Characterization of cereal ß-glucan extracts from oat and barley and quantification of proteinaceous matter.

An extraction method for mixed-linkage ß-glucan from oat and barley was developed in order to minimize the effect of extraction on the ß-glucan structure. ß-Glucan were characterized in terms of molecular size and molar mass distributions using asymmetric flow field-flow fractionation (AF4) coupled to multiangle light scattering (MALS), differential refractive index (dRI) and fluorescence (FL) detection. The carbohydrate composition of the extracts was analysed using polysaccharide analysis by carbohydrate gel electrophoresis (PACE) and high-performance anion-exchange chromatography (HPAEC). Whether there were any proteinaceous moieties linked to ß-glucan was also examined. Purified extracts contained 65% and 53% ß-glucan for oats and barley, respectively. The main impurities were degradation products of starch. The extracts contained high molecular weight ß-glucan (105-108 g/mol) and large sizes (root-mean-square radii from 20 to 140 nm). No proteins covalently bound to ß-glucan were detected; therefore, any suggested functionality of proteins regarding the health benefits of ß-glucan can be discounted.

Interactions of PAMAM dendrimers with negatively charged model biomembranes.

We have investigated the interactions between cationic poly(amidoamine) (PAMAM) dendrimers of generation 4 (G4), a potential gene transfection vector, with net-anionic model biomembranes composed of different ratios of zwitterionic phosphocholine (PC) and anionic phospho-L-serine (PS) phospholipids. Two types of model membranes were used: solid-supported bilayers, prepared with lipids carrying palmitoyl-oleoyl (PO) and diphytanoyl (DPh) acyl chains, and free-standing bilayers, formed at the interface between two aqueous droplets in oil (droplet interface bilayers, DIBs) using the DPh-based lipids. G4 dendrimers were found to translocate through POPC:POPS bilayers deposited on silica surfaces. The charge density of the bilayer affects translocation, which is reduced when the ionic strength increases. This shows that the dendrimer-bilayer interactions are largely controlled by their electrostatic attraction. The structure of the solid-supported bilayers remains intact upon translocation of the dendrimer. However, the amount of lipids in the bilayer decreases and dendrimer/lipid aggregates are formed in bulk solution, which can be deposited on the interfacial layers upon dilution of the system with dendrimer-free solvent. Electrophysiology measurements on DIBs confirm that G4 dendrimers cross the lipid membranes containing PS, which then become more permeable to ions. The obtained results have implications for PAMAM dendrimers as delivery vehicles to cells.

Complexes formed between DNA and poly(amido amine) dendrimers of different generations--modelling DNA wrapping and penetration.

This study deals with the build-up of biomaterials consisting of biopolymers, namely DNA, and soft particles, poly(amido amine) (PAMAM) dendrimers, and how to model their interactions. We adopted and applied an analytical model to provide further insight into the complexation between DNA (4331 bp) and positively charged PAMAM dendrimers of generations 1, 2, 4, 6 and 8, previously studied experimentally. The theoretical models applied describe the DNA as a semiflexible polyelectrolyte that interacts with dendrimers considered as either hard (impenetrable) spheres or as penetrable and soft spheres. We found that the number of DNA turns around one dendrimer, thus forming a complex, increases with the dendrimer size or generation. The DNA penetration required for the complex to become charge neutral depends on dendrimer generation, where lower generation dendrimers require little penetration to give charge neutral complexes. High generation dendrimers display charge inversion for all considered dendrimer sizes and degrees of penetration. Consistent with the morphologies observed experimentally for dendrimer/DNA aggregates, where highly ordered rods and toroids are found for low generation dendrimers, the DNA wraps less than one turn around the dendrimer. Disordered globular structures appear for high generation dendrimers, where the DNA wraps several turns around the dendrimer. Particularly noteworthy is that the dendrimer generation 4 complexes, where the DNA wraps about one turn around the dendrimers, are borderline cases and can form all types of morphologies. The net-charges of the aggregate have been estimated using zeta potential measurements and are discussed within the theoretical framework.

DNA compaction induced by a cationic polymer or surfactant impact gene expression and DNA degradation.

There is an increasing interest in achieving gene regulation in biotechnological and biomedical applications by using synthetic DNA-binding agents. Most studies have so far focused on synthetic sequence-specific DNA-binding agents. Such approaches are relatively complicated and cost intensive and their level of sophistication is not always required, in particular for biotechnological application. Our study is inspired by in vivo data that suggest that DNA compaction might contribute to gene regulation. This study exploits the potential of using synthetic DNA compacting agents that are not sequence-specific to achieve gene regulation for in vitro systems. The semi-synthetic in vitro system we use include common cationic DNA-compacting agents, poly(amido amine) (PAMAM) dendrimers and the surfactant hexadecyltrimethylammonium bromide (CTAB), which we apply to linearized plasmid DNA encoding for the luciferase reporter gene. We show that complexing the DNA with either of the cationic agents leads to gene expression inhibition in a manner that depends on the extent of compaction. This is demonstrated by using a coupled in vitro transcription-translation system. We show that compaction can also protect DNA against degradation in a dose-dependent manner. Furthermore, our study shows that these effects are reversible and DNA can be released from the complexes. Release of DNA leads to restoration of gene expression and makes the DNA susceptible to degradation by Dnase. A highly charged polyelectrolyte, heparin, is needed to release DNA from dendrimers, while DNA complexed with CTAB dissociates with the non-ionic surfactant C12E5. Our results demonstrate the relation between DNA compaction by non-specific DNA-binding agents and gene expression and gene regulation can be achieved in vitro systems in a reliable dose-dependent and reversible manner.

Adsorption of α-synuclein to supported lipid bilayers: positioning and role of electrostatics.

An amyloid form of the protein α-synuclein is the major component of the intraneuronal inclusions called Lewy bodies, which are the neuropathological hallmark of Parkinson's disease (PD). α-Synuclein is known to associate with anionic lipid membranes, and interactions between aggregating α-synuclein and cellular membranes are thought to be important for PD pathology. We have studied the molecular determinants for adsorption of monomeric α-synuclein to planar model lipid membranes composed of zwitterionic phosphatidylcholine alone or in a mixture with anionic phosphatidylserine (relevant for plasma membranes) or anionic cardiolipin (relevant for mitochondrial membranes). We studied the adsorption of the protein to supported bilayers, the position of the protein within and outside the bilayer, and structural changes in the model membranes using two complementary techniques-quartz crystal microbalance with dissipation monitoring, and neutron reflectometry. We found that the interaction and adsorbed conformation depend on membrane charge, protein charge, and electrostatic screening. The results imply that α-synuclein adsorbs in the headgroup region of anionic lipid bilayers with extensions into the bulk but does not penetrate deeply into or across the hydrophobic acyl chain region. The adsorption to anionic bilayers leads to a small perturbation of the acyl chain packing that is independent of anionic headgroup identity. We also explored the effect of changing the area per headgroup in the lipid bilayer by comparing model systems with different degrees of acyl chain saturation. An increase in area per lipid headgroup leads to an increase in the level of α-synuclein adsorption with a reduced water content in the acyl chain layer. In conclusion, the association of α-synuclein to membranes and its adsorbed conformation are of electrostatic origin, combined with van der Waals interactions, but with a very weak correlation to the molecular structure of the anionic lipid headgroup. The perturbation of the acyl chain packing upon monomeric protein adsorption favors association with unsaturated phospholipids preferentially found in the neuronal membrane.

On the ability of PAMAM dendrimers and dendrimer/DNA aggregates to penetrate POPC model biomembranes.

Poly(amido amine) (PAMAM) dendrimers have previously been shown, as cationic condensing agents of DNA, to have high potential for nonviral gene delivery. This study addresses two key issues for gene delivery: the interaction of the biomembrane with (i) the condensing agent (the cationic PAMAM dendrimer) and (ii) the corresponding dendrimer/DNA aggregate. Using in situ null ellipsometry and neutron reflection, parallel experiments were carried out involving dendrimers of generations 2 (G2), 4 (G4), and 6 (G6). The study demonstrates that free dendrimers of all three generations were able to traverse supported palmitoyloleoylphosphatidylcholine (POPC) bilayers deposited on silica surfaces. The model biomembranes were elevated from the solid surfaces upon dendrimer penetration, which offers a promising new way to generate more realistic model biomembranes where the contact with the supporting surface is reduced and where aqueous cavities are present beneath the bilayer. The largest dendrimer (G6) induced partial bilayer destruction directly upon penetration, whereas the smaller dendrimers (G2 and G4) leave the bilayer intact, so we propose that lower generation dendrimers have greater potential as transfection mediators. In addition to the experimental observations, coarse-grained simulations on the interaction between generation 3 (G3) dendrimers and POPC bilayers were performed in the absence and presence of a bilayer-supporting negatively charged surface that emulates the support. The simulations demonstrate that G3 is transported across free-standing POPC bilayers by direct penetration and not by endocytosis. The penetrability was, however, reduced in the presence of a surface, indicating that the membrane transport observed experimentally was not driven solely by the surface. The experimental reflection techniques were also applied to dendrimer/DNA aggregates of charge ratio = 0.5, and while G2/DNA and G4/DNA aggregates interact with POPC bilayers, G6/DNA displays no such interaction. These results indicate that, in contrast to free dendrimer molecules, dendrimer/DNA aggregates of low charge ratios are not able to traverse a membrane by direct penetration.

Interactions between DNA and poly(amido amine) dendrimers on silica surfaces.

This study increases the understanding at a molecular level of the interactions between DNA and poly(amido amine) (PAMAM) dendrimers on solid surfaces, which is a subject of potential interest in applications such as gene therapy. We have used in situ null ellipsometry and neutron reflectometry to study the structure of multilayer arrangements formed by PAMAM dendrimers of generation 2 (G2), 4 (G4), and 6 (G6) and DNA on silica surfaces. Specifically, we adsorbed cationic dendrimer layers, then we condensed DNA to form dendrimer-DNA bilayers, and last we exposed further dendrimer molecules to the interface to encapsulate DNA in dendrimer-DNA-dendrimer trilayers. The dendrimer monolayers formed initially result in the deformation of the cationic adsorbates as a result of their strong electrostatic attraction to the hydrophilic silica surface. The highest surface excess and most pronounced deformation occurs for the G6 molecules due to their relatively large size and high surface charge density. G6-functionalized surfaces give rise to the highest surface excess of DNA during the bilayer formation process. This result is explained in terms of the high number of charged binding sites in the G6 monolayer and the low electrostatic repulsion between DNA and exposed patches of silica surface due to the relatively thick G6 monolayer. The binding strengths of the silica-dendrimer and dendrimer-DNA interactions are demonstrated by the high stability of the interfacial bilayers during rinsing. For the formation of trilayers of dendrimers, DNA, and dendrimers, G2 adsorbs as a smooth layer while G4 and G6 induce the formation of less well-defined structures due to more complex DNA layer morphologies.

DNA binding to zwitterionic model membranes.

This study shows that DNA (linearized plasmid, 4331 base pairs and salmon sperm, 2000 base pairs, respectively) adsorbs to model membranes of zwitterionic liquid crystalline phospholipid bilayers in solutions containing divalent Ca(2+) cations, and also in solutions containing monovalent Na(+). The interaction between DNA and surface-supported model membranes was followed in situ using null ellipsometry, quartz crystal microbalance with dissipation, as well as neutron reflectometry. In the presence of Na(+) (in the absence of multivalent ions), DNA adopts an extended coil conformation upon adsorption. The solvent content in the adsorbed layer is high, and DNA is positioned on top of the membrane. In the presence of divalent Ca(2+), the driving force for the adsorption of DNA is electrostatic, and the adsorbed DNA film is not as dilute as in a solution containing Na(+). Cryo-TEM and SANS were further used to investigate the interaction in bulk solution using vesicles as model membrane systems. DNA adsorption could not be identified in the presence of Na(+) using SANS, but cryo-TEM indicates the presence of DNA between neighboring unilamellar vesicles. In the presence of Ca(2+), DNA induces the formation of multilamellar vesicles in which DNA intercalates the lamellae. Possible electrostatic and hydrophobic mechanisms for the adsorption of DNA in solutions containing monovalent salt are discussed and compared to the observations in divalent salt.

Watching DNA condensation induced by poly(amido amine) dendrimers with time-resolved cryo-TEM.

The condensation of DNA by poly(amido amine) dendrimers of generation 1, 2, and 4 has been followed by time-resolved cryogenic transmission electron microscopy (cryo-TEM). The recorded images show that significant morphological rearrangement occurs for DNA condensed with the lower generation dendrimers leading to the formation of toroidal aggregates. Higher charge density dendrimers, on the other hand, give rise to globular aggregates, where no transient morphologies are observed. We suggest that the dendrimers in this case are kinetically trapped as soon as they bind to the DNA strand.

Analytical model study of dendrimer/DNA complexes.

The interaction between positively charged poly(amido amine) (PAMAM) dendrimers of generation 4 and DNA has been investigated for two DNA lengths; 2000 basepairs (bp; L = 680 nm) and 4331 bp (L = 1472.5 nm) using a theoretical model by Schiessel for a semiflexible polyelectrolyte and hard spheres. The model was modified to take into account that the dendrimers are to be regarded as soft spheres, that is, the radius is not constant when the DNA interact with the dendrimer. For the shorter and longer DNA, the estimated optimal wrapping length, l(opt) is ≈15.69 and ≈12.25 nm, respectively, for dendrimers that retain their original size (R(o) = 2.25 nm) upon DNA interaction. However, the values of l(opt) for the dendrimers that were considered to have a radius of (R = 0.4R(o)) 0.9 nm were 9.3 and 9.4 nm for the short and long DNA, respectively, and the effect due to the DNA length is no longer observed. For l(opt) = 10.88 nm, which is the length needed to neutralize the 64 positive charges of the G4 dendrimer, the maximum number of dendrimers per DNA (N(max)) was ≈76 for the shorter DNA, which is larger than the corresponding experimental value of 35 for 2000 bp DNA. For the longer DNA, N(max) ≈ 160, which is close to the experimental value of 140 for the 4331 bp DNA. Charge inversion of the dendrimer is only observed when they retain their size or only slightly contract upon DNA interaction.