**3. Discussion**

Polysaccharide PEC are well-tolerated macroorganism systems and can be used in various fields, such as drug delivery systems, cell cultivation and enzyme immobilization, or tissue reparation and regeneration [41]. However, despite the numerous studies devoted to the preparation and application of PEC, their biological activity and the modification of the activity of the initial polysaccharides after PEC formation have been studied insufficiently.

CH and CGN have pronounced immunostimulatory properties [1,20,34–36]. Therefore, this study was aimed at the comparative analysis of the effects of PEC and initial compounds on some components of the immune system associated with immunomodulating activity.

Immunity is the host physiological defense of an organism against pathogenic factors such as toxins, infectious microorganisms and viruses. The immune response passes on the collective and coordinated cooperation of specific cells and humoral mediators including vascular and cell adhesion molecules and proteins of the complement system. The most relevant cells involved in the immune response are lymphocytes, monocytes/macrophages, polymorphonuclear granulocytes, and mast cells. Macrophages and antigen-presenting cells (APC) are involved in all stages of non-specific immune responses and are responsible for phagocytosis, antigen processing and presentation, secretion of NO and cytokines such as and TNF-<sup>α</sup>, as well as antibody-dependent and cell-mediated cytotoxicity [42]. The effect of complexation on some cellular immunity reactions mediated by B- and T-lymphocytes in vitro, ex vivo and in vivo models have been considered.

In this study a comparative analysis of the immunotropic activity of polysaccharides and their complexes was carried out. The soluble complexes obtained by mixing the starting components at given ratios of κ-CGN-CH 1:10 *w*/*w* or κ-CGN-CH 10:1 *<sup>w</sup>*/*<sup>w</sup>*. The complexes formation was accompanied changes in the physicochemical parameters of the initial polysaccharides. After complex formation, the polydispersity of the initial polysaccharides was reduced significantly (Figure 1). According to our earlier data [15] the macromolecular structure of PEC was different from the structures of the initial components in the appropriate concentrations. The mechanism of PEC complex particle formation is determined by the nature of the prevailing polymer. The particles in the soluble complexes κ-CGN-CH 1:10 *w*/*w* has surface localization of the polycation and stabilized by unreacted CH amine groups. The incorporation of CH into the network structure of CG for complexes κ-CGN-CH 10:1 *w*/*w* was assumed [15].

NO is a highly reactive free radical involved in a number of physiological and pathological processes [43]. NO acts immunologically as a cytotoxic agen<sup>t</sup> on invading microorganisms in macrophages or on tumor cells [44]. Our studies on the antioxidant capacity of CGN [17] and studies on other polyionic polysaccharides have shown that antioxidant activity is determined by the location of ionic groups in the carbohydrate chain and by the molecular weight of this chain [38,45]. According to our results, the activity of PEC to scavenge of NO depends on the content of κ-CGN in the complex: PECs with a high content of κ-CGN exhibited activity comparable to the activity of the κ-CGN alone.

Sulfated polysaccharides are commonly reported as strong antioxidants, which is partly due to its ordered, extended structure. Sulfated polysaccharides usually trap free radicals in an electrostatic manner since the sulfate groups usually generate a highly acidic environment and the sulfur substitution may also weaken hydrogen bond interactions between polysaccharides [46]. This explains the high activity of the initial κ-CGN and the PEC κ-CGN:CH 10:1 *<sup>w</sup>*/*<sup>w</sup>*. It is noteworthy that the complex with a high content of CH, which, according to our early data [29], had surface localization of the polycation, also showed a low ability to scavenge NO, unlike the initial CH.

As already described in the literature, polysaccharides have an important immunostimulatory capacity through the production of cytokines [8,9,16]. The data presented in this study clearly demonstrate that κ-CGN and PEC at a low concentration (1 ng/mL) do not induce the pro-inflammatory TNF-<sup>α</sup>, which is an important mediator for the induction of many biological responses [47]. The initial CH did not have the ability to induce cytokine synthesis, but the PEC κ-CGN:CH 1:10 *w*/*w* was able to induce the synthesis of TNF-α and IL-10 in cells, although to a lesser extent than the original κ-CGN. Du et al. [48] suggested that the solubility and assembly of the components influence the immuno-pharmacological activities of glucans. Wang et al. [49] postulated that the relatively high bioactivity of polysaccharides can be attributed to good water solubility and expanded chain conformation. So, it can be assumed, that κ-CGN-CH 1:10 *w*/*w* PEC, with a small particle size and low polydispersity detected by dynamic light scattering (Figure 1) can facilitate contact with the cell compared to the initial more heterogeneous and tightly packed CH.

The results of this investigation indicate that PEC has anti-inflammatory activity against phagocytes, activated by histamine (Table 1). Histamine is the main mediator of the initial phase of acute inflammation. Intra-plantar injection of histamine induces the activation of resident macrophages and mast cells in the tissue of the mouse paw. The pro-inflammatory effects of histamine are also achieved through interactions with its receptors on the endothelium and blood leukocytes. Binding H1 receptors causes an increase in vascular permeability, as well as the formation of prostaglandin E2 (PGE2), leukotrienes and chemokines, which attract eosinophils and neutrophils to the site of inflammation [50]. Preventive i.p. administration of CH, κ-CGN and their water-soluble PEC of the 1:10 and 10:1 *w*/*w* compositions significantly delayed the development of acute local inflammation. Under these conditions, their activity was comparable to non-steroidal anti-inflammatory drugs, the effect of which is based on the inhibition of cyclooxygenase 1 and 2 on the membranes of endothelial and blood cells, followed by impaired synthesis of prostaglandins E2 and F2α, thromboxane A2, prostacyclin, leukotrienes, and the release of lysosomal enzymes. It can be assumed that polysaccharides use a similar mechanism as a result of direct contact with the membranes of effector cells. This is confirmed by our recent data on the ability of CGN to inhibit of PGE2 synthesis at low concentrations [51]. On the other hand, the scavenger abilities of κ-CGN and its water-soluble PEC could attenuate damage associated with highly active NO-species, which are produced by phagocytes, as shown in ex vivo experiments described here (Figure 2).

The inflammatory response induced by concanavalin A is an example of type I or immediate hypersensitivity, which is due to IgE antibody production and the development allergic sensitization. This type of inflammation is associated with the activation of Th2 cells and adaptive immunity. The polyvalent lectin concanavalin A stimulates the release of histamine and leukotrienes from mast cells and basophils, activates neutrophils, increases the activity of cyclooxygenase-2 (COG-2) and stimulates the proliferation and differentiation of Th2 lymphocytes. Lectin-activated cells secrete various inflammatory mediators, quickly causing severe edema and inflammation in the form of an allergic-like reaction, but without contact with a native allergen [52–54]. Intraperitoneal administration of CH and κ-CGN reduced mast cell degranulation and the subsequent development of inflammation. However, the PEC of the 1:10 and 10:1 *w*/*w* compositions had no anti-inflammatory activity. It is suggested that free CH and κ-CGN could occupy the membrane surface of mast cells and interrupt the binding of lectin molecules, or could block the transfer of pro-inflammatory signals from primary activated mast cells to the membrane receptors of effector cells (macrophages, neutrophils, or lymphocytes), thereby inhibiting downstream cytokine secretion and inflammation.

A similar effect was found with the DTH test which is based on T-cell immunity. In this case, T-lymphocytes, preliminary primed with antigen, stimulate the blast transformation and proliferation of cytotoxic T-lymphocytes. The secreted pro-inflammatory cytokines secreted by them stimulate the activity of macrophages and cytotoxic lymphocytes, which induce an inflammatory response and the development of a granuloma in the mouse paw [42]. Among the tested samples only κ-CGN alone decreased the DTH reaction, while the others did not (Table 1).

The particle surface charge could potentially control biopolymers binding to tissue both in vivo and in vitro. Cellular surfaces are dominated by negatively charged sulfated proteoglycans, molecules that play pivotal roles in cellular proliferation, migration, and motility [55], therefore, interactions between proteoglycans and positively charged particles are preferred [56]. At the same time negatively charged nanoparticles (NP) show highly increased bioadhesive properties and are absorbed by both M cells and absorptive enterocytes [57]. Jung et al. 57] has also shown that charges on the NP surface are not the only requirement, a combination of both NP surface charges and increased hydrophilicity of the matrix material seem to affect the gastrointestinal uptake in a positive sense. Unfortunately, to date, information on the correlation of activity depending on the charge of natural polysaccharides is very limited. We earlier have shown [29], that CH, CGN and their positively and negatively charged complexes had the different ability to suppress the biofilm formation by Gram-negative and Gram-positive microorganisms. CH and positively charged complexes inhibited only biofilm formation by Gram positive microorganisms (*B. subtilis*). CGN and negatively charged PEC prevented the formation of biofilm by Gram negative microorganisms (*E. coli*) and Gram positive microorganisms.

It could be expected that the activity of the studied PEC would be determined by the prevailing polysaccharide. However, only in the in vitro test, we observed some dependence of the ability to scavenge NO on the polysaccharide content in the PEC. In ex vivo test which is more complex systems, including not only cells but also different soluble serum components, the direct dependency of the activity of the PEC on the polysaccharide contents was not observed. It is much more difficult to carry out any correlations between the activity and surface characteristics of the objects in in vivo experiments. The overall protein concentrations in typical body fluids (e.g., blood, lung, gut) and intracellular environments can be as high as 0.35 gmL−<sup>1</sup> [58]. These fluids may contain more than 3000 different proteins at widely differing concentrations. It was shown that nanomaterials adsorb biomolecules on contact with biological fluids [59]. Thus the components of the biological fluids can affect the manifestation of a biological activity of studied compounds and an activity determined in experiments in vitro and in vivo can manifest itself in different ways.

A comparison of in vivo test results suggests that the immunomodulatory effect of polysaccharides and the PEC was directly related to their ability to bind to the membrane receptors of immune cells and to influence signal transmission between components of the inflammatory process. The greatest activity of PEC, as well as the initial polysaccharides κ-CGN and CH, was observed in acute exudative

inflammation, directly related to the activation of phagocytic cells, i.e., macrophages and neutrophils. The macrophage surface bears pattern recognition receptors (PRRs), which can recognize and bind the molecules of bacterial polysaccharides [42]. Moreover several members of the TLR (toll-like receptor) family interact with exogenous peptidoglycans and polysaccharides to induce adaptive immunity and modulate immune response [60]. However, in tests mediated by B- and T-lymphocyte stimulation, PEC did not show activity, unlike the free polysaccharides. Thus, the complexation of the CH and κ-CGN led to a decrease in their immunological activity in terms of B- and T-cell immunity. It is assumed that this is due to specific physicochemical limitations that impede PEC interactions with membrane receptors on lymphocytes.

#### **4. Materials and Methods**
