V I Gorbach's research while affiliated with Botanical Garden-Institute of the Far Eastern Branch of the Russian Academy of Sciences and other places

Complexes of lipopolysaccharide (LPS) from the bacterium Escherichia coli and chitosan (CN) with a molecular weight of 5 kDa were obtained and their supramolecular organization was studied. Using atomic force microscopy, it was shown that during the formation of complexes there is a transition from the micellar structure of the original LPS to linear network structures uniformly distributed over the surface of mica. The stability of LPS-CN complexes of various stoichiometries in biological media in the presence of serum proteins was investigated. It was shown that complexes with an LPS–CN ratio of 1 : 1 in the presence of serum proteins lost their surface charge and tended to aggregate while complexes with the maximum saturation of CN (1 : 5) did not aggregate under these conditions and maintained their surface charge. The effect of CNs of different molecular weights on the ability of LPS to interact with neutrophils of human whole blood was studied. It was observed that LPS-CN complexes were capable of binding to neutrophils and internalazing the cell, and this ability was enhanced in the presence of serum proteins. Chitosan exhibited the ability to suppress the synthesis of the proinflammatory cytokine TNF-α induced by LPS, not only as part of the complex but also when cells were pretreated with a polycation.

Herpes simplex virus (HSV) infections, the incidence of which is still widespread throughout the world, are actualizing the search and development of new, more effective antiherpetic drugs. The development of multifunctional drug delivery systems, including liposome-based ones, has become a relevant and attractive concept in nanotechnology. The ability of complexes of κ- and Σ-carrageenans (CRGs)—sulfated polysaccharides of red algae, with echinochrome A (Ech), as well as the liposomal form of the Σ-CRG/Ech complex—to inhibit different stages of HSV-1 infection in Vero cells was studied. By quantum chemical calculations, it was shown that CRG forms stable complexes with Ech. We have shown that complexes of κ-CRG/Ech and Σ-CRG/Ech exhibit highest virucidal activity with a selectivity index (SI) of 270 and 350, respectively, and inhibition of virus-cell interaction (SI of 83 and 32, respectively). The liposomal form of the Σ-CRG/Ech complex after virus adsorption and penetration to cells effectively reduced the HSV-1 plaque formation. The virus-inhibiting activity of the liposomal form of the Σ-CRG/Ech complex was three times higher than that of the Σ-CRG/Ech complex itself. Obtaining CRGs/Ech complexes and their liposomal forms can become the basis of a successful strategy for the development of promising antiherpetic drugs.

Carrageenan (CRG) and carrageenan/chitosan (CH) gel beads (CRG/CH) were prepared as a release delivery system for echinochrome A (Ech). According to spectral data, the Ech was dispersed in the polymer matrix, interacted with CRG, was not oxidised, and remained stable after encapsulation in CRG beads. Carrageenan beads containing Ech were coated with CH by layering. The influence of the structural features of CRG on the formation of beads and the beads morphology, swelling behaviour, mucoadhesive properties and drug release were evaluated. The polysaccharide matrices with Ech showed different swelling characteristics depending on the pH of the medium and the structure of the CRG used. The slow drug release from polysaccharide matrixes was observed for κ- and κ/β-CRG beads, that contained 3,6-anhydro-α-d-galactopyranose units and had high molecular weight. The obtained results showed the prospects of using polysaccharide beads to include Ech.

Inclusion of drugs in liposomes offers the potential for localized and sustained delivery to mucosal surfaces. The inclusion of the carrageenan matrix with echinochrome A ((Ech)—the active substance of the drug Histochrome) in liposomes was studied. According to the spectral characteristics, Ech was not oxidized and retained stability after encapsulation in the liposomes and the lyophilization process. Loading the liposomes with negatively charged polysaccharide results in the increase in the zeta potential to more negative values (from −14.6 to −24.4 mV), that together with an increasing in the sizes of liposomes (from 125.6 ± 2.5 nm to 159.3 ± 5.8 nm) propose of the formation of the polymer coating on liposomes. The interactions of liposomes with porcine stomach mucin was determined by the DLS and SEM methods. The changes in the zeta-potential and size of the mucin particles were observed as the result of the interaction of liposomes with mucin. To evaluate the mucoadhesive properties of liposomes and the penetration of Ech in the mucosa, a fresh-frozen inner surface of the small intestine of a pig as a model of mucous tissue was used. Polysaccharide-coated liposomes exhibit very good mucoadhesive properties −50% of Ech remains on the mucosa.

To gain a mechanistic insight in the functioning of the OmpF-like porin from Yersinia pseudotuberculosis (YOmpF), we compared the effect of pH variation on the ion channel activity of the protein in planar lipid bilayers and its binding to lipid membranes. The behaviour of YOmpF channels upon acidification was similar to that previously described for Escherichia coli OmpF. In particular, a decrease in pH of the bathing solution resulted in a substantial reduction of YOmpF single channel conductance, accompanied by the emergence of subconductance states. Similar subconductance substates were elicited by the addition of lysophosphatidylcholine. This observation, made with porin channels for the first time, pointed to the relevance of lipid-protein interactions, in particular, the lipid curvature stress, to the appearance of subconductance states at acidic pH. Binding of YOmpF to membranes displayed rather modest dependence on pH, whereas the channel-forming potency of the protein tremendously decreased upon acidification.

The application of mass spectrometry towards the structural analysis of the most interesting sulfated biopolymers of the brown algae-fucoidans only developed relatively recently. During method development, many problems, both chemical and instrumental, have to be solved. For example, mass spectrometry has a limitation in the analysis of anionic high molecular weight (HMW) polysaccharides because of the labile nature of sulfate groups which cause the polysaccharide to desulfate rather than ionize. Thus, decomposition methods should be developed taking into account the structural features of such a complex and fragile compound. The selection of optimal instrument settings for the electrospray ionization mass spectrometry (ESIMS) and of matrix media for matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS) is also required. When optimal parameters for mass spectrometric analyses are found, the application of these methods to the elucidation of structural features of fucoidans (by studying their fragments) allows researchers to rapidly obtain new and unique data, often impossible to achieve by other techniques. Herein, we describe tandem mass spectrometry of sulfated fucooligosaccharides, obtained by an autohydrolysis technique from structurally different fucoidans.

The interaction of endotoxin (lipopolysaccharide – LPS) with low molecular weight chitosan (5.5 kDa), its N-acylated derivative and chitoliposomes were studied using a gravimetric piezoelectric quartz crystal microbalance biosensor. The optimal conditions for the formation of a biolayer based on immobilized LPS on the resonator surface and its regeneration were elaborated. The association and dissociation rate constants for LPS binding to chitosans were determined and the affinity constants (Kaf) were calculated based on the data on changes in the oscillation frequency of the quartz crystal resonator. The Kaf values correlated with the ones obtained using other methods. The affinity of N-acylated chitosan binding to LPS was higher than that of the parent chitosan binding to LPS. Based on the results obtained, we suggest that water-soluble N-acylated derivatives of chitosan with low degree of substitution of amino groups could be useful compounds for endotoxin binding and neutralization.