Chemical Sciences

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 β-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 β-aggregates. It is worth noticing that the structural conversion from helical monomer to β-aggregates, mimics β-amyloid peptide aggregation mechanisms.

A detailed structural investigation on mixed micelle aggregates as target-specific contrast agents for magnetic resonance imaging technique has been carried out by way of small angle neutron scattering measurements. These mixed micelles are formed by two new amphiphilic molecules formed by a bioactive peptide and a claw moiety. The first molecule, C 18 H 37 CONH(AdOO) x -G-CCK8 (C18LxCCK8, x ) 2, 5), contains an 18carbon-atom alkylic chain bound to the C-terminal of the cholecystokinin octapeptide amide (CCK 26-33 or CCK8) and is able to bind to the overexpressed CCK receptor of some tumor cells. The second molecule, C 18 H 37 CONHLys(DTPAGlu)CONH 2 5-(C18DTPAGlu) or its gadolinium complex [C 18 H 37 CONHLys(DTPAGlu)-CONH 2 Gd] 2-, C18DTPAGlu(Gd), contains the same 18-carbon-atom alkylic chain bound, through a lysine residue, to the DTPAGlu chelating agent. Small angle neutron scattering measurements have been performed on ternary systems at different total concentrations and at various ratios of the two molecules. The effect of the concentration on the aggregation number as well as on the shape of the micelle has been investigated. Furthermore in order to optimize the exposure of the peptide on the micelle surface, C18LxCCK8 having the spacer L of different length has been used. The pure binary systems of the synthesized molecules are also presented.

An efficient drug delivery strategy is presented for novel anticancer amphiphilic ruthenium anionic complexes, based on the formation of stable nanoparticles with the cationic lipid 1,2-dioleyl-3-trimethylammoniumpropane chloride (DOTAP). This strategy is aimed at ensuring high ruthenium content within the formulation, long half-life in physiological media, and enhanced cell uptake. An in-depth microstructural characterization of the aggregates obtained mixing the ruthenium complex and the phospholipid carrier at 50/50 molar ratio is realized by combining a variety of techniques, including dynamic light scattering (DLS), small angle neutron scattering (SANS), neutron reflectivity (NR), electron paramagnetic resonance (EPR), and zeta potential measurements. The in vitro bioactivity profile of the Ru-loaded nanoparticles is investigated on human and non-human cancer cell lines, showing IC 50 values in the low μM range against MCF-7 and WiDr cells, that is, proving to be 10−20-fold more active than AziRu, a previously synthesized NAMI-A analog, used for control. Fluorescence microscopy studies demonstrate that the amphiphilic Ru-complex/DOTAP formulations, added with rhodamine-B, are efficiently and rapidly incorporated in human MCF-7 breast adenocarcinoma cells. The intracellular fate of the amphiphilic Ru-complexes was investigated in the same in vitro model by means of an ad hoc designed fluorescently tagged analog, which exhibited a marked tendency to accumulate within or in proximity of the nuclei.

The aggregation behavior of an amphiphilic supramolecular system, with potential application as a tumor-specific magnetic resonance imaging contrast agent, has been studied in detail by dynamic light scattering, small-angle neutron scattering and cryotransmission electron microscopy. The system was constituted of mixed aggregates formed by an anionic unimer containing the DTPAGlu, a chelating agent for the paramagnetic Gd 31 ion, and an uncharged unimer containing the bioactive peptide CCK8, capable of directing the assembly toward tumor cells. Mixed aggregates formed by both unimers, and in the case of the DTPAGlu unimer with the chelating agent as free base or as Gd 31 complex, have been investigated. A number of interesting features of the aggregation behavior were revealed: at physiological pH, micelles and bilayer structures were present, whereas upon decreasing solution pH or increasing ionic strength, the formation of bilayer structures was favored. On the basis of the above observations, the aggregating mechanism has been elucidated by considering the screening effect on intra-and interaggregate electrostatic repulsions.

Two novel amphiphilic unimers containing an aliphatic hydrophobic chain (PDA) with two C≡C triple bonds and hydrophilic heads presenting the chelating agent DTPAGlu and the CCK8 bioactive peptide, respectively, have been prepared by solid phase synthesis. Aggregates obtained by mixing together PDA-DTPAGlu, or its Gd(III) complex, and PDA-L2-CCK8 in 70/30 molar ratio before and after a polymerization process carried out by UV irradiation have been structurally characterized by means of small angle neutron scattering. The relaxivity properties of aggregates containing Gadolinium complexes have also been investigated. Elongated mixed micelles have been observed, in which the relaxivity value r 1p for each Gadolinium complex, measured at 20 MHz and 298 K, is around 12 mM -1 s -1 .

In continuation with our studies concerning the synthesis, characterization and biological evaluation of nucleolipidic Ru(III) complexes, a novel design for this family of potential anticancer agents is presented here. As a model compound, a new uridine-based nucleolipid has been prepared, named HoUrRu, following a simple and versatile synthetic procedure, and converted into a Ru(III) salt. Stable formulations of this highly functionalized Ru(III) complex have been obtained by co-aggregation with either the zwitterionic lipid POPC or the cationic DOTAP, which have been subjected to an in-depth microstructural characterization, including DLS, SANS and EPR measurements. The in vitro bioactivity profile of HoUrRu, as a pure compound or in formulation with POPC or DOTAP, reveals high antiproliferative activity against MCF-7 and WiDr human cancer cell lines.

Nanoparticles (NPs) are emerging as a potential medical tool for novel diagnostic, drug delivery, and therapeutic approaches. Among them, a spherical NP with a core-shell structure is a way to combine multiple functionalities on the nanoscale. In this paper, we describe the preparation characterization and applications of core-shell iron oxide-gold nanoparticles (Fe 3 O 4 @Au NPs). A comprehensive set of experiments, including transmission electron microscopy, dynamic light scattering, small angle neutron scattering, and ultraviolet visible spectroscopy is applied to characterize their chemical, physical, and optical properties. We also study their applicability as contrast agents for magnetic resonance imaging (MRI): the measurement of longitudinal and transverse relaxation times of Fe 3 O 4 @Au NPs in vitro and in vivo allowed the assessment of longitudinal (R 1 ) and transverse (R 2 ) relaxivities at 1.5 and 3 T. Finally, a procedure for functionalizing NPs with integrin targeting cyclic Arginine-Glycine-Aspartate peptidomimetic is reported, leading to the development of nanoscale probes for α v β 3 integrin, particularly attractive in terms of resolution and 3-D imaging capabilities. The resulting multifunctional nanoprobes offer suitable bloodcirculation time and contrast for microimaging as well as for gradient-echo MRI, and could enable new imaging magnetoplasmonic applications.

The phase behavior (temperature vs composition) and microstructure for the two binary systems Pluronic 25R4 [(PO) (EO) 33 (PO) ]water and Pluronic 25R2 [(PO) 21 (EO) 14 (PO) 21 ]-water have been studied by a combined experimental approach in the whole concentration range and from 5 to 80 • C. The general phase behavior has been identified by inspection under polarized light. Precise phase boundaries have been determined by analyzing 2 H NMR line shape. The identification and microstructural characterization of the liquid crystalline phases have been achieved using small-angle X-ray scattering (SAXS). The isotropic liquid solution phases have been investigated by self-diffusion measurements (PGSE-NMR method). 25R2 does not form liquid crystals and is miscible with water in the whole concentration range; with increasing temperature, the mixtures split into water-rich and a copolymer-rich solutions in equilibrium. 25R4 shows rich phase behavior, passing, with increasing copolymer concentration, from a water-rich solution to a lamellar and copolymer-rich solution. A small hexagonal phase, completely encircled in the stability region of the water-rich solution, is also present. In water-rich solutions, at low temperatures and low copolymer concentrations, the copolymers are dissolved as independent macromolecules. With increasing copolymer concentrations an interconnected network of micelles is formed in which micellar cores of hydrophobic poly(propylene oxide) are interconnected by poly(ethylene oxide) strands. In copolymer-rich solutions water is molecularly dissolved in the copolymer. The factors influencing the selfaggregation of Pluronic R copolymers (PPO-PEO-PPO sequence) are discussed, and their behavior in water is compared to that of Pluronic copolymers (PEO-PPO-PEO sequence). (G. D'Errico). the possible block sequences are commercially available: copolymers with a PEO-PPO-PEO sequence have the trade name Pluronic, while those with a PPO-PEO-PPO sequence are named Pluronic R. Water is a selective solvent for these copolymers; i.e., it is a good solvent for the PEO blocks and a relatively poor solvent for the PPO blocks. Consequently, in aqueous mixture, these copolymers behave similarly to the nonionic surfactants of the oligo(ethylene oxide) type, with formation of micellar solutions and lyotropic liquid crystalline (LLC) phases . Structural parameters such as the average molecular weight (M w ) and the copoly-

Intradiffusion coefficients, D, of n-alkyltrimethylammonium bromides [CH 3 -(CH 2 ) n−1 -N(CH 3 ) 3 Br, C n TAB] (n = 6, 8, 10, 12) in mixtures with heavy water were measured by the PGSE-NMR technique at 25 • C. The experimental data permitted evaluation of the influence of the alkyl chain length on the surfactant selfaggregation process. For all the surfactants considered, the D trend showed a slope change corresponding to the critical micellar composition (cmc). In the premicellar composition range, D decreased linearly with the square root of the surfactant molality. The D values extrapolated at infinite dilution were related to the limiting mutual diffusion coefficients, determined through the Taylor dispersion technique. In the micellar composition range, solubilized tetramethylsilane (TMS) molecules were used to determine the micelle intradiffusion coefficient, D M , from which the aggregate radii and the aggregation numbers were obtained. The decreasing trend of D M with increasing surfactant molality was interpreted in terms of interparticle electrostatic repulsion. D M values allowed evaluation of the Gouy-Chapman layer thickness. The solvent intradiffusion coefficient in the heavy water-C n TAB mixtures, D w , was also measured. It decreased with increasing surfactant molality. For n = 8, 10, 12 the D w trend presented a slope change at the cmc, which could be ascribed to the strong decrease in hydration of surfactant molecules upon micellization. Because of its short hydrophobic tail, C 6 TAB exhibited peculiar aggregation behavior. Its cmc, which is poorly marked, is lower than the value predicted by extrapolating the cmc values obtained for the other terms of the series. The C 6 TAB aggregates do not solubilize TMS molecules; the estimated aggregation number is extremely low (∼3). Finally, no abrupt slope change in the solvent intradiffusion coefficient trend was detected. This evidence suggests that C 6 TAB molecules do not micellize in aqueous solution, but form trimers in which the surfactant hydrophobic tails are not hidden from contact with water molecules. C 2001 Academic Press

. For this reason we thought it useful to present a set Apparent enthalpies of mixing were measured for various of microcalorimetric titrations on a series of C n E m surfactant surfactants of the general formula CH 3 -(CH 2 ) n01 -(OCH 2aqueous solutions; the apparent molar enthalpies of mixing CH 2 ) m OH(C n E m ) in aqueous solution at 25ЊC. From the experiwere determined for the following systems: C 10 E 5 , C 8 E 5 , mental data the enthalpies of micellization were obtained. The C 6 E 5 , C 6 E 3 , and C 6 E 2 . results are briefly discussed. ᭧ 1998 Academic Press

The interaction between pentaethylene glycol n-octyl ether (C 8 E 5 ) and low-molecular-weight poly(acrylic acid) (PAA, M w = 2000) in aqueous solution has been investigated by various experimental techniques at constant polymer concentration (0.1% w/w) with varying surfactant molality. Spectrofluorimetry, using pyrene as molecular probe, shows (i) the formation of surfactant-polymer aggregates at a surfactant molality (T 1 ) lower than the critical micelle concentration (cmc) of C 8 E 5 in water and (ii) the formation of free micelles at a surfactant molality (T 2 ) slightly higher than the cmc. Fluorescence quenching measurements indicate that the presence of PAA induces a lowering of the C 8 E 5 aggregation number. Calorimetry confirms spectrofluorimetric evidence; in addition, it shows the presence of weak interactions below T 1 between monomeric surfactant molecules and the polymer chains. Tensiometry shows that, above T 1 , only a low fraction of surfactant molecules interact with the polymer and that free micelle formation occurs before polymer saturation. The peculiarities of the interaction between surfactants and low-molecular-weight polymers have been discussed.