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Ruthenium complexes are attracting increasing attention as second-generation metal-based anticancer agents, with NAMI-A and KP1019 as the major representatives of this class having undergone clinical trials. Our recent interest has been focused on the synthesis and characterization of new amphiphilic derivatives of nucleosides (nucleolipids). These compounds have been selected as core scaffolds linked to RuIII complexes and capable of self-assembly into stable nanostructures in aq. solutions so to transport the metal ions efficiently into the cell. Through the use of ribo- and deoxyribonucleosides as starting building blocks, a mini-library of nucleolipidic RuIII complexes decorated with diverse hydrophilic and lipophilic chains and incorporating the NAMI-A analogue AziRu has been prepared. When co-aggregated with biocompatible lipids, these RuIII-containing nucleolipids proved to be stable for months under physiological conditions. Detailed microstructural characterization, carried out by a combined approach including different physico-chemical techniques, allowed their stability, size and shape to be determined. Tested on a panel of human and non-human cells, all of the studied RuIII complexes showed potent in vitro anticancer activity, significantly higher than that of NAMI-A-like analogues, and minimal toxicity. Here we summarize the design and synthetic procedures developed to prepare these new Ru-containing candidate drugs, discussing the beneficial effects achievable through exploiting nucleolipid appendages in the delivery of metal-based drugs.

The synthesis, formulation and a complete physico-chemical characterization, by dynamic light scattering and small angle neutron scattering techniques, of new liposomal aggregates obtained by co-assembling an amphiphilic molecule containing a platinum complex, Peg1500 -Lys(Pt-aminoEtGly)-Lys(C18)2, (abbreviated as (C18)2-PKAG-Pt), with a second amphiphilic monomer, (C18H37)2NCO(CH2)2CO(AdOO)5-Oct ((C18)2 L5-Oct), containing the octreotide bioactive peptide, is reported. Liposomes of (C18)2-PKAG-Pt present a radius of 48 nm, whereas the mixed aggregates (C18)2-PKAG-Pt/(C18)2L5-Oct at 90/10 M ratio give larger liposomes with a radius of 84 nm. In both cases, the bilayer thickness is ~5.3 nm. Encapsulation of doxorubicin in mixed liposomes is also obtained by using the pH gradient method. The obtained liposomes could represent a new target selective cargo system for delivery of cisplatin based drugs and/or doxorubicin on cells overexpressing the sstr2 and sstr5 somatostatin receptors.

Natural polyamines (putrescine, spermidine, and spermine) self-assemble in a simulated physiological environment (50 mm sodium phosphate buffer, pH 7.2), generating in vitro nuclear aggregates of polyamines (ivNAPs). These supramolecular compounds are similar in structure and molecular mass to naturally occurring cellular nuclear aggregates of polyamines, and they share the ability of NAPs to interact with and protect the genomic DNA against nuclease degradation. Three main ivNAP compounds were separated by gel permeation chromatography. Their elution was carried out with 50 mm sodium phosphate buffer supplemented with 150 mm NaCl. Freezing and thawing of selected chromatographic fractions obtained by GPC runs in which the mobile phase was sodium phosphate buffer not supplemented with NaCl yielded three different microcrystallites, specifically corresponding to the ivNAPs, all of which were able to bind DNA. In this study, we demonstrated that in vitro self-assembly of polyamines and phosphates is a spontaneous, reproducible and inexpensive event, and provided the indications for the production of the ivNAPs as a new tool for manipulating the genomic DNA machinery.

Self-complementary synthetic peptides, composed by 8 and 16 residues, were analyzed by CD, NMR and small angle neutron scattering (SANS) techniques in order to investigate the relevance of charge and hydrophobic interactions in determining their self-assembling properties. All the sequences are potentially able to form fibrils and membranes as they share, with the prototype EAK16, a strictly alternating arrangement of polar and nonpolar residues. We find that 16-mer peptides show higher self-assembling propensities than the 8-mer analogs and that the aggregation processes are favored by salts and neutral pH. Peptide hydrophobic character appears as the most relevant factor in determining self-assembling. Solution conformational analysis, diffusion and SANS measurements all together show that the sequences with a higher self-assemble propensity are distributed, in mild conditions, between light and heavy forms. For some of the systems, the light form is mostly constituted by monomers in a random conformation, while the heavy one is constituted by beta-aggregates. In our study we also verified that sequences designed to adopt extended conformation, when dissolved in alcohol-water mixtures, can easily fold in helix structures. In that media, the prototype of the series appears distributed between helical monomers and beta-aggregates. It is worth noticing that the structural conversion from helical monomer to beta-aggregates, mimics beta-amyloid peptide aggregation mechanisms.

The chemical synthesis by solid-phase methods of a novel amphiphilic peptide, peptide-conjugate amphiphile (PCA), containing in the same molecule three different functions: (i) the N,N-bis[2-[bis(carboxy-ethyl)amino]ethyl]-L-glutamic acid (DTPAGlu) chelating agent, (ii) the CCK8 bioactive peptide, and (iii) a hydrophobic moiety containing four alkyl chains with 18 carbon atoms each, is reported. In water solution at pH 7.4, PCA self-assembles in very stable micelles at very low concentration [critical micellar concentration (cmc) values of 5 x 10(-7) mol kg(-1)] as confirmed by fluorescence spectroscopy. The structural characterization, obtained with small-angle neutron scattering (SANS) measurements, indicates that the aggregates are substantially represented by ellipsoidal micelles with an aggregation number of 39 +/- 2 and the two micellar axes of about 52 and 26 A.

The structural and relaxometric characterization of a novel class of supramolecular aggregates, as potential tumor-specific contrast agents in magnetic resonance imaging (MRI), is reported. The aggregates are based on a new monomer with an upsilon shape (MonY) that contains, in the same molecule, all three fundamental tasks that are required: 1) a hydrophobic moiety that allows the formation of supramolecular aggregates; 2) the bioactive CCK8 peptide for target recognition; and 3) a chelating agent able to give stable gadolinium complexes. As indicated by dynamic light scattering and small-angle neutron scattering (SANS) measurements, MonY and its gadolinium complex MonY(Gd) aggregate in aqueous solution to give ellipsoidal micelles with a ratio between the micellar axes of approximately 1.7 and an aggregation number N(agg) of approximately 30. There are no differences in the aggregation behavior of MonY and MonY(Gd), which indicates that the presence of metal ions, and therefore the reduction of the net charge, does not influence the aggregation behavior. When MonY or MonY(Gd) are blended with dioleoyl phosphatidylcholine (DOPC), the aggregation behavior is dictated by the tendency of DOPC to give liposomes. Only when the amount of MonY or MonY(Gd) is higher than 20 % is the coexistence of liposomes and micelles observed. The thickness d of the bilayer is estimated by SANS to be approximately 35-40 A, whereas cryogenic transmission electron microscopy images show that the diameter of the liposomes ranges from approximately 50 to 150 nm. Self-assembling micelles of MonY(Gd) present high relaxivity values (r(1p)=15.03 mM(-1) s(-1)) for each gadolinium complex in the aggregate. Liposomes containing MonY(Gd) inserted in the DOPC bilayer at a molar ratio of 20:80 present slightly lower relaxivity values (r(1p)=12.7 mM(-1) s(-1)), independently of their internal or external position in the liposome.

The subject of this article is the combined interpretation of intradiffusion and mutual-diffusion data for polymer–solvent mixtures in terms of integrals over velocity self-correlation functions and velocity cross-correlation functions. The combination of mutual-diffusion, intradiffusion, and activity data allows the evaluation of velocity-correlation coefficients (VCCs) and distinct-diffusion coefficients in systems containing one monodisperse solute. This study is the first attempt to extend these approaches to polymers that are polydisperse solutes. Because of the polydispersity, this correlation analysis may become critical for polymers. Its application to polydisperse samples requires the reduction of intradiffusion and mutual-diffusion coefficients to the same average. After such a reduction, the VCCs and distinct-diffusion coefficients are evaluated for a homologous series of poly(ethylene glycol)s (PEGs). Attractive PEG–PEG interactions depend on the chain length and concentration of PEG. In this analysis, network formation in PEG–water systems appears to be a smooth process. © 2001 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 40: 43–51, 2002

This chapter presents a summary of papers published in the period June 2008–May 2009 focusing on the use and implementation of NMR techniques to elucidate the microstructure and dynamics of self-assembled systems.

Diffusion coefficients in the ternary system α-cyclodextrin (at one concentration)-(DL) norleucine (at three concentrations)-water have been measured using the Gouy interferometric technique. ― The effect of the inclusion equilibrium on the cross-term diffusion coefficients was observed. ― The measured diffusion coefficients in the ternary systems were used to calculate values of the binding constant. These values are in good agreement with that obtained from calorimetric studies