**1. Introduction**

Natural polysaccharides are promising compounds for use in biomedicine and pharmaceuticals. The main prerequisites for this are the combination of their abundance and simplicity of preparation with biocompatibility and a broad spectrum of biological e ffects, such as immunostimulating, antioxidant, antitumor, antimicrobial, and antiviral.

One of the most well-known polysaccharides of red algae is carrageenan (CGN). CGN is a sulfated galactose copolymer composed of alternating units of D-galactose and 3,6-anhydro-galactose joined by α-1,3 and β-1,4-glycosidic linkages [1]. CGN is classified into various types such as λ, κ, ι, ε, μ, depending on the amount and location of sulfate groups as well as the presence or absence of 3,6-anhydro-galactose units [2]. CGNs have diverse activities including immunomodulatory [3], anticoagulant [4], antithrombotic [5], antiviral [6], and antitumor e ffects [7]. In recent years, CGNs have been increasingly used for pharmaceutical purposes. CGNs are one source of soluble dietary fibers [8]. Standard animal safety studies in which CGN was administered in diet showed no adverse effects [9]. Due to their biocompatibility, safety (USP35-NF30S1, BP2012, EP7.0), availability, wide range of biological activity, a simple thermo-reversible gelation mechanism, viscoelastic properties and the

ability to form complexes with polycations via electrostatic interactions, CGNs are ideal components to obtain new vehicles for the delivery therapeutic substances that can be retained at mucosal surfaces and release the drug slowly [10].

Chitosan (CH) is a natural polycation, β-1,4-linked glucosaminoglycan, which have some acetylated amino groups. CH is a non-toxic, biodegradable and non-immunogenic agen<sup>t</sup> [11] used widely as a biomaterial with an established safety profile in humans, as pharmaceutical excipient, weight loss supplement [11], and as a major component of hemostatic dressings [12]. At relatively low pH (<6.5), chitosan is positively charged and soluble in dilute aqueous solutions [13] CGN.

Marine polysaccharides stimulate di fferent types of immune system cells, both in vitro and in vivo, to produce and secrete molecules with immunostimulatory e ffects [14,15]. CGNs have been demonstrated to play an important role as free-radical scavengers and antioxidants for the prevention of oxidative damage in living organisms [16]. Our studies on the antioxidant capacity of CGN [17] have shown that the activity of carrageenans depends on the polysaccharide structure. Hence, they have therapeutic potential for the treatment of immunological disorders.

Inflammation is the first biological response of the immune system to infection or irritation. However, in some cases, it can become chronic, and lead to tissue damage [18]. Non-steroidal anti-inflammatories drugs are often used to treat of inflammation. However, their prolonged usage is followed by complications, including damage to the gastrointestinal tract and other side e ffects [19]. Therefore, the search for new bioactive compounds with anti-inflammatory activities with minimal adverse e ffects is of grea<sup>t</sup> importance. CGN. We have shown that chitosan with molecular weight of 110 kDa ex vivo inhibits the synthesis of anti-inflammatory cytokine, the tumor necrosis factor alpha (TNFα) induced by endotoxin and stimulates synthesis of the anti-inflammatory cytokine interleukene (IL-10) by oral administration in the blood serum of mice [20]. We have also determined that κ-CGN [21] and chitosan [20] possess anti-inflammatory activity in a model of acetic acid induced colitis in mice. Recently, we have shown the inhibitory e ffect of CGNs on inflammation caused by endotoxin [22].

The mechanism of action of CGNs is not ye<sup>t</sup> completely understood; however, dietary studies done in animals with food-grade CGN shown no intestinal inflammation [23]. There are some conflicting reports on the e ffects of CGN on the gastrointestinal tract what, may be in part due to the fact that a food-grade CGN is often confused with a degraded carrageenan, termed poligeenan [24]. In contrast to CGN, poligeenan is not produced biologically but only in the laboratory or commercially by subjecting CGN to very low pH at 0.9–1.3 and non-physiological temperatures of >80 ◦C for several hours [24]. High molecular weight or food-grade CGN is ingested at low dosages; is not absorbed across the intestinal epithelium, is wholly excreted in feces, does not enter the systemic circulation, and does not cause intestinal ulceration or inflammation. The safety of CGN is supported by a large number of animal oral safety studies in which no adverse e ffects were reported at high doses (up to 5% in diet) [9].

The obtaining of polyelectrolyte complexes (PEC) by binding two opposite charged polymers is the way to improve and expand the range of properties of polyionic polysaccharides. This allows the modification of their functional properties to improve stability and facilitate application. For example, stable forms of PEC CH-CGN can be obtained in soluble form, in the form of gels, films, sponges, and microparticles. PEC are promising materials for use in biomedicine and pharmaceuticals, since the combination of the favorable properties of each constituent polymer leads to new systems with improved properties, which quite often substantially di ffer from those of the individual polymers [25]. These properties can be modulated by adjusting the conditions of complex formation, i.e., varying pH, ionic strength, polysaccharide concentration, ratio of the biopolymers, and temperature [26]. In addition, PEC can easily adapt to the requirements dictated by specific applications, for example, by changing the composition of the monomer, the density of the formed bonds, particle size, surface charge density, or type of solvent [27]. We can expect synergies between the components of the complex, the predominance of the activity of one of the components, or the loss of one or another activity of the

starting polyions. Although the biological activity of polysaccharides has been actively studied, there is very little data on the physiological effects of complexes based on them.

We previously studied the conditions for the formation of a soluble form of PEC and found that the complexes have gastroprotective [28] and antibacterial [29] activity. The activity of the complexes in these tests exceeded the activity of the starting components [28].

The aim of this work was to study the anti-inflammatory activity of PEC, consisting of various compositions of κ-CGN and CH, in comparison with the initial polysaccharides taken in comparable doses. Tests of acute inflammation in mouse paw associated with the induction of a predominantly cellular immune response mediated by phagocytes (histamine test), B-lymphocytes (concanavalin A test) or T-lymphocytes (HRT reaction) were used. In ex vivo experiments, the effect of agents on the cytokine profile of donated blood was also evaluated.
