A theoretical approach has been developed here to describe the slow diffusion of small charged molecules of sodium dithionite (S 2 O 4 2− ) in polyelectrolyte multilayers (PEMs) composed of polyallylamine hydrochloride (PAH) and polystyrene sulfonate (PSS), which is demonstrated here to be a case of subdifussion. Diffusion is measured experimentally by recording the quenching of the fluorescence of (7-nitrobenz-2-oxa-1,3-diazol-4yl) amino (NBD) labelled PAH layers assembled on silica particles by flow cytometry. NBD is reduced when it encounters dithionite leading to the disappearance of the fluorescence. The fluorescence decay curves show a slow diffusion of dithionite, that does not follow classical Fickean law. Dithionite diffusion in the PEMs is shown to be a non-Markovian process and the slow diffusion can be described via diffusion equations with fractional time derivatives. Results are explained assuming subdifussion of dithionite in the PEMs, as a result of the trapping of the negatively charged dithionite in the positively charged layers of PAH.

The diffusion of sodium dithionite (S2O42-) through polyelectrolyte multilayers of poly(diallyl dimethyl ammonium chloride) (PDADMAC)/poly(styrene sodium sulfonate) (PSS) assembled on colloidal particles with the layer-by-layer technique is studied by means of flow cytometry and quenching assay. Fluorescence is provided by a layer of (7-nitrobenz-2-oxa-1,3-diazol-4yl) amino) hexanoate (NBD)-labeled poly(allyl amine hydrochloride) assembled below the PDADMAC/PSS multilayer. NBD is quenched by a redox reaction with S2O42-. NBD quenching is fast at short times but strongly retarded at longer times. Quenching is faster for PDADMAC as the top layer and for increasing concentrations of S2O42-. The quenching kinetics of NBD is described with a model assuming a non-Fickean diffusion of S2O42-, with diffusion coefficients that depend on time with an inverse power law. Diffusion coefficients show little dependence on the number of layers but are highly dependent on the concentration of S2O42-. Increasing the concentration of S2O42- over 10 mol/m3 results in a decrease of the diffusion coefficient, more evident at longer times. The non-Fickean behavior for S2O42- diffusion is explained on the basis of the trapping of dithionites in the multilayers.

An innovative approach has been developed to measure small molecule diffusion in polyelectrolyte multilayers (PEMs) assembled on colloidal particles by means of flow cytometry (FACS). FACS allows changes in fluorescence emission as a function of time to be recorded per particle in a colloidal dispersion. Dithionite, S2O42, diffusion in PEMs composed of polyallylamine hydrochloride (PAH) and poly styrene sulfonate (PSS) assembled on silica particles has been studied by recording the quenching of (7-nitrobenz-2-oxa-1,3-diazol-4yl)amino (NBD) labelled PAH layers by FACS. NBD is reduced when it encounters dithionite, and is therefore no longer fluorescent. The decay in fluorescence will be used to follow the kinetics of dithionite diffusion. The fluorescence decay curves show slow diffusion that does not follow classical Fickean law. However, by assuming that the diffusion coefficient is time dependent and follows an inverse power law in an atypical diffusion case, it was possible to obtain an excellent fit for the decay curves.

The impact of polyanions on the formation of lipid bilayers on top of polyelectrolyte multilayers (PEMs) with poly(allylamine hydrochloride) (PAH) as the top layer is studied for the deposition of vesicles of mixed lipid composition, 50:50 molar ratio of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and negatively charged 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS). PEMs are assembled with polystyrene sulfonate (PSS), poly(acrylic acid) (PAA), and alginic acid sodium salt (Alg) as polyanions. The assembly of the vesicles on the PEMs is followed by means of the quartz crystal microbalance with dissipation. Fluorescence recovery after photobleaching measurements are applied to evaluate bilayer formation. Whereas a bilayer is formed on top of PAH/PSS multilayers, the vesicles are adsorbed on top of PAH/Alg and PAH/PAA multilayers, remaining unruptured or only partially fused. The influence of the surface composition of the PEM and of the bulk properties of the film are analyzed. The phosphate ions present in phosphate-buffered saline (PBS) play a fundamental role in bilayer formation on top of PAH/PSS as they complex with PAH and render the surface potential close to zero. For PAH/PAA and PAH/Alg, PBS renders the surface negative. X-ray photoelectron spectroscopy shows that the dibasic phosphate ions from PBS complex preferentially with PAH in PAH/PAA and PAH/Alg multilayers, whereas monobasic phosphates complex with PAH in PAH/PSS. An explanation for the absence of bilayer formation on PAH/PAA and PAH/Alg is given on the basis of the different affinities of phosphate ions for PAH in combination with the different polyanions.

In this manuscript we review work of our group on the assembly of lipid layers on top of polyelectrolyte multilayers (PEMs). The assembly of lipid layers with zwitterionic and charged lipids on PEMs is studied as a function of lipid and polyelectrolyte composition by the Quartz Crystal Microbalance. Polyelectrolyte lipid interactions are studied by means of Atomic Force Spectroscopy. We also show the coating of lipid layers for engineering different nanomaterials, i.e., carbon nanotubes and poly(lactic-co-glycolic) nanoparticles and how these can be used to decrease in vitro toxicity and to direct the intracellular localization of nanomaterials.

: The search for strategies to improve the performance of bioelectrochemical platforms based on supramolecular materials has received increasing attention within the materials science community, where the main objective is to develop low-cost and fl exible routes using self-assembly as a key enabling process. Important contributions to the performance of such bioelectrochemical devices have been made based on the integration and supramolecular organization of redox-active polyelectrolyte − surfactant complexes on electrode supports. Here, we examine the in fl uence of the processing solvent on the interplay between the supramolecular mesoorganization and the bioelectrochemical properties of redox-active self-assembled nano-particle − polyelectrolyte − surfactant nanocomposite thin fi lms. Our studies reveal that the solvent used in processing the supramolecular fi lms and the presence of metal nanoparticles not only have a substantial in fl uence in determining the mesoscale organization and morphological characteristics of the fi lm but also have a strong in fl uence on the e ffi ciency and performance of the bioelectrochemical system. In particular, a higher bioelectrochemical response is observed when nanocomposite supramolecular fi lms were cast from aqueous solutions. These observations seem to be associated with the fact that the use of aqueous solvents increases the hydrophilicity of the fi lm, thus favoring the access of glucose, particularly at low concentrations. We believe that these results improve our current understanding of supramolecular nanocomposite materials generated via polyelectrolyte − surfactant complexes, in order to use the processing conditions as a variable to improve the performance of bioelectrochemical devices.

The influence of the surface chemistry of polyelectrolyte multilayers (PEMs) on the formation of lipid bilayers is studied here for PEMs with either polyallylamine hydrochloride (PAH) or polydiallyldimethylammonium chloride (PDADMAC) as a polycation as a top layer, and polystyrene sulfonate (PSS) as a polyanion. Small unilamellar vesicles (SUVs) composed of phosphatidyl choline and phosphatidyl serine at a 50 : 50 molar ratio are deposited on top of the PEM films. The assembly of the SUVs into bilayers is studied via a quartz crystal microbalance with dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP). SUV deposition on PDADMAC/PSS results in vesicle adsorption while on PAH/PSS under the same conditions a bilayer is formed mainly due to weak interactions between the quaternary amines of PDADMAC. FRAP measurements confirm that SUVs are not fused on top of PDADMAC/PSS. The effect of phosphate ions, in solution, on the formation of lipid bilayers is also analysed. X-ray photoelectron spectroscopy shows the complexation of phosphate salts to the primary amines of PAH and no interaction with the quaternary amines of PDADMAC. ζ-potential measurements show a potential close to 0 for the PAH/PSS multilayers in PBS while PDADMAC/PSS displays a potential of 25 mV. A model is presented for the formation of lipid bilayers on PAH/PSS PEMs taking into account the role of phosphate ions in decreasing the electrostatic interactions between SUVs and PEMs and the formation of hydrogen bonds between the phospholipids and the primary amines of PAH.

Immunostimulating reconstituted influenza virosomes (IRIVs) are liposomes with functional viral envelope glycoproteins: influenza virus hemagglutinin (HA) and neuraminidase intercalated in the phospholipid bilayer. Here we address the fusion of IRIVs to artificial supported lipid membranes assembled on polyelectrolyte multilayers on both colloidal particles and planar substrates. The R18 assay is used to prove the IRIV fusion in dependence of pH, temperature and HA concentration. IRIVs display a pH-dependent fusion mechanism, fusing at low pH in analogy to the influenza virus. The pH dependence is confirmed by the Quartz Crystal Microbalance technique. Atomic Force Microscopy imaging shows that at low pH virosomes are integrated in the supported membrane displaying flattened features and a reduced vertical thickness. Virosome fusion offers a new strategy for transferring biological functions on artificial supported membranes with potential applications in targeted delivery and sensing.

A great number of stimuli-responsive particles have been developed and used for biomedical applications such as intracellular drug delivery. It is of paramount importance to study the intracellular responsive process of these particles, offering insight into the understanding of their structure variation and design criteria for better performance. In this study polyethyleneimine (PEI)-coated poly(lactide-co-glycolide) (PLGA) particles with a diameter of 430 nm were prepared via a one-step emulsion method. The amino groups in the PEI molecules allowed further covalent linking of fluorescein isothiocyanate (FITC), bifunctional coupling agents 3,3'-dithiobispropionimidate, amino-ended polyethylene glycol (NH2-PEG-NH2) and tetramethylrhodamine isothiocyanate (TRITC), resulting in fluorescence resonance energy transfer (FRET) pairs on the particles. The particles exhibited glutathione-responsive ability, and lost the FRET effect due to the separation of FITC/TRITC pairs from the particle surface as a result of the cleavage of disulfide bonds. The particles showed different FRET change rates in A549 cells and HEK293 cells depending on the intracellular GSH concentration. Moreover, a much slower degradation rate was found inside cells than in simulated buffer with a similar GSH concentration. The results suggest that the responsive behaviors of the particles obtained in simulated buffer may not match fully/correctly with the real situation in a complicated intracellular environment.

Polymer brushes of poly[2-(methacryloyloxy)ethyl]trimethylammonium chloride (PMETAC) and poly(sulfo propyl methacrylate) (PSPM) were synthesized by Atomic Transfer Radical Polymerization from planar and colloidal surfaces. Polymer brush growth was followed by QCMD and the water content determined by combined QCMD and elipsometry. From the water content the percentage of water lost during the brush collapse with the ionic strength could be obtained.Highly charged PSPM brushes were indented by Atomic Force Microscopy at different ionic strengths. The force response was fitted to a phenomenological equation analogous to the equation of state of a compressible fluid. Internal energy and brush compressibility were obtained as a function of ionic strength.Spherical brushes of PMETAC and PSPM display an invariance of the zeta potential with ionic strength in the range from 20 mM to 200 mM NaCl, the zeta potential remains almost constant. This invariance can be explained applying a hairy surface approach.

Cerium Oxide nanoparticles (CeO(2-x) NPs) are modified with polymer brushes of negatively charged poly (3-sulfopropylmethacrylate) (PSPM) and positively charged poly (2-(methacryloyloxy)ethyl-trimethylammonium chloride) (PMETAC) by Atom Transfer Radical Polymerisation (ATRP). CeO(2-x) NPs are fluorescently labelled by covalently attaching Alexa Fluor® 488/Fluorescein isothiocyanate to the NP surface prior to polymerisation. Cell uptake, intracellular distribution and the impact on the generation of intracellular Reactive Oxygen Species (ROS) with respect to CeO(2-x) NPs are studied by means of Raman Confocal Microscopy (CRM), Transmission Electron Microscopy (TEM) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). PSPM and PMETAC coated CeO(2-x) NPs show slower and less uptake compared to uncoated Brush modified NPs display a higher degree of co-localisation with cell endosomes and lysosomes after 24 h of incubation. They also show higher co-localisation with lipid bodies when compared to unmodified CeO(2-x) NPs. The brush coating does not prevent CeO(2-x) NPs from displaying antioxidant properties.

The self-assembly of mixed vesicles of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylserine (PS) phospholipids on top of polyelectrolyte multilayers (PEMs) of poly(allylamine hydrochloride) (PAH), as a polycation, and polystyrenesulfonate (PSS), as a polyanion, is investigated as a function of the vesicle composition by means of the quartz crystal microbalance with dissipation (QCM-D), cryo-transmission electron microscopy (Cryo-TEM), atomic force microscopy (AFM), and atomic force spectroscopy (AFS). Vesicles with molar percentages of PS between 50% and 70% result in the formation of lipid bilayers on top of the PEMs. Vesicles with over 50% of PC or over 80% of PS do not assembly into bilayers. AFS studies performed with a PAH-modified cantilever approaching and retracting from the lipid assemblies reveal that the main interaction between PAH and the lipids takes place through hydrogen bonding between the amine groups of PAH and the carboxylate and phosphate groups of PS and with the phosphate groups of PC. The interaction of PAH with PS is much stronger than with PC. AFS measurements on assemblies with 50% PC and 50% PS revealed similar adhesion forces to pure PS assemblies, but the PAH chains can reorganize much better on the lipids as a consequence of the presence of PC. QCM-D experiments show that vesicles with a lipid composition of 50% PC and 50% PS do not form bilayers if PSS is replaced by alginate (Alg) or poly(acrylic acid) (PAA).

Noble-metal nanoparticles (NPs) especially prepared from gold and silver have been combined on the surface of graphene to obtain graphene-based nanocomposites for novel functions in enhanced performance in bio-imaging, cancer detection and therapy. However, little is known about their cellular uptake, especially the intracellular quantity which plays a critical role in determining their functions and safety. Therefore, we prepared covalently conjugated GO/Au and GO/Ag composites by immobilizing Au and Ag nanoparticles on GO sheets pre-functionalized with disulfide bonds, respectively. The cellular uptake of these composites was quantitatively studied by means of an ion beam microscope (IBM) to determine the metal content in human lung cancer cells (A549 cells) and liver hepatocellular carcinoma cells (HepG2 cells). The cell uptake was also studied by inductively coupled plasma mass spectrometry (ICP-MS), which is one of the most sensitive techniques being applied to cell suspensions, for comparison. Toxicity, one of the consequences of cellular uptake of GO based composites, was studied as well. The potential toxicity mechanism was also suggested based on the results of intracellular quantification of the nanomaterials.

The thickness of a poly(sulfo propyl methacrylate) (PSPM) brush is determined by Atomic Force Microscopy (AFM) imaging as a function of the loading force at different ionic strengths, ranging from Milli-Q water to 1 M NaCl. Imaging is performed both with a sharp tip and a colloidal probe. The brush thickness strongly depends both on the applied load and on the ionic strength. A brush thickness of 150 nm is measured in Millipore water when applying the minimal loading force. Imaging with an 8 μm silica particle as a colloidal probe results in a thickness of 30 nm larger than that measured with the tip. Increasing the ionic strength causes the well known reduction of the thickness of the brush. The apparent thickness of the brush decreases with increasing loading forces. An empirical model analogous to that of a compressible fluid is applied to describe the dependence of the apparent thickness of the brush with loading force. The model comprises three ionic strength dependent parameters for the brush: thickness at infinite compression, energy, and cohesive force. The meaning and significance of these parameters are discussed. A particular advantage of the model is that it allows for determination of the brush thickness at zero loading force.

Studies of the uptake, biological fate, and toxicity of several metal oxide nanoparticles (NPs), such as Al2O3, TiO2, CeO2‐x, and ZnO NPs undertaken in the European Project “Health Impact of Engineered Metal and Metal Oxide Nanoparticles: Response, Bioimaging and Distribution at Cellular and Body Level” are reviewed here. Metal oxide NPs are radiolabeled by direct proton bombardment of commercially available NPs or enriched during synthesis with 18O to generate 18F after‐proton bombardment. Size, degree of aggregation, and zeta potential of the metal oxide NPs are studied in the presence of proteins and cell media. NP uptake and intracellular fate are followed by ion beam microscopy (IBM), transmission electron microscopy, confocal Raman microscopy and confocal laser scanning microscopy. IBM allows for the quantification of the intracellular dose of NPs. Cell viability studies and the immune response are studied “in vitro” in primary alveoli, and immortalized cell lines. Biodistribution studies in rodents are performed with positron emission tomography following different exposure routes: intravenous, oral, topical, and inhalation using radiolabelled NPs. Activity per organ is quantified for the different uptake routes and with the time.

A strategy of encapsulation of the antiTNF-α antibody on top of poly(lactide-co-glycolide) nanoparticles (PLGA NPs) is presented on the basis of the complexation of antiTNF-α with alginate (Alg) and subsequent assembly layer by layer with poly(L-lysine) (PLL). The assembly of the antiTNF-α/Alg complex with PLL and its stability in PBS and lysozymes are monitored on a planar support using a quartz crystal microbalance with dissipation. The assembly of the antiTNF-α/Alg complex on PLGA NPs is followed by zeta potential measurements. AntiTNF-α release from the PLGA NPs is measured in PBS at 37 and 60 °C and in the HepG2 cell line following NP uptake, using the Q-ADA kit detection kit. The release follows first-order kinetics with an initial burst. Intracellular release of antiTNF-α is confirmed by confocal Raman microscopy.

Highly charged dense poly(sulfopropyl methacrylate) polyelectrolyte brushes were indented with an atomic force microscopy (AFM) tip as well as with an 8 mu m silica colloidal probe at different ionic strengths ranging from Millipore water to 1 M NaCl. The force response during indentation was fitted to a phenomenological equation analogous to the equation of state of a compressible fluid. In this way, internal energy and brush thickness were obtained as a function of ionic strength. Long-range forces decayed exponentially with distance. The characteristic decay lengths were much larger than the Debye screening lengths at the respective ionic strengths. It was therefore concluded that long-range repulsion was due to compression of a loose corona of polymers in front of the dense part of the brush. The size of the indentor determines which region of the brush can be explored by AFM. The tip probes the denser parts of the brush, while with the colloidal probe the corona of the brush can be investigated. The obtained fits of the experimentally measured force distance curves were used as regularization tools for obtaining the brush swelling pressure or "force per unit area" as a function of brush compression. The swelling pressure as a function of brush thickness, h, followed over a wide range a power law close to similar to h(-2). This approach allowed deriving fundamental brush parameters on a thermodynamical basis like the compressibility as a function of thickness.

The zeta-potential of spherical brushes of poly[2-(methacryloyloxy) ethyl] trimethylammonium chloride (PMETAC) and poly(potassium sulfopropyl methacrylate) (PSPM) was measured as a function of ionic strength. The zeta-potential of PMETAC brushes varies from +30 mV at 10 mM NaCl to +20 mV at 200 mM, and the zeta-potential of PSPM changes from -30 mV to -25 mV, at their respective ionic strengths. This unusual weak dependence of the zeta-potential on ionic strength is quantitatively explained on the basis of the responsiveness of brushes toward changes in the ionic strength as well as taking into account the specific hydrodynamics of the hairy brush solution interface. The electric potential distribution is described in the framework of the Debye-Huckel approximation. An analytical equation describing the dependence of the zeta-potential of polyelectrolyte brushes on ionic strength is provided.