**2. Crystallinity of 1,3-β-glucans**

### **2.1. Structure**

1,3-β-glucans are semi-crystalline polysaccharides comprising a combination of single helices, triple helices and random coils. The crystallinity of these polysaccharides has been

© 2012 Verma and Gu, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

studied using X-ray diffraction (XRD). In this study, curdlan, which is a linear 1,3-β-glucan, was used as a model polysaccharide [3]. Different forms and states of curdlan demonstrate different crystallinity. One example that was studied in detail is the annealed "dry" state, where the curdlan is dissolved in dimethyl sufoxide, extracted in methanol and annealed in the presence of water at 145 °C. Curdlan is then dried *in vacuo* to obtain the sample for XRD experiments. The results from XRD measurements conclude that six-fold triple-helices are formed with an advance of 2.935 Å per monomer unit. The model of this structure confirms that the three strands of triple helices are held together by hydrogen bonding between O(2) hydroxyls while the helices are brought together by O(4) and O(6) hydrogen bonding [3].

An alternate structure of curdlan helices is presented based on semi-empirical modeling. It is proposed that hydrogen bonding of the strands occurs along the helix axis rather than perpendicular to it. The different structures are illustrated in Figure 1. It is demonstrated that this alternate structure provides a more stable structure of curdlan and hence is likely to have higher population [25].

**Figure 1.** Illustration of possible orientations of curdlan triple helices: a) hydrogen bonding perpendicular to helix axis; b) hydrogen bonding along helix axis. Figure adapted from [25].

1,3-β-glucans have also been complexed with nucleotides to form crystalline structures. In the example of curdlan and poly(cytidylic acid) complex, semi-empirical modeling suggests that two glucose units of different curdlan chains form hydrogen bonds with one base of the nucleotide chain [26]. This property of curdlan complexing with nucleotides has been exploited in forming liquid crystalline gels with deoxyribonucleic acid (DNA). Such structures could be synthesized at varying scales ranging from nanometers to centimeters [27].

### **2.2. Liquid crystalline gels**

Curdlan can be used to form liquid crystalline gels when it is exposed to transition metal salts [28,29]. The crystallinity of these gels depends on the molecular weight of the gels [30]. DNA has also been used to synthesize gel beads [31]. When used together, DNA and curdlan provide control over the size and morphology of the synthesized hybrid structures. Various structures can be obtained by modifying the concentration of curdlan and DNA [27].

556 The Complex World of Polysaccharides

have higher population [25].

[27].

**2.2. Liquid crystalline gels** 

studied using X-ray diffraction (XRD). In this study, curdlan, which is a linear 1,3-β-glucan, was used as a model polysaccharide [3]. Different forms and states of curdlan demonstrate different crystallinity. One example that was studied in detail is the annealed "dry" state, where the curdlan is dissolved in dimethyl sufoxide, extracted in methanol and annealed in the presence of water at 145 °C. Curdlan is then dried *in vacuo* to obtain the sample for XRD experiments. The results from XRD measurements conclude that six-fold triple-helices are formed with an advance of 2.935 Å per monomer unit. The model of this structure confirms that the three strands of triple helices are held together by hydrogen bonding between O(2) hydroxyls while the helices are brought together by O(4) and O(6) hydrogen bonding [3].

An alternate structure of curdlan helices is presented based on semi-empirical modeling. It is proposed that hydrogen bonding of the strands occurs along the helix axis rather than perpendicular to it. The different structures are illustrated in Figure 1. It is demonstrated that this alternate structure provides a more stable structure of curdlan and hence is likely to

**Figure 1.** Illustration of possible orientations of curdlan triple helices: a) hydrogen bonding perpendicular to helix axis; b) hydrogen bonding along helix axis. Figure adapted from [25].

1,3-β-glucans have also been complexed with nucleotides to form crystalline structures. In the example of curdlan and poly(cytidylic acid) complex, semi-empirical modeling suggests that two glucose units of different curdlan chains form hydrogen bonds with one base of the nucleotide chain [26]. This property of curdlan complexing with nucleotides has been exploited in forming liquid crystalline gels with deoxyribonucleic acid (DNA). Such structures could be synthesized at varying scales ranging from nanometers to centimeters

Curdlan can be used to form liquid crystalline gels when it is exposed to transition metal salts [28,29]. The crystallinity of these gels depends on the molecular weight of the gels [30]. DNA has also been used to synthesize gel beads [31]. When used together, DNA and curdlan Curdlan is insoluble in water but it dissolves in alkaline solutions. Thus, DNA and curdlan are mixed together in a basic solution and then this mixture is added to a solution of calcium chloride salt either directly or through a dialysis membrane. Direct addition leads to formation of structures at the nanometer and millimeter scales. Dialysis allows for the formation of centimeter sized gels. The macroscopic structures are assessed by using crossed nicols (Figure 2), while the nano- and micro-structures are characterized using transmission electron microscopy (TEM, Figure 4). When viewing the centimeter scale gels between two perpendicularly placed polarizers, orthogonal dark lines are observed on the gels. These lines are known as isogyres and indicate the anisotropy in liquid crystalline gels. It is observed that increasing the concentration of DNA decreases the crystallinity of the gel as the isogyres become less defined. This is illustrated in Figure 2 [27].

**Figure 2.** Liquid crystalline gels of curdlan and DNA: 100% curdlan, 5% DNA, 10% DNA and 20 % DNA as seen under visible light (top) and crossed nicols (bottom). Scale bars are 1 cm each. Figure obtained from [27].

A similar phenomenon was observed at the milimeter scale when the structure was observed under the microscope. It was seen that although DNA provided rigidity and welldefined shape to the structure, it reduced the crystallinity. This is likely because DNA forms a less crystalline structure compared to curdlan. It is possible that DNA might not be forming helices with curdlan, but instead forming a gel with microphase separation. The results from millimetre scale are highlighted in Figure 3. The opacity and lack of isogyres in DNA sample implies low crystallinity. Thus, a simple method is presented to determine degree of crystallinity of gels qualitatively [27].

**Figure 3.** Spherical gels of curdlan and DNA observed under visible light (top and middle) and polarized light (bottom). Scale bars are 1 mm each. Figures obtained from [27].

**Figure 4.** TEM images of micro- and nano-structures of curdlan and DNA. White scale bars are 500 nm and black scale bars are 2000 nm. Figure reproduced from [27].

At the micro- and nano-scale levels, the morphology of the gels could be changed between spheres and fibers by modulating the concentration of the DNA or curdlan. This is summarized in Figure 4. These hybrid liquid crystalline gel systems have the potential of creating advanced drug delivery vehicles where the crystalline regions of the system prevent degradation of the biomolecule and the amorphous regions maintain functionality of the encapsulated moiety. This hybrid system also serves as a tool for further studies of the molecular structure undertaken by 1,3-β-glucan and DNA in various microenvironments [27].
