Bioconjugation of gold-polymer core-shell nanoparticles with bovine serum amine oxidase for biomedical applications.

Core-shell gold nanoparticles [AuNPs], stabilized with a hydrophilic polymer, poly(3-dimethylammonium-1-propyne hydrochloride) [PDMPAHCl], have been used for the immobilization of bovine serum amine oxidase [BSAO]. The functionalized surface of the hybrid nanoparticles is pH responsive, due to the presence of aminic groups that carry out a double role: on one hand they act as ligands for the gold nanoparticle surface, allowing the colloidal stabilization and, on the other hand, they give a hydrophilic characteristic to the whole colloidal suspension. The core-shell nanoparticles [Au@PDMPAHCl] have been characterized by using UV-vis and X-ray photoelectron spectroscopy, DLS, ζ-potential measurements and by FE-TEM microscopy. BSAO enzyme can be loaded by non-covalent immobilization onto Au@PDMPAHCl nanoparticles up to 70% in weight, depending on the pH values of the environmental medium. Activity tests on Au@PDMPAHCl-BSAO bioconjugates confirm an enzymatic activity up to 40%, with respect to the free enzyme activity. Moreover, our results show that loading and enzymatic activity are rather interrelated characteristics and that, under appropriate polymer concentration and pH conditions, a satisfactory compromise can be reached. These results, as a whole, indicate that Au@PDMPAHCl-BSAO bioconjugate systems are promising for future biomedical applications.

Properties of mixed DOTAP-DPPC bilayer membranes as reported by differential scanning calorimetry and dynamic light scattering measurements.

We have investigated the effect of a cationic lipid [DOTAP] on both the thermotropic phase behavior and the structural organization of aqueous dispersions of dipalmitoyl-phosphatidylcholine [DPPC] by means of high-sensitivity differential scanning calorimetry and dynamic light scattering measurements. We find that the incorporation of increasing quantities of DOTAP progressively reduces the temperature and the enthalpy of the gel-to-liquid crystalline transition. We are further showing that, in mixed DOTAP-DPPC systems, the reduction of the phase transition temperature is accompanied by a reduction of the average size of the structures present in the aqueous mixtures, whatever the DOTAP concentration is. These results, which extend a previous investigation by Campbell et al. (Campbell, R. B.; Balasubramanian, S. V.; Straubinger, R. M.; Biochim. Biosphys. Acta 2001, 27, 1512.) limited to a DOTAP concentration below 20 mol %, confirm that the insertion of cationic head groups in zwitterionic phosphatidylcholine bilayers facilitates the formation of stable, relatively small, unilamellar vesicles. This self-assembling restructuring from an aqueous multilamellar structure toward a liposomal phase is favored by decreasing the phospholipid phase transition temperature and by increasing the temperature of the system. This reduction of the average size and the appearance of a stable liposomal phase is also promoted by a heating and cooling thermal treatment.

Direct evidence of multicompartment aggregates in polyelectrolyte-charged liposome complexes.

By means of the combined use of dynamic light scattering and transmission electron microscopy measurements, we provide a direct evidence for the existence of an equilibrium cluster phase in the polyion-induced liposome aggregation, where the liposomes maintain their integrity, with the ability of preserving the aqueous core content from the external medium. We prepared single liposomes containing, in their interior, different CsCl electrolyte solutions at different concentrations (0.1 and 0.01 M, respectively). During the polyion-induced complexation of a mixture of these two differently loaded liposomes, reversible aggregates form, whose multicompartmental structure reveals the simultaneous presence of nonfused liposomes. Clusters composed by mesoscopic-sized vesicles and realized by charged lipids coupled to oppositely charged polyions are playing an increasingly important role as model systems in a variety of phenomena in soft matter and for their potential use in biomedical applications as drug delivery systems. Aggregates of liposomes such as those described in this article, where the electrostatic interactions are the primary driving forces promoting aggregation, may represent a new and interesting class of colloids which give rise to a rich phenomenology with several unusual colloidal behaviors that deserve to be further investigated.

Large equilibrium clusters in low-density aqueous suspensions of polyelectrolyte-liposome complexes: a phenomenological model.

In this paper, we revisit some of our previous results together with further experimental evidences for the existence of large equilibrium clusters in low-density aqueous colloidal suspensions, resulting from the screening of charged spherical macroions by oppositely charged linear polyelectrolytes. The aggregation process is described by a simple phenomenological model where aggregates interact via a long-range repulsion Yukawa potential and some supports to the equilibrium properties of the resulting aggregates, on the basis of dynamic light scattering, zeta potential, and transmission electron microscopy techniques, have been provided.

Charge patch attraction and reentrant condensation in DNA-liposome complexes.

We investigated the formation of complexes between cationic liposomes built up by DOTAP and three linear anionic polyions, with different charge density and flexibility, such as a single-stranded ssDNA, a double-stranded dsDNA and the polyacrylate sodium salt [NaPAA] of three different molecular weights. Our aim is to gain further insight into the formation mechanism of polyion-liposome aggregates of different sizes (lipoplexes), by comparing the behavior of DNA with a model polyelectrolyte, such as NaPAA, with approximately the same charge density but with a higher flexibility. We employed dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements, in order to explore both the hydrodynamic and structural properties of the aggregates resulting from polyion-liposome interaction and to present a comprehensive picture of the complexation process. The phenomenology can be summarized in a charge ratio-dependent scenario, where the main feature is the formation of large equilibrium clusters due to the aggregation of intact polyion-coated vesicles. At increasing polyion-liposome ratio, the size of the clusters continuously increases, reaching a maximum at a well-defined value of this ratio, and then decreases ("reentrant" condensation). The aggregation mechanism and the role of the polyion charge density in the complex formation are discussed in the light of the recent theories on the correlated adsorption of polyelectrolytes at charged interfaces. Within this framework, the phenomena of charge inversion and the reentrant condensation, peaked at the isoelectric point, finds a simple explanation.

Low frequency ultrasound induces aggregation of porcine fumarase by free radicals production.

Hydrogen peroxide and hydroxyl-free radicals determine a diffuse aggregation of porcine fumarase and a loss of its enzymatic activity. In this study, hydroxyl-free radicals were generated "in situ" by irradiation with ultrasound (US) at 38 kHz. The structural characteristics of aggregated fumarase were studied using circular dichroism spectroscopy (CD) and steady state fluorescence spectroscopy. Enzyme aggregation is caused by the formation of intermolecular disufide bridges, originated by the oxidation of cysteine residues, together with a diffuse increase in beta-turn in the protein's secondary structure. These conformational changes lead to a fibrous, amyloid-like aggregation which appears ordered and regular under TEM microscopy.

In vitro effects on macrophages induced by noncytotoxic doses of silica particles possibly relevant to ambient exposure.

The RAW 246.7 macrophage cell line was exposed in vitro to aged crystalline silica particles of respirable size for 24 h at a range of doses starting from 15 microg/2 x 10(6) cells, which is a realistic exposure level of macrophages in the airways of ambiently exposed individuals. The particle sample used for the experiments was prepared to mimic some aspects of ambient crystalline silica particles: size distribution, morphology, and surface reactivity. Our purpose was to determine whether a nontoxic quartz load comparable to that of ambient exposure would be able to induce macrophage activation and impairment of the phagocytic ability, factors altering the lung's capacity to deal with increased particle loads (as occurs during high-pollution episodes) or infections and affecting the local and systemic responses through the release of biologically active compounds (cytokines, reactive oxygen species, NO, isoprostanes). Exposure of RAW 264.7 cells to aged silica particles induced macrophage activation (evidenced by the morphological features observed with scanning electron microscopy and by the release of TNF-alpha and IL-6) and impairment of phagocytosis of test particles, even at noncytotoxic doses. The reduction of the phagocytic function of the cells after silica treatment was dose-dependent, as evidenced by an increase of the population of unphagocytic cells, paralleled by a decrease of the actively phagocytizing cell population. We evaluated the oxidative stress induced by aged silica particles, quantifying the peroxidation products (8-isoprostanes) in the culture media of treated cells, and found a strong release at low doses. Isoprostanes are a complex family of compounds which have been used as in vivo markers of lipid peroxidation in human disorders, but that, as far as we know, have never been evaluated in relation to airborne particulate matter exposure. Lipid peroxides are involved in various cellular events in the inflammatory response, and isoprostanes are also supposed to exert important biological actions on airway and pulmonary vascular smooth muscles and on platelets.

Complexation of anionic polyelectrolytes with cationic liposomes: evidence of reentrant condensation and lipoplex formation.

We have studied the complexation process taking place in cationic liposomes in the presence of anionic polyelectrolytes, in the polyion concentration range from the dilute to the concentrated regime, by combining dynamic light scattering and transmission electron microscopy techniques. We employed as the cationic lipid a two-chained amphiphile (Dioleoyltrimethylammoniumpropane) and sodium polyacrylate salt as the flexible anionic polyelectrolyte. The results evidence a variety of different structures, mainly depending on the liposome-polyion charge ratio, whose peculiar dynamical and structural features are briefly described. In particular, three different polyion concentration regions are found, within which a monomodal or bimodal distribution of aggregates, with a well-defined time evolution, is present. At low polyion content, close to the isoelectric point, large aggregates are formed, deriving from the collapse of the liposomal bilayers into extended charged surfaces, where adsorbed polyions form a two-dimensional strongly correlated array and organize into a two-dimensional Wigner liquid. At high polyion content, above a critical concentration, the size distributions of the complexes are clearly bimodal and a large-component aggregate, continuously increasing with time, coexists with a population of smaller-size aggregates. At an intermediate polyion concentration, spherical, small-size vesicular structures are reformed, connected in a network by polymer chains. A brief discussion tries to summarize our results into a consistent picture.

Immunogold localisation of P-glycoprotein in supported lipid bilayers by transmission electron microscopy and atomic force microscopy.

In this study, purified P-glycoprotein molecules, a membrane drug pump responsible for the multidrug resistance phenomenon, were incorporated in model membranes deposited onto solid supports, according to the method described by Puu and Gustafson (1997). The insertion of proteins into planar supported model membranes is of interest, as the films are fundamental in biosensor applications and for the investigation of how proteins conform and aggregate in a lipid environment. In our investigation, two model membranes were prepared by transferring liposomes containing P-glycoprotein to different hydrophobic supports: (a) thin amorphous carbon films; (b) Langmuir-Blodgett lipid monolayers on mica. After the labelling of P-glycoprotein with two well-characterised monoclonal antibodies, MM4.17 and MRK-16, samples (a) were observed by transmission electron microscopy (TEM) and samples (b) by atomic force microscopy (AFM). The comparative analysis performed by TEM and AFM allowed us to demonstrate the successful insertion of P-glycoprotein in the model membranes and their stability under different environmental conditions (vacuum, air and water). P-glycoprotein appeared to maintain, after purification and insertion in lipid bilayers, a good part of its conformational features as shown by the P-glycoprotein segments bearing the specific monoclonal antibody epitopes.

The two PM(2.5) (fine) and PM(2.5-10) (coarse) fractions: evidence of different biological activity.

Recent studies have shown that an increased concentration of environmental particulate matter (PM(10)) is related to many respiratory diseases. One major issue is whether the toxicity of the particles resides in some particular fraction as defined by chemical composition and size. The overall purpose of this study was to compare the in vitro toxicity of coarse (PM(2.5-10)) and fine (PM(2.5)) particulate matter, collected in an urban area of Rome, in relation to their physicochemical composition as assessed by analytic electron microscopy and atomic absorption spectroscopy. In particular, our aim was to evaluate the importance of particle physicochemical components in the induced toxicity. The in vitro toxicity assays used included human red blood cell hemolysis, cell viability, and nitric oxide (NO) release in the RAW 264.7 macrophage cell line. The hemolytic potential has been widely used as an in vitro toxicity screen and as a useful indicator of oxidative damage to biomembranes. We found that human erythrocytes underwent dose-dependent hemolysis when they were incubated with varying concentrations of fine and coarse particles. The hemolytic potential was greater for the fine particles than for the coarse particles in equal mass concentration. However, when data were expressed in terms of PM surface per volume unit of suspension, the two fractions did not show any significant hemolytic differences. This result suggested that the oxidative stress induced by PM on the cell membranes could be due mainly to the interaction between the particle surfaces and the cell membranes. RAW 264.7 macrophage cells challenged with particles showed decreased viability and an increased release of NO, a key inflammatory mediator, and both effects were not dose dependent in the tested concentration range. The fine particles were the most effective and the differences between the two size fractions in inducing these biological effects remained unchanged when the basis of comparison was changed from weight to surface measures. It seemed therefore that these differences relied on the different physicochemical nature of the particles. The main chemical difference between the two fractions resided in a greater abundance of C-rich particles with S traces in the fine fraction. Therefore, we cautiously suggest a role for these particles in the induction of toxicity.

Induction of P-glycoprotein expression on the plasma membrane of human melanoma cells.

Melanoma cells exhibit, both in vivo and in vitro, intrinsic drug resistance to various chemotherapeutic agents. Cultured human melanoma cells (M14) intrinsically express significant amounts of multidrug resistance-related protein (MRP1) and P-glycoprotein (P-gp) in the Golgi apparatus, but do not express these drug transporters on the plasma membrane. A panel of multidrug resistant (MDR) melanoma cell lines (M14Dx), showing different degrees of resistance to doxorubicin (DOX), were isolated. In M14Dx lines, the appearance of surface P-gp, but not of MRP1 or lung resistance related protein (LRP), occurred in cells grown in the presence of DOX concentrations higher than 60 nM. Furthermore, P-gp levels appeared to be dose-dependent. Flow cytometry, laser scanning confocal microscopy and cytotoxicity studies demonstrated that the activity of the drug extrusion system was related to both surface P-gp expression and resistance to DOX. In conclusion, P-gp, but not MRP1 or LRP, might play a pivotal role in the pharmacologically-induced MDR phenotype of melanoma cells.

Characterization of surface layer proteins from different Clostridium difficile clinical isolates.

In a previous study we suggested that two surface proteins of a Clostridium difficile strain were involved in the formation of a regularly assembled surface layer (S-layer) external to the cell wall. In the present paper six C. difficile strains isolated from cases and healthy carriers were studied. By using freeze-etching and negative staining techniques two superimposed structurally different lattices were detected on the cell surface of the different C. difficile strains. In each strain, the outer S-layer lattice was arranged in a square symmetry and the inner S-layer lattice in hexagonal symmetry. The S-layer proteins from the different strains were isolated and characterized. Each strain showed two distinct S-layer glycoproteins ranging in molecular mass 36-56 kDa. Antigenic cross-reactivity among the S-layer proteins of higher molecular masses extracted from each strain was demonstrated whereas no antigenic relationship was observed among the different S-layer proteins of lower molecular masses. N-terminal sequence analysis showed the presence of common structural motifs conserved among the high S-layer proteins as well as among the low S-layer proteins. These data indicate that the presence of S-layer on C. difficile strains is common and that its glycoprotein subunits show a certain degree of heterogeneity.

Morphological and functional alterations of human erythrocytes induced by SiO2 particles: An electron microscopy and dielectric spectroscopy study.

The interaction of aerosil particles with human erythrocytes was investigated by electron microscopy methods complemented with hemolysis and radio wave dielectric spectroscopy to elucidate the extent of morphological and functional modification induced by aerosil surface. Scanning electron microscopy and freeze-fracturing techniques were used to follow morphological and ultrastructural modifications and hemolysis tests and radio wave dielectric spectroscopy to monitor the membrane damage. All experimental results indicate that there is an effect depending on both silica concentration and incubation time. Our results are in good agreement with an interaction model based on membrane protein denaturation due to the electrostatic attraction between (-SiO-) groups at the silica surface and proteins embedded in the membrane. The process is time-limited and reaches saturation after about 20 min. The extent of the damage is determined mainly by the ratio between cell and aerosil surface, that is, aerosil concentration. Limited damage is observed, especially when little aerosil surface per cell is available. Conversely, strong membrane damage is obtained when aerosil surface is considerable. In any case, due to the high surface/volume of aerosil particles used in our experiments we obtained considerable membrane damage with small weight concentrations.

Interactions of anthracyclines with zwitterionic phospholipid monolayers at the air-water interface.

The present note describes the use of surface pressure measurements (Langmuir monolayer technique) for the analysis of interactions of two different anthracyclines (adriamycin and daunorubicin) with a non-ionic, zwitterionic phospholipid monolayer, at the air-water interface. Because the surface membrane of the cell is the first barrier encountered by the anthracyclines in the treatment of cancer, drug-membrane interactions studied in model (monolayers or bilayers) and natural systems play an important role in the understanding of the bioactivity properties of these molecules. We report here the rate constants of the adsorption process of adriamycin and daunorubicin in the presence of a zwitterionic phospholipid monolayer at the air-water interface. Because interactions with the lipid monolayer strongly depend on the molecular packing of the lipid, we investigated this process at a relatively low surface pressure (7 mN/m), the interactions being favoured by the gaseous and liquid expanded structure of the lipid monolayer. The apparent molecular area of these molecules during the insertion into the lipid film and their interactions with the phospholipid polar head groups was evaluated and the estimated percentage of anthracyclines at the interface after adsorption into the lipid monolayer is briefly discussed. The rate constants for the adsorption and desorption process at the water-monolayer interface have been calculated on the basis of a single-exponential model. The observed difference of these parameters for daunorubicin and adriamycin suggests a different interaction of these anthracyclines during the adsorption to and/or penetration across the phospholipid monolayer.

Intracellular mapping of 4'-deoxy-4'-iododoxorubicin in sensitive and multidrug resistant cells by electron spectroscopic imaging.

Electron spectroscopic imaging (ESI) was employed to study, with high spatial resolution, the intracellular distribution of the halogenated derivative of doxorubicin 4'-deoxy-4'-iododoxorubicin (IDX) in sensitive and multidrug resistant human breast carcinoma cells. Both ESI and electron energy loss spectroscopy (EELS) observations confirmed results obtained with flow cytometry (FC), laser scanning confocal microscopy (LSCM) and secondary ion mass spectrometry (SIMS). Moreover, ESI allowed us to obtain a more detailed intracellular localization of IDX. Our results confirm that nuclear DNA represents the main intracellular target for IDX and that the Golgi apparatus is involved in the intracellular transport of the drug.

Ultrastructural and spectroscopic methods in the study of anthracycline-membrane interaction.

Observations on the interaction of anthracycline antibiotics with the plasma membrane, performed by morphological, ultrastructural, microanalytical and spectroscopic methods, are reported and discussed in this review. Scanning and transmission electron microscopy analysis indicates that doxorubicin (DOX) induces dose-dependent modifications of the cell morphology and membrane ultrastructure of human erythrocytes. The formation of intramembrane particle-free domains on both the fracture faces of the plasma membrane suggests that the DOX molecules become incorporated within the lipid bilayer. Electron energy-loss spectroscopy measurements reveal a reduction in the P/C ratio in treated membranes, probably due to a phospholipid "dilution" following the incorporation of DOX molecules. The radiowave dielectric spectroscopy indicates modifications induced by DOX in the passive electrical properties of the membrane. In particular, the decrease in membrane conductivity suggests that the interaction of the drug with the membrane lipids can affect the function of specific ion channels. The results obtained allow us to propose a structural model of the DOX-membrane interaction, in which DOX molecules self-associate in the phospholipid bilayer. The DOX incorporation induces remarkable changes in the structural and functional properties of the plasma membrane, strengthening the hypothesis that this drug can also exert its cytotoxic action at the membrane level.

Electron energy-loss spectroscopy analysis of adriamycin-plasma membrane interaction.

Our previous studies on the mechanism of cytotoxic action of the anti-tumour drug adriamycin (ADR) indicated that this anthracyclinic antibiotic strongly modified the molecular architecture of the plasma membrane of human erythrocytes, presumably becoming incorporated within both lipid layers. In order to verify this hypothesis, electron energy-loss spectroscopy (EELS) has been used to compare the P content in control and ADR-treated erythrocyte ghosts. EELS measurements allowed us to reveal a significant reduction in the P/C ratio in erythrocyte ghosts after ADR treatment. This finding seems to reflect a phospholipid 'dilution' produced by the incorporation of the drug molecules in the membrane layers. A structural model of the ADR-membrane interaction is proposed.