**2. Results**

#### *2.1. Characterisation of the Initial Polysaccharides*

For the study of the anti-inflammatory activity of the polysaccharides, κ-CGN, CH and soluble forms of their PEC were prepared. κ-CGN from red algae *Chondrus armatus* was obtained as described earlier, it structure was determined according to [30]. The molecular weight (MW) of κ-CGN was determined by viscometry and was found to be 250 kDa.

CH was obtained by the alkaline deacetylation of crab chitin as described in [31]. The MW determined by viscometry was 110 kDa, and the deacetylation degree (DA), determined according [32] by FTIR-spectroscopy was 96%.

#### *2.2. Characterization of PEC* κ*-CGN-CH*

Activity of biopolymers is determined by numerous parameters, among which are the size of the molecule, its charge, density, solubility, the molecule conformation and flexibility of polysaccharide chain. The formation of the complex affects all these parameters; it is obvious that the activity of the complexes will differ from that of the original compounds.

The process of obtaining complexes of CH with CGN in soluble form was studied by us in detail earlier. It was shown that soluble complexes were obtained preferentially by mixing the starting components at given ratios with an excess of one of the components [33]. The complex formation of κ-CGN:CH 1:10 and 10:1 *w*/*w* was shown by centrifugation in a Percoll gradient [34].

In this study the soluble PEC with a κ-CGN:CH weight ratios of 10:1 and 1:10 were prepared by mixing solutions of the initial polysaccharides and characterized by dynamic light scattering (Figure 1).

The initial CH and κ-CGN presented as polydisperse particles, heterogeneous in size, with Z-average of about 1 μm (data not shown), which corresponds to their polysaccharide nature [35]. The nature of polysaccharides creates certain difficulties for their study by hydrodynamic methods. The mutual repulsion of charged groups along the polymer chain is the reason why the molecule becomes strongly extended, while its hydrophilic part is in the hydrate shell. This makes it difficult to study by the DLS (dynamic light scattering) and we could not obtain a size distribution for CGN and CH with an acceptable value of polydispersity index PdI. The surface potential of CH was +26.2 ± 2.3 mV, while that of κ-CGN was −50.7 ± 1.4 mV.

After complex formation, the polydispersity of the system was reduced significantly. The complex with an excess of CH (κ-CGN-CH 1:10 *w*/*w*) was fairly composed of homogeneous, positively charged particles with an average diameter of about 123 nm. Negatively charged particles of the complex with an excess of κ-CGN (κ-CGN-CH 10:1 *w*/*w*) was more polydisperse in size with an average diameter of about 325 nm.


**Figure 1.** Intensity particle size distribution of κ-CGN-CH 10:1 *w*/*w* (black line) and κ-CGN-CH 1:10 *w*/*w* (grey line) complexes measured on a Nano ZS (Malvern PANalytical, Malvern, UK) using scattering detection at 173◦. The hydrodynamic diameters of the particles were automatically calculated with the instrument's software based on analysis of the autocorrelation function.

The charge of the PEC was determined by the polymer, that was in excess. The charge of CGN in the complex with its excess (κ-CGN-CH 10: 1 *w*/*w*) was partially neutralized, but the charge of the complex with an excess of CH (κ-CGN-CH 1:10 *w*/*w*) corresponded to the charge of the initial polycation, which may indicate surface localization of CH in the complex, shown by us earlier [29].

#### *2.3. In Vitro Activity of the Initial Polysaccharides and Their Complexes*

In biomedicine, much attention has been paid to natural antioxidants and their association with health benefits [36]. Taking into account the complexity of antioxidants action in vivo, different in vitro methodologies have been developed to estimate, in a simple experimental way, the capacity of antioxidants and their complex mixtures to neutralize the reactive oxygen and nitrogen species (ROS/RNS) [37].

We considered the potential antioxidant activity of κ-CGN, CH, and PEC determined by their ability to bind nitric oxide (NO) (Figure 2).

**Figure 2.** Nitric oxide scavenging effect of κ-carrageenan (κ-CGN), chitosan (CH) and their complexes. Statistical analysis was done by one-way ANOVA (analysis of variance). \*—Differences between samples and the control were significant, *p* < 0.05; #—Differences between polyelectrolyte complexes (PEC) and κ-CGR were significant, *p* < 0.05; +—Differences between PEC and CH were significant, *p* < 0.05.

According to Figure 2, κ-CGN showed a pronounced ability to scavenge NO, and its activity (at 1 mg/mL, 0.5 mg/mL) was comparable to the action of the standard (ascorbic acid). CH did not show the ability to scavenge NO (Figure 2). The low activity of this polysaccharide in this test was noted earlier [38]. It was previously reported that with an increase in the MW of chitosan up to 100 kDa, its antioxidant activity have decreased [39]. Together with this, the CH used here had a MW of 110 kDa. The PEC with a high content of κ-CGN exhibited activity comparable to the activity of the initial κ-CGN. Unlike the initial polycation, the complex with a high CH content (κ-CGN-CH 1:10 *w*/*w*) exhibited antioxidant activity, although it was less pronounced than κ-CGN and the complex κ-CGN-CH 10:1 *<sup>w</sup>*/*<sup>w</sup>*.

#### *2.4. Ex Vivo Activity of the Initial Polysaccharides and Their Complexes*

The ability of polysaccharides and their complexes to induce the synthesis of pro-inflammatory cytokines, i.e., tumor necrosis factor-α (TNF-α), and anti-inflammatory cytokine, i.e., interleukine-10 (IL-10), in peripheral blood mononuclear cell (PBMCs) was studied. Both of these cytokines play important roles in the immune response, as they activate circulating cells and stimulate the production of chemokines and adhesion molecules [40]. Figure 3 shows that the ability of the polysaccharides and their PEC to activate cells and to induce cytokine synthesis has been correlated to the activity of LPS. At a high concentration (10 μg/mL) κ-CGN caused a strong increase in the level of cytokines, in comparison with the control, while at a low concentration (1–10 ng/mL) the activity was insignificant. CH was inert in this test.

The CGN and PEC did not show an anti-inflammatory activity in the presence of LPS (Figure 3c,d). Only chitosan at a concentration of 100 ng/mL had the ability to suppress of LPS-induced TNF-α and IL-10 production.

According to Figure 3, the ability of PEC to induce the synthesis of pro-inflammatory (TNF-α) and anti-inflammatory (IL-10) cytokines in the whole blood cell assay in both conditions (with and without LPS) is determined as the activity of the initial κ-CGN, regardless of their composition. The effect of the complex with an excess of CGN was similar to that the initial κ-GRG. The activity of the complex with an excess of CH was slightly lower than that of the initial κ-GRG, that possibly due to the presence of CH.

**Figure 3.** (**a**) Tumor necrosis factor-α (TNF-α) and (**b**) interleukine-10 (IL-10) level stimulated by κ-CGR, CH and complexes. TNF-α (**c**) and IL-10 (**d**) level stimulated by preliminary incubation of peripheral blood mononuclear cell (PBMCs) with *Escherichia coli* LPS (10 min), then by κ-CGN, CH, and complexes. Contents of a cytokine in serum are presented as a mean ± SD. Whole blood samples were obtained from 5 healthy subjects and incubated with the polysaccharides and PEC at different concentrations. The cytokine level in serum of normal donors (**<sup>a</sup>**,**b**) or the cytokine level in serum after preliminary incubation of PMBC with *E. coli* LPS (**<sup>c</sup>**,**d**) were considered as a negative control used for statistical calculation. Statistical analysis was done using one-way ANOVA. \*—Differences between samples and the control were significant, *p* < 0.05; #—Differences between PEC and κ-CGR were significant, *p* < 0.05; +—Differences between PEC and CH were significant, *p* < 0.05.

#### *2.5. In Vivo Anti-Inflammatory Activity of the Initial Polysaccharides and Their Complexes*

The anti-inflammatory activity of PEC and the initial compounds was evaluated using histamine-, concanavalin A- and sheep erythrocyte immunization-induced paw edema in mice. The results are expressed as the inflammatory edema index (IEI) and percentage of anti-inflammatory activity (AIA) relative to the control group (Table 1).

In experiments in vivo, polysaccharides and complexes were administered intraperitoneally in the form of aqueous solutions to provide more efficient delivery of compounds. The doses of the complexes were selected taking into account their low toxicity and the ability to dissolve in the volume of the solvent (water) allowed for intraperitoneal administration to mice (0.2 mL/10 g of body weight). Doses of polysaccharides were taken in accordance with their maximum proportion in the composition of the complexes.

The data indicate that preventive i.p. injections of PEC and both polysaccharides significantly decreased local inflammation induced by histamine in mice. CH and PEC κ-CGN:CH 1:10 demonstrated the most potent decrease IEI values relative to the control group (1.8 and 1.7 times, correspondingly), which are insignificantly different from that of indomethacin (2.3 times) (Table 1). κ-CGN alone and PEC κ-CGN:CH 10:1 were less active in decreasing edema (1.4 and 1.3 times, correspondingly).


**Table 1.** Anti-inflammatory activity of κ-carrageenan (κ-CGN), chitosan (CH), and their polyelectrolyte complexes (PECs) in mouse paw edema tests.

\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 differences with control group are significant; # *p* < 0.05, ## *p* < 0.001 differences with reference group are significant; IEI—inflammatory edema index, AIA—anti-inflammatory activity. Data represent as mean ± standard error (SEM); *n* = 8 mice in group. Statistical analyses were performed using "Statistica 6" software. The significant difference between groups was calculated by one-way ANOVA by Fisher test (at parametric distribution) or not—parametric Kruskal–Wallis test. A *p*-value of ≤0.05 was considered as statistically significant.

The results presented in Table 1 show a downward trend in activity of tested agents with higher level of the κ-CGN. It correlates with the data from the ex vivo experiments, indicating an increase in TNF-α production at a high dose of κ-CGN. TNF-α is a pro-inflammatory cytokine secreted by activated macrophages in an early stage of acute inflammation. The low level of TNF-α indicates attenuation of the inflammatory reaction in response to a low dose of κ-CGN.

In the concanavalin A inflammation test, κ-CGN and CH showed a moderate anti-inflammatory effect (lowering the edema index by 1.9 and 1.7 times, respectively, versus control) in comparison with diclofenac (2.6 times), while the complexes were not active (Table 1). In the DTH reaction, only κ-CGN alone inhibits inflammation. Both PECs and CH were inactive (Table 1).
