Liposomes derivatized with tetrabranched Neurotensin peptide via click chemistry reactions

Liposomes decorated with Neurotensin tetramers are obtained by using a post-liposomal derivatization method in which a click-chemistry reaction between liposomes containing azido functions on the external surface and branched neurotensin peptides modified for the presence of a C-C triple-bond is performed

Conformational disorder in phosphopeptides: solution studies by CD and NMR techniques

In the last few years intrinsically disordered proteins (IDPs) have received great attention from the scientific community as they participate in several important biological processes and diseases. The intrinsic disorder and flexibility of IDPs grant them a number of advantages with respect to ordered proteins, such as conformational plasticity to bind several targets, a large interaction surface, involvement in high specificity/low affinity interactions, enhanced binding kinetics. It is assumed that post-translational modifications such as phosphorylation can stimulate structural rearrangement in IDPs and facilitate their binding to partners. To better understand at a structural level the multifaceted mechanisms that govern molecular recognition processes involving IDPs, we designed, synthesized by solid phase methods, and structurally characterized unstructured peptides. These molecules contain a putative disordered module, flanked at either the N- or C-terminal ends by a different phosphorylated amino acid (serine or threonine) to mimick the effects of phosphorylation. The absence of an ordered state in the designed peptides was proved experimentally by CD and NMR conformational studies that were carried out under different solution conditions

Pre-clinical evaluation of eight DOTA coupled gastrin-releasing peptide receptor (GRP-R) ligands for in vivo targeting of receptor-expressing tumors.

BACKGROUND: Overexpression of the gastrin-releasing peptide receptor (GRP-R) has been documented in several human neoplasms such as breast, prostate, and ovarian cancer. There is growing interest in developing radiolabeled peptide-based ligands toward these receptors for the purpose of in vivo imaging and radionuclide therapy of GRP-R-overexpressing tumors. A number of different peptide sequences, isotopes, and labeling methods have been proposed for this purpose. The aim of this work is to perform a direct side-by-side comparison of different GRP-R binding peptides utilizing a single labeling strategy to identify the most suitable peptide sequence. METHODS: Solid-phase synthesis of eight derivatives (BN1-8) designed based on literature analysis was carried out. Peptides were coupled to the DOTA chelator through a PEG4 spacer at the N-terminus. Derivatives were characterized for serum stability, binding affinity on PC-3 human prostate cancer cells, biodistribution in tumor-bearing mice, and gamma camera imaging at 1, 6, and 24 h after injection. RESULTS: Serum stability was quite variable among the different compounds with half-lives ranging from 16 to 400 min at 37 °C. All compounds tested showed K d values in the nanomolar range with the exception of BN3 that showed no binding. Biodistribution and imaging studies carried out for compounds BN1, BN4, BN7, and BN8 showed targeting of the GRP-R-positive tumors and the pancreas. The BN8 compound (DOTA-PEG-DPhe-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2) showed high affinity, the longest serum stability, and the highest target-to-background ratios in biodistribution and imaging experiments among the compounds tested. CONCLUSIONS: Our results indicate that the NMeGly for Gly substitution and the Sta-Leu substitution at the C-terminus confer high serum stability while maintaining high receptor affinity, resulting in biodistribution properties that outperform those of the other peptides

Self-assembled or mixed peptide amphiphile micelles from herpes simplex virus glycoproteins as potential immunomodulatory treatment

The use of micelle aggregates formed from peptide amphiphiles (PAs) as potential synthetic self-adjuvant vaccines to treat Herpes simplex virus (HSV) infection are reported here. The PAs were based on epitopes gB409-505 and gD301-309, selected from HSV envelope glycoprotein B (gB) and glycoprotein D (gD), that had their N-terminus modified with hydrophobic moieties containing two C18 hydrocarbon chains. Pure and mixed micelles of gB and/or gD peptide epitopes were easily prepared after starting with the synthesis of corresponding PAs by solid phase methods. Structural characterization of the aggregates confirmed that they were sufficiently stable and compatible with in vivo use: critical micelle concentration values around 4.0 · 10-7 mol · Kg-1; hydrodynamic radii (RH) between 50-80 nm, and a zeta potential (ζ) around -40 mV were found for all aggregates. The in vitro results indicate that both peptide epitopes and micelles, at 10 μM, triggered U937 and RAW 264.7 cells to release appreciable levels of cytokines. In particular, interleukin (IL)-23-, IL-6-, IL-8- or macrophage inflammatory protein (MIP)-2-, and tumor necrosis factor (TNF)-α-release increased considerably when cells were treated with the gB-micelles or gD-micelles compared with the production of the same cytokines when the stimulus was the single gB or gD peptid

Synthetic heme-proteins in biosensors development

A biosensor is an analytical device that relies on a biological recognition element, such as an enzyme, an antibody, a DNA fragment, which communicates with a signal transducer, thus providing a measurable response related to the concentration of an analyte. Currently, electrochemical biosensors, which exploit an electron transfer (ET) event at a solid electrode for the signal transduction, are the larger and most promising biosensor category in terms of ease of construction, costs, versatility, tunability and miniaturization (up to the nanoscale) for in vivo applications. A real breakthrough toward the development of electrochemical systems with superior selectivity, stability and durability is represented by the third generation biosensors. These devices exploit the unmediated direct electron transfer occurring at interfaces made up of an electrode immobilized redox-active specie, with a current transduction of the biological interaction, resulting in an amperometric detection of the analyte. An attractive feature of systems based on direct electron transfer is the possibility of modulating the properties of the analytical device using protein modification with genetic or chemical engineering techniques. Efficient direct electron transfer (DET) reactions have been reported for a restricted number of redox enzymes. The majority of these redox enzymes contain metal sites and particularly heme. Nevertheless, several studies based on heme-proteins adsorbed or immobilized on various electrodes showed that high-molecular weight enzymes are not amenable to direct electrical communication with the electrode. Our strategy to overcome the above limitations is the exploitation of artificial low-molecular weight proteins, designed on rational bases, to possess the required activity. In our laboratory, a new class of heme-peptide conjugate, named Mimochromes, have been developed, with the aim of understanding the effects of the peptide chain composition and conformation in modulating the heme redox potential. The main features of these molecules are the covalent structure and the well defined helical conformation of the peptide chains linked to the deuteroporphyrin ring. The presented work exploits the use of two different class of Mimochromes, the penta-coordinated Fe(III)-Mimochrome VI and the esa-coordinated Fe(III)-Mimochrome IV Lys3, in biosensor technology. These two artificial molecules were used as bio-recognition elements/signal transducers for biosensing. The aim of this thesis was to develop and optimise catalytic and affinity-based biosensors for the rapid screening of target analytes in complex matrices. The electrochemical properties of the two Mimochromes were studied by voltammetric techniques, then they were suitably immobilized on a gold electrode surface to achieve the biosensors construction and finally the analyte recognition was performed

Supramolecular Delivery Systems for Non-Platinum Metal-Based Anticancer Drugs

Stimulated by the enormous success of the inorganic complex cisplatin in tumor treatment, interest in metal complexes has recently grown. Within cells, metal complexes can participate in reactions that are not possible with conventional organic substances, and most of them have promising efficacy as anticancer drugs. However, to be effective in vivo metal complexes need adequate delivery systems able to increase their water solubility, the in vivo bioavailability, and the safe delivery to target organs. The present review reports on the state of the art of these new, nonplatinum, anticancer metallodrugs delivered by nanosized vehicles. The development of complexes of ruthenium, gold, cobalt, copper, gallium, and others that show promising antitumor efficacy is reported, and we emphasize the different approaches in the individuation of the most appropriate delivery system for each of them

Influence of PEG Length on Conformational and Binding Properties of CCK Peptides Exposed by Supramolecular Aggregates

Five novel peptide amphiphiles, PA-[700] ÷ PA-[3000], with common formula (C18)2-PEGx-CCK8 are described as potential target selective nanocarriers towards tumor cells overexpressing cholecistokynin receptors. DLS measurements indicate that PA-[700] ÷ PA-[2000] self-assemble in in supramolecular aggregates with a hydrodynamic radius ranged between 63 and 83 nm, while PA-[3000] shows a RH of 104 nm with an increase of ≈ 30% with respect to the other peptide derivatives. Fluorescence studies suggested that, irrespective from the PEG length on the PA, the tryptophan residue located at the center of the CCK8 sequence, is completely surrounded from water molecules at high mobility. This result indicates a potential capability of all formulated nanovectors to recognize the over-expressed CCK-2 receptors. Also CD data suggest that CCK8 peptide in all PAs in their aggregate form, with the exception of PA-[700], adopt a conformation allowing the interaction with the receptor. Anyway biological data obtained by flow cytometry analysis indicate that the five PAs with different PEG spacers have a different behavior in the binding ability of PA towards to the CCK2-R, with higher binding properties shown by PA-[2000]. Finally, proteolitic cleavage experiments indicate that in PA-[2000] supramolecular aggregates the CCK8 peptide present the Tyr-Met and Trp-Met cleavable amide bonds available for the enzymatic process, while in PA-[3000] the longer PEG moiety masks the active site in CCK8, thus preventing both enzymatic cleavage of the Tyr-Met bond and receptor interaction. Based on these results, PEG2000 can be considered as the best spacer for CCK8 peptide amphiphiles

The influence of liposomal formulation on the incorporation and retention of PNA oligomers

Liposomal formulations composed of phospholipids with different unsaturation degrees, head groupsand at different cholesterol content have been tested for the encapsulation of Peptide Nucleic Acid (PNA)oligomers. The best loading capability (177 g, ER% = 87.2) was obtained for pure liposomes of phos-phatidylglycerol (DOPG) with negatively charged head group. The insertion of a 10–20% of cholesterol inDOPG based liposomes provides a slight decrease (∼160 g) of the PNA loading. On the other hand, thecholesterol addition (20–30%) slows down the PNA’s release (∼27%) in fetal bovine serum from the lipo-somal formulation. Based on the encapsulation and the release properties, PEGylated DOPG liposomeswith a percentage of cholesterol of 10–20% are the optimal formulation for the loading of PNA-a210