Transient Anomalous Diffusion MRI Measurement Discriminates Porous Polymeric Matrices Characterized by Different Sub-Microstructures and Fractal Dimension.

Considering the current development of new nanostructured and complex materials and gels, it is critical to develop a sub-micro-scale sensitivity tool to quantify experimentally new parameters describing sub-microstructured porous systems. Diffusion NMR, based on the measurement of endogenous water's diffusion displacement, offers unique information on the structural features of materials and tissues. In this paper, we applied anomalous diffusion NMR protocols to quantify the subdiffusion of water and to measure, in an alternative, non-destructive and non-invasive modality, the fractal dimension dw of systems characterized by micro and sub-micro geometrical structures. To this end, three highly heterogeneous porous-polymeric matrices were studied. All the three matrices composed of glycidylmethacrylate-divynilbenzene porous monoliths obtained through the High Internal Phase Emulsion technique were characterized by pores of approximately spherical symmetry, with diameters in the range of 2-10 µm. Pores were interconnected by a plurality of window holes present on pore walls, which were characterized by size coverings in the range of 0.5-2 µm. The walls were characterized by a different degree of surface roughness. Moreover, complementary techniques, namely Field Emission Scanning Electron Microscopy (FE-SEM) and dielectric spectroscopy, were used to corroborate the NMR results. The experimental results showed that the anomalous diffusion α parameter that quantifies subdiffusion and dw = 2/α changed in parallel to the specific surface area S (or the surface roughness) of the porous matrices, showing a submicroscopic sensitivity. The results reported here suggest that the anomalous diffusion NMR method tested may be a valid experimental tool to corroborate theoretical and simulation results developed and performed for describing highly heterogeneous and complex systems. On the other hand, non-invasive and non-destructive anomalous subdiffusion NMR may be a useful tool to study the characteristic features of new highly heterogeneous nanostructured and complex functional materials and gels useful in cultural heritage applications, as well as scaffolds useful in tissue engineering.

Diffusion through skin in the light of a fractional derivative approach: progress and challenges.

This review is focussed on modelling the transport processes of different drugs across the intact human skin by introducing a memory formalism based on the fractional derivative approach. The fundamental assumption of the classic transport equation in the light of the Fick's law is that the skin barrier behaves as a pseudo-homogeneous membrane and that its properties, summarized by the diffusion coefficient D,  do not vary with time and position. This assumption does not hold in the case of a highly heterogeneous system as the skin is, whose outermost layer (the stratum corneum) is comprised of a multi-layered structure of keratinocytes embedded in a lamellar matrix of hydrophobic lipids, followed by the dermis that contains a network of capillaries that connect to the systemic circulation. A possible way to overcome these difficulties resides in the introduction of mathematical models which involve fractional derivatives to describe complex systems with interactions in space and time, following the model originally developed by Caputo in order to consider the memory effects in materials. Although the introduction of fractional derivatives to model memory effects is completely phenomenological, i.e., characterized by a single parameter, i.e., the fractional derivative order [Formula: see text] a number of authors have found that this approach can provide a better comparison to experimental data and that this technique may be alternative to integer-order derivative models. In this review, we aim to summarize some our recent results, concerning the transport of different diffusing compounds of different structural complexity across the intact skin.

Fractional derivatives in the diffusion process in heterogeneous systems: The case of transdermal patches.

In this note, we present a simple mathematical model of drug delivery through transdermal patches by introducing a memory formalism in the classical Fick diffusion equation based on the fractional derivative. This approach is developed in the case of a medicated adhesive patch placed on the skin to deliver a time released dose of medication through the skin towards the bloodstream.The main resistance to drug transport across the skin resides in the diffusion through its outermost layer (the stratum corneum). Due to the complicated architecture of this region, a model based on a constant diffusivity in a steady-state condition results in too simplistic assumptions and more refined models are required.The introduction of a memory formalism in the diffusion process, where diffusion parameters depend at a certain time or position on what happens at preceeding times, meets this requirement and allows a significantly better description of the experimental results.The present model may be useful not only for analyzing the rate of skin permeation but also for predicting the drug concentration after transdermal drug delivery depending on the diffusion characteristics of the patch (its thickness and pseudo-diffusion coefficient).

Direct interaction of hydrophilic gold nanoparticles with dexamethasone drug: loading and release study.

Water-soluble gold nanoparticles functionalized by sodium 3-mercapto-1-propansulfonate (Au-3MPS) were synthesized with different Au/thiol molar ratios for their ability to interact with biomolecules. In particular, a synthetic glucocorticoid steroid, i.e. dexamethasone (DXM) was selected. Herein, the formation of the Au-3MPS/DXM bioconjugate is reported. Au-3MPS nanoparticles show a plasmon resonance at 520 nm, have a spherical morphology and average size of 7-10 nm. The total number of gold atoms was estimated to be about 10600, with a surface component of 8800 atoms and a number of thiol ligands of about 720, roughly one anchored thiol every 10 surface gold atoms. The drug-nanoparticle interaction occurs through the fluorine atom of DXM and Au(I) atoms on the gold nanoparticle surface. The 3MPS ligands closely pack apart each other to leave room for the DXM, that lies at the gold surface in an unusual, almost parallel feature. The loading efficiency of DXM on Au-3MPS was assessed in the range 70-80%, depending on the thiol content. Moreover, our studies confirmed the drug release of about 70% in 5 days. Thanks to their unique properties, i.e. high water solubility, small size and almost monodispersity, Au-3MPS display high potential in biotechnological and biomedical applications, mainly for the loading and release of water insoluble drugs.

Hyaluronic acid and alginate covalent nanogels by template cross-linking in polyion complex micelle nanoreactors.

Hyaluronic acid (HA) and alginate (AL) covalent nanogels cross-linked with l-lysine ethyl ester were prepared by template chemical cross-linking of the polysaccharide in polyion complex micelle (PIC) nanoreactors. By using this method we were able to prepare HA and AL nanogels without organic solvents. PICs were prepared by using poly(ethylene oxide)-block-poly[(3-acrylamidopropyl)-trimethylammonium chloride] (PEO-b-PAMPTMA) or poly[(N-isopropylacrylamide)-block-PAMPTMA] (PNIPAAM-b-PAMPTMA). Only PNIPAAM-b-PAMPTMA block copolymers allowed to prepare PIC with small and controlled size. Short polysaccharide chains (Xn=50 and 63 for AL and HA, respectively, where Xn is the number of monosaccharidic units present in the polysaccharide) where used to optimize PIC formation. The remarkable difference in charge density and rigidity of HA and AL did not have a significant influence on the formation of PICs. PICs with small size (diameter of about 50-80 nm) and low polydispersity were obtained up to 5mg/mL of polymer. After cross-linking with l-lysine ethyl ester, the nanoreactors were dissociated by adding NaCl. The nanogels were easily purified and isolated by dialysis. The dissociation of the nanoreactors and the formation of the nanogels were confirmed by (1)H NMR, DLS, TEM and ζ-potential measurements. The size of the smallest nanogels in solution in the swollen state was 50-70 nm in presence of salt and 80-100 nm in water.

Chitosan-coated PLGA nanoparticles: a sustained drug release strategy for cell cultures.

A recently patented one-step methodology was used for the formulation of chitosan (CS) coated polylactic-co-glycolic acid (PLGA) nanoparticles containing dexamethasone (DXM) as a model drug. SEM investigations showed that nanoparticles (NPs) were spherical in shape with smooth surface. CS coating switched NPs ζ-potential from negative to positive, without modifying particle size distribution. Moreover, CS coating allowed a significant modulation of in vitro drug release, providing a sustained drug delivery in cultured cells. The uptake of fluorescent CS-coated PLGA NPs by hepatocytes (C3A) and fibroblasts (3T6) as well as the fate of internalized NPs were investigated by confocal microscopy. 3T6 and C3A cells were treated with DXM-loaded NPs and experiments were addressed to analyze the specific cell response to DXM, in order to evaluate its functional efficiency in comparison with conventional addition to culture medium. CS-coating of DXM loaded PLGA NPs allowed their uptake by cultured cells without inducing cytotoxicity.

Functional polymeric nanoparticles for dexamethasone loading and release.

Poly(phenylacetylene) (PPA) and poly(phenylacetylene-co-acrylic acid) (P(PA-co-AA)), nanoparticles bioconjugated with dexamethasone (DXM) during the synthesis, named PPA@DXM and P(PA-co-AA)@DXM, were prepared by a modified surfactant free emulsion method. The loading was studied as a function of different functionality grades of the copolymer and different amounts of drug, obtaining up to 90% of drug loading for P(PA-co-AA)@DXM with 8/1 PA/AA monomer ratio. The SEM images and DLS measurements showed spheres with average diameters in the range 190-500 nm, depending on the content of acrylic acid monomer units in the copolymer and of DXM loading. ζ-potential and surface charge density of DXM loaded nanoparticles were also investigated and confirm the charge density modulation in the range 0.62-2.68σ (µC/m(2)). The results highlight the enhanced capability of our copolymer of hosting DXM, with the advantage of a control of size, surface functionality, charge and release. Moreover we demonstrate for the first time the ability of P(PA-co-AA) DXM loaded nanoparticles to be used in the apoptosis inhibition of human tumor cells (HeLa). On the basis of the results obtained by comparing the effects elicited in HeLa cells by free DXM versus DXM loaded nanoparticles we confirmed the biological efficacy of our preparation.

Core shell hybrids based on noble metal nanoparticles and conjugated polymers: synthesis and characterization.

Noble metal nanoparticles of different sizes and shapes combined with conjugated functional polymers give rise to advanced core shell hybrids with interesting physical characteristics and potential applications in sensors or cancer therapy. In this paper, a versatile and facile synthesis of core shell systems based on noble metal nanoparticles (AuNPs, AgNPs, PtNPs), coated by copolymers belonging to the class of substituted polyacetylenes has been developed. The polymeric shells containing functionalities such as phenyl, ammonium, or thiol pending groups have been chosen in order to tune hydrophilic and hydrophobic properties and solubility of the target core shell hybrids. The Au, Ag, or Pt nanoparticles coated by poly(dimethylpropargylamonium chloride), or poly(phenylacetylene-co-allylmercaptan). The chemical structure of polymeric shell, size and size distribution and optical properties of hybrids have been assessed. The mean diameter of the metal core has been measured (about 10-30 nm) with polymeric shell of about 2 nm.

Dielectric properties of micellar aggregates due to the self-assembly of thermoresponsive diblock copolymers.

The radiowave dielectric properties of aqueous solutions of thermosensitive copolymers, consisting of poly(2-acrylamido-2-methylpropanesulfonate) [PAMPS] and poly(N-isopropylacrylamide) [PNIPAAM] with different block lengths, have been investigated over a broad temperature and frequency range. These copolymers PAMPS(n)-b-PNIPAAM(m) form temperature responsive aggregates (micelles) that represent a class of self-assembled structures in water of great interest because of their potential use as drug delivery formulations and in diverse biotechnological applications. Copolymers formed by hydrophilic segments covalently attached to a hydrophobic segments are capable of forming a micellar structure as soon as the temperature is raised above their lower critical solution temperature. We have investigated the dielectric properties of PAMPS(n)-b-PNIPAAM(m) diblock copolymers with different lengths of the hydrophilic and hydrophobic segments during the whole aggregation process driven by the progressive increase of temperature. The process has been followed by the changes resulting in the dielectric parameters (the dielectric increment Δε and the relaxation frequency ν(0)) of the whole aqueous solution. The dielectric response of the micelles has been described within the framework of the standard electrokinetic model for charged colloidal particles, and the main characteristic parameters have been evaluated. Subsequent cross-linking of these diblock copolymers by a cationic PEO(x)-b-PAMPTMA(y) polyelectrolyte yields hybrid core-shell-corona systems, with the PNIPAAM hydrophobic blocks collapsed in the core, an interpolyelectrolyte chain complex forming the shell, and the hydrophilic PEO chains as an external corona. In this case too, the dielectric spectra can be appropriately accounted for within the same theoretical framework.

Self-assembled nanoparticles of functional copolymers for photonic applications.

A modified emulsion copolymerization of phenylacetylene (PA) with hydrophilic monomers having different functions, i.e., acrylic acid (AA) and N,N-dimethylpropargylamine (DMPA) respectively, yields functionalized polymeric P(PA-co-AA) and P(PA-co-DMPA) nanoparticles. The systematic investigation on the experimental parameters affecting size, surface charge and polydispersity of the copolymers (initiator concentration, reaction time, cosolvent and PA/comonomer ratios) allows to modulate the nanoparticle physico-chemical properties. Spherical shaped particles with diameters in the range 80-500 nm, low polydispersity (PI values in the range 1.11-1.30) and different surface charge densities, between 0.44 and 2.87 microC/cm(2), have been consistently obtained and characterized by means of Dynamic Light Scattering (DLS), laser Doppler electrophoretic and Scanning Electron Microscopy (SEM) studies. XPS measurements have provided information on the nanoparticles chemical surface structure and suggest that AA and DMPA units are preferentially distributed on the surface of the spheres. The nanospheres self-assemble giving large domains (9.5 x 14.5 microm). Photonic analysis of the self-assembled copolymeric nanoparticles has been performed by means of Spectroscopic Ellipsometry (SE) and Bragg reflection spectroscopy, both of them demonstrating a three-dimensional photonic crystal property of these systems.

Hemocompatibility of carotid artery stents: alterations of the electrical parameters of erythrocyte cell membrane--a word of caution.

The hemocompatibility of standard surgical treatment of carotid artery disease through the insertion of metallic stents is investigated by means of radio wave dielectric spectroscopy technique that allows the measurements of the electrical parameters of the red blood cell membrane. Our measurements suggest that both the membrane permittivity and the membrane conductivity, which characterize the overall electrical behavior of the cell membrane, undergo an appreciable alteration of their standard values as a consequence of the stent insertion. These alterations persist over long period of time, up to 4 weeks. Even if these effects could not cause any evident damage at physiological or clinical level to the patient, the presence of a host response to the stent implant suggests that a full hemocompatibility has not yet reached, and a word of caution is necessary.

Dielectric properties of thermo-reversible hydrogels: the case of a dextran copolymer grafted with poly(N-isopropylacrylamide).

We investigated the dielectric properties of aqueous solutions of a grafted copolymer, consisting of a polysaccharide, Dextran, grafted with a thermo-sensitive polymer, poly(N-isopropylacrylamide), [pNIPAAM], over broad temperature and frequency ranges. The graft copolymers, prepared by atom-transfer radical polymerization [ATRP], form temperature-responsive materials that represent a class of self-assembled structures in water of great interest because of their potential use as drug delivery formulations and in diverse biotechnological applications. In these systems, in the dilute regime and below the lower critical solution temperature, relaxation modes corresponding to two different length-scales have been observed and analyzed in terms of ion fluctuation dielectric models specifically developed to describe the dielectric relaxation in highly charged polyion aqueous solutions. Regardless of whether the ions were produced by the ionization of the polymer chain, as in polyelectrolyte solutions, or not, as in the present case, they represent a probe at a microscopic level that is expected to reveal the structural characteristics of the system at different scales. We have identified a characteristic length associated with the size of the polymer coil in the dilute regime and a length due to fixed cross-links, where ions are partially localized by the local profile of the Coulombic field. These lengths are in reasonable agreement with analogous lengths derived from structural information and from the hydrodynamic radius of the polymer coils, measured by means of a dynamic light-scattering technique.

L-DOPA preloading increases the uptake of borophenylalanine in C6 glioma rat model: a new strategy to improve BNCT efficacy.

PURPOSE: Boron neutron capture therapy (BNCT) is a radiotherapeutic modality based on (10)B(n,alpha)(7)Li reaction, for the treatment of malignant gliomas. One of the main limitations for BNCT effectiveness is the insufficient intake of (10)B nuclei in the tumor cells. This work was aimed at investigating the use of L-DOPA as a putative enhancer for (10)B-drug 4-dihydroxy-borylphenylalanine (BPA) uptake in the C6-glioma model. The investigation was first performed in vitro and then extended to the animal model. METHODS AND MATERIALS: BPA accumulation in C6-glioma cells was assessed using radiowave dielectric spectroscopy, with and without L-DOPA preloading. Two L-DOPA incubation times (2 and 4 hours) were investigated, and the corresponding effects on BPA accumulation were quantified. C6-glioma cells were also implanted in the brain of 32 rats, and tumor growth was monitored by magnetic resonance imaging. Rats were assigned to two experimental branches: (1) BPA administration; (2) BPA administration after pretreatment with L-DOPA. All animals were sacrificed, and assessments of BPA concentrations in tumor tissue, normal brain, and blood samples were performed using high-performance liquid chromatography. RESULTS: L-DOPA preloading induced a massive increase of BPA concentration in C6-glioma cells only after a 4-hour incubation. In the animal model, L-DOPA pretreatment produced a significantly higher accumulation of BPA in tumor tissue but not in normal brain and blood samples. CONCLUSIONS: This study suggests the potential use of L-DOPA as enhancer for BPA accumulation in malignant gliomas eligible for BNCT. L-DOPA preloading effect is discussed in terms of membrane transport mechanisms.

Diffusion with memory in two cases of biological interest.

The complexity of a biological structure, such as membrane where the transport process may carry solid particles which may obstruct some of the pores, diminishing their size and making the permeability dependent on the local structure of the medium, suggests the introduction of a space-dependent diffusion constant. In this note, the profile concentration of diffusing solutes inside a cell membrane has been calculated on the basis of the Fick diffusion equation modified by introducing a memory formalism (diffusion with memory). This approach has been employed to describe the concentration profile inside the membrane when a sudden change of the concentration in the medium bathing one of its face is applied for a limited interval of time. A further application of the method concerns the so-called concentration boundary layer that occurs at the membrane-aqueous medium interface, where the solute concentration depends, even at considerable depth, on the local structure of the interface. These profiles are compared to some recent experiments concerning the diffusion of ethanol in a layer close to a nephrophane membrane. This approach generalizes the diffusion models based on the Fick equation to more complex systems, where a space-independent diffusion coefficient could be inappropriate to take into account the large variety of diffusion processes in biological systems.

Hybrid niosome complexation in the presence of oppositely charged polyions.

We have investigated the formation of complexes between negatively charged niosomal vesicles (hybrid niosomes), built up by dicethylphosphate [DCP], Tween 20 and Cholesterol, and three linear differently charged cationic polyions, such as alpha-polylysine, epsilon-polylysine, and polyethylvinylpyridinium bromide [PEVP], with two different substitution degrees. Our aim is to investigate the interaction mechanism between anionic-nonionic vesicles (hybrid niosomes) and linear polycations, characterizing the resulting aggregates in view of possible applications of these composite colloidal particles as vectors for multidrug delivery. In order to explore the aggregation behavior of the complexes and to gain information on the stability of the single niosomal vesicles within the aggregates, we employed dynamic light scattering (DLS), laser Doppler electrophoretic measurements, and fluorescence measurement techniques. The overall phenomenology is well described in terms of the re-entrant condensation and charge inversion behavior, observed in different colloidal systems. The aggregate size and overall charge depend on the charge ratio between vesicles and polyions, and the aggregates reach their maximum size at the point of charge inversion (re-entrant condensation). While the overall phenomenology is similar for all three polycations investigated, the stability and the integrity of the hybrid niosomal vesicles forming the aggregates strongly depend on the chemical structure of the polycations. The role of the polycations in the aggregation process is discussed by identifying specific interactions with the niosomal membrane, pointing out their importance for possible applications as drug delivery vectors.

Liposomes from a new chiral cationic lipid based on iridoidic template.

A new cationic polyhydroxylated lipid, characterized by a chiral template, was synthesized. It comes from an iridoid glucoside, as polyhydroxylated moiety. This lipid affords liposomes using cholesterol-like co-lipid. The liposomes had a spheroidal shape with a small size distribution.

Are aortic endograft prostheses fully hemo-compatible? A dielectric spectroscopy investigation of the electrical alterations induced on erythrocyte cell membranes.

In this paper we present a new approach directed to ascertain the full hemo-compatibility of aortic endograft prostheses based on the measurement of the passive electrical parameters of the erythrocyte cell membrane. The red blood cell membrane, from an electric point of view, is characterized by an electrical permittivity, (s), which takes into account the structural charged organization of the lipid double layer, and by the electrical conductivity, sigma(s), which accounts for the ionic transport processes across the membrane. These parameters can be easily measured by means of a radiowave dielectric spectroscopy technique, analyzing the dependence of the electrical impedance of an erythrocyte suspension on the frequency of the applied electric field. In this preliminary report, we investigate the alterations induced, at a membrane level, by two different devices commonly employed for endovascular abdominal aortic aneurysm exclusion, i.e., Excluder and Zenith devices, implanted in ten patients. We observe, in all the cases investigated, a statistically significant increase of both the permittivity (s) and electrical conductivity sigma(s) of the erythrocyte membrane upon the prosthesis implant, this increase being higher than about 20% of the un-treated values. Moreover, these alterations remain roughly unaffected 30 days after surgery. These findings suggest that a complete hemo-compatibility of these prostheses is lacking, even if the observed alterations may not have a clinical relevance.

Role of cholesterol, DOTAP, and DPPC in prostasome/spermatozoa interaction and fusion.

Prostasomes are membranous vesicles present in ejaculated human semen. They are very rich in cholesterol and can interact with spermatozoa. Their physiological roles are still under study. Prostasomes were mixed with liposomes prepared from various lipids, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), DOTAP/1,2-dipalmytoyl-sn-glycero-3-phosphorylcholine (DPPC, 4:1 molar ratio) and DOTAP/cholesterol (4:1, molar ratio) at different pH values (5-8). The mixing of the lipid phases (fusion) was determined by the relief of octadecyl rhodamine B chloride (R(18)) self-quenching and the radii of the vesicles, by light scattering measurements. The mixing of lipids and the radii of prostasomes were both influenced by the addition of liposome, although in a different manner. The ability of prostasomes (modified by previous treatment with liposomes) to transfer lipid to spermatozoa was also measured. Pretreatment with DOTAP decreased the phenomenon and addition of DPPC abolished it. On the other hand, pretreatment of prostasomes with DOTAP/cholesterol liposomes did not affect the transfer of lipid between prostasome and spermatozoa. Therefore, the ability of vesicles to fuse (or, at least, to exchange the lipid component) was affected by the enrichment in either natural or artificial lipid. This may open new possibilities for the modulation of spermatozoa capacitation and acrosome reaction.

Charge renormalization in planar and spherical charged lipidic aqueous interfaces.

The charge renormalization in planar and spherical charged lipidic aqueous interfaces has been investigated by means of thermodynamic and electrokinetic measurements. We analyzed the behavior of mixed DOTAP/DOPE monolayers at the air-electrolyte solution interface and DOTAP/DOPE liposomes 100 nm in size dispersed in an aqueous phase of varying ionic strength. For the two systems, we have compared the "effective" surface charge derived from the measurements of surface potential and zeta-potential to the "bare" charge based on the stoichiometry of the lipid mixture investigated. The results confirm that a strong charge renormalization occurs, whose strength depends on the geometry of the mesoscopic system. The dependence of the "effective" charge on the "bare" charge is discussed in light of an analytical approximation based on the Poisson-Boltzmann equation recently proposed.

Tetracycline inhibits W7FW14F apomyoglobin fibril extension and keeps the amyloid protein in a pre-fibrillar, highly cytotoxic state.

A significant number of fatal diseases are classified as protein deposition disorders, in which a normally soluble protein is deposited in an insoluble amyloid form. It has been reported that tetracycline exhibits anti-amyloidogenic activity by inhibiting aggregate formation and disaggregating preformed fibrils. In this work, we examined the effect induced by the presence of tetracycline on the fibrillogenesis and cytotoxicity of the amyloid-forming apomyoglobin mutant W7FW14F. Like other amyloid-forming proteins, early prefibrillar aggregates formed by this protein are highly cytotoxic, whereas insoluble mature fibrils are not. The effect induced by tetracycline on the fibrillation process has been examined by atomic force microscopy, light scattering, DPH staining, and thioflavin T fluorescence. The cytotoxicity of the amyloid aggregates was estimated by measuring cell viability using MTT assay. The results show that tetracycline acts as anti-aggregating agent, which inhibits the fibril elongation process but not the early aggregation steps leading to the formation of soluble oligomeric aggregates. Thus, this inhibition keeps the W7FW14F mutant in a prefibrillar, highly cytotoxic state. In this respect, a careful usage of tetracycline as fibril inhibitor is indicated.