Interaction between collagen and adenosine 5'-triphosphate.

A quantitative study was made of the adsorption of adenosine 5′-triphosphate (ATP) on collagen by following the change in the absorbance at 258 nm of ATP in the soaking solution. The amount of ATP adsorbed decreased exponentially with the increase of pH up to pH 8 and fell off more rapidly at higher pH values. At a given pH, when the concentration of ATP was increased, the amount of ATP adsorbed increased following the pattern of a Langmuir isotherm. The adsorption was independent of the cation present. The adsorption of adenosine 5′-diphosphate was essentially the same as that for ATP. For tendons deposited with calcium phosphate, the amount of ATP adsorbed decreased compared to natural tendons. The adsorption of ATP on collagen fibers inhibited the contraction caused by calcium chloride, calcium bromide, and lithium bromide. In solution, ATP had very little effect on the denaturation of acid-soluble collagen caused by calcium chloride.     

Interaction of polyanions with basic fuchsin and formaldehyde.

Basic fuchsin and formaldehyde react readily with a number of polyanions in aqueous solutions. Deoxyribonucleic acid, ribonucleic acid, several synthetic polynucleotides and polyvinylsulfate all convert buffered solutions of basic fuchsin and formaldehyde from a magenta to a purple color at ambient temperature. The formation of product is maximal in about 20 min and the resulting complexes are stable enough to be isolated by gel filtration. Microgram amounts of polyanion suffice to produce the color changes and the spectral shifts vary with the type of polyanion. These results are interpreted to indicate that polymerization of basic fuchsin and formaldehyde is an essential aspect of color formation.     

Polyelectrolyte complexes based on pectin-NH2 and chondroitin sulfate

This work reports on the formation and characterization of a polyelectrolyte complex based on pectin (PT), functionalized with primary amine groups (PT-NH₂), and chondroitin sulfate (CS). From the simple mixture of PT-NH₂ and CS, in acid conditions, it was formed a polyelectrolyte complex, labeled as PT-NH₂/CS complex, which was confirmed through FTIR spectroscopy. The electrostatic interactions among the protonated amine groups from PT-NH₂ and the sulfate groups from CS are responsible by complex formation. XRD patterns and thermal analysis showed that the complex formation disrupts some interactions present on the PT-NH₂ and CS, but on the other hand, others are created. SEM images showed that the PT-NH₂/CS complex presents a porous and rough morphology. PT-NH₂/CS complex is new material that maintains the properties of CS with synergic association of properties from both polymers, which could maximize its applicability as biomaterial, for example.     

Interaction between hepatocytes and collagen gel in hollow fibers

Gel entrapment culture of primary mammalian cells within collagen gel is one important configuration for construction of bioartificial organ as well as in vitro model for predicting drug situation in vivo. Gel contraction in entrapment culture, resulting from cell-mediated reorganization of the extracellular matrix, was commonly used to estimate cell viability. However, the exact influence of gel contraction on cell activities has rarely been addressed. This paper investigated the gel contraction under varying culture conditions and its effect on the activities of rat hepatocyte entrapped in collagen gel within hollow fibers. The hepatocyte activities were reflected by cell viability together with liver-specific functions on urea secretion and cytochrome P450 2E1. Unexpectedly, no gel contraction occurred during gel entrapment culture of hepatocyte under a high collagen concentration, but hepatocytes still maintained cell viability and liver-specific functions at a similar level to the other cultures with normal gel contraction. It seems that cell activities are unassociated with gel contraction. Alternatively, the mass transfer resistance induced by the combined effect of collagen concentration, gel contraction and cell density could be a side effect to reduce cell activities. The findings with gel entrapment culture of hepatocytes would be also informative for the other cell culture targeting pathological studies and tissue engineering.     

Anionic complexes of MWCNT with supergiant cyanobacterial polyanions

Multi‐walled carbon nanotubes (MWCNTs) were well dispersed in an aqueous solution of the cyanobacterial polysaccharide, sacran, with an ultra‐high molecular weight >10 million g/mol. MWCNTs powder was put into aqueous solutions of various polysaccharides including sacran and was dispersed under sonication. As a result of the turbidity measurement of the supernatant, it was found that sacran showed the highest MWCNT‐dispersion efficiency of all the polysaccharides used here. Cryogenic transmission electron microscopic (Cryo‐TEM) studies directly demonstrated the existence of MWCNTs in the supernatant, and high‐resolution TEM observation revealed that MWCNTs covered by sacran chains made their efficient dispersion in water. Raman spectroscopy demonstrated the existence of MWCNT in dried sample from supernatant and the interaction between MWCNT and sacran. The ζ‐potential measurement of the dispersion indicated the negative surface charges of the sacran/MWCNT complexes. Then the MWCNT complexes were able to fabricate by ionic interaction; electrophoresis of the anionic complex formed the sacran/MWCNT gels on the anode while the droplet of sacran/MWCNT dispersion formed gel beads in the presence of the lanthanoid cations. © 2012 Wiley Periodicals, Inc.     

Interaction between proteins and salt solutions. IV. Enrichment of salts in collagen during denaturation

Using ¹⁴C-labeled KSCN and ³⁶Cl-labeled KC1, we have determined the selective removal of ions and water by fibrous, crosslinked collagen both below and above the denaturation temperatures at several molarities under or near isoelectric conditions. The results for KSCN show there is an increase in ion binding at the denaturation temperature. These data, in conjunction with date from the literature, have been used to evaluate binding constants and mole fractions of available binding sites, as well as the free energy of binding various salts to collagen and gelatin. The values so obtained correlate satisfactorily with similar quantities obtained from the experimental dependence of the shrinkage temperature of collagen on salt concentration using a simple theory for the melting point depression which includes the effect of binding in the denatured state. Salting-out agents show negligible binding to the protein, and this confirms the earlier finding that the interaction between these agents and the protein can be approximately described by conventional polymer–diluent theories which do not consider ion binding. Also, an analysis of the role of unequal anion and cation binding is presented.     

Polyelectrolyte complexes of glycol chitosan with some polysaccharides. II. Electrical conductivity

The dc electrical conductivity of films of the polyelectrolyte complexes of glycol chitosan (GlChi) with the sodium salts of dextran sulfate (DS), carboxymethyl cellulose (CMC), polygalacturonic acid (GalUA)_n, and alginic acid (AlgA) was measured at temperatures above and below room temperature. The maximum field strength in the thinnest film used amounted to 3 × 10⁴ V/cm. A plot of normalized current against the reciprocal of the absolute temperature revealed two regions with different slopes, and activation energies in these two regions have been obtained for all the complexes. The activation energies in the high-temperature region vary from 0.85 to 1.18 eV and in the low-temperature region from 0 to 0.22 eV. Reasons are given to show that the conductivity is probably ionic. Near room temperature, the current–voltage relation is almost linear in the GlChi–DS complex, while in the other three complexes the current varies as a power n of the voltage with the value of n ranging from 1.7 to 2.5. A rise in temperatures causes an increase in the slope of the log I vs log V plot in GlChi–DS and GlChi–CMC complexes. The nonlinear current–voltage relation is ascribed to a space-charge-limited conductivity.     

Interaction between albumin-bound dinitrosyl iron complexes and reactive oxygen species

It has been established that albumin-bound dinitrosyl iron complexes can be destroyed by superoxide radicals generated in a xanthine-xanthine oxidase system. It was shown that peroxynitrite also effectively destroyed albumin-bound dinitrosyl iron complexes. At the same time, hydrogen peroxide and tert-butyl hydroperoxide did not stimulate the destruction of albumin-bound dinitrosyl iron complexes up to concentrations one order higher than the content of NO. The data have been obtained indicating that dinitrosyl iron complexes possess the vasodilatory activity. It has been proposed that peroxynitrite and superoxide radical, by causing the destruction of albumin-bound dinitrosyl iron complexes, affect the physiological properties of nitric oxide.     

Interaction between albumin-bound dinitrosyl iron complexes and reactive oxygen species

Dinitrosyl iron complexes (DNIC) bound to BSA are shown to be destroyed by superoxide radicals generated in the xanthine oxidase-xanthine system. Peroxynitrite is also efficient in this respect. By contrast, neither hydrogen peroxide nor tert-butyl hydroperoxide appreciably destroy BSA-DNIC even at a tenfold molar excess. Evidence is obtained for the vasodilatory properties of BSA-DNIC. It is suggested that in this way peroxynitrite and superoxide radical can affect the physiological functions of nitric oxide.     

Polyelectrolyte complexes and layer-by-layer capsules from chitosan/chitosan sulfate

Polyelectrolyte complex formation of chitosans of varying average molecular weight and degree of acetylation with chitosan sulfate or poly(styrene sulfonate) was studied by static light scattering in dilute solution at various ionic strengths. Unlike the molecular weight, the degree of acetylation was found to have a significant effect on the resultant structural densities of the complexes. The same system was applied to the preparation of micrometer-sized hollow shells by means of a layer-by-layer technique (in total eight layers). Their behavior toward fluorescent probes such as fluorescein and rhodamin 6G or fluorescein isothiocyanate labeled dextrans at various ionic strengths and pH (observed by confocal laser light scanning microscopy) could be understood through a discussion of electrostatic forces between the highly charged shells and the probes to be dominant. At an ionic strength of 0.1 M and above, charge effects are largely suppressed (screening effect) and a size-dependent "cutoff" for the permeation of the macromolecular fluorophore was observed.     

Interaction of amino acyl-tRNA-synthetases from the rabbit liver with RNA and polyanions

The interaction of aminoacyl-tRNA synthetase with RNA and polyanions was studied. The inhibition of the enzymes by polyU, polyI and heparin was demonstrated. It was found that this interaction is of limited specificity and is typical of single-stranded RNAs which possess no orderly secondary structure as well as of other polyanions possessing similar polyelectrolytic properties. Data from kinetic analysis and lysyl-tRNA synthetase modification by pyridoxal phosphate are suggestive of participation of the tRNA binding site in the enzyme interaction with polyanions.     

Polyelectrolyte complexes of lactoferrin and pH-sensitive microparticles on their basis

Suspensions of insoluble polyelectrolyte complexes of dextran sulfate (DS) of different molecular masses with lactoferrin (LF) have been fabricated and characterized. The encapsulation efficiency of LF and DS in a complex at pH 3.0 and 4.0 was assessed, and particles were characterized by their sizes and ζ-potential. The complexes formed at pH 3.0 differed by a higher stability level. The interaction with DS resulted in a twofold decrease in the antioxidant activity of LF, although the formation of complexes was not accompanied by conformational changes in LF molecules according to IR-spectrometry data. Microencapsulation was carried out by treating the suspensions with negatively charged LF-DS complexes with protamine and chitosane solutions with different molecular masses. The composition, size, and the ζ-potential of interaction products were assessed which allowed us to select the conditions for the preparation of pH-sensitive polyelectrolyte microparticles loaded with LF which would be able to gradually release glycoprotein under conditions that model the passage through the gastrointestinal tract of humans. These data indicate that this approach is promising for the creation of pH-sensitive biopolyelectrolytes suitable for oral administration of LF to target cells.     

Polyelectrolyte complexes as a tool for purification of plasmid DNA. Background and development

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.     

Polyelectrolyte complexes stabilize and controllably release vascular endothelial growth factor

Angiogenesis has long been a desired therapeutic approach to improve clinical outcomes of conditions typified by ischemia. Vascular endothelial growth factor (VEGF) has demonstrated the ability to generate new blood vessels in vivo, but trials using intravenous delivery have not yet produced clinical success. Localized, sustained delivery of VEGF has been proven necessary to generate blood vessels as demonstrated by implantable, controlled release devices. Ultimately, nanoparticles delivered by intravenous injection may be designed to accumulate in target tissues and sustain the local VEGF concentration; however, injectable nanosuspensions that control the release of stabilized VEGF must first be developed. In this study, we utilize the heparin binding domain of VEGF to bind the polyanion dextran sulfate, resulting in an enhanced thermal stability of VEGF. Coacervation of the VEGF-bound dextran sulfate with selected polycations (chitosan, polyethylenimine, or poly-L-lysine) produced nanoparticles approximately 250 nm in diameter with high VEGF encapsulation efficiency (50-85%). Release of VEGF from these formulations persisted for >10 days and maintained high VEGF activity as determined by ELISA and a mitogenic bioassay. Chitosan-dextran sulfate complexes were preferred because of their biodegradability, desirable particle size ( approximately 250 nm), entrapment efficiency ( approximately 85%), controlled release (near linear for 10 days), and mitogenic activity.