Current Chemical Biology, 2009, 3, 203-230

203

Functional Characterization of Chitin and Chitosan
Inmaculada Aranaz, Marian Mengíbar, Ruth Harris, Inés Paños, Beatriz Miralles, Niuris Acosta, Gemma Galed and Ángeles Heras*
Department of Physical Chemistry II, Faculty of Pharmacy, Institute of Biofunctional Studies, Complutense University, Paseo Juan XXIII, nº 1. Madrid 28040, Spain
Abstract: Chitin and its deacetylated derivative chitosan are natural polymers composed of randomly distributed -(1-4)linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitin is insoluble in aqueous media while chitosan is soluble in acidic conditions due to the free protonable amino groups present in the D-glucosamine units. Due to their natural origin, both chitin and chitosan can not be defined as a unique chemical structure but as a family of polymers which present a high variability in their chemical and physical properties. This variability is related not only to the origin of the samples but also to their method of preparation. Chitin and chitosan are used in fields as different as food, biomedicine and agriculture, among others. The success of chitin and chitosan in each of these specific applications is directly related to deep research into their physicochemical properties. In recent years, several reviews covering different aspects of the applications of chitin and chitosan have been published. However, these reviews have not taken into account the key role of the physicochemical properties of chitin and chitosan in their possible applications. The aim of this review is to highlight the relationship between the physicochemical properties of the polymers and their behaviour. A functional characterization of chitin and chitosan regarding some biological properties and some specific applications (drug delivery, tissue engineering, functional food, food preservative, biocatalyst immobilization, wastewater treatment, molecular imprinting and metal nanocomposites) is presented. The molecular mechanism of the biological properties such as biocompatibility, mucoadhesion, permeation enhancing effect, anticholesterolemic, and antimicrobial has been updated.

Keywords: Chitin, chitosan, molecular weight, deacetylation degree, crystallinity, functional characterization. 1. INTRODUCTION Among the novel families of biological macromolecules, whose relevance is becoming increasingly evident, are chitin and its main derivative, chitosan. Potential and usual applications of chitin, chitosan and their derivatives are estimated to be more than 200 [1]. This wide range of applications includes biomedicine, food, biotechnology, agriculture and cosmetics, among others. The importance of chitin and chitosan in the last years is evident in Table 1. Chitin and chitosan are described as a family of linear polysaccharides consisting of varying amounts of (1 4) linked residues of N-acetyl-2 amino-2-deoxy-D-glucose (denoted in this review as A residues) and 2-amino-2-deoxy-Dglucose residues (denoted in this review as D residues). Chitin samples have a low amount of D units and hence the polymer is insoluble in acidic aqueous media (Fig. 1a). On the other hand, the amount of D units in chitosan samples is high enough to allow the polymer to dissolve in acidic aqueous media. Some authors consider that chitosan is the polymer with at least 60% of D residues [2]. Chitin is the second most abundant natural polymer in nature after cellulose and it is found in the structure of a wide number of invertebrates (crustaceans’ exoskeleton, insects’ cuticles) and the cell walls of fungi, among others. On the other hand, chitosan only occurs naturally in some fungi (Mucoraceae) [3].
*Address correspondence to this author at the Institute of Biofunctional Studies, Complutense University, Paseo Juan XXIII, nº 1, Madrid 28040, Spain; Tel/Fax: +34-913943284; E-mail: aheras@farm.ucm.es; inma@ieb.ucm.es 1872-3136/09 $55.00+.00

Chitosan can also be prepared by cleavage of N-acetyl groups of the chitin A residues. This reaction is rarely conducted to full completion; hence chitosan polymeric chain is generally described as a copolymeric structure comprised of D-glucosamine (D residues) along with N-acetyl residues (Fig. 1b). The fine structure of chitosan is defined by the overall or bulk content of D-hexosamine residues as well as their distribution along the polymeric chain. The molar fraction of residual A groups in chitosan is expressed as a degree of Nacetylation (DA) or fraction of acetylation (Fa). The molar fraction of D residues, deacetylation degree (DD), is also very frequently used. In contrast to chitin, the presence of free amine groups along the chitosan chain allows this macromolecule to dissolve in diluted aqueous acidic solvents due to the protonation of these groups, rendering the corresponding chitosan salt in solution. So, there are important experimental variables that should be taken into account when working with chitosan solutions such as the nature of the salt counterion, degree of acetylation, Mw, pH, ionic strength and the addition of a non-aqueous solvent. The aim of the present review is to present a state-of-theart study of the relationship between the physico-chemical properties of these two polymers and their biological activities, as well as their applications. Since this aim is very ambitious, due to the extension of the topic, chitin and chitosan derivatives are not considered. The review has been divided into the following sections: the first part is devoted to the preparation, characterization,
© 2009 Bentham Science Publishers Ltd.

204 Current Chemical Biology, 2009, Vol. 3, No. 2

Aranaz et al.

Table 1.

Number of Scientific Publications Related to Chitin and Chitosan. Source: Scopus. Publication Year After 2000
Search Chitin and not chitosan Chitosan and not chitin Chitin and chitosan Reviews 182 401 119 Articles 2741 5959 2040 Patents 9064 20041 11804

effects of the preparation process on the properties of chitin and chitosan and regulatory aspects. The second part covers the main biological properties of the polymers and relates these properties to the physicochemical characteristics. Finally, several applications of both polymers are reviewed emphasizing the effect of the polymers’ characteristics on these applications.
A
H OH HO HO H O H O H NH C H CH3 HO O H H O CH3 C H NH H OH O HO H n O HO H OH NH H C CH3

H O H OH

B
H OH HO HO H O H O H NH C H CH3 HO O H H H NH2 H O H OH n O HO

H OH HO H H OH NH2 H

Fig. (1). Chemical structure of 100% acetylated chitin (A) and chitosan (B).

2. METHODS OF PREPARATION A schematic representation of the processes to prepare chitin and chitosan from raw material is shown in Scheme 1. 2.1. Chitin Extraction As mentioned above, chitin is present within numerous taxonomic groups. However, commercial chitins are usually isolated from marine crustaceans, mainly because a large amount of waste is available as a by-product of food processing. In this case, -chitin is produced while squid pens are used to produce -chitin. The structure of -chitin has been investigated more extensively than that of either the - or - form, because it is the most common polymorphic form. Very few studies have been carried out on - chitin. It has been suggested that chitin may be a distorted version of either - or -chitin rather than a true third polymorphic form [3]. In –chitin, the chains are arranged in sheets or stacks, the chains in any one sheet having the same direction or ‘sense’. In -chitin, adjacent sheets along the c axis have the same direction; the sheets are parallel, while in -chitin adjacent sheets along the c axis have the opposite direction, they are antiparallel .In - chitin, every third sheet has the opposite direction to the two preceding sheets [3]. A schematic representation of the three structures is shown in Fig. (2).
Scheme 1. Preparation of chitin and chitosan from raw material.

Crustacean shells consist of 30-40% proteins, 30-50% calcium carbonate, and 20-30% chitin and also contain pigments of a lipidic nature such as carotenoids (astaxanthin, astathin, canthaxanthin, lutein and -carotene). These proportions vary with species and with season. On the other hand, -chitin is associated with a higher protein content but lower carbonate concentration. Chitin is extracted by acid treatment to dissolve the calcium carbonate followed by alkaline extraction to dissolve the proteins and by a depigmentation step to obtain a colourless product mainly by removing the astaxantine [4]. 2.2. Chitin Deacetylation Chitosan is prepared by hydrolysis of acetamide groups of chitin. This is normally conducted by severe alkaline hydrolysis treatment due to the resistance of such groups imposed by the trans arrangement of the C2-C3 substituents in the sugar ring [5]. Thermal treatments of chitin under strong aqueous alkali are usually needed to give partially deacetylated chitin (DA lower than 30%), regarded as chitosan. Usually, sodium or potassium hydroxides are used at a concentration of 30-50% w/v at high temperature (100ºC). In general, two major different methods of preparing chitosan from chitin with varying degree of acetylation are

Functional Characterization of Chitin and Chitosan

Current Chemical Biology, 2009, Vol. 3, No. 2

205

Fig. (2). Three polymorphic configurations of Chitin (A) -chitin, (B) ß-chitin and (C) -Chitin.

known. These are the heterogeneous deacetylation of solid chitin and the homogeneous deacetylation of pre-swollen chitin under vacuum (by reducing pressure) in an aqueous medium. Heterogeneous deacetylation, which is the preferred industrial treatment, involves preferential reaction in the amorphous regions of the polymer, leaving almost intact the intractable crystalline native regions in the parent chitin. Alternatively, homogeneous modification is conducted by use of moderately concentrated alkali (13% w/w) acting on pre-swollen chitin to improve the interaction with the alkali and left to react at 25-40ºC for 12-24 hours. In both, heterogeneous or homogeneous conditions, the deacetylation reaction involves the use of concentrated alkali solutions and long processing times which can vary depending on the heterogeneous or homogeneous conditions from 1 to nearly 80 hours. Factors that affect the extent of deacetylation include concentration of the alkali, previous treatment, particle size and density of chitin. The last two factors affect penetration rate of the alkali into the amorphous region and to some extent also into the crystalline regions of the polymer, needed for the hydrolysis to take place. In practice, the maximal DD that can be achieved in a single alkaline treatment is about 75-85% [3]. In general, during deacetylation, conditions must be the proper ones to deacetylate, in a reasonable time, the chitin to yield a chitosan soluble in diluted acetic acid. Thiophenol and NaBH4 have been used as oxygen scavenger and reducing agents, respectively, thus effectively resulting in a product of greater viscosity [6]. Also, treatments with concentrated NaOH in the presence of water-miscible diluents such as 2-propanol, 2-methyl-2-propanol, polyethylene glycol dimethyl ether, acetone or paraffin oil have enabled the volume of concentrated NaOH required to be reduced by at least 85%. Several alternative processing methods have also been developed to reduce the long processing times and large amounts of alkali typically needed to deacetylate chitin to an acid-soluble derivative. Examples of these include the use of successive alkali treatments using thiophenol in DMSO [7]; thermo-mechanical processes using a cascade reactor operated under low alkali concentration [8]; flash treatment under saturated steam [9]; use of microwave dielectric heating [10]; and intermittent water washing [11]. There is evidence that in certain bacteria and fungi, enzymatic deacetylation can take place [12]. Deacetylases have been isolated from various types of fungi, namely Mucor rouxii, Aspergillus nidulans and Colletotrichum lindemuthianium. However, the activity of these deacetylases is severely limited by the insolubility of the chitin substrate. There have been some attempts to use amorphous chitin of high DA as a substrate for the deacetylase enzyme, however no acid-soluble chitosan could be isolated and characterized

[13]. The lack of solubility of chitinous substrates with high DA in aqueous solvents still represents a practical limitation for the preparation of chitosan using the chitin deacetylase system, a process which so far has been achieved in vivo [14]. 2.3. Chitosan Depolymerization The main limitations in the use of chitosan in several applications are its high viscosity and low solubility at neutral pH. Low molecular weight (Mw) chitosans and oligomers can be prepared by hydrolysis of the polymer chains. For some specific applications, these smaller molecules have been found to be much more useful. Chitosan depolymerization can be carried out chemically, enzymatically or physically. Chemical depolymerization (Fig. 3) is mainly carried out by acid hydrolysis using HCl or by oxidative reaction using HNO2 and H2O2 [15]. It has been found to be specific in the sense that HNO2 attacks the amino group of D-units, with subsequent cleavage of the adjacent glycosidic linkage. In the case of enzymatic depolymerization, low molecular weight chitosan with high water solubility were produced by several enzymes such as chitinase, chitosanase, gluconase and some proteases. Non-specific enzymes including lysozyme, cellulase, lipase, amylase and pectinase that are capable of depolymerizing chitosan are known [16]. In this way, regioselective depolymerization under mild conditions is allowed. Physical depolymerization yielding dimers, trimers and tetramers has been carried out by radiation (Co-60 gamma rays) but low yields have been achieved [17].
NHR' HO RO HOH2C O HOH2C O HO O O NH3 R = H, GlcN, GlcNAc R' = H, Ac Chitosan HNO2 HOH2C O HO CHO NHR' HO OR HO O HOH2C NHR' HO O HOH2C OR

HOH2C RO HO

O NHR'

O

Fig. (3). Chemical depolymerization of chitosan.

2.4. Influence of the Preparation Methods on the Physicochemical Characteristics The preparation method is a factor that affects the sample characteristics. Early studies have clearly demonstrated that specific characteristics of these products (Mw, DD) depend on the process conditions.

206 Current Chemical Biology, 2009, Vol. 3, No. 2

Aranaz et al.

Typically commercial chitins are prepared by a first step of deproteinisation followed by a second step of demineralization. In these conditions a “collapsed chitin”, in which the native structure of the chitin is lost, is extracted. On the other hand, “compacted chitin”, in which the native chain and fibrous structures are intact and stabilized, is extracted when demineralization occurred in the first step. Another way to damage chitin structure was found to be even brief exposure to bleaching agents [18]. The DA value of the bulk molecules depends directly on the process conditions. Early studies by Kurita and coworkers showed that chitosan produced under homogeneous conditions presented broad X-ray diffraction patterns, which was interpreted as a consequence of a more randomly distributed fine structure in terms of A and D groups [19,20]. It has become evident that the overriding factor regarding the fine structure of chitosan is the chemical polydispersion of the DA value [21]. During chitosan deacetylation, the degradation of the polymeric chain takes place. At the same time, the crystallinity of chitosan can be damaged by using harsh reaction conditions [22]. Taking these two facts into account, the reaction conditions must be controlled when preparing chitosan [23]. Our findings have shown that the proper conditions to deacetylate chitin avoiding high degradation involve using heterogeneous conditions with NaOH 75% (w/v) and a temperature of 110ºC [24]. The type of crustacean and the chitin isolation process are also factors that affect chitosan quality [25]. 3. METHODS OF CHARACTERIZATION As will be shown in this review, chitin and chitosan characteristics have a great effect on their properties and hence on their possible applications. In fact, not every chitin or chitosan sample can be used for the same applications. That is why a complete characterization of the samples is mandatory. Three crystalline forms are known for chitin: -, - and -chitins. Chitosan is also crystalline and shows polymorphism depending on its physical state. Depending on the
Table 2.

origin of the polymer and its treatment during extraction from raw resources, the residual crystallinity may vary considerably. Crystallinity is maximal for both chitin (i.e. 0% deacetylated) and fully deacetylated chitosan (i.e. 100%). Rinaudo has reported in a recent review that the origin of chitin influences not only its crystallinity and purity but also its polymer chain arrangement, and hence its properties [26]. It has also been reported that the surface area of the material is related to the source (i.e., crab>lobster >shrimp). The main parameters affecting the polymer properties are DD, Mw, polydispersity and crystallinity. For applications related to human consumption such as food and medical applications, the purity (ash content), the moisture and the content of heavy metals, endotoxin and proteins must be determined. It has been reported that the DD is one of the most important chemical characteristics, [27] which could influence the performance of chitosan in many of its applications [28]. The influence of average Mw on the viscosity development of aqueous solutions plays a significant role in the biochemical and biopharmacological significance of chitosan [29]. It is important to note that due to its low solubility chitin Mw is not easily determined. As is shown in Table 2, various methods have been reported for the determination of chitin and chitosan characteristics [30-45]. Different results are obtained when using methods based on different principles. Therefore, it is important to indicate the characterization method. Today, even the best characterized chitosans available in the market are usually described only with regard to their average degree of acetylation and their average degree of polymerization (DP), their ash content and the absence of contaminating bacteria, in some cases also indicating the polydispersity index. In addition to the above criteria, the distribution of the acetyl groups along the linear backbone of the chitosan molecules may be of crucial importance in defining the interactions with the biological systems [46]. For further information about preparation of chitin and chitosan, characterization and

Physicochemical Characteristics of Chitin and Chitosan and the Determination Methods
Physicochemical Characteristics DD Determination Methods Infrared spectroscopy [30,31,35] First derivative UV-spectrophotometry [32, 33] Nuclear magnetic resonance spectroscopy ( 1HNMR) and (13 CNMR) [34-37] Conductometric titration [37] Potentiometric titration [38] Differential scanning calorimetry [39]

Average Mw and/or Mw distribution

Viscosimetry [40] Gel Permeation chromatography [41] Light scattering [42]

Crystallinity Moisture content Ash content Protein
DD: deacetylation degree.

X-ray Diffraction [3, 43] Gravimetric analysis [44] Gravimetric analysis [44] Bradford method [45]

Functional Characterization of Chitin and Chitosan

Current Chemical Biology, 2009, Vol. 3, No. 2

207

chemistry Rinaudo and Kurita’s reviews are recommended [26,47]. 4. REGULATORY ASPECTS Chitosan has been approved as functional food in some Asian countries (Japan, Korea) during the last decade. The inclusion of chitin and chitosan was considered in 2003 by the Codex Alimentarius Commission but it is not currently listed in the General Standard for Food Additives nor has it been authorized as a food ingredient in the EU. Although several studies have shown that this compound is not toxic, no long-term studies of human safety have been reported. In the field of medical applications, chitosan has not been approved yet by the FDA. However, The American Society of Testing Materials (ASTM F04 division IV) is making a concerned effort to establish standard guidelines for tissueengineered medical products (TEMPs). The F2103 guide covers the evaluation of chitosan salts suitable for use in medical applications considering aspects such as control of protein content and, hence, potential for hypersensitivity, endotoxin content, and total bioburden [44]. The F2260-03 guide covers the determination of DD while the WK965 guide covers the determination of Mw of chitosan and chitosan salts [48,49]. A derivative of chitosan (chitosan hydrochloride) has been included in the European Pharmacopoeia in 2002 [50]. This monograph includes tests for heavy metal as contaminats but bioburden, sterility and bacterial endotoxins are not addressed. Taking into account that purity, which is quantified as the remaining ashes, proteins, insolubles and also the bio-burden (microbes, yeasts and moulds, endotoxins,...), is vital particularly for high value products, a more detailed characterization is needed. Further information regarding this topic is found in reference [51]. 5. BIOLOGICAL PROPERTIES OF CHITIN AND CHITOSAN Chitin and chitosan are currently receiving a great deal of interest as regards medical and pharmaceutical applications because they have interesting properties that make them suitable for use in the biomedical field, such as biocompatibility, biodegradability and non toxicity. Moreover, other properties such as analgesic, antitumor, hemostatic, hypocholesterolemic, antimicrobian, and antioxidant properties have also been reported [1,52,53]. A deeper understanding of the mechanism of these properties makes it necessary for chitosan to be well characterized and purified from accompanying compounds [54]. In addition, chitins and chitosans derivatized in a variety of fashions can be used to prove molecular hypothesis for the biological activity. Since the majority of the biological properties are related to the cationic behaviour of chitosan, the parameter with a higher effect is the DD. However, in some cases, the Mw has a predominant role. In addition to the DD and Mw, other properties such as chain conformation, solubility or degree of substitution have been considered. Chitosans produced by heterogenous deacetylation, with a block arrangement of acetylated and deacetylated units, have a tendency to form aggregates in aqueous solutions [55]. Extensive aggregation and intermolecular

interactions may reduce available sites on the chitosan molecule. This may account for some of the differences between reported effects of chitosan, especially if the authors did not pay close attention to the preparation of chitosan dispersions or if the preparation procedure in these studies was different [56]. The relationship between some chitin and chitosan biological properties and their physicochemical characteristics are shown in Table 3.
Table 3. Relationship Between Chitin and Chitosan Biological Properties and their Characteristics
Property Biodegradability Biocompatibility Mucoadhesion Hemostatic Analgesic Adsorption enhancer Antimicrobian Anticholesterolemic Antioxidant
DD: deacetylation degree. Mw: molecular weight.

Characteristic DD, distribution of acetyl groups, Mw DD DD, Mw (only chitosan) DD, Mw DD DD (only Chitosan) Mw DD, Mw, viscosity DD, Mw

5.1. Biodegradability Chitin and chitosan are absent from mammals but they can be degraded in vivo by several proteases (lysozyme, papain, pepsin…). Their biodegradation leads to the release of non-toxic oligosaccharides of variable length which can be subsequently incorporated to glycosaminoglycans and glycoproteins, to metabolic pathways or be excreted [57]. Lysozyme, a non-specific protease present in all mammalian tissues, seems to play a degradation role on chitin and chitosan. The degradation kinetics seem to be inversely related to the degree of crystallinity which is controlled mainly by the degree of deacetylation. Moreover, the distribution of acetyl groups also affects biodegradability since the absence of acetyl groups or their homogeneous distribution (random rather than block) results in very low rates of enzymatic degradation [2,58]. Finally, several studies reported that the length of the chains (Mw) also affects the degradation rate [59-61]. The understanding and control of the degradation rate of chitin and chitosan-based devices is of great interest since degradation is essential in many small and large molecule release applications and in functional tissue regeneration applications. Ideally, the rate of scaffold degradation should mirror the rate of new tissue formation or be adequate for the controlled release of bioactive molecules. Thus, it is important to understand and control both the mechanism and the rate by which each material is degraded. The degradation rate also affects the biocompatibility since very fast rates of degradation will produce an accumulation of the amino sugars and produce an inflammatory response. Chitosan samples with low DD induce an acute inflammatory response while chitosan samples with high DD

208 Current Chemical Biology, 2009, Vol. 3, No. 2

Aranaz et al.

induce a minimal response due to the low degradation rate. Degradation has been shown to increase as DD decreases [62-64]. Kofuji et al. investigated the enzymatic behaviours of various chitosans by observing changes in the viscosity of chitosan solution in the presence of lysozyme [65]. They found that chitosan with a low DD tended to be degraded more rapidly. However, other authors reported that differences in degradation are due to variations in the distribution of acetamide groups in the chitosan molecule [2,66]. This occurs due to differences in deacetylation conditions which influences viscosity of the chitosan solution by changing the inter- or intra-molecular repulsion forces [64]. Therefore, It can be concluded that it is impossible to estimate biodegradation rate from the DD alone. 5.2. Biocompatibility Both chitin and chitosan show very good compatibility but this property depends on the characteristics of the sample (natural source, method of preparation, Mw and DD). Due to its higher versatility and biological properties the majority of the assays have been carried out on chitosan samples. Although the gastrointestinal enzymes can partially degrade both chitin and chitosan, when both polymers are orally administered they are not absorbed. For this reason, they are considered as not bioavailable. Chitosan shows a LD50 of around 16g/kg, very similar to the salt and glucose values in assays carried out on mice [67]. Toxicity of chitosan is reported to depend on DD. Schipper et al. reported that chitosans with DD higher than 35% showed low toxicity, while a DD under 35% caused dose dependant toxicity. On the other hand, Mw of chitosan did not influence toxicity [68] . Chitosan presents higher cytocompatibility in vitro than chitin. The cytocompatibility of chitosan has been proved in vitro with myocardial, endothelial and epithellial cells, fibroblast, hepatocytes, condrocytes and keratinocytes [69]. This property seems to be related to the DD of the samples. When the positive charge of the polymer increases, the interactions between chitosan and the cells increase too, due to the presence of free amino groups. The adhesion and proliferation of keratinocytes and fibroblasts on several chitosan films with different DDs depend on both, DD and cell type. In both cells, the percentage of cell adhesion was strongly dependent of the DD, increasing with this parameter. The type of cell was a factor that also affected the adhesion, being more favourable for fibroblasts which exhibit a more negative charge surface than for keratinocytes. On the other hand, the proliferation decreased considerably by increasing the DD. Residual proteins in chitin and chitosan could cause allergic reactions such as hypersensitivity. The protein content in a sample depends on the source of the sample and, especially, on the method of preparation. 5.3. Haemostatic It has been reported that chitosan, as well as sulphated chitosan oligomers, presents anticoagulant activity tested in vitro [70]. The anticoagulant activity of chitosan seems to be related to its positive charge since red blood cells’ membranes are negatively charged and chitin is less effective than chitosan [71, 72]. Chitosan Mw also affects the binding or

agglutination of red blood cells [73]. In a recent paper, a comparative study has been carried out among solid-state chitosan and chitosan acetic acid physiological saline solution. Several chitosan samples with Mw from 2000 to 400 kDa and DD from 90 to 70% were tested. It was found that solid-state chitosan and chitosan acetic acid physiological saline solution followed different haemostatic mechanisms. When blood was mixed with chitosan acetic acid physiological saline solution, the erythrocytes aggregated and they were deformed. The DD, especially a high DD, in the chitosan acetic acid physiological saline solution, had a significant effect on the unusual aggregation and deformation of erythrocytes, compared with the effect of Mw within a range between 105 and 106. However, this phenomenon could not be observed in solid-state chitosan soliquoid. Solid-state chitosan with a high DD bound more platelets and was more haemostatic [74]. 5.4. Analgesic Effect Several authors have reported that both chitin and chitosan show analgesic effects [75-77]. Okamoto et al. have studied the analgesic effect of chitin and chitosan on inflammatory pain due to intraperitoneal administration of acetic acid and have proposed a mechanism for this analgesic effect [78]. These authors found that chitosan showed a greater effect than chitin. This difference was explained by the different action mechanism of the two polymers. The results suggested that the main analgesic effect of chitosan is the absorption of proton ions released in the inflammatory area. Due to its polycationic nature, the free primary amino groups of chitosan can protonate in the presence of proton ions and the reduction in the pH is the main cause of the analgesic effect. On the other hand, chitin was also able to slightly absorb the proton ions but the concentration needed to show the same effect as chitosan was lower than expected. From experimental data, it was concluded that the analgesic effect was due to the absorption of bradykinin, one of the main substances related to pain. 5.5. Antitumor Activity An antitumor activity of chitosan has been claimed by inhibition of the growth of tumor cells mainly due to an immune stimulation effect. However, this property is very controversial [73]. Jeon and Kim have found that chitosan oligomers possess antitumor activities tested both in vitro and in vivo [79]. Studies carried out using mice that had ingested low-Mw chitosan revealed significant antimetastatic effects of chitosan against Lewis lung carcinoma. Partially deacetylated chitin as well as chitin with a carboxymethyl group have also been effective to demote tumor progression [80]. The suggested mechanism involves immunostimulating effects of chitin and its carboxymethyl derivatives via stimulation of cytolytic T-lymphocytes. This activity increases with smaller molecular sizes and it is suggested that they have immunostimulating effects that activate peritoneal macrophages and stimulate non-specific host resistance. However, higher Mw oligomers have also exhibited antitumor activity. The same mechanism has been suggested for their activity via increased production of lymphokines by activated lymphocytes [81].

Functional Characterization of Chitin and Chitosan

Current Chemical Biology, 2009, Vol. 3, No. 2

209

Ueno et al. studied the effect of chitosan on tumor growth and metastasis. The activation of macrophages by chitosan is suggested to mediate its antitumor effects in vivo, while its angiogenic inducing properties may be the harmful effects of chitosan, such as promotion of tumor growth and invasion [82]. 5.6. Mucoadhesion Several factors affect chitosan mucoadhesion, such as physiological variables and the physicochemical properties of chitosan. The mucus is composed of a glycoprotein called mucin, which is rich in negative charges since it has sialic acid residues. In the stomach, chitosan is positively charged due to the acidic environment and, therefore, it can interact with mucin by electrostatic forces. The extent of this union depends on the amount of sialic acid present in the mucin and on the Mw and DD of chitosan. It has been found that when the Mw of chitosan increases, the penetration in the mucin layer also increases and hence the mucoadhesion is stronger [83]. On the other hand, a higher DD leads to an increase in charge density of the molecule and the adhesive properties become more relevant [84]. Huang et al. evaluated the effects of Mw and DD on the cellular uptake and in vitro cytotoxicity of chitosan molecules and nanoparticles [59]. They found that the binding affinity and uptake capacity of chitosan nanoparticles decreased when decreasing polymer Mw and degree of deacetylation. The effect of the degree of deacetylation was greater than the effect of Mw because of its effect on the zeta potential of the nanoparticles. However, the uptake of chitosan molecules was less dependent on Mw and degree of deacetylation. El-Kamel et al. developed mucoadhesive micromatricial chitosan/poly( -caprolactone) films for the treatment of periodontal diseases [85]. These authors found that films containing different Mw chitosans had different forces of adhesion but statistical analysis revealed that there was no significant difference in bioadhesion force between the films. On the contrary, Roldo et al. showed that the maximal detachment force of medium Mw chitosan was higher than that of both low and high Mw chitosans [86]. 5.7. Permeation Enhancing Effect It has been reported that chitosan acts as a permeation enhancer by opening epithelial tight junctions [87, 88]. The mechanism underlying this behaviour is based on the interaction of positively charged chitosan and the cell membrane resulting in a reorganization of the tight junction-associated proteins [89]. Schipper et al. investigated the effect of chitosan structural characteristics (Mw and DD) on their absorption enhancing properties in vitro (Caco-2 cell monolayers), using chitosan hydrochloride salts at pH 5.5 [68]. It was found that the capacity of chitosan to improve mannitol transport is dependent on Mw and the DD; accordingly, while chitosans with a high DD were efficient as permeation enhancers at low and high Mw, those with low degrees of deacetylation were efficient only at high Mws. Subsequently published articles in this field agree that > 80% deacetylation affords the greatest promoter effect on cells in culture [89,90].

Soane et al. investigated the effect on mucociliary transport velocity of five different types of chitosan with varying Mws and degrees of deacetylation. The five types of chitosan tested were shown to have no toxic effect on the frog palate clearance mechanism [91]. The cilia beat frequency in guinea pigs after nasal administration of chitosan solution was also studied for 28 days and none of the chitosans used showed any effect on the cilia frequency, which suggests that using various types of chitosan for nasal delivery applications is not harmful. 5.8. Anticholesterolemic There are several proposed mechanisms for cholesterol reduction by chitosan. The latest findings in this field consider more than one hypothesis. The entrapment caused by a viscous polysaccharide solution is thought to reduce the absorption of fat and cholesterol in the diet. On the other hand, the presence of the amino group in its structure determines the electrostatic force between chitosan and anion substances, such as fatty acids and bile acids. Muzzarelli et al. propose a spontaneous formation of insoluble chitosan salts from bile acids whose hydrophobic nature should permit the collection of cholesterol and lipids via hydrophobic interaction [92]. A commercial food grade chitosan of DD 87 and average Mw of 150 kDa was used to demonstrate this theory. The interaction between chitosan and anionic surfaceactive materials (phospholipids, bile acids) depends on its three types of reactive functional groups: the amino group at the C2 position and primary and secondary hydroxyl groups at the C-3 and C-6 positions, respectively. Thongngam et al. have demonstrated the formation of micelle-like clusters within the chitosan structure in its interactions with a model bile salt [93,94]. Another mechanism accounts for the adsorption of chitosan to the surface of the emulsified lipid and the formation of a protective coating that might prevent the lipase/co-lipase from adsorbing to the droplet surfaces and gaining access to the lipids inside the droplets [95]. Although great effort has been made to make a correlation between the physicochemical characteristics of chitosan and its fat-binding capacity, only some significant relationships have been demonstrated. No et al. used six commercially available chitosans with varying physicochemical characteristics and showed that the fat binding capacity was negatively correlated to the bulk density in a significant way whereas it showed a trend to positively correlate with the Mw [96]. The same group studied the fat binding capacity of five chitosans of increasing Mw (range 500-800 kDa) prepared by different depolymerization times, keeping a similar DD, and found that the sample showing significant higher activity was the one with the second lowest Mw [97]. In another study, a chitosan sample was submitted to degradation with irradiation and sonolysis, and five decreasing Mw where produced in the range 25-400 kDa. Samples showed a trend to increase the fat-binding activity with decreasing Mw using a biopharmaceutical model of digestive track [98]. Different experimental designs have been used with the aim of mimicking the reactions taking place in the stomach and duodenum. A digestive chemical model has been used to study the interaction between chitosans of different viscosity and DD and sunflower oil. Although a negative correlation was found between the percentage of en-

210 Current Chemical Biology, 2009, Vol. 3, No. 2

Aranaz et al.

trapped oil and increasing oil addition, no significant differences where found in chitosan behaviour according to its characteristics [99]. Another in vitro human digestion model was used to check the adsorption of chitosan to the fat droplets. It was observed that the high Mw chitosan adsorbed to the droplet more strongly than the low Mw. The reasons proposed for this phenomenon were the different conformations of the chitosan molecule, with cationic loops and tails in the high Mw and its higher surface activity [100]. A recent contribution has examined and compared eleven chitosan preparations for their in vitro fat-binding capacity, potency to bind individual bile acids, DD, solution viscosity, and swelling volume. It was noted that the chitosan sample having the strongest binding capacity against a selected bile acid did not necessarily exhibit the strongest binding capacity against other bile acids. No correlation was detected between individual bile acid-binding capacity and any other tested physico-chemical properties of chitosan. These data suggested that Mw, as reflected by solution viscosity, DD, or swelling capacity might not be used to predict the bile acidbinding capacity of chitosan [101]. 5.9. Antimicrobial Activity The antimicrobial activity of chitin, chitosan, and their derivatives against different groups of microorganisms, such as bacteria, yeast, and fungi, has received considerable attention in recent years. Two main mechanisms have been suggested as the cause of the inhibition of microbial cells by chitosan. The interaction with anionic groups on the cell surface, due to its polycationic nature, causes the formation of an impermeable layer around the cell, which prevents the transport of essential solutes. It has been demonstrated by electron microscopy that the site of action is the outer membrane of gram negative bacteria. The permeabilizing effect has been observed at slightly acidic conditions in which chitosan is protonated, but this permeabilizing effect of chitosan is reversible [102]. The second mechanism involves the inhibition of the RNA and protein synthesis by permeation into the cell nucleus. Liu et al. have observed labelled chitosan oligomers with Mw from 8 to 5 kDa inside the E. coli cell and they showed good antibacterial activities [103]. In this case the Mw is the decisive property (Table 4).
Table 4. Influence of Chitosan DD and Mw on Antimicrobial Activity
Effect on Antimicrobial Activity electrostatic binding to membrane permeabilizing effect Mw
DD: deacetylation degree. Mw: molecular weight.

proposed that the antimicrobial action of chitosan against filamentous fungi could be explained by a more direct disturbance of membrane function [105]. However, it is not clear whether the antimicrobial activity of chitosan is caused by growth inhibition or cell death. Antibacterial activities were found to increase in the order of N,O-CM-chitosan, chitosan, and O-CM-chitosan. The first product, where amino and hydroxyl groups have been substituted by carboxymethyl groups, contains fewer amino residues. In the case of O-CM-chitosan, its number of amino groups is not changed. Moreover, its carboxyl group may have reacted with the amino groups intra- or intermolecularly and charged these groups. The authors concluded that the antibacterial activities of chitosan and carboxymethylated derivatives depend on the effective number of –NH3+ groups [103]. Several studies prove that an increase in the positive charge of chitosan makes it bind to bacterial cell walls more strongly [106]. The relationship between Mw, number of charges and antimicrobial activity has been pointed out by Kim et al. [107]. They showed that O-CM chitosan derived from degraded chitosan was more effective than plain chitosan. This was attributed to the interaction of the COOH group with the NH2 group intra-or intermolecularly to impart a charge, the number of –NH3 groups becoming larger. In the case of native chitosan, an excessive concentration of amino groups on O-CM chitosan promotes a structure that involves cross-linking through strong intramolecular hydrogen bonding, where the number of amino groups that are available to attach bacterial surfaces is reduced. In contrast, some authors have not found a clear relationship between the degree of deacetylation and antimicrobial activity. These authors suggest that the antimicrobial activity of chitosan is dependent on both the chitosan and the microorganism used [108, 109]. Park et al. studied the antimicrobial activity of hetero-chitosans and hetero-COs with different degrees of deacetylation and Mws against three Gramnegative bacteria and five Gram-positive bacteria and found that the 75% deacetylated chitosan showed more effective antimicrobial activity compared with that of 90% and 50% deacetylated chitosan [110]. 5.10. Antioxidative Activity Chitosan has shown a significant scavenging capacity against different radical species, the results being comparable to those obtained with commercial antioxidants. Samples prepared from crab shell chitin with DD of 90, 75 and 50% where evaluated on the basis of their abilities to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, superoxide radical and alkyl radical. The results revealed that chitosan with higher DD exhibited the highest scavenging activity [111]. On the other hand, chitosans of different size as well as their sulphate derivatives were assayed against superoxide and hydroxyl radicals. A negative correlation was found between chitosan Mw and activity (Table 5). The chitosan sulphated derivatives presented a stronger scavenging effect on peroxide radicals but the chitosan of lowest Mw showed more considerable ferrous ion-chelating potency than others [112]. The chelation of metal ions is one of the reasons why chitosan may be considered as a potential natural antioxidant

Physico-Chemical Property DD

permeation into the cell nucleus

Other mechanisms have also been proposed. Chitosan may inhibit microbial growth by acting as a chelating agent rendering metals, trace elements or essential nutrients unavailable for the organism to grow at the normal rate. Chitosan is also able to interact with flocculate proteins, but this action is highly pH-dependent [104]. Several authors have

Functional Characterization of Chitin and Chitosan

Current Chemical Biology, 2009, Vol. 3, No. 2

211

for stabilizing lipid containing foods to prolong shelf life. Chitosans may retard lipid oxidation by chelating ferrous ions present in the system, thus eliminating their prooxidant activity or their conversion to ferric ion [113].
Table 5. Influence of Chitosan DD and Mw on Antioxidative Activity
Effect on Antioxidant Activity scavenging effect radical scavenging effect ion-chelating potency
DD: deacetylation degree. Mw: molecular weight.

Physico-Chemical Property DD Mw

study shows that in addition to chitin and chitosan, their oligomers and monomers enhance wound healing acceleration. Wound break strength and collagenase activity of the chitosan group (D-glucosamine (GlcN), chito-oligosaccharide (COS), chitosan) were higher than the chitin group (Nacetyl-D-glucosamine (GlcNAc), chiti-oligosaccharide (NACOS), chitin). Collagen fibres run perpendicular to the incisional line in the oligosaccharide group (NACOS, COS) and many activated fibroblasts were observed in the histological studies around the wound in the chitosan groups. The break strength was stronger and more activated fibroblasts were observed at higher DD. The potential use of chitin oligosaccharides (DP2, DP3, DP4, DP5 and DP7) in wound healing as well as their capacity against chronic bowel disease has been studied. For the first time, a mucin-stimulating effect of chitin oligomers DP3 and DP5 has been observed in an ex-vivo model [122]. The wound healing effect of chitin and chitosan oligomers and monomers is of great interest because in vivo lysozyme degrades chitin and chitosan to these smaller molecules. 6.2. Drug Delivery Systems An important application of chitosans in industry is the development of drug delivery systems such as nanoparticles, hydrogels, microspheres, films and tablets (Fig. 4). As a result of its cationic character, chitosan is able to react with polyanions giving rise to polyelectrolyte complexes [123124]. Pharmaceutical applications include nasal, ocular, oral, parenteral and transdermal drug delivery. Three main characteristics of chitosan to be considered are: Mw, degree of acetylation and purity. When chitosan chains become shorter (low Mw chitosan), it can be dissolved directly in water, which is particularly useful for specific applications in biomedical or cosmetic fields, when pH should stay at around 7.0. In drug delivery, the selection of an ideal type of chitosan with certain characteristics is useful for developing sustained drug delivery systems, prolonging the duration of drug activity, improving therapeutic efficiency and reducing side effects. Kofuji et al. suggested that the physicochemical characteristics of chitosan are important for the selection of the appropriate chitosan as a material for drug delivery vehicles [65]. Investigations have indicated that DD and Mw of chitosan have significantly affected the role of chitosan in therapeutic and intelligent drug delivery systems [125, 126]. Mi et al. studied the gelation properties of microspheres cross-linked with glutaraldehyde as it had significant effect on drug incorporation [127]. Microspheres prepared with a high Mw chitosan gelled faster than those prepared with a low Mw because they have different activation energies of gelation. Chitosan with short chains have higher activation energy and need more time to interact with the other chains and to gelate with glutaraldehyde. Gupta and Jabrail studied the effect of degree of deacetylation and cross-linking on physical characteristics, swelling and release behaviour of centchroman loaded chitosan microspheres [128]. The DD controls the degree of crystallinity and hydrophobicity in chitosan due to variations in hydrophobic interactions which control the loading and release characteristics of chitosan matrices. The DD also controls the degree of cross-linking of chitosan in the presence of any

This activity has been also studied in chitooligosaccharides (COS). Chitobiose and chitotriose have proved to be more potent than three reference compounds (aminoguanidine, pyridoxamine and trolox) in scavenging hydroxyl radicals while glucosamine and the corresponding Nacetylchito-oligosaccharides did not show any capacity [114]. Electron spin resonance spectrometry has been used to follow the scavenging activity of chitooligosaccharide mixtures fractionated by ultrafiltration. This activity was shown to be dependent on the Mw, the fraction 1-3 kDa having the highest radical scavenging effect [115]. When the DD was considered, a correlation between scavenging activity over all tested free radicals with the increment of deacetylation values of COS was found. Therefore, it has been pointed out that the free amino groups in the hetero COS play an important role in free radical scavenging activity, probably by forming stable macromolecule radicals [116]. This capacity of oligosaccharides has been further assayed in vivo. Yang et al. assayed two different Mw COS (1.1 and 0.5 kDa) against H202 released from polymorphonuclear leukocytes stimulated by phorbol-12-myristate-13-acetate in rats [117]. They found that the radical scavenging capacity was higher for the first COS. 6. BIOMEDICAL APPLICATIONS OF CHITIN AND CHITOSAN Due to its high biocompatibility, chitosan has been employed in drug delivery systems, implantable and injectable systems such as orthopaedic and periodontal composites, wound healing management and scaffolds for tissue regeneration [118,119]. 6.1. Wound Healing Chitin and chitosan activate immunocytes and inflammatory cells such as PMN, macrophage, fibroblasts and angioendothelial cells. These effects are related to the DD of the samples, chitin presenting a weaker effect than chitosan [82]. Chitosan oligomers have also exhibited wound-healing properties, it is suggested that their wound-healing properties are due to their ability to stimulate fibroblast production by affecting the fibroblast growth factor. Subsequent collagen production further facilitates the formation of connective tissue [120]. Recently, the effects of chitin and chitosan oligomers and monomers on wound healing have been studied [121]. This

212 Current Chemical Biology, 2009, Vol. 3, No. 2

Aranaz et al.

Fig. (4). (A) High Mw Chitosan (640 kDa) microspheres crosslinked with 0.2% TPP obtained by spray-drying. (B) Detail of the microspheres.

suitable cross-linker. The higher the DD is, the higher the number of free amino groups and therefore the degree of covalent cross-linking increases [129]. When analyzing the influence of cross-linking degree and degree of deacetylation on size and morphology of the microspheres, these authors reported that the size and the surface roughness decreased on increasing the degree of cross-linking and the degree of deacetylation. Zhang et al. also reported that a high degree of chitosan deacetylation and narrow polymer Mw distribution were shown to be critical for the control of particle size distribution [130]. A higher degree of cross-linking and a higher DD in chitosan increase the compactness of matrices and its hydrophobicity, thus controlling the degree of swelling and diffusivity of the drug entrapped in chitosan matrixes. It was observed that a DD between 48-62% promotes maximal loading capacity, due to the size of the cross-links and pores formed. Regarding the release properties, a very low DD can induce burst release [128]. In another study with chitosan microspheres loaded with centchroman and crosslinked with glutaraldehyde, Gupta and Jabrail observed that the lower Mw of chitosan employed, the lower sphericity of the microspheres obtained and these microspheres were larger in size than those prepared with medium-high Mw chitosan due to the poorer molecular packing and crosslinking [131]. These results are in agreement with those presented by Desai and Park, who studied the influence of Mw of chitosan on chitosan-TPP microspheres prepared by spray-drying [132]. They observed that an increase of Mw also produced more spherical microspheres, with greater size homogeneity and a smoother surface. In addition they found that an increase in molecular weight gave bigger microspheres as a result. Gupta and Jabrail in their study also found that microspheres prepared with high Mw chitosan presented a very low degree of swelling and a high degree of crosslinking, thus, those microspheres prepared with medium Mw chitosan that lead to less strong intermolecular interactions being more appropriate for sustained release [131]. These results were also in agreement with Desai and Park who observed that the release rate of vitamin C was much lower as the Mw of chitosan used for preparing microspheres increased [132]. They studied the

release kinetics and found that it followed Fick’s law of diffusion. Low Mw chitosan leads to poor retention of centchroman in microspheres due to a high degree of swelling and a fragile network structure. The microspheres with medium Mw chitosan showed an optimum loading efficiency [131]. Microspheres with medium Mw chitosan are more efficient in releasing the centchroman in a controlled manner in comparison to low and high Mw chitosan microspheres. The initial burst release of centchroman from microspheres with different Mws and different degrees of deacetylation of chitosan varied linearly with the square root of the release time indicating a diffusion-controlled release of centchroman from these microspheres (n = 0.5). However, the release of centchroman in the controlled stage of drug release was anomalous [133]. The initial slope of these curves was used to calculate the diffusion coefficient (D) for centchroman from chitosan microspheres. The value of the diffusion coefficient for centchroman from microspheres decreased on increasing the Mw of chitosan, and decreased on increasing the DD in chitosan. This clearly indicates that the release of centchroman from these microspheres is diffusion controlled and the variation in the diffusion coefficient (D) of centchroman on varying the Mw and degree of deacetylation in chitosan is due to the variations in the structure of microspheres [131]. The influence of chitosan DD and Mw on the microspheres properties prepared as matrix for drug delivery is shown in Table 6. Desai and Park in the study of the influence of chitosan Mw on chitosan-TPP microspheres found that it does not affect the spray drying yield [132]. However, it has influence on some parameters of the microspheres that have already been commented on. In addition they studied the influence on zeta potential and observed some differences that were not very significant. Chiou et al. investigated the effect of post-coating PLLA microspheres with different chitosans on the initial burst and controlling the drug release of the microspheres [134]. Without chitosan, 20% lidocaine was released within the first hour and the time of 50% release was 25 hours. This period was extended to 90 hours after coating with chitosan. They observed that when applying chitosan of the same Mw, the

Functional Characterization of Chitin and Chitosan

Current Chemical Biology, 2009, Vol. 3, No. 2

213

efficacy of reducing the initial burst of drug release was higher for a lower degree of deacetylation. With chitosan in acetic acid solution, coating the microspheres with high Mw and high viscosity could most effectively reduce the initial burst and control drug release of PLLA microspheres. The study indicated that manipulating the viscosity of the chitosan solution was the most important factor in contributing to controlling the drug release of chitosan post-coated PLLA microspheres.
Table 6. Influence of Chitosan DD and Mw on Microspheres Properties
Effect on Microsphere Properties covalent crosslinking size surface roughness swelling compactness and hydrophobicity loading capacity burst release Mw sphericity morphology homogeneity crosslinking swelling release rate diffusion coefficient (D)
DD: deacetylation degree. Mw: molecular weight.

films containing higher Mw chitosan. These authors attributed this to the increased viscosity of the chitosan solution as the Mw increased. After studying water absorption capacity, results revealed that there was no statistically significant difference in percentage water uptake with different Mw chitosans. This result was in agreement with Roldo et al. [86], who found no correlation between the Mw of chitosan and its swelling behaviour. The mechanical properties of films with different Mw chitosans were also measured by El- Kamel et al. The tensile strength (TS), the percentage elongation at break (% EB) and the elastic modulus (EM) are important parameters to indicate the strength and elasticity of the film [85]. They found that medium Mw chitosan films had highest values for TS and EM, followed by high Mw and low Mw chitosan films. On the other hand, the highest % EB was obtained for low Mw chitosan films, followed by high and medium Mw chitosan films. With regard to in vitro release studies, they found that the amount of drug released from prepared films was similar for films that contained low and medium Mw chitosan and lower for the ones prepared with high Mw chitosan. This behaviour was predictable, taking into account the direct relationship between the molar mass of chitosan and the viscosity of its solution. By increasing the viscosity of the polymer, the diffusion of the drug through the formed gel layer into the release medium was retarded [135]. The high polymer viscosity may also affect the size of particles formed by reducing the homogenization efficiency, leading to the formation of larger PCL microparticles, as indicated by the particle-size analysis studies. Therefore, the exposed surface area is reduced and the release of the entrapped drug is decreased. 6.3. Gene Delivery Due to its positive charge, chitosan has the ability to interact with negative molecules such as DNA. This property was used for the first time to prepare a non-viral vector for a gene delivery system by Mumper in 1995 [136]. The use of chitosan as non-viral vector for gene delivery offers several advantages compared to viral vectors. Mainly, chitosan does not produce endogenous recombination, oncogenic effects or immunological reactions [137]. Moreover, chitosan/pDNA complexes can be easily prepared at low cost. The Mw of chitosan is a key parameter in the preparation of chitosan/pDNA complexes since transfection efficiency correlates strongly with chitosan Mw. High molecular weight chitosan renders very stable complexes but the transfection efficiency is very low. To improve transfection efficiency, recent studies have examined the use of low Mw chitosans [138-146] and oligomers [147-149] in gene delivery vectors. It appears that a fine balance must be achieved between extracellular DNA protection (better with high Mw) versus efficient intracellular unpackaging (better with low Mw) in order to obtain high levels of transfection. Lavertu et al. studied several combinations of Mw and DA of chitosan finding two combinations of high transfection efficiency using a chitosan of 10 kDa and DD of 92 and 80%, respectively [150]. Kiang et al. studied the effect of the degree of chitosan deacetylation on the efficiency of gene transfection in chito-

Physico-Chemical Property DD

Kofuji et al. studied the relationship between physicochemical characteristics and functional properties of chitosan such as the ability to form spherical gel, control of drug release from chitosan gel and biodegradation of chitosan [65]. They found that the formation of spherical chitosan gels in aqueous amino acid solution or aqueous solution containing metal ions was affected mainly by viscosity of the chitosan solution. High concentration of chitosan species with a high Mw could not be used to prepare chitosan spherical gel due to its high viscosity and the use of very low concentration of chitosan did not result in instantaneous spherical gel formation because the diffusion of chitosan within the preparative medium was too rapid. The degree of deacetylation also had an effect on spherical gel formation in the case of gelation of chitosan by chelation with metal ions. Chitosan with high degree of deacetylation was able to form spherical gel by chelation due to higher availability of amino groups that chelated with metal ions better than chitosan of low DD. Only in the case of chelation with metal ions was the extent of deacetylation related to drug release. El-Kamel et al. developed mucoadhesive micromatricial chitosan/poly( -caprolactone) films for the treatment of periodontal diseases [85]. They examined the effect of different molar masses of chitosan on morphology of microparticles trapped in the films, water absorption, in vitro bioadhesion, mechanical properties and in vitro drug release. The mean size of entrapped caprolactone particles was higher in

214 Current Chemical Biology, 2009, Vol. 3, No. 2

Aranaz et al.

san-DNA nanoparticles [151]. Highly deacetylated chitosan (above 80%) releases DNA very slowly. They suggest that the use of chitosan with a DD below 80% may facilitate the release of DNA since it lowers the charge density, may increase steric hindrance in complexing with DNA, and is known to accelerate degradation rate. They reported an increase in luciferase expression when the degree of deacetylation was decreased from 90% to 70%. Formulations with 62% and 70% deacetylation led to luciferase transgenic expression two orders of magnitude higher than chitosan with 90% deacetylation. 6.4. Tissue Engineering Recent studies in regenerative tissue engineering suggest the use of scaffolds to support and organize damaged tissue because three-dimensional matrices provide a more favourable ambient for cellular behaviour. Due to their low immunogenic activity, controlled biodegradability and porous structure, chitosan scaffolds are promising materials for the design of tissue engineered systems [152-154]. Tı lı et al. studied the influence of DD on some structural and biological properties of chitosan scaffolds for cell culture and tissue engineering [155]. They observed that chitosan scaffolds with low DD (75-85%) displayed a more regular structure and the pores were fairly uniform and parallel with a polygonal cross section. The lateral pore connectivity was much lower than for scaffolds with high deacetylation degrees (>85%). It is known that the microstructure such as pore size, shape and distribution, has prominent influence on cell intrusion, proliferation and function in tissue engineering. Swelling studies were also performed but no relationship was found between DD and swelling ratio. Mechanical testing of chitosan scaffolds showed that mechanical strength was higher with higher DD. Biodegradability of the scaffolds also depends on the DD. Cell attachment studies on the scaffolds showed that higher DD favoured cell adhesion. Other authors also reported that a lower degree of acetylation favoured cell adhesion [69,156]. The viability of fibroblasts on chitosan scaffolds with different DD was evaluated. A significant increase in cell number was observed on >85% deacetylated chitosan scaffolds. A high proliferation trend was suggested when compared to low deacetylated chitosan scaffolds. Chitin and chitosan tubes for nerve regeneration were prepared by Freier et al. [157]. The compressive strength of these tubes was found to increase with decreasing acetylation. Both chitin and chitosan support adhesion and differentiation of primary chick dorsal root ganglion neurons in vitro, with significantly enhanced neurite outgrowth on chitosan than on chitin films. The effect of DA on the cell adhesion and biodegradation of chitin and chitosan films was studied to find the most suitable conditions for cell compatibility and optimum biodegradation [158]. Injectable thermosetting chitosan hydrogels are attractive systems for drug delivery and tissue engineering that combine biodegradability, biocompatibility and the ability to form in situ gel-like implants. Thermally-induced gelation relies advantageously on biopolymer secondary interactions, avoiding potentially toxic polymerization reactions that may occur with in situ polymerizing formulations. Besides -

glycerophosphate [159], other molecules such as 1,3propanediol, 1,2-propanediol as well as glycerol, mannitol or polyoses such as trehalose have been reported to induce the thermogelation of chitosan [160]. Schuetz et al. studied the effect of the Mw of chitosan on the properties of the thermosetting chitosan hydrogels during storage and sterilization by autoclaving [160]. The autoclaving process produced a reduction of the Mw of the chitosan samples which was affected by the initial Mw of the sample. The authors concluded that chitosans exhibiting highly reduced Mw when autoclaved might not be adapted to this sterilization method in specific applications where maximal mechanical performance is essential for implant function. With regard to the freeze storage, low Mw chitosan thermogels or those prepared with low enough concentration might be kept frozen for prolonged storage. Porous scaffolds were prepared by freeze-drying a solution of collagen and chitosan, followed by cross-linking by dehydrothermal treatment. The effect of the chitosan Mw and the blending ratio was studied. The lysozyme biodegradation test demonstrated that the presence of chitosan, especially the high-molecular-weight species, could significantly prolong the biodegradation of collagen/chitosan scaffolds. In vitro culture of L929 mouse connective tissue fibroblast evidenced that low-molecular-weight chitosan was more effective for promoting and accelerating cell proliferation, particularly for scaffolds containing 30% (w/w) chitosan. The results elucidated that the blends of collagen with low-Mw chitosan have a high potential to be applied as new materials for skin-tissue engineering [161]. Apart from the aforementioned characteristics, which are specific for each application, there is a degree of consensus regarding general characteristics that must be present in chitosan samples to be used in the field of biomedical applications (Table 7) [162, 163]. 7. FOOD APPLICATIONS OF CHITOSAN Chitosan offers a wide range of unique applications in the food industry, including preservation of foods from microbial deterioration, formation of biodegradable films, and recovery of material from food processing discards. Moreover, it can act as a dietary fibre and as a functional food ingredient. 7.1. Dietary Ingredient Chitosan has been used in multiple nutritional supplement products due to its ability to bind fat. The in vivo studies are intended to demonstrate a significant reduction in the body weight gain or the plasma lipid content of humans or animals. Recently, Liu et al. have reported that rats fed diets containing the highest deacetylated chitosan significantly lowered plasma cholesterol and LDL-C, and increased HDL-C level [164]. Chitosan with high Mw limited the body weight gain of adult rats significantly. When the DD and particle size were considered, chitosan with higher Mw also exhibited better cholesterol-binding capacity in vitro. These results indicated that the viscosity in the upper gastrointestinal tract was not the major factor influencing the hypocholesterolaemic effect of chitosan. Nonetheless, they concluded that

Functional Characterization of Chitin and Chitosan

Current Chemical Biology, 2009, Vol. 3, No. 2

215

Table 7.

Characterization of Chitosan for Medical Application [162, 163]
Variable Under Study Moisture Content Ash content Protein content Insolubility Turbidity Viscosity DD Heavy metal content Bioburden, aerobic bacteria plus fungi Organoleptic properties Appropiate Characteristic