Nature and significance of the interactions between amyloid fibrils and biological polyelectrolytes

Charged polyelectrolytes such as glycosaminoglycans and nucleic acids have frequently been found associated with the proteinaceous deposits in the tissues of patients with amyloid diseases. We have investigated the nature and generality of this phenomenon by studying the ability of different polyanions, including DNA, ATP, heparin, and heparan sulfate, to promote the aggregation of amyloidogenic proteins and to bind to the resulting aggregates. Preformed amyloid fibrils of human muscle acylphosphatase and human lysozyme, proteins with a net positive charge at physiological pH values, were found to bind tightly to the negatively charged DNA or ATP. The effects of the polyelectrolytes on the kinetics of aggregation were studied for acylphosphatase, and the presence of ATP, DNA, or heparin was found to increase its aggregation rate dramatically, with a degree dependent on the net charge and size of the polyanion. Magnesium or calcium ions were found to attenuate, and ultimately to suppress, these interactions, suggesting that they are electrostatic in nature. Moreover, heparin was found to stabilize the aggregated state of acylphosphatase through compensation of electrostatic repulsion. Noteworthy, differences in affinity between native and aggregated acylphosphatase with heparin suggest that amyloid fibrils can themselves behave as polyelectrolytes, interacting very strongly with other polyelectrolytes bearing the opposite charge. Within an in vivo context, the strengthening of the electrostatic interactions with other biological polyelectrolytes, as a consequence of protein misfolding and aggregation, could therefore result in depletion of essential molecular components and contribute to the known cytotoxicity of amyloid fibrils and their precursors.     

Flocculating performances of exopolysaccharides produced by a halophilic bacterial strain cultivated on agro-industrial waste

This study reports the first systematic investigation of the flocculation dynamics of exopolysaccharides (EPSs) produced by a halophilic bacterial strain grown on pretreated molasses as fermentation substrate. The potential use of these EPSs as an easily biodegradable, natural alternative for synthetic polyelectrolytes which are widely used and contain toxic and carcinogenic monomers was investigated. Flocculating activities of the EPS samples in synthetic water, synthetic sea water and natural sea water media which were used as model raw waters were monitored via the Photometric Dispersion Analyser (PDA 2000) instrument and removals were determined by measuring residual turbidities. One of the six EPS specimens, which formed the largest flocs thus performed highest turbidity removal, exhibited flocculation performance and particle removal efficiency comparable with commercial cationic, nonionic and anionic synthetic polyelectrolytes.     

Self-cross-linking polyelectrolyte complexes for therapeutic cell encapsulation

Self-cross-linking polyelectrolytes are used to strengthen the surface of calcium alginate beads for cell encapsulation. Poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), containing 30 mol % 2-aminoethyl methacrylate, and poly(sodium methacrylate), containing 30 mol % 2-(methacryloyloxy)ethyl acetoacetate, were prepared by radical polymerization. Sequential deposition of these polyelectrolytes on calcium alginate films or beads led to a shell consisting of a covalently cross-linked polyelectrolyte complex that resisted osmotic pressure changes as well as challenges with citrate and high ionic strength. Confocal laser fluorescence microscopy revealed that both polyelectrolytes were concentrated in the outer 7-25 microm of the calcium alginate beads. The thickness of this cross-linked shell increased with exposure time. GPC studies of solutions permeating through analogous flat model membranes showed molecular weight cut-offs between 150 and 200 kg/mol for poly(ethylene glycol), suitable for cell encapsulation. C 2C 12 mouse cells were shown to be viable within calcium alginate capsules coated with the new polyelectrolytes, even though some of the capsules showed fibroid overcoats when implanted in mice due to an immune response.     

Development of an equation for potentiometric titration of polyelectrolytes using a periodic lattice model. A numerical analysis of interactions among ionizable groups

A numerical method is presented for analysing the potentiometric titration behavior of linear polyelectrolytes. A polyelectrolyte molecule is treated as a one-dimensional lattice containing a large number of lattice points, each of which has an identical ionizable group. In this method, the polyelectrolyte model lattice is divided into identical repeating unit cell systems with a finite number of ionizable groups to calculate the thermodynamic partition function of the system of polyelectrolyte solution. The electrostatic interaction between ionized groups adopted in the present study is given by the Debye-Hückel type screened Coulombic potential. The titration behavior as well as several thermodynamic quantities is derived by making a canonical ensemble summation of all states in a unit cell system under an appropriate periodic boundary condition. This method serves as a model of the two-step ionization often appearing in polyions with strong neighboring interactions such as homo- and copolymers of maleic acid. Several characteristics of the titration behavior of these polyelectrolytes are well reproduced by using a lattice model with pairwise intervals, including the effects of hydrogen bond formation and change in dielectric constant of the medium around an ionizable group. In addition, this method is valid for a more detailed analysis of the titration behavior of polyelectrolytes with various kinds of arrangements of charged groups.     

Inhibitory effect of polyelectrolytes on oligomeric enzymes

The effect of polyelectrolytes on the stability and catalytic characteristics of oligomeric enzymes--pig muscle lactate dehydrogenase (LDH) and bovine liver glutamate dehydrogenase (GDH)--was studied by fluorescent spectroscopic and steady state kinetic methods. It was shown that the binding of negatively charged polyelectrolytes--polystyrene sulfonate, polymethacrylate, and polyphosphate--destroys the tertiary and partially the secondary structure of LDH and GDH, resulting in their complete inactivation at pH < 7. The concentrations of polyelectrolytes needed for inhibition of the enzymes were in this case by two or more orders of magnitude lower than the corresponding concentrations for monomers--toluene sulfonate, methacrylate, and phosphate. The affinity of the substrate (pyruvate) for LDH did not vary in the presence of the polyelectrolytes, but the inhibition was removed by excess of substrate. We propose that the oligomeric state of enzymes causes polyelectrolytes to act on them in a special manner, this special effect differing significantly from the effect of polyelectrolytes on monomeric enzymes. The effect consists in that polyelectrolytes cleave the oligomeric structure of the enzymes, this "cleaving" effect being higher the greater the hydrophobicity of the polyelectrolyte chain.     

Anisotropic nucleation growth of actin bundle: a model for determining the well-defined thickness of bundles

Biopolymers such as DNA, F-actins, and microtubules, which are highly charged, rodlike polyelectrolytes, are assembled into architectures with defined morphology and size by electrostatic interaction with multivalent cations (or polycations) in vivo and in vitro. The physical origin to determine their morphology and size is not clearly understood yet. Our results show that the actin bundle formation consists of two stages: the thickness of actin bundles is determined nearly at the initial stage, while the length of actin bundles is determined later on. It is also found that the thickness of actin bundles decreases with the increase of polycation-mediated attraction between F-actins. From these results, we propose the anisotropic nucleation-growth mechanism, in which the thickness of actin bundles is determined by critical nucleus size, whereas the length of actin bundles is determined by the concentration of free actins relative to nucleus concentration. Observing that polycations are concentrated in some sites of actin bundles, which are thought to be nucleation sites to initiate the formation of actin bundles, supports this model. This anisotropic nucleation-growth mechanism of actin bundles can be broadly applied to the self-assembly of rodlike polyelectrolytes.     

Biodegradable microcapsules with entrapped DNA for development of new DNA vaccines

In this study an universal method for preparation of biodegradable microcapsules for antigen entrapment was proposed and optimized. The multilayer microcapsules were prepared by layer-by-layer adsorption of various polyelectrolytes (such as alginate, poly-L-lysine, κ-carrageenan, chitosan and dextran derivatives). High entrapment efficiency of protein and plasmid DNA (non less than 90%) was shown. To carry out in vivo tests, a set of microcapsules with entrapped pTKShi plasmid encoding the E2 polypeptide of classical swine fever was prepared. It was shown that an injection of these microcapsules into mice induced an immune response. The highest antibody titers of mouse blood sera were got after immunization by microcapsules based on modified dextran/carrageenan and modified chitosan/carrageenan. The proposed method for antigen entrapment in biodegradable microcapsules could be used for development of encapsulated vaccines of a new generation (DNA-vaccines).     

Robust and responsive silk ionomer microcapsules

We demonstrate the assembly of extremely robust and pH-responsive thin shell LbL microcapsules from silk fibroin counterparts modified with poly(lysine) and poly(glutamic) acid, which are based on biocompatible silk ionomer materials in contrast with usually exploited synthetic polyelectrolytes. The microcapsules are extremely stable in an unusually wide pH range from 1.5 to 12.0 and show a remarkable degree of reversible swelling/deswelling response in dimensions, as exposed to extreme acidic and basic conditions. These changes are accompanied by reversible variations in shell permeability that can be utilized for pH-controlled loading and unloading of large macromolecules. Finally, we confirmed that these shells can be utilized to encapsulate yeast cells with a viability rate much higher than that for traditional synthetic polyelectrolytes.     

The role of the ionic environment in the orientation of nucleic acids in electric fields

The relationship between polyelectrolyte theories based on linear charge density models and the electric-field induced orientation of the polyelectrolytes, poly(A), poly(C) and DNA is examined by varying their ionic environment with respect to ionic strength and acidity. The degree of counterion condensation on the polyelectrolytes predicted by the theories of Manning and Record is shown to be related linearly to the orientation as measured by their dichroism in the field. Micro-structural differences between poly(A) and poly(C) account for the differences in their dependence on the pH of the medium which affects the counterion condensation and thus the polarization in the orienting electric fields. The results consequently support recent treatments of linear polyelectrolytes having a high charge density which model them as smoothly charged linear polyions, but indicate that these models are insufficient to account for some of the effects of microstructural variations.     

Microvillar ion channels: cytoskeletal modulation of ion fluxes

The recently presented theory of microvillar Ca(2+)signaling [Lange, K. (1999) J. Cell. Physiol.180, 19-35], combined with Manning's theory of "condensed counterions" in linear polyelectrolytes [Manning, G. S. (1969). J. Chem. Phys.51, 924-931] and the finding of cable-like ion conductance in actin filaments [Lin, E. C. & Cantiello, H. F. (1993). Biophys. J.65, 1371-1378], allows a systematic interpretation of the role of the actin cytoskeleton in ion channel regulation.Ion conduction through actin filament bundles of microvilli exhibits unique nonlinear transmission properties some of which closely resemble that of electronic semiconductors: (1) bundles of microfilaments display significant resistance to cation conduction and (2) this resistance is decreased by supply of additional energy either as thermal, mechanical or electromagnetic field energy. Other transmission properties, however, are unique for ionic conduction in polyelectrolytes. (1) Current pulses injected into the filaments were transformed into oscillating currents or even into several discrete charge pulses closely resembling that of single-channel recordings. Discontinuous transmission is due to the existence of counterion clouds along the fixed anionic charge centers of the polymer, each acting as an "ionic capacitor". (2) The conductivity of linear polyelectrolytes strongly decreases with the charge number of the counterions; thus, Ca(2+)and Mg(2+)are effective modulator of charge transfer through linear polyelectrolytes. Field-dependent formation of divalent cation plugs on either side of the microvillar conduction line may generate the characteristic gating behavior of cation channels. (3) Mechanical movement of actin filament bundles, e.g. bending of hair cell microvilli, generates charge translocations along the filament structure (mechano-electrical coupling). (4) Energy of external fields, by inducing molecular dipoles within the polyelectrolyte matrix, can be transformed into mechanical movement of the system (electro-mechanical coupling). Because ionic transmission through linear polyelectrolytes is very slow compared with electronic conduction, only low-frequency electromagnetic fields can interact with the condensed counterion systems of linear polyelectrolytes.The delineated characteristics of microvillar ion conduction are strongly supported by the phenomenon of electro-mechanical coupling (reverse transduction) in microvilli of the audioreceptor (hair) cells and the recently reported dynamics of Ca(2+)signaling in microvilli of audio- and photoreceptor cells. Due to the cell-specific expression of different types and combinations of ion channels and transporters in the microvillar tip membrane of differentiated cells, the functional properties of this cell surface organelle are highly variable serving a multitude of different cellular functions including receptor-mediated effects such as Ca(2+)signaling, regulation of glucose and amino acid transport, as well as modulation of membrane potential. Even mechanical channel activation involved in cell volume regulation can be deduced from the systematic properties of the microvillar channel concept. In addition, the specific ion conduction properties of microfilaments combined with their proposed role in Ca(2+)signaling make microvilli the most likely cellular site for the interaction with external electric and magnetic fields.     

Biomedical applications of electrostatic layer-by-layer nano-assembly of polymers,enzymes, and nanoparticles

The introduction of electrostatic layer-by-layer (LbL) self-assembly has shown broad biomedical applications in thin film coating, micropatterning, nanobioreactors, artificial cells, and drug delivery systems. Multiple assembly polyelectrolytes and proteins are based on electrostatic interaction between oppositely charged layers. The film architecture is precisely designed and can be controlled to 1-nm precision with a range from 5 to 1000 nm. Thin films can be deposited on any surface including many widely used biomaterials. Microencapsulation of micro/nanotemplates with multilayers enabled cell surface modification, controlled drug release, hollow shell formation, and nanobioreactors. Both in vitro and in vivo studies indicate potential applications in biology, pharmaceutics, medicine, and other biomedical areas.     

The possibilities for enhancing the immunogenicity of adenovirus hexon

Synthetic polyelectrolytes and muramyldipeptide derivatives are tested as immunostimulators. It is established that the level of antibody-forming cells and specific (virus-neutralizing and precipitating) antibodies considerably rises during the immunization of human adenovirus I hexon in the combination with N-acetylglucosaminyl-muramyldipeptide and conjugation with a copolymer including acrylic acid and N-vinylpyrrolidone. A more expressed intensification of the antibody-formation is observed with the use of polyelectrolytes.     

FMT-XCT: in vivo animal studies with hybrid fluorescence molecular tomography-X-ray computed tomography

The development of hybrid optical tomography methods to improve imaging performance has been suggested over a decade ago and has been experimentally demonstrated in animals and humans. Here we examined in vivo performance of a camera-based hybrid fluorescence molecular tomography (FMT) system for 360° imaging combined with X-ray computed tomography (XCT). Offering an accurately co-registered, information-rich hybrid data set, FMT-XCT has new imaging possibilities compared to stand-alone FMT and XCT. We applied FMT-XCT to a subcutaneous 4T1 tumor mouse model, an Aga2 osteogenesis imperfecta model and a Kras lung cancer mouse model, using XCT information during FMT inversion. We validated in vivo imaging results against post-mortem planar fluorescence images of cryoslices and histology data. Besides offering concurrent anatomical and functional information, FMT-XCT resulted in the most accurate FMT performance to date. These findings indicate that addition of FMT optics into the XCT gantry may be a potent upgrade for small-animal XCT systems.     

New catalytic properties of monoamine oxidase immobilized in Langmuir-blodgett films with amphiphilic polyelectrolytes

Langmuir-Blodgett (LB) films of monoamine oxidase (MAO) have been formed on the surface f a polypropylene membrane using amphiphilic polyelectrolytes. The enzyme activity of such protein-polyelectrolyte films was measured by a Clark electrodes. It was shown that in LB films thus formed the use of amphiphilc polyelectrolytes, MAO activity was higher than in polyelectrolyte-free LB films. Immobilization of MAO with branched polyethylenimine modified on 12% by laurylchain led to pronounced changes in its catalytic properties. The dependence of the enzyme's kinetic parameters on amphiphilic polyelectrolyte structures was discussed. (c) 1994 John Wiley & Sons, Inc.     

Review: tissue engineering: reconstitution of human hematopoiesis ex vivo

The reconstruction of functioning human tissues ex vivo is becoming an important part of biotechnology. There are compelling scientific, clinical, and biotechnological reasons for fully or partially reconstituting human tissues such as skin, bone marrow, and liver ex vivo. In particular, bone marrow is a tissue of much importance, and there are significant societal and health benefits derived from a successfully constructed ex vivo hematopoietic system. In this article, we review the current status of this effort. The topics covered include the current understanding of the biology of human hematopoiesis, the motivation for reconstructing it ex vivo, the current state of ex vivo human hematopoietic cultures, the development of important metrics to judge culture performance, and an approach based on in vivo mimetics to accomplish this goal. We discuss some applications of functional ex vivo hematopoietic cultures and the biological and engineering challenges that face research in this area. (c) 1993 John Wiley & Sons, Inc.     

Ultrathin antibiotic walled microcapsules

Ultrathin microcapsules comprised of anionic polyelectrolytes (PE) and a polycationic aminoglycoside (AmG) antibiotic drug were prepared by depositing PE/AmG multilayers on zinc oxide (ZnO) colloid particles using the layer-by-layer self-assembly technique and subsequently dissolving the ZnO templated cores. The polyelectrolytes, dextran sulfate sodium (DxS) and poly(styrenesulfonate) (PSS), were selected owing to their different backbone structure. An aminoglycoside, tobramycin sulfate (TbS), was used for studying DxS/TbS or PSS/TbS multilayer films. The multilayer growth on ZnO cores was characterized by alternating zeta potential values that were different for the DxS/TbS and PSS/TbS multilayers due to the PE chemistry and its interaction with Zn(2+) ions. Transmission and scanning electron microscopy provide evidence of PE/TbS multilayer coating on ZnO core particles. The slow acid-decomposition of the ZnO cores using weak organic acids and the presence of sufficient quantity of Zn(2+) in the dispersion were required to produce antibiotic multilayer capsules. There was no difference in the morphological characteristics of the two types of capsules; although, the yield for [PSS/TbS](5) capsules was significantly higher than for [DxS/TbS](5) capsules which was related to the physicochemical properties of DxS/TbS/Zn(2+) and PSS/TbS/Zn(2+) complexes forming the capsule wall. The TbS quantity in the multilayer films was determined using a quartz crystal microbalance and high performance liquid chromatography techniques which showed less TbS loading in both, capsules and multilayers on planar gold substrate, than the theoretical DxS:TbS or PSS:TbS stoichiometric ratio. The decomposition of the [PE/TbS](6) multilayers was fastest in physiological buffer followed by mannitol and water. The decomposition rate of the [PSS/TbS](6) multilayers was slower than [DxS/TbS](6) monolayers. The incomplete decomposition of DxS/TbS under saline conditions suggests the major role of hydrogen bonding for stability of DxS/TbS multilayers. A combination of hydrogen bonding and hydrophobic interaction between phenyl rings in PSS was responsible for PSS/TbS multilayer stability. In vivo studies in rabbits highlight the safety and sustained drug delivery potential of the PE/AmG microcapsules. The antibiotic walled ultrathin capsules presented here are suitable for sustained ophthalmic antibiotic delivery.     

Investigation of selective protein immobilization on charged protein array by wavelength interrogation-based SPR sensor

We have investigated the use of multilayer films of polyelectrolytes as selective surfaces to analyze protein interactions with a self-assembled SPR wavelength-shift sensor. Charged arrays were prepared by alternating adsorption of the charged polyelectrolytes, poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS). Multilayer formation was monitored with the SPR wavelength-shift sensor and a Spreeta SPR sensor. Protein immobilization on the charged surfaces, which was also analyzed by the SPR sensors, was dependent on the pI of the proteins. Tissue transglutaminase (tTGase) and beta-galactosidase (pIs, 5.1 and 5.3, respectively) were preferentially bound to the positively charged PDDA surface, whereas lysozyme (pI, 11.0) was selectively bound to the negatively charged PSS surface. Immobilization of tTGase on the PDDA surface was also dependent on the buffer pH. The interaction of tTGase with RhoA(V14), a constitutively active form of Rho, could be detected on the charged arrays with the wavelength-shift sensor. The arrays could be reutilized at least 5 times. Thus, it is likely that charged surfaces, assembled by the layer-by-layer method using polyelectrolytes, will prove useful for preparing selective protein arrays.     

Structure and interactions of aggrecans: statistical thermodynamic approach

Weak polyelectrolytes tethered to cylindrical surfaces are investigated using a molecular theory. These polymers form a model system to describe the properties of aggrecan molecules, which is one of the main components of cartilage. We have studied the structural and thermodynamical properties of two interacting aggrecans with a molecular density functional theory that incorporates the acid-base equilibrium as well as the molecular properties: including conformations, size, shape, and charge distribution of all molecular species. The effect of acidity and salt concentration on the behavior is explored in detail. The repulsive interactions between two cylindrical-shaped aggrecans are strongly influenced by both the salt concentration and the pH. With increasing acidity, the polyelectrolytes of the aggrecan acquire charge and with decreasing salt concentration those charges become less screened. Consequently the interactions increase in size and range with increasing acidity and decreasing salt concentration. The size and range of the forces offers a possible explanation to the aggregation behavior of aggrecans and for their ability to resist compressive forces in cartilage. Likewise, the interdigitation of two aggrecan molecules is strongly affected by the salt concentration as well as the pH. With increasing pH, the number of charges increases, causing the repulsions between the polymers to increase, leading to a lower interdigitation of the two cylindrical polymer layers of the aggrecan molecules. The low interdigitation in charged polyelectrolytes layers provides an explanation for the good lubrication properties of polyelectrolyte layers in general and cartilage in particular.     

Synthesis and preliminary characterisation of charged derivatives and hydrogels from scleroglucan

The synthesis of negatively and positively charged polyelectrolytes from scleroglucan is described. Polycarboxylates were synthesised through nucleophilic substitution with chloroacetic acid or through a selective 2,2,6,6-tetramethyl-l-piperidinyloxy (TEMPO)-mediated oxidation of the primary alcohol groups. Amine groups were introduced through nucleophilic substitution with 2-chloroethylamine or 3-chloropropylamine. Reaction conditions were varied to obtain insight into the influence of variables on the degree of substitution. The conformational behaviour of the obtained polyelectrolytes was studied as a function of pH, temperature and solvent. For the products with a low degree of modification, evidence of an ordered conformation was found, whereas the polymers with a higher degree of modification behaved as random coils in solution. The negatively charged polymers were reticulated using the Ugi four-component condensation, obtaining negatively charged hydrogels. The positively charged polymers were reticulated using diethyl squarate (3,4-diethoxy-3-cyclobutene-1,2-dion, DES) to obtain positively charged hydrogels.     

Different measures of human hematopoietic cell culture performance are optimized under vastly different conditions

Hematopoiesis, the formation of mature blood cells from stem (LTC-IC) and progenitor (CFU-GM) cells in the bone marrow, is a complex tissue-forming process that leads to many important physiological functionalities. Consequently, a functioning ex vivo hematopoietic system has a variety of basic scientific and clinical uses. The design and operation of such a system presents the tissue engineer with challenges and choices. In this study, three culture variables were used to control ex vivo human hematopoiesis. Systematic variation of inoculum density (ID), medium exchange interval (MEI), and the use of preformed stroma (PFS) showed that (1) all three variables significantly influenced culture performance, (2) the three variables interacted strongly, and (3) the variables could be manipulated to achieve the optimization of different performance criteria. Donor-to-donor variability in culture performance was great at low ID but was minimized at higher ID. PFS had a large positive effect on cell and CFU-GM output at low ID, but had minimal effect at higher ID. In fact, PFS caused a decrease in LTC-IC output at high ID. The effects of PFS indicated that stromal cell elements became more limiting than proliferative cell elements as ID was reduced.In cultures without PFS, maximum cell output was obtained with high ID using a short MEI, whereas the greatest cell expansion ratio was obtained at low ID with an intermediate MEI. Maximum CFU-GM output was obtained from cultures with high ID using a short to intermediate MEI, whereas the greatest CFU-GM expansion ratio was obtained at intermediate ID with an intermediate MEI. The addition of PFS altered the locations of these maxima. In general, PFS moved the maxima to lower ID, and culture output became more sensitive to MEI. Therefore, the optimization of one performance criterion always resulted in a decline of the others. This study demonstrates that ex vivo tissue function is sensitive to many culture variables in an interactive fashion and that systematic multivariable studies are required to characterize tissue function. Once the effects of individual variables and their interactions are known, this knowledge can be used to optimize tissue performance with respect to desired criteria. (c) 1996 John Wiley & Sons, Inc.