Selenium-Containing Polysaccharides—Structural Diversity, Biosynthesis, Chemical Modiﬁcations and Biological Activity

: Selenosugars are a group of sugar derivatives of great structural diversity (e.g., molar masses, selenium oxidation state, and selenium binding), obtained as a result of biosynthesis, chemical modiﬁcation of natural compounds, or chemical synthesis. Seleno-monosaccharides and disaccharides are known to be non-toxic products of the natural metabolism of selenium compounds in mammals. In the case of the selenium-containing polysaccharides of natural origin, their formation is also postulated as a form of detoxiﬁcation of excess selenium in microorganisms, mushroom, and plants. The valency of selenium in selenium-containing polysaccharides can be: 0 (encapsulated nano-selenium), IV (selenites of polysaccharides), or II (selenoglycosides or selenium built into the sugar ring to replace oxygen). The great interest in Se-polysaccharides results from the expected synergy between selenium and polysaccharides. Several plant- and mushroom-derived polysaccharides are potent macromolecules with antitumor, immunomodulatory, antioxidant, and other biological properties. Selenium, a trace element of fundamental importance to human health, has been shown to possess several analogous functions. The mechanism by which selenium exerts anticancer and immunomodulatory activity differs from that of polysaccharide fractions, but a similar pharmacological effect suggests a possible synergy of these two agents. Various functions of Se-polysaccharides have been explored, including antitumor, immune-enhancement, antioxidant, antidiabetic, anti-inﬂammatory, hepatoprotective, and neuroprotective activities. Due to being non-toxic or much less toxic than inorganic selenium compounds, Se-polysaccharides are potential dietary supplements that could be used, e.g., in chemoprevention.


Introduction
Polysaccharides are macromolecules that are extremely interesting due to their remarkable structural diversity and, consequently, functional versality. Their structure is much more complex than that of other natural macromolecules, proteins, and nucleic acids. Polysaccharides are composed of more than 10 monomers-monosaccharides; their number ranges from 11 up to several thousands. Polysaccharides may consist of a single type of monomer (homoglycans) or of different monomers (heteroglycans), and each unit can be linked to others in multiple ways. Consequently, they can form straight or branched chains, create cyclic forms, contain a protein component, lipid, etc. It can be therefore concluded that there is an infinite number of combinations of polysaccharide structures [1,2].
The result of the structural diversity of polysaccharides is their functional versatility. There are great differences in physical and chemical properties of polysaccharides, including solubility; viscosity; swelling capacity; resistance to acids, bases, and enzymes; and chemical character (acidic, basic, neutral) [1], which also contribute to their versality.
The modification of the polysaccharides' structure carried out by biotechnological or chemical methods may cause a significant change in their biological activity [6][7][8][9]. For example, incorporation of selenium (Se) in the polysaccharide molecules is one of the currently explored methods of modifying the structure and activity of these compounds [10][11][12][13].
Selenium is one of the trace elements that are of fundamental importance to human health, being part of the antioxidant enzymes that protect cells against the effects of free radicals [14][15][16][17][18]. It plays an important role in a number of biochemical functions in humans and animals, such as antioxidant, immune function, reproduction, and thyroid hormone metabolism. Selenium appears to be a key nutrient in cancer prevention and inhibition of HIV progression to AIDS [19][20][21][22][23][24][25]. The important health effect of selenium is related to its impact on the immune system. Numerous reports on the immunomodulatory and antiviral activity of preparations containing selenium show that supplementation with this micronutrient may be beneficial for both prevention and treatment of viral infections, including coronaviruses [26,27]. The mechanism by which selenium exerts antioxidant, anticancer, and immunomodulating activity differs from that of polysaccharides, but a similar pharmacological effect suggests a possible synergy of these two agents. This has led to a growing research interest in Se-polysaccharides in recent years, particularly the methods of incorporating selenium into the carbohydrate structure and the structureactivity relationship of these compounds.
The approach to the problem of the Se-polysaccharides presented in the current review is based precisely on the analysis of the structure of these compounds and related changes in biological activity. To our knowledge, an extensive analysis of the chemical structures of seleno-carbohydrates (with a broader analysis of possible ways of incorporating selenium into the sugar structure), and the reference of these data to the currently known selenopolysaccharides, has not yet been presented.

Biologically Active Polysaccharides-Structure and Function
In the recent decades, natural polysaccharides isolated from bacteria, algae, fungi, and higher plants have attracted attention in the fields of nutrition and medicine [28]. The reasons include their broad spectrum of biological and pharmacological activity combined with low toxicity and rare negative side effects [29]. Many of them have been used for hundreds of years in traditional medicine. Further, some of the natural polysaccharides are already used as auxiliary substances, drug carriers, blood substitutes, and pharmacologically active compounds [30][31][32].
The polysaccharides showing immunomodulatory and anti-tumor properties are of particular interest [4]. Biological activity of polysaccharides results from direct inhibition of the neoplastic cells survival (cytotoxic activity) and from indirect mechanisms involving the activation of the immune system (immunostimulating activity). Detailed characterization of the structure of polysaccharides showing immunomodulatory effect is challenging; however, the most frequently mentioned parameters include molecular weight, monosaccharide composition, type of glycosidic bond, degree of cross-linking, conformation of the molecule, and modification in the structure of the molecule (carboxymethylation, sulfonation, aminopropylation, hydroxyethylation, phosphorylation, element incorporation into the molecule) [13,[33][34][35][36][37]. The higher the molecular weight of a polysaccharide, the greater its chance of effective binding to a receptor or binding protein, which triggers the immune response or tumor cell apoptosis [38]. According to the literature data, the polymers of glucose and mannose show the highest immunomodulatory activity [39], which can bind to the receptors specific to these polysaccharides [40].
Additionally, polysaccharides with a predominance of β-(1-6) glycosidic bonds show weaker activity than those with predominant β-(1-3) bonds in the main chain [41,42]. Both too low and too high a degree of branching of the molecule results in a decrease of the activity of the polysaccharide [43]. A greater number of side chains are associated with a tighter conformation of the helix, which makes polysaccharides difficult for the receptors to recognize [44]. Moreover, the degree of branching of the most biologically active β-(1-3)-Appl. Sci. 2021, 11, 3717 3 of 40 glucans is 0.20-0.33 [45]. Furthermore, the conformation of polysaccharides is related to the spatial arrangement of the atoms that determine the shape of the chain. The triple-helix conformation provides strong immunostimulatory effects of β-D-glucans by promoting the release of TNF-α by macrophage cells [46]. It must be admitted that the structure-activity relationships proposed by the above-cited authors work well only for a fairly homogeneous group of β-D-glucans, but not for all currently known immunoactive polysaccharides.
The section below describes the biological activity of the polysaccharides isolated from various natural sources.

Bacterial Polysaccharides
Bacteria have the ability to synthesize biopolymers, which are divided into intracellular polysaccharides (CPS) and extracellular polymeric substances (EPS) according to their localization and function in the cell [47]. The main function of CPS is the structural support of the bacterial cell wall, but EPS have strong antioxidant properties and application in cancer therapy, antimicrobial and antiviral activities, and antioxidant and cholesterol-lowering effects [48]. K5PS is a heparin-like envelope polysaccharide of Escherichia coli that inhibits the metastasis of human breast cancer and murine melanoma cells [49].
The source of bioactive polymers showing antiproliferation activity is Paenibacillus polymyxa rods, which secrete polysaccharides of the levan-type, consisting mainly of β-D-fructofuranosyl residues connected by 2,6-bonds with 2,1-branches [50]. Moreover, Rhizobium papillary bacteria contain polysaccharides with the structure of a-glucans and a skeleton composed of glucose molecules linked by 1,4-bonds with 1,6-branches, which inhibit the formation of neoplasms in mice [51].
In recent years, there has been an increased interest in curdlan, insoluble in water linear beta-1,3-glucan, a high-molecular-weight polymer of glucose, obtained for the first time from Alcaligenes faecalis [52]. Curdlan has anti-cancer and anti-inflammatory properties and also supports wound healing [53]; its reaction mechanism is based on the interaction with the dectin-1 receptor present in dendric cells, macrophages, and monocytes. Further, it stimulates the immune system and enhances the phagocytosis process and production of TNF-α, IL-6, and ROS [54]. In addition, symbiotic polysaccharides of Bacerioides fragilis, presented in intestinal dendritic cells, activate CD4 T cells and stimulate the production of cytokines [55], while the polysaccharide from Paenibacillus polymyxa has an anti-proliferative effect, and its derivatives formed as a result of acetylation show even stronger biological activity [28].
A commercially used biopolymer is dextran, a high molecular weight water-soluble glucose polymer released by Leuconostoc mesenteroides. Dextran is used as a drug carrier to inhibit cancer cells and reduce the toxic effects of drugs [56].
Many cyanobacteria are able to release easily recoverable polysaccharides into the culture medium. Most cyanobacterial polysaccharides show an anionic nature, due to the presence of uronic acids and other charged groups such as pyruvyl or sulfate [57]. Numerous publications describe exopolysaccharides produced by cyanobacteria Spirulina platensis, considered by some researchers to be microalgae. Polysaccharides extracted from of S. platensis are water-soluble; it has been proven that they have antitumor, antioxidation, antiaging, and antivirus properties [58]. Rajasekar et al. investigated the structural characterization and bioactive potential of sulfated polysaccharides from S. platensis and isolated a fraction containing glucose, rhamnose, xylose, fucose, mannose, and galactose as main sugars with Mw of 1016 kDa [59]. every fourth glucose unit [103,104]. Another source of therapeutic (immunomodulatory and antidiabetic) polysaccharides is the edible medicinal Tremmella mushrooms. Tremella polysaccharides are composed of a linear backbone of α-(1,3)-D-rhamnose, with side groups of xylose and glucuronic acid. These polysaccharides are acidic, with a pH of 5.1-5.6 in aqueous solutions [105][106][107]. Extracellular acidic heteroglycans from filtrates of Tremella species, in turn, are characterized by an α-(1-3)-mannan backbone with β-linked side chains. They contain xylose, arabinose, mannose, galactose, glucose, and glucuronic acid residues [106].
A list of so-called "medicinal mushrooms" capable of biosynthesis biologically active polysaccharides is long, and includes nearly 270 species [96]; therefore, it is impossible to list all types of active polysaccharides of fungal origin in a short paragraph in this review.
Numerous natural polysaccharides found in herbs, often components of the plant cell wall, are known to stimulate the human immune system. The activation of macrophages by plant polysaccharides results from recognition of polysaccharide by TLR4 (toll-like receptors), CR3, and scavenger receptors. The studies on the polysaccharides from Aloe barbadenis showed that acetylated mannan isolated from aloe has the ability to increase the production of monocytes [110]. In addition, Astragalus membranaceus polysaccharides (α-(1-4)-D-Glucans with α-(1-6)-branches) have the ability to inhibit the proliferation of tumor cells by regulating p53 expression. Additionally, they strongly induce the process of erythropoiesis through gene modulation, expression of γ-globin mRNA, and the synthesis of fetal hemoglobin [28]. Further, angelan is a pectin polysaccharide isolated from the roots of Angelica gigas, which reduces the adhesive capacity of neoplastic cells and has the ability to induce the maturation of dendritic cells [28], while fructan isolated from the root of Achyranthes bidentata shows a dose-dependent effect on the growth of Lewis lung cancer in vivo. Lower doses of this polymer inhibited tumor growth, as anti-tumor activity of A. bidentata is associated with inhibition of the tumor cell cycle. However, in higher doses, it can also cause stimulation of tumor growth due to NK dysfunction and an increase in the amount of interleukin-6 and TNF-α [111]. Polysaccharides isolated from P. ginseng, co-responsible for the biological activity of ginseng preparations, are mainly composed of starch-like glucans: α-D-(1,4)-glucans, 6-branched α-D-(1,4)-glucans, 3-branched α-D-(1,6)-glucans, α-D-(1,6)-glucans without side chains, and pectin, mainly composed of homogalacturonan, rhamnogalacturonan, and arabinogalactans [112,113].
It was reported that the PS, isolated from the root of Panax ginseng, can inhibit the proliferation of human bladder cancer T24 tumor cells and lactate dehydrogenase release. In addition, they have also significantly hampered cell migration and invasion. It was reported that polysaccharides isolated from P. ginseng induced the peritoneal macrophages (PMs) activity against K562, HL-60, and KG1α cells. The mechanism was involved with increasing the expressions of CD68, increasing the level of TNF-α, interleukin-1 (IL-1), and IL-6 [114][115][116]. In a like manner, the polysaccharides and glycoproteins are compounds partly responsible for the immunostimulatory effect of Echinacea sp. The polysaccharides and glycoproteins, which have been isolated from E. purpurea and E. angustifolia, are inulin-type fructans, acidic arabinogalactans, arabinogalactan-protein complexes, rhamnoarabinogalactans, and xyloglucans [117].
The toxicity and bioavailability of selenium strongly depend on its chemical form [119]. Numerous studies show that organic Se sources, such as selenomethionine, are assimilated more efficiently than inorganic Se, such as selenite, and inorganic Se species are more toxic than organic Se species. At the same time, naturally occurring Se compounds, such as animal and plant metabolites, are less toxic than artificial Se compounds [120,121]. Consequently, it can be predicted that Se-polysaccharides with a natural origin might be characterized by low toxicity and good selenium accessibility.

Physiological Functions of Se
Selenium is one of the key micronutrients required to maintain a cellular redox state as well as to control cell proliferation and survival. As a component of the amino acid selenocysteine, selenium is incorporated in about 35 selenoproteins [122]. Some of them have important enzymatic functions [123], but the roles of many others have not yet been fully elucidated [124]. The selenium-dependent enzymes with selenocysteine at the active site, such as glutathione peroxidases, thioredoxin reductases, and iodothyronine deiodinases, have biological functions in oxidoreductions, redox signaling, antioxidant defense, and thyroid hormone metabolism [125]. Selenium also appears to be important for the methylation of DNA, which is known to control gene expression and thus proliferation and differentiation; however, the exact mechanism of this interaction is not yet known [126,127]. Selenium has additional important effects on the immune response that are not exclusively associated to enzymatic activity. Even in a selenium non-deficient population, the supplementation of this micronutrient appears to stimulate the immune response [128,129]. This may be attributed to the stimulation of many cellular immune functions, including increased natural killer cell activity, as well as protection against oxidative stress-induced damage to immune cells [130][131][132]. Selenium appears to be a key nutrient in cancer prevention, although reports on this subject are not clear [133,134]. Nevertheless, Se is important in the prevention and treatment of several viral infections including RNA viruses [135,136]: it boosts Th1-type host immunity against viral infections [137] and appears to prevent the evolution of more virulent strains of some viral pathogens [138,139].

Selenium Species, Bioavailability, and Effects of Excess and Deficiency
Selenium is an essential element with a narrow safety margin: essential to humans in trace amounts, but highly toxic when in excess. It has one of the narrowest ranges among all the elements between dietary deficiency (less than 40 µg/day) and toxic levels (over 400 µg/day) [140]. The recommended dietary allowance (RDA) of selenium for adults is 55 µg/day, according to the Linus Pauling Institute [141]. The tolerable upper intake level advised by the USA is 400 µg/day in the USA [142,143] and 300 µg/day in Europe [144]. However, the recommendations do not take into account that Se can be present in food (or food supplements) under different chemicals forms, organic or inorganic. These forms differ in bioavailability and therefore exert different effects on the organism [145,146].
To address this problem, several methods are used to determine selenium bioavailability. These include the measurement of plasma selenium concentration, measurement of glutathione peroxidase (GPx) activity, and absorption-retention tests following intake of a specific dose of a selenium compound [147]. Plasma selenium is the most commonly measured biomarker, reflecting different levels of Se exposure that also respond positively to intervention. According to the Mayo Clinic Laboratories, the normal concentration of Se in adult human blood serum is 70 to 150 micrograms per liter [148]. This is indicative of the adequate dietary intake, above which selenium supplementation is not recommended.
When concentration of selenium is lower than the recommended 70 µg/L, the selenium intake is inadequate, and there is a risk of diseases related to selenium deficiency. In this case, supplementation of this element is recommended. Conversely, excessive selenium intake leads to a serum concentration higher than the recommended Se (over 150 µg/L −1 ), and there is an increased risk of diseases related to selenium excess. The U-shaped relationship between the concentration of selenium in human serum and the biological effects expressed as disease risk is particularly interesting [17]. The optimal selenium serum concentration (70-150 µg/L) is indicative of the adequate dietary intake, in which case selenium supplementation is not recommended. Both the excess and deficiency of selenium have several common negative health effects, i.e., increased mortality, type 2 diabetes, and increased prostate cancer risk [18,[149][150][151].
A selenium overdose can occur during chronic exposure to high levels of selenium in food and water. This may occur, for example, during regular snacking on Brazil nuts, which may contain up to 90 µg of selenium per nut, or participation in clinical trials where high doses of selenium are given for a long period of time [18], and overdose of Se food supplements [158]. The effects are similar for both organic and inorganic forms of selenium [144]. Symptoms of selenium overdose include hair loss, abnormal nails, nausea, vomiting, diarrhea, rash, fatigue, and nerve damage [159].
To summarize, the beneficial and harmful effects of selenium depend on its dose and form (speciation). The introduction of the new forms of selenium (e.g., selenopolysaccharides) to be used as selenium supplementation or treatment of various diseases requires extensive studies on the bioavailability, absorption, and metabolism of this element.

Organoselenium Natural and Synthetic Compounds
Natural organoselenium compounds are products obtained from the metabolism of bacteria, fungi, plants, and animals [160]. In the environment (soil, rocks, water), selenium is present in the inorganic forms, as selenate (SeO 4 2− ), selenite (SeO 3 2− ), elemental (Se), and selenide (Se 2− ) [161]. During the bioaccumulation in plants, fungi, and bacteria, the inorganic forms of selenium are incorporated into amino acids like selenomethionine, selenocystine, methylselenocystine, selenocystathionine, γ-glutamyl-Se-methylselenocysteine, and Se-adenosyl-selenohomocysteine [161]. These forms of selenium can also be methylated and form dimethylselenide, dimethyldiselenide, dimethyl selenone, methylselenol, and dimethyl selenyl-sulfide [162,163]. Several plant and mushroom species are known to be good Se-accumulators [164]; however, selenium is not considered an essential micronutrient for those organisms as it is for humans. Their ability to accumulate large amounts of organoselenium compounds may be the result of the detoxification process [165]. In humans, the inorganic forms of selenium are converted to selenocysteine and incorporated into selenoproteins or transformed into methylated metabolites, which are excreted through exhalation, urine, and faeces [166].
Mammals have no efficient mechanism of methionine synthesis. Therefore, they are also unable to synthesize selenomethionine, which is produced along with methionine in fungi and plants in quantities dependent on the amount of Se available. Consequently, selenomethionine, which can be incorporated nonspecifically in body proteins, constituting the main selenium store, must be supplied in the human diet.
According to Kuehnelt et al., two other selenocarbohydrates may be present in human urine after ingestion of selenium supplements, and they are 1β-methylseleno-Dgalactosamine and 1β-methylseleno-N-acetyl-D-glucosamine [169].
Several fungi and plant species, grown on the selenium-enriched substrates mainly as inorganic forms, such as sodium selenite, are able to incorporate selenium into the cell wall polysaccharides [170,171]. The knowledge about their structures is still fragmentary, and its current status will be presented in more detail in Section 5.
The number of known synthetic organoselenium compounds is incomparably greater than that of known natural compounds. In the past three decades, an exponential growth of organoselenium chemical studies was observed, as well as of the number of reactions and the variety of selenium compounds [172]. For example, organoselenium compounds were already used in organic chemistry as an electrophile, nucleophile, or even as a radical in chemo-, regio-, and stereo-selective reactions [173,174].

Selenium in Therapy and Chemoprevention-Traditional and Next Generation of Se Supplements
Due to the aforementioned beneficial health effects (Section 3.1), selenium compounds are used in prevention and treatment (as adjuvant) of various human diseases, such as several types of cancers, immune disorders, bacterial and viral infections, and cardiovascular diseases [211,212]. Nevertheless, the previously described U-shaped relationship between selenium dose and disease (Section 3.2) indicates that a daily intake of Se higher than that recommended leads to disease promotion.
The recommended plasma selenium concentration (an indicator of recent selenium intake) assures a balanced expression of bioactive selenoproteins, which act mainly as oxidoreductases, redox signal regulators, or thyroid hormone activators [213,214]. However, the overexpression of selenium enzymes and excess formation of bioactive selenium metabolites results in pathophysiological effects [214,215].
As stated above, selenium bioavailability depends largely on the form (organic versus inorganic) and the origin of the supplement (natural versus synthetic). However, the health implications of selenium intake are also related to its epigenetic effects [127,216]. All those factors suggest the need for a more personalized selenium supplementation; however, this approach is difficult to implement, as selenium preparations are freely available on the market as dietary supplements and nonprescription medicine.
Currently, the most widely used selenium supplements are (i) inorganic forms, such as sodium selenate and selenite; (ii) elemental selenium; and (iii) several organic selenium forms, such as selenomethionine and selenized yeast (rich in selenomethionine). Some reports describe other types of synthetic organic selenium supplements, such as phenylselenocysteine, methylseleninic acid, and selenocyanate [217,218]. These supplements, however, are not optimal and sometimes are even hazardous, given that selenium is easy to overdose on [160,219,220].
The search for selenium preparations with reduced toxicity, higher bioavailability, and controlled release led to the development of a "new generation" of selenium supplements [214]. Such next-generation selenium dietary supplements are based on selenium zerovalent nanoparticles encapsulated into polysaccharides [221] and selenium polysaccha-rides [13,222]. These selenium forms are not yet officially used as selenium supplements; however, the available information suggests that they have a history of safe use [214]. The big advantage of these selenium species is the slow release of active selenium forms. which results in a low risk of overdose. Synergistic biological effects of polysaccharides and selenium [223] could also make them more suitable for personalized supplementation.

Selenosugars: How Selenium May Be Bound to the Carbohydrate Structure
Selenosugars are a complex group of organoselenium compounds. They differ considerably in the type of selenium binding, valency, and degree of oxidation. They can beclassified as Se-alkylselenides and Se-arylselenides, selenopyranoses, selenofuranoses, selenoglycosides, selenonucleosides, selenoesters, and others. Selenosugars are derivatives of mono-, di-, oligo-, and finally, polysaccharides, that can be obtained by various methods (synthesis, biosynthesis, chemical modification of natural compounds), and differ in their biological activity. Several of them are currently being intensively studied as a non-toxic and highly bioavailable source of organic selenium [224].
Currently, the largest and best studied group of selenosugars are Se-monosaccharides and their derivatives. However, the exact knowledge of the structure and selenium bonding in many of the currently explored Se-polysaccharides is still missing. The following brief overview of the types of selenium bonding in selenosugars may be used for prediction of the probable structures of novel Se-polysaccharides.

Selenopyranoses and Selenofuranoses
Selenopyranoses and selenofuranoses are cyclic forms of carbohydrate derivatives, with the ring oxygen replaced by a selenium atom: a polyhydroxytetrahydroselenophene or selenane derivatives [224].
In general, selenopyranose and selenofuranose derivatives are obtained by chemical synthesis, starting from appropriate monosaccharides [225][226][227]. Most of them were synthesized as potent water-soluble selenium derivatives able to act as potential antioxidants with expected good oral bioavailability [228,229].
Numerous selenosugars have also been investigated for their capacity as glycosidase inhibitors. Glycosidases catalyze the hydrolysis of glycosidic linkages and are suitable targets for development of anti-type 2 diabetes, anticancer, and antiviral agents [230][231][232]. The selenosugars bearing selenium atoms in their ring positions, synthesized by Davies and Shiesser (2019) as part of the program aiming at developing powerful water-soluble antioxidants, are particularly interesting [233]. Several of them, as expected, proved to be powerful antioxidants. 1,4-anhydro-4-seleno-D-tallitol ( Figure 1c) seems to have a privileged structure: it acts not only as an efficient scavenger of multiple biologically important oxidants, but also exhibits the ability to repair damaged skin tissue, including accelerating healing of diabetic wounds, and is capable of decreasing endothelial dysfunction arising from diabetes [233].

Selenopyranoses and Selenofuranoses
Selenopyranoses and selenofuranoses are cyclic forms of carbohydrate derivatives, with the ring oxygen replaced by a selenium atom: a polyhydroxytetrahydroselenophene or selenane derivatives [224].
In general, selenopyranose and selenofuranose derivatives are obtained by chemical synthesis, starting from appropriate monosaccharides [225][226][227]. Most of them were synthesized as potent water-soluble selenium derivatives able to act as potential antioxidants with expected good oral bioavailability [228,229].
Numerous selenosugars have also been investigated for their capacity as glycosidase inhibitors. Glycosidases catalyze the hydrolysis of glycosidic linkages and are suitable targets for development of anti-type 2 diabetes, anticancer, and antiviral agents [230][231][232]. The selenosugars bearing selenium atoms in their ring positions, synthesized by Davies and Shiesser (2019) as part of the program aiming at developing powerful water-soluble antioxidants, are particularly interesting [233]. Several of them, as expected, proved to be powerful antioxidants. 1,4-anhydro-4-seleno-D-tallitol ( Figure 1c) seems to have a privileged structure: it acts not only as an efficient scavenger of multiple biologically important oxidants, but also exhibits the ability to repair damaged skin tissue, including accelerating healing of diabetic wounds, and is capable of decreasing endothelial dysfunction arising from diabetes [233].
. Se-nucleosides are a specific group of carbohydrates with selenium bound in the sugar backbone. They cannot be converted into the corresponding nucleotides due to hampered capacity for enzymatic phosphorylation. Most of the Se-nucleosides are biologically not active; however, the selenonucleoside developed by Jeong et al. showed antitumor activity against paclitaxel-resistant prostate cancer [234,235]. Regarding the seleno- Se-nucleosides are a specific group of carbohydrates with selenium bound in the sugar backbone. They cannot be converted into the corresponding nucleotides due to hampered capacity for enzymatic phosphorylation. Most of the Se-nucleosides are biologically not active; however, the selenonucleoside developed by Jeong et al. showed antitumor activity against paclitaxel-resistant prostate cancer [234,235]. Regarding the selenopyranose derivatives of natural origin, the polysaccharides containing selenium atoms in their ring positions were investigated using the XAS (X-ray absorption spectroscopy) method [39].

Se-Glycosides
Selenoglycosides are members of the selenosugar family in which the bridging oxygen of the glycosidic bond is replaced by selenium [236]. (Figure 2).

Se-Glycosides
Selenoglycosides are members of the selenosugar family in which the bridging oxygen of the glycosidic bond is replaced by selenium [236]. (Figure 2). The Se-glycosidic bond is resistant to enzymatic hydrolysis; therefore, selenoglycosides can act as inhibitors of glycosidase enzymes [237,238]. This type of sugars found application in disaccharide and oligosaccharide synthesis as glycosyl donors, where an arylselenyl group introduced at the anomeric position functions during glycosylation as a leaving group ( Figure 3). Using similar synthesis methods, it is possible to obtain selenodisaccharides in which the bridging oxygen of the glycosidic bond is replaced by selenium (Figure 4), as it happened in Nanami et al. experiment [239]; the first examples of the synthesized α-selenoglycosides that link to the carbon in the sugar ring were obtained.  The Se-glycosidic bond is resistant to enzymatic hydrolysis; therefore, selenoglycosides can act as inhibitors of glycosidase enzymes [237,238]. This type of sugars found application in disaccharide and oligosaccharide synthesis as glycosyl donors, where an arylselenyl group introduced at the anomeric position functions during glycosylation as a leaving group (Figure 3).

Se-Glycosides
Selenoglycosides are members of the selenosugar family in which the bridging oxygen of the glycosidic bond is replaced by selenium [236]. (Figure 2). The Se-glycosidic bond is resistant to enzymatic hydrolysis; therefore, selenoglycosides can act as inhibitors of glycosidase enzymes [237,238]. This type of sugars found application in disaccharide and oligosaccharide synthesis as glycosyl donors, where an arylselenyl group introduced at the anomeric position functions during glycosylation as a leaving group (Figure 3). Using similar synthesis methods, it is possible to obtain selenodisaccharides in which the bridging oxygen of the glycosidic bond is replaced by selenium (Figure 4), as it happened in Nanami et al. experiment [239]; the first examples of the synthesized α-selenoglycosides that link to the carbon in the sugar ring were obtained.  Using similar synthesis methods, it is possible to obtain selenodisaccharides in which the bridging oxygen of the glycosidic bond is replaced by selenium (Figure 4), as it happened in Nanami et al. experiment [239]; the first examples of the synthesized αselenoglycosides that link to the carbon in the sugar ring were obtained. Using similar synthesis methods, it is possible to obtain selenodisaccharides in which the bridging oxygen of the glycosidic bond is replaced by selenium (Figure 4), as it happened in Nanami et al. experiment [239]; the first examples of the synthesized α-selenoglycosides that link to the carbon in the sugar ring were obtained.  Most of selenoglycosides are obtained by chemical synthesis. However, three natural selenoglycosides, identified as derivatives of galactosamine and glucosamine, have been detected in human urine [167][168][169]. Kobayashi et al. (2002) stated that selenium was excreted into mammals' urine in the form of these selenosugars, when selenium intake was lower than the toxic level [168] ( Figure 5). Most of selenoglycosides are obtained by chemical synthesis. However, three natural selenoglycosides, identified as derivatives of galactosamine and glucosamine, have been detected in human urine [167][168][169]. Kobayashi et al. (2002) stated that selenium was excreted into mammals' urine in the form of these selenosugars, when selenium intake was lower than the toxic level [168] ( Figure 5). Another example of natural Se-glycoside is a selenium metabolite in the liver-a glycoconjugated molecule with the unusual Se-S linkage, known as "hepatic metabolite A" [168] (Figure 6).

Se-Alkylselenides and Se-Arylselenides
Se-alkyl-and Se-arylselenides derived from carbohydrates contain an organoselenium moiety at the non-anomeric position of sugars [  Another example of natural Se-glycoside is a selenium metabolite in the liver-a glycoconjugated molecule with the unusual Se-S linkage, known as "hepatic metabolite A" [168] (Figure 6). Most of selenoglycosides are obtained by chemical synthesis. However, three natural selenoglycosides, identified as derivatives of galactosamine and glucosamine, have been detected in human urine [167][168][169]. Kobayashi et al. (2002) stated that selenium was excreted into mammals' urine in the form of these selenosugars, when selenium intake was lower than the toxic level [168] ( Figure 5). Another example of natural Se-glycoside is a selenium metabolite in the liver-a glycoconjugated molecule with the unusual Se-S linkage, known as "hepatic metabolite A" [168] (Figure 6).

Se-Alkylselenides and Se-Arylselenides
Se-alkyl-and Se-arylselenides derived from carbohydrates contain an organoselenium moiety at the non-anomeric position of sugars [200,240,241] (Figure 7). Many of these synthetically obtained sugar derivatives show biological activity, e.g., antioxidant activity. Further, Vargas et. al. (2015) reported synthesis of simple alkylseleno-carbohydrates with different sugar scaffolds. Screening of the selected compounds for antioxidant activity revealed that several of them express free radical scavenging activity and therefore can potentially prevent oxidative damage of proteins and lipids [242].

Se-Alkylselenides and Se-Arylselenides
Se-alkyl-and Se-arylselenides derived from carbohydrates contain an organoselenium moiety at the non-anomeric position of sugars [200,240,241] (Figure 7). Many of these synthetically obtained sugar derivatives show biological activity, e.g., antioxidant activity.
Further, Vargas et. al. (2015) reported synthesis of simple alkylseleno-carbohydrates with different sugar scaffolds. Screening of the selected compounds for antioxidant activity revealed that several of them express free radical scavenging activity and therefore can potentially prevent oxidative damage of proteins and lipids [242].

Se-Esters
Carbohydrates, as polyhydroxy aldehydes or ketones, contain several hydroxyl groups that may react with an appropriate acidic compound, resulting in the formation of sugar esters. Among them are the selenious acid esters. The selenylation (esterification/acylation with selenious acid) reaction may be conducted, for example, using the HNO3-Na2SeO3 method [243] (Figure 8). Formation of the selenious acid sugar esters is a method widely used for the preparation of Se-polysaccharides containing selenium at the fourth oxidation state and will be further described in the next section (Section 5).

Divalent, Tetravalent, and Zerovalent Selenium in Polysaccharide Structure
The current literature shows that selenium in Se-polysaccharides may be di-, tetra-, or zero-valent, depending on the nature of the compound and the type of chemical bonds or interactions. Divalent selenium was found in selenoglycosides or selenopyranoses [13], tetravalent in selenites of polysaccharides (selenious acid esters) [244], and zerovalent in selenium nanoparticles encapsulated into polysaccharides [245]. However, the structures of numerous selenium-containing polysaccharides described in the literature have not been conveniently studied yet. Certain structural analyses were limited to examining only the amount of selenium bound to the polysaccharide structure, without taking into consideration its chemical environment. Moreover, conclusions regarding selenium binding (oxidation degree, chemical bonds, chemical environment) in some of the published reports were based on insufficient data (e.g., only IR spectra). Unraveling the chemical environment of selenium in selenoorganic compounds requires advanced analytical methods. The principal techniques that should be used for the characterization of selenium-

Se-Esters
Carbohydrates, as polyhydroxy aldehydes or ketones, contain several hydroxyl groups that may react with an appropriate acidic compound, resulting in the formation of sugar esters. Among them are the selenious acid esters. The selenylation (esterification/acylation with selenious acid) reaction may be conducted, for example, using the HNO 3 -Na 2 SeO 3 method [243] (Figure 8).

Se-Esters
Carbohydrates, as polyhydroxy aldehydes or ketones, contain several hydroxyl groups that may react with an appropriate acidic compound, resulting in the formation of sugar esters. Among them are the selenious acid esters. The selenylation (esterification/acylation with selenious acid) reaction may be conducted, for example, using the HNO3-Na2SeO3 method [243] (Figure 8). Formation of the selenious acid sugar esters is a method widely used for the preparation of Se-polysaccharides containing selenium at the fourth oxidation state and will be further described in the next section (Section 5).

Divalent, Tetravalent, and Zerovalent Selenium in Polysaccharide Structure
The current literature shows that selenium in Se-polysaccharides may be di-, tetra-, or zero-valent, depending on the nature of the compound and the type of chemical bonds or interactions. Divalent selenium was found in selenoglycosides or selenopyranoses [13], tetravalent in selenites of polysaccharides (selenious acid esters) [244], and zerovalent in selenium nanoparticles encapsulated into polysaccharides [245]. However, the structures of numerous selenium-containing polysaccharides described in the literature have not been conveniently studied yet. Certain structural analyses were limited to examining only the amount of selenium bound to the polysaccharide structure, without taking into consideration its chemical environment. Moreover, conclusions regarding selenium binding (oxidation degree, chemical bonds, chemical environment) in some of the published reports were based on insufficient data (e.g., only IR spectra). Unraveling the chemical environment of selenium in selenoorganic compounds requires advanced analytical methods. The principal techniques that should be used for the characterization of selenium- Formation of the selenious acid sugar esters is a method widely used for the preparation of Se-polysaccharides containing selenium at the fourth oxidation state and will be further described in the next section (Section 5).

Divalent, Tetravalent, and Zerovalent Selenium in Polysaccharide Structure
The current literature shows that selenium in Se-polysaccharides may be di-, tetra-, or zero-valent, depending on the nature of the compound and the type of chemical bonds or interactions. Divalent selenium was found in selenoglycosides or selenopyranoses [13], tetravalent in selenites of polysaccharides (selenious acid esters) [244], and zerovalent in selenium nanoparticles encapsulated into polysaccharides [245]. However, the structures of numerous selenium-containing polysaccharides described in the literature have not been conveniently studied yet. Certain structural analyses were limited to examining only the amount of selenium bound to the polysaccharide structure, without taking into consideration its chemical environment. Moreover, conclusions regarding selenium binding (oxidation degree, chemical bonds, chemical environment) in some of the published reports were based on insufficient data (e.g., only IR spectra). Unraveling the chemical environment of selenium in selenoorganic compounds requires advanced analytical methods. The principal techniques that should be used for the characterization of selenium-containing molecules are mass spectrometry, X-ray crystallography, Se NMR spectroscopy, X-ray absorption spectroscopy (XAS), and several others [118,214].
However, the structurally undefined Se-polysaccharides are a relevant research subject, as the introduction of selenium into their structure often has significant effects on the biological activity.

Se-Glycosides and Selenopyranoses
As explained in Section 4, in most of the selenosugars and their derivatives, e.g., selenopyranoses, selenofuranoses, selenoglycosides, selenonucleosides, and Se-alkylselenides, selenium is divalent. Most of the selenosugars containing divalent selenium are small molecules obtained by chemical synthesis. However, as previously described, four natural selenoglycosides, identified as derivatives of galactosamine and glucosamine, have been detected in human urine [167][168][169]. The results of research conducted by Kobayashi et al. [168] suggested that the major selenium metabolite in mammals urine is a selenoglycoside (Figure 5a). Therefore, since mammals are capable of selenium sugars biosynthesis, in which selenium is glycosidically bound, it is highly probable that plants, fungi, and microorganisms may also have such capability. In fact, multiple reports describe the ability of these organisms to accumulate selenium in the structure of polysaccharides.
In a series of studies on selenium-enriched mycelial cultures of the medicinal mushroom Lentinula edodes, Turlo et al. proved that mycelium effectively accumulated Se from the cultivation medium enriched in sodium selenite [252,253] and that selenium was also incorporated into the mycelial polysaccharides [171,254]. Further, Malinowska et al. (2018) [39] isolated a selenium-containing polysaccharide-protein fraction from the Se-enriched mycelial cultures of L. edodes composed of glucose and mannose, containing 190 µg Se/g dry weight. X-ray absorption of fine structure (XAFS) spectra analysis in the near edge region (XANES) confirmed that selenium in the Se-polysaccharide structure is divalent and organically bound. The simulation analysis in the EXAFS (extended X-ray absorption fine structure) suggested that selenium is bound in a β-1,3-, α-1,4-glycosidic linkage or substitutes oxygen in a pyranosidic ring (Figure 9). containing molecules are mass spectrometry, X-ray crystallography, Se NMR spectros copy, X-ray absorption spectroscopy (XAS), and several others [118,214]. However, the structurally undefined Se-polysaccharides are a relevant research sub ject, as the introduction of selenium into their structure often has significant effects on th biological activity.

Se-Glycosides and Selenopyranoses
As explained in Section 4, in most of the selenosugars and their derivatives, e.g., se lenopyranoses, selenofuranoses, selenoglycosides, selenonucleosides, and Se-alkylselen des, selenium is divalent. Most of the selenosugars containing divalent selenium are sma molecules obtained by chemical synthesis. However, as previously described, four natura selenoglycosides, identified as derivatives of galactosamine and glucosamine, have bee detected in human urine [167][168][169]. The results of research conducted by Kobayashi et a [168] suggested that the major selenium metabolite in mammals urine is a selenoglycosid (Figure 5a). Therefore, since mammals are capable of selenium sugars biosynthesis, i which selenium is glycosidically bound, it is highly probable that plants, fungi, and m croorganisms may also have such capability. In fact, multiple reports describe the abilit of these organisms to accumulate selenium in the structure of polysaccharides.
In a series of studies on selenium-enriched mycelial cultures of the medicinal mush room Lentinula edodes, Turlo et al. proved that mycelium effectively accumulated Se from the cultivation medium enriched in sodium selenite [252,253] and that selenium was als incorporated into the mycelial polysaccharides [171,254]. Further, Malinowska et al. (2018 [39] isolated a selenium-containing polysaccharide-protein fraction from the Se-enriche mycelial cultures of L. edodes composed of glucose and mannose, containing 190 μg Se/ dry weight. X-ray absorption of fine structure (XAFS) spectra analysis in the near edg region (XANES) confirmed that selenium in the Se-polysaccharide structure is divalen and organically bound. The simulation analysis in the EXAFS (extended X-ray absorptio fine structure) suggested that selenium is bound in a β-1,3-, α-1,4-glycosidic linkage o substitutes oxygen in a pyranosidic ring (Figure 9).  According to the calculations performed with Gaussian 03 software, incorporation of selenium cause deformations in the polysaccharide structure, including change in bond lengths and torsion angles, and, as a result, disappearance of hydrogen bonds in the vicinity of the selenium atoms [13]. This modification may affect the interaction with specific polysaccharide receptors and introduce changes in the biological activity [11].

Se-Esters-Selenites and Diselenites of Polysaccharides
The esterification of polysaccharides with selenious acid results in formation of polysaccharide selenites, esters of selenious acid (Figure 8). Under appropriate conditions, practically all native polysaccharides, as polyhydroxy aldehydes or ketones, may react with H 2 SeO 3 or Na 2 SeO 3 in an acidic environment to yield the polysaccharide selenites. In these compounds, selenium is tetravalent and in the fourth oxidation state, so it retains the redox properties typical for this oxidation state [118]. Depending on the acylation method used and the degree of polysaccharide substitution (DS), the content of selenium in the polysaccharide molecule may be significantly different.
Currently, the selenylation of natural polysaccharides to selenious acid esters is a frequent method to obtain selenium-containing polysaccharides. The method most commonly used to obtain a selenite from a polysaccharide is using nitric acid and sodium selenite [255].
In 2009, Ji et al. described the use of this method to introduce selenium into polysaccharides isolated from Cynomorium songaricum. The maximum selenic content determined by ICP-AES was 2925 µg/g [256]. In 2012, Wang et al. reported the synthesis of seleniumcontaining polysaccharides derivatives from Artemisia sphaerocephala polysaccharide with a high Se content (1703 µg/g) using H 2 SeO 3 /HNO 3 and BaCl 2 as a catalyst [244]. The C-6 substitution was predominant in selenized polysaccharide, as proven by Raman and (13)C NMR spectroscopy. The authors have observed a sharp decrease in the molecular weight of the polysaccharide caused by a degradation of the polymer. The structure of obtained Se-polysaccharides was studied by using size exclusion chromatography combined with multi angle laser light scattering (SEC-MALLS) [257].

Polysaccharide Encapsulated Nanoselenium
Zero-valent selenium (0 oxidation state) in the form of selenium nanoparticles (SeNPs) has attracted research attention thanks to its high bioavailability, lower toxicity-it is lower than that of inorganic and organic forms of selenium (e.g., SeNPs have seven-fold lower toxicity than sodium selenite), and controlled Se release [214,[282][283][284][285]. As a result, SeNPs carry a lower risk of excess Se supplementation.
According to the IUPAC definition, a nanoparticle is a particle of any shape with dimensions in the 1 × 10 −3 to 1 × 10 −1 micron range [286]. This wide range of nanoparticle sizes results in differences in their properties, with smaller particles showing a greater cellular intake and activity [287][288][289]. Hence, Desai et al., (1997) observed that in vitro absorption of 0.1 µm particles of SeNPs was found to be 2.5 and six times higher when compared to 1 and 10 µm particles, respectively [288]. Thus, when considering preparation of the potential drugs and dietary supplements, an appropriate particle size should be chosen. However, selenium in the form of nanoparticles is unstable and easily transformed into an inactive form [289], which necessitates the use of stabilizing agents in SeNPs solutions. These solutions may be stabilized and modified by various polymers, such as chitosan [290,291] and other polysaccharides [44,45,[292][293][294][295][296][297], proteins [293,294], or their combinations [298][299][300].
The stabilizing agents, such as polysaccharides, may play a dual role in the SeNPs formulations, as they can also improve the bioavailability and biological activity due to the synergistic effect of SeNPs and polysaccharides [301]. Polysaccharide-stabilized SeNPs may be obtained by chemical methods, e.g., by reduction of selenites in the solution containing polysaccharides [302], or by biological and biotechnological methods-extraction from the plant, fungal, algal, or microbial material grown in media enriched in selenium compounds, preferably sodium selenite [303,304]. The reduction of selenium to elemental nanoselenium in organisms is thought to be a form of detoxification and elimination of selenium excess, where part of the reduced elemental selenium becomes linked to the polysaccharides in the cell wall of the cultured organism [39].

Methods for Obtaining Selenium-Containing Polysaccharides
The low molecular weight selenosugars may be obtained by a biosynthesis, a multistep, enzyme-catalyzed process occurring in living organisms, chemical modification of natural compounds (semisynthesis, i.e., partial chemical synthesis that uses chemical compounds isolated from natural sources as starting material) [314], or by a complete chemical synthesis from simple precursors [315,316]. Due to the enormous complexity of the polysaccharide molecules, not all of the above-mentioned methods are applicable to the preparation of the high-molecular weight polysaccharides and Se-polysaccharides: most of the methods of chemical synthesis of polysaccharides yield compounds with a limited molecular weight (typically <10,000 g/mol) [317]. The total synthesis of selenium polysaccharides has not yet been described. However, selenosaccharides with low molar mass (selenoglycosides) have already shown an application in the disaccharide and oligosaccharide synthesis that was described in Section 4 ( Figure 3).
In conclusion: Se-polysaccharides may be obtained by extraction from natural products, mainly plants and fungi, but also from microorganisms, by biosynthesis with biotechnological methods or chemical modification of natural polysaccharides. A separate subject is the preparation of nanoselenium-polysaccharide complexes, i.e., nanoselenium particles stabilized (encapsulated) into polysaccharides. All these methods will be discussed below.

Extraction from Natural Products
As stated in the previous paragraph, chemical synthesis of high molecular-weight polysaccharides is practically impossible. Therefore, these compounds are either isolated from natural sources or obtained by biotechnological methods and, in certain cases, chemically modified.
Depending on the biological material from which the polysaccharides are extracted, slightly different extraction conditions have to be chosen [318] due to the different cell wall structure of bacteria, fungi, and plants. Polysaccharides are mostly soluble in water, but insoluble in organic solvents; therefore, water is the most common extraction medium [319] and the base solvent in the acid or alkali extraction and the enzymolysis method [320].
Basically, the Se-polysaccharides are isolated and purified using the same methods as non-selenium-enriched polysaccharides. Certain differences, observed in our research on the biosynthesis, extraction, and purification of these compounds, consist in different solubilities and limitations in the use of aggressive reagents in derivatization processes (strong acids and bases) due to the instability of some selenium compounds in such an environment [252,320]. As it has been observed, selenium polysaccharides have lower water solubility than non-selenated analogues, which thus affects their extraction efficiency [39].
In plants, breaking the cell wall with ultrasound or microwave methods is required prior to the polysaccharide extraction [321,322]. In mushrooms, on the other hand, the biologically active polysaccharides (mainly β-glucans) are components of the outer layer of the cell wall [323]. In this case the extraction of cell wall polysaccharides commonly involves a pre-extraction with 80% ethanol for elimination of low molecular substances [324].
However, apart from those slight differences, the basic extraction methods are similar and include hot water extraction, precipitation with alcohol (ethanol or methanol), deproteinization, and fractionation, utilizing different methods.
Taking into account the differences in the physical and chemical properties of the various polysaccharides (Se-polysaccharides), an appropriate extraction method should be chosen based on the structure and solubility (in water or alkali) of the target compounds.
During the extraction, a transition from gentle to aggressive extraction conditions (pH, temperature, pressure) is essential. This way the cell wall is gradually destroyed, resulting in the release of Se-polysaccharides from deeper cell wall layers. Procedures are often time-consuming and multiphasic [321].
The example procedure for the extraction of the active polysaccharides from mushroom cultures may involve the following steps. The initial water extraction is carried out with organic solvents, mainly ethanol, acetone, or a mixture of chloroform and methanol in order to remove non-polar compounds such as lipids, phenols, and terpenes. The next step is centrifugation, which produces a supernatant called cold water extract. The residue is then extracted in boiling water, and the biomass is isolated from the hot water extract by another centrifugation. The extraction is repeated to increase the yield [325]. The aqueous extraction residue is extracted with an aqueous base solution (with 2% NaOH or KOH solutions) at the boiling point [326]. The precipitate is separated by a centrifugation using NaBH 4 to protect against degradation of the end units of the polysaccharide chains. Exactly the same procedures were used to extract Se-polysaccharides from selenium enriched mycelium of L. edodes and Hericium erinaceus [11,13,39,249].
After the extraction, the polysaccharides are purified from proteins, amino acids, monosaccharides, phenolic derivatives, and other molecules. Protein contaminants are removed using a 20% trichloroacetic acid solution by an enzymatic process using a protease enzyme, by the Sevage method, or by extraction with a phenolic reagent [326].
The extracted polysaccharide is in fact a polysaccharide fraction-a mixture of chains with different degrees of polymerization, sometimes varying monosaccharide composition, and chemical character. Therefore, fractionation and deep purification are necessary to obtain a homogeneous polysaccharide (Se-polysaccharide). Based on the purification techniques used, the methods can be divided into three categories: physical, chromatographic, and chemical precipitation. In most cases, the combination of several separation methods has to be employed to improve the purification results.
Fractional precipitation may be used to separate polysaccharides with large differences in solubility and molecular weight [329]. The long-chain quaternary ammonium salt chemical precipitation method may also be applied [318].
Chromatographic separation by column chromatography is also used, with several modifications, mostly as an anion-exchange column chromatography and gel column chromatography [330]. In most cases, anion-exchange chromatography is used in the first step, followed by gel column chromatography.
Design of the methods for extracting polysaccharide fractions from biological material may have to consider some unexpected factors. For example, significant differences were found between the structure of the cell walls of the mycelium of L. edodes cultured by biotechnological methods and the fruiting bodies of this fungus. These differences were expressed as a low chitin content and higher exopolysaccharide content in the mycelial cells, which determine the conditions for the polysaccharide extraction [171]. The enrichment of the mycelium with selenium also causes significant changes in the structure of the mycelial cell walls, which is related to the content of polyphenols. These differences also have an impact on the polysaccharides and ergosterol in the biomass extracts. Such differences significantly affect the extractability of Se-polysaccharides from the biological material [171].

Biotechnological Methods
When considering the biotechnological methods to obtain Se-polysaccharides, the cultures of plants, fungi, algae, or microorganisms are grown in media enriched with soluble selenium compounds. The culture is then used to isolate selenium-enriched polysaccharide fractions from the selenium-enriched biomass of the cultivated species (cell wall polysaccharides and endo-polysaccharides) or from the culture medium (exo-polysaccharides). In order to isolate and purify Se-polysaccharides from those cultured organisms, the methods used are the same as for other natural sources. As a result of the transport into the cell and following enzymatic processes, selenium compounds supplementing the medium may be incorporated into the structure of polysaccharides, as happens with plants or fungi grown on substrates enriched with selenium compounds. However, the biotechnological methods are in many aspects advantageous over the crops cultivation [331].
A major advantage is the shorter time of cultivation in bioreactors, which gives a significant reduction in the time needed to obtain a comparable biomass. Moreover, culturing in bioreactors is carried out under repeatable conditions, resulting in a stable composition of the grown biomass. This facilitates standardization of the preparations, for example for pharmaceutical use.
Biotechnological processes also use optimized composition of the culture media and physico-chemical conditions of the culture. This enables regulation of the metabolism of the cultivated organism and results in a significant increase in efficiency of the biologically active compounds. Finally, the technology of the biotechnological processes provides monitoring and maintenance of the biochemical and genetic identity of the strain cultivated in a fermenter.
However, there are also serious disadvantages of using of modern biotechnological methods when compared with crops cultivation or hydroponics. Primarily, not all species of plants and fungi are capable of growing as efficiently as cell, callus, or mycelial cultures in bioreactors. Moreover, the cultivation of plants (cell, callus, or tissue cultures) and higher fungi (mostly mycelial cultures) in bioreactors faces more challenges than the culture of single-celled organisms. Additionally, the use of biotechnological methods is mostly more expensive than crops. When using biotechnological methods of cultivation, the costs of specialized equipment and apparatus, energy, the required specific constituents of the culture media, highly specialized service, maintaining sterile conditions, etc., are relatively high. However, in the case of protected plants or fungi, plants growing slowly in crops, or hydroponic cultivation, coming from climatic zones/regions, where cropping is very difficult, the choice of the biotechnological methods is favorable. Biotechnological cultures of ginseng (Panax ginseng CA Mayer) are a good example. Field cultivation of Panax ginseng is a slow and laborious process; therefore, cell and tissue culture methods have been explored as potentially more efficient for ginseng biomass and its bioactive components production [332]. Regardless, the decisive factors in choosing the method of obtaining biological material, for example, for the polysaccharide isolation, are its implementability and of course the cost.
A great number of reports have been published on the preparation of Se-polysaccharides (exo-and endo-polysaccharides) by biotechnological methods. Only a few examples will be given below.

Chemical Modification of Natural Polysaccharides
In order to improve the physico-chemical or biological properties of natural polysaccharides, various chemical modification methods, known as for functionalization or diversification, may be applied [336]. However, the basic properties of the polysaccharide should be maintained during chemical modification: the polymeric chain has to remain intact or, in case of its partial degradation, the products still have to be polysaccharides [337]. One of the widely used methods that may interfere with functionalization and/or diversification of polysaccharides is esterification or acylation with different reagents [336]. To our knowledge, acylation with selenious acid is currently the only method used for the chemical selenylation of natural polysaccharides (stabilization of SeNP with polysaccharides is strictly a non-chemical modification of the polysaccharide structure, and therefore will be discussed separately). In general, esterification involves the reaction of an alcohol (one of the saccharide hydroxyl groups) with an acylating agent. Regarding the diversification reactions, the extent of the process is evaluated by the degree of substitution (DS), defined as a number of substitutions per monomer unit [336]. The maximum DS depends on the structure of the polysaccharide and the type of the diversification reaction. For example, the polyglucopyranose has three hydroxyl groups per monomer that may be acylated; therefore, the maximum DS achievable by esterification of this polysaccharide would be 3 ( Figure 10). In order to introduce a selenium-containing moiety into the polysaccharide molecule, maintaining the favorable redox properties of selenium in the oxidation state 4, the polysaccharides should be esterified with selenious acid. Appl. Sci. 2021, 11 Assuming that the highest DS value for a linear selenite-polyglucan is 3 (Figure 10), it can be calculated that the theoretical highest amount of selenium accumulated in a molecule of such Se-polysaccharide is close to 48 × 10 3 mg/g. A similar DS value and theoretical maximum amount of accumulated selenium is for each hexose polymer (polyglycan). In principle, these values depend on the number of hydroxyl groups per mer of the polysaccharide and may be calculated theoretically for each glycan. This is only a theoretical value, because complete esterification of all hydoxyl groups of the polysaccharide is very unlikely; acylation of the primary hydroxyl group at the C-6 position of the monomer is preferred. In addition, the efficiency of the acylation reaction depends on the structure of the polysaccharide, including the spatial secondary and tertiary structure.
The most commonly used method to obtain the selenite-polysaccharide is the chemical reaction with nitric acid and sodium selenite [281]. The general procedure includes the following steps [281]: native polysaccharides are dissolved in nitric acid and react with sodium selenite in the presence of BaCl2. Then, the solution is neutralized with NaOH, and Ba 2+ ions are removed by precipitation. The product is purified by dialysis, at room temperature, in a dialysis sack with 1kDa ultrafiltration membrane, against distilled water [255], yielding the selenite-polysaccharide (selenylated polysaccharide). During the preparation of the selenites, several modifications of the acylation method may be carried out, including chemical reactions with nitric acid and sodium selenite [255,265,[267][268][269]271,272,[338][339][340], nitric acid and selenious acid [258], glacial acetic acid and selenious acid, glacial acetic acid and sodium selenite [258], and selenium oxychloride [260,261]. In order to improve the efficiency of selenylation, some additional techniques, such as microwave [341,342] and ultrasound [262], were already tested. For example, Wei et al. (2019) [343] observed some beneficial effects after using acidic ionic liquids as catalyst that promoted the selenylation of polysaccharides. Interestingly, their work showed that a controlled regulation of Se content and MW (weight averaged molecular weight) could be achieved by adjusting anions in acidic ionic liquids [343].
In turn, based on the available literature [4,344], Huang et al. [345] suggested that it is also possible to obtain diselenite or cyclic forms of selenite-polysaccharides ( Figure 11) by use of selenium oxychloride as acylating agent. Assuming that the highest DS value for a linear selenite-polyglucan is 3 (Figure 10), it can be calculated that the theoretical highest amount of selenium accumulated in a molecule of such Se-polysaccharide is close to 48 × 10 3 mg/g. A similar DS value and theoretical maximum amount of accumulated selenium is for each hexose polymer (polyglycan). In principle, these values depend on the number of hydroxyl groups per mer of the polysaccharide and may be calculated theoretically for each glycan. This is only a theoretical value, because complete esterification of all hydoxyl groups of the polysaccharide is very unlikely; acylation of the primary hydroxyl group at the C-6 position of the monomer is preferred. In addition, the efficiency of the acylation reaction depends on the structure of the polysaccharide, including the spatial secondary and tertiary structure.
The most commonly used method to obtain the selenite-polysaccharide is the chemical reaction with nitric acid and sodium selenite [281]. The general procedure includes the following steps [281]: native polysaccharides are dissolved in nitric acid and react with sodium selenite in the presence of BaCl 2 . Then, the solution is neutralized with NaOH, and Ba 2+ ions are removed by precipitation. The product is purified by dialysis, at room temperature, in a dialysis sack with 1 kDa ultrafiltration membrane, against distilled water [255], yielding the selenite-polysaccharide (selenylated polysaccharide). During the preparation of the selenites, several modifications of the acylation method may be carried out, including chemical reactions with nitric acid and sodium selenite [255,265,[267][268][269]271,272,[338][339][340], nitric acid and selenious acid [258], glacial acetic acid and selenious acid, glacial acetic acid and sodium selenite [258], and selenium oxychloride [260,261]. In order to improve the efficiency of selenylation, some additional techniques, such as microwave [341,342] and ultrasound [262], were already tested. For example, Wei et al. (2019) [343] observed some beneficial effects after using acidic ionic liquids as catalyst that promoted the selenylation of polysaccharides. Interestingly, their work showed that a controlled regulation of Se content and M W (weight averaged molecular weight) could be achieved by adjusting anions in acidic ionic liquids [343].
In turn, based on the available literature [4,344], Huang et al. [345] suggested that it is also possible to obtain diselenite or cyclic forms of selenite-polysaccharides ( Figure 11

Encapsulation of Nanoselenium Particles into Polysaccharides
Three methods for preparation of SeNPs are currently used: chemical synthesis [304], physical procedures [346], and biogenic methods utilizing enzymatic reactions in microorganisms, mushrooms, or plants [347]. The polysaccharide-stabilized SeNPs are currently obtained by chemical synthesis and biological and biotechnological methods.
Chemical methods using elemental nano-selenium employ the reduction of this element, mostly from the +4-oxidation state, with a reducing agent in the presence of a polysaccharide as a capping agent. Encapsulated nanoparticles form in a colloidal suspension of a characteristic red color. Some selenium precursors are selenious acid, sodium selenite, or sodium hydrogen selenite; reducing agents include ascorbic acid or reducing mono and disaccharides, like glucose and galactose [286,302,[306][307][308][309][310][348][349][350][351][352]. The process is conducted in the presence of polysaccharides such as chitosan, glucomannan, acacia gum, carboxymethylcellulose, or other polysaccharides with different molecular properties and chain conformations [302,353]. The polysaccharides are used as stabilizers and coating agents to obtain stable and water-dispersible selenium nanoparticles.
The procedure to obtain encapsulated SeNPs is simple and includes several steps [354]: 1) adding a vitamin C solution to an aqueous solution of polysaccharides, and mixing uniformly; 2) adding dropwise, with mixing, a selenium dioxide solution or a selenite solution; 3) adding water to the solution to obtain a polysaccharide functionalized nanoselenium hydrosol. The product is stored in the form of sol in an aqueous solution.
Chemical methods of SeNPs preparation are more effective than biological and biotechnological methods; however, the risk of presence of toxic by-products unacceptable in medicinal preparations and dietary supplements led to the development of novel biogenic methods, typical of green chemistry. In green synthesis, a bioreductive capacity of plant extracts is utilized for preparation of selenium nanoparticles. The components of the complex plant extracts (among others polysaccharides) may act as good encapsulating stabilizing agents and facilitate the formation of stable colloidal nanoparticles [355]. Several plant extracts were used for the synthesis and stabilization of the SeNPs, e.g., tea extract [45], aqueous extract of Allium sativum [356], leaf extract of Clausena dentata [357], polysaccharide solutions extracted from Undaria pinnatifida [358], and leaf extract of Terminalia arjuna [359]. Biosynthesis of encapsulated SeNPs using plant extracts is inexpensive and does not require any special conditions [304].
Several organisms such as plants, fungi, or bacteria have the ability to convert some toxic ions to less toxic forms including metal precipitants or formation of NPs [360,361]. This capacity is based on the involvement of oxidoreductases in the detoxification process,

Encapsulation of Nanoselenium Particles into Polysaccharides
Three methods for preparation of SeNPs are currently used: chemical synthesis [304], physical procedures [346], and biogenic methods utilizing enzymatic reactions in microorganisms, mushrooms, or plants [347]. The polysaccharide-stabilized SeNPs are currently obtained by chemical synthesis and biological and biotechnological methods.
Chemical methods using elemental nano-selenium employ the reduction of this element, mostly from the +4-oxidation state, with a reducing agent in the presence of a polysaccharide as a capping agent. Encapsulated nanoparticles form in a colloidal suspension of a characteristic red color. Some selenium precursors are selenious acid, sodium selenite, or sodium hydrogen selenite; reducing agents include ascorbic acid or reducing mono and disaccharides, like glucose and galactose [286,302,[306][307][308][309][310][348][349][350][351][352]. The process is conducted in the presence of polysaccharides such as chitosan, glucomannan, acacia gum, carboxymethylcellulose, or other polysaccharides with different molecular properties and chain conformations [302,353]. The polysaccharides are used as stabilizers and coating agents to obtain stable and water-dispersible selenium nanoparticles.
The procedure to obtain encapsulated SeNPs is simple and includes several steps [354]: (1) adding a vitamin C solution to an aqueous solution of polysaccharides, and mixing uniformly; (2) adding dropwise, with mixing, a selenium dioxide solution or a selenite solution; (3) adding water to the solution to obtain a polysaccharide functionalized nanoselenium hydrosol. The product is stored in the form of sol in an aqueous solution.
Chemical methods of SeNPs preparation are more effective than biological and biotechnological methods; however, the risk of presence of toxic by-products unacceptable in medicinal preparations and dietary supplements led to the development of novel biogenic methods, typical of green chemistry. In green synthesis, a bioreductive capacity of plant extracts is utilized for preparation of selenium nanoparticles. The components of the complex plant extracts (among others polysaccharides) may act as good encapsulating stabilizing agents and facilitate the formation of stable colloidal nanoparticles [355]. Several plant extracts were used for the synthesis and stabilization of the SeNPs, e.g., tea extract [45], aqueous extract of Allium sativum [356], leaf extract of Clausena dentata [357], polysaccharide solutions extracted from Undaria pinnatifida [358], and leaf extract of Terminalia arjuna [359]. Biosynthesis of encapsulated SeNPs using plant extracts is inexpensive and does not require any special conditions [304].
Several organisms such as plants, fungi, or bacteria have the ability to convert some toxic ions to less toxic forms including metal precipitants or formation of NPs [360,361]. This capacity is based on the involvement of oxidoreductases in the detoxification process, in case of an excess of harmful factors in the environment. The ability to reduce excess amounts of selenium in the oxidation state +6 or +4 to elemental selenium, often to red selenium in the form of nanoparticles, has been observed in many organisms. Due to that ability, the cultures of microorganisms or mushroom may be used for synthesizing SeNPs with an ecofriendly approach [362][363][364][365].
Several types of bacteria and cyanobacteria cultivated in submerged cultures have already been used for the biosynthesis of SeNPs, and these include Escherichia coli [366], Enterobacter cloacae [367], Pseudomonas aeruginosa [368], Klebsiella pneumoniae [369], Zooglea ramigera [370], Lactobacillus casei [371], Streptomyces sp. [372], Arthrospira [Spirulina] platensis [373], and many others. Further, fungal cultures may be used for in vivo synthesis of SeNPs. For example, several filamentous fungi species [374], yeast Saccharomyces cerevisiae [375], Aspergillus terreus [376], Phanerochaete chrysosporium [377], and even higher fungi such as Lentinula edodes [378] were already used. The list of species used for the production of SeNPs is much longer, and the species presented above should be treated as examples. The nanoselenium particles in fungal and microbial cultures may be present inside the cell or secreted into the culture medium. Depending on the location, they interact with either the components of the culture medium or the cytosol, leading to natural stabilization of SeNPs through the interactions with proteins and polysaccharides. Thus, SeNPs stabilized with polysaccharides can be obtained after extraction from the cultures of mushrooms or microbes enriched with high concentrations of selenium, as previously described for L. edodes mycelia [13]. When the selenium content in the L. edodes Se-polysaccharides was equal to 1045µg/g, the X-ray absorption fine structure (XAFS) spectra analysis in the near edge region (XANES) confirmed that selenium in the Se-polysaccharides structure is zerovalent (at 0 oxidation state). The disadvantage of synthesis in vivo by macro-or microorganisms are the changes of the particle morphology and shape caused by the diversity of the reducing enzymes in organisms [289].

Biological Activity of Se-Polysaccharides
Natural Se-polysaccharides are not common, and the selenium content in their molecules is very low. Therefore, there is a growing interest in the synthesis of polymers with a high selenium index [262]. In the process of chemical modification of polysaccharides, the ability of organisms to absorb the microelements present in the culture medium has been used. Hence, the best way to improve that absorption is the enrichment of the culture medium with inorganic selenium (e.g., sodium selenite), leading to the incorporation of the element into the biopolymer structure and turning it into an organic form. Selenium compounds resulting from the time-consuming biotransformation method in fungi, bacteria, plants, and algae are attracting increasing attention due to their high bioavailability and low toxicity [13,262]. These selenated polysaccharides can thus serve not only as a new source of selenium, but also as compounds with a new, higher bioactivity. Se-polysaccharides show a higher immunomodulating, anticancer, antioxidant, and hepatoprotective bioactivity in relation to the native polysaccharides [10,281] due to the synergistic effect of selenium and polysaccharides (despite their different mechanisms of action) [11,39]. In general, along with the increase in selenium content, the bioactivity of the modified polysaccharide also increases, but this relation is still a subject of research. The following subsections outline the biological activity of Se-polysaccharides isolated from bacteria, algae, fungi, and plants.

Bacterial Se-Polysaccharides
Selenium-enriched exopolysaccharide isolated from Enterobacter cloacae culture medium increased the proliferation of lymphocytes in mice, indicating that it might be used as an immunomodulator to increase the cellular and humoral immune response [250]. In addition, studies by Lu et al. on Enterobacter cloacae confirmed the immunological activity of Se-polysaccharides after administration of 840 mg/kg in broilers [12]. Further, Se-polysaccharides isolated from Lactococcus lactis showed a stronger effect on the immune system than non-selenated polysaccharides, which was manifested by enhanced macrophage phagocytosis and hemolytic complement activity in mice [379]. Pseudomonas PT-8 is another strain used in the biosynthesis of selenium-rich polymers. The obtained Se-exopolysaccharides (SePEPS) showed higher antioxidant and antiradical activity when compared to the unmodified biopolymers [334]. In turn, the Se-polysaccharides isolated from selenium-enriched cultures of cyanobacterium, Spirulina platensis, showed strong biological activity, dependent on the concentration and chemical form of selenium. It was discovered that Se-polysaccharide fraction had a protective effect against cadmium-induced toxicity in vitro and in vivo-in a rat model. The protective effect for Se-polysaccharide was superior compared to the reference polysaccharide or Na 2 SeO 3 alone. The authors hypothesize that this effect may be due to the covalent bonding of selenium to polysaccharide [380]. The results of another study confirmed that supplementing S. platensis with selenium increases the production of phycocyanins in the bacterial cells [381], and that those products showed antioxidant, anti-inflammatory, and antitumor properties, including against a very aggressive non-small-cell lung carcinoma (NSCLC). The last was investigated in vitro on three typical NSCLC cell lines, NCI-H1299, NCI-H460, and LTEP-A2; however, without the control (normal cell line) [382,383]. On the other hand, Zhu et al. found that the Se-polysaccharides isolated from S. platensis reduce the symptoms of sodium dextran sulfate-induced colitis in mice [384].

Algal Se-Polysaccharides
The antioxidant and antitumor properties of selenium polysaccharides were also investigated in algae, such as Sargassum fusiforme (Harv) Setch. In mice with S 180 tumor, selenium-enriched polysaccharides reduced the level of malondialdehyde, while increasing the activity of catalase, superoxide dismutase, and glutathione peroxidase in the blood, liver, and heart cells [385]. Additionally, Se-polysaccharides isolated from Ulva fasciata showed antiproliferative activity against human lung cancer cells A549 [386]. The selenium nanoparticles encapsulated in Ulva lactuca polysaccharides have also been observed to reduce the symptoms of sodium dextran sulfate-induced colitis in mice [295].
Data on Se-polysaccharides in microalgae are very limited and concern mainly selenium metabolism in these organisms, as in the work cited below [387].

Fungal Se-Polysaccharides
Compounds isolated from Hercium erinaceus were modified in order to obtain Sepolysaccharides. In vitro studies have shown that the use of the modified polysaccharides induced morphological changes in dendritic cells, increasing the expression of the surface molecules, key for antigen presentation [271]. Additionally, the Se-polysaccharide isolated from the mycelium of Ganoderma lucidum showed a tenfold higher inhibitory effect on the proliferation of human cancer cell lines when compared to the natural polysaccharide [388]. Further, in a study conducted by Wang et al., Se-polysaccharides from Hohenbuehelia serotina were isolated after the esterification of the polysaccharide with selenious acid [389]. Again, it was shown that the chemically modified compound had a greater antioxidant capacity than the natural polysaccharide [251]. Furthermore, Se-polysaccharides can be obtained by supplementing the culture medium used in the biosynthetic process with selenium, as was the case of Lentinula edodes mycelial cultures, which accumulated selenium from the culture medium. The incorporation of selenium into the L. edodes mycelium increased the immunomodulatory activity of the produced Se-polysaccharides that showed no cytotoxic effects and increased the cell survival [171]. Moreover, the protection test against oxidative stress induced by the H 2 O 2 exposure showed the antioxidant activity of a Se-polysaccharide fraction and the reference polysaccharides in HeLa cells. At the same time, the viability of cells treated with selenium polysaccharides was higher than that of the native polysaccharides, and the selenium polysaccharide fraction showed a greater effect on the modulation of the immune response [252].

Plant Se-Polysaccharides
The immunomodulatory properties of the natural garlic polysaccharides and those modified with selenium have been compared. The selenation process significantly increased the bioactivity of the polysaccharides. Hence, Se-polysaccharides showed a stronger stimulating effect of the cellular and humoral immune system. Further, chickens administered these Se-polysaccharides showed a higher level of lymphocyte proliferation and a higher serum antibody titer along with a higher stimulation of Th1 helper lymphocytes to produce IFN-γ and IL-2 [280]. In addition, the native polysaccharides isolated from the roots of Hedysarum polybotrys also showed an immunomodulatory effect [261]. However, the research demonstrated that the H. polybotrys polysaccharides modified with selenium had a stronger antioxidant effect, reflected by the removal of superoxide and hydroxyl radicals [390]. In another study with a group of rats administered CCl 4 , the biological activity of Se-polysaccharides isolated from the root of Astragalus membranoceus was compared with those of the natural polysaccharides. The results showed that selenopolysaccharides had a greater efficiency in increasing the activity of SOD, GPx, and MDA. Additionally, histopathological examination of the liver tissue of the rats confirmed the effectiveness of Se-polysaccharides in alleviating the inflammation and necrosis of hepatocytes, induced by the administration of CCl 4 by inactivating the Kupffer cells [270]. Research on selenium polysaccharides derived from Pyracantha fortunaeana showed that they can enhance the cytotoxic effect of doxorubicin on human breast cancer cells in vitro [391]. Furthermore, investigation in Se-polysaccharides from Platycodon grandiflorum indicated that seleniumenriched biopolymers had the ability to protect against oxidative damage induced by hydrogen peroxide in the pheochromocytoma cells of rats [392]. In another study, tea was identified as another plant source of bioactive Se-polysaccharides, and its ability to inhibit the proliferation of S 180 in mice was reported to be significantly higher than that of the Se-yeast (Se-enriched Saccharomyces cerevisiae) [392]. Green tea Se-polysaccharides may have a positive effect in the treatment of human osteosarcoma and greater antioxidant activity when compared to the native polysaccharides [170].

Se-Polysaccharides-Perspectives
For nearly twenty years, the selenium-enriched polysaccharides have been attracting great interest among scientists and have become a subject of comprehensive research. As a result, a large number of selenium-containing polysaccharides of diversified structure, origin, selenium binding, and oxidation state were obtained with a range of experimental methods. The biological activity of these compounds was also a subject of detailed investigation.
These compounds had proven to be very promising in terms of the potential use in the selenium supplementation and in the adjunctive therapy of many diseases. Sepolysaccharides are characterized by a low toxicity and a slow release of selenium, which is a great advantage over the "traditional" selenium supplements. The incorporation of selenium into a molecule of biologically active polysaccharides significantly enhances their action, which suggests the synergy of selenium and polysaccharides.
The main weakness of the current research is the lack of a broader look at the problem of the relationship between the structure and activity of Se-polysaccharides, and especially, the impact of the selenium binding method into the polysaccharide molecule and its oxidation degree on its activity. The treatment of seleno-polysaccharides as a structurally homogeneous group of compounds whose activity is affected only by the amount of selenium introduced into the molecule, regardless of its chemical environment or the degree of oxidation, is in our opinion an inappropriate approach. For example, it is difficult to expect a similar antioxidant effect from Se-polysaccharides containing selenium in the second, fourth, or zero oxidation state. Selenium compounds are often antioxidants, but can also act as pro-oxidants depending on their redox properties.
To summarize, it is necessary to use the standard methods of medicinal chemistry in the research on the preparation and activity of selenium-containing polysaccharides. The study of the structure-activity relationship requires preparation of a series of compounds with methodically introduced structural changes. To take a simple example: one could biosynthesize a selenium analogue of a natural polysaccharide containing glycosidically bound (divalent) selenium, then obtain a semisynthetic analog of the same polysaccharide containing ester-bound selenium (selenite with tetravalent selenium), and, finally, obtain selenium nanoparticles encapsulated with the same polysaccharide (zero-valent selenium) by a chemical or a biological method. A comparison of the activity of such seleniumcontaining analogues of a biologically active polysaccharide would provide conclusive data on the influence of "selenium" on the activity of the investigated polysaccharide. Of course, the study of the structure-activity relationship should consider the complex analysis of not only the primary structure of the Se-polysaccharide, but also the secondary and tertiary structure, which are very important in shaping biological activity other than simple redox properties. The published data shows that the incorporation of selenium into a polysaccharide molecule causes far-reaching deformation of the molecule. In case of the receptor activity (binding to the receptor), such changes are extremely important and should also be carefully investigated.
Regarding the wide scope and diversity of this research subject, the comprehensive research on the effect of selenium binding on the activity of Se-polysaccharides would require cooperation across various research centers, mostly specialized in certain methods of "selenating" polysaccharides.