A study of the subdiffusion of small molecules in charged polyelectrolyte multilayers.

A theoretical approach has been developed here to describe the slow diffusion of small charged molecules of sodium dithionite (S2O42-) 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.

A typical diffusion monitored by flow cytometry: slow diffusion of small molecules in polyelectrolyte 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 effect of top-layer chemistry on the formation of supported lipid bilayers on polyelectrolyte multilayers: primary versus quaternary amines.

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.

Lipid Layers on Polyelectrolyte Multilayers: Understanding Lipid-Polyelectrolyte Interactions and Applications on the Surface Engineering of Nanomaterials.

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.

Virosome engineering of colloidal particles and surfaces: bioinspired fusion to supported lipid layers.

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.

Role of Hydrogen Bonding and Polyanion Composition in the Formation of Lipid Bilayers on Top of Polyelectrolyte Multilayers.

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).

Lipid/Polyelectrolyte coatings to control carbon nanotubes intracellular distribution.

Carbon Nanotubes have been functionalized with a layer of poly (sulfopropyl methacrylate) synthesized from silane initiators attached to the walls of the Carbon nanotubes. On top of the poly sulfo propyl methacrylate, lipid vesicles composed of 75% 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine and 25% 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] were assembled. The surface modification of the Carbon Nanotubes and lipid assembly were followed by TEM. Confocal Raman Microscopy was used to study the uptake and localization of the surface modified Carbon Nanotubes in the HepG2 cell line. The localization of the Carbon Nanotubes in the cells was affected by the surface coating. It was found that poly (sulfopropyl methacrylate) and lipid modified Carbon Nanotubes were present in the region of the lipid bodies in the cytoplasm.

Size and mechanical stability of norovirus capsids depend on pH: a nanoindentation study.

Norovirus-like particles were imaged using atomic force microscopy. The mechanical stability of the virus-like particles (VLPs) was probed by nanoindentation at pH values ranging from 2 to 10. This range includes pH values of the natural environment during the life cycle of noroviruses. The resistance of VLPs to indentation was constant at acidic and neutral pH. The Young's modulus was of the order of 30 MPa. At basic pH the compliance of the capsid increased along with an increase in diameter. This specific pH-dependent mechanical response of the capsid may be related to mechanisms controlling uptake and release of the RNA during infection. Consecutive indentations with pressures ≤ 300 bar demonstrated the ability of the capsids to fully recover from deformations comparable with the size of the capsid. The capsids can be viewed as nanocontainers with an inbuilt self-repair mechanism. At pH 10 the capsids lost their stability and were irreversibly destroyed after one single indentation.

Spectroscopic studies on the competitive interaction between polystyrene sodium sulfonate with polycations and the N-tetradecyl trimethyl ammonium bromide surfactant.

The interaction of N-tetradecyl trimethyl ammonium bromide (TTAB) surfactants with poly(sodium styrene sulfonate) (PSS), PSS/poly(allylamine hydrochloride) (PAH), and PSS/poly(diallyl dimethyl ammonium chloride) (PDADMAC) complexes has been studied by means of Raman and IR spectroscopy. The stoichiometry of the polyelectrolyte complexes and of the complexes with TTAB has been established. TTAB molecules bind to single PSS molecules in a coiled liquid-like alkyl configuration up to a molar fraction of 67% in dry state. At higher concentrations, TTAB shows a transition to a crystalline phase. In the case of PSS being complexed with PAH, surfactant binds to PSS with a stoichiometry of 2 molecules of TTAB per sulfonic acid group. Spectroscopic data show that TTAB interacting with PSS/PDADMAC complexes is capable of disassembling this polyelectrolyte complex, but when TTAB interacts with the PSS/PAH complexes this polyelectrolyte pair remains stable. Spectroscopic measurements performed at different humidity showed that dry PSS/PAH complexes display the nu(SO(2)) and nu(s)(SO(3)(-)) bands at positions, which are indicative of the presence of hydrogen bonds between PSS and PAH. Red shifts of these bands when mixing the PSS/PAH complexes with TTAB point to structural rearrangements of the complex when interacting with the surfactant.

Specific zeta-potential response of layer-by-layer coated colloidal particles triggered by polyelectrolyte ion interactions.

The zeta-potential of PSS/PAH and PSS/PDADMAC coated silica particles was studied in the presence of ClO4(-) and H2PO4(-) salts. In the presence of ClO4(-), layer-by-layer (LbL) coated silica particles with PDADMAC as the top layer show a reversal in the surface charge with increasing salt concentration but remain positive in phosphate solutions. LbL particles with PAH as the top layer become, however, negative in the presence of H2PO4(-) but retain their positive charge in the presence of ClO4(-). Charge reversal was explained by specific interaction of ClO4(-) ions with the quaternary amine groups and of H2PO4(-) with the primary amines through hydrogen bonding. Atomic force microscopy (AFM) and quartz crystal microbalance with dissipation (QCM-D) were employed to study the corresponding layer stability on planar surfaces.

Composite lipid polyelectrolyte capsules templated on red blood cells: fabrication and structural characterisation.

Charged phospholipids and mixtures of charged phospholipids with zwitterionic lipids were adsorbed onto polyelectrolyte capsules templated on erythrocytes. The assembly was proved by means of electrophoretic mobility measurements, confocal laser scanning microscopy and flow cytometry. Freeze-fracture electron microscopy proved that the phospholipids assemble as bilayers or multilayers. Single particle light scattering showed that bilayers composed of anionic lipids can be intercalated between subsequent polyelectrolyte inter-layers in a regular manner. Neutral lipids can form multilayers. A pronounced decrease in capsule permeability for small polar dyes upon lipid adsorption was followed by confocal laser scanning microscopy.

Freeze-fracture electron microscopy of lipid membranes on colloidal polyelectrolyte multilayer coated supports.

Lipid membranes were assembled on polyelectrolyte (PE)-coated colloidal particles. The assembly was studied by means of confocal microscopy, flow cytometry, scanning force microscopy, and freeze-fracture electron microscopy. A homogeneous lipid coverage was established within the limits of optical resolution. Flow cytometry showed that the lipid coverage was uniform. Freeze-fracture electron microscopy revealed that the lipid was adsorbed as a bilayer, which closely followed the surface profile of the polyelectrolyte support. Additional adsorption of polyelectrolyte layers on top of the lipid bilayer introduced inhomogeneities as evident from jumps in the fracture plane. Characteristic lipid multilayers have not been seen with freeze-fracture electron microscopy.

Permeation of macromolecules into polyelectrolyte microcapsules.

Polyelectrolyte microcapsules (PEMCs) have been prepared by coating red blood cells with the polyelectrolytes poly(styrenesulfonate), poly(allylamine hydrochloride), and dextran sulfate applying the layer-by-layer technique with subsequent dissolution of the core. The capsule permeability for human serum albumin (HSA) was studied as a function of the ionic strength and pH by means of confocal microscopy. PEMCs produced with dextran sulfate and poly(allylamine hydrochloride) show a significant increase in permeability for HSA at salt concentrations over 1 mM. For PEMCs prepared with poly(styrenesulfonate) and poly(allylamine hydrochloride) the limiting salt concentration is 5 mM. No pH dependence for permeation was observed. A correlation between the permeation and adsorption of HSA on the PEMC walls was investigated. Finally, a mechanism for the permeability, combining electrostatic interactions, and the presence of pores in the polymer layers is presented confirmed by the considerable increase of permeation of charged molecules in the presence of salt and the permeation of neutral molecules regardless of the ionic strength.

Encapsulation of proteins by layer-by-layer adsorption of polyelectrolytes onto protein aggregates: factors regulating the protein release.

A novel method of protein encapsulation is proposed. Preformed protein aggregates are covered with polyelectrolyte layers by means of layer-by-layer adsorption. The polyelectrolyte membrane prevents protein leakage out of the capsule. Using chymotrypsin as a model enzyme the capsule wall selective permeability was demonstrated for substrates and inhibitors of different molecular weight and solubility.

Fabrication of micro reaction cages with tailored properties.

Hollow polyelectrolyte capsules in micro- and submicrometer size were prepared. Their interior was functionalized by a "ship in bottle" synthesis of copolymers. While the monomers permeated the capsule wall easily, the formed polymers remained in the capsule cage. The physicochemical properties of the capsule interior such as ion strength, pH, light absorption, and fluorescence could be controlled independently from the surrounding solvent by means of the chemical nature of the captured polymer. In case of polyelectrolytes the osmotic pressure of the counterions led to a swelling of the capsules which can be important for micromechanics. The functionalization with light-sensitive materials allowed selective photoreactions inside the capsules. Synthesis of polyelectrolytes at high concentration resulted in an intertwining of the capsule wall with the polymer. The modified walls behaved like ion exchange membranes and showed selectivity toward adsorption and permeation of organic ions. The modified capsules offer many possibilities for novel applications as containers for controlled precipitation, as nanoreactors for catalyzed reactions, or as sensors.

Biological cells as templates for hollow microcapsules.

Microcapsules in the micrometer size range with walls of nanometer thickness are of both scientific and technological interest, since they can be employed as micro- and nano-containers. Liposomes represent one example, yet their general use is hampered due to limited stability and a low permeability for polar molecules. Microcapsules formed from polyelectrolytes offer some improvement, since they are permeable to small polar molecules and resistant to chemical and physical influences. Both types of closed films are, however, limited by their spherical shape which precludes producing capsules with anisotropic properties. Biological cells possess a wide variety of shapes and sizes, and, thus, using them as templates would allow the production of capsules with a wide range of morphologies. In the present study, human red blood cells (RBC) as well as Escherichia coli bacteria were used; these cells were fixed by glutardialdehyde prior to layer-by-layer (LbL) adsorption of polyelectrolytes. The growth of the layers was verified by electrophoresis and flow cytometry, with morphology investigated by atomic force and electron microscopy; the dissolution process of the biological template was followed by confocal laser scanning microscopy. The resulting microcapsules are exact copies of the biological template, exhibit elastic properties, and have permeabilities which can be controlled by experimental parameters; this method for microcapsule fabrication, thus, offers an important new approach for this area of biotechnology.

Assembly of Alternated Multivalent Ion/Polyelectrolyte Layers on Colloidal Particles. Stability of the Multilayers and Encapsulation of Macromolecules into Polyelectrolyte Capsules.

Alternating adsorption of multivalent ions and oppositely charged polyelectrolytes on colloid particles has been investigated. Multilayer films composed of Tb(3+)/polysterene sulfonate (PSS) and 4-pyrene sulfate/polyallylamine (PAH) were successfully assembled on polysterene sulfonate (PS) and melamine formaldehyde (MF) latex particles. The amount of assembled material was estimated by fluorescence and the linear growth of the film versus the number of layers was demonstrated. These multilayers are not stable and can be decomposed by salt and temperature. Dissolution of MF particles leads to formation of hollow capsules consisting of multivalent ion/polyelectrolyte multilayers. Comparative analysis of the capsules was done by confocal and scanning force microscopy. Complex hollow spheres consisting of Tb(3+)/PSS or 4-PS/PAH as an inner shell and stable PSS/PAH as an outer shell were produced. Due to selective permeability of the outer shell after degradation of the inner shell the multivalent ions are released out of the capsule while the polyelectrolytes fill the capsule interior. This is indicative of swelling of the capsule by osmotic pressure. The filled capsules were studied by confocal and scanning electron microscopy. Possibilities of encapsulating macromolecules in defined amounts per capsule are discussed. Copyright 2000 Academic Press.

Microencapsulation by means of step-wise adsorption of polyelectrolytes.

Step-wise adsorption of polyelectrolytes is used for the fabrication of micro- and nanocapsules with determined size, capsule wall composition and thickness. The capsule walls made of polyelectrolyte multilayers exclude high molecular weight compounds. Assembling of lipid layers onto these polyelectrolyte capsules prevents the permeation of small dyes. Encapsulation of magnetite nanoparticles is demonstrated and the features of these novel capsules are discussed.

Microencapsulation of Organic Solvents in Polyelectrolyte Multilayer Micrometer-Sized Shells.

Hollow-shell micrometer-sized particles were fabricated in aqueous media by stepwise deposition of oppositely charged polyelectrolytes onto melamine latex particles and biological cells with a dissolution of the core afterward. It is demonstrated that these shells can be suspended in various organic media, such as methanol, ethanol, pentanol, hexanol, octanol, octane, and decane, by a gradual solvent exchange. At this stage of the procedure the shells contain the respective organic solvent. Oil suspensions in water are then formed by transferring the particles from the organic media into water, without the use of any further surfactant addition. By an additional adsorption step employing phospholipids, it is possible to obtain a dispersion of shells in organic solvents containing an aqueous solution inside. AFM measurements are provided which show that the shells preserve their integrity in the different solvents. Confocal microscopy is employed to demonstrate encapsulation of solvents and the presence of lipids. Copyright 1999 Academic Press.