Chitosan as an oral antimicrobial agent - Formatex · Chitosan as an oral antimicrobial agent ... Chitin and chitosan have shown ... These peculiar properties provide a variety of

Chitosan as an oral antimicrobial agent

Morgana Maria Souza Gadelha de Carvalho1,2,3, Thayza C. Montenegro Stamford2, Emerson Pereira dos Santos1, Pedro Tenório2 and Fabio Sampaio2 1 Santa Emilia de Rodat Faculty, 58020-560, João Pessoa, Brasil 2 Oral Biology Laboratory, Health Science Center, Federal University of Paraiba, 58051-900, João Pessoa , Brasil 3 Biological Department, Biological Science Center, State University of Paraiba, 58429-500, Campina Grande, Brasil.

Chitosan (1-4, 2-amino-2-deoxi-b-D-glucana) is a deacetylated derivative from the biopolysaccharide chitin which is present in insects’ exoskeletons, crustaceans’ shells and fungi cell walls. While chitin is considered an abundant and undesirable polysaccharide since it is related to environmental contamination, the chitosan has shown: an excellent biocompatibility; almost no toxicity to human beings and animals; high bioactivity; biodegradability; reactivity of the deacetylated amino group; selective permeability; polyelectrolyte action; antimicrobial activity; ability to form gel and film; chelation ability and absorptive capacity. The applicability of the chitosan is directly related with its physicochemical features, since the distinct obtaining sources (crustaceans, mollusks, fungi), different processes of extraction and purification have caused changes in the deacetylation degree, molar weight, thermal stability and crystallinity level in the chitosan molecule. Regarding its antimicrobial activity, chitosan has low toxicity and the development of resistance has being reported. The antimicrobial activity of chitosan regarding gram-positive and gram-negative bacteria, ranges from 100 up to 100,000 mg l-1 and 100 up to 1,250 mg l-1 for gram-negative and gram-positive bacteria, respectively. To date there is enough evidence to support that chitosan molecular mass can influence the solubility and its antibacterial activity. Some hypothesis have been tested to verify if the lower molar weight chitosan penetrates more easily in the microorganisms and could immediately affect vital components of the cells and their physiological activities. For the high molecular weight chitosan the studies have indicated that its mechanism of action may be related to the formation of films around the cell which inhibits nutrients absorption. Throughout this review our aim is to present and discuss the antimicrobial activity of several chitosan types against oral bacteria particularly the Streptococcus mutans and the challenges for developing commercial products such as mouthwashes and toothpastes based on chitosan antimicrobial activity.

Key-words: polymer, pathogenic bacteria, dental caries, antibacterial activity

1. Introduction

It is universally accepted that the dental caries is a multifactorial disease. It is also well known that the carious lesion which is characterized by the tooth structure demineralization is diet dependent. In this complex process, the microorganisms, particularly Streptococcus species, have an important role in its ethiology [1,2]. The Streptococcus mutans is a member of the oral microbial community which plays a key role in modulating the transition of the non-pathogenic state, highly cariogenic biofilms. The key factors that contribute to the pathogenesis caused by S. mutans are the production of a great variety of carbohydrates, which generate low pH and cause the consequent demineralization of the tooth enamel [3]. Since microorganisms are active participants in the caries development, the searches of substances which may control the growth of these cells which are organized as dental biofilms are of great relevance. These substance and compounds are suppose to keep the same level of lower pathogenicity, to reduce the existing dental biofilm, to inhibit the associates bacteria to the disease in a selective way, as well as to reduce virulence determinants [4]. The substance that presents throughout the years the best antimicrobial effect is chlorhexidine. This is a cationic detergent, belonging to the bisbiguanidas class, available in acetate forms, hydrochloride and digluconate, being this last one, the salt more commonly used in formulas and products. It possesses a wide action specter, acting on gram-positive, gram-negative bacteria, fungi, yeast and lipophilic viruses [5]. As a mechanism of action for chlorhexidine, it has been seen that its cationic molecule is quickly attracted by the negative charge of the bacterial surface, being adsorbed to the cell membrane through electrostatic interactions, probably by hydrophobic bonds or hydrogen bridges, being this adsorption concentration-dependent. Thus, in high dosages, it causes precipitation and coagulation of cytoplasmic proteins and bacterial death and, in lower dosages, the integrity of the cell membrane is modified, resulting in an overflow of the bacterial components of low molecular weight [6-8]. Unfortunately, the use of chlorhexidine is limited due to the notorious adverse effects, such as: teeth, restorations and prosthesis staining, taste alterations (mainly for salt), formation of the supra-gum stone, and seldom, reversible swelling in the lips or parotid glands, peeling off the oral mucosa, hives, dyspinea and anaphylactic shock [9-11]. Amongst these effects, the dental staining is highlighted as the main complaint on behalf of the patients [12] being the main limiting factor of the use of chlorhexidine for extended periods [5].

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Science against microbial pathogens: communicating current research and technological advances A. Méndez-Vilas (Ed.)______________________________________________________________________________

2. Chitosan: General considerations

Chitin and chitosan are natural co-polymers, composed by units of 2-amino-2-desoxi-D-glycopyranose and of 2-acetamide-2-desoxi-D-glycopyranose interconnected by glycosidic bonds β-1,4 in variable proportions. The first type of units is frequently present in chitosan. Except celluloses, chitin is the most abundant polysaccharide largely distributed in nature, being the main component of the exoskeleton of crustaceans and insects, also occurs, in nematodes and in the cell wall of yeast and fungi [13-15]. Chitosan is naturally found in the cell wall of fungi, mainly in the order Mucorales. Usually chitosan is achieved through chitin deacetylation in alkaline environment; the group N-acetil may suffer several degrees of deacetylation (Figure 1). The chitin conversion process into chitosan must be performed in appropriate way to guarantee the production of chitosan with high quality and purity, free of contaminants like proteins, endotoxins and toxic metals. The polymer obtained must be characterized according to its acetylation level and molar mass, once such features may influence the degradability and in the polysaccharide hydrolysis [16,17]. According to the medium acetylation level (AL), chitosan may be obtained with physical-chemical properties differentiated regarding the solubility parameters, pKa and viscosity [18,19]. It is difficult to obtain chitosan with high deacetylation level because as long as the process increases, the degradation of the polymer also increases [16].

Source: authors

Fig. 1: Scheme of N- deacetylation of chitin into chitosan through hydrolisis by sodium hidroxide. Other derivatives from chitosan can be achieved through modification reactions like deacetylation, N-acetylation, acylation, O-acetylation, O-e N-ftalation, O-carboxymethylation, oxidation among others, to prepare several formulations (products), as tablets, micro-nanoparticles, microspheres, granules, liposomes, films, polymeric membranes, gels and hydrogels, with specific features for certain applications. However, due the strong intermolecular interactions and because it is a semi-crystalline structure, chitosan is less accessible to chemical reagents when compared to celluloses [16, 20]. Chitin and chitosan have shown ordered crystalline phase, being observed higher crystallinity in the chitin structure. The chains of these polymers are linked through hydrogen bonds in three different automorphic structures, thus showing polymorphism, assuming conformations α, β and γ, according to the acquired polarity by the sugar chains [21-23]. The α form is the most abundant in nature, being found in the cell wall of fungi, carapace of crustaceans and in the cuticle of some insects. The β form is rare and difficult to be achieved; may be found associated with proteins in synthesized tubes [24]. Some authors have cited that the α form predominates in fungi while the β form in crustaceans [25,26]. In the α conformation, individual chains show anti-parallel arrangement, in a way that the adjacent chains are oriented in the opposite direction. The β conformation corresponds to a unit with polymers of arranged chains in parallel form. The third form, γ, is characterized by three units of chains, in which two chains “up” are followed by a chain “down”. These forms have been the subject of detailed analysis with x-ray diffraction and spectrum of NMR, but is not yet known if the γ form is a third true structure or a variation between the α and β forms [27]. It is believed that the three polymorphic structures are related to the different functions in the body. An important physical aspect of the chitosan to be highlighted is that, despite the polymorphism, this polymer presents optical activity to 25°C. After temperature increase, a change occurs in the rotation angle. The temperature increase also favors the occurrence of hydrophobic interactions and new inter and intra-molecular hydrogen bonds present in the chitosan [28,29].

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Science against microbial pathogens: communicating current research and technological advances A. Méndez-Vilas (Ed.)_______________________________________________________________________________

The α chitosan creates a strong inter-molecular hydrogen bond, while the chitosan β is characterized by weaker intermolecular effects [29,30]. The crystallinity of the chitosan, as evaluated by studies of x-ray, depends of the acetylation level and process which the polysaccharide was achieved. The spatial structure is related to the form how the chitosan has been found in solid state, in other words, it depends if the chitosan is in its hydrate form, anhydrous, as complexes or salts of chitosan [14].

3. Properties and uses for chitosan

Chitosan has specific properties that reveal its potential for numerous applications in several commercial products. This polymer shows as main properties an excellent biocompatibility; almost any toxicity to human beings and animals; high bioactivity; biodegradability; reactivity of the group amino deacetylated; selective permeability; poluelectrolyte action; antimicrobial activity; ability to form gel and film; chelation ability and absorptive capacity [19,31]. These peculiar properties provide a variety of applications to the chitosan, such as: drug carrier of controlled release [32], anti-bacterial [33,34] and anti-acid [35]; inhibits the bacterial plaque formation and decalcification of dental enamel [36,37]; promotes the osteogenesis [38]; fat absorbent action [39] and promotes the healing of ulcers and lesions [40,41]. The applicability of the chitosan is directly related with its physical-chemical features, once the distinct attainment sources (crustaceans, fungi, and clams), and different processes of extraction and purification cause alterations in the deacetylation level, molar weight, thermal stability and crystallinity level in the chitosan molecule. Several derivatives of the chitosan are told in literature, which can be differentiated for the deacetylation level, molar weight, group arrangement N-acetil residual in the polymer chain, by the specific reaction of the group - NH2 in carbon 2 (C2) or nonspecific with the group - OH in carbons of position 3 or 6 (C3 or C6) of polymer with other functional groups [16]. Chitosan is an insoluble weak base in water, but soluble in diluted watery solutions of several acid - the ascetic and hydrochloric acid solutions are the most commonly used [42]. This characteristic limits some pharmaceutical applications of the polymer, once some physiological enzymes exert their activity in watery and neutral environment. Aiming to increase the applications of the chitosan, soluble derivatives in water and in neutral to a basic environment (up to pH 10) can be prepared, due the formation of the carbamate ion after adding ammonium carbamate (NH4HCO3). The production of the salt occurs when chitosan amino group reacts with an aldehyde to form an imine, or base of Schiff. This intermediary reacts with methyl iodide to form the quaternary salt of the chitosan [16]. The solubility in acid conditions is explained by the protonation of the free amino group, characteristic in the chitosan in natura, which passes from NH2 to NH3

+, whereas in alkaline way, the hydro solubility is due to the formation of carboxylate, from the introduced carboxylic group [20,22]. The polymer derivatives provide differences regarding the solubility, thermal stability, reactivity with other substances and specificity regarding the binding site, providing several biological applications of the chitosan [16]. Amongst some applications of the chitosan, it is highlighted its use in the pharmaceutical industry, more specifically related with dental clinic. This polymer usually is gotten in the flake or powder form, it does not possess high porosity and it is soluble in acid way. Other forms of chitosan presentation, like gel, sponge, paste and solution, are being studied [43].

4. Antimicrobial activity of the chitosan: action mechanisms

Chitosan has a recognized antimicrobial activity, being this, one of the main properties of the polymer. Several researchers tell that this polysaccharide has antimicrobial action in a great variety of microorganisms, including algae, fungi and bacteria [44]. This glycosaminoglycan has shown antimicrobial activity for some pathogen microorganisms, being highlighted its performance against gram-positive bacteria and various species of yeast [45]. It is assumed that chitosan acts in the cellular wall of the microorganism modifying the electric potential of the cellular membrane [46]. This polysaccharide also acts potentiating other inhibition drugs, as the chlorhexidine gel, once it increases the drug permanence time action place [47,48]. According to Kong et al [33], regarding the antimicrobial activity, the chitosan has demonstrated low toxicity and the resistance development has not occurred. The antimicrobial action of the chitosan and its derivatives suffers influence from factors, that depending on the performed role may be classified in four main categories: 1. Microbial factors (microbial species, age of the cell); 2. Intrinsic factors of the chitosan (positive charge density, molecular weight, hydrophobic and hydrophilic characteristics, chelation capacity); 3. Physical state factors (soluble and solid state) and 4. Environmental factors (pH, ionic forces, temperature, time). The antimicrobial action mechanism of the chitosan is not yet fully elucidated, being several action mechanisms suggested in literature. Some authors tell that the amino groups of the chitosan when in contact with physiological fluids are protonated and if bind to anionic groups of the microorganisms, resulting in the agglutination of the microbial cells and inhibition of growth [49,50]. On the other hand, Yadav, Bhise (2004) report that when interacting with the bacterial cell, the chitosan, promotes displacement of Ca++ of the anionic sites of the membrane resulting in cell damages. Other

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studies suggest that the antimicrobial action is closely related to the physical-chemical properties of the polymer and the features of the cell wall of the microorganism [16]. Another postulate is the interaction between the positive load of the chitosan and the negative load of the microbial cell wall, because it causes the rupture and loss of important intracellular constituent of the microorganism life. Chitosan with low molecular weight penetrates in the cell and is linked to the microorganism DNA inhibiting the transcription and consequently the translation, whereas the chitosan of high molecular weight acts as a chelant agent, binding to the cell membrane [17]. In literature it is evidenced that the molecular mass of the chitosan influences the solubility and the antibacterial activity of the derivatives. Chitosan of low molecular mass results in derivatives endowed with bigger solubility. On the other hand, the bactericidal power increases with the increase of the molecular mass of the hydro soluble chitosan, these results are opposing to the ones gotten in studies with chitosan in natura with low molar weight which have demonstrated greater effectiveness in the inhibition of the fungi growth when compared with the chitosan with high molar weight. Other authors suggest that this polymer with lower molar weight easily easily penetrates the cellular wall of fungi, which affects faster the vital components of the cells and physiological activities [16,51-53]. When investigating the relation between antimicrobial activity of the chitosan and the characteristics of the cellular wall of bacteria, Chung et al [46], have observed that the polysaccharide presents a better bactericidal and bacteriostatic action for Gram-negative bacteria than Gram-positive due the composition of phospholipids and carboxylic acids of the bacterial cellular wall. These results suggest that the effects of the chitosan are distinct in the two types of bacteria: in the case of the gram-positive, the hypothesis is that chitosan of high molecular mass may form films around the cell that inhibit the absorption of nutrients, while chitosan of low molecular mass penetrates more easily in gram-negative bacteria, causing riots in the metabolism of these microorganisms. Tsai and Su [54] and Zheng and Zhu [52] have verified that the antimicrobial activity of the chitosan is directly related with the absorption of the polysaccharide to the bacterium, which will cause alterations in the cellular wall structure, consequently, in the permeability of the cellular membrane. Studies with electronic micrographs demonstrate that in Gram positive bacteria the chitosan weakens or even though breaks up the bacterial cellular wall, while in the Gram negative the cytoplasm is concentrated and the cell interstice, extended. The Gram-negative bacteria possess the external membrane of the cellular wall composed by lipopolysaccharides (LPS) that provide a hydrophilic surface to the bacterium. The LPS also have anionic groups (phosphate, carboxyl), which contribute for the stability of the LPS through divalent electrostatic interactions with cations). The removal of these cátions for chelant agents result in the run down and molecule release of the LPS. On the other hand, the cellular wall of the Gram-positive bacteria is composed mainly for peptidoglican (PG) and teichoic acid -TA (polymer polyanion), as visualized in (Figure 2). These components are linked covalently to the acid N-acetylmuramic of the PG or anchored in direction to the cytoplasmic membrane, via glycolipid (lipoteichoic acid -LTA), which provides a binding site with the chitosan, causing functional riots in the membrane [33,55].

Source: Zong et. al. (2010) modified

Fig.2 Scheme showing the cell wall differences between Gram-negative (A) and Gram-positive bacteria (B).

5. Streptococcus mutans and caries

Despite the significant role of the Streptococcus sp. in the development of the dental caries, the microbial community is responsible by the mineral imbalance which features the caries development. Technically, the term biofilm describes communities of microorganisms which are attached to a surface, specially organized in a three-dimensional structure and included in a matrix of extracellular material, derived from the cells metabolism and the environment [56]. The dental biofilms are peculiar in their condition, because have been exposed to numerous challenges such as: nutrients

A B

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offering, transitions in aerobic and anaerobic, changes in the pH, as well as, exposition to detergent substances which act as regulators in the biofilm development [57]. According to Marsh [58], the dental biofilm formation may be divided into the following stages: 1. Formation of the acquired film; 2. Microorganisms transport to the tooth surface; 3. Long distance physical-chemical interactions between the microbial surface and tooth. Work in this phase, the attractive power of Van der Waals forces and the electrostatic repulsive forces; 4. Short space interactions, involving specific electrochemical interaction among the adhesins in the microbial cell surface and receptors of the acquired film; 5. Coagregation of microorganisms to cells which are already linked; 6. Multiplication of linked microorganisms and extracellular polymers production; 7. Biofilm cells displacement to new places of colonization (Figure 3). Independent of the dental surface type, the initial settlers constitute a highly selective part of the oral microbiota, which are the Streptococcus sanguis, Streptococcus oralis and Streptococcus mitis, corresponding to 95% of the microbiota of Streptococcus besides the Actinomyces sp and gram negative bacteria like Haemophilus sp and Neisseria sp [59].

Source: authors

Figure 3 Scheme of a biofilm formation on a tooth surface

The Streptococcus mutans plays an important role in the caries process due to its high capacity in the carbohydrates

metabolism under the harshest conditions. In addition, extracellular polysaccharides promote the adhesion of the oral microorganisms facilitating the episodes of demineralization instead of remineralization [60].

The confocal laser microscopy has shown that the supragingival biofilm structure has a more open architecture than that one suggested in the beginning of the studies with optical microscopy, and allows the passage of substances through canals which cross the biofilm from a surface to another [61]. In this sense, antimicrobial products, for example the chitosan which may interfere in initial biofilms as well as in well established biofilms. However, independent of the involved antimicrobial factors, the microbial organization in biofilms increases the resistance practically against all type of antimicrobial agents. This has lead to consider that the minimum inhibitory concentrations are below the ideal one for clinical practice [62]. This factor promotes natural products as antimicrobial agents due their biological activity with lower risk of deleterious effects.

6. Chitosan as antimicrobial for Streptococcus mutans

The gel of chitosan achieved through the polymer dilution in acetic acid has been suggested as a preventive and therapeutic material for dental caries [63,64]. In caries prevention, study, in vivo, with mouthwash solution containing chitosan has confirmed the effectiveness of this product to oral hygiene, reducing the bacterial plaque formation [36]. Regarding the hydro soluble derivatives of the chitosan, some authors have reported the production of quaternary ammonium salts of chitosan soluble in water endowed with antibacterial activity [16]. Association studies of the chitosan with dental materials, such as tetrafluotileno and hidroxidoapatita suggest that chitosan increases the biocompatibility of materials, favors the cell migration and inhibit the absorption of oral bacteria (S. sanguis, S. mutans, S, mitus, S. salivarius) to the tooth [63,65]. To the studies, in vitro, carried out in the Microbiology laboratory from UFPB to determine the minimum inhibitory concentration (CIM) and minimum bactericidal concentration (CFM) of chitosan and its derivatives, from different origins, and varying the parameters, deacetylation level, molecular weight and solubility to Streptococcus mutans, it has

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been observed greater influence of the solubility and molecular weight of the polymer. The water soluble chitosans, in hydrochloride form, as well as those with low molar weight have shown greater antimicrobial activity, with lower CIM and CBM, when compared to chitosan in gel, soluble in acetic acid 1% and with greater molecular weight, as visualized in (Table 1).

Table 1. Studies in vitro performed by the karyology research group, Microbiology laboratory from UFPE, demonstrating the minimum inhibitory concentration (CIM) and minimum bactericidal concentration (CBM) of the chitosan and its derivatives from different origins, and varying the parameters, deacetylation level (DA), molecular weight (MW) and solubility to Streptococcus mutans

CHITOSAN Origin Form DA

(%) MW

(g/mol) Solubility Antimicrobial

CIM (mg/mL)

CBM (mg/mL)

Crustaceans GEL 65 3,24x 105 Acetic acid 1% 1.25 2.50 Crustaceans GEL 90 3,24x 105 Acetic acid 1% 1.25 2.50 Crustaceans Solution 85 3,24x 105 Water- chitosan

hydrochloride 0.06 0.03

Fungi GEL 85 2,72x 106 Acetic acid 1% 1.25 2.00 Fungi GEL 85 3,14x 104 Acetic acid 1% 0.06 1.25 Fungi Solution 85 3,14x 104 Water- chitosan

hydrochloride 0.03 0.01

Fungi Solution 85 2,36x 105 Water- chitosan hydrochloride

0.06 0.03

Fungi Solution 85 2,72x 106 Water- chitosan hydrochloride

0.06 0.03

The use of chitosan in different formulations, such as toothpastes (Chitodent®), mouthwash solutions and chewing gums, is mentioned in literature. In all forms the chitosan has shown antibacterial activity for Streptococcus bacteria groups. The chitosan inhibits the bacterial plaque formation and stimulates salivation in vivo. These effects suggest the application of chitosan as preventive and therapeutic agent to control dental caries [36,66-69]. Experiments were performed in the Laboratory of Oral Biology (LABIAL) at UFPB regarding the search of safe concentrations of chitosan in acetic acid. Chitosan was tested regarding its antibacterial activity when diluted in acetic acid. Therefore the essay was performed taking into account the antimicrobial activity of the acetic acid. The technique of microdilution in broth was selected and this procedure indicated the safety range to evaluate chitosan in the mentioned acid as diluent. The results showed that concentrations below or equal to 200 ppm are safe. There was an evident oxidation of the resazurin in row A in pits 2 to 6, the MIC (minimum inhibitory concentration) of the acetic acid was equal to 400 ppm (Figure 4). The results found in this study are compatible with the experiment of Liu et al. [70], where the author has perforned tests of the antibacterial activity of the acetic acid, being different the studied microorganism, because the study used strains of Escherichia coli. The acetic acid concentrations used have varied from 20 to 1000 ppm, the results have shown that concentrations above 200 ppm are toxic to E. coli. All these data support the view that chitosan is a promissing material for dental products.

Source: Authors

Fig. 4 Micro plaque of the antibacterial activity of the acetic acid

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Acknowledgements The support by CNPq and CAPES are gratefully acknowledged

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