**2.1.2 Ulvan conformation**

78 Biomaterials – Physics and Chemistry

(Brand & Morand, 1997) but as it will be stressed in the following part of the chapter, the chemicals and polymers of this underexploited biomass along with their abundance,

Green algae such as *Ulva sp.* are known to contain high amounts of good-quality protein, carbohydrate, vitamins and minerals (Taboada et al., 2010). Among these, polysaccharides are gaining increasing attention as they possess unique physical and chemical properties

Ulvan represents a class of sulphated heteropolysaccharide extracted from the cell wall of green seaweeds belonging to *Ulva sp.* whose composition has been extensively debated (Lahaye & Robic, 2007; Robic et al., 2009; ) and showed to vary according to several factors including the period of collection, the ecophysiological growth conditions, the taxonomic

Four types of polysaccharides are reported to be contained in the biomass of *Ulva sp.*, including the water soluble Ulvan and insoluble cellulose as major one and an alkali-soluble linear xyloglucan and glucuronan in minor amounts (Lahaye & Robic, 2007). Ulvan represents the major biopolymeric fraction of the cell wall having the function of maintaining the osmolar stability and protection of the cell (Paradossi et al., 2002). As usually found in polysaccharides present into the cell walls, Ulvan is present in close association with proteins and the conventional methods of extraction and purification resulted not completely effective in the removal of the protein fraction even after a specific

Extraction is conventionally achieved by using warm water solution (80-90°C) containing ammonium oxalate as divalent cation chelator and the recovery of Ulvan is generally obtained by precipitation in ethanol. The yield of extraction usually ranges from 8% to 29% of the algal dry weight depending on the applied purification procedure (Lahaye & Axelos,

The sugar composition of Ulvan is extremely variable but rhamnose, xylose, glucuronic and iduronic acid and the presence of sulphate groups have been identified as the main constituents of the polymer (Paradossi et al., 2002; Robic et al., 2009). These monomers are arranged in an essentially linear fashion even though a slight degree of branching has been found (Lahaye & Robic, 2007). The chemical heterogeneity of Ulvan is partially striken by a "structural motif" found within the heteropolymer chain essentially given by the presence of repeating dimeric sequences constituted by aldobiuronic acid disaccharides designated as type A (glucurorhamnose 3-sulphate, A3s) and type B (iduronorhamnose 3-sulphate, B3s) (Figure 3).

Fig. 3 Structure of the main disaccharide repeating units in Ulvan.

biological properties and "renewability" represent a potential source to be explored.

representing a versatile material platform for potential biological applications.

origins and the post-collection treatment of the algal sources (Lahaye & Robic, 2007).

**2.1 Chemical-physical properties** 

deproteinization protocol (Alves et al., 2010).

1993; Lahaye et al., 1994).

**2.1.1 Ulvan composition** 

The physical properties of polymeric materials are deeply affected by the association and conformation assumed by the constituting chains in the final product. The balance between ordered crystalline and disordered amorphous structures dictates the ultimate mechanical properties of the polymeric material. Indeed the possibility of forming crystalline regions inside a polymeric structure could even generate physical crosslinks between the chains inducing ultimately to the formation of stiff networks, as in the case of polyvinyl alcohol (Ricciardi et al, 2005). The achievement of suitable mechanical properties for a material to be used in biomedical applications, namely tissue engineering, represent a key requirement to fulfil since the final product must provide a physical support for the cell growth and differentiation.

Past investigations on this issue revealed an essentially disordered conformation of Ulvan (Paradossi et al., 1999) mainly induced by the heterogeneous chemical composition of this polysaccharide. The local regularity given by the repeating aldobiuronic units, denominated as A3s and B3s (Figure 3), is believed to be sufficient for the formation of transient "junctionzones" responsible for the formation of the weak gel that ulvan is known to perform in nature (Paradossi et al., 2002). The stability of these ordered structures can be affected by the attractive and repulsive interactions that form between the functional groups of the polysaccharide, and in particular by the electrostatic forces. Ulvan is an anionic polyelectrolyte as it contains carboxylic and sulphate groups inside its structure, so that its net charge strongly depends on the pH and ionic strength of the working medium. The net charge on Ulvan is found to affect the conformation of its polymeric chains and ultimately controls the order to disorder transitions given by the locally regular sequences (Paradossi et al., 2002). The conformational change from an ordered structure present in the uncharged chain, i.e. the protonated form of ulvan, toward a disordered state, happens when a critical charge density is reached and is induced only in the chemically regular portions of the chains. The structures of the ordered sequences have been hypothesized on the basis of molecular modelling calculations and are compatible with the formation of helical conformations inside homogeneous portions of the chains containing the repeating units A3s and B3s (Paradossi et al., 2002).

The presence of ordered structures limited only in the regular sequences of the Ulvan polymeric chains is not sufficient to provide enough "junction-zones" for the preparation of a material with mechanical properties suitable for biomedical applications. For this purpose Ulvan has to be modified through the introduction of chemical groups or molecules that increase the number of "junction-zones".

### **2.1.3 Ulvan morphology and solubility**

The possibility of chemically modifying Ulvan is strongly dependent on the physical availability of its functional groups so that its solubility and morphology in the working

Ulvan: A Versatile Platform of Biomaterials from Renewable Resources 81

induce a beneficial biological activity on the host organism or on the environment can be considered even more intriguing. This may be the case of Ulvan. Most of the positive health effects induced by this polysaccharide are generated by the presence of sulphate groups in its structure (Wijesekara et al., 2011). A wide list of beneficial biological effects reported by the literature span from antioxidant (Qi et al., 2006) to anticoagulant (Zhang et al., 2008), antitumor (Kaeffer et al., 1998) antihyperlipidemic (Yu et al., 2003) and immunomodulating

A brief discussion about the chemical mechanisms that trigger this bioactivity can be worth of mentioning in order to have a deeper insight on the potentiality of using this biomaterial in biomedical applications, and possibly find the "keys" to improve its

The research of new antioxidant from renewable natural resources able to scavenge free

In recent years, several classes of sulphated polysaccharides have been demonstrated to show antioxidant activity. Among them Ulvan extracted from *Ulva pertusa* is reported to play an important role as free radical scavenger *in vitro* and displayed antioxidant activity for the prevention of oxidative damage in living organisms (Qi et al., 2005). As found with other sulphated polysaccharides (Wijesekara et al., 2011) the antioxidant activity is deeply affected by the amount and distribution of sulphate groups inside the

The possibility to increase the antioxidant activity of Ulvan can be useful according to the envisaged application and has been successfully investigated both by increasing the degree of sulphation through a sulphur trioxide/N,N-dimethylformamide treatment (Qi et al., 2005) and by introducing suitable groups (acetyl and benzoyl) that can boost the activity of

Heparin, a glycosaminoglycan of animal origin containing carboxylic acid and sulphate groups, has been identified and used for more than fifty years as a commercial anticoagulant and it is widely used for the prevention of venous thromboembolic disorders (Pereira et al., 2002). The heparinoid-like structure of Ulvan makes it also able to provide anticoagulant activity. Indeed this class of polysaccharides displayed the inhibition of both the intrinsic pathways of coagulation or thrombin activity and the conversion of fibrinogen to fibrin (Zhang et al., 2008). The molecular weight of the polysaccharide showed an important effect on the anticoagulant activity indicating that longer chains were necessary to

This behavior has been found to be typical of sulphated polysaccharides of marine origins whose anticoagulant activity has been correlated to the content and position of the sulphate

The importance of finding sources of anticoagulants alternative to heparin has been arising due to the associated harmful side effects and the complex steps of purification required to face the immunological concerns and disease transmission associated with its use (Stevens, 2008). Thus the increasing demand for a safer anticoagulant therapy could be potentially

radicals can represent a virtuous strategy for preventing ROS-induced diseases.

(Leiro et al., 2007) activities, proved both *in vitro* and *in vivo*.

biological activity.

Ulvan structure.

the native polysaccharide (Qi et al., 2006).

**2.2.2 Anticoagulant activity** 

achieve thrombin inhibition.

groups inside the polymer chains (Melo et al., 2004).

**2.2.1 Antioxidant activity** 

medium could affect deeply its reactivity. Ulvan has been shown to dissolve only in water due to its charged and highly hydrophilic nature. Nevertheless, the obtained solutions are not transparent, indicating the formation of microaggregates of polymeric material not fully dispersed in the solvent. Indeed TEM analysis of Ulvan revealed the presence of aggregates of spherical shaped forms partially linked by strands-like filaments (Robic et al., 2009). This necklace-like ultrastructure is usually formed by polyelectrolyte material in poor solvent conditions (Dobrynin, 2008) so that even water can not be considered a good solvent for Ulvan. The large presence of methyl groups provided by the rhamnose repeating unit has been considered responsible for the unusual hydrophobic behavior of this highly charged polysaccharide (Robic et al., 2009).

The unusual low intrinsic viscosity of Ulvan in solution can also be ascribed to the presence of condensed spherical shaped aggregates not typical for polyelectrolytes whose conformation usually expands in the form of charged filaments and leads to an increase in the viscosity (Dobrynin et al., 1995). The formation of microaggregates in solution does not allow also a reliable mass analysis of Ulvan, whose different type of aggregation affects deeply the peak distributions usually found on the GPC chromatograms (Robic et al., 2009). Being a polyelectrolyte, both the ionic strength and the pH of the dissolving medium would affect the solubility and the morphology of Ulvan. Indeed the association of the bead-like aggregates in a necklace-type ultrastructure is promoted by the ionic interactions of carboxylated groups as demonstrated by its rupture at pH below the pKa of glucuronic acid (3.28) (Robic et al. 2009). In basic conditions (pH 13) the bead-like structures resulted to collapse into a dense homogeneous network likely prompted by the ionic interactions of carboxylate and sulphate groups. The type and amount of counter-ion in solution could also contribute to chain expansion or condensation as demonstrated by the aggregative propensity of Ulvan at low NaCl concentration observed by light scattering and rheological measurements (Lahaye & Robic, 2007).

The tendency of Ulvan to form aggregates in aqueous solution and its insolubility in almost every organic solvents limit the number of functional groups available for chemical modifications thus hampering its potential versatility. But its great number of reactive groups still present on the "free" surface exposed outside the aggregate and the possibility to optimize the solvent variables (pH and ionic strength) that affect the dispersion of the polymer in solution make Ulvan a suitable reactive platform, tailorable according to the envisaged application.

#### **2.2 Biological activity**

The possibility of using bio-based materials in almost every technological field and particularly in biomedical applications is challenging and can be considered the strategy of election for limiting environmental concerns and create a virtuous circle of sustainability.

Biomaterials possess the essential prerequisite of renewability and biocompatibility and as such are worth of deep investigations as main candidates for the substitution of synthetic petroleum-based materials, well known for being not renewable and often not biocompatible.

Biodegradability represents also an important property possessed by biomaterials and it is especially required in materials used for biomedical applications with specific reference to tissue engineering and regenerative medicine. Not only the material has to be safe but also the products of degradation should be non-toxic and easily cleared from the body. Biomaterials that other than being renewable, biocompatible and biodegradable are able to induce a beneficial biological activity on the host organism or on the environment can be considered even more intriguing. This may be the case of Ulvan. Most of the positive health effects induced by this polysaccharide are generated by the presence of sulphate groups in its structure (Wijesekara et al., 2011). A wide list of beneficial biological effects reported by the literature span from antioxidant (Qi et al., 2006) to anticoagulant (Zhang et al., 2008), antitumor (Kaeffer et al., 1998) antihyperlipidemic (Yu et al., 2003) and immunomodulating (Leiro et al., 2007) activities, proved both *in vitro* and *in vivo*.

A brief discussion about the chemical mechanisms that trigger this bioactivity can be worth of mentioning in order to have a deeper insight on the potentiality of using this biomaterial in biomedical applications, and possibly find the "keys" to improve its biological activity.
