Short peptides containing Norbornene amino acid (NRB): role of the NRB scaffold in self-assembly

A number of papers report on spontaneous assembly of peptides into ordered nanostructures with a variety of morphologies and this number is still expanding.[1] Besides the numerous advantages of using peptides as building blocks for different types of nanostructures, some limitations are well-known such as a low stability in biological medium and their unstable conformation especially when they are short or medium-sized. The insertion of unnatural amino acids in the peptide sequences is a well-known tool to overcome these problems. Both theoretical and experimental studies on this subject have been published and, in particular, the group of C\u3b1,\uf061-tetrasubstituted residues, in which the quaternary \u3b1-carbon is part of a ring has been the object of extensive investigation.[2] Notwithstanding this interest, studies on the self-assembly of short peptides containing cyclic C\u3b1-tetrasubstituted amino acids are very rare.[3] The two diastereoisomeric pentapeptides AcAla-NRB-Ala-Aib-AlaNH2 1 and 2, containing the two enantiomers of the non-proteinogenic C\u3b1-tetrasubstituted norbornene amino acid (NRB), were synthesized and their conformational analysis was performed. Interestingly, despite they are made of hydrophobic amino acids, they resulted insoluble in organic solvent, but completely soluble in water. The formation of supramolecular assemblies in water was assessed by TEM and DLS. Moreover, the stability of the aggregates in fetal bovine serum was evaluated and tested by DLS. A comparison between NRB containing peptides and other peptides containing cyclic and non-cyclic C\u3b1-tetrasubstituted residues (i.e. Aib, Ac5AA) was done in order to better understand the role played by the NRB residue in aggregation phenomena

Silver nanoparticles in complex biological media : assessment of colloidal stability and protein corona formation

Engineered silver nanoparticles (AgNPs) are among the most used nanomaterials in consumer products, therefore concerns are raised about their potential for adverse effects in humans and environment. Although an increasing number of studies in vitro and in vivo are being reported on the toxicity of AgNPs, most of them suffer from incomplete characterization of AgNPs in the tested biological media. As a consequence, the comparison of toxicological data is troublesome and the toxicity evaluation still remains an open critical issue. The development of a reliable protocol to evaluate interactions of AgNPs with surrounding proteins as well as to assess their colloidal stability is therefore required. In this regard, it is of importance not only to use multiple, easy-to-access and simple techniques but also to understand limitations of each characterization methods. In this work, the morphological and structural behaviour of AgNPs has been studied in two relevant biological media, namely 10 % FBS and MP. Three different techniques (Dynamic Light Scattering, Transmission Electron Microscopy, UV\u2013Vis spectroscopy) were tested for their suitability in detecting AgNPs of three different sizes (10, 40 and 100 nm) coated with either citrate or polyvinylpyrrolidone. Results showed that UV\u2013Vis spectroscopy is the most versatile and informative technique to gain information about interaction between AgNPs and surrounding proteins and to determine their colloidal stability in the tested biological media. These findings are expected to provide useful insights in characterizing AgNPs before performing any further in vitro/in vivo experiment

Amine-modified Poly(vinyl alcohol) as a novel surfactant to modulate size and surface charge of Poly-Lactic-co-Glycolic Acid nanoparticles

Poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) represent a promising tool for effective delivery of biomacromolecules, thanks to their biodegradability and biocompatibility properties. PLGA NPs are often synthesized by emulsion-solvent evaporation method and Poly(vinyl) alcohol (PVA) represents one of the most commonly used surfactant. Although PVA-mediated synthesis of PLGA NPs is effective in tailoring NPs size and stability, the resulting negative surface charge can prevent both endosomal escape and biomacromolecules release in the cells cytosol. To overcome this limit, a novel surfactant (amino-PVA) with a cationic charge was synthesized and its potential for PLGA NPs formulation investigated. In either single (oil-in-water) or double (water-in-oil-in-water) emulsion synthesis, different mixtures of PVA and amino-PVA were studied, by monitoring their effects on NPs size and surface charge. The optimized properties were obtained by the combination of 0.975% w/v of PVA with 0.025% w/v of amino-PVA. This formulation has been further investigated for degradation properties and cytocompatibility. High stability and low cytotoxicity made the system promising for the encapsulation and release of hydrophilic drugs and biomacromolecules

Spherical shape supramolecular structure of serum stable self- assembled pentapeptides containing the constrained norbornene amino acid

Self-assembly is a spontaneous process by which unordered systems of monomers organize into ordered structures as the result of non-covalent interactions and lies behind a number of biological nanostructures. The concept of self-assembly has also been used in many disciplines for constructing useful materials. Supramolecular spontaneous assembly of high molecular weight peptides or peptides conjugated with non-peptidic molecules has been reported. Less is known on the self-assembly of short peptides alone which self-organize predominantly into nanotubes and nanofibers. Spherical (micellar and vesicle-like) architectures are rarely described although they appear very attractive, due to promising applications in biomedicine and nanotechnology.1 Besides the numerous advantages of using peptides containing natural amino acids for nanostructures, they present some limitations such as low bio-stability and unstable conformation especially when they are short or medium-sized. The insertion of unnatural amino acids in the peptide sequences is a well-known tool to overcome these problems.2 Here we report on the preparation and self-assembly of short hydrophobic peptides able to stabilize the formation of supramolecular spherical shape assemblies in water. Two diastereoisomeric pentapeptides AcAla-NRB-Ala-Aib-AlaNH2 1 and 2, containing the unnatural constrained norbornene amino acid (NRB) 3, were prepared. Interestingly, peptides 1 and 2 are insoluble in organic solvent but completely soluble in water despite the presence of hydrophobic non polar norbornene scaffold. The formation of a supramolecular assembly in water was assessed by DLS analyses for both 1 and 2 either as pure compounds or as a mixture. In all cases, the obtained assemblies showed almost monomodal distributions in the size range of 320-370 nm with low polidispersity. To assess their stability in conditions mimicking the in vivo environment, they were suspended in fetal bovine serum. Interestingly, peptide assemblies were found to keep their size and shape. An interesting feature of these peptides is also the presence of the C-C double bond in the norbornene scaffold, which could allow the easy labeling of the system introducing a variety of useful biotag. 1. Panda, J. J.; Chauhan, V. S. Polym. Chem. 2014, 5, 4418 2. Ruffoni, A. et al. RSC Adv., 2015, 5, 32643-3265

Aqueous self-assembly of short hydrophobic peptides containing norbornene amino acid into supramolecular structures with spherical shape

The preparation and self-assembly of short hydrophobic peptides able to solubilize in water through the formation of supramolecular assembly is reported. The two diastereoisomeric pentapeptides AcAla-NRB-Ala-Aib-AlaNH21 and 2 containing the two enantiomers of non-proteinogenic norbornene amino acid (NRB) were synthesized in an efficient way and in good yields. They were insoluble in organic solvent, except MeOH and DMSO, but completely soluble in water despite that they are made of hydrophobic amino acids. The formation of a supramolecular assembly in water was assessed by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) using 1 and 2 individually or in a mixture. Conformational analysis on the two diastereoisomers, performed in CD3CN, CD3OH and in H2O/D2O, indicated the formation of a stable 310-helix structure for both peptides: the helix structure is more stable in CD3CN and CD3OH than in H2O/D2O where a helix/random coil transition was observed. Apparently, the norbornene moiety plays a role in the stabilization, in fact 1R2R3R-norbornene AA present in peptide 2 induces a more stable secondary structure with respect to the 1S2S3S-isomer present in peptide 1

Confronto degli effetti tossici e della biodistribuzione di nanoparticelle d’argento di 10 nm in seguito a somministrazione endovenosa ed orale

I nanomateriali vengono attualmente impiegati in numerose applicazioni e tra di essi le nanoparticelle d'argento (AgNPs) risultano quelle maggiormente utilizzate grazie alle loro propriet\ue0 antibatteriche. I campi di applicazione in cui vengono utilizzate le AgNPs includono i dispositivi medici (cerotti medicati, antisettici, disinfettanti, strumenti chirurgici), e numerosi prodotti di consumo (cosmetici, deodoranti, contenitori per cibi, ecc). Questo ampio utilizzo di AgNPs pone crescentiinterrogativisullaloropotenzialetossicit\ue0,mairisultatideglistudicondottisinoadorarisultanodiscrepantiper cui al momento non \ue8 possibile trarre definitive conclusioni. Studi precedenti condotti dal nostro gruppo hanno messo in evidenza una marcata tossicit\ue0 epatobiliare delle AgNPs di 10 nm dopo 24 ore dalla loro somministrazione per via endovenosa in topi maschi CD1. Con l'intento di valutare gli effetti tossici di queste nanoparticelle in un modello di somministrazione orale ripetuta, lo scopo di questo lavoro \ue8 la valutazione preliminare della tossicit\ue0 e della biodistribuzione di AgNPs di 10 nm in seguito a somministrazione IV e orale, per confrontare i risultati e selezionare il rivestimento delle nanoparticelle da utilizzare per future prove. Per gli esperimenti sono state utilizzate nanoparticelle di 10 nm, con 2 differenti rivestimenti (citrato, CT e polyvinylpyrrolidone,PVP),disponibiliincommercio(NanoComposix,SanDiego,USA).Primadellasomministrazione\ue8 stato condotto un controllo di qualit\ue0 interno per confermare la loro dimensione ed escludere la presenza di eventuali aggregazioni.TopimaschiCD1(ICR)sonostatitrattaticon5mg/kginunasingolasomministrazioneIVoPO,oppureper 5 giorni PO. 24 ore dopo la fine della somministrazione sono stati prelevati il sangue per la valutazione dei parametri ematochimici e gli organi principali per la valutazione istopatologica e la quantificazione dell'argento presente nei tessuti tramite inductively coupled plasma mass spectrometry (ICP-MS). A seguito di somministrazione IV, la tossicit\ue0 e la biodistribuzione delle AgNPs di 10 nm \ue8 risultata essere indipendente dal rivestimento. Negli animali trattati sono stati osservati l'aumento dei valori di GLDH e ALT, marcata necrosi epatocellulare ed emorragie della cistifellea, indicativi di una grave tossicit\ue0 epatobiliare acuta. Per ci\uf2 che concerne la biodistribuzione,gliorganiincuileconcentrazionidiargentosonorisultatimaggiorisonolamilza,ilfegatoeilpolmone. L'ausiliodiunacolorazioneistochimica,l'autometallografia,hapermessodivisualizzarelapresenzadiargentoneitessuti analizzati e di osservare che le AgNPs si localizzano prevalentemente nel citoplasma delle cellule del sistema reticoloendoteliale. Diversamente da quanto osservato negli animali trattati IV, non sono stati osservati effetti avversi in seguitoallasomministrazioneoralesingolaoripetutadiAgNPs.Labiodistribuzionedell'argento\ue8risultatadipendentedal rivestimento, infatti dopo 5 giorni di somministrazione orale la concentrazione di argento negli organi analizzati (milza e fegato) risulta maggiore negli animali trattati con le nanoparticelle rivestite di citrato. Infine a parit\ue0 di dosi IV e PO somministrate, la concentrazione di argento nei tessuti analizzati \ue8 circa 10 volte inferiore dopo somministrazione PO

Single particle optical extinction and scattering allows real time quantitative characterization of drug payload and degradation of polymeric nanoparticles

The behavior of nanoparticles in biological systems is determined by their dimensions, size distribution, shape, surface chemistry, density, drug loading and stability; the characterization of these parameters in realistic conditions and the possibility to follow their evolution in vitro and in vivo are, in most of the cases, far from the capabilities of the standard characterization technologies. Optical techniques such as dynamic light scattering (DLS) are, in principle, well suited for in line characterization of nanoparticle, however their fail in characterizing the evolution of nanoparticle in solution where change in particle dimension and density is present. Here we present an in-line optical technique based on single particle extinction and scattering (SPES) overcoming the limitations typical of DLS and allowing for the efficient characterization of nanoparticle polydispersity, index of refraction and degradation dynamics in solution. Using SPES, we characterized the evolution of PLGA nanoparticles with different structures and drug payloads in solution and we compared the results with DLS. Our results suggest that SPES could be used as a process analytical technology for pharmaceutical nanoparticle production