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.

Dielectric response of shelled toroidal particles carrying localized surface charge distributions. The effect of concentric and confocal shells.

Dielectric models of biological cells are generally based on spherical or ellipsoidal geometries, where the different adjoining dielectric media are arranged as distinct core and shells, representing the cytosol and the cell membrane. For ellipsoidal particles, this approach implies the assumption of confocal shells that, in turn, means a cell membrane of ill-defined thickness. A quantitative analysis of the influence of a non-uniform thickness of the cell membrane has been not considered so far. In the case of a toroidal particle, this problem can be conveniently addressed by considering the solution of the Laplace equation in two different coordinate systems, i.e., toroidal coordinates (confocal shells and hence non-uniform thickness of the shell membrane) and toroidal polar coordinate, (concentric shells and hence a uniform thickness of the shell membrane). In the present paper, we compare the dielectric spectra of a toroidal particle aqueous suspension obtained from the two above stated solutions of the Laplace equation and we furnish a first quantitative estimate of the differences arising from considering the presence of confocal or concentric shells. This approach offers a complete view of the influence of the membrane thickness on the whole dielectric spectrum of a biological particle suspension, at least as far as toroidal objects are concerned.

Gold nanoparticles and gold nanoparticle-conjugates for delivery of therapeutic molecules. Progress and challenges.

This article reviews the most recent literature data on the applications of gold nanoparticles and their various conjugates which make them suitable structures towards biomedical and clinical purposes, with an emphasis on their use as drug delivery vehicles for selective targeting of cancer cells. With the rapid surge in the development of nanomaterials, new methodologies and treatment strategies have been explored and these topics should be taken into consideration when a current scenario is required in the design of new experimental approaches or in a comprehensive data interpretation. We present here a summary of the main properties of gold nanoparticles and their conjugates and the state-of-the-art of non-conventional treatment in targeted drug delivery based on gold nanoparticles as carriers, with the aim to give the reader an overview of the most significant advances in this field.

Anomalous Debye-like dielectric relaxation of water in micro-sized confined polymeric systems.

While it is well known that spatial confinement on a nm scale affects the molecular dynamics of water resulting in a hindered dipolar reorientation, question of whether these effects could result at length scales larger than these, i.e., in confined regions of the order of µm or more, is still under debate. Here we use dielectric relaxation spectroscopy techniques to study the relaxation orientation dynamics of water entrapped in different polymeric matrices with pore sizes of the order of 100 µm, analyzing the frequency relaxation behaviour of the dielectric response. Our results show that, contrary to what has been generally thought, even in confinements which are not particularly high such as those realized here, regions typically hundred micrometers in size can affect the water structure, inducing a water phase with properties different from those of bulk water. In particular, we observe a dielectric dispersion centered in the range 10(5)-10(7) Hz, in between the one characteristic of ice (8.3 kHz at T = 0 °C) and the one of bulk water (19.2 GHz at T = 25 °C). The analysis of the dependence on temperature of the relaxation time of this unexpected contribution rules out the possibility that it can be attributed to an interfacial polarization (Maxwell-Wagner effect) and suggests a dipolar Debye-like origin due to a slow-down of the hydrogen-bonded network orientational polarization. Also at these scales, the confinement alters the structure of water, leading to a hindered reorientation. These properties imply that water confined within these polymeric porous matrices is more ordered than bulk water. These findings may be important in order to understand biological processes in cells and in different biological compartments, where water is physiologically confined.

Dielectric characterization of hepatocytes in suspension and embedded into two different polymeric scaffolds.

The dielectric and conductometric properties of hepatocytes in two different environments (in aqueous suspension and embedded into polymeric scaffolds) have been investigated in the frequency range from 1 kHz to 2 GHz, where the interfacial electrical polarization gives rise to marked dielectric relaxation effects. We analyzed the dielectric behavior of hepatocytes in complete medium aqueous suspensions in the light of effective medium approximation for heterogeneous systems and hepatocytes cultured into two different highly porous and interconnected polymeric structures. In the former case, we have evaluated the passive electrical parameters associated with both the plasmatic and nuclear membrane, finding a general agreement with the values reported elsewhere, based on a partially different analysis of the experimental spectra. In the latter case, we have evaluated the cell growth into two different polymeric scaffolds made of alginate and gelatin with a similar pore distribution and similar inter-connectivity. Based on a qualitative analysis of the dielectric spectra, we were able to provide evidence that alginate scaffolds allow an overall survival of cells better than gelatin scaffold can do. These indications, confirmed by biological tests on cell viability, suggest that hepatocytes embedded in alginate scaffolds are able to perform liver specific functions even over on extended period of time.

Lysozyme hydration in concentrated aqueous solutions. Effect of an equilibrium cluster phase.

Water close to proteins plays a key role in determining their structural and functional properties. Despite being a subject of considerable interest, the characterization of hydration water, as far as its total amount is concerned, is still controversial and its influence on protein structure and folding is not yet fully understood. In this work, we have investigated the dielectric properties of lysozyme aqueous solutions over the frequency range where the orientational polarization relaxation of the aqueous phase occurs (from 500 MHz to 50 GHz). Measurements extend over a wide concentration range, up to 300 mg/mL, corresponding to a volume fraction of the order of 0.4. The analysis of the dielectric spectra, based on the decrease of the dielectric increment of the γ-dispersion as a function of protein concentration, allows us to estimate the total amount of hydration water (both bound water and loosely bound water) present in the system investigated. We observe a decrease of the hydration number as a function of the protein concentration. This behavior is well accounted for by considering the formation of small equilibrium clusters with aggregation number of some units, as recently reported by Stradner et al. (1) on the basis of small-angle X-ray and neutron scattering measurements.

Radiowave dielectric investigation of water confined in channels of carbon nanotubes.

Structure and dynamics of water confined in channels of diameter of few nanometer in size strongly differ from the ones of water in the bulk phase. Here, we present radiowave dielectric relaxation measurements on water-filled single-walled carbon nanotubes, with the aim of highlighting some aspects on the molecular electric dipole organization of water responding to high spatial confinement in a hydrophobic environment. The observed dielectric spectra, resulting into two contiguous relaxation processes, allow us to separate the confined water in the interior of the nanotubes from external water, providing support for the existence in the confinement region of water domains held together by hydrogen bonds. Our results, based on the deconvolution of the dielectric spectra due to the presence of a bulk and a confined water phase, furnish a significantly higher Kirkwood correlation factor, larger than the one of water in bulk phase, indicating a strong correlation between water molecules inside nanotubes, not seen in bulk water.

Heat-denatured lysozyme aggregation and gelation as revealed by combined dielectric relaxation spectroscopy and light scattering measurements.

The dielectric behavior of native and heat-denatured lysozyme in ethanol-water solutions was examined in the frequency range from 1 MHz to 2 GHz, using frequency-domain dielectric relaxation spectroscopy. Because of the conformational changes on unfolding, dielectric methods provide information on the denaturation process of the protein and, at protein concentration high enough, on the subsequent aggregation and gelation. Moreover, the time evolution of the protein aggregation and gelation was monitored measuring, by means of dynamic light scattering methods, the diffusion coefficient of micro-sized polystyrene particles, deliberately added to the protein solution, which act as a probe of the viscosity of the microenvironment close to the particle surface. All together, our measurements indicate that heat-induced denaturation favors, at high concentrations, a protein aggregation process which evolves up to the full gelation of the system. These findings have a direct support from IR measurements of the absorbance of the amide I band that, because of the unfolding, indicate that proteins entangle each other, producing a network structure which evolves, in long time limit, in the gel.

PLGA-based nanoparticles: effect of chitosan in the aggregate stabilization. A dielectric relaxation spectroscopy study.

Chitosan-modified polylactic-co-glycolic acid (PLGA) nanoparticles with average diameter of 200 nm in PBS buffer solution have been investigated by means of dielectric relaxation spectroscopy measurements in the frequency range (1 MHz-2 GHz) where interfacial polarizations occur. PLGA-based nanoparticles offer remarkable advantages in different biotechnological fields, such as their biocompatibility, easiness of administration and rather complete biodegradation. However, despite the use of these drug delivery systems is increasing, little is known about the basic process involved in the formation of complexes and in the subsequent release kinetics. In the present work, we have characterized the colloidal behavior of PLGA-based nanoparticles in the presence of oppositely charged chitosan polyelectrolyte by means of dynamic light scattering, electrophoretic mobility and radiowave dielectric relaxation measurements. In particular, we have emphasized how the presence of a coating layer at the nanoparticle surface could exert a marked slowing-down in the drug release. The consequence of this finding is briefly discussed at the light of some biological implications.

Hemocompatibility of stent materials: alterations in electrical parameters of erythrocyte membranes.

BACKGROUND: It is presently unknown if stents used in the correction of artery stenosis are fully hemocompatible or if their implantation causes alterations at the level of the plasma membrane in red blood cells. METHODS: We addressed this important issue by measuring the passive electrical properties of the erythrocyte membrane before and after stent insertion by means of dielectric relaxation spectroscopy in the radiowave frequency range in a series of patients who were undergoing standard surgical treatment of arterial disease. RESULTS: Our findings provide evidence that full hemocompatibility of stents has not yet been reached, and that there are some measurable alterations in the passive electrical behavior of the red blood cell membrane induced by the presence of the stent. CONCLUSION: It is possible that these changes do not have any physiological significance and simply reflect the intrinsic variability of biological samples. However, caution is urged, and the technique we describe here should be considered when investigating the hemocompatibility of a medical device at a cell membrane level.

Assemblies of thermoresponsive diblock copolymers: micelle and vesicle formation investigated by means of dielectric relaxation spectroscopy.

The dielectric properties of aqueous solutions of two different thermoresponsive mixed copolymers, (3-acrylamidopropyl)trimethylammonium chloride and N-isopropylacrylamide, PAMPTMA-b-NIPAAM, and sodium 2-acrylamido-2-methylpropanesulfonate and N-isopropylacrylamide, PAMPS-b-PNIPAAM, have been investigated in the frequency range where marked interfacial polarization mechanisms occur, both below and above the lower critical solution temperature. In the presence of poly(ethylene oxide)-PNIPAAM block polymers, PEO-b-PNIPAAM, these classes of copolymers give rise to different types of aggregates with different compositions and different architectures. By the combined results from dielectric relaxation spectroscopy, dynamic light scattering, and ζ-potential measurements, we give evidence for assembling into two different composite structures, a core-shell-type micellar structure built up by a hydrophobic core surrounded by a hydrophilic charged layer, in the case of the PEO-b-PNIPAAM + PAMPS-b-PNIPAAM system, and a vesicular structure encompassing an aqueous core in the case of the PEO-b-PNIPAAM + PAMPTMA-b-PNIPAAM system. These different structures, governed by a delicate interplay between electrostatic and hydrophobic interactions, are characterized by dielectric parameters (dielectric increments and relaxation frequencies) that can be properly deduced from suitable dielectric models, in the framework of heterogeneous system theory. These structures and in particular hallow particles with a large internal aqueous core, where large hydrophilic compounds can be encapsulated, offer novel and interesting properties in biomedical technologies and in particular in drug delivery as drug mesoscopic carriers.

Spatio-temporal anomalous diffusion in heterogeneous media by nuclear magnetic resonance.

In this paper, we describe nuclear magnetic resonance measurements of water diffusion in highly confined and heterogeneous colloidal systems using an anomalous diffusion model. For the first time, temporal and spatial fractional exponents, α and µ, introduced within the framework of continuous time random walk, are simultaneously measured by pulsed gradient spin-echo NMR technique in samples of micro-beads dispersed in aqueous solution. In order to mimic media with low and high level of disorder, mono-dispersed and poly-dispersed samples are used. We find that the exponent α depends on the disorder degree of the system. Conversely, the exponent µ depends on both bead sizes and magnetic susceptibility differences within samples. The new procedure proposed here may be a useful tool to probe porous materials and microstructural features of biological tissue.

Dielectric relaxations of ionic thiol-coated noble metal nanoparticles in aqueous solutions: electrical characterization of the interface.

The radiowave dielectric properties of organothiol monolayer-protected Au and Ag metallic nanoparticles have been investigated in the frequency range of 10 kHz to 2 GHz, where a dielectric relaxation, due to the polarization of the ionic atmosphere at the aqueous interface, occurs. The simultaneous measurement of the particle size, by means of dynamic light scattering technique, and of the particle electrical charge, by means of laser microelectrophoresis technique, allow us to describe the whole dielectric behavior at the light of the standard electrokinetic model for charged colloidal particles. Au and Ag metallic nanoparticles experience a large charge renormalization, in agreement with the counterion condensation effect for charged spherical colloidal particles. The value of the effective valence Z(eff) of each nanoparticle investigated has been evaluated thanks to the dielectric parameters of the observed relaxation process and further confirmed by direct current electrical conductivity measurements. All in all, these results provide support for the characterization of the electrical interfacial properties of metallic nanoparticles by means of dielectric relaxation measurements.

Dielectric relaxation spectroscopy of lysozyme aqueous solutions: analysis of the δ-dispersion and the contribution of the hydration water.

The dielectric properties of lysozyme aqueous solutions have been investigated over a wide frequency range, from 1 MHz to 50 GHz, where different polarization mechanisms, at a molecular level, manifest. The dielectric relaxation spectra show a multimodal structure, reflecting the complexity of the protein-water interactions, made even more intricate with the increase of the protein concentration. The deconvolution of the spectra into their different components is not unambiguous and is generally a delicate process which requires caution. We have analyzed the whole relaxation region, on the basis of the sum of simple Debye-type relaxation functions, considering three main contributions. Particular attention has been payed to the δ-dispersion, intermediate between the ß-dispersion (rotational dynamics of the protein) and the γ-dispersion (orientational polarization of the water molecules). This intermediate contribution to the dielectric spectrum is attributed to the orientational polarization of water molecules in the immediate vicinity of the protein surface (hydration water). Our measurements clearly demonstrate that, at least at high protein concentrations, the δ-dispersion has a bimodal structure associated with two kinds of hydration water, i.e., tightly bound and loosely bound hydration water. In the concentration range investigated, the existence of a three-modal δ-dispersion, as recently suggested, is not supported, on the basis of statistical tests, by the analysis of the dielectric relaxations we have performed and a bimodal dispersion is accurate enough to describe the experimental data. The amount of the hydration water has been evaluated both from the dielectric parameters associated with the δ-dispersion and from the decrement of the loss peak of the γ-dispersion. The relative weight of tightly bound and loosely bound hydration water is briefly discussed.

On the dielectric relaxation of biological cell suspensions: the effect of the membrane electrical conductivity.

Due to the mismatch of the electrical parameters (the permittivity ϵ' and the electrical conductivity σ) of the membrane of a biological cell with the ones of the cytosol and the extracellular medium, biological cell suspensions are the site, under the influence of an external electric field, of large dielectric relaxations in the radiowave frequency range. However, a point still remains controversial, i.e., whether or not the value of membrane conductivity σ(s) might be extracted from the de-convolution of the dielectric spectra or otherwise if it would be more reasonable to assign to the membrane conductivity a value equal to zero. This point is not to be considered with superficiality since it concerns an a priori choice which ultimately influences the values of the electrical parameters deduced from this technique. As far as this point is concerned, the opinion of the researchers in this field diverges. We believe that, at least within certain limits, the membrane conductivity can be deduced from the shape of the relaxation spectra. We substantiate this thesis with two different examples concerning the first a suspension of human normal erythrocyte cells and the second a suspension of human lymphocyte cells. In both cases, by means of an accurate fitting procedure based on the Levenberg-Marquardt method for complex functions, we can evaluate the membrane conductivity σ(s) with its associated uncertainty. The knowledge of the membrane electrical conductivity will favor the investigation of different ion transport mechanisms across the cell membrane.

Many facets of the polyelectrolyte and oppositely charged colloidal particle complexation: counterion release and electrical conductivity behavior.

The lateral correlated adsorption of polyions onto oppositely charged vesicles, leading to the formation of stable equilibrium clusters of mesoscopic size, is associated to the release of a fraction of counterions, initially condensed on the polyion chains. This ulterior release of counterions provokes an increase of the number of free ions, besides the ones due to the partial ionization of both charged particles and polyions, that can be appropriately monitored by means of electrical conductivity measurements of the whole system. We have investigated this behavior in a suspension of cationic vesicles made up by dioleoyl trimethyl ammonium propane (DOTAP) liposomial vesicles interacting with an anionic polyelectrolyte composed by polyacrylate sodium salt. This system has been in the past extensively studied by us by means of different experimental techniques, and its behavior has been sufficiently characterized, as far as hydrodynamic and electrical properties are concerned. In this note, we report on the dc electrical conductivity behavior during the whole aggregation process, from the single polyion-coated liposomal particles, to polyion-induced liposome clusters, to finally polyion-fully covered liposomes, in polyion excess conditions. We have evaluated the excess of released counterions on the basis of the standard theory of the electrical properties of aqueous charged solutions and compared this quantity with the one predicted by the lateral correlation adsorption model. The agreement is quite good, offering strong experimental evidence of the role played by the release of counterions in the aggregation process. Finally, we have considered a similar liposomial system, where the lateral correlation adsorption was inhibited by structural reasons, having replaced the polyion by a simple electrolyte, whose dissociated ions will adsorb randomly at the particle surface, rather than in a correlated manner. In this case, no counterion release upon complexation occurs, and the electrical conductivity of the suspension approaches the one theoretically expected.

Polarizability of spherical biological cells in the presence of localized surface charge distributions at the membrane interfaces.

The electrical polarizability α(ω) of a biological cell in the presence of a layer of localized, partially bounded, charges at the two cell membrane interfaces has been calculated within the dipolar approximation. The cell is modeled in the light of the single-shell spherical model, but the results can be easily extended to shelled particles of more complex shape. Under the influence of an external electric field, the presence of these charge distributions, which added to the ones originated by the mismatch of the complex dielectric constants of the different media, produces a further dielectric relaxation, besides the one due to the usual Maxwell-Wagner effect. We explicitly find the contribution that must be added to the electrical polarizability α(ω) in order to take into account the surface electrical currents originated by the localized charges free to move on the membrane surfaces. Our results, maintaining their validity whatsoever the values of the surface charge distributions and, moreover, whatsoever the values of the membrane conductivity are, extend the applicability of the model recently proposed by Prodan [Biophys. J. 95, 4174 (2008)], who developed an analytical solution which offers reliable results only in the case of weak surface charge distributions and, moreover, for negligible small values of the membrane conductivity. Our approach, removing these constrains, represents a valuable improvement toward more realistic biological cell models and widens the use of dielectric relaxation methods to a larger class of biological systems.

The dielectric behavior of nonspherical biological cell suspensions: an analytic approach.

The influence of the cell shape on the dielectric and conductometric properties of biological cell suspensions has been investigated from a theoretical point of view presenting an analytical solution of the electrostatic problem in the case of prolate and oblate spheroidal geometries. The model, which extends to spheroidal geometries the approach developed by other researchers in the case of a spherical geometry, takes explicitly into account the charge distributions at the cell membrane interfaces. The presence of these charge distributions, which govern the trans-membrane potential DeltaV, produces composite dielectric spectra with two contiguous relaxation processes, known as the alpha-dispersion and the beta-dispersion. By using this approach, we present a series of dielectric spectra for different values of the different electrical parameters (the permittivity epsilon and the electrical conductivity sigma, together with the surface conductivity gamma due to the surface charge distribution) that define the whole behavior of the system. In particular, we analyze the interplay between the parameters governing the alpha-dispersion and those influencing the beta-dispersion. Even if these relaxation processes generally occur in well-separated frequency ranges, it is worth noting that, for certain values of the membrane conductivity, the high-frequency dispersion attributed to the Maxwell-Wagner effect is influenced not only by the bulk electrical parameters of the different adjacent media, but also by the surface conductivity at the two membrane interfaces.

Radiowave dielectric properties of sodium maleate copolymers in aqueous solutions in light of a scaling approach.

The concentration dependence of the dielectric properties of aqueous solutions of sodium maleate copolymers with comonomers of different hydrophobicities have been investigated by means of frequency domain dielectric spectroscopy in the frequency range from 1 kHz to 2 GHz. The dielectric relaxation falling between the process at low frequencies (polarization involving the whole polymer chain) and the one at high frequencies (polarization of the bulk aqueous phase) has been analyzed in light of the scaling properties of polyelectrolyte solutions. Within this framework, the dielectric properties are governed by two characteristic lengths, the distance R(cm) between chains in the dilute regime and the correlation length xi in the semidilute regime. We find that, for both these regimes, the exponents of the scaling laws, which describe the dielectric increment Deltaepsilon and the relaxation time tau(ion) are in a reasonably good agreement with the ones experimentally observed. This analysis gives further support to the scaling approach of the dynamic behavior of polyelectrolytes, appearing very suitable to modeling our dielectric results.

Dielectric spectra of ionic water-in-oil microemulsions below percolation: frequency dependence behavior.

We have investigated the dielectric properties of water-in-oil microemulsions composed of sodium bis(2-ethyl-hexyl)sulfosuccinate, water, and decane, using radiofrequency impedance spectroscopy, below the percolation threshold, where the system behaves as surfactant-coated individual water droplets dispersed in a continuous oil phase. The analysis of the dielectric spectra has evidenced that the whole dielectric response below percolation is due to two different contributions, which give rise to two partially overlapping dielectric relaxations, approximately in the frequency range from 10 to 500 MHz. The first of these mechanisms is originated by the bulk polarization of counterions distributed in the electrical double layer of the droplet interior. The second mechanism is associated with a correlated motion of the anionic head groups SO3- at the surfactant-water interface. The introduction of this latter contribution allows us to justify the experimentally observed increase in the low-frequency permittivity as a function of temperature up to temperatures very close to percolation. The present study shows that deviations from the expected values on the basis of dielectric theories of heterogeneous systems (Maxwell-Wagner effect) observed when percolation is approaching can be accounted for, in a reasonable way, by the introduction of a further polarization mechanism, which involves the anionic surfactant groups. Only very close to percolation, when microemulsions undergo a scaling behavior, deviations of the permittivity (and electrical conductivity as well) are a print of the structural rearrangement of the whole system and models based on colloidal particle suspension theories fail. Even if the whole picture of the dielectric properties of microemulsion systems does not change in deep, nevertheless, the refinement introduced in this paper demonstrates how different polarization mechanisms could be simultaneously present in these rather complex systems and, above all, how the individual particle colloidal properties are maintained up to very close to the percolation threshold.