Paola Ringhieri

Redox and Electrocatalytic Properties of Mimochrome VI, a Synthetic Heme Peptide Adsorbed on Gold

Mimochrome VI (MC-VI) is a synthetic heme peptide containing a helix-heme-helix sandwich motif designed to reproduce the catalytic activity of heme oxidases. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of the electron-transfer process for MC-VI immobilized through hydrophobic interactions on a gold electrode coated with a nonpolar SAM of decane-1-thiol have been determined through cyclic voltammetry. Immobilization slightly affects the reduction potential of MC-VI, which under these conditions electrocatalytically turns over molecular oxygen. This work sets the premise for the exploitation of totally synthetic mimochrome-modified electrode surfaces for clinical and pharmaceutical biosensing.

Target selective micelles for bombesin receptors incorporating Au(III)-dithiocarbamato complexes

Pure sterically stabilized micelles (SSM) of DSPE-PEG2000, and sterically stabilized mixed micelles (SSMM) containing PC or DOPC phospholipids (5, 10 or 20% mol/mol with respect to DSPE-PEG2000) are developed as delivery systems for the gold based cytotoxic drug Au(III)-dithiocarbamato complex AuL12. In particular, SSMM containing 5% of PC at 5mM of lipid concentration encapsulates 61.0 μg of AuL12 with a DL% of 1.13. The gold complex remains stable up to 72 h when incorporated in the aggregate, as indicated by UV-vis measurements. Incorporation in micelle composition of a low amount of the peptide derivative MonY-BN-AA1, containing a bombesin peptide analogue does not influence structural parameters of the micelles (diameter around 20 nm) neither the AuL12 loading parameters. Target selective properties of the peptide containing full aggregate on PC-3 cells overexpressing the GRP/bombesin receptors are observed by in vitro cytotoxic studies: a decrease of cell viability, ∼ 50%, is obtained in cells treated with AuL12-targeted micelles at 10 μM drug concentration for 48 h with respect to untargeted micelles.

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

Incorporation of Naked Peptide Nucleic Acids into Liposomes Leads to Fast and Efficient Delivery

The delivery of peptide nucleic acids (PNAs) to cells is a very challenging task. We report here that a liposomal formulation composed of egg PC/cholesterol/DSPE-PEG2000 can be loaded, according to different encapsulation techniques, with PNA or fluorescent PNA oligomers. PNA loaded liposomes efficiently and quickly promote the uptake of a PNA targeting the microRNA miR-210 in human erythroleukemic K562 cells. By using this innovative delivery system for PNA, down-regulation of miR-210 is achieved at a low PNA concentration.

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 peptide.

Liposomes derivatized with tetrabranched neurotensin peptides 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, DOPC/(C18)2-Peg9-N3 (90/10), and branched neurotensin peptides modified for the presence of a CC triple-bond [(NT8-13)4-alkyne] is performed. Results show that the post liposomal derivatization method is very efficient and that click-chemistry procedures are very attractive for nanoparticle functionalization. A structural characterization of liposomes has been performed by dynamic light scattering (DLS) measurements. The hydrodynamic radii of pure DOPC and mixed DOPC/(C18)2-Peg9-N3 and DOPC/(C18)2-Peg-triazole-(NT8-13)4 liposomes (at 90 : 10 molar ratio) are 61 ± 21 nm, 60 ± 22 nm and 82 ± 43 nm, respectively. A very efficient doxorubicin loading has been observed, specially for DOPC/(C18)2-Peg9-N3 liposomes, with a drug loading content of 90%.

Liposomes derivatized with multimeric copies of KCCYSL peptide as targeting agents for HER-2-overexpressing tumor cells

Mixed liposomes, obtained by coaggregation of 1,2-dioleoyl-sn-glycero-3-phosphocholine and of the synthetic monomer containing a gadolinium complex ([C18]2DTPA[Gd]) have been prepared. Liposomes externally decorated with KCCYSL (P6.1 peptide) sequence in its monomeric, dimeric, and tetrameric forms are studied as target-selective delivery systems toward cancer cells overexpressing human epidermal growth factor receptor-2 (HER-2) receptors. Derivatization of liposomal surface with targeting peptides is achieved using the postmodification method: the alkyne-peptide derivative Pra-KCCYSL reacts, through click chemistry procedures, with a synthetic surfactant modified with 1, 2, or 4 azido moieties previously inserted in liposome formulation. Preliminary in vitro data on MDA-MB-231 and BT-474 cells indicated that liposomes functionalized with P6.1 peptide in its tetrameric form had better binding to and uptake into BT-474 cells compared to liposomes decorated with monomeric or dimeric versions of the P6.1 peptide. BT-474 cells treated with liposomes functionalized with the tetrameric form of P6.1 showed high degree of liposome uptake, which was comparable with the uptake of anti-HER-2 antibodies such as Herceptin. Moreover, magnetic MRI experiments have demonstrated the potential of liposomes to act as MRI contrast agents.

CCK8 peptide-labeled Pluronic® F127 micelles as a targeted vehicle of gold-based anticancer chemotherapeutics

The bioavailability and target selectivity of chemotherapeutics are significant issues in drug development. Here, we report the loading of the antiproliferative gold(III) complex, dibromo[ethyl-N-(dithiocarboxy-kS,kS′)-N-methylglycinato] gold(III) (AuL12), into the lipophilic core of micelles produced from the surfactant Pluronic® F127 (PF127). When AuL12 is encapsulated in PF127-based micelles it remains stable in saline solution up to 72 h with the gold center in the +3 oxidation state. PF127-based aggregates are efficient carriers as they enhance the water solubility of the gold complex. In vitro studies indicate that after micelle encapsulation, AuL12 gold complex preserves its antiproliferative efficacy. Moreover, by labeling the hydrophilic shell of micelles with the bioactive CCK8 peptide, the aggregates act as target-selective vehicles. In fact, cytotoxic activity towards the A431 cells overexpressing the CCK2 receptors is 10-fold higher than that towards the control cells.

Influence of PEG length on conformational and binding properties of CCK peptides exposed by supramolecular aggregates

Five novel peptide amphiphiles (PAs), with common formula (C18)2‐PEGx‐CCK8 in which the CCK8 peptide and the (C18)2‐hydrophobic moiety are spaced by polyethylene linkers of different length (PEG moieties with molecular weights of 700, 1000, 1500, 2000, and 3000 Daltons) are described. They act as potential target‐selective nanocarriers towards tumor cells overexpressing cholecistokynin receptors. PAs self‐assemble in supramolecular aggregates, with hydrodynamic radius ranging between 63 and 104 nm, as indicated by DLS measurements. Fluorescence studies suggested that, irrespective from the PEG length, the tryptophan residue located at the center of the CCK8 sequence is completely surrounded by water molecules at high mobility. This result indicates a potential capability of all formulated nanovectors to recognize the overexpressed CCK‐2 receptors. CD data suggest that CCK8 peptide, in most of PAs in their aggregate form, adopts a conformation allowing the interaction with the receptor. Anyway, biological data obtained by flow cytometry analysis indicate that the five PAs have a different binding ability towards the CCK‐2 receptors, with higher binding properties shown by PA containing PEG with MW of 2000 Dalton. Therefore, PEG2000 can be considered as the best spacer in the formulation of nanovectors based on CCK8 peptide amphiphiles.

A biocompatible process to prepare hyaluronan-based material able to self-assemble into stable nano-particles

New self-assembled nano-particles were developed by chemical conjugation of natural fatty acids to the backbone of hyaluronan (HA). The chemical structure and the self-association behavior of them were studied by FT-IR, NMR, fluorescence and dynamic light scattering. The HA derivatives form stable spherical shape aggregates, as assessed by transmission electron microscopy.