Functionalization of multilayered DNA-coatings with bone morphogenetic protein 2.

The focus of the present study was to functionalize multilayered DNA-coatings with the osteoinductive factor bone morphogenetic protein 2 (BMP-2) using different loading modalities. The multilayered DNA-coatings were built up from either poly-d-lysine (PDL) or poly(allylamine hydrochloride) (PAH) and DNA using electrostatic self-assembly (ESA). The amounts of BMP-2 loaded into the multilayered DNA-coatings and its subsequent release characteristics were determined using radiolabeled BMP-2. Additionally, the effect of BMP-2 functionalized multilayered DNA-coatings on the in vitro behavior of bone marrow-derived osteoblast-like cells was evaluated in terms of proliferation, differentiation, mineralization, and cell morphology. The results demonstrate the feasibility of multilayered DNA-coatings to be functionalized by embedding BMP-2 according to three different loading modalities: superficial (s), deep (d), and double-layer (dl). BMP-2 was incorporated proportionally into the multilayered DNA-coatings as: s+(4*d)=dl. All differently loaded multilayered DNA-coatings showed an initial burst release followed by an incremental sustained release of the remaining BMP-2. In vitro experiments demonstrated that the loaded factor remained biologically active, as an accelerated calcium deposition was observed on s- and dl-loaded multilayered DNA-coatings, without affecting cell proliferation. In contrast, d-loaded multilayered DNA-coatings influenced osteoblast-like cell behavior by decreasing the deposition of calcium.

Antifungal activity of DNA-lipid complexes and DNA-lipid films against Candida species.

In this study amphiphilic lipids, DNA-lipid complexes, and DNA-lipid films were prepared, and their antifungal activity against Candida species was examined. The amphiphilic lipids were synthesized from a reaction of glycine or L-alanine with n-alkyl alcohol in the presence of p-toluene sulfonic acid. DNA-lipid complexes, which were prepared by the simple mixing of DNA and amphiphilic lipids, were insoluble in water. Self-standing, water-insoluble DNA-lipid films were prepared by casting the DNA-lipid complexes from a chloroform/ethanol solution. The antifungal activities of the lipids and DNA-lipid complexes against the Candida species were evaluated by minimum inhibitory concentrations (MICs); those of DNA-lipid films were evaluated by the disk diffusion method. The seven kinds of lipids, DNA-lipid complexes, and DNA-lipid films showed antifungal activity, and no differences were seen in the antifungal activities between glycine and L-alanine derivatives. The lipids, DNA-lipid complexes, and DNA-lipid films, which have shorter alkyl chain length in lipids, showed antifungal activity against all Candida species. However, the effect of antifungal activity against Candida species decreased with increased alkyl chain length in lipids. In this study, it was found that lipids, DNA-lipid complexes, and films with a decyl or dodecyl group exhibit more favorable antifungal activity.

Binding of influenza A virus to monosialoganglioside (GM3) reconstituted in glucosylceramide and sphingomyelin membranes.

The binding of influenza A virus to GM3-containing monolayers at an air/water interface was quantitatively investigated by use of a quartz crystal microbalance (QCM). A QCM was horizontally attached to the monolayer from the air phase and the binding behavior of influenza virus was followed by the frequency changes of the QCM. GM3 was reconstituted in the momolayer of sphingomyelin (SM) or glucosylceramide (GlcCer). When the mole fraction of GM3 was below 30 mol%, the binding rate of the influenza A virus to the GM3/GlcCer membrane was significantly faster than that to GM3/SM membranes. When the mole fraction of GM3 in SM was below 20 mol%, specific binding of influenza virus was not observed at all. Binding of the virus to the GM3/GlcCer mixed membrane was inhibited by the addition of sialyllactose (Neu5Ac alpha 2-3Gal beta 1-4Glc). The virus binding was also visually observed by scanning electron microscopy. Viruses selectively bound to GM3/GlcCer (20:80, by mol%) membrane, but not to GM3/SM (20:80, by mol%) membrane. Furthermore, it was suggested that specific binding of influenza virus to the GM3/GlcCer membrane induced the changes in morphology of virus. It was clearly demonstrated that binding of influenza virus to GM3 was influenced by matrix lipids surrounding GM3.

Mechanism of cell transfection with plasmid/chitosan complexes.

Chitosan is useful as a non-viral vector for gene delivery. Although there are several reports supporting the use of chitosan for gene delivery, studies regarding effects on transfection and the chitosan-specific transfection mechanism remain insufficient. In this report, the level of expression with plasmid/chitosan was observed to be no less than that with plasmid/lipofectin complexes in SOJ cells. The transfection mechanism of plasmid/chitosan complexes as well as the relationship between transfection activity and cell uptake was analyzed by using fluorescein isothiocyanate-labeled plasmid and Texas Red-labeled chitosan. In regard to effects on transfection, there were several factors to affect transfection activity and cell uptake, for example: the molecular mass of chitosan, stoichiometry of complex, as well as serum concentration and pH of transfection medium. The level of transfection with plasmid/chitosan complexes was found to be highest when the molecular mass of chitosan was 40 or 84 kDa, ratio of chitosan nitrogen to DNA phosphate (N/P ratio) was 5, and transfection medium contained 10% serum at pH 7.0. We also investigated the transfection mechanism, and found that plasmid/chitosan complexes most likely condense to form large aggregates (5-8 microm), which absorb to the cell surface. After this, plasmid/chitosan complexes are endocytosed, and possibly released from endosomes due to swelling of lysosomal in addition to swelling of plasmid/chitosan complex, causing the endosome to rupture. Finally, complexes were also observed to accumulate in the nucleus using a confocal laser scanning microscope.

Quantitative measurements of the interaction between monosialoganglioside monolayers and wheat germ agglutinin (WGA) by a quartz-crystal microbalance.

Monosialogangliosides (GM1, GM2, GM3 and GM4) were reconstituted in lipid monolayers at the air-water interface. The binding amounts and the initial binding rates of wheat germ agglutinin (WGA) to the monosialoganglioside monolayers were quantitatively studied by use of a quartz-crystal microbalance (QCM). A QCM was horizontally attached to the monolayer from the air phase, and the binding behavior (mass increase) was followed by the frequency decrease of the QCM. WGA binding affinities for the ganglioside monolayers were influenced by hydrophilic head groups of lipid matrices, densities of gangliosides, and sequences of oligosaccharide in gangliosides. Binding of WGA to the gangliosides reconstituted in a phosphatidylcholine (sphingomyelin and distearoylphosphatidylcholine) matrix was strongly suppressed, but not in a neutral glycolipids (GlcCer, GalCer, and LacCer), dipalmitoylphosphatidylethanolamine, and dipalmitoylphosphatidylethanolamine matrix. WGA showed high affinity for monolayers containing 20 mol% gangliosides, but only low affinity for 100% ganglioside monolayers. WGA preferably binds to gangliosides in the following sequence: GM3 > GM4 >> GM2 = GM1. No affinities of WGA for GM2 and GM1 were observed. The combined techniques of monolayer and QCM have the advantages of investigating recognition properties of gangliosides.

Adsorption behaviors of poly(N-p-vinylbenzyl-4-o-beta-d-galactopyranosyl-[1-->4]-d-gluconamide) by quartz-crystal microbalance.

Adsorption behaviors of amphiphilic poly(N-p-vinylbenzyl-4-o-beta-d-galactopyranosyl-[1-->4]-d-gluconamide) (PVLA) on the polystyrene (PS) surface was studied using 27 MHz quartz-crystal microbalance (QCM). The amount of adsorbed PVLA on PS surface was increased with an increase of PVLA concentration as a Langmuir-type in a monolayer. The saturated mass change (DeltaM(max)) and association constant (K(a)) of PVLA on PS surface were 498.6 ng/cm(2) and 1.93 x 10(7)M(-1), respectively. The adsorbed PVLA on PS surface was specifically recognized by Allo A lectin due to specific interaction between galactose moieties in the PVLA and Allo A. The hydrophobic interaction between hydrophobic main chain of PVLA and hydrophobic surface of PS was reduced in the presence of urea and the diameter of PVLA aqueous solution was decreased with an increase of urea concentration.

Selection of ganglioside GM1-binding peptides by using a phage library.

Ganglioside Gal beta1 --> 3GalNAc beta1 --> 4(NeuAc alpha2 --> 3) Gal beta1 --> 4Glc beta1 -->1'Cer (GM1)-binding peptides were obtained from a phage-displayed pentadecapeptide library by an affinity selection. The selection processes were in situ-monitored by a quartz-crystal microbalance method, on which a ganglioside GM1 monolayer was transferred. After five rounds of biopanning, the DNA sequencing of 18 selected phages showed that only three individual clones were selected. The peptide sequences of the random region were found to be DFRRLPGAFWQLRQP, GWWYKGRARPVSAVA and VWRLLAPPFSNRLLP. Binding constants of these phage clones to the GM1 monolayer were 10(10) M(-1). Three synthetic pentadecapeptides inhibited the binding of cholera toxin B subunit to the GM1 monolayer with an IC50 of 24, 13 and 1.0 microM, respectively. These peptides will be useful for searching functional roles of ganglioside GMI.

In vitro gene delivery mediated by chitosan. effect of pH, serum, and molecular mass of chitosan on the transfection efficiency.

Aminopolysaccharides such as chitosan and polygalactosamine (pGalN) were used to transfer luciferase plasmid into tumor cells. Chitosan largely enhanced the transfection efficiency of luciferase plasmid (pGL3), while pGalN did not at all. Transfection efficiencies of the pGL3/chitosan complexes were dependent on pH of culture medium, stoicheometry of pGL3:chitosan, serum, and molecular mass of chitosan. Transfection efficiency at pH 6.9 was higher than that at pH7.6. Optimum charge ratio of the pGL3:chitosan was 1:5. Chitosan polymer of 15 and 52 kDa largely promoted luciferase activities. Transfection efficiency mediated by chitosan of > 100 kDa was less than that by chitosan of 15 and 52 kDa. Heptamer (1.3 kDa) did not show any gene expression. Cationic liposome (lipofectin)-associated gene expression was inhibited by serum, while chitosan showed resistance to serum.

Dynamic analyses on induced-fit gaseous guest binding to organic crystals with a quartz-crystal microbalance

The inclusion behavior of gaseous guest molecules in a solid apohost, an orthogonal anthracene-bis(resorcinol)tetraol (1), was investigated with a quartz-crystal microbalance (QCM). Compound 1 forms crystals composed of molecular sheets bound together by an extensive hydrogen-bonded network. An apohost of 1 was cast onto a QCM and the binding of gaseous guest molecules was followed as a function of time by observing the decrease in the oscillation frequency, which is directly related to the increase in mass. Ethyl acetate and methyl ethyl ketone were significantly included into the apohost, whereas benzene and cyclohexane were simply adsorbed onto the surface of the solid; all these guests have similar vapor pressures at 25 degrees C. On the other hand, a host analogue 2, a tetramethoxy derivative of 1, barely included these guest molecules. The inclusion amount and the rate of inclusion of ethyl acetate or methyl ethyl ketone showed a drastic increase above a threshold concentration of guests in the gas phase. Thus, the structure of the apohost changed cooperatively in order to bind guest molecules above the threshold guest concentration. This cooperativity of the binding behavior was kinetically analyzed.

Intramolecular electron conduction along DNA strands and their temperature dependency in a DNA-aligned cast film.

Electroconductivity along a long DNA strand (ca. 10 microns length) in a DNA-aligned cast film of DNA-lipid complex was measured on a comb-type electrode (5 microns distance), and it could be reversibly regulated by temperatures across 70 degrees C.

Preparation of and tissue response to DNA-lipid films.

DNA-containing films have the potential to form complexes with antibiotics or cytokines by intercalation or groove-binding. This principle can be highly relevant for regenerative wound-healing around oral implants and in periodontology. In this study, we prepared DNA-lipid films and examined tissue responses to them as an indicator of their biological properties. The lipids were synthesized from the reaction of L-alanine, n-alkyl alcohol, and p-toluenesulfonic acid. We prepared the self-standing, water-insoluble DNA-lipid films by casting the DNA-lipid complex from chloroform/ethanol solution. The DNA-lipid complexes, which had 1:1 ratios of phosphate anions to cationic lipid, were found to have a double-helical B-form structure. The DNA-lipid films were almost dissolved 3 days after subcutaneous implantation in the backs of rats. There were no inflammatory reactions or inhibition of new tissue formation. We concluded that DNA-lipid films can be prepared by simple methods, and that they do not cause an unfavorable tissue response.

Antibacterial characteristics of newly developed amphiphilic lipids and DNA-lipid complexes against bacteria.

The purpose of this study was to investigate the antibacterial activity of newly developed amphiphilic lipids and DNA/lipid complexes against two types of oral bacteria and two types of hospital infection bacteria. Nine amphiphilic lipids were quantitatively prepared from the reaction of n-alkyl alcohol, alpha-amino acids, and p-toluenesulfonic acid. Nine DNA-lipid complexes were prepared by the simple mixing of DNA and amphiphilic lipids. The DNA-lipid complexes were insoluble in water. The antibacterial activity of lipids and DNA-lipid complexes against Porphyromonas gingivalis, Streptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa were evaluated by the disk-diffusion method. Seven artificial lipids showed antibacterial behavior; in particular, the lipids prepared from n-decyl alcohol and glycine and from n-decyl alcohol and L-alanine showed antibacterial activity against the four bacterial strains used in this study. On the other hand, the lipids of glutamic acid derivatives did not show any antibacterial activity against the four bacteria strains except for the lipid with an n-octyl group. Five DNA-lipid complexes also had an antibacterial effect. The complex prepared from DNA and glycine decyl ester p-toluenesulfonic acid salt exhibited antibacterial activity against the four types of bacteria strains. In this study it was found that lipids and DNA-lipid complexes with a mono-decyl group or a mono-dodecyl group have more favorable antibacterial activity.

Transglycosylation in a two-phase aqueous-organic system with catalysis by a lipid-coated beta-D-galactosidase.

A lipid-coated beta-D-galactosidase was prepared in which the enzyme surface is covered with a lipid monolayer and two long alkyl lipophilic tails serve to solubilize the enzyme in organic solvents. In a two-phase aqueous-organic system, a lipid-coated enzyme exists in the organic (2-propyl ether) phase and acts as an efficient transgalactosylation catalyst for various hydrophobic alcohols with lactose in the aqueous buffer solution. When a native beta-D-galactosidase was employed in the two-phase system, neither the transgalactosylation nor the hydrolysis reaction proceeded due to denaturation of the enzyme at the interface. Effects of coating lipid molecules, origins of enzymes, reaction in organic solvents, and chemical structures of acceptor alcohols on the transgalactosylation catalyzed by the lipid-coated enzyme were studied. This system could also be applied in a large-scale synthesis on the 0.1-1 g scale.

Adsorption to or desorption of beer components from a lipid membrane related to sensory evaluation.

The relationship between the adsorption to or desorption of beer from a lipid membrane and sensory evaluation was studied using a lipid-coated quartz crystal microbalance connected to a flow injection system. The adsorption and duration of adsorption of commercial beers showed a significant correlation with their body and smoothness in a sensory evaluation, respectively. Isohumulones, tartaric acid, NaCl, glutamic acid, and tannic acid were adsorbed onto the lipid membrane. Di- and trihydroxyoctadecenoic acids increased the duration of adsorption of the beer components onto the lipid membrane but not the extent of adsorption. They decreased the astringent duration of beer and the smoothness in the sensory evaluation but did not affect the intensity of bitterness or astringency or the body. It seems that this system, which modifies the electrostatic and hydrophobic interactions of the beer components with the tongue and throat surfaces, can mainly evaluate bitterness and/or astringency which significantly affect the body and smoothness of beer.

Novel instrumentation monitoring in situ platelet adhesivity with a quartz crystal microbalance.

The quartz crystal microbalance (QCM) in a solution is capable of sensing an extremely small mass change in the nanogram range. In this article, the authors attempted to apply QCM to in situ continuous monitoring of platelet adhesion in plasma. The instrumentation consisted of a piezoelectric quartz crystal vacuum-deposited with gold and connected to two electrodes, an oscillation circuit, a frequency counter, and a DC source (5 V), coupled with a personal computer, a television monitor, and a printer. The authors noted resonant frequency shifts to determine weight increase upon cell adhesion. A QCM sensor, with the sensitivity of 1 ng/Hz, was placed horizontally in platelet poor plasma. The authors did not observe any measurable frequency shift upon adding a suspension of non-adherent cells, such as red blood cells and prostaglandin I2-sensitized platelets, indicating that QCM does not count the increase in the mass of cells that simply settled on the quartz. They did observe, however, a time-dependent frequency shift upon addition of platelet rich plasma. Coupled with visual determination of numbers of adherent platelets and their morphology under scanning electron microscopy, they found that the magnitude of shift and its time dependence seem to correlate not only numbers of adherent platelets, but to their spreading state, indicating that QCM detects only the weight at the focal contact region of adherent cells. This suggested that the former contributes to the early phase of the frequency shift, and the latter contributes to a shift change after a longer period of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

[Development of gene delivery system by using DNA complexes and their application to antisense drug].

Recently, development of delivery system for oligonucleotides and genes into mammalian cells has been remarkably improved. Basic requirements for the therapeutic use of nucleotides are both of increase in their stability and efficient cell uptake. In this paper, we newly prepared DNA complexes with cationic lipoglutamates such as dibutyl glutamate (2C4N+), dihexyl glutamate (2C6N+) and dioctyl glutamate (2C8N+). Formations of the DNA/lipoglutamate complexes were confirmed by gel chromatography, elemental analysis, CD spectra, and light scattering measurement. Compaction of DNA by binding with the cationic lipoglutamate was revealed by means of multi-angle light scattering. The DNA/lipoglutamate complexes showed the increase in the stability against enzymatic hydrolysis by DNase I. The DNA/lipoglutamate complexes showed increase in cell uptake and transfection efficiency (CAT assay). The transfection efficiency of DNA/2C8N+ complex was comparable with that of commercially available lipofectin. Furthermore, a DNA complex with lipoglutamide (2EO4C10N+) having tetraethyleneglycol tails was prepared. This DNA/2EO4C10N+ complex showed high water solubility. And, the complexes of DNA or antisense (S-oligo) with 2EO4C10N+ showed efficient uptake by Hera cells, and showed intracellular distribution to the cytoplasmic reagion.

Adsorption behaviors of recombinant E-cadherin-IgG Fc fusion protein on polystyrene surface.

Adsorption behaviors of recombinant E-cadherin-IgG Fc (E-cad-Fc) fusion protein and mutated E-cad-Fcs on the polystyrene (PS) surface were investigated using a 27 MHz quartz-crystal microbalance (QCM) and ELISA. The amount of adsorbed E-cad-Fc on PS surface was increased with an increase of E-cad-Fc concentration as a Langmuir-type in a monolayer. Adsorbed E-cad-Fc on PS surface was stable even after washing if calcium ions are absent in the washing solution due to the calcium ion dependence in the adsorption. E-cadherin homophilic adhesion among E-cadherins during adsorption of E-cad-Fc was involved. Deglycosylation of the E-cad in the E-cad-Fc did not affect adsorption of E-cad-Fc on the PS surface although deglycosylation of the E-cad in the E-cad-Fc enhanced cell adhesion compared with E-cad-Fc.

Controlled release from a large capsule membrane corked with phosphatidylethanolamine bilayers: effect of temperature, pH and concentration of Ca2+ ions.

Permeations of a fluorescent probe entrapped in the inner aqueous phase of a large nylon capsule membrane corked with phosphatidylethanolamine bilayers were reversibly controlled by (i) the phase transition of bilayers, (ii) the ambient pH change, and (iii) the addition of Ca2+/EDTA from outside. Their signal-receptive permeation control could be reproduced repeatedly without damaging corking bilayers, in contrast to that of liposomal membranes.

Kinetic analyses of ATP-dependent deoxyribonuclease (DNase) reactions on a quartz-crystal microbalance.

We report here kinetic analyses of the hydrolysis of DNA by the ATP-dependent DNase using a DNA-immobilized quartz-crystal microbalance (QCM), which enables in situ real-time monitoring both the binding of enzyme and the hydrolysis reaction on DNA strands, as mass changes.