29
`Macromolecular Complexes of Chitosan
`
`Naoji Kubota and Kei Shimoda
`Oita University, Oita, Japan
`
`I.
`
`INTRODUCTION
`
`A. Macromolecular Complexes
`
`Macromolecular complexes are molecular aggregates of
`two or more complementary polymers, which have definite
`composition and characteristics and arise from intermo-
`lecular interactions, such as Coulomb forces, hydrogen-
`bonding forces, charge-transfer forces, and van der Waals
`forces. The complexes can be divided into the following
`four classes according to the nature of their interactions [1]:
`polyelectrolyte complexes, hydrogen-bonding complexes,
`charge-transfer complexes, and stereocomplexes.
`It is rare that these intermolecular interactions take
`place singly, and hydrophobic interactions are also signif-
`icant factors in aqueous media. Although these secondary
`binding forces, among which Coulomb forces are stron-
`gest, are weaker than primary binding forces, they play
`very important roles in the biological systems (e.g., molec-
`ular recognition, accumulation and interpretation of ge-
`netic information, and formation of higher-order structure
`of proteins).
`Biological systems comprise different kinds of macro-
`molecules, such as nucleic acids, enzymes, proteins, and
`polysaccharides, and most of them have definite structure
`and specific functions. Antigen–antibody reactions and
`enzymic reactions, for example, are very specific. In these
`cases, intermacromolecular interactions play important
`roles. Many bioreactions proceed via formation of macro-
`molecular complexes in the beginning of the reaction, and
`the concerted interactions are included. As a simplified
`model of these bioreactions, it is rational to investigate the
`macromolecular complexes. The majority of researches on
`macromolecular complexes have been directed toward a
`better understanding of biological systems and of structure
`and properties of functional units. However, macromolec-
`ular complexes have recently found their way into practical
`applications, in particular, as biomaterials.
`
`B. Structure and Properties of Chitosan
`Chitin, poly[h(1!4)-2-acetoamido-2-deoxy-D-glucopyra-
`nose], is one of the most abundant natural polysaccharides
`and is present in crustacea, insects, fungi, and yeasts. The
`total annual global estimates of accessible chitin amount
`to 1  109 tons [2]. It is obtained primarily as a by-product
`of the seafood industry. Deacetylation of chitin by alkali
`readily affords chitosan (Ch), poly[h(1!4)-2-amino-2-de-
`oxy-D-glucopyranose]. Ch is also found in various fungi.
`However, the molecular structure of Ch is believed to be a
`copolymer of N-acetyl-glucosamine and glucosamine; usu-
`ally the glucosamine content is more than 90%. It is
`known that 50% N-deacetylated chitin (50% N-acetylated
`Ch) is water soluble [3]. Kubota and Eguchi showed that
`the water solubility of half N-acetylated Ch increased
`with decreasing the molecular weight, as shown in Fig. 1
`[4]. In addition, the solubility of half N-acetylated Ch
`with the molecular weight lower than 10,000 is rather high
`even in aqueous dimethylacetamide and aqueous dimethyl
`sulfoxide (DMSO) [5].
`Because Ch has an amino group in the repeating unit,
`it affords ammonium group in aqueous acidic media.
`Owing to its cationic nature, Ch spontaneously forms
`water-insoluble complexes with anionic polyelectrolytes.
`Therefore, Ch has been used mainly as a flocculant for the
`treatment of wastewater. However, it has recently been
`used in biomedical and pharmaceutical fields because of its
`favorable properties of good biocompatibility, low toxici-
`ty, and biodegradability.
`The intrinsic viscosity [g] of Ch depends on the pH and
`the ionic strength, as shown in Fig. 2 [6]. If the pH of the
`solution is increased, the intermolecular and intramolecu-
`lar electrostatic repulsions between cationic charges are
`reduced. This allows the Ch chains to come closer and thus
`lowers the hydrodynamic volume of the Ch molecules. This
`effect may enhance the interchain and intrachain hydrogen
`bonding. Similarly, as the ionic strength increases, [g]
`
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`680
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`Kubota and Shimoda
`
`decreases due to the shielding effect of the counterions.
`Although Co¨ lfen et al. suggested that the hydrodynamic
`behavior of Ch was consistent with wormlike chain model
`[7], the Ch molecule is rather stiff. The flexibility of some
`polysaccharides has been investigated in terms of a ‘‘stiff-
`ness parameter’’ B (i.e., the dependence of the intrinsic
`viscosity on the ionic strength). The B values for a variety
`of polymers are compiled in Table 1. The B value of Ch was
`estimated as 0.08 [8]. Terbojevich et al. obtained B values
`from 0.043 to 0.091 for Ch with degrees of acetylation
`ranging from 52.2% to 12.1% [9]. These values are essen-
`tially the same as those for carboxymethyl cellulose and
`hyaluronic acid, greater than DNA, and less than poly-
`acrylate. These solution properties of Ch greatly affect the
`formation of macromolecular complexes.
`
`II. FORMATION OF MACROMOLECULAR
`COMPLEXES OF CHITOSAN
`
`The formation of macromolecular complexes in solution,
`depending on the intensity of polymer–polymer and poly-
`mer–solvent interactions, leads to the separation of com-
`plexes as a solid or liquid phase. Because Ch is a cationic
`polyelectrolyte, most studies on the macromolecular com-
`plexes of Ch are concerned with polyelectrolyte complexes
`(PECs). However, only a few articles dealt with the poly-
`ionic interaction between polysaccharides, until Kikuchi
`
`Figure 1 Dependence of water solubility of half N-acety-
`lated Ch on the molecular weight. (From Ref. 4.)
`
`Figure 2 Effects of pH and ionic strength on intrinsic viscosity [g] of Ch solution:
`L; n, 0.200 mol/L; ., 0.300 mol/L; z, 0.500 mol/L NaCl. (From Ref. 6.)
`
`x
`
`, 0.050 mol/L; Ez, 0.075 mol/L; E, 0.100 mol/
`
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`Macromolecular Complexes of Chitosan
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`Table 1 Stiffness Parameter, B, for Ch and Some Polymers
`
`Polymer
`
`Ch
`
`Polyphosphate
`Polyacrylate
`Amylose xanthate
`Carboxymethylamylose
`Carboxymethylcellulose
`Hyaluronic acid
`Sodium pectinate
`DNA
`
`Degree of acetylation
`(%)
`
`12.1
`20.1
`42.1
`52.2
`—
`—
`—
`—
`—
`—
`—
`—
`
`B
`
`0.091
`0.060
`0.061
`0.043
`0.44
`0.23
`0.22
`0.20
`0.065
`0.07
`0.044
`0.006
`
`Source: Refs. 9 and 8. Reproduced by permission of Routledge, Inc.,
`part of The Taylor & Francis Group.
`
`first reported the PEC containing Ch as a component [10].
`Several reviews on PEC have been published so far [11,12].
`The formation of PEC is governed not only by the nature of
`the individual polyelectrolyte components, such as charac-
`teristics of polyions (strong or weak), position of ionic sites,
`charge density, molecular weight, flexibility, functional
`group structure, hydrophilicity and hydrophobicity, and
`stereoregularity, but also by the reaction conditions, such
`as pH, ionic strength, polymer concentration, mixing ratio,
`and temperature. This, therefore, may lead to a diversity of
`physical and chemical properties of the complexes. PECs
`arise due to interactions between oppositely charged poly-
`mers and can be additionally stabilized through short-
`
`range interactions such as hydrophobic interactions and
`hydrogen bonds.
`
`A. Thermodynamics and Stoichiometry
`of Complex Formation
`
`The complexation reaction of macromolecules is signifi-
`cantly different from that of low-molecular weight mole-
`cules due to the polymer effects by which a relatively stable
`complex occurs, although the force of each bound pair is
`small. This implies that the apparent equilibrium constant
`of the polymer–polymer complex is extremely large and the
`reaction is apparently irreversible. The free-energy change
`DGo for macromolecular complexation is a function of the
`degree of polymerization, and the equilibrium constant
`abruptly increases at the critical chain length [13]. Pe´ rez-
`Gramatges et al. revealed that complexation of Ch with
`poly(acrylic acid) (PAA) proceeded cooperatively and the
`larger degree of conversion, h, was obtained for the higher-
`molecular weight Ch [14]. The relationship between the
`stability constant, K, and h (Fig. 3) indicates that this
`complex consists of a long sequence of bound pairs of
`repeating units. The slope of this curve shows the influence
`of neighboring functional groups on their reactivity.
`Kanbayashi and Arai determined thermodynamic
`parameters for PEC formation in the methyl glycol Ch
`(MGC)–carboxymethyl cellulose (CMC) system [15]. The
`formation of PEC gained a large negative DGo, but the
`factors were dependent on the charge density of CMC. The
`PEC formation using CMC with low charge density
`showed a marked exothermic tendency and was classified
`as an enthalpy-dominating reaction. On the other hand, the
`reaction using CMC with high charge density showed a
`smaller exothermic tendency and gained large positive
`
`Figure 3 Linear relationship between the stability constant, K, and the degree of conversion h for Ch–PAA complexes: D, Mv =
`o
`8.5  104;
`, Mv = 1.1  105; 5, Mv = 2.3  105. (From Ref. 14.) Copyright 1996 Springer-Verlag.
`
`Copyright 2005 by Marcel Dekker
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`
`entropy, indicating an entropy-dominating type. They also
`investigated the dependence of thermodynamic parameters
`on ionic strength using the MGC–partially hydrolyzed
`poly(acrylamide) system [16]. The degree of complexation
`did not change much with the ionic strength (0.001–0.2),
`and a gain of a large negative DGo was accompanied by the
`PEC formation. However, DHo became a large positive
`value with increasing ionic strength. Therefore, the higher
`ionic strength suggested the entropy-dominating-type PEC
`formation reaction. A much higher ionic strength affects
`PEC formation; for example, when Ch and n-carrageenan
`(Car) were mixed in the presence of 5.7% NaCl at pH 2.8
`on a boiling water bath, such a phase separation did not
`occur, but a viscous and macroscopically homogeneous
`PEC mixture was obtained [17]. The presence of Na+ and
`Cl reduces the electrostatic attraction between oppositely
`charged polyelectrolytes. This mixture gelled as its temper-
`ature or ionic strength decreased.
`In general, the composition of the reaction mixture, Z,
`is unity at the equivalence point, suggesting a 1:1 stoichi-
`ometry for the complex, no matter what order of mixing is
`chosen. However, h varied with Z in the case of Ch–
`polygalacturonate (PGal) complex [18]. It fell from unity
`to 0.8 as Z increased from 0.2 to 0.5 and then rose again up
`to 0.85 for Z = 1. If the complexation reaction is termi-
`nated before a 1:1 stoichiometry of charge neutralization is
`reached, water-soluble complexes are possible. In addition,
`when the PEC involves two polyelectrolytes of different
`molecular size, soluble nonstoichiometric PEC can be
`formed. In such a complex, the larger polymer chain
`behaves as a host macromolecule to the shorter one, and
`the host polymer should be a strong polyelectrolyte. For
`weak polyelectrolytes, not all of the repeating units need to
`have a 1:1 stoichiometry.
`
`B. Complexes with Polysaccharides
`
`The interaction between oppositely charged polyelectro-
`lytes involving weak polyacid and/or weak polybase is
`affected by the solution pH because of the change in the
`degree of dissociation. In this sense, it is of great interest to
`examine the properties of complexes involving weak poly-
`electrolytes under various pH conditions. When Ch, a
`weak polybase, is reacted with a weak polyacid, the insol-
`uble complex formation occurs only in the narrow pH
`range. Argu¨ elles-Monal et al. reported the complex forma-
`tion reaction of the Ch–CMC system [19]. At pH 3.6, the
`PEC was rich in CMC, whereas at pH 4.8 the excess
`component was Ch. At pH 4, the yield of PEC increased
`with the addition of one polyelectrolyte solution to the
`other, and the maximum yield corresponded to the ratio
`[CMC]/[Ch] = 1. Beyond this point, the yield remained
`constant, and the composition of the PEC obtained showed
`the stoichiometry. Similar results were obtained in the case
`using PAA, a synthetic polymer, as a polyacid [20,21]. The
`mixing ratio Ch/(Ch + PAA) for maximum insoluble
`complex formation, Rmax, increased with increasing solu-
`tion pH. Interestingly, at the initial pH = 6, the superna-
`tant pH of the reaction mixture increased as the complex
`was formed. At the initial pH=3, the opposite behavior
`
`was observed. As shown in Fig. 4, pKa of Ch increased as
`the addition of the polyanion increased, suggesting that the
`charged carboxylate group induced the ionization of the
`amino group of Ch [22]. Therefore, the supernatant pH
`increased at pH 6 as the yield of the complex increased:
`
`NH2 þ OOC þ Hþ ! NHþ3 OOC
`The supernatant pH decreased at pH 3 as the yield of
`complex increased:
`3 þ HOOC ! NHþ 3 OOC þ Hþ
`
`NHþ
`However, changes in the supernatant pH by complexation
`was dependent on the type of polyanions [23]. For Ch–Car,
`Ch–alginate (Alg), and Ch–pectin (Pec) complexes, no
`significant pH change occurred at the initial pH = 3. At
`pH 4.5, there was a slight increase in pH as a result of
`complexation of Ch with Alg and Pec, and a more signif-
`icant increase with Car. At pH 5.4, there was an increase in
`pH analogous to that at pH 6 for the Ch–PAA system.
`However, unlike PAA, a maximum pH change was main-
`tained in the wide mixing ratio from 0.2 to 0.4. Differences
`in polyanion conformation are responsible for the differ-
`ences in pH changes between Ch–PAA and Ch–Car, Ch–
`Alg, Ch–Pec complex formation. For example, structural
`similarity between Ch and Car, Alg, and Pec or flexibility of
`PAA is possible.
`Takahashi et al. observed the difference in basic
`properties between Ch–Alg and Ch–PAA complexes [24].
`In the Ch–Alg system, the insoluble complexes were
`formed at a constant unit molecular ratio of 1:1.3 (Ch:Alg)
`at pH 3.7–4.7. However, the unit molecular ratio of the
`Ch–PAA system was greatly affected by pH, showing a
`change from 1:2.4 to 1:1.7 (Ch:PAA) with an increase in
`pH from 3.7 to 4.7. They concluded that this was due to
`the higher flexibility of the polymer chain of PAA than
`that of Alg.
`On the other hand, owing to the ease of interfacial
`PEC formation, the fitness of the structures of the back-
`bones of Ch and some polyanions was estimated as fol-
`lows [25]:
`heparin ðHepÞ > Alg > carboxymethyl
`chitin ðCMChitinÞ > PAA
`
`Nakajima and Shinoda showed that the backbone chain
`conformations of component polymers together with the
`kind and location of ionizable groups were important
`factors to discuss the formation and structure of PEC
`[26]. They used glycol Ch (GC), which is water soluble at
`all pHs, as a polycation and hyaluronic acid (HA), chon-
`droitin sulfate (CS), Hep, and sulfated cellulose (SCS) as
`polyanions. In the GC–HA and GC–CS systems, the
`experimental curves of complex composition, R, to pH
`crossed the theoretical curves at R = 0.5 (Fig. 5 shows the
`GC–HA system as an example.) In both complexes, the
`neutral complex appears only at R = 0.5. The positive and
`negative charges may remain in the regions R < 0.5 and
`R > 0.5, respectively. This result shows that the dominant
`factor in these cases is the pyranose structure itself rather
`
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`Macromolecular Complexes of Chitosan
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`Figure 4 Degree of ionization for amino groups of Ch in the presence of xanthan. The mole ratio of carboxyl groups to amino
`groups: ., 0.30; E, 0.60; n, 1.19. (From Ref. 22.)
`
`than the kind and location of the ionizable groups on the
`pyranose ring. In the case of the GC–Hep system, the
`discrepancy between theoretical and experimental curves
`was rather small, and complex formation seemed to pro-
`ceed almost stoichiometrically. However, the complex
`composition was GC/(GC + Hep) = 0.65 at the lower
`
`pH region. They suggested that one of the possible struc-
`tures having these compositions was the ladder form
`sandwiching a Hep molecule between two GC molecules.
`Moreover, the experimental curve of the GC–SCS system
`was remarkably different from the theoretical curve, as
`shown in Fig. 6. However, the complex composition at the
`
`Figure 5 Composition of complex plotted against pH for GC–HA system. (From Ref. 26.) Copyright 1976, reprinted with
`permission from Elsevier Science.
`
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`Figure 6 Composition of complex plotted against pH for GC–SCS system. (From Ref. 26.) Copyright 1976, reprinted with
`permission from Elsevier Science.
`
`lower pH region was the same value as that of the GC–Hep
`system. In this case, it is thought that the GC–SCS system is
`similar to the GC–Hep system in structure, but negative
`charges are always conserved. The reason for such differ-
` groups on SCS and
`ence may be the location of the –OSO3
`Hep molecules. Similarly, the theoretical and experimental
`curves of the mixing ratio intersect at 0.46, 0.52, and 0.53,
`for the GC–CMC, GC–Alg, and GC–PGal complexes,
`respectively [27]. On the contrary, in the case of the GC–
`dextran sulfate (DS) complex, the stoichiometric compo-
`sition occurs at R = 0.62 at pH < 3.5. It was concluded
`that the GC–DS complex had a different structure from the
`other three [28].
`In the case of n-Car, K+ ions induce helix conforma-
`tion and promote helix–helix aggregation. The decisive
`factor of the Ch–Car complex formation seems to be
`whether n-Car is in a helix–helix aggregated state [29].
`The interaction between Ch and n-, L-, and E-Car in their
`coil or nonaggregating helical conformation normally
`resulted in the formation of PEC with stoichiometric
`charge ratios of unity. Nevertheless, the formation of
`PEC was significantly affected by the fraction of n-Car in
`K+-induced helical conformation. If the n-Car exists in the
`helix–helix aggregated state then the interaction with Ch
`produces PEC with a charge ratio below unity, thereby
`providing a means of complexes with a surplus of negative
`charge. In this case, Ch molecules act as binding elements
`between helix–helix aggregated n-Car.
`Partially N-acylated Ch samples were used to examine
`the effect of the N-acyl groups on the complex formation
`with CS [30]. The Rmax values became larger with increas-
`ing substitution degree of N-acyl groups but were not
`dependent on the kind of N-acyl group. Broad turbidity
`
`curves appeared at pH 4.5 at the higher substitution degree
`of N-propionyl, N-hexanoyl, and N-myristoyl. The higher
`the acyl group, the stronger the effect, due to the insolu-
`bility of the complexes and the hydrophobic interactions of
`N-fatty acyl groups. It is difficult to form a ladderlike
`structure in this system owing to bulky acyl groups.
`Therefore, some of the free groups that are not involved
`in the PEC formation may exist inside the complexes.
`
`C. Complexes with Proteins
`
`Remunˇ a´ n-Lo´ pez and Bodmeier investigated optimal con-
`ditions for the complexation between Ch and type B
`gelatin (Gel) [31]. All of the optimal preparation condi-
`tions were essentially coincident with those for Ch–poly-
`saccharide complexes. In addition, complexation was
`found to depend on the molecular weight of Ch and Gel;
`higher-molecular weight Ch resulted in higher amounts of
`complex formed, whereas higher-molecular weight Gel
`solubilized the complexes.
`When a protein reacts with an oppositely charged
`polyelectrolyte to form PEC, a conformational change
`occurs. Park et al. reported the conformational change of
`a-keratose (Ker) by complexing with Ch at pH 5.2 [32].
`The a-helical structure in Ker was transformed into a
`random structure by complexing with Ch at Rmax, whereas
`the h-sheet was not affected. It seemed that helix-
`favoring amino acid residues, aspartic and glutamic acids,
`participated in forming electrostatic linkages. As shown in
`Fig. 7, the lower the molecular weight of Ch, the higher
`the destruction of the a-helix in Ker.
`low-molecular
`weight Ch seems to cause easier or complete complexa-
`tion reaction.
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`Copyright 2005 by Marcel Dekker
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`Macromolecular Complexes of Chitosan
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`Figure 7 Effect of molecular weight of Ch on the secondary structure of keratose at Rmax: (A) Mv = 5.91  105; (B) Mv =
`4.83  105; (C) Mv = 1.91  105; (D) Mv = 1.08  105; (E) Mv = 0.31  105; n, a-helix; m, h-sheet; n, random structure.
`(From Ref. 32.)
`
`Taravel and Domard investigated the interaction be-
`tween Ch and bovine atelocollagen (Col) in detail [33,34].
`When a solution of Ch HCl was added to a Col solution of
`pH 7.8, a pure PEC was formed. Interestingly, the aggre-
`gation of the triple helices of Col was not influenced by this
`interaction. During the formation of PEC, Col behaves like
`dispersions of encapsulated microgels. Accordingly, only
`some of the negative charges of Col can participate in the
`PEC. The weight proportion of Ch in these complexes was
`14.2% and 10.2% for low-molecular weight and high-
`molecular weight Col, respectively. These values are much
`lower than that of the theoretical value (28.5%). This
`limitation is attributed to a competition between the for-
`mation of PEC and Col triple helices. Moreover, they
`attempted to prepare a 1:1 complex by two different
`methods [35]. The first method was to form the complex
`at a temperature higher than the denaturation temperature
`of Col to avoid gelation of Col. Denaturation allowed the
`formation of a pure PEC with much higher Ch/Col ratio
`than the values obtained at lower temperature. However, a
`theoretical complex was not formed, as denaturation was
`not entirely completed. The presence of a large excess of Ch
`was the second method. The solution of Ch HCl was added
`to a Col solution of pH 3.6. The solutions thus prepared
`could contain a great excess of Ch up to 1200%. By in-
`creasing the pH of these mixture solutions to pH 5.8, the
`complexation between the two polymers was achieved. A
`large excess of Ch could destabilize the gelation of Col, and
`Col did not precipitate in the intermolecular associations
`
`of triple helices. The presence of an infrared (IR) absorp-
`tion band at 1600 cm1, the shift of the amide I band, and
`the insolubility at pH 5.6–5.8 of this complex suggested the
`formation of a hydrogen-bonding complex (HBC). HBC is
`often found in polymer blends, such as Ch–poly(viny
`alcohol) (PVA) [36] and N-acetylated Ch–PVA systems
`[37].
`
`Hydrophobic interactions are also important in the
`complexes with proteins. a-, h-,and n-caseins (Cas), which
`are phosphoproteins in milk, are precipitated by Ch [38].
`NaCl up to 1 mol/L was ineffective to prevent interaction
`between Ch and Cas, and a nonionic detergent, Tween 20,
`up to 2% was unable to prevent the Ch–Cas interactions.
`However, the complex could be dissolved in a mixture of
`increased NaCl and Tween 20. In this case, hydrophobic
`and electrostatic interactions participate in the association
`and coagulation of Cas with Ch. The fraction of hydro-
`phobic surface on a Ch molecule was estimated as
`51.5% at pH 6.3, and it increased slightly to 52.4% on
`increasing the ionization degree to 100% [39]. Therefore,
`an increase in the ionic strength would reinforce the
`hydrophobic interactions between Ch and hydrophobic
`residues of Cas. The complexation with Ch also imparts to
`faba bean legumin (FBL), a seed storage protein, the
`solubility at the isoelectric point, pI = 5.2, and higher
`pH [40]. Only at pH > 7.0 did the Ch–FBL complex
`precipitate from the solution due to the insolubility of Ch,
`and the complex was stable even at high ionic strength as
`1 M NaCl. This result points to a substantial contribution
`
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`of noncoulombic interactions to the Ch–FBL complex.
`The increase in negative deviations of limited viscosity
`numbers from additive values with increasing NaCl con-
`centration may be evidence for a substantial role of hydro-
`phobic interactions.
`
`D. Complexes with DNA
`
`As expected, Ch interacts very securely with DNA in
`solution and causes DNA to be precipitated from solution.
`This complexation is very similar to the Ch–polyphosphate
`(PP) [41], GC–metaphosphate (MP) [42], and MGC–MP
`[42] systems. Phosphoric acid is a slightly stronger acid
`than carboxylic acids. Therefore, Ch–DNA complex is
`stable in 0.1 mol/L HCl, but unstable in 0.1 mol/L NaOH.
`Kendra and Hadwiger showed that the Ch must be 7 or
`more sugar units in length both to optimally induce plant
`genes and to inhibit fungal growth [43]. This length re-
`quirement suggests that a series of positive charges match
`up with phosphate negative charges in the grooves of the
`DNA helix in the B form.
`Hayatsu et al. added Ch solution (pH 5) to the solu-
`tion of DNA, RNA, and homopolynucleotides (pH 7.2)
`to form insoluble complexes [44]. The double strandedness
`of DNA was retained in the complex because the PEC
`preparation process is mild enough not to inflict any
`damage on DNA. In this system, DNA molecule was
`accessible to enzymes and reagents having an affinity to
`
`DNA. For example, DNA in the complex could be
`digested with a mixture of DNase I and phosphodiester-
`ase, and cytosine residues in the DNA (denatured DNA)
`could be deaminated by treatment with sodium bisulfate.
`In addition, carcinogenic heterocyclic amines showed ad-
`sorption to DNA and RNA in the complex. On the
`contrary, it was reported that DNA complexed with Ch,
`at all charge ratios, resulted in a significant decrease in
`degradation by DNase II; the lower the molecular weight
`of Ch, the higher the inhibition effect [45].
`N-Dodecylated Ch (Ch12) also reacted with DNA to
`form a PEC [46]. Although DNA in the Ch–DNA complex
`was not sufficiently protected when it was exposed to
`DNase, DNA in the Ch12–DNA remained intact due to
`the protection from nuclease offered by Ch12. In addition,
`complexation with Ch12 enhanced the thermal stability of
`DNA. However, the complex was dissociated by the addi-
`tion of microions; the ability of Mg2+ to break the PEC
`was greater than that of Na+ and K+. This is related to the
`different affinity of ions to DNA; Mg2+ has a much higher
`affinity to DNA compared with Na+ and K+ [47].
`After complexation between Ch and DNA, phase
`separation occurs to yield coacervates that represent the
`aggregated colloidal complexes. The Ch–DNA particles
`had a negative surface charge when the complexes were
`made at N/P ratio below 2, and became positively charged at
`N/P ratio above 2 through neutral at N/P = 2, as shown in
`Fig. 8 [48]. The pH of the solution at 5–5.8 and a temperature
`
`Figure 8 N/P ratio dependence of zeta potential of Ch–DNA complex. Measurements were performed with 20 mg of DNA in
`1 mL of 0.15 mol/L NaCl. (From Ref. 48.)
`
`Copyright 2005 by Marcel Dekker
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`Macromolecular Complexes of Chitosan
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`687
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`Figure 9 Effect of pH on zeta potential of Ch–DNA nanoparticles. (From Ref. 49.) Copyright 2001, reprinted with permission
`from Elsevier Science.
`
`of the solution above 50jC resulted in Ch–DNA nano-
`particles [49]. The size of Ch–DNA nanoparticles was
`optimized to 150–250 nm with a narrow distribution at N/
`P ratio between 3 and 8 and Ch concentration of 100 Ag/mL.
`The zeta potential of the Ch–DNA nanoparticles was +22
`to +18 mV at pH < 6.5, and decreased dramatically to20
`mV at pH 8–8.5, as shown in Fig. 9. At pH 7.0–7.4, the
`nanoparticles appeared to be electrostatically neutral and
`may offer an effective protection to the encapsulated DNA
`from nuclease degradation.
`
`E. Ternary Complexes
`
`Kikuchi and his coworkers reported complexes consisting
`of three polyelectrolyte components, strong polybase–
`weak polybase–strong polyacid system and strong poly-
`base–strong polyacid–weak polyacid system.
`In the MGC–GC–poly(vinyl sulfate) (PVS) system,
`the molar ratio, S(PVS)/N(MGC+GC), to form insoluble
`complexes decreased with increasing solution pH [50,51].
`Experimental conditions and results of elemental analyses
`
`Table 2 Reaction Conditions and Elemental Analyses of MGCGCPVS Complexes
`
`Samplea
`
`1-A
`1-B
`1-C
`1-D
`1-E
`1-F
`2-A
`2-B
`2-C
`2-D
`2-E
`2-F
`
`[H+]
`
`7% HCl
`4% HCl
`1% HCl
`pH 2.0
`pH 6.5
`pH 13.0
`7% HCl
`4% HCl
`1% HCl
`pH 2.0
`pH 6.5
`pH 13.0
`
`Reacting conditions
`
`Molar ratio
`in mixture
`(S/N)
`
`Sulfur
`(%)
`
`Nitrogen
`(%)
`
`Molar ratio
`in PEC
`(S/N)
`
`2.00
`1.60
`1.20
`1.00
`0.80
`0.70
`2.00
`1.60
`1.20
`1.00
`0.50
`0.50
`
`6.70
`7.02
`8.15
`8.12
`8.10
`6.82
`6.83
`6.93
`7.68
`8.17
`6.51
`8.19
`
`2.63
`2.98
`3.13
`3.18
`3.04
`2.88
`2.08
`2.75
`3.10
`3.22
`3.19
`3.13
`
`1.11
`1.03
`1.14
`1.12
`1.16
`1.03
`1.43
`1.10
`1.08
`1.11
`0.89
`1.14
`
`a Series 1: PVS solution was added dropwise to MGC + GC solution. Series 2: MGC + GC solution was added dropwise to PVS solution.
`Source: Ref. 51. Copyright n 1988 Wiley. Reprinted by permission of John Wiley & Sons, Inc.
`
`Copyright 2005 by Marcel Dekker
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`Kubota and Shimoda
`
`for each PEC prepared are given in Table 2. The compo-
`sitions of sulfur and nitrogen are almost the same. Because
`coagulation did not occur at pH > 6.5 in the GC–PVS
`system [52,53], only MGC was expected to react with PVS
`in the solution of pH > 6.5. However, in IR spectra, the
`absorption band at 1540 cm1 assigned to –NH3
`+ in GC
`was present in the PEC except for the PEC prepared at pH
`13.0, and the absorption band assigned to –NH2 that
`should appear at 1600 cm1 was absent. This is due to
`the strong induction effect by charged PVS as described
`above, because the absorption band at 1230 cm1 assigned
` in PVS was present in each PEC.
`to –OSO3
`The MGC–poly(L-glutamate) (PLG)–PVS system is
`more convenient for investigating the composition of the
`complexes by IR spectrometry, as each component in this
`system has a different reactive group [54]. The intensity of
`the IR absorption based on the –COO group increased
`with increasing pH from 2.0 to 5.0, and was constant at pH
`6.0–13.0. These results inferred that PLG was absent in the
`PEC at pH < 2, increased with increasing pH from 2.0 to
`5.0, and was constant at pH > 6.0. The intensities of the
`
`absorption band around 1240 cm1 assigned to the –OSO3
`groups of PVS for the PEC prepared at pH < 2.0 were
`medium, and those for the PEC prepared at 2.0 < pH <
`5.0 were weak. The absorption band around 1740 cm1
`assigned to the –COOH group of PLG was present in the
`PEC prepared at pH 4.0 and 5.0. These results were
`identical with those of the elemental analyses. This kind
`of PEC was also prepared in the MGC–carboxymethyl
`dextran (CMD)–PVS system under various pH conditions
`and in a different order of mixing, and similar results were
`obtained [55]. The PECs obtained are classified into three
`groups by means of elemental analysis, IR spectroscopy,
`and solubilities. The first is the group of PECs prepared at
`pH < 3, which are composed of MGC and PVS connected
`by ionic bond with each other. The second is the group of
`PECs formed at pH > 6.5, which are made up of MGC,
`CMD, and PVS linked by ionic bond with one another. The
`last is the group of PECs prepared at intermediate pH,
`which also constitute MGC, CMD, and PVS, but have a
`more complex three-dimensional network structure.
`
`F. Complexes by Template Polymerization
`
`Water-insoluble PECs of Ch–PAA and Ch–poly(styrene
`sulfonate) (PSS) can be obtained from the radical polymer-
`
`ization of acrylic acid (AA) and sodium 4-styrene sulfonate
`(NaSS), respectively, in the presence of Ch as a template at
`pH 4–4.5 [56]. It was concluded that the template polymer-
`ization technique appeared advantageous only for the
`synthesis of the Ch–PAA complex. The molecular weight
`of PAA obtained increased with increasing molecular
`weight of Ch, as shown in Table 3 [57].
`However, the structure and conformation of Ch mol-
`+ groups are separated from each
`ecule, in which –NH3
`other by a sequence of four atoms and oriented towards
`opposite directions in space, are disadvantages for the
`coordination of AA and NaSS molecules. Therefore, the
`effect of Ch as a template seems to be quite poor in
`comparison with that of vinyl polymer such as polyallyl-
`amine. At least the first step of this template polymeriza-
`tion is not an ionic preadsorption of monomer molecules
`onto Ch template, but the formation of a growing oligomer
`in the solution, which complexes with the template only
`beyond a critical chain length. In other words, an oligomer
`formation seems more favored than the polymerization of
`monomer molecules adsorbed onto Ch molecules.
`Ch–PAA complex nanoparticles were also prepared
`by template polymerization of AA in Ch solution [57]. The
`diameter of the nanoparticles was 200–300 nm, and the
`surface of the nanoparticles had positive charges. At pH
`7.4, PAA was highly extended while Ch was insoluble,
`resulting in the phase separation of nanoparticles where
`Ch was coated on the nanoparticles. These nanoparticles
`seem to form a sort of core–shell structure.
`
`III. PROPERTIES OF MACROMOLECULAR
`COMPLEXES OF CHITOSAN
`
`The properties of macromolecular complexes depend not
`only on the component polymers but also preparation
`conditions as mentioned above. The stoichiometric com-
`plexes ar