F. Bordi

Dielectric spectroscopy and conductivity of polyelectrolyte solutions

The dielectric and conductometric properties of aqueous polyelectrolyte solutions present a very complex phenomenology, not yet completely understood, differing from the properties of both neutral macromolecular solutions and of simple electrolytes. Three relaxations are evident in dielectric spectroscopy of aqueous polyelectrolyte solutions. Near 17 GHz, water molecules relax and hence this highest frequency relaxation gives information on the state of water in the solution. At lower frequencies in the MHz range, free counterions respond to the applied field and polarize on the scale of the correlation length. This intermediate frequency relaxation thus provides information about the effective charge on the polyelectrolyte chains, and the fraction of condensed counterions. However, the presence of polar side chains adds a further polarization mechanism that also contributes in this intermediate frequency range. At still lower frequencies, the condensed counterions polarize in a non-uniform way along the polyelectrolyte chain backbone and dielectric spectroscopy in the kHz range may determine the effective friction coefficient of condensed counterions. In this review, we analyse in detail the dielectric and conductometric behaviour of aqueous polyelectrolyte solutions in the light of recent scaling theories for polyelectrolyte conformation and summarize the state-of-the-art in this field.

New cationic liposomes as vehicles of m-tetrahydroxyphenylchlorin in photodynamic therapy of infectious diseases.

Antimicrobial photodynamic therapy is emerging as a promising therapeutic modality for bacterial infections. For optimizing the antibacterial activity of the photosensitizer m-tetrahydroxyphenylchlorin, it has been encapsulated in mixed cationic liposomes composed of different ratios of dimyristoyl- sn-glycero-phosphatidylcholine and any of four cationic surfactants derived from l-prolinol. The delivery efficiency of the different liposomes formulations has been evaluated on a methicillin-resistant Staphylococcus aureus bacterial strain (MRSA), and one of the tested formulations shows a biological activity comparable to that of the free chlorin. In order to rationalize the physicochemical parameters of the carriers that control the biological activity, the new liposome formulations have been characterized by measuring (a) the zeta potential, (b) their capability of chlorin entrapping efficiency, i.e. entrapment efficacy, (c) the effect of storage on chlorin entrapment and (d) the localization of chlorin in the bilayer. The correlation of the physicochemical and biological features of formulations has allowed us to rationalize, to some extent, some of the parameters that may control the interactions with the biological environment.

Frequency domain electrical conductivity measurements of the passive electrical properties of human lymphocytes

An extensive set of electrical conductivity measurements of human lymphocyte suspensions has been carried out in the frequency range from 1 kHz to 100 MHz, where the surface polarization due to the Maxwell-Wagner effect occurs. The data have been analyzed according to well-established heterogeneous system theories and the passive electrical parameters of both the cytoplasmic and nuclear membranes have been obtained. Moreover, a further analysis to take into account the roughness of the membrane surface on the basis of a fractal model yields new estimates for the membrane conductivity and the membrane permittivity, independently of the surface architecture of the cell. These findings are confirmed by measurements carried out at higher frequencies, in the range from 1 MHz to 1 GHz, on lymphocytes dispersed in both hyperosmotic and hypoosmotic media, that influence the surface complexity of the membrane due to the microvillous protrusions. The surface roughness of the cell is described by a macroscopic parameter (the fractal dimension) whose variations are associated to the progressive swelling of the cell, as the osmolality of the solution is changed.

Lipase-supported synthesis of peptidic hydrogels

Self-assembling gelling peptides are increasingly being investigated as defined biomatrices for biomedical applications. Using an enzymatic reaction to convert a precursor into a hydrogelator one can control or modulate functions and responses of a hydrogel, depending on its preparation conditions. This work details the self-assembly, under physiological conditions, of amphiphilic building blocks consisting of tripeptides (Phe3) linked to fluorenylmethoxycarbonyl (Fmoc) obtained by using a lipase to link an Fmoc–Phe amino acid to the dipeptide diphenylalanine (Phe2). The viscoelastic properties of the tripeptides obtained were investigated. SEM and AFM images of Fmoc–peptides confirmed that they self-assemble to generate supramolecular aggregates driven by π–π stacking interactions of the Fmoc groups.

On-chip detection of multiple serum antibodies against epitopes of celiac disease by an array of amorphous silicon sensors

In this paper, we present the preliminary results of an ELISA-on-chip device, intended as a technological demonstrator of a novel analytical system suitable for the diagnosis and follow-up of celiac disease. The idea of the work is to combine an array of amorphous silicon photosensors with a pattern of a poly(2-hydroxyethyl methacrylate) polymer brush film, which acts as anchor for the immobilization of gliadin peptides containing the celiac disease epitopes. Recognition relies on a sandwich immunoassay between antibodies against the peptides and secondary antibodies marked with horseradish peroxidase to obtain a chemiluminescent signal. Detection is based on the measurement of photocurrent induced in the array of amorphous silicon photosensors by the chemiluminescent signal. An ad-hoc procedure has been developed in order to enable the fabrication of the photodiode array and the polymer brush pattern on the two sides of the same glass substrate ensuring the compatibility of the different technological steps. The sensitivity and the selectivity of the chip for multiplex immunoassays were demonstrated using two gliadin peptides (VEA and DEC). In particular, we found that the average amount of the bound HRP revealed by our analytical protocol is 3.5(±0.3) × 10−6 pg μm−2 and 0.85(±0.3) × 10−6 pg μm−2 for specific and non-specific interactions, respectively.

Structure–activity relationships of Candida rugosalipase immobilized on polylactic acid nanoparticles

Interfacing proteins with nanostructured materials offers the possibility to obtain novel bioconjugates for many applications. We report herein the ability of nanostructured poly-DL-lactic acid (PDLLA) based carriers to enhance enzymatic activity and stability. PDLLA was processed using an innovative patented methodology that permitted to obtain spherical nanoparticles with an average diameter of 220 nm that were used as carrier for the physical adsorption of Candida rugosalipase (CRL). Enzymatic activity and stability of CRL before and after conjugation to the nanopolymeric support were evaluated in different conditions (pH, T, organic solvents) and the conformational changes of CRL produced by its interaction with the nanopolymeric carrier were investigated by using Fourier Transform Infrared (FTIR) spectroscopy. A comparative study between X-ray diffraction data in the literature and experimental FTIR results gave deeper insight into the conformational features of the immobilized protein.

Distribution of GD3 in DPPC Monolayers: A Thermodynamic and Atomic Force Microscopy Combined Study

Gangliosides are the main component of lipid rafts. These microdomains, floating in the outer leaflet of cellular membrane, play a key role in fundamental cellular functions. Little is still known about ganglioside and phospholipid interaction. We studied mixtures of dipalmitoylphosphatidylcholine and GD3 (molar fraction of 0.2, 0.4, 0.6, 0.8) using complementary techniques: 1), thermodynamic properties of the Langmuir-Blodgett films were assessed at the air-water interface (surface tension, surface potential); and 2), three-dimensional morphology of deposited films on mica substrates were imaged by atomic force microscopy. Mixture thermodynamics were consistent with data in the literature. In particular, excess free energy was negative at each molar fraction, thus ruling out GD3 segregation. Atomic force microscopy showed that the height of liquid-condensed domains in deposited films varied with GD3 molar fraction, as compatible with a lipid aggregation model proposed by Maggio. No distinct GD3-rich domain was observed inside the films, suggesting that GD3 molecules gradually mix with dipalmitoylphosphatidylcholine molecules, confirming DeltaG data. Morphological analysis revealed that the shape of liquid-condensed domains is strongly influenced by the amount of GD3, and an interesting stripe-formation phenomenon was observed. These data were combined with the thermodynamic results and interpreted in the light of McConnells model.

Chemical and physical hydrogels: two casesystems studied by quasi elastic light scattering

A chemical hydrogel based on telechelic poly(vinyl alcohol) and a physical synergic hydrogel, xanthan–glucomannan, were studied by means of dynamic light scattering technique. Two different nonergodic methods, due to Pusey et al. (Physica A 157 (1989) 705, Phys. Rev. A 42 (1990) 2161) and Xue et al. (Phys. Rev. A 46 (1992) 6550), were used to obtain the correct dynamic structure factors f(q,t). Good agreement between the results obtained with the two procedures was found. The behavior of the f(q,t) for different q vectors is consistent with the hypothesis that the nonfluctuating component in the scattered light is not due to microscopic heterogeneities but to restricted motion of the scatterers. The model of Krall et al. (Phys. Rev. Lett. 80 (1998) 778, Physica A 235 (1997) 19) was successfully applied to both hydrogels to extract the characteristic parameters of the network from the f(q,t) functions.

IONIC TRANSPORT IN LIPID BILAYER MEMBRANES

The current-voltage relationships of model bilayer membranes have been measured in various phospholipid systems, under the influence of both a gradient of potential and an ionic concentration, in order to describe the ion translocation through hydrated transient defects (water channels) across the bilayer formed because of lipid structure fluctuations and induced by temperature. The results have been analyzed in the light of a statistical rate theory for the transport process across a lipid bilayer, recently proposed by Skinner et al. (1993). In order to take into account the observed I-V curves and in particular the deviation from an ohmic behavior observed at high potential values, the original model has been modified, and a new version has been proposed by introducing an additional kinetic process. In this way, a very good agreement with the experimental values has been obtained for all of the systems we have investigated (dimyristoylphosphatidyl ethanolamine bilayers and mixed systems composed by dimyristoylphosphatidyl ethanolamine/dimyristoylphosphatidylcholine mixtures and dimyristoylphosphatidyl ethanolamine/phosphatidic acid dipalmitoyl mixtures). The rate constants governing the reactions at the bilayer interfaces have been evaluated for K+ and Cl- ions, as a function of temperature, from 5 to 35 degrees C and bulk ionic concentrations from 0.02 to 0.2 M. Finally, a comparison between the original model of Skinner and the modified version is presented, and the advantages of this new formulation are briefly discussed.

On the phase diagram of reentrant condensation in polyelectrolyte-liposome complexation

Complexation of polyions with oppositely charged spherical liposomes has been investigated by means of dynamic light scattering measurements and a well-defined reentrant condensation has been observed. The phase diagram of charge inversion, recently derived [T. T. Nguyen and B. I. Shklovskii, J. Chem. Phys. 115, 7298 (2001)] for the complexation of DNA with charged spherical macroions, has been employed in order to define the boundaries of the region where polyion-liposome complexes begin to condense, forming larger aggregates, and where aggregates dissolve again, towards isolated polyion-coated-liposome complexes. A reasonable good agreement is observed in the case of complexes formed by negatively charged polyacrylate sodium salt polyions and liposomes built up by cationic lipids (dioleoyltrimethylammoniumpropane), in an extended liposome concentration range.