibilità degli idrogel nanostrutturati. Nel Capitolo 5, infine, sono state discusse tre diverse strategie utilizzabili per indurre un disassemblaggio controllato degli idrogel identificati, in modo da ottenere materiali intelligenti in grado di rispondere a stimoli esterni di tipo fisico, chimico o fotochimico. In particolare, la termoreversibiltà dei materiali è stata studiata per un approccio reversibile e indipendente dalla presenza dello zolfo. Quest’ultima è stata invece sfruttata per garantire un disassemblaggio esclusivo di peptidi contenenti cisteina, grazie alla loro selettiva reattività con bromomaleimide. Infine, un approccio fotochimico ha permesso di individuare un nuovo protocollo basato sulla desulfurizzazione della cisteina per garantire un disassemblaggio spazialmente controllato e irreversibile dell’idrogel nanostrutturato. L’andamento della reazione è stato seguito attraverso studi di 1H-NMR, 31P-NMR, LC-MS, e spettroscopia Raman.

Nanostrutture Supramolecolari di Tripeptidi Contenenti Zolfo in Acqua / Cringoli, MARIA CRISTINA. - (2020 Feb 20).

Nanostrutture Supramolecolari di Tripeptidi Contenenti Zolfo in Acqua

CRINGOLI, MARIA CRISTINA
2020-02-20

Abstract

Nanomaterials have attracted attention in several research fields, such as chemical catalysis, medicine, and engineering. Among the broad range of nanostructured materials that have been developed in recent years, self-assembling short-peptides are attractive minimalistic bioactive motifs and low-cost building blocks for supramolecular nanostructured hydrogels. They are inherently biocompatible and biodegradable functional nanomaterials, useful to the creation of novel therapeutic paradigms in nanomedicine. This PhD thesis studied the self-assembly of heterochiral tripeptides, where the presence of both D- and L-amino acids at specific positions along the sequences is evaluated as a useful tool to obtain supramolecular nanostructured hydrogels at physiological conditions. These sequences typically contain two phenylalanine residues for self-assembly, and thus far those reported to form supramolecular hydrogels were composed of hydrophobic amino acids devoid of functional groups in the side chain. However, the presence of a sulfur atom in the peptide sequence could give interesting added properties, such as the possibility to undergo redox cycles, metal coordination, or sulfur-based reversible chemistry. In this PhD thesis, new self-assembling heterochiral tripeptides that contain two phenylalanine residues (to drive the process) and either methionine or cysteine are described in Chapter 3 and Chapter 4, respectively. Peptides were synthesized via a standard Fmoc-based solid-phase peptide synthesis and then purified by reverse-phase HPLC. ESI–MS, 1H-NMR, and 13C-NMR have been carried out to have a spectroscopic characterisation of the molecules. After having obtained nanostructured hydrogels from peptide self-assembly, supramolecular behaviour of these materials has been characterised performing rheology analysis, circular dichroism analysis (CD), Fourier-transform infrared spectroscopy (FT-IR) and Thioflavin T fluorescence assay. Moreover, nanomorphology of the hydrogels, which are mainly composed of nanofibers, has been assessed by means of transmission electron microscopy (TEM). Single-crystal X-ray diffraction (XRD) studies on self-assembling peptides showed the ability of these peptides to form water channels, thanks to a clear segregation of the hydrophilic part (peptide backbone) and the hydrophobic part (side chains) of the tripeptides. Their biocompatibility was tested in fibroblast cell culture in vitro. After 72 hours, the cells presented a good viability and the hydrogels did not lead to cytotoxicity. The possibility to disassemble such nanostructured hydrogels, using an external stimulus to create new smart materials, has been discussed in Chapter 5. In particular, thermoreversibility has been investigated as a useful tool to disassemble a hydrogel by heating it up. The re-assembly has been easily achieved by cooling down the system. Moreover, sulfur-based chemistry has been exploited to induce selective disassembly of a hydrogel deriving from a cysteine containing peptide, by means of reaction with bromomaleimide. A photo-triggered desulfurization protocol in water has been reported to irreversibly disassemble a nanostructured hydrogel for its final disposal after use, by converting an assembling peptide into a non-assembling one. The reaction was followed by 1H-NMR, 31P-NMR, LC-MS, and Raman analyses. The use of light as trigger allowed spatial patterning by using a photomask.
20-feb-2020
FORNASIERO, Paolo
MARCHESAN, SILVIA
32
2018/2019
Settore CHIM/06 - Chimica Organica
Università degli Studi di Trieste
File in questo prodotto:
File Dimensione Formato  
PhD Thesis - Maria Cristina Cringoli.pdf

Open Access dal 19/02/2023

Descrizione: tesi di dottorato
Licenza: Digital Rights Management non definito
Dimensione 8.76 MB
Formato Adobe PDF
8.76 MB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/2960318
 Avviso

Registrazione in corso di verifica.
La registrazione di questo prodotto non è ancora stata validata in ArTS.

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact