Nanotubes and water-channels from self-assembling dipeptides

Dipeptides are attractive building blocks for biomaterials in light of their inherent biocompatibility, biodegradability, and simplicity of preparation. Since the discovery of diphenylalanine (Phe-Phe) self-assembling ability into nanotubes, research efforts have been devoted towards the identification of other dipeptide sequences capable of forming these interesting nanomorphologies, although design rules towards nanotube formation are still elusive. In this review, we analyze the dipeptide sequences reported thus far for their ability to form nanotubes, which often feature water-filled supramolecular channels as revealed by single-crystal X-ray diffraction, as well as their properties, and their potential biological applications, which span from drug delivery and regenerative medicine, to bioelectronics and bioimaging

Designing New Hybrid Antibiotics: Proline-Rich Antimicrobial Peptides Conjugated to the Aminoglycoside Tobramycin

Resistance to aminoglycoside antibiotics is a serious problem,typically arising from inactivating enzymes, reduced uptake, or increasedefflux in the important pathogens for which they are used as treatment.Conjugating aminoglycosides to proline-rich antimicrobial peptides(PrAMPs), which also target ribosomes and have a distinct bacterialuptake mechanism, might mutually benefit their individual activities.To this aim we have developed a strategy for noninvasively modifyingtobramycin to link it to a Cys residue and through this covalentlylink it to a Cys-modified PrAMP by formation of a disulfide bond.Reduction of this bridge in the bacterial cytosol should release theindividual antimicrobial moieties. We found that the conjugation oftobramycin to the well-characterized N-terminal PrAMP fragment Bac7(1-35)resulted in a potent antimicrobial capable of inactivating not onlytobramycin-resistant bacterial strains but also those less susceptibleto the PrAMP. To a certain extent, this activity also extends to theshorter and otherwise poorly active fragment Bac7(1-15). Althoughthe mechanism that allows the conjugate to act when its individualcomponents do not is as yet unclear, results are very promising andsuggest this may be a way of resensitizing pathogens that have developedresistance to the antibiotic

Self-Assembly of Homo- and Hetero-Chiral Cyclodipeptides into Supramolecular Polymers towards Antimicrobial Gels

There is an increasing interest towards the development of new antimicrobial coatings, especially in light of the emergence of antimicrobial resistance (AMR) towards common antibiotics. Cyclodipeptides (CDPs) or diketopiperazines (DKPs) are attractive candidates for their ability to self-assemble into supramolecular polymers and yield gel coatings that do not persist in the environment. In this work, we compare the antimicrobial cyclo(Leu-Phe) with its heterochiral analogs cyclo(D-Leu-L-Phe) and cyclo(L-Leu-D-Phe), as well as cyclo(L-Phe-D-Phe), for their ability to gel. The compounds were synthesized, purified by HPLC, and characterized by 1H-NMR, 13C-NMR, and ESI-MS. Single-crystal X-ray diffraction (XRD) revealed details of the intermolecular interactions within the supramolecular polymers. The DKPs were then tested for their cytocompatibility on fibroblast cells and for their antimicrobial activity on S. aureus. Overall, DKPs displayed good cytocompatibility and very mild antimicrobial activity, which requires improvement towards applications

SELF-ASSEMBLY OF MINIMALISTIC PHENYLALANINE DERIVATIVES INTO SUPRAMOLECULAR HYDROGELS

Nel corso degli anni, i materiali nanostrutturati costituiti da piccoli peptidi e con una buona biodegradabilità hanno suscitato un notevole interesse nella ricerca. Grazie ai numerosi vantaggi che presentano rispetto ad altri tipi di materiali (ad es. biocompatibilità, versatilità chimica di composizione e semplicità di preparazione con bassi costi e su grande scala), si è assistito ad un crescente ed attivo interesse verso lo sviluppo di biomateriali di natura peptidica a partire da sequenze il più semplici e brevi possibili, quali singoli amminoacidi o dipeptidi, secondo un approccio di tipo minimalista. Questa tesi di dottorato, per prima cosa, descrive la capacità di un derivato dell’amminoacido fenilalanina, protetta all’N-terminale in posizione para con un gruppo nitrobenzoile, di auto-assemblarsi in condizioni fisiologiche per formare un idrogel trasparente. Studi preliminari in vitro, hanno rivelato per questo composto una promettente attività antimicrobica ed una buona biocompatibilità verso colture cellulari di mammifero. Successivamente, è stato investigato l’effetto della sostituzione del gruppo N-terminale della fenilalanina con un altro amminoacido di natura idrofobica, quale fenilalanina, leucina, isoleucina o valina, considerando questa sostituzione come una strategia promettente per ottenere biomateriali a partire da dipeptidi capaci di auto-organizzarsi. È stata inoltre esplorata l’influenza della chiralità nei singoli amino acidi, sulla capacità di questi dipeptidi di formare idrogeli. L’eterochiralità si è rivelata una promettente strategia sia per guidare l’organizzazione gerarchica di piccole molecole quali i dipeptidi, specialmente nel caso della difenilalanina, sia per favorirne la loro auto-organizzazione in acqua, aumentando l’idrofobicità della sequenza peptidica. Poichè il fenomeno di auto-organizzazione è un processo cooperativo, anche minime differenze strutturali nella sequenza peptidica, quali la diversa ramificazione della catena alifatica nei tre regioisomeri leucina, isoleucina e norleucina, può essere amplificata e portare a significative differenze a livello sopramolecolare. In questa tesi, infine, è stata investigata anche la possibile influenza dell’alogenazione sull’organizzazione sopramolecolare della difenilalanina eterochirale. I peptidi sono stati sintetizzati in fase solida, purificati mediante HPLC in fase inversa e caratterizzati a livello molecolare tramite 1H-NMR, 13C-NMR e studi di massa ESI-MS. La capacità di ogni dipeptide sintetizzato di auto-organizzarsi e formare degli idrogeli è stata testata in condizioni fisiologiche (es. soluzioni di tampone fosfato) ed il comportamento sopramolecolare è stato investigato tramite l’utilizzo di diverse tecniche, quali analisi di reologia, dicroismo circolare (CD), spettroscopia infrarossa (FT-IR), saggi di fluorescenza (Th-T) amiloide, microscopia ottica ed a trasmissione elettronica (TEM), diffrazione a raggi X (XRD) su singolo cristallo e su polvere e spettroscopia visibile ed UV-Raman. Infine, la biocompatibilità di questi composti è stata testata in vitro attraverso saggi di citotossicità sia di tipo qualitativo (live/dead) che quantitativo (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) su culture cellulari di mammifero. Complessivamente, questa tesi di dottorato consente di definire le regole chiave che guidano l’auto-organizzazione di semplici e piccole molecole, costituite da 1 o 2 amminoacidi, per ottenere biomateriali con ideali caratteristiche di biodegradabilità e biocompatibilità. Inoltre, molti dei sistemi studiati in questa tesi dimostrano la loro capacità di organizzarsi in maniera ordinata a formare interessanti strutture a canale ed idrogel, con promettenti e potenziali applicazioni future nel campo della biomedicina.Biodegradable nanomaterials based on short-peptide building blocks have raised an increasing interest in research over the years. They present several advantages (i.e., biocompatibility, chemical diversity, low cost) relative to other types of materials, and there is a large scope to study minimalistic short sequences as active building blocks for biomaterials, with single amino acids and dipeptides standing out as ideal candidates, athough prediction of their supramolecular behaviour is very challenging. This PhD thesis aims to fill this gap firstly describing the self-assembly of a N-capped phenylalanine (Phe) derivative into a transparent gel under physiological conditions with promising antimicrobial activity and biocompatibility in vitro. Next, the substitution of the N-cap with another hydrophobic amino acid (i.e., Phe, Leu, Ile, or Val) has been explored as a preferable strategy towards biocompatible self-assembling dipeptides, and effects of amino acid chirality over gelation has been studied. Heterochirality revealed to be a successful strategy to control the hierarchical assembly of dipeptides, especially in the case of Phe-Phe, and to promote self-assembly in water through increased peptide hydrophobicity. As self-assembly is a cooperative process, small structural sequence differences (i.e., branching of the aliphatic amino acid sidechain for the regioisomers Leu and Ile) can be amplified and lead to significant supramolecular differences. Furthermore, halogenation was studied for its influence on the supramolecular organization of D-Phe-L-Phe. Peptides were synthesized by Fmoc-based solid-phase peptide synthesis, purified by reverse-phase HPLC and characterized by 1H-NMR, 13C-NMR and ESI-MS. Self-assembly was probed under physiological conditions (i.e., phosphate buffered solutions) and the supramolecular behaviour was investigated by means of rheology, circular dichroism (CD), Fourier-transformed infrared spectroscopy (FT-IR), Thioflavin T fluorescence, optical imaging, transmission electron microscopy (TEM), powder and single-crystal X-ray diffraction (XRD), visible and UV-resonant Raman spectroscopy. Finally, biocompatibility was assayed qualitatively (live/dead) and quantitatively (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). Overall, this PhD thesis allows to delineate design rules for the self-assembly of very simple building blocks based on 1-2 amino acids into biocompatible biomaterials and outlines several examples of supramolecular water-channels and hydrogels with potential applications in medicine

Peptide Nanostructured Materials as Drug Delivery Carriers

Peptides are ideal building blocks for biomaterials and nanostructures aimed at advanced drug delivery, with hydrogels playing an elected role for their ability to mimic natural soft tissues and provide a matrix for the sustained release of therapeutic cargo. In this chapter, we discuss the advantages and innovation potential of using peptide-based vehicles for drug delivery; we then describe recent examples in the literature where peptide nanostructures or hydrogels were designed for this type of application, using non-covalent or covalent approaches to load the therapeutics. Finally, we conclude with a perspective on the future of this exciting field that is moving towards life-like materials to address the unsolved challenges in clinical applications

Peptide Gelators to Template Inorganic Nanoparticle Formation

The use of peptides to template inorganic nanoparticle formation has attracted great interest as a green route to advance structures with innovative physicochemical properties for a variety of applications that range from biomedicine and sensing, to catalysis. In particular, short-peptide gelators offer the advantage of providing dynamic supramolecular environments for the templating effect on the formation of inorganic nanoparticles directly in the resulting gels, and ideally without using further reductants or chemical reagents. This mini-review describes the recent progress in the field to outline future research directions towards dynamic functional materials that exploit the synergy between supramolecular chemistry, nanoscience, and the interface between organic and inorganic components for advanced performance

Polymer Conjugates of Antimicrobial Peptides (AMPs) with d-Amino Acids (d-aa): State of the Art and Future Opportunities

In recent years, antimicrobial peptides (AMPs) have enjoyed a renaissance, as the world is currently facing an emergency in terms of severe infections that evade antibiotics’ treatment. This is due to the increasing emergence and spread of resistance mechanisms. Covalent conjugation with polymers is an interesting strategy to modulate the pharmacokinetic profile of AMPs and enhance their biocompatibility profile. It can also be an effective approach to develop active coatings for medical implants and devices, and to avoid biofilm formation on their surface. In this concise review, we focus on the last 5 years’ progress in this area, pertaining in particular to AMPs that contain d-amino acids, as well as their role, and the advantages that may arise from their introduction into AMPs

Dipeptide self-assembly into water-channels and gel biomaterial

Dipeptides are convenient building blocks for supramolecular gel biomaterials that can be produced on a large scale at low cost and do not persist in the environment. In the case of unprotected sequences, hydrophobicity is a key requirement to enable gelation, with Phe-Phe standing out for its self-assembling ability. Conversely, more hydrophilic sequences such as homochiral dipeptides Phe-Val and Val-Phe neither fibrillate nor gel aqueous buffers and their crystal structures reveal amphipathic layers. In this work, we test emerging rules for the design of self-assembling dipeptides using heterochiral Phe-Val and Val-Phe. Each dipeptide is characterized by H-1- and C-13-NMR, LC-MS, circular dichroism, infrared and Raman spectroscopies, rheology, electron microscopy, and single-crystal X-ray diffraction. In particular, d-Phe-l-Val is the first heterochiral dipeptide to self-assemble into supramolecular water-channels whose cavity is defined by four peptide molecules arranged head-to-tail. This minimalistic sequence is devoid of amyloid character as probed by thioflavin T fluorescence and it displays excellent biocompatibility in vitro. The dataset provided, through comparison with the literature, significantly advances the definition of molecular design rules for minimalistic unprotected dipeptides that self-assemble into water-channels and biocompatible gels, to assist with the future development of supramolecular biomaterials with fine control over nanomorphological features for a variety of applications

An SPR investigation into the therapeutic drug monitoring of the anticancer drug imatinib with selective aptamers operating in human plasma

The anticancer drug imatinib is often involved in therapeutic drug monitoring (TDM) studies aimed at improving the treatment of several forms of leukemia and gastrointestinal stromal tumors (GIST). To further implement the TDM of imatinib in clinical practice, we developed a detection assay by using an ssDNA aptamer, which demonstrated excellent selectivity and was not affected by interference from the components of human plasma samples. The efficient binding of imatinib to the aptamer was demonstrated by means of surface plasmon resonance (SPR) analysis, which allowed the development of a quantitative assay in the concentration range between 400 and 6000 ng mL 121 (0.7\u201310 \u3bcM), where a lower limit of quantification (LLOQ) of 400 ng mL 121 was achieved. The precision of the assay was found to be within 12.0%, whereas the accuracy was in a range between 97.1 and 101.5%. The sample preparation procedure displayed a recovery in the range of 48.8\u201352.8%. Solid validation data were collected according to the regulatory guidelines and the method was compared with standard analytical techniques, leading to the development of a feasible aptasensor for the TDM of patients administered with imatinib

Supramolecular hydrogel biomaterials and water-channels of differing diameters from dipeptide isomers

Dipeptides stereoisomers and regioisomers composed of norleucine (Nle) and phenylalanine (Phe) self-assemble into hydrogels in physiological conditions that are suitable for cell culture. The supramolecular behavior, however, differs as the packing modes comprise amphipathic layers, or water channels, whose diameter is defined by either four or six dipeptide molecules. A variety of spectroscopy, microscopy, and synchrotron-radiation based techniques unveil fine details of intermolecular interactions that pinpoint the relationship between chemical structure and ability to form supramolecular architectures that define soft biomaterials