Carlo Diaferia

Self-assembly of PEGylated tetra-phenylalanine derivatives: structural insights from solution and solid state studies

Water soluble fibers of PEGylated tetra-phenylalanine (F4), chemically modified at the N-terminus with the DOTA chelating agent, have been proposed as innovative contrast agent (CA) in Magnetic Resonance Imaging (MRI) upon complexation of the gadolinium ion. An in-depth structural characterization of PEGylated F4-fibers, in presence (DOTA-L6-F4) and in absence of DOTA (L6-F4), is reported in solution and at the solid state, by a multiplicity of techniques including CD, FTIR, NMR, DLS, WAXS and SAXS. This study aims to better understand how the aggregation process influences the performance of nanostructures as MRI CAs. Critical aggregation concentrations for L6-F4 (43 μM) and DOTA-L6-F4 (75 μM) indicate that self-aggregation process occurs in the same concentration range, independently of the presence of the CA. The driving force for the aggregation is the π-stacking between the side chains of the aromatic framework. CD, FTIR and WAXS measurements indicate an antiparallel β-sheet organization of the monomers in the resulting fibers. Moreover, WAXS and FTIR experiments point out that in solution the nanomaterials retain the same morphology and monomer organizations of the solid state, although the addition of the DOTA chelating agent affects the size and the degree of order of the fibers.

Hierarchical Analysis of Self-Assembled PEGylated Hexaphenylalanine Photoluminescent Nanostructures

Despite the growing literature about diphenylalanine-based peptide materials, it still remains a challenge to delineate the theoretical insight into peptide nanostructure formation and the structural features that could permit materials with enhanced properties to be engineered. Herein, we report the synthesis of a novel peptide building block composed of six phenylalanine residues and eight PEG units, PEG8 -F6. This aromatic peptide self-assembles in water in stable and well-ordered nanostructures with optoelectronic properties. A variety of techniques, such as fluorescence, FTIR, CD, DLS, SEM, SAXS, and WAXS allowed us to correlate the photoluminescence properties of the self-assembled nanostructures with the structural organization of the peptide building block at the micro- and nanoscale. Finally, a model of hexaphenylalanine in aqueous solution by molecular dynamics simulations is presented to suggest structural and energetic factors controlling the formation of nanostructures.

Liposomal doxorubicin doubly functionalized with CCK8 and R8 peptide sequences for selective intracellular drug delivery

A new dual‐ligand liposomal doxorubicin delivery system, which couples targeting to enhanced cellular uptake and may lead to a more efficient drug delivery system, is here designed and synthetized. Liposomes based on the composition 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine/1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐Peg2000‐R8/(C18)2‐L5‐SS‐CCK8 (87/8/5 mol/mol/mol) were prepared and loaded with doxorubicin. Presence of the two peptides on the external surface is demonstrated by fluorescence resonance energy transfer assay. The combination of the R8 cell‐penetrating peptide and of the CCK8 targeting peptide (homing peptide) on the liposome surface is obtained by combining pre‐modification and post‐modification methods. In the dual‐ligand system, the CCK8 peptide is anchored to the liposome surface by using a disulfide bond. This chemical function is inserted in order to promote the selective cleavage of the homing peptide under the reductive conditions expected in proximity of the tumor site, thus allowing targeting and internalization of the liposomal drug. Copyright

Gadolinium containing telechelic PEG-polymers end-capped by diphenylalanine motives as potential supramolecular MRI contrast agents

Telechelic PEG‐polymers end‐capped by diphenylalanine (FF) motives and containing a DOTA‐Gd complex, bound on a lysine side chain at the centre of peptide moiety, are studied for their assembling properties and for the relaxometric behavior. The observed variations in terms of relaxivity are correlated to the assembling properties of the aggregates by using several techniques: fluorescence, Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) for aggregation tendency and secondary structure determination; Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) for morphological definition. Self‐aggregation in water solution of the peptide conjugates, due to interaction of the phenylalanine frameworks, starts at concentration around 1 mg/ml with a first evidence of the coexistence of fibrillary networks and hydrogels at 10 mg/ml. Definitive presence of well‐structured fibrillary networks, dominated by an antiparallel β‐sheet arrangement, occurs at 50 mg/ml. At the latter concentration relaxivity values measured at 20 MHz and 298 K, are around 11 mM−1 s−1, in line with a possible use of the these aggregates as MRI contrast agents. Copyright

Cross-beta nanostructures based on dinaphthylalanine Gd-conjugates loaded with doxorubicin

Very recently we proposed novel di- and tetra-phenylalanine peptides derivatized with gadolinium complexes as potentials supramolecular diagnostic agents for applications in MRI (Magnetic Resonance Imaging). It was observed that in very short FF dipeptide building blocks, the propensity to aggregate decreases significantly after modification with bulky moiety such as Gd-complexes, thus limiting their potential as CAs. We hypothesized that the replacement of the Phe side chain with more extended aromatic groups could improve the self-assembling. Here we describe the synthesis, structural and relaxometric behavior of a novel water soluble self-assembled peptide CA based on 2-naphthylalanine (2Nal). The peptide conjugate Gd-DOTA-L6-(2Nal)2 is able to self-assemble in long fibrillary nanostructures in water solution (up to 1.0 mg/mL). CD and FTIR spectroscopies indicate a β sheet secondary structure with an antiparallel orientation of single strands. All data are in good agreement with WAXS and SAXS characterizations that show the typical “cross-β pattern” for fibrils at the solid state. Molecular modeling indicates the three-dimensional structure of the peptide spine of aggregates is essentially constituted by extended β-sheet motifs stabilized by hydrogen bonds and hydrophobic interactions. The high relaxivity of nanoaggregates (12.3 mM−1 s−1 at 20 MHz) and their capability to encapsulate doxorubicin suggest their potential application as supramolecular theranostic agents.

Photoluminescent Peptide‐Based Nanostructures as FRET Donor for Fluorophore Dye

A great interest has been recently generated by the discovery that peptide-based nanostructures (NSs) endowed with cross-β structure may show interesting photoluminescent (PL) properties. It was shown that NSs formed by PEGylated hexaphenylalanine (PEG8 -F6, PEG=polyethylene glycol) are able to emit at 460 nm when excited at 370 or 410 nm. Here, the possibility to transfer the fluorescence of these PEG8 -F6-based NSs by foster resonance electron transfer (FRET) phenomenon to a fluorescent dye was explored. To achieve this aim, the 4-chloro-7-nitrobenzofurazan (NBD) dye was encapsulated in these NSs. Structural data in solution and in solid state, obtained by a variety of techniques (circular dichroism, Fourier-transform infrared spectroscopy, wide-angle X-ray scattering, and small-angle X-ray scattering), indicated that the organization of the peptide spine of PEG8 -F6 NS, which consists of anti-parallel β-sheets separated by a dry interface made of interacting phenylalanine side chains, was maintained upon NBD encapsulation. The spectroscopic characterization of these NSs clearly showed a red-shift of the emission fluorescence peak both in solution and in solid state. This shift from 460 to 530 nm indicated that a FRET phenomenon from the peptide-based to the fluorophore-encapsulated NS occurred. FRET could also be detected in the PEG8 -F6 conjugate, in which the NBD was covalently bound to the amine of the compound. On the basis of these results, it is suggested that the red-shift of the intrinsic PL of NSs may be exploited in the bio-imaging field.

Insights into amyloid-like aggregation of H2 region of the C-terminal domain of nucleophosmin

Nucleophosmin (NPM1) is a multifunctional protein involved in a variety of biological processes including the pathogenesis of several human malignancies and is the most frequently mutated gene in Acute Myeloid Leukemia (AML). To deepen the role of protein regions in its biological activities, lately we reported on the structural behavior of dissected C-terminal domain (CTD) helical fragments. Unexpectedly the H2 (residues 264-277) and H3 AML-mutated regions showed a remarkable tendency to form amyloid-like assemblies with fibrillar morphology and β-sheet structure that resulted as toxic when exposed to human neuroblastoma cells. More recently NPM1 was found to be highly expressed and toxic in neurons of mouse models of Huntingtons disease (HD). Here we investigate the role of each residue in the β-strand aggregation process of H2 region of NPM1 by performing a systematic alanine scan of its sequence and structural and kinetic analyses of aggregation of derived peptides by means of Circular Dichorism (CD) and Thioflavin T (Th-T) assay. These solution state investigations pointed out the crucial role exerted by the basic amyloidogenic stretch of H2 (264-271) and to shed light on the initial and main interactions involved in fibril formation we performed studies on fibrils deriving from the related Ala peptides through the analysis of fibrils with birefringence of polarized optical microscopy and wide-angle X-ray scattering (WAXS). This analysis suggested that the presence of branched Ile269 conferred preferential packing patterns that, instead, appeared geometrically hampered by the aromatic side-chain of Phe268. Present investigations could be useful to deepen the knowledge of AML molecular mechanisms and the role of cytoplasmatic aggregates of NPM1c+.

Structural Characterization of PEGylated Hexaphenylalanine Nanostructures Exhibiting Green Photoluminescence Emission

Peptides containing aromatic residues are known to exhibit spontaneous phenomena of supramolecular organization into ordered nanostructures (NSs). In this work we studied the structural behavior and optoelectronic properties of new biocompatible materials obtained by the self-assembly of a series of hexaphenylalanines (F6) modified at the N terminus by a PEG chain of different lengths. PEG12 -F6, PEG18 -F6, and PEG24 -F6 peptides were synthesized by coupling sequentially two, three, or four units of amino-carboxy-PEG6 blocks, each one containing six oxyethylene repetitions. Changes in the length and composition of the PEG chain were found to modulate the structural organization of the phenylalanine-based nanostructures. An increase in the self-aggregation tendency was observed with longer PEG chains, whereas, independently of the PEG length, the peptide NSs display cross-β-like secondary structures with an antiparallel β-strand arrangement. WAXS/GIWAXS diffraction patterns indicate a progressive decrease in fiber order along the series. All the PEG-F6 derivatives present blue photoluminescent (PL) emission at 460 nm, with the adduct with the longest PEG chain (PEG24 -F6) showing an additional green emission at 530 nm.

Self-Assembling of Fmoc-GC Peptide Nucleic Acid Dimers into Highly Fluorescent Aggregates

The study of molecules that self-assemble through noncovalent interactions is one of the most attractive topics in supramolecular chemistry. The use of short peptides or modified nucleotides as building blocks for the aggregates is particularly intriguing because these are very easy to synthesize; moreover, subtle changes in the chemical structure of such building blocks may drastically affect the properties of the aggregates. The ability of peptide nucleic acids (PNA) to aggregate has been very little explored, despite its practical applications. In this work we investigated the self-assembling properties of a PNA dimer, conjugated at the N-terminus to a fluorenylmethoxycarbonyl group. This PNA dimer forms nano-aggregates at low concentration in CHCl3 /CH3 OH mixtures. The aggregates retain very interesting fluorescent properties (high quantum yield in the visible region with lifetimes on the nanosecond scale), which make them promising materials for applications in optoelectronics.

Structural Characterization of Self-Assembled Tetra-Tryptophan Based Nanostructures: Variations on a Common Theme

Over the years, a large number of multidisciplinary investigations has unveiled that the self-assembly of short peptides and even of individual amino acids can generate a variety of different biomaterials. In this framework, we have recently reported that polyethylene glycol (PEG) conjugates of short homopeptides, containing aromatic amino acids such as phenylalanine (Phe, F) and naphthylalanine (Nal), are able to form elongated fibrillary aggregates having interesting chemical and physical properties. We here extend these analyses characterizing the self-assembling propensity of PEG6 -W4, a PEG adduct of the tetra-tryptophan (W4) sequence. A comprehensive structural characterization of PEG6 -W4 was obtained, both in solution and at the solid state, through the combination of spectroscopic, microscopic, X-ray scattering and computational techniques. Collectively, these studies demonstrate that this peptide is able to self-assemble in fibrillary networks characterized by a cross β-structure spine. The present findings clearly demonstrate that aromatic residues display a general propensity to induce self-aggregation phenomenon, despite the significant differences in the physicochemical properties of their side chains.