Ultrasound delivery of Surface Enhanced InfraRed Absorption active gold-nanoprobes into fibroblast cells: a biological study via Synchrotron-based InfraRed microanalysis at single cell level.

Ultrasound (US) induced transient membrane permeabilisation has emerged as a hugely promising tool for the delivery of exogenous vectors through the cytoplasmic membrane, paving the way to the design of novel anticancer strategies by targeting functional nanomaterials to specific biological sites. An essential step towards this end is the detailed recognition of suitably marked nanoparticles in sonoporated cells and the investigation of the potential related biological effects. By taking advantage of Synchrotron Radiation Fourier Transform Infrared micro-spectroscopy (SR-microFTIR) in providing highly sensitive analysis at the single cell level, we studied the internalisation of a nanoprobe within fibroblasts (NIH-3T3) promoted by low-intensity US. To this aim we employed 20 nm gold nanoparticles conjugated with the IR marker 4-aminothiophenol. The significant Surface Enhanced Infrared Absorption provided by the nanoprobes, with an absorbance increase up to two orders of magnitude, allowed us to efficiently recognise their inclusion within cells. Notably, the selective and stable SR-microFTIR detection from single cells that have internalised the nanoprobe exhibited clear changes in both shape and intensity of the spectral profile, highlighting the occurrence of biological effects. Flow cytometry, immunofluorescence and murine cytokinesis-block micronucleus assays confirmed the presence of slight but significant cytotoxic and genotoxic events associated with the US-nanoprobe combined treatments. Our results can provide novel hints towards US and nanomedicine combined strategies for cell spectral imaging as well as drug delivery-based therapies.

Long-term physical evolution of an elastomeric ultrasound contrast microbubble.

HYPOTHESIS: One of the main assets of crosslinked polymer-shelled microbubbles (MBs) as ultrasound-active theranostic agents is the robustness of the shells, combined with the chemical versatility in modifying the surface with ligands and/or drugs. Despite the long shelf-life, subtle modifications occur in the MB shells involving shifts in acoustic, mechanical and structural properties. EXPERIMENTS: We carried out a long-term morphological and acoustic evolution analysis on elastomeric polyvinyl-alcohol (PVA)-shelled MBs, a novel platform accomplishing good acoustic and surface performances in one agent. Confocal laser scanning microscopy, acoustic spectroscopy and AFM nanomechanics were integrated to understand the mechanism of PVA MBs ageing. The changes in the MB acoustic properties were framed in terms of shell thickness and viscoelasticity using a linearised oscillation theory, and compared to MB morphology and to nanomechanical analysis. FINDINGS: We enlightened a novel, intriguing ageing time evolution of the PVA MBs with double behaviour with respect to a crossover time of ∼50 days. Before, significant changes occur in MB stiffness and shell thickness, mainly due to a massive release of entangled PVA chains. Then, the MB resonance frequency increases together with shell thickening and softening. Our benchmark study is of general interest for emerging viscoelastomeric bubbles towards personalised medicine.

Differential effects on membrane permeability and viability of human keratinocyte cells undergoing very low intensity megasonic fields.

Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies.

Solution behaviour of poly(N-isopropylacrylamide) stereoisomers in water: a molecular dynamics simulation study.

The water affinity of poly(N-isopropylacrylamide), PNIPAM, is tuned by tacticity, since the hydrophobicity rises with the increase of the degree of isotacticity. On the basis of this experimental evidence, atomistic molecular dynamics simulations of pairs of PNIPAM stereoisomers in 1.6% w/w polymer aqueous solution, a condition intermediate between the dilute and semidilute regimes, were carried out to comparatively investigate the solution behaviour and hydration of atactic and isotactic-rich PNIPAMs, both below and above the lower critical solution temperature, LCST. 30-mers with contents of meso dyads, m, of 45% and 59%, built assuming a Bernoullian dyad distribution, are used as models since their stereochemical composition corresponds to that of experimentally characterized PNIPAM stereoisomers. The simulation results at 283 K, below the LCST, show a slight influence of tacticity on the chain size, but a higher propensity for inter-chain association of the meso-dyad-rich system, in agreement with the experimental results. Junctions between chains are formed because of hydrophobic interactions and are stabilized by a layer of hydrogen bonded water molecules, whose mobility is reduced as compared to that observed for the same meso-dyad-rich stereoisomer at infinite dilution. At 323 K, above the LCST, simulations detect both the coil-globule transition and the aggregation of chains. Under these conditions, the influence of tacticity on the characteristics of PNIPAM aggregate is negligible.

A preliminary in vitro assessment of polymer-shelled microbubbles in contrast-enhanced ultrasound imaging.

This paper focuses on the use of poly (vinyl alcohol)-shelled microbubbles as a contrast agent in ultrasound medical imaging. The objective was an in vitro assessment of the different working conditions and signal processing methods for the visual detection (especially in small vessels) of such microbubbles, while avoiding their destruction. Polymer-shelled microbubbles have recently been proposed as ultrasound contrast agents with some important advantages. The major drawback is a shell that is less elastic than that of the traditional lipidic microbubbles. Weaker echoes are expected, and their detection at low concentrations may be critical. In vitro experiments were performed with a commercial ultrasound scanner equipped with a dedicated acquisition board. A concentration of 100bubbles/mm(3), excitation pressure amplitudes from 120kPa to 320kPa, and a central frequency of 3MHz or 4.5MHz were used. Three multi-pulse techniques (i.e., pulse inversion, contrast pulse sequence based on three transmitted signals, and contrast pulse sequence in combination with the chirp pulse) were compared. The results confirmed that these microbubbles produce a weaker ultrasound response than lipidic bubbles with a reduced second-order nonlinear component. Nevertheless, these microbubbles can be detected by the contrast pulse sequence technique, especially when the chirp pulse is adopted. The best value of the contrast-to-tissue ratio was obtained at an excitation pressure amplitude of 230kPa: although this pressure amplitude is higher than what is typically used for lipidic microbubbles, it does not cause the rupture of the polymeric contrast agent.

High-frequency dielectric study of side-chain dynamics in poly(lysine) aqueous solutions.

The high-frequency dielectric properties of poly(lysine) of different chirality in aqueous solutions have been measured in the frequency range from 1 MHz to 1.8 GHz. The dielectric spectra show the existence of relatively small dielectric dispersions at around 100 MHz that have been attributed to internal motion in the polymer chain, due to side-chain polar groups. Our results indicate that the local structure of the chain and its possibility to undergo a conformational transition induced by pH does not modify the main feature of the side-chain dynamics, the dielectric strength being largely proportional to the concentration of charged groups. A similar behavior has been found in poly(alpha-glutamate) and in poly(gamma-glutamate) aqueous solutions, where the dielectric parameters appear to be related to the change in the charge density on the main chain, rather than to the accompanying conformational helix-coil transition.

A physico-chemical study on the polysaccharide ulvan from hot water extraction of the macroalga Ulva.

Results of a study on the solution behaviour of the cell-wall polysaccharide named ulvan obtained from hot water extraction of a flour of Ulva 'rigida' are reported. In particular the spectroscopic properties and ion binding capacity of this charged polysaccharide were studied by circular dichroism and isothermal microcalorimetric titrations in order to gain information on the potential exploitation of this low cost biomass. A marked tendency of this polysaccharide to uptake water was evidenced by studying the proton spin-lattice relaxation times of the solvent, T1, embedded in this highly charged polysaccharide.

Study of the interactions of D- and L-polylysine enantiomers withpectate in aqueous solutions.

The interaction between D- and L-enantiomers of polylysine and potassium pectate was studied by means of CD, microcalorimetry, and osmometry. Upon binding with pectate, only poly(L-lysine) undergoes a coil to alpha-helix transition, while poly(D-lysine) remains in the disordered state. This suggest that the energetics of the interaction is influenced by stereochemical constraints besides electrostatic forces. Experimental findings from microcalorimetry suggest that a contribution to the overall enthalpy of binding comes from the polysaccharidic moiety. Stoichiometry of the macromolecular complexes studied by osmometry gives a polylysine:pectate ratio of 3:1, in agreement with the respective degree of polymerization of the two polyelectrolytes.

1H NMR relaxation study of a chitosan-cyclodextrin network.

The results of measurements of longitudinal and transverse proton relaxation times for a chemical network obtained by reacting chitosan with oxidized beta-cyclodextrin (beta-cyclodextrin polyaldehyde) are presented. The network was characterized by a 'two-component' transverse relaxation mechanism relative to structurally different environments experienced by water molecules. Different environments were also indicated by the temperature of the spin-spin relaxation times (T2) studied in the range 4-50 degrees C. Between 4 and 18 degrees C, proton exchange between the matrix and water prevails on the inter- and intra-molecular dipolar interactions of the water confined in the meshes of the network, resulting in a marked change in the slope of T2 with temperature. Stiffness of the matrix and reduced mobility of water in the gel meshes are prerequisites for observing such relaxation phenomena. Possible mechanisms contributing to the activation energy in the case of chitosan-cyclodextrin networks are discussed. The behaviour of the chitosan-cyclodextrin hydrogel is compared with that of a gellan gel.

A comparative study of the high-frequency dielectric properties of poly (alpha-glutamate) and poly (gamma-glutamate) aqueous solutions.

The dielectric properties of poly(alpha-glutamic acid) and poly(gamma-glutamic acid) aqueous solutions in the fully ionized state have been investigated in the frequency range from 1 MHz to 1 GHz by means of frequency-domain dielectric spectroscopy measurements. In this frequency range, micro-Brownian dynamics and internal motion of side-chain polar groups give rise to nonexponential relaxation processes resulting in an intermediate dielectric relaxation between that due to the counterion atmosphere polarization and that due to the orientational polarization of the water molecules. In this work, we study the influence of the polymer concentration in the dilute--semidilute regime on the dielectric parameters, i.e., the dielectric increment delta epsilon and the relaxation time tau, and discuss their relevance in the light of some scaling pictures recently proposed by Dobrynin, Colby, and Rubinstein to describe the unentangled regime of flexible polyelectrolyte solutions.

Molecular dynamics in sodium poly (L-glutamate) aqueous solutions analyzed by means of the stretched exponential decay of the Williams-Watts function.

The high-frequency dielectric dispersions of the sodium salt of poly(L-glutamic acid) aqueous solutions in the frequency range from 1 to 1,000 MHz have been analyzed by means of the Cole-Davidson relaxation function in the frequency domain and by means of the Williams-Watts relaxation function in the time domain. The analogies between the two descriptions are discussed on the basis of the analysis carried out by Lindsey and Patterson and the connections between the dipole-dipole correlation function based on the kinetic model for polyion dynamics proposed by Skinner and the Williams-Watts nonexponential decay function are briefly discussed.

Association complexes between Fe(III) or Cu(II) ions and chitosan derivatives. A thermodynamic and spectroscopic investigation.

Association complexes between iron(III) or copper(II) ions and deoxylactit-1-yl (1), 2-substituted pentanedioic acid (2), or 2-substituted propanoic acid (3) derivatives of chitosan were prepared and characterized by thermodynamic and spectroscopic measurements. Complex solutions did not show any precipitate or even opalescence, owing to the hydrolysis of free metal ions, within a wide range of [Me(n+)]/[P] molar ratio, even at a pH as high as 10.5 (Me(n+) = Fe3+ or Cu2+). Both equilibrium dialysis and Job plot experiments suggest that the functional groups in each monomeric residue are an effective site of binding for one metal ion. Reduction potentials, as obtained by cyclic voltammetric measurements, indicate that (i) coordination of the aforementioned polymeric ligands to Cu2+ ions stabilizes the oxidized species, and (ii) iron complexes have an oxidation power definitely higher than that of the corresponding copper compounds. Electron paramagnetic resonances (100 or 6 K) and Mössbauer (r.t.) spectra suggest that the order of increasing distortion from idealized geometry is Me(n+)-chitosan approximately Me(n+)-(3) < Me(n+)-(2) < or = Me(n+)-(1). These results are discussed briefly in the light of a few general considerations concerning the structural features of association complexes between macromolecules and transition metal ions.

Copper complexes immobilized to chitosan.

Polymeric ligands, such as 2-substituted pentanedioic acid (2), 2-substituted propanoic acid (3), and deoxylactit-1-yl (4) derivatives of chitosan (1), were used to prepare copper complexes that are widely soluble in aqueous solution. EPR results (100 K) show that all association complexes basically have a tetragonal symmetry. Visible CD spectra suggest, however, that the order of increasing departure from this geometry is Cu-(1) approximately Cu-(3) less than Cu-(2) less than or equal to Cu-(4), the lack of sterically constraining side-chains in (1) and (3) allowing a more symmetric arrangement of ligands around the central metal ion. Results on the catalytic activity of the association complexes for air oxidation of catechol derivatives are also presented.

Branched-chain analogues of linear polysaccharides: a spectroscopic and conformational investigation of chitosan derivatives.

The solution properties and conformational features of 2-substituted propanoic acid (I) and 2-substituted pentanedioic acid (II) derivatives of chitosan were investigated over a wide range of pH by potentiometric, optical and chiroptical measurements, and by theoretical conformational analysis. No significant change is observed in the solution properties of I upon pH variations, in agreement with computational results showing that the conformational features of the polymer do not vary with respect to the charge state of the ionizable groups. In contrast, spectroscopic titration and preliminary 1H-n.m.r. data indicate that conformational equilibria in II are pH-dependent. Consistently, computed models show that both the charge state of the ionizable groups and the chirality of the carbon atom in the side chain control the structural features of the polymer.

Stereoselective electron transfer between chiral substrates and metal chelates anchored to polypeptides.

Electron transfer from ortho-dihydroxy substrates, such as L(+)-ascorbic acid, L-adrenaline, and L-dopa, to iron(III) in [Fe(tetpy)(OH)2]+ ions anchored to sodium poly(L-glutamate) (FeTL) or poly(D-glutamate) (FeTD) was found to proceed stereoselectively when structurally ordered and partially shielded active sites prevent easy approach for redox partner. Oxidant-reductant interactions are then mediated by the polypeptide, whose conformational asymmetry ensures an efficient sterically discriminating environment. Evidence is produced that stereoselectivity chiefly arises from transition state effects, while thermodynamic discrimination is of minor importance. Theoretical models of the diastereomeric electron-transfer complexes were constructed by conformational energy calculations based on Coulombic, nonbonded, and hydrogen-bonded energy terms. The molecular parameters of the models enabled "differential" thermodynamic functions of the diastereomeric pairs and stereoselectivity to be evaluated and satisfactorily compared with those experimentally determined. The models give good insight into the observed topochemical phenomena and support the idea that stereoselectivity is coupled with a remote attack mechanism on the central metal ion where the peripheral tetpy ligand of the active sites acts as an electron-transfer agent.

The mechanism of thermal degradation of a high-molecular-weight glycoprotein complex from bovine cervical mucus.

Gel-like oestrus bovine cervical mucus can be brought to the point of dissolution by thermal treatment. The glycoprotein complex so produced was isolated on CsCl density gradients, and found to be of a size comparable with that of a complex purified from mucus that had been brought to the point of dissolution by mild mechanical stirring. The latter material (GP-S) had a mol.wt. of 15.9 X 10(6) and was used to study further the effect of thermal treatment. Time and temperature lead to a gradual breakdown of GP-S, which is characterized by a single activation energy of 93.3 kJ/mol (22.3 kcal/mol) over the temperature range of 21-99 degrees C. The process responsible is thermal hydrolysis of peptide bonds, particularly next to aspartic acid residues. This conclusion is consistent with the appearance of aspartic acid as a new N-terminal amino acid and the activation energy of the process. After thermal degradation there is an increase in the buoyant density of GP-S and a change in the amino acid composition. These findings were found to be consistent with the loss of the naked peptide region and the preponderance of aspartic acid residues in this region. Thermal degradation therefore does not involve dispersion of non-covalent bonds, and indeed GP-S is quite unaffected by media commonly used to disperse such bonds.