Structure of Chitosan Determines Its Interactions ... - ACS Publications

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Structure of Chitosan Determines Its Interactions with Mucin B. Menchicchi,† J. P. Fuenzalida,† Kishore Babu Bobbili,‡ A. Hensel,§ Musti J. Swamy,‡ and F. M. Goycoolea*,† †

Institute of Plant Biology and Biotechnology (IBBP), University of Münster, Schlossgarten 3, D-48149 Münster, Germany School of Chemistry, University of Hyderabad, Hyderabad-500046, Andra Pradesh, India § Institute for Pharmaceutical Biology and Phytochemistry (IPBP), University of Münster, Correnstrasse 48, D-48149 Münster, Germany ‡

S Supporting Information *

ABSTRACT: Synthetic and natural mucoadhesive biomaterials in optimized galenical formulations are potentially useful for the transmucosal delivery of active ingredients to improve their localized and prolonged effects. Chitosans (CS) have potent mucoadhesive characteristics, but the exact mechanisms underpinning such interactions at the molecular level and the role of the specific structural properties of CS remain elusive. In the present study we used a combination of microviscosimetry, zeta potential analysis, isothermal titration calorimetry (ITC) and fluorescence quenching to confirm that the soluble fraction of porcine stomach mucin interacts with CS in water or 0.1 M NaCl (at c < c*; relative viscosity, ηrel, ∼ 2.0 at pH 4.5 and 37 °C) via a heterotypic stoichiometric process significantly influenced by the degree of CS acetylation (DA). We propose that CS−mucin interactions are driven predominantly by electrostatic binding, supported by other forces (e.g., hydrogen bonds and hydrophobic association) and that the DA influences the overall conformation of CS and thus the nature of the resulting complexes. Although the conditions used in this model system are simpler than the typical in vivo environment, the resulting knowledge will enable the rational design of CS-based nanostructured materials for specific transmucosal drug delivery (e.g., for Helicobacter pylori stomach therapy).

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INTRODUCTION Mucoadhesion is defined as the ability of materials to adhere to the soft mucosal surface that lines the gastrointestinal, tracheobronchial, reproductive, and ocular systems.1 The mucoadhesive properties of natural and synthetic polymers are interesting because these can be used to develop carriers for transmucosal drug delivery that achieve a localized and prolonged effect of active ingredients (∼12−24 h). The underlying mechanisms that specifically control the mucoadhesive performance of different biomaterials (e.g., chitosan, alginate, hyaluronic acid, etc.) are not well understood, and the nature of their interactions with mucin at the molecular level has not been characterized in detail. Chemically, mucins comprise a complex and heterogeneous mixture of high-molecular-weight glycoproteins featuring dense O-linked glycans (predominantly on serine, threonine, and proline residues), N-linked glycans on asparagine residues, and nonglycosylated cysteine-rich domains.2−5 The glycans com© 2014 American Chemical Society

pose 50−80% of the glycoprotein mass and occur as covalently linked brush-like oligosaccharides with an average of 8−10 units, including N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, galactose, fucose, and sialic acids (N-acetylneuraminic acid).6,7 Disulfide bridges between the cysteine-rich domains generate a cross-linked three-dimensional polymer gel, which is further reinforced by hydrophobic association.8 Mucin fulfills a number of general biological roles, including lubrication, modulation of water and electrolyte absorption, and protection of the underlying epithelium against mechanical and chemical stress,9 but may also have specific functions such as providing attachment sites for commensal and pathogenic microorganisms, displaying ligands for leucocytes and endothelial cells,10 Received: May 31, 2014 Revised: August 11, 2014 Published: August 14, 2014 3550

dx.doi.org/10.1021/bm5007954 | Biomacromolecules 2014, 15, 3550−3558

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Germany). Four additional biomedical-grade CS samples as used in our previous studies (DA range = 1.6−56%, Mw ∼ 123−266 kDa, Table 1)27 were originally prepared from a parent ultrapure biomedical-grade chitosan sample from Mathani Chitosan Pvt. Ltd. (Kerala, India). All standard analytical grade chemicals were purchased from Sigma-Aldrich (Munich, Germany) and all solutions were prepared in ultrapure MilliQ water. Preparation of Mucin Solutions. The powdered mucin was hydrated in ultrapure MilliQ water (5 mg/mL) by gentle magnetic stirring for 3 h at room temperature and the insoluble fraction was removed by centrifugation at 25 000 × g for 50 min at 10 °C. Sodium azide (0.02%) was added and 15 mL aliquots were collected, lyophilized, and stored at 4 °C until further use. A completely clear solution of mucin was obtained after redissolving the lyophilized sample in MilliQ water to achieve a relative viscosity (ηrel) of ∼2 (8 mg/mL). Some aliquots were supplemented with 5 M NaCl to a final concentration of 0.1 M. The pH was adjusted to 4.5 with 5 M HCl. Preparation of Chitosan Solutions. The CS solutions were prepared by the dissolution of CS powders in a 5% stoichiometric excess of HCl in ultrapure MilliQ water with gentle magnetic stirring overnight. Each solution was filtered through a 5 μm pore membrane and the pH was adjusted to 4.5 with 1 M NaOH. Some aliquots were supplemented with 5 M NaCl to a final concentration of 0.1 M. Preparation of Chitosan−Mucin Mixtures. Stock solutions of CS and mucin of concentrations such that their relative viscosity match closely to ηrel ∼2.0 were mixed in different proportion to achieve mixtures of different composition ratios for the mass fraction of mucin relative to the total mass (defined here as f) was in the interval from f = 0 (i.e., only CS) to f = 1.0 (i.e., only mucin). The solutions were allowed to equilibrate at 37 °C by stirring at 400 rpm for 20 min before starting the viscosity measurements as described above. The relative viscosity (ηrel) of mixed CS−mucin solutions and its deviation from the individual stock solutions was determined by adapting two previously described methods for viscosity calculations.29,30 Microviscosimetry. The dynamic viscosity of CS and mucin solutions was measured as average values of four runs for each sample at 37 °C using an AMVn automated rolling ball microviscometer (Anton Paar, Ostfildern, Germany) with programmable tube angle based on the principle of the rolling ball time (the time that a steel ball needs to roll through the mixture inside a calibrated 1.6 mm diameter capillary). This was expressed as ηrel with respect to water or 0.1 M NaCl, both at pH 4.5. Hence concentration-dependence of the dynamic viscosity was determined to identify the concentration of CS or mucin with ηrel ∼ 2.0. At this concentration, the Newtonian regime was assessed by the null dependence of the values of dynamic viscosity at varying inclination angles in the range 20−80° (Supporting Information Figure S1). Thus, all the following measurements were registered at an inclination angle of 50°. The intrinsic viscosity, [η], of the CS and mucin samples was therefore determined in water and 0.1 M NaCl from experimental measurements of the dynamic viscosity of solutions with different concentrations of each component by the joint extrapolation of the Huggins (Eq. 1), Kraemer (Eq. 2), and ‘single point’ (Eq. 3) relationships.

and blocking specific adhesion protein on the surface of pathogenic bacteria.11,12 Polysaccharides are widely studied natural macromolecules because they are ideal for various drug delivery applications that rely on their mucoadhesive properties. They are generally stable, nontoxic, hydrophilic, and biodegradable13 and display reactive functional groups (e.g., carboxyl or amino groups) that promote mucoadhesion.14 Many polysaccharides are already approved for food and biomedical use (e.g., chitosan, carboxymethyl cellulose, alginate, dextran, etc.) and practical experience has shown that these polymers are easy to formulate as drug delivery nanocarriers for bioactive macromolecules.13 Chitosan (CS) has uniquely advantageous biological and physicochemical properties15,16 such as low toxicity and ability to promote wound healing,17 ability to form complexes with nucleic acids,18,19 and the fact that it is broken down by ubiquitous enzymes such as chitosanase, lysozyme, and human chitotriosidase.20 The mucoadhesive properties of CS are widely documented,21−24 but detailed mechanistic data on what happens between CS and mucin at molecular level are scarce. The interaction between CS with porcine stomach mucin in the presence of different additives confirmed that electrostatic interactions are complemented by hydrogen bonding and hydrophobic forces when CS and mucin are mixed in an aqueous environment,25 as also suggested by in vitro methods based on tensile and shear measurements.26 In the present investigation we revisited the interaction between biomedical-grade CS polymers with different properties (Mw and DA) and the nongelled purified soluble fraction of rehydrated crude porcine gastric mucin in dilute aqueous solutions. This model system provided insight into the underlying molecular mechanisms, particularly on the role of CS structure (as defined by the DA and molecular weight) on its interactions with mucin. We used a panel of biophysical techniques to monitor the interactions of macromolecules in solution. Our results are consistent with the hypothesis that CS−mucin interactions are controlled by the combined effects of electrostatic interactions, the conformational adaptation of the CS polymer (based on the DA), and other types of molecular forces such as Coulomb and hydrophobic associations.

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MATERIALS AND METHODS

Materials. Mucin from porcine stomach (type III, bound sialic acid 0.5−1.5%, partially purified powder) was purchased from SigmaAldrich (Munich, Germany). Two samples of pharmaceutical-grade CS (DA = 14.8% and 32.4%, Mw ∼ 10−30 kDa, Table 1) were sourced from Heppe Medical Chitosan (HMC+) GmbH (Halle/Saale,

Table 1. Origin and Characteristics of the CS Samples CS sample

supplier

HDP 1 HDP 11 HDP 27 HDP 56 HMC+15 HMC+30

Mathani Chitosan Mathani Chitosan Mathani Chitosan Mathani Chitosan HMC+ GmbH HMC+ GmbH

Pvt. Pvt. Pvt. Pvt.

Ltd. Ltd. Ltd. Ltd.

degree of N-acetylation (%)

Mw

1.6a 11.0a 27.5a 56a 14.8b 32.4b

123900c 122100c 143000c 266100c 27500d 17600d

ηsp /c = [η] + k′[η]2 c

(1)

(ln ηrel )/c = [η] + k″[η]2 c

(2)

[η] = {2 × (ηsp − ln ηrel )}1/2 /c

(3)

Turbidimetry. The turbidity of the CS−mucin complexes was measured in a 96-well microtiter plate using the Safire Tecan-F129013 Microplate Reader (Tecan, Crailsheim, Germany) at λ = 520 nm and 25 °C. Readings were taken at a range of f ratios in water and 0.1 M NaCl (pH 4.5). The turbidity was defined as

T = ln(I /I0)

a

Determined by 1H NMR spectroscopy. bData in accordance with specifications given by supplier. cDetermined by GPC-HPLC with multidetection (MALLS-DRI). dDetermined by intrinsic viscosity measurements in 0.3 M acetic acid/0.2 M sodium acetate at 20 °C.28

(4) −A

where (I/I0) = 10 and A is absorbance at λ = 520 nm. Size and Zeta Potential. The size distribution (Z-average diameter) of the CS−mucin complexes was determined by dynamic 3551


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light scattering with noninvasive back scattering (DLS-NIBS) at an angle of 173°, and the zeta potential (ζ) was measured by mixed laser Doppler electrophoresis and phase analysis light scattering (M3PALS), in both cases using a Malvern Zetasizer NanoZS (Malvern Instruments, Worcestershire, UK). Chitosan and Mucin Association. The net amount of CS associated with mucin was measured indirectly by determining the concentration of polymer remaining in solution after the separation of CS−mucin complexes by centrifugation at 25 000 × g for 40 min at 20 °C in a Mikro 220R centrifuge (Hettich, Bäch, Germany). The colorimetric assay based on the reaction between CS and Cibacron Brilliant Red 3B-A was used as previously described.31 Uncomplexed mucin was also estimated indirectly by means of a calibration curve constructed by spectrophotometry at λ = 280 nm. Isothermal Titration Calorimetry (ITC). ITC measurements were performed on a VP-ITC isothermal titration calorimeter from MicroCal (Northampton, MA, USA). The data were acquired and analyzed using MicroCal Origin ITC analysis software (OriginLab Corp.). Solutions of mucin for the ITC experiments were prepared by dissolving the lyophilized powder as described above, but in 20 mM acetate buffer rather than water. Prior to use, the reconstituted aqueous mucin solution was dialyzed for 24 h against four changes of a large volume of buffer. The final dialysate was filtered through 5 μm membranes and used as solvent to prepare HMC+15 and HMC+30 solutions (10 mg/mL). It was also used as a washing buffer to prepare the ITC experiment and as the blank for value correction. This exhaustive dialysis is necessary to ensure identical chemical potential conditions in the two solutions and thus reduce the occurrence of false-positive signals caused by the heat of mixing or dilution.32 After dialysis, the mucin was centrifuged at 10 000 × g for 10 min at 4 °C and the final concentration was determined by spectrophotometry at λ = 280 nm (UV-3600 Shimadzu) using a calibration curve obtained from the initial mucin stock solution prior to dialysis. The solutions were degassed under vacuum before use in order to prevent bubbling. We then injected 7 μL aliquots of HMC+15 or HMC+30 sequentially into 1.445 mL titration cells containing buffer at pH 4.5 or 5 mg/mL mucin. To ensure proper mixing after each injection, a constant stirring speed of 300 rpm was maintained during the experiment. Each individual injection lasted 30 s, and the interval between injections was 480 s, long enough to reach optimum compensation of the heat to the baseline. The temperature was fixed at 25 °C. Fluorescence Quenching Studies. Fluorescence spectroscopic measurements were carried out on a PC-1 fluorescence spectrometer from ISS (Champaign, IL, USA). The difference in fluorescence emission spectra between the mucin solutions before and after the addition of HMC+15 or HMC+30 was determined by preparing a mucin solution as described above for the ITC method, and transferring 2 mL of a 0.5 mg/mL solution into a 1 × 1 × 4.5 cm3 quartz cell, resulting in an absorbance of ∼ 0.1 at λ = 295 nm. Aliquots (10 μL) of 0.9 mg/mL CS were added subsequently. The titrated sample was excited at λex = 295 nm and the highest peak in the emission spectra was registered at λem = 338 nm (25 °C). The data were processed by subtracting the emission of CS added to buffer only, and applying the inner-filter correction. The values of R2, slope, and intercept were derived from the double log plot of (F0 − F)/F vs the CS concentration (Stern−Volmer equation, eq 5), and the two CS formulations were compared in terms of the binding affinity constant:33

log[(F0 − F )/F ] = log Ka + n log[L]

The mucoadhesive behavior of CS has been invoked to account for the ability of CS-based nanocarriers to transport macromolecular drugs across mucosal epithelia.35 This is consistent with the idea that CS-based nanocarriers accumulate on the mucosal surface, where they can contribute to the release and enhanced absorption of the drug. However, the underlying mechanisms are not clearly understood, particularly the role of the structural properties of CS, i.e., the degree and pattern of acetylation and the molecular weightoften abbreviated to DA, PA, and DP (degree of polymerization). The lack of concrete data reflects the diverse experimental methods used to investigate such interactions, the inherent structural complexity of the molecules, and the sui generis behavior of CS and mucin macromolecules in solution. To gain insight into the interaction mechanism, we studied model interactions between fully characterized forms of CS with different molecular weights and DA and the purified soluble fraction of porcine gastric mucin. Before investigating the interaction between CS and mucin in dilute solutions, the [η] of the chitosan samples in water and 0.1 M NaCl (pH 4.5, 37 °C) was determined. The HDP 1, HDP 11, HDP 27, and HDP 56 samples had Mw ranging between 123 and 266 kDa with no evident dependence on DA (Table 1). By contrast, the intrinsic viscosities of these samples in water and 0.1 M NaCl ([η]H2O and [η]NaCl, respectively) showed a clear dependence on DA that followed the sequence: HDP 1 > HDP 11 > HDP 27 > HDP56 (Table 2). CS solutions in water with only a 5% Table 2. Viscosity of Different CS and Mucin Samplesa sample

concentration at ηrel ∼2.0 in water (g/mL)

[η]H2Ob (mL/g)

[η]NaClb (mL/g)

ratio [η]H2O/ [η]NaClb

c· [η]H2Oc

HDP 1 HDP 11 HDP 27 HDP 56 HMC+15 HMC+30 Mucin

0.0003 0.0004 0.0005 0.0009 0.0033 0.0040 0.0080

3238 2155 2037 1260 349 313 69

858 480 449 389 174 147 51

3.80 4.49 4.54 3.24 2.01 2.13 1.35

0.97 0.86 1.02 1.13 1.15 1.25 0.55

a

Microviscosimetric measurements taken at pH 4.5 in water and 0.1 M NaCl at 37 °C, inclination angle 50°. b[η] = intrinsic viscosity (subscript denotes the solvent). cc·[η]H2O= Coil overlap parameter at concentration such that ηrel ∼ 2.0 in water at pH 4.5 and 37 °C.

stoichiometric excess of HCl with respect to the glucosamine monomolar concentration are likely to feature more than 75% protonation of the −NH2 groups.36 Therefore, the screening of long-range electrostatic interactions under such conditions should be much less prevalent in water than in 0.1 M NaCl, yielding a more expanded conformation.37 This is consistent with the observed tendency for the [η]H20 values to be greater than the [η]NaCl values (Table 2). In turn, the relationship between the intrinsic viscosity of the high-Mw CS samples and the DA suggests that CS adopts a more compact hydrodynamic volume as the chain becomes more acetylated, probably due to the presence of more hydrophilic structures as indicated by pyrene fluorescence studies that suggested CS behaves like a hydrophobic polyelectrolyte chainthe “pearl necklace model”.38 Independent DLS studies have shown that the aggregation of CS in solution is independent of the DA39 which contradicts reports suggesting that CS polymers with higher DA tend to form more hydrophobic aggregated structures.40 In addition, 1H relaxation NMR studies have indicated that CS

(5)

where F0 is starting fluorescence, F the measured fluorescence (corrected from the inner-filter effect) upon addition of a known amount of chitosan [L], Ka is the binding constant, and n the number of binding sites.

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RESULTS AND DISCUSSION The ability of CS to interact with mucosal epithelia has been investigated in vitro and in vivo and the interactions between CS and mucin have been studied in different model systems. 3552


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chain flexibility increases with the DA.41 Our results showed that increasing the DA increases the [η]H20/[η]NaCl ratio up to a DA of 27%, which concurs with studies suggesting the chain flexibility increases in higher-DA polymers. However, the samples with the highest DA (HDP 56) yielded the lowest [η]H20/[η]NaCl ratio, which suggests that the already compact conformation of HDP 56 may be less sensitive to long-rage electrostatic charge screening in the presence of NaCl. Low-Mw CS samples yielded lower [η]H20/[η]NaCl ratios than samples with a higher molecular weight. This supports previous research suggesting that there is a critical CS chain length that corresponds to a DP of ∼360 to ∼730 residues,41 below which the semirigid character of the polymer becomes dominant. CS samples HMC+15 and HMC+30, which have DP values of ∼100 to ∼150, are therefore expected to behave as semirigid polymers. Based on the complex behavior of CS in dilute solutions, we propose that the interplay between chain flexibility, hydrophilic/hydrophobic interactions, and the presence of charges in the CS chain, which combine to influence its conformation, determines the manner in which it interacts with mucin and other macromolecules in solution. We adapted a previously described microviscosimetry method to measure the ηrel of mixed solutions of CS and mucin in aqueous dilute acid (pH 4.5) in the presence and absence of 0.1 M NaCl with the peculiarity that the ηrel stock solutions for both components in aqueous dilute acid (pH 4.5) were similar (∼2.0, ηsp ≈ 1.0). The concentration of CS used to achieve these ηrel values was different depending on the kind of CS (Table 2) and corresponded with an equivalent low degree of volume occupancy c·[η]H2O < 1.45, thus, below the CS concentration threshold required for the onset of entanglement.42 Under such conditions, polymer exclusion effects are expected to be negligible, as previously demonstrated using mixed dilute solutions of plant polysaccharides.43 Mixing the two stock solutions of CS and mucin at increasing f ratio (mass proportion of mucin respect the total mass in the mixture) always reduced the ηrel to a minimum value ( fηmin) beyond which, upon a subsequent increase in f, the ηrel increased again to approach that of mucin stock solution. Such behavior described a skewed U-shaped curve both in water and in 0.1 M NaCl (pH 4.5) as shown in Figure 1a and b, respectively, for a representative CS−mucin system (HMC+30). For the sake of clarity, the variation in ηrel was subsequently expressed as a percentage of deviation from the theoretical additive line (or line of “no interaction”) calculated as additive viscosity contribution of the CS and mucin fractions at each composition (Figure 1c and d) as they would behave independently without macromolecular interactions. The observed reduction in viscosity must reflect either a net reduction in the concentration of macromolecules in solution relative to the pure solvent, resulting from the formation of larger (hence fewer in number) species such as complexes that segregate in one phase, or else an overall reduction in the hydrodynamic volume of the macromolecules when they are combined. The first explanation indicates the heterotypic interaction of the two species to form complexes, whereas the second might also reflect polymer exclusion effects that compact one or both components driven by the presence of the other.44 Previous studies of polysaccharide−polysaccharide interactions30 suggest that the use of a dilute polymer regime results in negligible polymer exclusion effects, indicating that the observed reduction in viscosity can likely be attributed to the formation

Figure 1. Relative viscosity (ηrel) of CS HMC+30−mucin mixtures of varying compositions expressed as the mass fraction of mucin (f) with respect to the total mass in (a) water and (b) 0.1 M NaCl (37 °C, pH 4.5, inclination angle 50°). The red dotted line in (a) and (b) represents the calculated values of ηrel of the mixtures assuming there is no interaction (additive line). The ηrel values at f = 0 and 1 are the relative viscosities of the CS (∼4.0 mg/mL) and mucin (∼8.0 mg/ mL) stock solutions, respectively. The lower panels show the normalized data expressed as percentage deviation from the additive line in (c) water and (d) 0.1 M NaCl, both at pH 4.5 (mean values ± minimum and maximum, n = 2). The blue shaded areas in plots (c) and (d) represent the integrated area under the curve calculated using a trapezoid approximation available in Origin 8.5 (Origin Lab Corp., Northampton, MA).

of heterotypic complexes of CS and mucin. In other previous studies using a similar microviscosimetric approach, a loss of viscosity was observed when dilute solutions of poly(acrylic acid) was mixed with vinyl alcohol/vinyl acetate copolymers with various degree of saponification in an acidic aqueous environment. The reduction in viscosity was attributed to the formation of a compact complex with a slightly turbid appearance.45 Figure 2 summarizes the normalized curves for all the CS− mucin interactions and reveals that the reduction in viscosity is invariably greater in water than in 0.1 M NaCl although the difference was less pronounced for the low-Mw CS (HMC+15 and HMC+30). Moreover, each CS−mucin system had a characteristic value of fηmin and curve shape. In fact while for the low-DA CS (HDP1) the maximum of interaction occurred in a well-defined mucin ratio (f = 0.9), in the case of high-DA CS (HDP56), the interaction was of similar magnitude in a larger range of compositions (from f = 0.5 to 0.8). In an attempt to evaluate quantitatively such a difference we calculated the integrated area under the curve (AUC) described by the sum of the trapezoids between the experimental points and the additive line (shown as the blue shaded area of Figure1c and d). The dependency of the AUC on DA and Mw in the presence or absence of 0.1 M NaCl is shown in Figure 3. The absolute value of AUC increases with CS’s DA both in water and in 0.1 M NaCl (Figure 3a and b, respectively). Such relationships can be fitted with a straight line in 0.1 M NaCl (R2 = 0.98) but not in water. On the other hand, without 0.1 M NaCl, a clearer dependency of AUC on the CS’s Mw was observed (Figure 3c). Indeed, in 0.1 M NaCl, when the longrange electrostatic forces are screened and the CS chains undergo coil contraction, the Mw has less impact on the 3553


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turbidity of the mixtures, were homogeneously dispersed and neither aggregation nor flocculation was observed. However, phase separation (precipitation) tended to occur during longterm storage. The turbidity data in the presence and absence of 0.1 M NaCl (pH 4.5, Figure 4) correlated well with the

Figure 4. Variation of the turbidity (λ = 520 nm) according to the mass fraction of mucin ( f) in CS−mucin complexes in (a) water and (b) 0.1 M NaCl (mean ± SD; n = 3).

microviscosimetric experiments in that the maximum turbidity was observed corresponding to the point of minimum viscosity ( fηmin). In 0.1 M NaCl a shift of the maximum turbidity to lower f values (less mucin per CS) was observed. This is due to favored complex formation between contracted coils of CS exposing higher surface charge density with less mucin.34 The low-Mw CS samples (HMC+15 and HMC+30) always produced more turbid mixtures than the high-Mw ones. This might be explained by the fact that these two CS were used at much higher concentration than that of high-Mw CS to achieve ηrel ≈ 2.0 (Table 2), thus offering a higher number of macromolecules and hence cationic groups which would favor the formation of a greater amount of complexes, thus resulting in higher turbidity. The overall greater turbidity observed in 0.1 M NaCl, compared to water, may be a consequence of charge screening by the added NaCl of the excess charge in the formed complexes, and it may also reflect the favorable entropic contribution of numerous small ions, which compensates for the unfavorable association with polyions.46 The turbidity results were confirmed by NIBS-DLS measurements that revealed higher derived count (DCR) values for mixtures containing HMC+15 or HMC+30 compared to the others (see Supporting Information Figure S3). NIBS-DLS analysis also revealed that the interaction between CS and mucin produced nanoscopic species whose Z-average ranged from ∼200 to ∼900 nm and represented true nanocomplexes (Figure 5). In mixtures with lowest proportion of mucin (f ≈ 0.2) the particle size of the nanocomplexes was proportional to the intrinsic viscosity values of the polymer in water, i.e., to the

Figure 2. Variation in the percent deviation of the relative viscosity from the additive line with the mass fraction of mucin ( f) for (a) HDP 1−mucin 1; (b) HDP 11−mucin; (c) HDP 27−mucin; (d) HDP 56− mucin; (e) HMC+15−mucin; and (f) HMC+30−mucin in water (full symbols) and 0.1 M NaCl (empty symbols) at 37 °C, pH 4.5, and an inclination angle of 50° (mean values ± minimum and maximum, n = 2).

Figure 3. Variation of the overall reduction in viscosity given as the area under the curve (AUC) calculated from the percentage deviation of the relative viscosity from the additive line (see Figure 2) as a function of DA (a and b) and Mw (c and d) in water (a and c) and 0.1 M NaCl (b and d). Mean values ± minimum and maximum, n = 2.

interaction (Figure 3d) while it appears to be determined by the DA and other properties such as hydrophobicity, conformation, and charge density that are collectively at play in determining the interaction with mucin (Figure 3b). The occurrence of CS−mucin interaction was accompanied by the appearance of turbidity (see Supporting Information, Figure S2) as also previously observed on CS−mucin mixtures or polycation/polyanion self-assembled complexes. The CS− mucin complexes formed which are responsible for the

Figure 5. Z-average size (diameter) of CS−mucin complexes based on the mass fraction of mucin ( f) in water (37 °C, pH 4.5) as determined by dynamic light scattering (mean ± SD; n = 3). 3554


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stoichiometric point, the amount present in the complexes slightly decreases for HDP 27 and HMC+30 but drastically declines for HDP 56. On the other hand, CS was incorporated into the complexes in higher percentage than mucin did, up to reaching 100% in some cases (Figure 7b). Interestingly, mixtures comprising high-Mw and high-DA CS (HDP 27 and 56) incorporated the largest amounts of CS, suggesting that mucin may have a preferential affinity for those CS, as recently proposed for the interaction of oligomers of alginate with mucin.47 The incorporation of lower amounts of the low-Mw CS (HMC+15 and HMC+30) into the complexes contradicts the derived count rates (DCR) we observed by NIBS-DLS and higher turbidity. One possible interpretation of this discrepancy is that such systems may form weaker complexes that dissociate during centrifugation, so that the apparent concentration of CS in the supernatant after isolating the complexes is higher than expected but does not reflect that of the originally created complexes. A similar phenomenon of instability during centrifugation has been reported for nanocomplexes formed between polyarginine and low-molecular-weight heparin.48 In the low-Mw CS it can be argued that the greater reduction in ηrel observed for the HMC+30 systems, the higher amount of CS complexed, and yet lowest turbidity than for HMC+15 systems is a consequence of the formation of complexes of a molecular architecture with a denser structure, hence with higher amount of chitosan retained inside and with smaller size. Instead, the lower DA CS (HMC+15) produces larger but less dense complexes, so that the overall concentration of free CS macromolecules contributing to the viscosity is bound to be higher than that achieved with chitosans with high DA. This explanation seems valid also for CS of high-Mw with DA values up to 30%. A similar behavior has been observed in synthetic polymers of increasing hydrophobicity.49,50 This is consistent with our proposal that complex formation is influenced by the interplay between heterotypic intermolecular interactions (electrostatic and nonelectrostatic) and the conformational adaptation of CS (governed by its intrinsic chain flexibility). We used ITC in two selected systems containing low-Mw CS to gain further insight into the mechanism of complex formation. The ITC data shown in Figure 8 did not fit to any classical molecular binding model (e.g., the one-site binding model). However, the observed switch from an exothermic to an endothermic process at a well-defined composition ratio (Figure 8c) defined by the proportion of chitosan (i.e., the 1 − f value) used as the titrant component, could be interpreted as the result of the aggregation and subsequent coacervation of the complexes.51 This characteristic multistage behavior was also observed when β-casein was titrated with 83% deacetylated chitosan at 50 °C in a study aiming to determine the effect of temperature on CS−protein nanoparticles.52 We envisage that the gradual addition of CS to mucin would result in the formation of complexes as an exothermic, enthalpy-driven process, resulting from heterotypic binding between CS and mucin. Although this process was characterized for HMC+15 by fewer picks (13 injections) of higher intensity, and more picks (17 injections) of less intensity for HMC+30, the overall magnitude of the net integrated area of the exothermic component was similar for both CS (Table 3). As the titration continues a saturation point is reached, beyond which heterotypic interactions no longer occur when more CS is added defining an inversion point (f inv) where endothermic peaks are observed. This process was largely higher for HMC+15 making the ratio of exothermic/endothermic

hydrodynamic volume of the CS coils (Table 2). As the proportion of mucin increased from f = 0.2 to 0.7−0.8, mixtures containing high-Mw CS with DA values of 1−27% underwent a monotonic contraction whereas there was no such contraction in mixtures containing low-Mw CS (HMC+15 and HMC+30), which is consistent with the view that in these CS the polysaccharide chains are inherently more rigid. In all cases an increase in the Z-average diameter was observed corresponding to the minimum in viscosity (fηmin) especially for the low-Mw CS where the increase was dramatic for HMC+15, and a similar albeit less extreme for the mixtures containing HMC+30. The large increase in size observed at specific f ratios was undoubtedly attributed to charge compensation between CS and mucin. Figure 6a illustrates that the ζ( f) traces were

Figure 6. (a) Variation of the zeta potential (ζ) with the mass fraction of mucin (f) in water at pH 4.5 for complexes of mucin and HDP 1 (■), HDP 11 (▲), HDP 27 (▼), HDP 56 (⧫), HMC+15 (●, blue), and HMC+30 (●, light blue)mean values ± minimum and maximum, n = 2; (b) Variation of the stoichiometric f values as determined by zeta potential analysis ( fζ=0) and microviscosimetry (fηmin) with the DA value of CS.

dependent on the DA of the CS in all the mixtures. In the range of f from 0 to 1, ζ decreases from highly positive values (only CS) to negative ones (only mucin) passing through a neutralization point ( fζ=0) which suggests CS−mucin complexes have a well-defined stoichiometry. This value becomes lower as the DA increases and was comparable to fηmin as shown in Figure 6b. However, the exact profile is highly dependent on the DA. For instance, ζ(f) curve for HDP 1 shows two distinct regions of performance, a plateau in the region f ≈ 0.2−0.7, and a steep slope at f ≈ 0.7−1.0. The same behavior was observed also for HDP 11 and HMC+15, whereas higher DA values (HDP 27; HMC+30) generate ζ(f) tending to a single linear slope as observed for HDP 56. At the stoichiometric point, the highest amount of mucin is sequestered into the complex (Figure 7a) up to ∼76% of the initial weight for HDP 1, HDP 11, and HMC+15 at f = 0.9, up to ∼70% for HDP 27 and HMC+30 at f = 0.8 and up to ∼ 60% for HDP 56 at both f = 0.6 and 0.7. When mucin is present in large excess beyond the

Figure 7. Variation of the composition of CS−mucin complexes with the mass ratio of mucin f in water at pH 4.5, in terms of the percentage of (a) mucin, calculated by spectrophotometry (λ = 280 nm), and (b) chitosan, determined by colorimetry (mean ± SD; n = 3). 3555


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Figure 8. Change in heat flow (kcal/s) over time when 7 μL of 10 mg/mL CS is injected into a reaction cell containing 1.445 mL of mucin (5 mg/ mL) in 20 mM acetate buffer at pH 4.5 and 25 °C for systems titrated with (a) HMC+15 and (b) HMC+30 (in both cases after buffer subtraction). Representative heat flow thermogram of two replicates: (c) variation of the integrated heat flow peaks with the mass fraction of CS (1 − f) of HMC+30 (■) and HMC+15 (●). Mean values ± minimum and maximum, n = 2.

constants (log Ka) were Ka = 4.58 (±1.51) × 104 and 2.62 (±1.33) × 104mol/L for HMC+30 and HMC+15, respectively. The two Ka values share the same order of magnitude and only reflect a slight greater affinity of the CS with a DA of 30% than that with DA of 15%.

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CONCLUSION We have gained insight into the mechanisms that determine the mucoadhesive properties of CS, particularly the role of the DA and Mw of the CS polymer. The microviscosimetric method employed here allowed us to distinguish the magnitude of interaction between different CS and mucin and was higher for high-Mw CS. Moreover, high-Mw and high-DA CS can interact with the same magnitude in a larger range of composition as compared to low-DA CS whose maximum interaction occurred only at a narrow range of composition. The point in which lowMw CS-containing systems show their maximum in viscosity reduction (fηmin) correlated with neutralization of the complex (zeta potential = 0, fζ=0) and the inversion from exothermic to endothermic process as found by ITC ( f inv). Even when the conditions of the present study represent only a model of interaction between chitosan and mucin in solution and are an oversimplification of the mucoadhesion phenomena occurring in vivo, the evidence gathered suggests that the high affinity of CS to interact with mucin may also contract the mucin gel network and thus create large pores throughout the gel mesh (i.e., between bundles of CS−mucin) as it has been suggested for alginate oligomers48a hypothesis that is yet to be tested. This may facilitate the diffusion of CS−based nano and microparticles and/or bioactive molecules through the mucus layer. Whether there are structural motifs in CS associated with the contents and distribution of N-acetyl-D-glucosamine residues with high specific affinity for mucin is also yet to be discovered. Based on the knowledge gained in this work, an accurate selection of the appropriate CS might be meaningful in the development of CS-based transmucosal drug delivery formulations with enhanced capacity to interact with mucin.

Figure 9. Stern−Volmer double logarithmic representation (see eq 5) of the data obtained by fluorescence measurement (λex = 295, λem = 338) during the titration of mucin with fixed aliquots of HMC+15 (●) and HMC+30 (■) CS. The line is the linear regression curve through the data points. The inset shows representative fluorescence quenching spectra of mucin at varying concentrations of HMC+30 chitosan (as shown by arrow).

Table 3. Integrated Values of the Single Exothermic and Endothermic Process Observed during the Isothermal Titration of Mucin with Chitosan CS sample

endothermic component (kcal/s)

exothermic component (kcal/s)

endothermic/ exothermic

HMC+15 HMC+30

592.67 ± 11.50 616.28 ± 0.36

142.34 ± 4.68 35.03 ± 0.00

4.2 17.6

components approximately 4-fold lower with respect to HMC+30. The occurrence of the endothermic components reveals the prevalence of the CS heat of dilution with respect to the total released heat, which is maximal as soon as the inversion point is reached and then declines due to further hydrophobic interactions with existing CS−mucin complexes.53 The observed differences in f inv at a well-defined proportion of added chitosan which corresponds to f inv = 0.91 and 0.88 for HMC+15 and HMC+30, respectively, confirms that the formation of CS−mucin complexes occurs at a well-defined CS−mucin proportion determined by the DA and occurs for the electrostatic neutralization between oppositely charged species (Table 4). Fluorescence spectroscopy revealed that the addition of CS to mucin induced fluorescence quenching that persisted after all relevant corrections, and the calculated values of the affinity

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures as described in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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Table 4. Comparison of Microviscosimetry ( fηmin), Zeta Potential (fζ=0) Analysis, and ITC Data (f inv)

AUTHOR INFORMATION

Corresponding Author HMC+15 HMC+30

fηmin

fζ=0

f inv

*E-mail: [email protected].

0.86 0.77

0.93 0.88

0.91 0.88

Notes

The authors declare no competing financial interest. 3556


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ACKNOWLEDGMENTS We acknowledge financial support and the PhD fellowships to B.M. and J.P.F. from German Research Council DFG (Project GRK 1549 International Research Training Group ‘Molecular and Cellular Glyco-Sciences’). The MicroCal VP-ITC equipment used in this study was obtained from the funds made available to the University of Hyderabad through the UPE program of the University Grants Commission (India). The ISS fluorescence spectrometer was procured from a grant from the DBT (India) to M.J.S. K.B.B. was supported by Senior Research Fellowship from the CSIR (India). We thank Dr. Richard M. Twyman for critical reading of the manuscript. We are also indebted to Prof. Edwin R. Morris for insightful discussions.

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NOTE ADDED AFTER ASAP PUBLICATION Due to an unintentional oversight, we failed to recognize the criticisms of Van de Weert and Stella33 and of Grossweiner54 on the model used to fit the titration data of the fluorescence quenching spectroscopic experiment shown in Figure 9 of our paper. These authors clearly pointed out that the use of the double log Stern−Volmer equation could have several pitfalls. First, the model assumes the formation of a non-fluorescence complex; second the amount of added ligand is usually set equal to the amount of free ligand. The deviation from both assumptions will affect the calculated values of the binding constant and number of binding sites (Ka and n parameters, respectively), obtained through the double logarithmic 3558


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