International Journal of Pharmaceutics 461 (2014) 280–285
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A mechanistic based approach for enhancing buccal mucoadhesion of chitosan Emil Meng-Lund 1 , Christian Muff-Westergaard 1 , Camilla Sander, Peter Madelung, Jette Jacobsen ∗ Section for Pharmaceutical Design and Drug Delivery, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
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Article history: Received 13 September 2013 Received in revised form 24 October 2013 Accepted 28 October 2013 Available online 27 November 2013 Keywords: Buccal mucoadhesion Chitosan Mucin Complex coacervation Isothermal titration calorimetry Texture analyzer
a b s t r a c t Mucoadhesive buccal drug delivery systems can enhance rapid drug absorption by providing an increased retention time at the site of absorption and a steep concentration gradient. An understanding of the mechanisms behind mucoadhesion of polymers, e.g. chitosan, is necessary for improving the mucoadhesiveness of buccal formulations. The interaction between chitosan of different chain lengths and porcine gastric mucin (PGM) was studied using a complex coacervation model (CCM), isothermal titration calorimetry (ITC) and a tensile detachment model (TDM). The effect of pH was assessed in all three models and the approach to add a buffer to chitosan based drug delivery systems is a means to optimize and enhance buccal drug absorption. The CCM demonstrated optimal interactions between chitosan and PGM at pH 5.2. The ITC experiments showed a significantly increase in affinity between chitosan and PGM at pH 5.2 compared to pH 6.3 and that the interactions were entropy driven. The TDM showed a significantly increase in strength of adhesion between chitosan discs and an artificial mucosal surface at pH 5.2 compared to pH 6.8, addition of PGM increased the total work of adhesion by a factor of 10 as compared to the wetted surface without PGM. These findings suggest that chitosan and PGM are able to interact by electrostatic interactions and by improving the conditions for electrostatic interactions, the adhesion between chitosan and PGM becomes stronger. Also, the three complementary methods were utilized to conclude the pH dependency on mucoadhesiveness. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Buccal drug administration has attracted attention as a means to achieve a fast onset and systemic delivery of drugs susceptible to the gastro intestinal or hepatic first pass metabolism (SalamatMiller et al., 2005). However, the continuous flow of saliva and swallowing reduces the amount of drug actually absorbed in the oral cavity, which is a limitation of this route of administration (Patel et al., 2012). Use of a buccal mucoadhesive formulation is an attractive approach to alleviate this limitation as provision of a prolonged high local concentration of the active pharmaceutical ingredient, will decrease the loss and even further facilitate fast absorption due to a steep concentration gradient (Haas and Lehr,
Abbreviations: PGM, porcine gastric mucin; CCM, complex coacervation model; ITC, isothermal titration calorimetry; TDM, tensile detachment model; CS, CHITOPHARM® S; CM, CHITOPHARM® M; CL, CHITOPHARM® L; MES, 2-(N-morpholino)ethanesulfonic acid; HWS, human whole saliva. ∗ Corresponding author at: Universitetsparken 2, 2100 Copenhagen, Denmark. Tel.: +45 3533 6299; fax: +45 3533 6030. E-mail address: [email protected] (J. Jacobsen). 1 Both authors contributed equally. 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.10.047
2002). Thus, lower and more available doses can be administered by the use of mucoadhesive formulations. The term mucoadhesion refers to the specific phenomenon of adhesion between a polymer and a mucus layer1−5 . The mucus layer comprise of a viscous loosely adherent layer mainly composed of mucins and water, it lines mucosal membranes, such as those found in the oral cavity (Andrews et al., 2009; Phillipson et al., 2008). The mucus layer lubricates, moistures and protects the epithelium from physical and chemical insults (Salamat-Miller et al., 2005; Schenkels et al., 1995). The ability of a polymer to attach to the mucus layer is dependent on several factors other than the formation of chemical bonding (Duchene et al., 1988). Swelling, molecular weight, and flexibility of polymer chains are all factors that have great influence on the strength and duration of adhesion (Gaserod et al., 1998; Hagesaether et al., 2009; Salamat-Miller et al., 2005). In the oral cavity, mucins are epithelial surface bound as well as solvated of salivary origin. Mucins are glycoproteins with a peptide backbone and oligosaccharide side chains, which often terminate in sialic acid residues (Salamat-Miller et al., 2005). With an isoelectric point of 2–3 (Lee et al., 2005), mucins will thus be overall negatively charged in the oral cavity, as pH is around 6.8. Chitosan is a non-toxic, biocompatible and biodegradable polysaccharide
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consisting of alternating glucosamine and N-acetyl-glucosamine units with a pKa of 6.5 (Illum, 1998; Liu et al., 2005; Sogias et al., 2008). Chitosan has received interest as a bioadhesive excipient due to its ability to interact with mucins in the mucus layer by electrostatic, hydrophobic, hydrophilic and by hydrogen bonding (Sogias et al., 2008). The following stepwise process of mucoadhesion has been proposed (Duchene et al., 1988): The initial contact stage of polymer and mucus, facilitating dehydration of the mucosa while hydration of polymer, forming a swelled polymeric network. The second step is the consolidation stage where polymer and mucin chains interdiffuse and entangle, thus facilitating non-covalent bonding and electrostatic interactions (Duchene et al., 1988; Smart, 2005). Mechanistic studies of chitosan and mucin have shown that the electrostatic interaction between the positively charged amines on the polymer chains and the negative sialic acid residues on the mucin glycoprotein play a role in the mucoadhesive properties of chitosan (He et al., 1998; Salamat-Miller et al., 2005; Sogias et al., 2008). The electrostatically driven reaction between chitosan and mucin can be interpreted as the process of complex coacervation. Complex coacervation is a process, where electrostatic interactions between a colloid suspension of a polymer and a protein will lead to the phase separation into a polymer-protein high concentration phase and polymer-protein low concentration phase (de Kruif et al., 2004). The formation of a protein-polymer rich phase will give rise to a sharp increase in turbidity of the solution. Typically, this reaction will take place in the pH range between the pKa value of the polymer and the isoelectric point of the protein with an optimum at the pH, where the molecules carry the same numeric charge (Liu et al., 2010). Thus, the maximal electrostatic interactions between chitosan and mucin should occur in the pH-range of 2–6.5. The process of complex coacervation is influenced by a number of factors, one of which is the ionic strength of the solution (Liu et al., 2010). The ionic strength influences the overall charge of the protein and polymer due to the screening effect of counter ions (Burgess, 1990). In this study, the interaction between chitosan and mucin was examined in a buffer solution resembling human saliva with regards to ionic composition and ionic strength. The aim of this study was to examine the pH-dependent interactions between chitosan polymers of increasing molecular weight and porcine gastric mucin (PGM) applying simulated oromucosal conditions. The mechanism of chitosan mucoadhesive properties was studied using complex coacervation model (CCM), isothermal titration calorimetry (ITC) and tensile detachment model (TDM).
2. Materials and methods 2.1. Materials All chemicals used were of analytical grade and were used as received unless otherwise described. Acetic acid (glacial), calcium chloride dihydrate, sodium chloride, sodium hydrogen carbonate and sodium hydroxide of analytical grade were purchased from Merck (Darmstadt, Germany). CHITOPHARM® L (CL) (MW 500–5000 kDa), CHITOPHARM® M (CM) (MW 100–2000 kDa), and CHITOPHARM® S (CS) (MW 50–1000 kDa) were provided as a free sample from Cognis GmbH (Monheim, Germany). 2(N-morpholino)ethanesulfonic acid (MES), potassium chloride, sodium phosphate dibasic, sodium phosphate monobasic and Type II porcine gastric mucin (PGM) purified from porcine stomach mucosa were all purchased from Sigma–Aldrich (St. Louis, MO). Deionized water was obtained from Millipore Milli-Q Ultrapure Water purification system (Billeria, MA).
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2.2. Complex coacervation model (CCM) The electrostatic interactions between chitosan (S, M and L) and PGM were studied by complex coacervate formation. A buffered electrolyte solution (pH 6.5) mimicking human whole saliva (HWS buffer) was prepared in accordance to (Gaserod et al., 1998):210 mg/L NaHCO3 , 430 mg/L NaCl, 750 mg/L KCl, 220 mg/L CaCl2 2H2 O and 910 mg/L NaH2 PO4 in deionized water. A stock solution of 2 mg/mL PGM was prepared by solvating PGM in HWS buffer at 5 ◦ C overnight to ensure complete solvation. Chitosan (CS, CM and CL) stock solutions of 1 mg/mL were prepared by dissolving chitosan in HWS buffer. Test solutions containing 0.5 mg/mL PGM in HWS buffer with 0.15 mg/mL CS, CM, or CL was prepared by mixing chitosan stock solutions and PGM stock solution and diluting with HWS. The control solution contained 0.5 mg/mL PGM in HWS buffer. The test solution (200 mL) was titrated at room temperature with 0.2 M HCl solution using a 842 Titrando titrator fitted with a Unitrode pH electrode with Pt1000 temperature sensor, a Dosino 10 mL dosing unit and a rod stirrer with a 20 mm wide propeller (Metrohm AG, Herisau, Switzerland). Tiamo software version 1.1 was used for controlling the titration as well as acquiring and analyzing the results. The titrations were performed in the pH range 6.5–2.7 and a sample (2.0 mL) was withdrawn for each 0.2 pH interval after reaching pH equilibrium. Sample turbidity was determined at a wavelength of 400 nm (Cary 100 UV, Agilent Technologies Denmark, Horsholm, Denmark) using the WinUV software. All samples were measured using disposable 1.5 mL plastic cuvettes from Brandtech Scientific Inc. (Essex, CT). The absorbance was zeroed against HWS buffer and the dilution effect of the titration was taken into account.
2.3. Isothermal titration calorimetry (ITC) The binding interactions involved in the mucoadhesive process were studied using ITC. To ensure uniform ionic strength, all test solutions were dialyzed. The test solutions consisted of 0.500 mg/mL PGM at pH 5.2 (0.0125 M acetic acid) and pH 6.3 (0.016 M MES). The solutions were dialyzed using dialysis bags with a cut-off of 12 kDa (Sigma, St. Louis) and were dialyzed against an excess of either acetic acid or MES buffer. The two different buffer concentrations ensured similar ionic strength of the buffer solutions. The dialysis fluid was changed seven times during approximately 20 h and was stable at the desired pH values after the fourth change of dialysis fluid. Before titration analysis, all samples were thoroughly degassed in vacuum, for a minimum of 30 min, under rapid magnetic stirring. Titration of 0.500 mg/mL PGM at pH 5.2 and pH 6.3 with 0.600 mg/mL CM were conducted on a NanoITC 2G, TA Instruments (Centennial Park, United Kingdom). Initially the reaction cell was rinsed thoroughly by flushing with degassed deionized water followed by PGM test solution. The CM solution to be tested was loaded in a 250 L syringe, which was carefully evacuated of air. The propeller was gently wiped clean of residual CM solution. The syringe was set to a propeller speed of 250 rpm, and the temperature in the reaction cell was 25.000◦ C. Each titration consisted of initial 3.15 L (i.e. lowest injection volume to omit residual air) and followed by 37 consecutive 5.15 L injections. The peak resulting from the first injection was subsequently ignored. All ITC data were analyzed using the NanoAnalyze Data Analysis software, version 2.3.6 (TA Instruments) by splitting the area of the heat peaks into two distinct curves and fitting the area of the heat peak of each curve to an independent site model, deriving a value for the affinity constant (K), the binding stoichiometry (n) and the enthalpy change (H). The amount of single sugar units of CM was calculated and used as molar concentration, assuming a degree of deacetylation of 70%. The molecular
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Table 1 Composition of chitosan liquid feeds for spray drying.
under the distance–force curve using the Exponent 32 program version 5.1.1 (Stable Micro Systems LTD, Godalming, UK).
Excipients
CS feed
CM feed
CL feed
Chitosan quality Deionized water (mL) HCl, 2.4 M (mL)
5 g CS 163 4.17
5 g CM 244 6.25
5 g CL 325 8.33
weight of PGM used for calculations was 1.25 × 106 Da (Jumel et al., 1996). 2.4. Tensile detachment model (TDM) 2.4.1. Preparation of chitosan discs The composition of chitosan liquid feeds is displayed in Table 1. The steady state flow viscosity of the feeds was measured as previously described (Sander et al., 2013) and was shown to be similar at shear rates in the range of 500–1000 s−1 . Chitosan was hydrated in water for 10 min before addition of HCl. Solutions were stirred overnight, allowing complete solvation. Spray dried chitosan particles were prepared from chitosan liquid feeds, using a Büchi Mini Spray Dryer B-290 (Büchi Labortechnik AG, Germany). Initially, the system was heated for approximately 30 min to reach an inlet air temperature of 200◦ C which lead to an outlet air temperature of 84–101◦ C. Atomizing air was set to 40 mm Hg and the nozzle was set to clean every 2 min using an automatic vacuum pump. A pump rate of 3 mL/min which corresponds to 10% of the capacity of the equipment was used together with an aspirator rate of 32 m3 /h, corresponding to 80% capacity of the equipment. The water content of the spray dried chitosan particles was measured by thermo gravimetric analysis (TAC 7/DX, PerkinElmer Inc., Waltham, MA). Samples of 5 mg spray dried chitosan particles were heated to 130◦ C for 30 min and water content was calculated as percentage weight loss to be approximately 10% (w/w). No difference in water content was found between the chitosan qualities. All discs were made from spray-dried chitosan-HCl particles of CS, CM, or CL (see above). Prior to the experiment, chitosan discs (diameter 11.3 mm) weighing approximately 50 mg were prepared using a hydraulic press (PerkinElmer Inc., Waltham, MA) with a pressure of 2 bar for 10 s. 2.4.2. Preparation of the artificial mucosal surface The preparation of the artificial mucosal surface was modified from (Hagesaether et al., 2009). An adsorbing paper with an inert backing layer (Bench surface protector VWR, Herlev, Denmark) was cut into pieces of 2 cm × 2 cm. The paper was fixed to a metal base surface with double adhesive tape and wetted with 90 L buffer solution (0.25 M acetic acid buffer pH 5.2 or 0.25 M phosphate buffer pH 6.8), which was allowed to soak into the paper for one min before the test was carried out. The adhesiveness of the disk to adsorbing paper was tested using buffers solutions both with 3% PGM and without (control). 2.4.3. Tensile detachment measurements Mechanical strength of the mucoadhesive bonds between compressed discs of chitosan (CS, CM or CL) and an artificial mucosal surface was tested at pH 5.2 and 6.8 using a texture analyzer (Texture Analyzer TX-XT plus, TA Instruments, UK) equipped with a 30 kg load cell. A disk of CS, CM or CL was fixated with double adhesive tape to the cylindrical probe of the Texture Analyzer and brought to contact with the artificial mucosal surface using pretest speed 0.5 mm/s, test speed 0.5 mm/s, post-test speed 5 mm/s, applied force 2000 mN, return distance of 20 mm and a contact time between disk and mucosal surface was 60 s. The force required to separate the chitosan disk from the artificial mucosal surface was recorded and total work of adhesion was computed as the area
2.5. Statistical analysis All data are presented as mean ± SD, unless otherwise stated. GraphPad Prism for Windows, version 6.00 from GraphPad Software Inc. (La Jolla, CA) was used for the statistical calculations. A one way analysis of variance for multiple comparisons was followed by a Student–Neuman–Keuls test. Two sided p-values below 5% (p < 0.05) were considered statistically significant. 3. Results and discussion The mechanism of mucoadhesion has not been fully elucidated and depends to a large extend on whether it is a solid, semi-solid or liquid formulation (Salamat-Miller et al., 2005). It is evident from literature that the method used for assessing the pH-dependency of the interaction between chitosan and mucin has a profound effect on the obtained results. Different techniques may focus on different stages of the mucoadhesive process leading to inconsistent results. 3.1. Complex coacervation model Methods for assessing the mucoadhesive properties of different polymers using either change in turbidity, particle size or zeta potential have previously been reported (He et al., 1998; Klemetsrud et al., 2013; Sogias et al., 2008). These methods could prove useful when comparing the mucoadhesive potential of either excipients or finished formulations, due to the high inaccuracy often found using more direct methods (Pongjanyakul et al., 2013). However, the validity of these methods needs to be established. The pH-dependency of the interaction between chitosan and mucin has been studied by a number of different methods, giving rise to several conflicting conclusions. A study using a fluorescence method has shown an increased interaction between chitosan and bovine submaxillary mucin (BSM) at more acidic pH values, highest degree of interaction at pH 2.9 (Qaqish and Amiji, 1999). Another study, using surface plasmon resonance, have shown an increase in interaction at pH 6 between chitosan and BSM, while yet another study have shown an increase in interaction between chitosan and BSM above pH 7 (Sigurdsson et al., 2006). PGM and BSM have been used as a model for human mucins when testing mucoadhesive properties and are different in terms of solubility and structure (Klemetsrud et al., 2013). PGM was preferred over BSM in this study as Teubl et al. showed that human mucin exhibits more similar chemical and morphological structures to PGM over BSM as determined by Fourier transform infrared spectroscopy and scanning electron microscopy, respectively (Teubl et al., 2013). For comparative studies, Sandberg et al. showed the importance of standardizing mucin, as significant batch to batch variation was observed for both PGM and BSM with respect to structure and mucin adsorption (Sandberg et al., 2009). Therefore, single batch PGM was used for the studies presented. The CCM showed that the interaction between PGM and CS, CM or CL was pH dependent and an optimal interaction was obtained around pH 5.2 (Fig. 1a). An increase in turbidity was observed for PGM mixed with any of the three chitosan test solutions, indicating a formation of a mucin–chitosan complex. The course of the titration curves was not dependent on the chain length of chitosan. At approximately pH 5, a turbidity maximum for all chitosan grades was reached. The turbidity of pure CS, CM, and CL solutions (control) was negligible. The turbidity of the control PGM dispersion showed a different curve track, with an increasing turbidity with decreasing pH, indicating an increase in particle size at low pH.
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Fig. 1. (a) Absorbance (400 nm) as a function of pH for 0.5 mg/mL PGM (), or 0.5 mg/mL PGM with 0.15 mg/mL CS (䊉), CM (), or CL (). (b) The -absorbance of CS (䊉), CM (), or CL () obtained by subtracting the absorbance of PGM from the absorbance of the PGM–chitosan complex (n = 2 ± mean difference).
This is consistent with the observation that PGM forms large aggregates at low pH (Bhaskar et al., 1991). In order to isolate the degree of interaction between chitosan and PGM, -absorbance curves of the three chitosan test solutions (Fig. 1b) were constructed by subtracting the absorbance of PGM from the absorbance values of the PGM–chitosan complexes. For a non-interacting system, the -absorbance upon mixing would be zero, while an increase in absorbance is an indirect quantitative measure of interaction (He et al., 1998). A pH optimum for the reaction between chitosan and PGM exist for all three chitosan grades. All three curves were fitted with a polynomial function of second degree (r2 valued 0.98, 0.99 and 0.99 for CS, CM and CL, respectively). By differentiating the function of the polynomial fit and solving the function y = 0, the exact pH optimum for each chitosan grade was calculated to be 5.01, 5.16, and 5.31 for CS, CM, and CL, respectively. In this pH range (5.0–5.3), mucin will have an overall negative charge due to the isoelectric point of 2–3 (Lee et al., 2005). Also, the sialic acid residues positioned on the oligosaccharide side chains will be fully ionized at pH 5.2 due to its pKa of 2.6 (Salamat-Miller et al., 2005). Likewise, a major proportion of the glucosamine residues of chitosan will carry a positive charge due to the pKa of 6.5 (Illum, 1998). However, the degree of ionization of the glucosamine residues would be even greater at, e.g. pH 4.9, due to the higher proportion of positively charged glucosamine residues, while the ionization of sialic acid residues of PGM remain unchanged. Therefore, it would have been expected that the optimal pH of interaction between chitosan and mucin would be at a slightly lower pH. The decreased interaction at pH < 5 could be explained by a reduced number of binding sites for chitosan due to aggregation of PGM. The decrease of turbidity at pH > 5 could be caused by the deprotonation of chitosan, thus reducing the ionization and thereby the ability to interact electrostatically with PGM. Another reason could be a decrease in particle size and thus a reduction in absorbance.
Fig. 2. Energy (J) as a function of the total concentration of sugar units (mM) in CM. Representative binding curves of CM and PGM at pH 5.2 () and pH 6.3 (䊉) for the titration of CM into a PGM solution.
molecular weight of 192 g/mol and assuming 70% degree of deacylation (manufacturer specifications). Fig. 2 shows representative binding curves of CM and PGM at pH 6.3 and 5.2. As can be seen, the interaction seems to happen in two steps at both pH 5.2 and pH 6.3, indicating two different modes for PGM binding to CM. Therefore, it was chosen to fit each step separately. As can be seen from Fig. 3, the apparent association constant (Kapp ) is significantly greater at pH 5.2 compared to pH 6.3 at both steps 1 and 2. The increase in affinity at pH 5.2 compared to pH 6.3 could, as shown using CCM, be explained by electrostatic
3.2. Binding interactions measured by ITC ITC allows for direct measurements of thermodynamic parameters during the course of binding interactions between molecules. It is therefore useful for studying whether the observed increase in turbidity at pH 5.2 is correlated with a higher affinity of chitosan to PGM compared to higher pH. Further, to study the mechanism of the interaction between PGM and chitosan. Because of insufficient solubility of chitosan at pH 6.8, which matches the pH of the human saliva most (Bardow et al., 2000), the affinity between chitosan and PGM was studied at pH 6.3 instead. The calculated thermodynamic parameters were derived using the mean value of molecular weight of PGM and since CM has a broad chain length range, the concentration of CM was calculated as the total concentration of sugar units using an average
Fig. 3. Apparent association constant (Kapp ) for the 1st and 2nd step of the titration curves for the interaction between chitosan and porcine gastic mucin at pH 5.2 (white) and pH 6.3 (pattern) by isothermal titration (mean ± SD, n = 4–5). *Significant difference (p < 0.05) between treatments.
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Table 2 Changes of enthalpy (H), entropy (S) and stoichiometry (n), for the interaction between chitosan (CM) and porcine gastic mucin at pH 5.2 and pH 6.3 by isothermal titration (mean ± SD, n = 4–5). Step
H (kJ/mol)
pH 5.2 1st step pH 5.2 2nd step pH 6.3 1st step pH 6.3 2nd step
1.66 6.63 3.62 9.73
± ± ± ±
0.133 0.430 1.24 0.222
S (J/mol K) 123 137 121 134
± ± ± ±
5.03 1.61 6.02 1.43
n (mol) 353 660 369 851
± ± ± ±
37.1 43.3 91.7 130
interactions between the positively charged glucosamine residues on CM and the negatively charged sialic acid residues on PGM, due to the pKa value of the glucosamine residues (around 6.5). Table 2 shows the changes in enthalpy and entropy associated with the reaction at pH 5.2 and pH 6.3. The data suggests that the reaction between CM and PGM is entropy driven, as H is positive and TS is much larger than H. An enthalpy driven reaction between CM and PGM could have been expected, as complex formation is usually connected to a loss in entropy (Ben-Tal et al., 2000). Therefore, the mechanism behind the interaction could be due to excluded volume or depletion interaction, where the reaction is dominantly driven by entropy (de Kruif and Tuinier, 2001). In the literature, contradictory claims regarding complex coacervation is an enthalpy or entropy driven reaction exist: the process could be entropy driven due to the liberation of counter ions and water molecules, but could also be enthalpy driven due to the decrease of electrostatically free energy (Turgeon et al., 2003). To the best of our knowledge, the interaction between chitosan and mucin has not before been studied using ITC. The reaction between chitosan and xanthan gum, a negatively charged polysaccharide, has been studied by ITC, and also showed, that the reaction was primarily entropy driven. No pH-dependency of the reaction was studied (Maurstad et al., 2012). 3.3. Tensile detachment model Evaluation of mucoadhesive properties is fundamental in order to choose the optimal composition of a mucoadhesive drug delivery system. The measured total work of adhesion gives information about the strength of the resulting combined mucin–chitosan complex (Edsman and Hagerstrom, 2005). TDM measures the work contributing from wetting at the initial stage to electrostatic interactions and diffusion of polymer chains at the consolidation stage. The effect of lowering pH on the mucoadhesive properties was studied utilizing a texture analyzer to measure the total work of adhesion. The total work of adhesion required to detach chitosan discs from an artificial mucosal surface wetted with and without mucin is shown in Fig. 4. Each chitosan chain length displayed significantly higher total work of adhesion at pH 5.2 compared to pH 6.8, but no difference was observed in the total work of adhesion between chitosan chain lengths. The control study without PGM showed that the majority of the total work of adhesion of chitosan discs was due to the interactions between chitosan and PGM as the total work of adhesion ranged from 0.3 to 0.4 mN, whereas the control study showed a total work of adhesion of approximately 10 times lower. The significantly increased total work of adhesion for pH 5.2 compared with pH 6.8 is in agreement with the data from the CCM and ITC experiments. This relationship was also seen when no mucin was present, which could be due to the higher solubility of chitosan at this pH, which could form a gel layer, thus facilitating adhesion (Mahrag Tur and Ch’ng, 1998). However, when comparing the moist surface with the mucosal surface, it is evident that these non-specific interactions are negligible. The measured total work of adhesion is the sum of all the forces between the chitosan discs and the moisten surface. Therefore, the TDM proved that rate
Fig. 4. Tensile detachment measurement displaying the total work of adhesion for removing chitosan discs (CS, CM, CL) from an artificial mucosal surface wetted with buffer with or without mucin at pH 5.2 (white) and pH 6.8 (pattern). Mean ± SD (n = 3–4). *Significant difference (p < 0.05) between treatments.
and degree of hydration, swelling, and entanglement are contributing factors in conjunction with electrostatic interactions for the adhesion to take place (Smart, 2005). Pilot studies were performed using fresh or pre-frozen porcine buccal mucosa, the epithelium was either manually or heat separated (data not shown). However, standard deviations were too large to draw any clear conclusions regarding the effect of pH on the chitosan–mucin interaction. This could be owing to the biological variation of the mucosal membranes (Pongjanyakul et al., 2013), as the buccal epithelium is a very elastic tissue composed of 20–40 cell layers forming a papillary structure. The used artificial mucosal surface with PGM has a limitation, as impact of full entanglement may not be examined. The degree of chain entanglement presumably only happens to a small degree using moisten adsorbent paper as this artificial mucosal surface has a lower thickness compared to native mucus layer in conjunction with surface bound mucins. Correlation of increasing chitosan chain length and an increase in mucoadhesive strength has been found previously (Lehr et al., 1992). However, this correlation was not observed in this experiment. One explanation could be that the range of polymer chain length in this study was rather large, e.g. CS had the range 50–1000 and therefore overlapping the range of both CM and CL with ranges of 100–2000 and 500–5000, respectively. Therefore, chitosan could exhibit a threshold of chain length dependent mucoadhesive strength. With increasing chain length, the polymer can impair the diffusion and penetration, thus no increase in mucoadhesion can be detected (Gurny et al., 1984). 4. Conclusions The mechanisms behind the mucoadhesive properties of chitosan were studied using a CCM, ITC, and TDM. A pH dependent interaction between chitosan and PGM was confirmed in this study by these three complementing techniques. The CCM showed an increase in interaction between PGM and CS, CM, and CL at pH 5.2, suggesting electrostatic interactions between chitosan and PGM. The ITC studies confirmed this pH-dependent binding between chitosan and PGM, and showed that the reaction is entropy driven. The TDM showed that pH, but not chitosan chain length was a significant factor for mechanical strength of mucoadhesion, and that hydration and swelling also are contributing factors. By understanding the mechanisms behind the chitosan–mucin interactions, it is possible to rationally optimize buccal mucoadhesive formulations.
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Klemetsrud, T., Jonassen, H., Hiorth, M., Kjoniksen, A.L., Smistad, G., 2013. Studies on pectin-coated liposomes and their interaction with mucin. Colloid Surface B 103, 158–165. Lee, S., Muller, M., Rezwan, K., Spencer, N.D., 2005. Porcine gastric mucin (PGM) at the water/poly(dimethylsiloxane) (PDMS) interface: influence of pH and ionic strength on its conformation, adsorption, and aqueous lubrication properties. Langmuir 21, 8344–8353. Lehr, C.-M., Bouwstra, J.A., Schacht, E.H., Junginger, H.E., 1992. In vitro evaluation of mucoadhesive properties of chitosan and some other natural polymers. Int. J. Pharm. 78, 43–48. Liu, S.H., Cao, Y.L., Ghosh, S., Rousseau, D., Low, N.H., Nickerson, M.T., 2010. Intermolecular interactions during complex coacervation of pea protein isolate and gum arabic. J. Agric. Food. Chem. 58, 552–556. Liu, W.G., Sun, S.J., Cao, Z.Q., Xin, Z., Yao, K.D., Lu, W.W., Luk, K.D.K., 2005. An investigation on the physicochemical properties of chitosan/DNA polyelectrolyte complexes. Biomaterials 26, 2705–2711. Mahrag Tur, K., Ch’ng, H.-S., 1998. Evaluation of possible mechanism(s) of bioadhesion. Int. J. Pharm. 160, 61–74. Maurstad, G., Kitamura, S., Stokke, B.T., 2012. Isothermal titration calorimetry study of the polyelectrolyte complexation of xanthan and chitosan samples of different degree of polymerization. Biopolymers 97, 1–10. Patel, V.F., Liu, F., Brown, M.B., 2012. Modeling the oral cavity: in vitro and in vivo evaluations of buccal drug delivery systems. J. Control Release 161, 746–756. Phillipson, M., Johansson, M.E.V., Henriksnas, J., Petersson, J., Gendler, S.J., Sandler, S., Persson, A.E.G., Hansson, G.C., Holm, L., 2008. The gastric mucus layers: constituents and regulation of accumulation. Am. J. Physiol. Gastrointest. Liver Physiol. 295, G806–G812. Pongjanyakul, T., Khunawattanakul, W., Strachan, C.J., Gordon, K.C., Puttipipatkhachorn, S., Rades, T., 2013. Characterization of chitosan–magnesium aluminum silicate nanocomposite films for buccal delivery of nicotine. Int. J. Biol. Macromol. 55, 24–31. Qaqish, R.B., Amiji, M.M., 1999. Synthesis of a fluorescent chitosan derivative and its application for the study of chitosan–mucin interactions. Carbohyd. Polym. 38, 99–107. Salamat-Miller, N., Chittchang, M., Johnston, T.P., 2005. The use of mucoadhesive polymers in buccal drug delivery. Adv. Drug Deliv. Rev. 57, 1666–1691. Sandberg, T., Blom, H., Caldwell, K.D., 2009. Potential use of mucins as biomaterial coatings. I. Fractionation, characterization, and model adsorption of bovine, porcine, and human mucins. J. Biomed. Mater. Res. A 91A, 762–772. Sander, C., Madsen, K.D., Hyrup, B., Nielsen, H.M., Rantanen, J., Jacobsen, J., 2013. Characterization of spray dried bioadhesive metformin microparticles for oromucosal administration. Eur. J. Pharm. Biopharm. 85, 682–688. Schenkels, L.C.P.M., Veerman, E.C.I., Amerongen, A.V.N., 1995. Biochemicalcomposition of human saliva in relation to other mucosal fluids. Crit. Rev. Oral Biol. Med. 6, 161–175. Sigurdsson, H.H., Loftsson, T., Lehr, C.M., 2006. Assessment of mucoadhesion by a resonant mirror biosensor. Int. J. Pharm. 325, 75–81. Smart, J.D., 2005. The basics and underlying mechanisms of mucoadhesion. Adv. Drug Deliv. Rev. 57, 1556–1568. Sogias, I.A., Williams, A.C., Khutoryanskiy, V.V., 2008. Why is chitosan mucoadhesive? Biomacromolecules 9, 1837–1842. Teubl, B.J., Absenger, M., Frohlich, E., Leitinger, G., Zimmer, A., Roblegg, E., 2013. The oral cavity as a biological barrier system: design of an advanced buccal in vitro permeability model. Eur. J. Pharm. Biopharm. 84, 386–393. Turgeon, S.L., Beaulieu, M., Schmitt, C., Sanchez, C., 2003. Protein-polysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects. Curr. Opin. Colloid Interface Sci. 8, 401–414.
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
A mechanistic based approach for enhancing buccal mucoadhesion of chitosan Emil Meng-Lund 1 , Christian Muff-Westergaard 1 , Camilla Sander, Peter Madelung, Jette Jacobsen ∗ Section for Pharmaceutical Design and Drug Delivery, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
a r t i c l e
i n f o
Article history: Received 13 September 2013 Received in revised form 24 October 2013 Accepted 28 October 2013 Available online 27 November 2013 Keywords: Buccal mucoadhesion Chitosan Mucin Complex coacervation Isothermal titration calorimetry Texture analyzer
a b s t r a c t Mucoadhesive buccal drug delivery systems can enhance rapid drug absorption by providing an increased retention time at the site of absorption and a steep concentration gradient. An understanding of the mechanisms behind mucoadhesion of polymers, e.g. chitosan, is necessary for improving the mucoadhesiveness of buccal formulations. The interaction between chitosan of different chain lengths and porcine gastric mucin (PGM) was studied using a complex coacervation model (CCM), isothermal titration calorimetry (ITC) and a tensile detachment model (TDM). The effect of pH was assessed in all three models and the approach to add a buffer to chitosan based drug delivery systems is a means to optimize and enhance buccal drug absorption. The CCM demonstrated optimal interactions between chitosan and PGM at pH 5.2. The ITC experiments showed a significantly increase in affinity between chitosan and PGM at pH 5.2 compared to pH 6.3 and that the interactions were entropy driven. The TDM showed a significantly increase in strength of adhesion between chitosan discs and an artificial mucosal surface at pH 5.2 compared to pH 6.8, addition of PGM increased the total work of adhesion by a factor of 10 as compared to the wetted surface without PGM. These findings suggest that chitosan and PGM are able to interact by electrostatic interactions and by improving the conditions for electrostatic interactions, the adhesion between chitosan and PGM becomes stronger. Also, the three complementary methods were utilized to conclude the pH dependency on mucoadhesiveness. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Buccal drug administration has attracted attention as a means to achieve a fast onset and systemic delivery of drugs susceptible to the gastro intestinal or hepatic first pass metabolism (SalamatMiller et al., 2005). However, the continuous flow of saliva and swallowing reduces the amount of drug actually absorbed in the oral cavity, which is a limitation of this route of administration (Patel et al., 2012). Use of a buccal mucoadhesive formulation is an attractive approach to alleviate this limitation as provision of a prolonged high local concentration of the active pharmaceutical ingredient, will decrease the loss and even further facilitate fast absorption due to a steep concentration gradient (Haas and Lehr,
Abbreviations: PGM, porcine gastric mucin; CCM, complex coacervation model; ITC, isothermal titration calorimetry; TDM, tensile detachment model; CS, CHITOPHARM® S; CM, CHITOPHARM® M; CL, CHITOPHARM® L; MES, 2-(N-morpholino)ethanesulfonic acid; HWS, human whole saliva. ∗ Corresponding author at: Universitetsparken 2, 2100 Copenhagen, Denmark. Tel.: +45 3533 6299; fax: +45 3533 6030. E-mail address: [email protected] (J. Jacobsen). 1 Both authors contributed equally. 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.10.047
2002). Thus, lower and more available doses can be administered by the use of mucoadhesive formulations. The term mucoadhesion refers to the specific phenomenon of adhesion between a polymer and a mucus layer1−5 . The mucus layer comprise of a viscous loosely adherent layer mainly composed of mucins and water, it lines mucosal membranes, such as those found in the oral cavity (Andrews et al., 2009; Phillipson et al., 2008). The mucus layer lubricates, moistures and protects the epithelium from physical and chemical insults (Salamat-Miller et al., 2005; Schenkels et al., 1995). The ability of a polymer to attach to the mucus layer is dependent on several factors other than the formation of chemical bonding (Duchene et al., 1988). Swelling, molecular weight, and flexibility of polymer chains are all factors that have great influence on the strength and duration of adhesion (Gaserod et al., 1998; Hagesaether et al., 2009; Salamat-Miller et al., 2005). In the oral cavity, mucins are epithelial surface bound as well as solvated of salivary origin. Mucins are glycoproteins with a peptide backbone and oligosaccharide side chains, which often terminate in sialic acid residues (Salamat-Miller et al., 2005). With an isoelectric point of 2–3 (Lee et al., 2005), mucins will thus be overall negatively charged in the oral cavity, as pH is around 6.8. Chitosan is a non-toxic, biocompatible and biodegradable polysaccharide
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consisting of alternating glucosamine and N-acetyl-glucosamine units with a pKa of 6.5 (Illum, 1998; Liu et al., 2005; Sogias et al., 2008). Chitosan has received interest as a bioadhesive excipient due to its ability to interact with mucins in the mucus layer by electrostatic, hydrophobic, hydrophilic and by hydrogen bonding (Sogias et al., 2008). The following stepwise process of mucoadhesion has been proposed (Duchene et al., 1988): The initial contact stage of polymer and mucus, facilitating dehydration of the mucosa while hydration of polymer, forming a swelled polymeric network. The second step is the consolidation stage where polymer and mucin chains interdiffuse and entangle, thus facilitating non-covalent bonding and electrostatic interactions (Duchene et al., 1988; Smart, 2005). Mechanistic studies of chitosan and mucin have shown that the electrostatic interaction between the positively charged amines on the polymer chains and the negative sialic acid residues on the mucin glycoprotein play a role in the mucoadhesive properties of chitosan (He et al., 1998; Salamat-Miller et al., 2005; Sogias et al., 2008). The electrostatically driven reaction between chitosan and mucin can be interpreted as the process of complex coacervation. Complex coacervation is a process, where electrostatic interactions between a colloid suspension of a polymer and a protein will lead to the phase separation into a polymer-protein high concentration phase and polymer-protein low concentration phase (de Kruif et al., 2004). The formation of a protein-polymer rich phase will give rise to a sharp increase in turbidity of the solution. Typically, this reaction will take place in the pH range between the pKa value of the polymer and the isoelectric point of the protein with an optimum at the pH, where the molecules carry the same numeric charge (Liu et al., 2010). Thus, the maximal electrostatic interactions between chitosan and mucin should occur in the pH-range of 2–6.5. The process of complex coacervation is influenced by a number of factors, one of which is the ionic strength of the solution (Liu et al., 2010). The ionic strength influences the overall charge of the protein and polymer due to the screening effect of counter ions (Burgess, 1990). In this study, the interaction between chitosan and mucin was examined in a buffer solution resembling human saliva with regards to ionic composition and ionic strength. The aim of this study was to examine the pH-dependent interactions between chitosan polymers of increasing molecular weight and porcine gastric mucin (PGM) applying simulated oromucosal conditions. The mechanism of chitosan mucoadhesive properties was studied using complex coacervation model (CCM), isothermal titration calorimetry (ITC) and tensile detachment model (TDM).
2. Materials and methods 2.1. Materials All chemicals used were of analytical grade and were used as received unless otherwise described. Acetic acid (glacial), calcium chloride dihydrate, sodium chloride, sodium hydrogen carbonate and sodium hydroxide of analytical grade were purchased from Merck (Darmstadt, Germany). CHITOPHARM® L (CL) (MW 500–5000 kDa), CHITOPHARM® M (CM) (MW 100–2000 kDa), and CHITOPHARM® S (CS) (MW 50–1000 kDa) were provided as a free sample from Cognis GmbH (Monheim, Germany). 2(N-morpholino)ethanesulfonic acid (MES), potassium chloride, sodium phosphate dibasic, sodium phosphate monobasic and Type II porcine gastric mucin (PGM) purified from porcine stomach mucosa were all purchased from Sigma–Aldrich (St. Louis, MO). Deionized water was obtained from Millipore Milli-Q Ultrapure Water purification system (Billeria, MA).
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2.2. Complex coacervation model (CCM) The electrostatic interactions between chitosan (S, M and L) and PGM were studied by complex coacervate formation. A buffered electrolyte solution (pH 6.5) mimicking human whole saliva (HWS buffer) was prepared in accordance to (Gaserod et al., 1998):210 mg/L NaHCO3 , 430 mg/L NaCl, 750 mg/L KCl, 220 mg/L CaCl2 2H2 O and 910 mg/L NaH2 PO4 in deionized water. A stock solution of 2 mg/mL PGM was prepared by solvating PGM in HWS buffer at 5 ◦ C overnight to ensure complete solvation. Chitosan (CS, CM and CL) stock solutions of 1 mg/mL were prepared by dissolving chitosan in HWS buffer. Test solutions containing 0.5 mg/mL PGM in HWS buffer with 0.15 mg/mL CS, CM, or CL was prepared by mixing chitosan stock solutions and PGM stock solution and diluting with HWS. The control solution contained 0.5 mg/mL PGM in HWS buffer. The test solution (200 mL) was titrated at room temperature with 0.2 M HCl solution using a 842 Titrando titrator fitted with a Unitrode pH electrode with Pt1000 temperature sensor, a Dosino 10 mL dosing unit and a rod stirrer with a 20 mm wide propeller (Metrohm AG, Herisau, Switzerland). Tiamo software version 1.1 was used for controlling the titration as well as acquiring and analyzing the results. The titrations were performed in the pH range 6.5–2.7 and a sample (2.0 mL) was withdrawn for each 0.2 pH interval after reaching pH equilibrium. Sample turbidity was determined at a wavelength of 400 nm (Cary 100 UV, Agilent Technologies Denmark, Horsholm, Denmark) using the WinUV software. All samples were measured using disposable 1.5 mL plastic cuvettes from Brandtech Scientific Inc. (Essex, CT). The absorbance was zeroed against HWS buffer and the dilution effect of the titration was taken into account.
2.3. Isothermal titration calorimetry (ITC) The binding interactions involved in the mucoadhesive process were studied using ITC. To ensure uniform ionic strength, all test solutions were dialyzed. The test solutions consisted of 0.500 mg/mL PGM at pH 5.2 (0.0125 M acetic acid) and pH 6.3 (0.016 M MES). The solutions were dialyzed using dialysis bags with a cut-off of 12 kDa (Sigma, St. Louis) and were dialyzed against an excess of either acetic acid or MES buffer. The two different buffer concentrations ensured similar ionic strength of the buffer solutions. The dialysis fluid was changed seven times during approximately 20 h and was stable at the desired pH values after the fourth change of dialysis fluid. Before titration analysis, all samples were thoroughly degassed in vacuum, for a minimum of 30 min, under rapid magnetic stirring. Titration of 0.500 mg/mL PGM at pH 5.2 and pH 6.3 with 0.600 mg/mL CM were conducted on a NanoITC 2G, TA Instruments (Centennial Park, United Kingdom). Initially the reaction cell was rinsed thoroughly by flushing with degassed deionized water followed by PGM test solution. The CM solution to be tested was loaded in a 250 L syringe, which was carefully evacuated of air. The propeller was gently wiped clean of residual CM solution. The syringe was set to a propeller speed of 250 rpm, and the temperature in the reaction cell was 25.000◦ C. Each titration consisted of initial 3.15 L (i.e. lowest injection volume to omit residual air) and followed by 37 consecutive 5.15 L injections. The peak resulting from the first injection was subsequently ignored. All ITC data were analyzed using the NanoAnalyze Data Analysis software, version 2.3.6 (TA Instruments) by splitting the area of the heat peaks into two distinct curves and fitting the area of the heat peak of each curve to an independent site model, deriving a value for the affinity constant (K), the binding stoichiometry (n) and the enthalpy change (H). The amount of single sugar units of CM was calculated and used as molar concentration, assuming a degree of deacetylation of 70%. The molecular
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Table 1 Composition of chitosan liquid feeds for spray drying.
under the distance–force curve using the Exponent 32 program version 5.1.1 (Stable Micro Systems LTD, Godalming, UK).
Excipients
CS feed
CM feed
CL feed
Chitosan quality Deionized water (mL) HCl, 2.4 M (mL)
5 g CS 163 4.17
5 g CM 244 6.25
5 g CL 325 8.33
weight of PGM used for calculations was 1.25 × 106 Da (Jumel et al., 1996). 2.4. Tensile detachment model (TDM) 2.4.1. Preparation of chitosan discs The composition of chitosan liquid feeds is displayed in Table 1. The steady state flow viscosity of the feeds was measured as previously described (Sander et al., 2013) and was shown to be similar at shear rates in the range of 500–1000 s−1 . Chitosan was hydrated in water for 10 min before addition of HCl. Solutions were stirred overnight, allowing complete solvation. Spray dried chitosan particles were prepared from chitosan liquid feeds, using a Büchi Mini Spray Dryer B-290 (Büchi Labortechnik AG, Germany). Initially, the system was heated for approximately 30 min to reach an inlet air temperature of 200◦ C which lead to an outlet air temperature of 84–101◦ C. Atomizing air was set to 40 mm Hg and the nozzle was set to clean every 2 min using an automatic vacuum pump. A pump rate of 3 mL/min which corresponds to 10% of the capacity of the equipment was used together with an aspirator rate of 32 m3 /h, corresponding to 80% capacity of the equipment. The water content of the spray dried chitosan particles was measured by thermo gravimetric analysis (TAC 7/DX, PerkinElmer Inc., Waltham, MA). Samples of 5 mg spray dried chitosan particles were heated to 130◦ C for 30 min and water content was calculated as percentage weight loss to be approximately 10% (w/w). No difference in water content was found between the chitosan qualities. All discs were made from spray-dried chitosan-HCl particles of CS, CM, or CL (see above). Prior to the experiment, chitosan discs (diameter 11.3 mm) weighing approximately 50 mg were prepared using a hydraulic press (PerkinElmer Inc., Waltham, MA) with a pressure of 2 bar for 10 s. 2.4.2. Preparation of the artificial mucosal surface The preparation of the artificial mucosal surface was modified from (Hagesaether et al., 2009). An adsorbing paper with an inert backing layer (Bench surface protector VWR, Herlev, Denmark) was cut into pieces of 2 cm × 2 cm. The paper was fixed to a metal base surface with double adhesive tape and wetted with 90 L buffer solution (0.25 M acetic acid buffer pH 5.2 or 0.25 M phosphate buffer pH 6.8), which was allowed to soak into the paper for one min before the test was carried out. The adhesiveness of the disk to adsorbing paper was tested using buffers solutions both with 3% PGM and without (control). 2.4.3. Tensile detachment measurements Mechanical strength of the mucoadhesive bonds between compressed discs of chitosan (CS, CM or CL) and an artificial mucosal surface was tested at pH 5.2 and 6.8 using a texture analyzer (Texture Analyzer TX-XT plus, TA Instruments, UK) equipped with a 30 kg load cell. A disk of CS, CM or CL was fixated with double adhesive tape to the cylindrical probe of the Texture Analyzer and brought to contact with the artificial mucosal surface using pretest speed 0.5 mm/s, test speed 0.5 mm/s, post-test speed 5 mm/s, applied force 2000 mN, return distance of 20 mm and a contact time between disk and mucosal surface was 60 s. The force required to separate the chitosan disk from the artificial mucosal surface was recorded and total work of adhesion was computed as the area
2.5. Statistical analysis All data are presented as mean ± SD, unless otherwise stated. GraphPad Prism for Windows, version 6.00 from GraphPad Software Inc. (La Jolla, CA) was used for the statistical calculations. A one way analysis of variance for multiple comparisons was followed by a Student–Neuman–Keuls test. Two sided p-values below 5% (p < 0.05) were considered statistically significant. 3. Results and discussion The mechanism of mucoadhesion has not been fully elucidated and depends to a large extend on whether it is a solid, semi-solid or liquid formulation (Salamat-Miller et al., 2005). It is evident from literature that the method used for assessing the pH-dependency of the interaction between chitosan and mucin has a profound effect on the obtained results. Different techniques may focus on different stages of the mucoadhesive process leading to inconsistent results. 3.1. Complex coacervation model Methods for assessing the mucoadhesive properties of different polymers using either change in turbidity, particle size or zeta potential have previously been reported (He et al., 1998; Klemetsrud et al., 2013; Sogias et al., 2008). These methods could prove useful when comparing the mucoadhesive potential of either excipients or finished formulations, due to the high inaccuracy often found using more direct methods (Pongjanyakul et al., 2013). However, the validity of these methods needs to be established. The pH-dependency of the interaction between chitosan and mucin has been studied by a number of different methods, giving rise to several conflicting conclusions. A study using a fluorescence method has shown an increased interaction between chitosan and bovine submaxillary mucin (BSM) at more acidic pH values, highest degree of interaction at pH 2.9 (Qaqish and Amiji, 1999). Another study, using surface plasmon resonance, have shown an increase in interaction at pH 6 between chitosan and BSM, while yet another study have shown an increase in interaction between chitosan and BSM above pH 7 (Sigurdsson et al., 2006). PGM and BSM have been used as a model for human mucins when testing mucoadhesive properties and are different in terms of solubility and structure (Klemetsrud et al., 2013). PGM was preferred over BSM in this study as Teubl et al. showed that human mucin exhibits more similar chemical and morphological structures to PGM over BSM as determined by Fourier transform infrared spectroscopy and scanning electron microscopy, respectively (Teubl et al., 2013). For comparative studies, Sandberg et al. showed the importance of standardizing mucin, as significant batch to batch variation was observed for both PGM and BSM with respect to structure and mucin adsorption (Sandberg et al., 2009). Therefore, single batch PGM was used for the studies presented. The CCM showed that the interaction between PGM and CS, CM or CL was pH dependent and an optimal interaction was obtained around pH 5.2 (Fig. 1a). An increase in turbidity was observed for PGM mixed with any of the three chitosan test solutions, indicating a formation of a mucin–chitosan complex. The course of the titration curves was not dependent on the chain length of chitosan. At approximately pH 5, a turbidity maximum for all chitosan grades was reached. The turbidity of pure CS, CM, and CL solutions (control) was negligible. The turbidity of the control PGM dispersion showed a different curve track, with an increasing turbidity with decreasing pH, indicating an increase in particle size at low pH.
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Fig. 1. (a) Absorbance (400 nm) as a function of pH for 0.5 mg/mL PGM (), or 0.5 mg/mL PGM with 0.15 mg/mL CS (䊉), CM (), or CL (). (b) The -absorbance of CS (䊉), CM (), or CL () obtained by subtracting the absorbance of PGM from the absorbance of the PGM–chitosan complex (n = 2 ± mean difference).
This is consistent with the observation that PGM forms large aggregates at low pH (Bhaskar et al., 1991). In order to isolate the degree of interaction between chitosan and PGM, -absorbance curves of the three chitosan test solutions (Fig. 1b) were constructed by subtracting the absorbance of PGM from the absorbance values of the PGM–chitosan complexes. For a non-interacting system, the -absorbance upon mixing would be zero, while an increase in absorbance is an indirect quantitative measure of interaction (He et al., 1998). A pH optimum for the reaction between chitosan and PGM exist for all three chitosan grades. All three curves were fitted with a polynomial function of second degree (r2 valued 0.98, 0.99 and 0.99 for CS, CM and CL, respectively). By differentiating the function of the polynomial fit and solving the function y = 0, the exact pH optimum for each chitosan grade was calculated to be 5.01, 5.16, and 5.31 for CS, CM, and CL, respectively. In this pH range (5.0–5.3), mucin will have an overall negative charge due to the isoelectric point of 2–3 (Lee et al., 2005). Also, the sialic acid residues positioned on the oligosaccharide side chains will be fully ionized at pH 5.2 due to its pKa of 2.6 (Salamat-Miller et al., 2005). Likewise, a major proportion of the glucosamine residues of chitosan will carry a positive charge due to the pKa of 6.5 (Illum, 1998). However, the degree of ionization of the glucosamine residues would be even greater at, e.g. pH 4.9, due to the higher proportion of positively charged glucosamine residues, while the ionization of sialic acid residues of PGM remain unchanged. Therefore, it would have been expected that the optimal pH of interaction between chitosan and mucin would be at a slightly lower pH. The decreased interaction at pH < 5 could be explained by a reduced number of binding sites for chitosan due to aggregation of PGM. The decrease of turbidity at pH > 5 could be caused by the deprotonation of chitosan, thus reducing the ionization and thereby the ability to interact electrostatically with PGM. Another reason could be a decrease in particle size and thus a reduction in absorbance.
Fig. 2. Energy (J) as a function of the total concentration of sugar units (mM) in CM. Representative binding curves of CM and PGM at pH 5.2 () and pH 6.3 (䊉) for the titration of CM into a PGM solution.
molecular weight of 192 g/mol and assuming 70% degree of deacylation (manufacturer specifications). Fig. 2 shows representative binding curves of CM and PGM at pH 6.3 and 5.2. As can be seen, the interaction seems to happen in two steps at both pH 5.2 and pH 6.3, indicating two different modes for PGM binding to CM. Therefore, it was chosen to fit each step separately. As can be seen from Fig. 3, the apparent association constant (Kapp ) is significantly greater at pH 5.2 compared to pH 6.3 at both steps 1 and 2. The increase in affinity at pH 5.2 compared to pH 6.3 could, as shown using CCM, be explained by electrostatic
3.2. Binding interactions measured by ITC ITC allows for direct measurements of thermodynamic parameters during the course of binding interactions between molecules. It is therefore useful for studying whether the observed increase in turbidity at pH 5.2 is correlated with a higher affinity of chitosan to PGM compared to higher pH. Further, to study the mechanism of the interaction between PGM and chitosan. Because of insufficient solubility of chitosan at pH 6.8, which matches the pH of the human saliva most (Bardow et al., 2000), the affinity between chitosan and PGM was studied at pH 6.3 instead. The calculated thermodynamic parameters were derived using the mean value of molecular weight of PGM and since CM has a broad chain length range, the concentration of CM was calculated as the total concentration of sugar units using an average
Fig. 3. Apparent association constant (Kapp ) for the 1st and 2nd step of the titration curves for the interaction between chitosan and porcine gastic mucin at pH 5.2 (white) and pH 6.3 (pattern) by isothermal titration (mean ± SD, n = 4–5). *Significant difference (p < 0.05) between treatments.
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Table 2 Changes of enthalpy (H), entropy (S) and stoichiometry (n), for the interaction between chitosan (CM) and porcine gastic mucin at pH 5.2 and pH 6.3 by isothermal titration (mean ± SD, n = 4–5). Step
H (kJ/mol)
pH 5.2 1st step pH 5.2 2nd step pH 6.3 1st step pH 6.3 2nd step
1.66 6.63 3.62 9.73
± ± ± ±
0.133 0.430 1.24 0.222
S (J/mol K) 123 137 121 134
± ± ± ±
5.03 1.61 6.02 1.43
n (mol) 353 660 369 851
± ± ± ±
37.1 43.3 91.7 130
interactions between the positively charged glucosamine residues on CM and the negatively charged sialic acid residues on PGM, due to the pKa value of the glucosamine residues (around 6.5). Table 2 shows the changes in enthalpy and entropy associated with the reaction at pH 5.2 and pH 6.3. The data suggests that the reaction between CM and PGM is entropy driven, as H is positive and TS is much larger than H. An enthalpy driven reaction between CM and PGM could have been expected, as complex formation is usually connected to a loss in entropy (Ben-Tal et al., 2000). Therefore, the mechanism behind the interaction could be due to excluded volume or depletion interaction, where the reaction is dominantly driven by entropy (de Kruif and Tuinier, 2001). In the literature, contradictory claims regarding complex coacervation is an enthalpy or entropy driven reaction exist: the process could be entropy driven due to the liberation of counter ions and water molecules, but could also be enthalpy driven due to the decrease of electrostatically free energy (Turgeon et al., 2003). To the best of our knowledge, the interaction between chitosan and mucin has not before been studied using ITC. The reaction between chitosan and xanthan gum, a negatively charged polysaccharide, has been studied by ITC, and also showed, that the reaction was primarily entropy driven. No pH-dependency of the reaction was studied (Maurstad et al., 2012). 3.3. Tensile detachment model Evaluation of mucoadhesive properties is fundamental in order to choose the optimal composition of a mucoadhesive drug delivery system. The measured total work of adhesion gives information about the strength of the resulting combined mucin–chitosan complex (Edsman and Hagerstrom, 2005). TDM measures the work contributing from wetting at the initial stage to electrostatic interactions and diffusion of polymer chains at the consolidation stage. The effect of lowering pH on the mucoadhesive properties was studied utilizing a texture analyzer to measure the total work of adhesion. The total work of adhesion required to detach chitosan discs from an artificial mucosal surface wetted with and without mucin is shown in Fig. 4. Each chitosan chain length displayed significantly higher total work of adhesion at pH 5.2 compared to pH 6.8, but no difference was observed in the total work of adhesion between chitosan chain lengths. The control study without PGM showed that the majority of the total work of adhesion of chitosan discs was due to the interactions between chitosan and PGM as the total work of adhesion ranged from 0.3 to 0.4 mN, whereas the control study showed a total work of adhesion of approximately 10 times lower. The significantly increased total work of adhesion for pH 5.2 compared with pH 6.8 is in agreement with the data from the CCM and ITC experiments. This relationship was also seen when no mucin was present, which could be due to the higher solubility of chitosan at this pH, which could form a gel layer, thus facilitating adhesion (Mahrag Tur and Ch’ng, 1998). However, when comparing the moist surface with the mucosal surface, it is evident that these non-specific interactions are negligible. The measured total work of adhesion is the sum of all the forces between the chitosan discs and the moisten surface. Therefore, the TDM proved that rate
Fig. 4. Tensile detachment measurement displaying the total work of adhesion for removing chitosan discs (CS, CM, CL) from an artificial mucosal surface wetted with buffer with or without mucin at pH 5.2 (white) and pH 6.8 (pattern). Mean ± SD (n = 3–4). *Significant difference (p < 0.05) between treatments.
and degree of hydration, swelling, and entanglement are contributing factors in conjunction with electrostatic interactions for the adhesion to take place (Smart, 2005). Pilot studies were performed using fresh or pre-frozen porcine buccal mucosa, the epithelium was either manually or heat separated (data not shown). However, standard deviations were too large to draw any clear conclusions regarding the effect of pH on the chitosan–mucin interaction. This could be owing to the biological variation of the mucosal membranes (Pongjanyakul et al., 2013), as the buccal epithelium is a very elastic tissue composed of 20–40 cell layers forming a papillary structure. The used artificial mucosal surface with PGM has a limitation, as impact of full entanglement may not be examined. The degree of chain entanglement presumably only happens to a small degree using moisten adsorbent paper as this artificial mucosal surface has a lower thickness compared to native mucus layer in conjunction with surface bound mucins. Correlation of increasing chitosan chain length and an increase in mucoadhesive strength has been found previously (Lehr et al., 1992). However, this correlation was not observed in this experiment. One explanation could be that the range of polymer chain length in this study was rather large, e.g. CS had the range 50–1000 and therefore overlapping the range of both CM and CL with ranges of 100–2000 and 500–5000, respectively. Therefore, chitosan could exhibit a threshold of chain length dependent mucoadhesive strength. With increasing chain length, the polymer can impair the diffusion and penetration, thus no increase in mucoadhesion can be detected (Gurny et al., 1984). 4. Conclusions The mechanisms behind the mucoadhesive properties of chitosan were studied using a CCM, ITC, and TDM. A pH dependent interaction between chitosan and PGM was confirmed in this study by these three complementing techniques. The CCM showed an increase in interaction between PGM and CS, CM, and CL at pH 5.2, suggesting electrostatic interactions between chitosan and PGM. The ITC studies confirmed this pH-dependent binding between chitosan and PGM, and showed that the reaction is entropy driven. The TDM showed that pH, but not chitosan chain length was a significant factor for mechanical strength of mucoadhesion, and that hydration and swelling also are contributing factors. By understanding the mechanisms behind the chitosan–mucin interactions, it is possible to rationally optimize buccal mucoadhesive formulations.
E. Meng-Lund et al. / International Journal of Pharmaceutics 461 (2014) 280–285
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