Surface active properties of chitosan and its derivatives

Colloids and Surfaces B: Biointerfaces 74 (2009) 1–16

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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Review

Surface active properties of chitosan and its derivatives Maher Z. Elsabee a,∗ , Rania Elsayed Morsi b , A.M. Al-Sabagh c a

Department of Chemistry, Faculty of Science, Cairo University, 12613 Cairo 12613, Egypt Central Lab Egyptian Petroleum Research Institute (EPRI), 1 Ahmed El-Zomor St., Nasr City, 11727 Cairo, Egypt c Department of Petroleum Applications, Egyptian Petroleum Research Institute (EPRI), 1 Ahmed El-Zomor St., Nasr City, 11727 Cairo, Egypt b

a r t i c l e

i n f o

Article history: Received 18 April 2009 Received in revised form 16 May 2009 Accepted 16 June 2009 Available online 23 June 2009 Keywords: Chitosan Chitosan derivatives Hydrophobic substitution Surface active properties Aggregation behavior Drug encapsulation Surface interactions

a b s t r a c t This review discusses the definition of surface active agents and specifically natural polymeric surface active agents. Chitosan by itself was found to have weak surface activity since it has no hydrophobic segments. Chemical modifications of chitosan could improve such surface activity. This is achieved by introducing hydrophobic substituents in its glucosidic group. Several examples of chitosan derivatives with surfactant activity have been surveyed. The surface active polymers form micelles and aggregates which have enormous importance in the entrapment of water-insoluble drugs and consequently applications in the controlled drug delivery and many biomedical fields. Chitosan also interacts with several substrates by electrostatic and hydrophobic interactions with considerable biomedical applications. © 2009 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Zwitterionic and amphoteric surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Polymeric surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Natural polymeric surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Surface activity of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Aggregation behavior in chitosan solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Carboxymethyl chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Hydrophobic modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Interaction of chitosan with various substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Layer-by-layer assembly technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Chitosan and lung surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The term surfactant is short for “surface active agent”. Surfactant is a molecule with an amphiphilic character that has a tendency to accumulate at the interfaces.

∗ Corresponding author. Tel.: +20 2 26352316. E-mail address: [email protected] (M.Z. Elsabee). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.06.021

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Surfactants have unique properties which are a direct result of incorporating both hydrophobic and hydrophilic groups in their structure. This dual functionality permits it to accumulate at interfaces such as those between a solution and air as well as solutions and solids, even at the walls of a container and of practical interest is the interface between two liquids such as water and oil. The association behavior of surfactants in solution and their affinity for interfaces is determined by the physical and chemical properties of the hydrophobic and hydrophilic groups. The size and shape of the hydrocarbon moiety and the size, charge and hydration

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Fig. 1. Surfactants on surface and formation of the (CMC).

of the hydrophilic head group are the dominant factors governing the self-assembled structures, both at the interfaces and in the bulk solution. Amphiphilic molecules display distinct behavior as it can arrange itself at the surface of the water such that the polar part interacts with the water and the non-polar part is held above the surface (either in the air or in a non-polar liquid). The presence of these molecules on the surface disrupts the cohesive energy at the surface and thus lowers the surface tension. The proportion of molecules present at the surface or as aggregates in the bulk of the liquid depends on the concentration of the amphiphile. At low concentrations, the amphiphiles will favor arrangement on the surface. As the concentration of surfactant increases, the surface tension decreases reaching a certain value after which the surface becomes crowded and completely loaded with amphiphiles and any further additions will lead to no decrease in the surface tension because they have to be directed to the bulk and orient themselves in an arrangement that allows each component to interact with its favored environment (Fig. 1). Molecules can form aggregates in which the hydrophobic portions are oriented within the cluster and the hydrophilic portions are exposed to the solvent. The concentration above which surfactant starts to form micelles or aggregates is called the critical micelle concentration (CMC) or the critical aggregate concentration (CAC) and the determination of this value is a very important step in using surfactants.

Surfactants have a wide range of uses in both biological and industrial applications. For example, the aggregation of surfactant molecules in bulk solution as micelles can be used as a carrier in drug delivery for the poorly soluble drugs while the accumulation of surfactant molecule at the interfaces between two immiscible liquids play an important role in emulsifying or solubilizing these immiscible phases. 2. Types of surfactants It is a longstanding practice to classify commercial surfactants as anionic, cationic, or nonionic based on the nature of their ionic charges in solution. This classification applies to the hydrophilic group on the molecule, since the hydrophobic portion is always nonionic and differences in the nature of the hydrophobic groups are usually less pronounced than in the nature of the hydrophilic groups. Generally the hydrophobic portion may be a long straight or branched alkyl group [1] of high molecular weight propylene oxide [2,3] or a long fluoro-alkyl group [4]. Anionic refers to negatively charged surfactants as carboxylates, sulfates, sulfonates or phosphates while poly alkyl methacrylateb-sulfonated glycidyl methacrylates are an example of anionic polymeric surfactants. Cationic are those that are positively charged. The majority of cationic polymeric surfactants are based on nitrogen atom carrying

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Scheme 1. Schematic diagram for polymeric surfactants with (a) hydrophilic backbone and hydrophobic side chains, (b) hydrophobic backbone and hydrophilic side chains and (c) alternating hydrophobic and hydrophilic blocks.

the cationic charge. Both amine and quaternary ammonium-based products are common but amines only function as a surfactant in the protonated state; they thus cannot be used at high pH. On the other hand quaternary ammonium compounds are not pHsensitive [5]. Nonionic refers to those that are uncharged in solution. Usually nonionic surfactants have either a polyether or polyhydroxyl moiety as the polar group. Ethylene oxide condensates form a major part of nonionic surfactants [6] giving a very broad spectrum of compounds than other surfactant types. The most common examples of these substances are that built up of long chain alkyl or alkyl aryl hydrophobic moiety connected with hydrophilic, mostly, polyethylene oxide. Nonionic polymeric surfactants find wide applications in emulsion polymerization; [7] liquid crystalline phases [8], miceller enhanced ultrafiltration [9], surface modifications, [10] destabilization of water in crude oil emulsions [11,12] and other industrial applications. 2.1. Zwitterionic and amphoteric surfactants In the zwitterionic case, both negative and positive charges may be present in the surface active portion. The positive charge is almost ammonium and the source of negative charge may vary, although carboxylate is the most common. Some referred to the zwitterionic as amphoteric and this is not always correct. An amphoteric surfactant is one that, depending on pH, can be cationic, zwitterionic or anionic. Among normal organic substances, simple amino acids are well known examples of amphoteric compounds [5]. 3. Polymeric surfactants The growing interest in using surface active polymer “polymeric surfactants” instead of the traditional surfactant molecules can be said to emanate from having a very strong driving force to go to the interfaces and the tendency to collect at these interfaces is not as dependent on physical variables as for normal, low molecular weight surfactants. This means that the polymeric surfactants are effective at low total concentrations. In addition, they can have long polyoxyethylene (or polysaccharide (chains and still be retained at the interfaces. (Low molecular weight surfactants with long hydrophilic chains tend to be desorbed from the interface and dissolve in the aqueous phase) [5]. Recently, the polymer micelle is being regarded as one of the most promising candidates for carrying and for delivering bioactive materials such as water-insoluble drugs, hormones and plasmid DNA [13]. A polymer with surface active properties can be constructed by three routes: hydrophobic chains grafted to a hydrophilic backbone, with hydrophilic chains grafted to a hydrophobic backbone or with alternating hydrophilic and hydrophobic segments as seen

in Scheme 1. In reality two or more types may be combined into one product. For example, a surface active macromolecule may have a backbone polymer consisting of alternating hydrophilic and hydrophobic segments and, in addition can contain hydrophilic or hydrophobic side chains, i.e. the molecule may at the same time be a block and a graft copolymer. A graft copolymer may also contain both hydrophilic and hydrophobic grafts. The important feature from the physicochemical point of view is that the molecule is able to orient itself so as to expose the hydrophilic regions to the polar environment and the hydrophobic segments to the other phase. 4. Natural polymeric surfactants Chitin is a naturally abundant polysaccharide and the supporting material of crustaceans such as crabs, shrimps, cuttlefish, insects, etc. Chitin is the second most abundant natural polymer after cellulose and estimated to be produced annually almost as much as 10 billion tons. It is a highly insoluble material resembling cellulose in its solubility and low chemical reactivity. Chitosan is the modified N-deacetylated derivative of chitin (Scheme 2). Recently, much attention has been paid to chitosan as a potential polysaccharide resource. It has become of great interest not only as an under utilized resource, but also as a new functional material of high potential in various fields and recent progress in chitin chemistry is quite noteworthy. Chitosan is a random copolymer, containing ␤ (1 → 4) dglucosamine and N-acetyl-d-glucosamine units. Chitosan carries the free amino and hydroxyl groups along its backbone, and it is soluble in dilute acid aqueous solutions. The interaction between water molecules and chitosan chains is primarily limited by intra- and inter-molecular interactions, e.g., hydrogen bonding and van der Waals forces [14,15]. Since chitosan is a positively charged linear polysaccharide [at low pH <6.5], it is a polyelectrolyte having numerous applications. The physicochemical properties of chitosan solutions can be controlled by manipulations of the solution conditions (temperature, pH, ionic strength, concentration, and solvent). Because of that, chitosan exhibits pH dependent solubility; at pH > 6.5 chitosan solutions exhibit phase separation, while for pH < 6.5 it is soluble and carries a positive charge due to the presence of protonated amino groups [16]. At higher pH of the solutions, i.e. 6.0 and 6.5, the free amino groups of chitosan molecules become less protonated and the hydrophobic character along the chitosan chain becomes stronger. The conformation of the chitosan polymer is a function of its chain flexibility and solution conditions [17]. Chain flexibility facilitated inter- and/or intra-molecular interaction between chitosan chains [18,19]. The interest on chitosan is not limited only to the academic science, but also demonstrated by the thousands of patents related to its applications. The largest part of them is related to the ability of

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Scheme 2. Structures of cellulose, chitin and chitosan.

this polymer to bind a lot of substances for the contemporary presence on its backbone of hydrophobic units, the acetamide groups, and hydrophilic units, the amino groups, and also to the capacity of chitosan to self-aggregate giving rise to gel systems [20].

5. Surface activity of chitosan Although there are a large number of papers related to chitosanbased surfactants, only a few papers relate to the surface activity of chitosan itself can be found in the literature. This may be due to the low surface activity of pure chitosan which agrees with its chemical structure; a polysaccharide with cationic–NH3 + and alcoholic–OH groups distributed along the hydrocarbon structure. The structure has no large hydrophobic groups which may be adsorbed at the air/solution interface. Schulz et al. [21] have measured the surface tension of chitosan in 1% acetic acid and they observed that the surface tension of chitosan is higher than that of the pure solvent. The surface activity and the aggregation properties of chitosan in water were reported [22]. The surface tension of chitosan in water at 25 ◦ C and pH 5.06 was 71.45 mN m−1 and at 34 ◦ C and pH 5.21 was 70.26 mN m−1 . These values are very close to the surface tension of pure water, 71.99 mN m−1 at 25 ◦ C and 70.52 mN m−1 at 34 ◦ C. The constant values of surface tension indicate that chitosan molecules are excluded from the air/solution interface, indicating thus the absence of surface activity. A slight decrease of the surface tension of chitosan in acetate buffer solutions (pH 6.38) was observed and the decrease in surface tension was equal to 2.47 mN m−1 at 25 ◦ C and to 2.17 mN m−1 at 34 ◦ C. Higher decrease in the surface tensions was obtained at lower pH value reaching 6.07 m Nm−1 at 25 ◦ C and 7.14 m Nm−1 at 34 ◦ C. These observations were explicable because at a relatively lower pH, the amino groups of chitosan molecules are protonated, which gave the chitosan a stronger hydrophilic character in bulk solutions. Schulz et al. [21] described analogous result explaining that chitosan molecule has no large hydrophobic groups that may be

adsorbed on the air/solution interface. At higher pH value of the solutions (6.0 and 6.5), the free amino groups of chitosan molecules become less protonated and the hydrophobic character along the chitosan chain becomes stronger. Therefore, the chitosan selfaggregates could be formed in acetate buffer solutions by intra- and inter-molecular hydrophobic interactions [22]. On the other hand, the decrease of surface tension with increased chitosan concentration in acetate buffer solutions may be explained by adsorption of the hydrophobic sequences of chitosan molecules, at least partially, on the air/solution interface. Geng et al. [23] studied the electrospinning of chitosan in concentrated acetic acid solution. They reported that the surface tension is one of the most important parameters in this study because it determines the upper and lower boundaries of the electrospinning window if all other parameters are constant; lowering the surface tension of the spinning solution helps electrospinning to occur at lower electric field. One of the most interesting results from this study is that the acetic acid concentration in water strongly influenced the surface tension of chitosan solutions; as the acetic acid concentration increased from 10% to 90%, the surface tension decreased from 54.6 mN m−1 to 31.5 mN m−1 without significant viscosity change as shown in Fig. 2. On the other hand Pepic´ et al. [22] mentioned that a similar effect in chitosan/acetate buffer solutions could not be expected, due to the almost constant pH values in the systems. Self-aggregation properties of chitosan have been found by fluorescent measurements using dye solubilization methods [24,25]. The concentration at which aggregates started to form was around 0.1% (w/v) chitosan in acetic acid solutions (0.1 mol dm−3 ). Katanchalee Mai-ngam [26] used the Langmuir film balance technique to confirm the surface activity of low molecular weight chitosan but a Langmuir isotherm at water interface for this low Mw chitosan hydrochloride (Mn = 5000) could not be obtained due to its poor solubility in the used solvents. However, by measuring the surface tension of the aqueous solutions of the low Mw chitosan hydrochloride solutions against log C (concentration), it was

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Fig. 2. Effect of acetic acid concentration on the surface tension and viscosity of chitosan.

found that it exhibits surface active properties and the result follows classic surfactant behavior as shown in Fig. 3. The water surface tension decreases linearly with increasing logarithm of chitosan concentration and then levels off. Chitosan hydrochloride with a molecular weight of 5000 may be partially accessed by water molecules to the degree that creates an appropriate hydrophobic/hydrophilic balance to facilitate the formation of a chitosan monolayer at the air–water interface. From the slope of the surface tension–log concentration d/dlog C plot and the simplified Gibbs adsorption equation d = −2.303 RT  dlog C, where R is the gas constant, T is absolute temperature (K) and  is the maximum surface density of the glucosamine repeating unit, the surface area per glucosamine unit [the surface area occupied by each glucosamine = (1/ N)] can be calculated and was found to be 0.43 nm2 . This value is close to the area obtained for a close packed monolayer (∼0.5 nm2 per monomer unit) that has been previously reported by Li et al. [27]. This suggests that the film of low Mw chitosan hydrochloride could form a monolayer structure on the water surface. 6. Aggregation behavior in chitosan solution It is well established that surfactants form micelles in solution in order to decrease the free energy when the concentration is higher than the critical micelle concentration (CMC). Analogously, aggregates should also be formed for amphiphilic polymers such as block polymers in aqueous media. As it is difficult to form micelle-like

Fig. 3. A plot of surface tension of aqueous solution vs. log concentration (mg/ml) of low molecular weight chitosan hydrochloride (Mn 5000).

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aggregates due to the stiffness of the molecules of chitosan and its derivatives [28], the transition concentration is consequently denoted as the critical aggregation concentration (CAC) rather than the (CMC) customary for surfactants and amphiphilic block copolymers [29]. The CAC of chitosan and its derivatives is usually measured by surface tension methodology or fluorescence spectroscopy, in addition to viscosity. The quite low solubility of chitosan in neutral water limits its applications. In order to overcome the poor solubility of chitosan in water or in organic solvents, chemical modification of chitosan has been carried out in different ways among which are the chemical reactions and grafting. Not all of the these modifications give rise to surface active agents that can form micelles. Different ways for insertion of hydrophilic groups, to increase solubility, and hydrophobic groups, to increase the surface aggregation and the self-aggregation of hydrophobized water-soluble chitosan are summarized below.

7. Carboxymethyl chitosan Carboxymethyl chitosan, CMC an important water-soluble chitosan derivative, has many attractive chemical, physical and biological properties. Carboxymethyl chitosan is an efficient metal chelator [30,31] and exhibits high adsorption capacities for dyes [32]. Due to its unique properties, particularly its biocompatibility, CMC has been extensively used in many biological fields [33–35]. Since it has been shown to possess a variety of unique properties, the compound has attracted worldwide attention particularly in the biomedical field. However, there are few reports dealing with the water soluble CMC in solution as a surface active agent and its interactions in water. Chena et al. [36] investigated the chemical characteristics of Ocarboxymethyl chitosan O-CMC in order to find out how they are affected by the preparation conditions. They found that the water solubility of the O-CMC had close relationships to the modifying conditions; the degree of carboxymethylation, the reaction temperature, the ratio of water:isopropanol in the reaction solvent. The yields of O-CMC prepared in the mixed solvents were higher than in water alone or in isopropanol alone. The highest yields were close to 100% at water:isopropanol ratios between 1:4 and 1:1 at 50 ◦ C. The carboxymethyl groups were mostly substituted on the –OH groups, with a small amount on the –NH2 groups. The 6-OH group had the highest degree of substitution. When the reaction temperatures were at 0 and 10 ◦ C, the O-CMC had good water solubility, and at higher temperatures, O-CMC was insoluble at near neutral pHs. Zhu et al. [29] investigated the aggregation behavior of O-CMC in dilute aqueous solution using surface tension method. The concentration dependence of the surface tension for O-CMC in aqueous solution is shown in Fig. 4. It is obvious that this is a conventional surface tension behavior curve, since the surface tension dropped significantly with increasing the concentration. After a transition concentration of about 0.050 mg/ml, the surface tension tended to become independent of the concentration. The transition point is attributed to the critical aggregation concentration CAC of O-CMC in aqueous solution. The CAC value of about 0.050 mg/ml, is much smaller than the CMC of common surfactants due to the high molecular weight of O-carboxymethyl chitosan. The aggregation behavior of O-CMC in aqueous solution was further examined by steady-state fluorescence spectroscopy which strongly indicates that in aqueous solution, the hydrophobic O-CMC microdomains that could incorporate the similarly hydrophobic pyrene were formed and confirmed the CAC value measured by

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Fig. 6. Concentration dependence of the intensity ratio I1 /I3 of pyrene in OCMCS aqueous solutions.

Fig. 4. Concentration dependence of the surface tension for O-carboxymethyl chitosan in aqueous solution.

surface tensionmetry. Also viscosity was used as a third method to determine the CAC value. Fig. 5 shows the relationship between the relative viscosity (r ) and O-CMC concentration in which, the viscosity increased with O-CMC concentration until a transition concentration of about 0.042 mg/ml, beyond which, the viscosity increased linearly with concentration with a relatively steeper slope. This was explained as follows; as O-CMC concentration increased, the solution viscosity increased and there was no aggregation until 0.042 mg/ml; beyond that, aggregation occurred, leading to the sharp increase in the solution viscosity as observed after the transition point. Clearly, the transition concentration could then be interpreted as the CAC, and the value of 0.042 mg/ml obtained is close to those from both steady-state fluorescence and surface tension studies. The hydrophobic nature of polymers and surfactants can be monitored by measuring the emission spectra of pyrene in solution. Pyrene spectrum has five peaks and the emission intensity of the first peak (374 nm) and of the third peak (385 nm) is sensitive to the microenvironment. Thus, the intensity ratio (I1 /I3 ) of the emission at 374 and 385 nm has been used to monitor the solution behavior of

Fig. 5. Dependence of the relative viscosity (r ) on the O-carboxymethyl concentration.

surfactants and/or polymers. A bigger ratio of (I1 /I3 ) means a greater polarity of the solution around the pyrene molecules. Therefore, the formation of the aggregates with a hydrophobic inner core can be detected by means of plotting (I1 /I3 ) versus polymer concentration [37]. The sharp decrease in the I1 /I3 ratio points to a transformation from a hydrophilic surrounding around the pyrene molecules to a hydrophobic one, in other words, to the formation of hydrophobic aggregates. Fig. 6 shows that this drastic drop in the I1 /I3 ratio occurs at around 0.050 mg/ml. The authors explained the reasons for the surface activity of OCMC. They reported that the H-bonding between water and the polymer and the presence of carboxylic groups on the O-CMCs chain make it soluble in water while the intermolecular H-bonding of OCMC and the electrostatic repulsion between them were the main driving forces for its aggregation in solution. This aggregation was dominated by inter-chain aggregation, with the glucose backbones of O-CMC forming the hydrophobic domains, and the dissociated carboxylic groups and the hydrophilic groups around the backbone forming the hydrophilic ones. All these properties are important for developing O-CMC for potential applications in many fields.

8. Hydrophobic modifications The low degree of hydrophobic substitution on chitosan as well as carboxymethyl chitosan (or its hydrophilic modified derivatives) may result in weakening stability of the micelles in the solution. Therefore, the preparation of a chitosan-based amphiphilic compound having more hydrophobic substituents is expected to improve the surface activity or lead to a higher stability of the polymer micelles. It is also expected to lower the critical aggregate concentration CAC or the critical micelle concentration CMC [37–42]. Sui et al. [38] reported that O-CMC has few surface active properties because there are no hydrophobic groups in the polymer. Therefore O-CMC was hydrophobically modified using dodectyl glycidol ether to give (2-hydroxyl-3-dodecanoxyl) propyl carboxymethyl chitosan, HDP-CMCHS an amphiphilic derivative, which can concentrate on the surface with their hydrophobic chains pointing to the air while the hydrophilic backbones lay on the surface to reduce surface tension. Fig. 7 shows the surface tension–concentration plots of CMC and its modified derivatives with different degrees of substitution. From the curve it is obvious that the introduction of hydrophobic substituents decreases the surface tension and as the degree of substitution increases, the surface tension decreases.

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Fig. 7. Surface tension–concentration plots of O-CMC and its modified derivatives with different degrees of substitution.

Fig. 7 shows that the decrease of surface tension is slow and there are no clear transition points on the surface tension concentration curve. The reason may be that for the anionic amphiphilic polymer, the hydrophobic chains of different molecules can associate to form aggregates in the solution while they concentrate on the surface before the surface tension gets to the lowest value. These phenomena were confirmed by the fluorescence measurements [38], i.e. aggregate can form at very low concentration while the surface tension decreases. As the surface tension reaches an equilibrium value, the additional excess polymer can only make the inner core of the aggregates a little more compact. The addition of a strong electrolyte e.g. NaCl was found to increase the hydrophobic character of the prepared polymers as shown in Fig. 8. The authors attributed these results to the fact that the electrolyte can compress the electric double-layer of the anionic carboxymethyl group on the hydrophilic backbone of the HDP-CMCHS. It is easy therefore for the polymers to concentrate on the surface of the solution which will reduce the surface tension, and can easily form aggregates by hydrophobic group association in solution. An analogous conclusion was reached by Amiji [29]. Surface pressure measurements (using the Langmuir balance technique) indicated that for carboxymethyl chitosan (CMCHS) with the decrease of the surface area, the relative compressed pressure increases slowly because there is little CMCHS concentrated at the surface. But for HDP-CMCHS, the relative compressed pressure increases dramatically with the decrease of the surface area, this is due to the increased hydrophobic interaction at the surface leading to the formation of a monolayer while for CHCTHS the molecules

Fig. 8. Surface tension–concentration plots of HDP-CMCHS (DS = 11.0%) in the presence and absence of NaCl.

Fig. 9. (I1 /I3 )–concentration plots of CMCHS and HDP-CMCHS at different degrees of substitution.

may arrange in a more compact structure or even form multilayer on the surface. Fig. 9 shows that CMCHS forms no hydrophobic domain around the pyrene molecules and that the values of the I1 /I3 ratio are almost equal to that in water, while the values for the HDP-CMCHS decrease with increasing both the concentration and the degree of substitution indicating an increase of the hydrophobicity of the inner core of the polymer with increasing the concentration and the DS. Another water-soluble anionic polymeric compound, Nsuccinyl-chitosan (NSCS), has been prepared and investigated. It can self-assemble in regular nanosphere morphology in distilled water [43]. The mechanism of self-assembly of NSCS in distilled water is due to the intermolecular H-bonding and hydrophobic interaction among the hydrophobic moieties such as –CH2 CH2 –, acetyl groups and glucosidic rings in NSCS. However, NSCS has little surface activity for there are weak hydrophobic groups in the polymer but after being modified with (2-hydroxypropyl-3-butoxy) propyl group (Scheme 3), the resulting derivatives are converted to amphiphilic polymers with the hydrophilic backbones substituted by hydrophobic groups [39]. These polymers can adsorb on the surface with the hydrophilic backbone in the solution, while the hydrophobic groups point up toward the air to reduce the surface tension of water. When the concentration is big enough, the surface adsorption is completed and the surface tension no longer descends as shown in Fig. 10. It is also obvious from the figure that with the increase of the degree of substitution of the (2-hydroxypropyl-3-

Fig. 10. Surface tension–concentration plots of succinyl chitosan and its hydrophobically modified derivative with different degrees of substitution.

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Scheme 3. Synthesis of succinyl chitosan and its hydrophobically modified derivative (R = C4 H9 ).

butoxy) propyl groups, a more pronounced decrease in the surface tension takes place. Jiang et al. [42] obtained micelles based on amphiphilic chitosan derivatives synthesized by grafting hydrophobic stearoyl, palmitoyl and octanoyl aliphatic chains onto molecules of chitosan with degrees of substitution from 0.9% to 29.6% by reacting carboxylic anhydride with chitosan in dimethyl sulfoxide. The micelle formation was confirmed by transmission electron microscope (TEM) and it was found that the micelles were spherical in shape and their sizes were measured by dynamic light scattering (DLS) and were in the range of 140–278 nm. The micellar critical aggregation concentration CAC can reach 1.99 × 10−3 mg/mL which therefore are more stable upon dilution compared to a previous study in which, Lee et al. [40,41] obtained micelles through chitosan grafted with deoxycholic acid. The degree of substitution (DS) of latter chitosan derivatives was relatively low (2.8–5.1%), leading to a high critical aggregation concentration (CAC 1.7 × 10−2 to 4.1 × 10−2 mg/ml), therefore, the formed micelles were unstable against dilution. To further augment these results, Li and Kwon [43] reported that the increase in the level of hydrophobic attachment leads to high stability of the polymer micelles. Ortona et al. [20] introduced linear aliphatic chains of variable length from 5 to 12 carbon atoms on the chitosan backbone at 10% in moles of the glycosidic units and analyzed the intra- and inter-aggregation properties of these modified chitosans and compared them with those of the chitosan itself. Viscosity in the dilute concentration range showed that the insertion of short aliphatic chain (C5-chitosan) does not modify the rigidity and/or the interaction with the solvent. For C6, C8, and C10-chitosan, the increasing length of the pendant groups promotes a progressively more efficient intra-aggregation of the polymer as shown by the reduction of its hydrodynamic radius. The hydrophobic nature of C8, C9, C12-chitosan was evident from pyrene fluorescence emission measurements. The emission of pyrene increased as if it was surrounded by a hydrocarbon solvent thus indicating the formation of well defined pools. Dilauryl chitosan pentamer, has been synthesized by reacting chitosan and lauryl aldehyde [27]. The obtained polymer showed surface activity. It was spread on a water surface from chloroform solution to investigate its monolayer behavior. The condensed monolayer formed had a collapse pressure of 50 mN m−1 and was transferred successfully onto a solid support by the

Langmuir–Blodgett (LB) technique giving a chitosan derivative ultrathin LB film with a highly ordered layer structure and smooth surface. The characterization of the LB film and the monolayer on the water are discussed. Lee et al. [44] studied the partial N-acylation of chitosan by acid anhydrides with different carbon chains from 6 to 16 carbon atoms to evaluate the number and length of carbon chains on chitosan required for micellar conformation. The surface tension of the modified samples was measured in aqueous solution at pH 4.0 as a function of their concentrations. As the substitution ratio and length of carbon chain increased, the surface tension of the modified samples tended to decrease. In addition, an increase in concentration of the modified samples decreased surface tension but most of these samples had quite high values of surface tension, presumably because they formed polymeric micelles. They also found that it is necessary to have a maximum length of carbon chain to observe micellar conformation. Ngimhuang et al. [13] synthesized a chitosan-based polymeric surfactant that had a tetrahydroxy alkyl linker and a dodecyl hydrophobic residue via the reaction of amphiphilic compound, 3-O-dodecyl-d-glucose (DG), with the amino group of chitosan through formation of Schiff’s base and subsequent reduction that achieved a high degree of substitution (Scheme 4). It is reported that the introduction of saccharide residues in the side chain of a polymer increased the water solubility [45], so it is expected that the glucose residue in chitosan–DG will increase its water solubility. The lower DS (9.8%) derivative was dissolved in the water to give a transparent solution. However, DS 27% swelled in water under neutral conditions. These results might suggest that the hydrophilic–hydrophobic balance of the entire molecule was essential for their water solubility. The increased substitution with hydrophobic dodecyl group was supposed to suppress the hydrophilicity of the glucose residue. A transparent solution of (DS 27%) was obtained in 0.1% (v/v) aqueous acetic acid. In order to investigate the surface activity of chitosan–DG, the critical aggregate concentration, CAC, was determined from the change of the quotient of vibrational band intensities in fluorescence emission spectrum of pyrene in the conventional way. The results suggested that the stability of the polymer micelle was highly dependent on the density of hydrophobic subsistent. Higher degrees of hydrophobic substitution in the macromolecule of chitosan may facilitate its self-aggregation. Poor ability to

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Scheme 4. Preparation of chitosan with a saccharide side group.

form micelle aggregate was observed with the low substituted DG–chitosan (DS 9.8%). CAC values determined in neutral and acidic (0.1% aqueous acetic acid) aqueous media were 100 and 1.2 × 103 mg/l, respectively. In contrast to this, the CAC value of DS 27% was equal to 28.1 mg/l in 0.1% (v/v) aqueous acetic acid. To obtain good solubility in water, a large number of hydrophilic groups must be introduced to provide chitosan derivatives where most of the glucosamine units have to be modified. But to obtain a highly water-soluble chitosan derivative by a low degree of substitution, a high-molecular weight hydrophilic modifier is preferable [46]. Katanchalee Mai-ngam [26] has prepared a series of comblike chitosan poly(ethylene oxide)/hydrophobic surfactants. The surfactant polymers consist of low molecular weight (Mw ) chi-

tosan backbone with hydrophilic polyethylene oxide (PEO) and hydrophobic hexyl pendant groups. Chitosan was depolymerized by nitrous acid deaminative cleavage. Hexanal and aldehydeterminated PEO chains were simultaneously attached to low Mw chitosan hydrochloride via reductive amination. The surfactant polymers were prepared with various ratios of the two side chains according to Scheme 5. It is well known that PEO has surface active properties in spite of the fact that it is highly soluble in water [47]. The hexyl group when added to chitosan will provide the hydrophobic part. The Langmuir –A isotherm for two chitosan hexyl derivatives in different mole percentage (25% and 42%) yield stable films as shown in Fig. 11. By extrapolating the isotherm to zero pressure, the surface area of each glucosamine monomer unit in chitosan–hexyl

Scheme 5. Synthesis pathways to (i) low molecular weight chitosan hydrochloride, (ii) PEO–CHO and (iii) chitosan-based surfactant polymer.

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Fig. 11. Surface pressure–area isotherms of chitosan-based surfactant polymers with only hexyl side chains in different mole percentages (42 mol% and 25 mol%) plotted as a function of the area per glucosamine monomer.

25% and chitosan–hexyl 42% films could be calculated to be 0.25 and 0.1 nm2 , respectively. These values are approximately two times and four times, respectively, smaller than that of low Mw chitosan hydrochloride. The authors concluded therefore that incorporation of hydrophobic C6 chains onto low Mw chitosan apparently induces molecular aggregation, resulting in loss of a true monolayer of surfactant polymers at the air–water interface. The –A isotherms of chitosan–PEO–hexyl surfactant polymers grafted with hydrophilic PEO and hydrophobic hexyl side chains in different ratios are shown in Fig. 12. The PEO grafted chitosan (Chito/PEO:Hex 1:0) exhibits an isotherm with no characteristic conformational transition known for PEO The author explained the behavior of the grafted chitosan as follows: at low surface pressure, the air–water interface is occupied by all surfactant components, i.e., chitosan backbones, hydrophilic PEO side chains and hydrophobic hexyl side chains. As the surface pressure increases, the chitosan–PEO–hexyl monolayers are forced to closely pack and, at a lateral pressure of ∼10 mN m−1 , the chitosan and PEO components start to dissolve into the water subphase. With further compression ( > 10 mN m−1 ), the hexyl side chains prevent the surfactant polymer from collapsing, resulting in a steep increase in surface pressure similar to that found in the isotherm for the polymer with only hexyl side chains (Fig. 12). At this point, the PEO and chitosan chains are forced into a subsurface layer while the hexyl side chains remain at the interface. Poly(ethylene glycol) (PEG) is a highly water-soluble amphipathic polymer and it is frequently used for chemical modification

Fig. 12. Surface pressure–area isotherms of chitosan-based surfactant polymers grafted with hydrophilic PEO and hydrophobic hexyl ligands in different ratios. The isotherm for hexyl free surfactant polymers (Chito/PEO:Hex 1:0) is shown for comparison.

of natural and artificial macromolecules. Grafting PEG onto chitosan should be a promising approach to obtain watersoluble chitosan derivatives. Ouchi et al. [46] for example, prepared 6-O-triphenylmethyl-chitosan firstly which was then reacted through coupling reaction with MeO–PEG acid using the water-soluble carbodiimide (WSC)-hydroxybenzotriazole (HOBO) method in N,N dimethylformamide (DMF) to give PEG-grafted6-O-triphenylmethyl-chitosan. The product was soluble in water. Although the introduction of PEG chains onto chitosan allows the dissolution of the modified chitosan molecule by interaction with water, the unmodified glucosamine units of PEG-g-chitosan should still possess strong inter- or intra-molecular interaction with other unmodified glucosamine units by way of hydrogen bonds as found in native chitosan. The attention therefore is focused on the formation of a new type of PEG-g-chitosan aggregate due to its intermolecular hydrogen bonds in aqueous media. The aggregation phenomenon was investigated in aqueous solution by measuring transmittance, and light scattering. These aggregates were used to release N-phenyl-l-naphthyl amine (PNA). On the other hand, Kulkarni Anandrao et al. [48] prepared methoxy polyethylene glycol MeO–PEG linked chitosan with different degrees of substitution using a novel yet simple method in the presence of formaldehyde in a solvent of formic acid and dimethyl sulfoxide and it was found that, the aqueous solubility of chitosan increased after chemically linking with MeO–PEG and the solubility was found to depend on the degree of substitution. With a proper degree of substitution of MeO–PEG on chitosan, the product may undergo inter- and/or intramolecular entanglements to produce nanoaggregates. The CAC as determined by the fluorescence emission spectra was found to be 0.003 mg/ml. The results of the size distribution and zeta potential of the prepared nanoaggregates suggested that as the degree of MPEG substitution increased, the size and polydispersity index of the prepared nanoaggregates decreased. The prepared nanoaggregates showed a pH-sensitive property and thus it may be suitable for the development of drug delivery devices for tumors treatment. A novel chitosan (CS) derivative conjugated with multiple galactose residues in an antennary fashion (Gal-m-CS) was synthesized [49]. A galactosylated CS (Gal-CS) was also prepared by directly coupling lactobionic acid on CS. Using an iontropic gelation method, CS and the synthesized Gal-CS and Gal-m-CS were used to prepare nanoparticles NPs (CS, Gal-CS, and Gal-m-CS NPs) for targeting hepatoma cells (HepG2). TEM examinations showed that the morphology of all three types of nanoparticles NPs was spherical in shape. No aggregation or precipitation of the NPs in an aqueous environment was observed during storage for all studied groups, as a result of the electrostatic repulsion between the positively charged NPs. Little fluorescence was observed in HepG2 cells after incubation with the FITC-labeled CS NPs. The intensity of fluorescence observed in HepG2 cells incubated with the Gal-m-CS NPs was stronger than that incubated with the Gal-CS NPs. These results indicated that the prepared Gal-m-CS NPs had the highest specific interaction with HepG2 cells among all studied groups, via the ligand-receptor-mediated recognition. During the past decade there has been a growing interest in the investigation of polymeric micelles as a potential carrier for drug delivery. It is well known that polymeric micelles have unique core–shell architecture composing hydrophobic segments as internal core and hydrophilic segments as surrounding corona in aqueous medium. The hydrophobic core provides a loading space for poorly water-soluble drugs. The hydrophilic shell allows polymeric micelle to gain stability in aqueous environment, and active targeting to tumor cells by further ligand modification [50]. Comparing to traditional micelles of low-molecular weight surfactant, polymeric micelles are generally more stable with a relatively lower CMC, and show slower dissociation in an aqueous environment

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Scheme 6. Preparation of chitosan–linolenic acid derivative.

[51]. Fortunately, micellar core can store the insoluble drugs to increase their solubility. Moreover, the special structure of polymeric micelles can defend the drug from decomposition in order to retain drug stability and decrease drug toxicity. The release behavior of drugs in vitro can be controlled by regulating the dissolubility, pH, zeta potential of the material. The nano-scale dimensions of polymeric micelles permit efficient accumulation in tumor tissue via the enhanced permeability and retention (EPR) effect, which is termed as passive targeting [52,53]. So polymeric micelles have been employed widely for different kinds of drugs, such as antitumor drug [54,55], anti-inflammatory agent [56], antifungal agent [57], antipsychotic [58] and so on (Scheme 6). Linoleic acid (LA)–grafted chitosan oligosaccharide (CSO) (CSO–LA) has been synthesized in the presence of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC), and the effects of molecular weight of CSO and the charged amount of LA on the physicochemical properties of CSO–LA were investigated, such as CMC, graft ratio, size, and zeta potential [50]. The results showed that these chitosan derivatives were able to self-assemble and form spherical shape polymeric micelles with the size range of 150.7–213.9 nm and the zeta potential range of 57.9–79.9 mV, depending on molecular weight of CSO and the charged amount of LA. Using doxorubicin (DOX) as a model drug, the DOX-loaded CSO–LA micelles were prepared. The drug encapsulation efficiencies (EE) of DOX-loaded CSO–LA micelles were as high as about 75%. The sizes of DOX-loaded CSO–LA micelles with 20% charged DOX (relating the mass of CSO–LA) were near 200 nm, and the drug loading (DL) capacity could reach up to 15%. The in vitro release studies indicated that the drug release from the DOX-loaded CSO–LA micelles was reduced with increasing the graft ratio of CSO–LA, due to the enhanced hydrophobic interaction between hydrophobic drug and hydrophobic segments of CSO–LA. Moreover, the drug release rate from CSO–LA micelles were faster with the drug loading. These data suggested the possible utilization of the amphiphilic micellar chitosan derivatives as carriers for hydrophobic drugs for improving their delivery and release properties. Although many antitumor drugs, such as paclitaxel (PTX), are widely used in cancer chemotherapy [58] their clinical use is limited by systemic toxicity, rapid blood clearance, and the occurrence of resistance. To increase the therapeutic index of these drugs, the antitumor drug PTX was encapsulated in novel micelles with

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glycolipid-like structure, which were formed by stearate grafted chitosan oligosaccharide in aqueous medium [58]. The micelles could load the poorly soluble antitumor drug (PTX) with high entrapment efficiency and drug loading. PTX release was retarded as a result of the encapsulation of the micelles. PTX loaded micelles present excellent internalization into tumor cells as well as resistant cells and subsequently reside in the cytoplasm, which results in increased intracellular accumulation of PTX at the moleculartarget site. Consequently, cytotoxicity of PTX loaded micelles was improved sharply and resistant cells were reversed. In conclusion, high cytotoxicity can be obtained and resistant cells can be reversed by enhancing PTX’s molecular-target delivery and accumulation via the encapsulation of the micelles. The present micelles are promising carrier candidates for effective therapy of antitumor drugs with the target molecules in cytoplasm. Several other researches on modified chitosan micelles have been carried out and most of these micelles can solubilize waterinsoluble drugs to a high concentration in water by self-assembly [59–63] Chitosan derivatives with both amphiphilic group and a brain-targeting group have been reported by Yao et al. [64]. In their study, a series of novel chitosan derivatives with octyl, sulfate and polyethylene glycol monomethyl ether (mPEG) groups as hydrophobic and hydrophilic moieties, respectively were designated to have the solubilization and self-assembly characteristics of polymeric micelles, and were expected at the same time to have brain-targeting characteristics. Such a new series of chitosan derivatives with multi-functions was obtained primarily by attaching octyl to amino groups, sulfate to hydroxyl groups, and mPEG (mPEG1100, mPEG2000 and mPEG5000) to the amino groups of chitosan molecules, providing hydrophobic, hydrophilic, and both brain-targeting and hydrophilic moieties, respectively. The micellar particle size was found to be around 100–130 nm. Some morphological characteristics of modified chitosan micelles were observed with fluorescence microscope and atomic force microscope (AFM). In addition, the potential of PEGylated amphiphilic chitosan derivatives in entrapping paclitaxel, in the polymeric micelles and enhancing its water solubility was investigated. The critical micelle concentrations of the modified chitosans determined by using fluorescence spectroscopy were found to be 0.011–0.079 mg/ml, and the log CMC was linearly relative to four structure parameters, which are the degree of substitution of chitosan unit, sulfate group, PEG unit and octyl group by mole per kilogram. The highest paclitaxel concentration of 3.94 mg/ml was found in micellar solution, which was much higher than that in water (less than 0.001 mg/ml). This study may lead to a kind of novel carriers for improving the efficiency of water-insoluble anticancer drugs.

9. Interaction of chitosan with various substances 9.1. Lipids Chitosan has been proposed for weight loss treatment by binding lipids and fats in the stomach where chitosan swells and acquires positive charges then will attract all negatively charged particles like fatty acid, bile acids forming ionic complexes and cholesterol (as a neutral lipid) forming hydrophobic complexes by hydrophobic interaction [65]. These complexes will pass to the small intestine where it will solidify and subsequently be excreted from the body. In this way the body will get rid of fats, lipids, and cholesterol. In vivo studies in animals were indicative of the lipid binding ability of chitosan [66–68] however contradicting results were obtained for humans [69–71].

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M.Z. Elsabee et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 1–16 Table 1 Characteristics of the Langmuir monolayers of the fatty acids on acetate buffer subphase.

Fig. 13. Structural formula of the investigated lipids.

The binding ability of chitosan with several lipids and cholesterol has been investigated by Wydro et al. [72] using the Langmuir Monolayer technique in vitro. The fatty acids used in that work were stearic, oleic, linoleic, and R-linolenic acids. Their structural formulas are presented in Fig. 13. The acids have the same length of the hydrocarbon chains but are unsaturated to different degrees. Saturated fatty acids are known to form straight chains resembling rods. Unsaturated fatty acids, by contrast, have one or more double bonds which in most naturally occurring acids are in the cis conformation. The numerical characteristics of the pressure–area (–A) isotherms for the fatty acids spread on an acetate buffer subphase are given in the following Table 1. The stearic film collapsed at sur-

Fatty acid

A2 /acid molecule

coll , mN m−1

Stearic Oleic Linoleic A-linolenic Cholesterol

20.0 34.5 40.5 40.0 37.2

62 32 29 28 44

face pressure  = 62 mN m−1 and area 17 A2 while the unsaturated fatty acids formed liquid-type monolayers which collapsed in a less sharp manner at surface pressures lower than that of stearic acid. The monolayers of the fatty acids were spread again on the buffer subphase which contained various concentrations of chitosan from 0.025 to 0.3 mg/ml. The –A isotherms of these fatty acids are shown in Fig. 14 [72]. It can be seen that the presence of chitosan in the subphase strongly influenced the shape and location of the isotherms, proving that there existed attractions between chitosan and lipid molecules. The attractions were revealed by changes of the molecular organization of the monolayers. The common feature of these changes was that all the monolayers studied underwent expansion, in each case reaching saturation with increasing chitosan concentration. In agreement with the lipid molecular structures, the highest expansions were observed for the most unsaturated fatty acids, linoleic and ␣-linolenic, the lowest for stearic acid, with oleic acid and cholesterol being the intermediate cases. By contrast, the main distinguishing feature of these changes was that, although none of the monolayers studied changed its state when completely saturated with chitosan, compared to the parent ones, the compactness of the monolayers was modified. The solid monolayers of stearic acid and cholesterol were loosened, whereas those of all the unsaturated acids, liquid in nature, were tightened. On the basis of these results the authors [72] proposed a mechanism of chitosan action that includes, (i) the molecules of fatty acids may form electrostatic

Fig. 14. Surface pressure–area (–A) isotherms of the fatty acids monolayers on the buffer subphase containing chitosan (Ref. [72]).

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complexes with chitosan through interactions of their carboxylic groups with –NH3 + groups of chitosan, (ii) chitosan molecules may get accommodated in the monolayers, possibly through hydrophobic interactions, (iii) hydrogen bonds between hydroxylic groups of cholesterol and of chitosan may be formed, and possibly (iv) chitosan may also have an effect on the conformation of the lipids in the monolayers. Of these, as the most likely, contributions of electrostatic and hydrophobic interactions have been most extensively discussed in the literature. A similar conclusion has been obtained by Pavinatto et al. [73] in which they proposed that chitosan has a strong affinity for phospholipids at the air–water interface, despite its lack of surface activity. Chitosan promotes a local distortion of the phospholipid tails, in a process governed by a combination of electrostatic, dipole, and hydrophobic interactions. As a consequence, chitosan causes disruption of the phospholipid layer, from which a possible implication is that chitosan may affect the stability of cell membranes. Taking together data from various techniques employed with Langmuir monolayers and LB films and from the pendent drop method, the authors proposed a model in which the surface activity of chitosan was promoted by the presence of a phospholipid at the air–water interface. At large phospholipid areas per molecule, chitosan is located at the interface, interacting with the phospholipid molecules via electrostatic and hydrophobic interactions. At small areas per phospholipid molecule, on the other hand, interaction is predominantly electrostatic, with chitosan forming a subsurface, with negligible contribution to the surface pressure or surface potential [73]. The isotherms of cholesterol and stearic acid at the air–water interface modified by different chitosans (chitosan chloride, hydrophobic modified chitosan, and medium and high molecular weight chitosans) in the aqueous subphase have also been investigated [74]. The Langmuir–Blodgett films of the complexes cholesterol–chitosan and stearic acid–chitosan are analyzed by atomic force microscopy (AFM), and a molecular simulation was performed to visualize the chitosan–lipid interactions. The isotherms have been strongly affected as a result of the chitosan interactions with cholesterol and stearic acid at the air–water interface. These modifications were dependent on the type and concentration of chitosan. Several modifications of all phases were noticed with larger molecular areas, and the observed changes in the compressional modulus were dependent on the type of chitosan used. The AFM images demonstrated that chitosan was disaggregated by the cholesterol and stearic acid interactions producing more homogeneous surfaces in some cases. The hydrophobic chitosan showed more affinity with stearic acid, while both medium and high molecular weight chitosans produced homogeneous surfaces with cholesterol. The complexes of chitosan–stearic acid were more flexible than the ones of chitosan–cholesterol. Adsorption of cholesterol on the different powdered chitosans, performed by HPLC, showed that the medium and high molecular weight chitosans could retain higher proportions of cholesterol compared with the other analyzed samples. The interaction between chitosan and dipalmitoyl-sn-glycero3-phosphocholine (DPPC) bilayer was examined with crosspolarization microscopy, differential scanning calorimetry (DSC), atomic force microscopy (AFM) and Fourier transform- (FT-) [75,76]. Raman spectroscopy, cross-polarized images showed that the direct hydration of the DPPC/chitosan mixture led to the formation of larger DPPC multilamellar vesicles (MLV), and pure chitosan also induced fusions of individual MLV. Under the influence of chitosan, the calorimetric enthalpy of DPPC was reduced in a concentration-dependent manner, and a new phase appeared at 28 ◦ C during sample cooling. Even the lowest chitosan mole fraction of 0.04% reduced the cooperative unit of the DPPC bilayer by more than 70%. In addition, the electrostatic effect between chitosan and

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DPPC tuned the degree of membrane bilayer perturbation. Reduction of pH increased the number of protonated amines on the chitosan backbone and caused further disruption on the membrane organization. Mixing DPPC with chitosan in an organic medium before hydration enhanced the hydrophobic interactions between the two molecules and greatly reduced the cooperative unit among individual lipids during the main phase transition. The increase of chitosan molecular weight also affected the cooperativity in the thermotropic transition of DPPC bilayer. FT-Raman spectroscopy provided additional evidence that chitosan directly perturbed the organizations of the hydrophobic inner core of the DPPC bilayer. In situ AFM measurement indicates that nucleation of chitosan occurs around the membrane defects at the initial stage of chitosan incubation [76]. Eventually, DPPC-chitosan binding and chitosan intermolecular association lead to chitosan aggregation on the membrane surface which is quantified by average height measurement and RMS roughness analysis. Adsorption of chitosan with the complex lipid, 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) has been investigated by Quemeneur et al. [77,78]. The authors found that variation of the zeta potential of the large unilamellar vesicle LUVs membranes as a function of chitosan concentration is independent on the chitosan molecular weight (Mw ). This result is interpreted in terms of electrostatic interactions, which induce a flat adsorption of the chitosan on the surface of the membrane. The role of electrostatic interactions is further studied by observing the variation of the zeta potential as a function of the chitosan concentration for two different charge densities tuned by the pH. Results show a stronger chitosan–membrane affinity at pH 6 (lipids are negatively charged, and 40% chitosan amino groups are protonated) than at pH 3.4 (100% of protonated amino groups but zwitterionic lipids are positively charged) which confirms that adsorption is of electrostatic origin. Since liposomes are used as protective capsules therefore they have to be stable in the biological environment and be resistant to external constraints before reaching the target. Numerous studies show that the “decoration” of liposomes with polymers affects their resistance to the in vivo environment, as well as their adsorption and specific targeting properties: for example, LUVs covered or grafted with polyethylene glycol are demonstrated to be furtive and stable in the intravenous environment [79–81]. A comparative study by zeta potential as a function of the pH (2.0 < pH < 12.0) reveals a difference in behavior between naked and chitosan–decorated LUVs. This result is further confirmed by a comparative observation by optical microscopy of naked and chitosan–decorated giant unilamellar GUVs in basic conditions (6.0 < pH < 12.0): At pH > 10.0, in the absence of chitosan, the vesicles present complex shapes, contrary to the chitosan-decorated vesicles which remain spherical, confirming thus that chitosan remains adsorbed on vesicles in basic conditions up to pH 12.0 as shown in Fig. 15. These results show that the chitosan-decorated vesicles are stable over a very broad range of pH (2.0 < pH < 12.0), which holds promise for their in vivo applications. It has been shown that chitosan-coated liposomes have superior quality compared to non-coated when the delivery of drugs to the lungs by nebulisation is considered [82]. Empty SUV (small unilamellar) liposomes were initially prepared (with different lipid compositions) and coated with chitosan by drop wise addition of chitosan solution in the liposome dispersion. After establishing the best conditions for chitosan-coating Rifampicin RIF-loaded chitosan-coated liposomes, with different lipid compositions (negatively charged and non-charged) were constructed, and their encapsulation efficiency (EE) and nebulisation efficiency (NE%)/stability (NER%) were evaluated. Charged liposomes (containing phosphatidylglycerol [PG]) can be coated with chitosan better compared to non-charged ones. The EE of chitosan-

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Fig. 15. Behaviors of naked GUVs (sequences A and B) and chitosan-decorated GUVs (sequence C) as a function of pH (6.0 < pH < 11.0, produced by NaOH shocks). The delay between each picture is 10 s. The scale bars represent 10 ␮m (Ref. [77]).

coated liposomes (that contain PG) is slightly increased while their stability after nebulisation is significantly increased (NER%). Mucoadhesive properties of chitosan-coated liposomes were substantially better (compared to non-coated ones) while the toxicity of liposomal RIF towards A549 epithelial cells was lower compared to free drug for all the types of vesicles evaluated, and especially the chitosan-coated ones. 10. Layer-by-layer assembly technique The layer-by-layer (LbL) assembly technique has shown remarkable capability for building multilayer film in recent years [83–85]. The distinguished advantage of LbL technique over cast and dipcoating methods is that the layer composition and thickness of LbL films can be precisely tailored by controlling the type of charged species and the number of adsorption cycles according to the predesigned architecture. LbL assembly between cationic chitosan and anionic dextran sulfate was analyzed quantitatively by a quartz crystal microbalance technique in the absence and presence of 0.2, 0.5, and 1 M NaCl in the polymer solution. The apparent film thickness increased upon increasing the NaCl concentration [86,87]. The anti- versus procoagulant activity of these films against whole human blood was studied by the immersion of a substrate into blood for 30 min incubation time at 37 ◦ C. The substrate was coated with films of varying NaCl concentrations and assembly step numbers. There was a critical concentration for the alternating activity. Above a concentration of 0.5 M NaCl, both anti- and pro-coagulation could be observed on the dextran sulfate and chitosan surfaces, respectively. The underlying layer of the assembly was necessary for this alternating activity. After a five-step assembly, the activity was realized. An assembly was also constructed from a combination of chitosan and heparin, but the activity was different from that of the former system. Strong anticoagulant activity was observed even on the chitosan surface. The authors suggested that the polymer species and/or the assembly conditions are key factors for realizing

the alternating bioactivities of films prepared by the layer-by-layer assembly [87]. The chemical structure of heparin is shown in Fig. 16. The multilayer film assembled by heparin and chitosan could offer both antibacterial and anti-coagulational function. Multifunctional systems based on the heparin/chitosan composite film may find wide application in the intervention therapy. Hyaluronan (HA) and chitosan (CH), were employed to engineer bioactive coatings for endovascular stents using layer-by-layer selfassembly technique [88]. A polyethyleneimine (PEI) primer layer was adsorbed on the metallic surface to initiate the sequential adsorption of the weak polyelectrolytes. The multilayer growth was monitored using a radio-labeled HA and shown to be linear as a function of the number of layers. The chemical structure, interfacial properties, and morphology of the self-assembled multilayer were investigated by time-of-flight secondary ion mass spectrometry (ToF-SIMS), contact angle measurements, and atomic force microscopy (AFM), respectively. The enhanced thromboresistance of the self-assembled multilayer together with the anti-inflammatory and wound healing properties of hyaluronan and chitosan are expected to reduce the neointimal hyperplasia associated with stent implantation. An interesting application of chitosan derivatives as protecting coatings of liposomes used as colloidal drug delivery system for topical ocular administration has been reported by Zhang and Wang

Fig. 16. Chemical structure of heparin.

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[89]. There is growing evidence that oxidative stress, mediated by active oxygen species in the lens and lipid peroxides, produced in the crystalline lens, is responsible for a breakdown of lens homeostasis leading to lens opacification. Coenzyme Q10 (CoQ10) is a fat-soluble vitamin-like substance present in many organisms and it consists of a redox active quinoid moiety and 10 isoprenoid units in the hydrophobic tail. CoQ10 functions as a well-known electron carrier in the electron transport chain, thus contributing to energy conservation, and the reduced form of CoQ10 (ubiquinol) is capable of scavenging free radical oxygen intermediates [90]. Its excellent ability to scavenge free radicals makes CoQ10 attractive as a potential anti-cataract agent. However, instability to light and extreme lipophilicity of CoQ10 hamper its bioavailability as a therapeutic agent for topical ocular administration. The use of colloidal drug delivery systems, such as liposomes, is a suitable strategy to obtain enhanced bioavailability in comparison with liquid formulations [91]. However, liposomes are generally rather unstable and tend to degrade or aggregate and fuse, which leads to leakage of entrapped drug during storage or after administration. To minimize these disruptive influences, many attempts have been made, including surface modification of liposomes which is an attractive method to improve liposomal stability both in vitro and in vivo. The authors used partially quaternized derivative, N-trimethyl chitosan chloride (TMC) (since it is soluble over a wide pH range) as a protective coating for the liposomes [89]. The obtained positively charged liposome is assumed to reduce rapid precorneal drug loss and improve poor corneal permeability due to the electrostatic interaction between the positively TMC and the negative charges present at the corneal surface [92]). In addition, TMC exhibits excellent absorption enhancing effect even at neutral pH values [93] by opening the tight junctions between adjacent cells of epithelial cell monolayers through ionic interaction [94]. It has been suggested that TMC with a medium to high degree of quaternization is capable of significantly enhancing the permeability of ofloxacin across a stratified epithelium, for example the cornea [95]. The data presented in this work demonstrated that the presence of TMC with two different Mw values both significantly prolonged the retention time of the formulations on the corneal surface which was attributed to the interaction between the polymer and the mucus layer covering the corneal surface involving electrostatic forces. In addition, TMC with the higher Mw allowed the liposomes to achieve better contact with the cornea offering a suitable viscosity and enabling easy manipulation for instillation.

11. Chitosan and lung surfactants The surface activities of chitosan have been found to play an important role in improving lung functions. The surface tension control imposed by lung surfactant (LS), a unique mixture of lipids and proteins that lowers the interfacial tension in the lungs and facilitates normal breathing [96], is compromised during acute respiratory distress syndrome (ARDS). Lung surfactant is a mixture of lipids (primarily dipalmitoylphosphatidylcholine) and four lung surfactant-specific proteins (SP-A, B, C, and D) that lines the interior of the lung alveoli and acts to lower the interfacial tension in the lungs, thereby insuring a negligible work of breathing and uniform lung inflation [97]. The competitive adsorption of serum proteins to the air–water interface can inhibit the adsorption of lung surfactant, leading to poor surfactant performance. Many serum proteins (including albumin) are surface-active and have a surface pressure, between 18 and 25 mN m−1 ( ∼ 47–54 mN m−1 ) [98], which is much lower than  ∼ 70 ( near zero) required for proper respiration. This albumin-induced energy barrier to surfactant adsorption is primarily electrostatic [99]; double-layer repulsion arises due to the

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negative lipids in lung surfactant and the net negative charge on albumin (and other surface active serum proteins) at the interface. In analogy to its effects on colloid stability, chitosan improves the performance of lung surfactant in vitro [100]. Stenger et al. [101] have shown that only 0.001 mg/ml chitosan is able to reverse albumin-induced surfactant inhibition to the same extent as ∼10 mg/ml 10 kDa polyethylene glycol (PEG) or ∼1 mg/ml 1240 kDa hyaluronic acid [102–104] under otherwise identical conditions. These chitosan concentrations are much too low to induce a depletion attraction, which is proportional to the polymer volume fraction [103]. 12. Conclusion Chitosan a biodegradable and biocompatible polymer is obtained from the deacetylation of chitin a naturally abundant polymer. Due to the presence of two functional groups chitosan can undergo many chemical modifications which render it a very attractive material with tremendous applications in various fields. One of these areas is the surface activity property. It has been shown that chitosan by itself exhibits weak surface activity due to the hydrophilic nature of its structure; however by introducing a hydrophobic group to one or both of its reactive functional groups (the NH2 or the OH group) it can develop surface activity. For example, carboxymethylation and succinyl chitosan formation lead to compounds with the ability of micelle formation. These reactions can be followed by introduction of hydrophobic groups to enhance the surface activity by the formation of aggregates with hydrophobic heads pointing to the air and hydrophilic side directed to the water bulk. Many trials have been performed to introduce various hydrophobic and other hydrophilic groups (e.g. polyethylene oxide) into the chitosan chain. These modified polymers could be used in many industrial fields as emulsifiers and due to the biocompatibility of chitosan its modified hydrophobic derivatives have found important applications as encapsulation of the water-insoluble antitumor and brain-targeting drugs, a fast growing trend in research. Chitosan was found to form layer-by-layer self-assembly with many other polymers especially those having different charged surfaces. Chitosan is also capable of interacting with different substances as for example heparin and several lipids a property which could find wide application in drug delivery systems (as for example liposomes coating protection). The surface activity of chitosan plays a pronounced role in treatment of lung surfactant malfunction. References [1] J.H. Manssen, E. Peeters, M.F. van Zundert, M.H.P. van Genderen, E.W. Meijer, Macromolecules 30 (1997) 8113–8128. [2] N.R. Cameron, D.C. Sherrington, Macromolecules 30 (1997) 5860–5869. [3] M.J. Newman, M. Balusubramanianm, C.W. Todd, Adv. Drug Deliv. Rev. 32 (1998) 199–223. [4] H. Sawada, A. Wake, M. Oue, T. Kawase, Y. Hayakawa, Y. Minoshima, M. Mitani, J. Colloid Interface Sci. 178 (1996) 379–381. [5] K. Holmberg, B. Jönsson, B. Kronberg, B. Lindman, Surfactants and polymers in aqueous solution, in: Surfactant–Polymer Systems, John Wiley and Sons, Chichester, 1998. [6] M.J. Schick, Nonionic Surfactants, Marcel Dekker, New York, 1967. [7] M.M.H. Ayoub, J. Elastomer Plastics 30 (1998) 207–229. [8] C. Schwarzwälderm, W. Meierm, Macromolecules 30 (1997) 4601–4607. [9] Y.-K. Choi, S.-B. Lee, D.-J. Lee, Y. Ishigami, T. Kajiuchi, J. Membr. Sci. 148 (1998) 185–194. [10] C. Yang, J.F. Rathman, Polymer 37 (1996) 4621–4627. [11] Z. Zhang, G.Y. Xu, F. Wang, S.L. Dong, Y.M. Li, J. Colloid Interface Sci. 277 (2004) 464–470. [12] A.M. Al-Sabagh, M.R. Noor El-Din, R.E. Morsi, M.Z. Elsabee, J. Appl. Polym. Sci. 108 (2008) 2301–2311. [13] J. Ngimhuanga, J.-I. Furukawab, T. Satoha, T. Furuikea, N. Sakairia, Polymer 45 (2004) 837–841. [14] N. Kubota, N. Tatsumoto, T. Sano, K. Toya, Carbohydr. Res. 324 (2000) 268–274. [15] M.W. Anthonsen, K.M. Varum, O. Smidsrød, Carbohydr. Polym. 22 (1993) 193–201.

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