**2. Structure of mannans**

tomannan / galactoglucomannan). The mannose and glucose residues in the backbone are

In plants, mannans have a structural role by binding cellulose, but also they serve a storage function as a reserve carbohydrate in endosperm walls and vacuoles of seeds and vacuoles in vegetative tissues [5]. Recently, mannan has also been proposed as a signaling molecule in

Mannan is a biodegradable and bioactive polysaccharide that has been a focus of interest by various sectors due to its valuable properties. The film forming capacity and biodegradability of mannans make them an interesting alternative to the petroleum-based materials. Mannanbased films and coatings were shown to exhibit low oxygen and grease permeability and, in some cases, relatively high tensile strength [4]. There are also interesting reports on the successful use of mannan as a bioactive material in health related applications. Mannan conjugated to vaccine preparations are already in the clinic [7,8]. Tang et al. [9] utilized a mannan-based system to target DNA vaccines to antigen presenting cells and demonstrated that it could induce far stronger immune responses in mice compared to naked DNA immu‐ nization. By further studies, they could explain the molecular basis of the observed immune enhancing attributes of mannan-based DNA vaccination [9]. Successful use of carboxylic mannan-coated iron oxide nanoparticles in targeting immune cells for *in vivo* lymph nodespecific Magnetic Resonance Imaging was also reported recently [10]. Moreover, to target mannose receptor expressed on the surface of antigen-presenting cells, biocompatible selfassembled mannan nanogels were designed to provide a therapeutic or vaccine delivery platform [11,12]. In a recent review on oral drug delivery research in Europe, mannan based nanogels were considered as a new approach for the oral delivery of labile molecules [13].

In this chapter, after a brief description of mannan, its production by algae, fungi, bacteria and other eukaryotic microorganisms will be mentioned with special focus on microbial resources. Then, use of mannan as a bioactive material in nanocarrier systems for drug delivery appli‐ cations will be covered in detail by giving examples from literature and industry. The final

sometimes acetylated at C-2 or C-3 (3,5).

312 Application of Nanotechnology in Drug Delivery

**Figure 1.** Polysaccharide composition of plants

plant growth and development [6].

Mannan is one of the important member of the hemicellulose family and can be divided to four subfamilies: linear mannan, glucomannan, galactomannan, and galactoglucomanan [14]. Mannan is present in different forms, each having a β-1,4-linked backbone containing mannose (linear mannan) or a combination of glucose and mannose residues (glucomannan) and occasional side chains of α-1,6-linked galactose residues (galactomannan / galactoglucomann‐ an) (Figure 2). In the backbone, mannose and glucose units can also be acetylated at C-2 or C-3 (3,5) .

Glucomannan is mainly a straight-chain polymer, with a small amount of branching. The component sugars are β-(1,4)-linked D-mannose and D-glucose with a reported ratio of 1.6:1 [15], or 1.4:1 [16]. Softwoods and hardwoods consist of glucomannan with a glucose/mannose ratio of 1:3 and 1:1.5–2, respectively [17-20]. There is a significant similarity between confor‐ mation of glucomannan chains and those of cellulose A two-fold screw axis was observed because of the extended chains. Due to axial position of the hydroxyl group at C-2 of mannose, the interaction between C-6 and O-2 atoms of contiguous residues is prevented, and the chains are loosened, weakening the packing and organization [17]. Different structures were reported for glucomannans isolated from different sources. For example, (1 → 4)-linked structure, acetyl groups at C-2, C-3 positions and O-acetyl group at C-6 position were reported for glucomannan extracted from seeds of *Lupinus* [21]. Irregular distribution of acetyl groups was reported for pine glucomannan [22]. Studies on a nonionic glucomannan with a main chain of β-(1 → 4) linked mannopyranosyl units to which D-glucopyranosyl units are linked by α-(1 → 6) linkages, isolated from seeds of *Bryonia lacinosa* was also reported [23]. Galactomannans are polysaccharides consisting of 1,4-linked β-D-mannopyranose backbone with side chains of single 1,6-linked α-D-galactopyranose attached along the chain [24-26]. Galactose to mannose ratio show differences among different sources. More than 5% galactose residues can be considered as galactomannans [27].They are mainly found in the seeds of the family of *Leguminoseae* [28,29]. They are also present in the species of *Annonaceae*, *Convolvulaceae*, *Ebenaceae*, *Loganiaceae*, and *Palmae*[29]. Unusual backbone structure, containing (1 → 3)-linked residues together with a small proportion of (1 → 4)-linked β-D-mannopyranosyl residues with galactopyranosyl units attached at position 6, of galactomannan isolated from *Retama raetam* was reported in 2004 [30]. Presence of arabinosyl and glucosyl residues in the structure of galactomannans was observed in the studies of green and roasted coffee [31]. Several lichen species have been also reported as a source of galactomannan [32]. (1 → 6)-α-D-mannopyra‐ nosyl backbone with a different substitution pattern at O-2 and O-4 was observed in galacto‐ mannans isolated from lichens. The four major galactomannans of commercial importance in food and non-food industries are guar gum (GG, *Cyamopsis tetragonolobo*, M/G ratio: 2:1), tara gum (TG, *Caesalpinia spinosa*,M/G ratio: 3:1), locust bean gum (LBG, *Ceratonia siliqua*, M/G ratio: 3.5:1) and Fenugreek (*Trigonella foenum-graecum* L., M/G ratio: 1:1) [33].

addition to carbohydrate storage and structure, mannans serve a variety of other functions. In fern roots, mannans are deposited as constituents of cell wall appositions as a defense mechanism to limit microbial ingress [45]. Besides plants, algae are also a viable resource for mannan polysaccharides. In particular, the Dasycladalean alga Acetabularia acetabulum, also known as 'mannan weed', has long been known to contain mannan-rich walls [46]. Moreover, mannans are a common feature of fungal walls and a recent review points to the importance of cell surface mannans of pathogenic Candida species since they were found to participate in the adhesion to the epithelial cells, recognition by innate immune receptors and development of pathogenicity. Hence, clarification of the precise chemical structure of Candida mannan was reported as indispensable for understanding the mechanism of pathogenicity, and for development of new antifungal drugs and immunotherapeutic procedures [47]. Also, some yeast species stand out for their capability for excreting mannan to the fermentation medium. Yeast Rhodotorula acheniorum MC bioreactor cultures have been reported to produce 6.2 g/L mannan when grown for 96 hours in sucrose containing media [48]. Moreover, psychro‐ philic Antarctic yeast Sporobolomyces salmonicolor AL1 reached maximum glucomannan yield of 5.64 g/L in medium containing sucrose after a 5 days of fermentation [49]. Mannan synthesized by R. acheniorum MC, as well as the glucomannan, synthesized from strain *S. salmonicolor* AL1 were both found to form stable emulsions making them suitable for various applications in pharmaceutical and cosmetic sectors [50]. On the other hand, studies also point

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Although mannan production is established by numerious algal, fungi and other eukaryotic microorganisms, they are not normally products of bacteria [52]. There are only very few reported examples on extracellular mannan production by bacteria. Gram negative phytopa‐ thogenic bacterium *Pseudomonas syringae pv. ciccaronei* was reported to produce a highly branched phytotoxic mannopyranose polymer, which consisted of a backbone of α-(1,6)-linked mannopyranose units with 80% substituted at C-2 by mono-, di-and trisaccharide side chains [53]. Then, to understand the role of this mannan polymer in the activation of plant defence responses, various concentrations of the polymer was infiltrated in the abaxial side of tobacco leaves. Mannan polysaccharide was found to induce chlorotic and necrotic symptoms even at very low concentrations very effectively suggesting that it was identified by the plant cells as a signal of pathogen attack or environmental perturbation [54]. Two mannans at different chain lengths were reported to be produced by the marine bacterium *Edwardsiella tarda*, an oppor‐ tunistic pathogen in human, and the polysaccharides were found to have good antioxidant and hydroxyl and DPPH radicals scavenging activities [55]. The lower molecular weight mannan was associated with higher antioxidant activity than the longer mannan and could be used as possible food supplement or ingredient in the pharmaceutical industry [55]. Recently, about 20-fold increase in mannan production has been reported in the pathogenic, constitutive biotin-producing *Pseudomonas mutabilis* bacteria [56]. The rheological properties of the highly branched mannan isolated from *P. mutabilis* T6 showed that its viscosity was over 30 times

to adverse toxic effects of fungal mannans when administered [51].

greater than that of the wild type *P. mutabilis* ATCC 31014.

Table 1. illustrates mannan producer organisms.

Galactoglucomannan consists of a backbone of randomly distributed (1 → 4)-linked mannose and glucose units with (1 → 6)-linked galactose units attached to mannose units. The hydroxyl groups in locations C2 and C3 in mannose are partially substituted by acetyl groups [34,18]. Molar ratio of mannose, glucose and galactose was reported as 3:1:1 in the study of Puls and Schuseil [35]. Some of the mannosyl units are partially substituted by O-acetyl groups, equally distributed between C-2 and C-3 on the average one group per three to four hexose units [18, 36]. 5.9%-8.8% acetyl content was also observed [18].

The acetylated galactoglucomannan is mainly found in hemicellulose of softwoods. They can be either galactose rich or galactose poor with 10-15% and 5-8% of the dry woods respectively [36-38]. Acetylation at C-2 and C-3 positionsin the ratio of 2.2:1 was reported for galactoglu‐ comannan backbone from native Norway spruce wood [36]. Formation of strong hydrogen bonds due to large content of D-galactose side-chains prevents the macromolecules from aligning themselves and hence galactoglucomannan is soluble in water [39].

## **3. Sources of mannans**

Mannan is the predominant hemicellulosic polysaccharide in softwoods from gymnosperms, but is the minor hemicellulose in hardwood from angiosperms [35]. Unsubstituted beta-1,4 mannan, composed of a main chain of beta mannopyranose residues, is an important structural component of some marine algae [40] and terrestrial plants such as ivory nut [41] and coffee bean [42].

A variety of plants store energy in the form of mannans in their endosperm tissue, including members of the Palmae, Liliaceae, Iridaceae, and Leguminosae families [43,44]. Glucomannans also are used for energy storage in corms of plants within the genus Amorphophallus. In addition to carbohydrate storage and structure, mannans serve a variety of other functions. In fern roots, mannans are deposited as constituents of cell wall appositions as a defense mechanism to limit microbial ingress [45]. Besides plants, algae are also a viable resource for mannan polysaccharides. In particular, the Dasycladalean alga Acetabularia acetabulum, also known as 'mannan weed', has long been known to contain mannan-rich walls [46]. Moreover, mannans are a common feature of fungal walls and a recent review points to the importance of cell surface mannans of pathogenic Candida species since they were found to participate in the adhesion to the epithelial cells, recognition by innate immune receptors and development of pathogenicity. Hence, clarification of the precise chemical structure of Candida mannan was reported as indispensable for understanding the mechanism of pathogenicity, and for development of new antifungal drugs and immunotherapeutic procedures [47]. Also, some yeast species stand out for their capability for excreting mannan to the fermentation medium. Yeast Rhodotorula acheniorum MC bioreactor cultures have been reported to produce 6.2 g/L mannan when grown for 96 hours in sucrose containing media [48]. Moreover, psychro‐ philic Antarctic yeast Sporobolomyces salmonicolor AL1 reached maximum glucomannan yield of 5.64 g/L in medium containing sucrose after a 5 days of fermentation [49]. Mannan synthesized by R. acheniorum MC, as well as the glucomannan, synthesized from strain *S. salmonicolor* AL1 were both found to form stable emulsions making them suitable for various applications in pharmaceutical and cosmetic sectors [50]. On the other hand, studies also point to adverse toxic effects of fungal mannans when administered [51].

Although mannan production is established by numerious algal, fungi and other eukaryotic microorganisms, they are not normally products of bacteria [52]. There are only very few reported examples on extracellular mannan production by bacteria. Gram negative phytopa‐ thogenic bacterium *Pseudomonas syringae pv. ciccaronei* was reported to produce a highly branched phytotoxic mannopyranose polymer, which consisted of a backbone of α-(1,6)-linked mannopyranose units with 80% substituted at C-2 by mono-, di-and trisaccharide side chains [53]. Then, to understand the role of this mannan polymer in the activation of plant defence responses, various concentrations of the polymer was infiltrated in the abaxial side of tobacco leaves. Mannan polysaccharide was found to induce chlorotic and necrotic symptoms even at very low concentrations very effectively suggesting that it was identified by the plant cells as a signal of pathogen attack or environmental perturbation [54]. Two mannans at different chain lengths were reported to be produced by the marine bacterium *Edwardsiella tarda*, an oppor‐ tunistic pathogen in human, and the polysaccharides were found to have good antioxidant and hydroxyl and DPPH radicals scavenging activities [55]. The lower molecular weight mannan was associated with higher antioxidant activity than the longer mannan and could be used as possible food supplement or ingredient in the pharmaceutical industry [55]. Recently, about 20-fold increase in mannan production has been reported in the pathogenic, constitutive biotin-producing *Pseudomonas mutabilis* bacteria [56]. The rheological properties of the highly branched mannan isolated from *P. mutabilis* T6 showed that its viscosity was over 30 times greater than that of the wild type *P. mutabilis* ATCC 31014.

Table 1. illustrates mannan producer organisms.

linkages, isolated from seeds of *Bryonia lacinosa* was also reported [23]. Galactomannans are polysaccharides consisting of 1,4-linked β-D-mannopyranose backbone with side chains of single 1,6-linked α-D-galactopyranose attached along the chain [24-26]. Galactose to mannose ratio show differences among different sources. More than 5% galactose residues can be considered as galactomannans [27].They are mainly found in the seeds of the family of *Leguminoseae* [28,29]. They are also present in the species of *Annonaceae*, *Convolvulaceae*, *Ebenaceae*, *Loganiaceae*, and *Palmae*[29]. Unusual backbone structure, containing (1 → 3)-linked residues together with a small proportion of (1 → 4)-linked β-D-mannopyranosyl residues with galactopyranosyl units attached at position 6, of galactomannan isolated from *Retama raetam* was reported in 2004 [30]. Presence of arabinosyl and glucosyl residues in the structure of galactomannans was observed in the studies of green and roasted coffee [31]. Several lichen species have been also reported as a source of galactomannan [32]. (1 → 6)-α-D-mannopyra‐ nosyl backbone with a different substitution pattern at O-2 and O-4 was observed in galacto‐ mannans isolated from lichens. The four major galactomannans of commercial importance in food and non-food industries are guar gum (GG, *Cyamopsis tetragonolobo*, M/G ratio: 2:1), tara gum (TG, *Caesalpinia spinosa*,M/G ratio: 3:1), locust bean gum (LBG, *Ceratonia siliqua*, M/G ratio:

Galactoglucomannan consists of a backbone of randomly distributed (1 → 4)-linked mannose and glucose units with (1 → 6)-linked galactose units attached to mannose units. The hydroxyl groups in locations C2 and C3 in mannose are partially substituted by acetyl groups [34,18]. Molar ratio of mannose, glucose and galactose was reported as 3:1:1 in the study of Puls and Schuseil [35]. Some of the mannosyl units are partially substituted by O-acetyl groups, equally distributed between C-2 and C-3 on the average one group per three to four hexose units [18,

The acetylated galactoglucomannan is mainly found in hemicellulose of softwoods. They can be either galactose rich or galactose poor with 10-15% and 5-8% of the dry woods respectively [36-38]. Acetylation at C-2 and C-3 positionsin the ratio of 2.2:1 was reported for galactoglu‐ comannan backbone from native Norway spruce wood [36]. Formation of strong hydrogen bonds due to large content of D-galactose side-chains prevents the macromolecules from

Mannan is the predominant hemicellulosic polysaccharide in softwoods from gymnosperms, but is the minor hemicellulose in hardwood from angiosperms [35]. Unsubstituted beta-1,4 mannan, composed of a main chain of beta mannopyranose residues, is an important structural component of some marine algae [40] and terrestrial plants such as ivory nut [41] and coffee

A variety of plants store energy in the form of mannans in their endosperm tissue, including members of the Palmae, Liliaceae, Iridaceae, and Leguminosae families [43,44]. Glucomannans also are used for energy storage in corms of plants within the genus Amorphophallus. In

3.5:1) and Fenugreek (*Trigonella foenum-graecum* L., M/G ratio: 1:1) [33].

aligning themselves and hence galactoglucomannan is soluble in water [39].

36]. 5.9%-8.8% acetyl content was also observed [18].

**3. Sources of mannans**

314 Application of Nanotechnology in Drug Delivery

bean [42].


**4. Biosynthesis of mannans**

Mannans are synthesized from activated nucleotide sugars such as GDP-mannose, GDPglucose, and UDP-galactose [75]. Enzymes necessary for the nucleotide sugar conversion from sucrose to GDP-mannose and UDP-galactose have been reported in planta. However, the enzyme for the formation of GDP-glucose has not been identified [76]. Golgi-localized glycosyltransferases (GTs) utilize the activated nucleotide sugars and synthesize the polymer

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The cellulose synthase-like family A (CSLA) genes are considered the best candidates to encode enzymes that polymerize the backbones of β-linked noncellulosic polysaccharides [79,80]. Experimental evidence to support this hypothesis for the CslA family came first from Dhugga et al. [81]. In this research, the first β-mannan synthase (ManS), a member of the cellulose synthase-like family A (CSLA) from GT family 2, was identified in guar seeds (CtManS in Cyamopsis tetragonoloba, a AtCSLA9 ortholog) including the demonstration of its in vitro ManS activity [82]. One year later, three Arabidopsis CSLA genes were expressed in Droso‐ phila Schneider 2 (S2) cells and demonstrated that the resulting CSLA proteins were capable of producing mannans when supplied with GDP-Man and glucomannans when provided with a mixture of GDP-Man and GDP-Glc [75]. CSLA genes appear to be present in all land plants, and ancestral genes with characteristics similar to CSLA sequences have been identified in a number of green algal genomes, in which they are thought to represent a homolog of the progenitor gene from which CSLA genes evolved [76]. In developing Trigonella foenumgraecum (Fenugreek) endosperm, a deep sequencing approach was used to identify genes involved in galactomannan biosynthesis [83]. This research reported a CSLA family protein involved in mannan backbone synthesis and a preference towards GDP-mannose as a donor substrate was observed from the activity assays with the heterologously expressed protein. Heterologously expressed CSLA proteins from a variety of species show mannan or gluco‐ mannan synthase activity in vitro [6,75,81,83]. Analysis of Arabidopsis CSLA mutants and over-expressing plants further confirmed that CSLA proteins function as glucomannan synthases *in vivo* [84]. Despite this progress in identifying and characterizing the enzymes responsible for galactoglucomannan biosynthesis, it is likely that other important enzymes are

by facilitating the formation of the specific linkage between the monomers [77,78].

required, and many aspects of this process need to be better understood.

stability or activity of a mannan synthase complex [93].

In tissues of *Arabidopsis*, that take role in tip-growth such as root hairs CSLD, (*At*CSLD2, 3 and 5) proteins were found to mediate mannan biosynthesis [85-92]. In Fenugreek, it was found that additional genes were involved in mannan biosynthesis, such as a golgi-localized mannan synthesis-related (*MSR*) gene that was observed in the fenugreek endosperm [83,93]. *Tf*MSR protein in Fenugreek and its homologs *AtMSR1* and *AtMSR2* in *Arabidopsis* were highly coexpressed with the ManS of the CSLA family. Glucomannan and ManS activity were signifi‐ cantly decreased in stems of *AtMSR* knock-out mutants [93]. While the biochemical activity of MSR proteins remains unknown, hypotheses include a role in primer synthesis to initiate mannan biosynthesis, the synthesis of oligosaccharides linked to CSLA or promoting folding,

**Table 1.** Mannan producer organisms
