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6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems
J. DRUG DEL. SCI. TECH., 20 (3) 181-186 2010
6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems G. Millotti1, H. Hoyer1, J.F.J. Engbersen2, A. Bernkop-Schnürch1* Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University, Innrain 52c, Josef Möller Haus, A-6020 Innsbruck, Austria 2 Department of Biomedical Chemistry, Faculty of Science and Technology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands *Correspondence: [email protected] 1
Among mucoadhesive polymers, thiolated polymers showed to be superior over non-thiolated polymers. Recently a novel thiolated chitosan named chitosan-6-mercaptonicotinamide has been generated. Its heteroaromatic structure confers a pH-independent reactivity to the thiol group and introduces a hydrophobic entity in the hydrophilic polymer. Within this study the potential of this novel polymer as excipient for drug delivery systems was evaluated with a special focus on mucoadhesion. The highest coupling degree was 975 µmol thiol groups in reduced form per gram of polymer. Mucoadhesion was increased by at least 69-fold compared to unmodified chitosan. This chemical modification of chitosan, altered the water uptake of the polymer by lowering it up to 75 %. The new polymer was effective in stabilizing a matrix tablet compared to an unmodified chitosan tablet. Cumulative release studies from a matrix tablet comprising the newly synthesized chitosan showed controlled release of a hydrophilic macromolecular model compound (FD4). Furthermore, the polymer showed improved biodegradability. These advantageous features render chitosan-6-mercaptonicotinic acid a useful excipient for various drug delivery systems Key words: Thiomers – Mucoadhesion – Drug release – Swelling properties – Biodegradability.
The potential of many drug delivery systems can be improved by mucoadhesive polymers. By prolonging the residence time of dosage forms at the site of drug absorption, the bioavailability of the drug can be increased and the frequency of administration lowered [1]. Mucoadhesive properties of polymers have been extensively studied. Chitosan, for instance, is a cationic polymer with mucoadhesive properties [2]. However, its adhesive properties are only weak as they result from electrostatic interactions of the positively charged chitosan and negatively charged mucus substructures. By covalently attaching thiol bearing ligands to chitosan its mucoadhesive properties are highly increased. This was confirmed by various thiolated chitosans that have already been tested for mucoadhesion [3-5]. In this case mucoadhesion is the result of the formation of covalent bonds between thiol groups present on the polymer and mucus glycoproteins via disulfide exchange reactions [6]. This reaction proceeds most efficiently when the thiol groups are in deprotonated state, i.e. present as thiolate anions. However, the intestinal pH is around 6-7 and pKa of alkyl thiols is around 8-10. This means that the thiomers developed so far do not exhibit their full mucoadhesive properties at intestinal pH and their full potential for oral drug delivery systems is not reached. Recently, novel types of thiomers, namely 6-mercaptonicotinamde-functionalized chitosan conjugates (chitosan-6-MNA) have been generated. The particularity of these novel polymers is their pH-independent reactivity to form disulfide bonds, due to the tautomeric properties of the mercaptonicotinamide ligand. This means that at physiological pH, the ability to interact with thiol/disulfides on the mucosa will be maximized. Another important parameter that has been pointed out to be advantageous for prolonged mucoadhesion is low swelling behavior. Due to the hydrophobic nature of the conjugated aromatic substructure to the chitosan, this polymer is prone to less water absorption which consequently may lead to improved mucoadhesive properties [7, 8]. Moreover, an increased hydrophobic character of the polymer should also contribute to a more sustained release of the incorporated drug [9]. According to these considerations, chitosan-6-MNA can provide various advantages as an excipient for mucoadhesive drug delivery
systems. So far, however, their potential for this application has not been investigated. It was the aim of this study to evaluate the essential properties of these newly synthesized polymers as an excipient for drug delivery systems focusing on mucoadhesion, swelling behavior, drug release, and biodegradability.
I. Materials and Methods 1. Materials
6-Mercaptonicotinic acid (6-MNA), dioxane, N-3(dimethyl aminopropyl) –N –ethylcarbodiimide hydrochloride (EDAC) and fluorescein isothiocyanate-dextran (FD4) were purchased from Sigma-Aldrich. Chitosan medium molecular mass (400 kDa) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were obtained from Fluka. Lysozyme from chicken egg white (185,000 U/mg) was purchased from Serva.
2. Synthesis of the polymer
The synthesis was carried out as described in detail previously [10]. Briefly, 2.5 g of 6-mercapto nicotinic acid dissolved in 100 mL of a dioxane-water mixture (80 mL + 20 mL) was slowly added to 1 g of chitosan (medium molecular mass: 400 kD) in a 1 % (m/v) aqueous solution under stirring. Then EDAC was added in two concentrations in order to obtain two conjugates with different coupling degrees, more precisely 12.5 mM for conjugate A and 25 mM for conjugate B. The pH was adjusted to 6 and the reaction was allowed to proceed for 7 h. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in a final concentration of 10 mM was added at pH 5 and incubated under vigorous stirring for 30 min to reduce possible disulfide bonds formed during the reaction. The resulting reduced conjugated polymer was dialyzed in tubings (molecular mass cut-off 12 kDa) first against 4.5 L of demineralized water with 7 mM HCl, twice against 7 mM HCl containing 1 % NaCl, once against 5 mM HCl and finally against 1 mM HCl at 10 °C in the dark. In order to check the purification steps, controls were prepared following the same protocol but omitting EDAC. The frozen aqueous solutions of all the conjugates and 181
J. DRUG DEL. SCI. TECH., 20 (3) 181-186 2010
6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems G. Millotti, H. Hoyer, J.F.J. Engbersen, A. Bernkop-Schnürch
controls were freeze-dried at -77 °C (Virtis Bench top freeze-drier, Bartelt, Graz, Austria) and stored at 4 °C until use. The conjugates A and B were then analyzed to determine the reduced and total amount of thiols present on the polymers as previously described [10].
dissolved in demineralized water was added in a final concentration of 1 % (m/v). The final polymer concentration was 3 % (m/v). The samples were kept at 37 °C. At predetermined time points the viscosity of 600 µL aliquots was measured at 37 °C with a plate-plate viscosimeter (Haake MARS) connected to a personal computer for setting the analysis parameters and for processing and recording the data with the Haake Rheowin program. Polymer solutions (3 % m/v in demineralized water), without the addition of lysozyme, were treated in the same way and served as controls.
3. Tablet manufacture
Freeze-dried chitosan-6-MNA conjugates and unmodified chitosan were compressed into 30 mg, 5.0 mM diameter flat-faced tablets (single-punch eccentric press, Paul Weber). The compaction pressure was kept constant during the preparation of all tablets (2.5 kN).
II. Results and discussion 1. Chitosan-6-mercaptonicotoniamide characterization
4. In vitro mucoadhesion studies with the rotating cylinder method
Unmodified chitosan and chitosan-6MNA conjugate tablets in dry form were placed on the intestinal porcine mucosa with a pair of tweezers. The mucosa was previously fixed on a stainless steel cylinder using a cyanoacrylate glue. The cylinder with the mucosa and tablets was immediately placed in the dissolution apparatus (Erweka DT 700) containing 1 L of 0.1 M phosphate buffer pH 6.8 at 37 °C. The cylinder was rotated at 100 rpm. The detachment of the tablets was determined visually from the beginning until the detachment time.
The structure of the newly synthesized thiolated chitosan can be seen in Figure 1. Two conjugates have been synthesized by changing the amount of EDAC added to the reaction mixture. In the presence of air, the thiol groups in the 6-mercaptonicotinamide-chitosan can undergo oxidation to form intra- and intermolecular disulfide bonds. Therefore, the amount of sulfide groups in these novel materials was determined. These two are named conjugate A and B. The results are summarized in Table I.
5. Evaluation of the swelling behavior
The water absorbing capacity was determined gravimetrically. Unmodified chitosan and chitosan-6MNA were fixed on a needle and submersed in a test-tube containing 0.1 M phosphate buffer pH 6.8 at 37 ºC. At pre-determined time intervals the swollen test tablets were taken out of the incubation medium and weighted. The amount of uptaken water was calculated subtracting the original weight of the tablet before the test, and the weight of the tablet taken at the scheduled times.
Figure 1 - Structure of the 6-mercaptonicotinamide-functionalized chitosan.
6. Disintegration studies
Firstly, 30 mg tablets in 5 mM diameter of unmodified chitosan and chitosan-6-MNA conjugates were placed in the disintegration apparatus (Erweka G.m.b.H., Germany) containing 1 L of 0.1 M phosphate buffer pH 5.8 and 6.8 at 37 ºC. The oscillating frequency was set to 0.5/s. The time was recorded when the tablets disintegrated completely.
Table I - Summary of the synthesised conjugates and controls.
7. Release study
2. Mucoadhesion studies
Conjugate A Conjugate B
Total thiol groups (μmol/g)
Reduced thiol groups (μmol/g)
950 1850
520 940
Mucoadhesion studies were performed with the rotating cylinder method. This method is assumed to simulate in vivo situation better than simple tensile studies, as it imitates the adhesion and cohesiveness of the polymer in physiological medium [4]. The results are shown in Figure 2. Unmodified chitosan detached from the intestinal mucosa after approximately 3 h, while both conjugates remained attached for more than 200 h. Therefore, mucoadhesive properties were increased at least 69-fold. Compared with other thiolated chitosans, only the adhesion time of chitosan-4-thiobutylamidine was in the same range confirming the effectiveness of this new generation of thiolated chitosans making it one of the most mucoadhesive polymers to date [11]. The strong mucoadhesion can be explained by the reaction of the thiol and disulfide groups of the polymer with cysteine rich-subdomains of glycoproteins in the mucus layer, leading to the formation of covalent disulfide linkages between the two layers [12]. The formation of disulfide bonds is a complicated process that can occur either by oxidative coupling of two thiol groups and/or by thiol-disulfide exchange reaction of the groups present in both polymer and mucus [13, 14]. The 6-mercaptonicotinamide group fulfils a special role in this process as it is reactive already at neutral pH and has a higher reactivity than normal alkyl and aryl thiol groups. The unusual reactivity may be attributed to the tautomeric properties of the 6- mercaptonicotinamide group, i.e. the occurrence of fast proton exchange equilibrium between the thiol group and the pyridine nitrogen (Figure 3). The possibility for
Firstly, 87 mg of each chitosan-6MNA and unmodified chitosan were dissolved in 15 mL of demineralized water and homogenized with 3 mg of fluorescein isothiocyanate-dextran (FD4) dissolved in 2 mL demineralized water. Samples were freeze-dried at -80 ºC. The lyophilized polymer/FD4 mixture was compressed into 30 mg tablets 5 mM in diameter. The release rate of FD4 from tablets was analyzed in vitro. The tablets were placed in the dissolution apparatus (Erweka DT 700) containing 500 mL of 0.1 M phosphate buffer pH 6.8 at 37 ºC. The agitation of the release media was provided by paddles. The speed was set at 100 rpm. Aliquots of 600 µL were withdrawn at 1 h intervals for 9 h, afterwards less frequently. The withdrawn aliquots were replaced with equal volume of the release medium equilibrated at 37 ºC. Samples were measured in fluorescent intensity (excitation wavelength: 485 nm; emission wavelength: 535 nm) (TECAN infinite M200). The amount of released FD4 was calculated from a calibration curve using FD4 (0.0025-5 µg/mL) in 0.1 M phosphate buffer pH 6.8, obtaining the following equation: y = 9169.6x - 34.06; R2 = 0.9991.
8. Degradation of chitosan and chitosan-6-MNA conjugates by lysozyme
Firstly, 300 mg of the polymers were hydrated in 8 mL of demineralized water. The pH was adjusted to 5.0 by the addition of 1 mL of 0.1 M acetic buffer pH 5.0. Afterwards, lysozyme previously 182
6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems G. Millotti, H. Hoyer, J.F.J. Engbersen, A. Bernkop-Schnürch
J. DRUG DEL. SCI. TECH., 20 (3) 181-186 2010
Figure 2 - Mucoadhesion studies via rotating cylinder method. Unmodified chitosan tablets are compared with both conjugate A and B tablets. Times of adhesion on the pig mucosa are expressed in hours. Data are means of at least three experiments ± SD. Both conjugates are significantly different from control (p < 0.001).
Figure 4 - Representation of the disulfide exchange mechanism of 6-mercaptonicotinamide and its dimer with thiol and disulfide groups in mucus, leading to formation of disulfide linkages between polymer and mucus at neutral pH. Scheme (a) represents the reaction of 6-mercaptonicotinamide disulfide dimer in the polymer with a thiol group in the mucus. Reaction (b) represents the formation of a disulfide linkage by reaction between 6-mercaptonicotinamide with a disulfide bond in the mucus.
Figure 3 - Tautomerism in 6-mercaptopyridine.
simultaneous proton donation and proton acceptance at the same face of this molecule enables bifunctional catalysis and makes it highly suitable for processes in which simultaneous proton transfer between reactants can avoid high energy intermediates, as is the case in the thiol-disulfide exchange mechanisms depicted in Figure 4a-b. In addition to the easy formation of disulfide linkages between polymer and mucus the low swelling behavior of the novel polymer conjugates can also contribute to the strong mucoadhesion. Correlation between the swelling rate and duration of adhesion was observed. The materials with the slowest swelling rates demonstrate the longest duration of adhesion [8]. Furthermore, other studies demonstrated that the attachment of mucin molecules to hydrophobic surfaces yield strong, stable complexes [15]. Therefore, the inclusion of hydrophobic entities in the mucoadhesive system is a possible way of prolonging mucoadhesion. Due to its aromatic ring, 6-mercaptonicoptinic acid contributes to increasing the lipophilic character of chitosan favoring hydrophobic interactions. Hence, this hydrophobic substructure combined with the ready disulfide bond formation with mucus glycoproteins would appear to be a promising strategy. Oral delivery of drugs, compared to buccal or nasal delivery bears the difficulty of a rapid turnover of the mucus [16], rendering necessary strong mucoadhesive properties to guarantee tight contact between the delivery system and the mucosa. Therefore, the novel thiomer could facilitate the oral uptake of many drugs due to its strongly mucoadhesive properties.
Figure 5 - Evaluation of the swelling behavior of chitosan-6 mercaptonicotinic acid conjugate A (n), conjugate B (s) and unmodified chitosan(u). The water uptake is expressed in mg over a time period of 420 min. Data are means of at least three experiments ± SD. *Differ significantly with p < 0.001.
3. Swelling behavior
The adhesive properties, cohesiveness and drug release profile of polymers are generally affected by their swelling behavior [7]. Mucoadhesive materials need to take up water from the underlying mucosal tissues by absorbing, swelling and capillary effects leading to a considerably stronger adhesion [17]. However, excessive water uptake will lead to overhydration forming slippery mucilage, which results in loss of adhesiveness. Therefore, slow swelling is necessary
to avoid overhydration and loss of adhesiveness [5]. Moreover, overhydration or very rapid hydration can also lead to a burst release of the incorporated drug. Water uptake studies as shown in Figure 5 demonstrated that the covalent attachment of 6-mercaptonicotinic acid to chitosan 183
J. DRUG DEL. SCI. TECH., 20 (3) 181-186 2010
6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems G. Millotti, H. Hoyer, J.F.J. Engbersen, A. Bernkop-Schnürch
significantly influenced the swelling behavior of the polymer. This observation is in contrast to almost all other thiolated chitosans that did not significantly alter their swelling properties in comparison to unmodified chitosan [4, 18] or that even increased the water uptake compared to unmodified chitosan [5, 19]. Conjugates A and B were almost 50 and 75 % less swollen than unmodified chitosan, respectively. This behavior is probably based on the lipophilic nature of the ligand. Eventually, lower water uptake could also be useful to protect the incorporated drug from an enzymatic degradation.
4. Disintegration studies
At pH of 6.8 both the conjugates and unmodified chitosan tablets did not disintegrate completely within a period of 3 days. At pH 5.8 the situation was different (Figure 6). Indeed unmodified chitosan tablets disintegrated in about 40 min, conjugate A tablets dissolved after about 2 h and conjugate B tablets proved stable for a period of approximately 50 h. Although conjugate A solutions showed much higher viscosity than unmodified chitosan, meaning a highly cross-linked structure, the disintegration occurred relatively quickly. However, it should be noted that the conditions of the viscosity and disintegration experiments were quite different as in this case the conjugates were tested in a solid tablet form and were agitated in a liquid medium, while the increase in viscosity was measured under non-agitated conditions and the polymer was initially in a solution. Nevertheless, the disintegration time of conjugate A is still 2-3 fold increased compared to unmodified chitosan. Conjugate B tablets were much more stable compared to unmodified chitosan and conjugate A. In addition to the oxidation process taking place in the conjugate network, an important factor is the lower swelling behavior and the lipophilicity of the ligand that will prevent the tablet from dissolving too fast. However, both conjugates stabilized the matrix tablet compared to the unmodified chitosan tablet.
Figure 6 - Disintegration times for tablets consisting of unmodified chitosan, conjugate A and conjugate B, in a 0.1 M phosphate buffer pH 5.8. The results are means of at least 3 experiments ± SD. *Differ significantly with p < 0.001. **Differ significantly with p < 0.05.
5. Drug release studies
It has been demonstrated that unmodified chitosan, in a phosphate buffer of pH 6.8, releases the total amount of drug very quickly, and the plateau is reached in less than 1 h [20]. Indeed, chitosan is a biopolymer with a limited solubility imposed by pH, as it is insoluble at pH > 6.5 [21]. Both conjugates A and B exhibited a significant delay in the release of the model drug FD4, making the release profile more linear (Figure 7). Reasons for the slower release profile can be found both in the presence of disulfide bonds within the matrix tablet, making it more compact, as well as the lipophilic character of the ligand that prevents extensive hydration, therefore lowering the velocity of drug molecule diffusion.
Figure 7 - Release profile of FD4 from unmodified chitosan (◊), Conjugate A (p) and conjugate B (D) tablets in phosphate buffer pH 6.8. The results are the mean of at least five experiments ± SD.
6. Susceptibility towards lysozyme degradation
It is known that chitosan is a biodegradable polymer which does not accumulate in the body. Therefore this test aimed to study how the thiolation with this new class of thiol ligands affects the biodegradability of the original polymer. Different studies have been undertaken to study the degradation of chitosan by lysozyme and the influence of different degrees of deacetylation, reacetylation and acylation [22, 23]. However, the results are not in full agreement with each other, most likely due to the different origin of the enzyme, the different degrees of substitution and the physical form of the samples [24]. Chitosanthioglycolic acid has been reported to have a much slower rate of degradation compared to unmodified chitosan in proportion to the coupling degree [19]. Chitosan –N-acetyl cysteine was also reported to exhibit a lower susceptibility towards lysozyme with respect to unmodified chitosan. Surprisingly, for the chitosan-6-MNA derivatives an opposite behavior was found as a significant decrease in viscosity was observed upon exposure of the newly synthesized polymers to lysozyme solution (Figure 8). The decrease in viscosity of the newly synthesized polymers is much higher in the conjugated chitosan than
Figure 8 - Susceptibility towards lysozyme degradation expressed as percent of loss in viscosity. Indicated values are means ± SD (n = 4). The polymers were used in a concentration of 3 % (m/v) while lysozyme in final concentration of 1 % (m/v). The experiment was performed at pH of 5.0. Unmodified chitosan (u), conjugate A (n), conjugate B (s), unmodified chitosan + lysozyme (◊), conjugate A + lysozyme (p) conjugate B + lysozyme (D).
in the unmodified chitosan. A correlation was observed between an amount of 6-mercaptonicotinamide groups present in the polymer and the susceptibility towards lysozyme. A number of effects may play a 184
6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems G. Millotti, H. Hoyer, J.F.J. Engbersen, A. Bernkop-Schnürch
J. DRUG DEL. SCI. TECH., 20 (3) 181-186 2010
4. 5. Figure 9 - Possible catalysis by 6-mercaptopyridine groups in the hydrolytic cleavage of the glycoside bonds in chitosan.
role in this behavior. Firstly, it is possible that the acylation of part of the amino groups in chitosan by 6-MNA and the concomitant increase in hydrophobic nature of the chitosan derivatives resulted in a higher affinity of the enzyme for the site of cleavage. It has been reported that N-acylation of chitosan leads to higher lysozyme susceptibility [24, 25]. It was suggested that acylation of primary amines in a subdomain of chitosan increases the accessibility of the active site of lysozyme to the glycosidic moieties present in this subdomain [25]. Other authors explained that by altering the hydrophilic nature of chitosan by introducing hydrophobic entities, the balance in the hydrophobic and hydrophilic properties thus improves the blood compatible properties [25]. Moreover, due to the presence of disulfide bonds in lysozyme, the three dimensional structure of lysozyme, and hence its catalytic activity, is sensitive to the concentration and nature of redox species in solution. Thus, the redox active groups in the chitosan derivatives may affect the lysozyme activity. Furthermore, as explained above, 6-mercaptopyridine (6-thiopyridone) is a potential bifunctional catalyst in processes in which simultaneous proton exchange can avoid highly energetic (charged) intermediates or transition stated. Thus, the tautomeric properties of 6-mercaptopyrydine by providing a proton and accepting a proton at different sites in the molecule may also be involved in hydrolysis of the glycoside group and takes into consideration as an additional effect in order to explain the enhanced degradation of the conjugate in comparison to unmodified chitosan (Figure 9).
6.
*
16.
In this work, two novel derivatives of chitosan, functionalized with different degrees of 6-mercapronicotinamide groups have been synthesized and characterized in terms of mucoadhesion, swelling behavior, controlled release, tablet disintegration behavior and biocompatibility. In vitro mucoadhesion was very high placing these new polymers among the most effective mucoadhesive thiomers. The lower swelling capacity compared to unmodified chitosan is advantageous for both the adhesion properties as well as a sustained drug release. Matrix tablets comprising this novel thiolated chitosan exhibited a prolonged disintegration time. Furthermore, the modification of chitosan with 6-mercaptonicotinic acid improved chitosan biodegradability. These properties could be useful in order to prolong the stability and adhesion time of various dosage forms on various mucosal tissues. Therefore this new conjugate has a potential to contribute to the development of more beneficial and specific pharmaceutical formulations.
17.
7. 8. 9. 10.
11. 12.
13. 14. 15.
18. 19. 20.
21.
22. 23.
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J. DRUG DEL. SCI. TECH., 20 (3) 181-186 2010
6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems G. Millotti, H. Hoyer, J.F.J. Engbersen, A. Bernkop-Schnürch
Acknowledgement
Manuscript
This work has been supported by the EC. NanoBioPharmaceutics is an Integrated Project funded within the 6th Framework Programme of the European Commission.
Received 4 February 2010, accepted for publication 15 March 2010.
186
6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems G. Millotti1, H. Hoyer1, J.F.J. Engbersen2, A. Bernkop-Schnürch1* Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University, Innrain 52c, Josef Möller Haus, A-6020 Innsbruck, Austria 2 Department of Biomedical Chemistry, Faculty of Science and Technology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands *Correspondence: [email protected] 1
Among mucoadhesive polymers, thiolated polymers showed to be superior over non-thiolated polymers. Recently a novel thiolated chitosan named chitosan-6-mercaptonicotinamide has been generated. Its heteroaromatic structure confers a pH-independent reactivity to the thiol group and introduces a hydrophobic entity in the hydrophilic polymer. Within this study the potential of this novel polymer as excipient for drug delivery systems was evaluated with a special focus on mucoadhesion. The highest coupling degree was 975 µmol thiol groups in reduced form per gram of polymer. Mucoadhesion was increased by at least 69-fold compared to unmodified chitosan. This chemical modification of chitosan, altered the water uptake of the polymer by lowering it up to 75 %. The new polymer was effective in stabilizing a matrix tablet compared to an unmodified chitosan tablet. Cumulative release studies from a matrix tablet comprising the newly synthesized chitosan showed controlled release of a hydrophilic macromolecular model compound (FD4). Furthermore, the polymer showed improved biodegradability. These advantageous features render chitosan-6-mercaptonicotinic acid a useful excipient for various drug delivery systems Key words: Thiomers – Mucoadhesion – Drug release – Swelling properties – Biodegradability.
The potential of many drug delivery systems can be improved by mucoadhesive polymers. By prolonging the residence time of dosage forms at the site of drug absorption, the bioavailability of the drug can be increased and the frequency of administration lowered [1]. Mucoadhesive properties of polymers have been extensively studied. Chitosan, for instance, is a cationic polymer with mucoadhesive properties [2]. However, its adhesive properties are only weak as they result from electrostatic interactions of the positively charged chitosan and negatively charged mucus substructures. By covalently attaching thiol bearing ligands to chitosan its mucoadhesive properties are highly increased. This was confirmed by various thiolated chitosans that have already been tested for mucoadhesion [3-5]. In this case mucoadhesion is the result of the formation of covalent bonds between thiol groups present on the polymer and mucus glycoproteins via disulfide exchange reactions [6]. This reaction proceeds most efficiently when the thiol groups are in deprotonated state, i.e. present as thiolate anions. However, the intestinal pH is around 6-7 and pKa of alkyl thiols is around 8-10. This means that the thiomers developed so far do not exhibit their full mucoadhesive properties at intestinal pH and their full potential for oral drug delivery systems is not reached. Recently, novel types of thiomers, namely 6-mercaptonicotinamde-functionalized chitosan conjugates (chitosan-6-MNA) have been generated. The particularity of these novel polymers is their pH-independent reactivity to form disulfide bonds, due to the tautomeric properties of the mercaptonicotinamide ligand. This means that at physiological pH, the ability to interact with thiol/disulfides on the mucosa will be maximized. Another important parameter that has been pointed out to be advantageous for prolonged mucoadhesion is low swelling behavior. Due to the hydrophobic nature of the conjugated aromatic substructure to the chitosan, this polymer is prone to less water absorption which consequently may lead to improved mucoadhesive properties [7, 8]. Moreover, an increased hydrophobic character of the polymer should also contribute to a more sustained release of the incorporated drug [9]. According to these considerations, chitosan-6-MNA can provide various advantages as an excipient for mucoadhesive drug delivery
systems. So far, however, their potential for this application has not been investigated. It was the aim of this study to evaluate the essential properties of these newly synthesized polymers as an excipient for drug delivery systems focusing on mucoadhesion, swelling behavior, drug release, and biodegradability.
I. Materials and Methods 1. Materials
6-Mercaptonicotinic acid (6-MNA), dioxane, N-3(dimethyl aminopropyl) –N –ethylcarbodiimide hydrochloride (EDAC) and fluorescein isothiocyanate-dextran (FD4) were purchased from Sigma-Aldrich. Chitosan medium molecular mass (400 kDa) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were obtained from Fluka. Lysozyme from chicken egg white (185,000 U/mg) was purchased from Serva.
2. Synthesis of the polymer
The synthesis was carried out as described in detail previously [10]. Briefly, 2.5 g of 6-mercapto nicotinic acid dissolved in 100 mL of a dioxane-water mixture (80 mL + 20 mL) was slowly added to 1 g of chitosan (medium molecular mass: 400 kD) in a 1 % (m/v) aqueous solution under stirring. Then EDAC was added in two concentrations in order to obtain two conjugates with different coupling degrees, more precisely 12.5 mM for conjugate A and 25 mM for conjugate B. The pH was adjusted to 6 and the reaction was allowed to proceed for 7 h. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in a final concentration of 10 mM was added at pH 5 and incubated under vigorous stirring for 30 min to reduce possible disulfide bonds formed during the reaction. The resulting reduced conjugated polymer was dialyzed in tubings (molecular mass cut-off 12 kDa) first against 4.5 L of demineralized water with 7 mM HCl, twice against 7 mM HCl containing 1 % NaCl, once against 5 mM HCl and finally against 1 mM HCl at 10 °C in the dark. In order to check the purification steps, controls were prepared following the same protocol but omitting EDAC. The frozen aqueous solutions of all the conjugates and 181
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6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems G. Millotti, H. Hoyer, J.F.J. Engbersen, A. Bernkop-Schnürch
controls were freeze-dried at -77 °C (Virtis Bench top freeze-drier, Bartelt, Graz, Austria) and stored at 4 °C until use. The conjugates A and B were then analyzed to determine the reduced and total amount of thiols present on the polymers as previously described [10].
dissolved in demineralized water was added in a final concentration of 1 % (m/v). The final polymer concentration was 3 % (m/v). The samples were kept at 37 °C. At predetermined time points the viscosity of 600 µL aliquots was measured at 37 °C with a plate-plate viscosimeter (Haake MARS) connected to a personal computer for setting the analysis parameters and for processing and recording the data with the Haake Rheowin program. Polymer solutions (3 % m/v in demineralized water), without the addition of lysozyme, were treated in the same way and served as controls.
3. Tablet manufacture
Freeze-dried chitosan-6-MNA conjugates and unmodified chitosan were compressed into 30 mg, 5.0 mM diameter flat-faced tablets (single-punch eccentric press, Paul Weber). The compaction pressure was kept constant during the preparation of all tablets (2.5 kN).
II. Results and discussion 1. Chitosan-6-mercaptonicotoniamide characterization
4. In vitro mucoadhesion studies with the rotating cylinder method
Unmodified chitosan and chitosan-6MNA conjugate tablets in dry form were placed on the intestinal porcine mucosa with a pair of tweezers. The mucosa was previously fixed on a stainless steel cylinder using a cyanoacrylate glue. The cylinder with the mucosa and tablets was immediately placed in the dissolution apparatus (Erweka DT 700) containing 1 L of 0.1 M phosphate buffer pH 6.8 at 37 °C. The cylinder was rotated at 100 rpm. The detachment of the tablets was determined visually from the beginning until the detachment time.
The structure of the newly synthesized thiolated chitosan can be seen in Figure 1. Two conjugates have been synthesized by changing the amount of EDAC added to the reaction mixture. In the presence of air, the thiol groups in the 6-mercaptonicotinamide-chitosan can undergo oxidation to form intra- and intermolecular disulfide bonds. Therefore, the amount of sulfide groups in these novel materials was determined. These two are named conjugate A and B. The results are summarized in Table I.
5. Evaluation of the swelling behavior
The water absorbing capacity was determined gravimetrically. Unmodified chitosan and chitosan-6MNA were fixed on a needle and submersed in a test-tube containing 0.1 M phosphate buffer pH 6.8 at 37 ºC. At pre-determined time intervals the swollen test tablets were taken out of the incubation medium and weighted. The amount of uptaken water was calculated subtracting the original weight of the tablet before the test, and the weight of the tablet taken at the scheduled times.
Figure 1 - Structure of the 6-mercaptonicotinamide-functionalized chitosan.
6. Disintegration studies
Firstly, 30 mg tablets in 5 mM diameter of unmodified chitosan and chitosan-6-MNA conjugates were placed in the disintegration apparatus (Erweka G.m.b.H., Germany) containing 1 L of 0.1 M phosphate buffer pH 5.8 and 6.8 at 37 ºC. The oscillating frequency was set to 0.5/s. The time was recorded when the tablets disintegrated completely.
Table I - Summary of the synthesised conjugates and controls.
7. Release study
2. Mucoadhesion studies
Conjugate A Conjugate B
Total thiol groups (μmol/g)
Reduced thiol groups (μmol/g)
950 1850
520 940
Mucoadhesion studies were performed with the rotating cylinder method. This method is assumed to simulate in vivo situation better than simple tensile studies, as it imitates the adhesion and cohesiveness of the polymer in physiological medium [4]. The results are shown in Figure 2. Unmodified chitosan detached from the intestinal mucosa after approximately 3 h, while both conjugates remained attached for more than 200 h. Therefore, mucoadhesive properties were increased at least 69-fold. Compared with other thiolated chitosans, only the adhesion time of chitosan-4-thiobutylamidine was in the same range confirming the effectiveness of this new generation of thiolated chitosans making it one of the most mucoadhesive polymers to date [11]. The strong mucoadhesion can be explained by the reaction of the thiol and disulfide groups of the polymer with cysteine rich-subdomains of glycoproteins in the mucus layer, leading to the formation of covalent disulfide linkages between the two layers [12]. The formation of disulfide bonds is a complicated process that can occur either by oxidative coupling of two thiol groups and/or by thiol-disulfide exchange reaction of the groups present in both polymer and mucus [13, 14]. The 6-mercaptonicotinamide group fulfils a special role in this process as it is reactive already at neutral pH and has a higher reactivity than normal alkyl and aryl thiol groups. The unusual reactivity may be attributed to the tautomeric properties of the 6- mercaptonicotinamide group, i.e. the occurrence of fast proton exchange equilibrium between the thiol group and the pyridine nitrogen (Figure 3). The possibility for
Firstly, 87 mg of each chitosan-6MNA and unmodified chitosan were dissolved in 15 mL of demineralized water and homogenized with 3 mg of fluorescein isothiocyanate-dextran (FD4) dissolved in 2 mL demineralized water. Samples were freeze-dried at -80 ºC. The lyophilized polymer/FD4 mixture was compressed into 30 mg tablets 5 mM in diameter. The release rate of FD4 from tablets was analyzed in vitro. The tablets were placed in the dissolution apparatus (Erweka DT 700) containing 500 mL of 0.1 M phosphate buffer pH 6.8 at 37 ºC. The agitation of the release media was provided by paddles. The speed was set at 100 rpm. Aliquots of 600 µL were withdrawn at 1 h intervals for 9 h, afterwards less frequently. The withdrawn aliquots were replaced with equal volume of the release medium equilibrated at 37 ºC. Samples were measured in fluorescent intensity (excitation wavelength: 485 nm; emission wavelength: 535 nm) (TECAN infinite M200). The amount of released FD4 was calculated from a calibration curve using FD4 (0.0025-5 µg/mL) in 0.1 M phosphate buffer pH 6.8, obtaining the following equation: y = 9169.6x - 34.06; R2 = 0.9991.
8. Degradation of chitosan and chitosan-6-MNA conjugates by lysozyme
Firstly, 300 mg of the polymers were hydrated in 8 mL of demineralized water. The pH was adjusted to 5.0 by the addition of 1 mL of 0.1 M acetic buffer pH 5.0. Afterwards, lysozyme previously 182
6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems G. Millotti, H. Hoyer, J.F.J. Engbersen, A. Bernkop-Schnürch
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Figure 2 - Mucoadhesion studies via rotating cylinder method. Unmodified chitosan tablets are compared with both conjugate A and B tablets. Times of adhesion on the pig mucosa are expressed in hours. Data are means of at least three experiments ± SD. Both conjugates are significantly different from control (p < 0.001).
Figure 4 - Representation of the disulfide exchange mechanism of 6-mercaptonicotinamide and its dimer with thiol and disulfide groups in mucus, leading to formation of disulfide linkages between polymer and mucus at neutral pH. Scheme (a) represents the reaction of 6-mercaptonicotinamide disulfide dimer in the polymer with a thiol group in the mucus. Reaction (b) represents the formation of a disulfide linkage by reaction between 6-mercaptonicotinamide with a disulfide bond in the mucus.
Figure 3 - Tautomerism in 6-mercaptopyridine.
simultaneous proton donation and proton acceptance at the same face of this molecule enables bifunctional catalysis and makes it highly suitable for processes in which simultaneous proton transfer between reactants can avoid high energy intermediates, as is the case in the thiol-disulfide exchange mechanisms depicted in Figure 4a-b. In addition to the easy formation of disulfide linkages between polymer and mucus the low swelling behavior of the novel polymer conjugates can also contribute to the strong mucoadhesion. Correlation between the swelling rate and duration of adhesion was observed. The materials with the slowest swelling rates demonstrate the longest duration of adhesion [8]. Furthermore, other studies demonstrated that the attachment of mucin molecules to hydrophobic surfaces yield strong, stable complexes [15]. Therefore, the inclusion of hydrophobic entities in the mucoadhesive system is a possible way of prolonging mucoadhesion. Due to its aromatic ring, 6-mercaptonicoptinic acid contributes to increasing the lipophilic character of chitosan favoring hydrophobic interactions. Hence, this hydrophobic substructure combined with the ready disulfide bond formation with mucus glycoproteins would appear to be a promising strategy. Oral delivery of drugs, compared to buccal or nasal delivery bears the difficulty of a rapid turnover of the mucus [16], rendering necessary strong mucoadhesive properties to guarantee tight contact between the delivery system and the mucosa. Therefore, the novel thiomer could facilitate the oral uptake of many drugs due to its strongly mucoadhesive properties.
Figure 5 - Evaluation of the swelling behavior of chitosan-6 mercaptonicotinic acid conjugate A (n), conjugate B (s) and unmodified chitosan(u). The water uptake is expressed in mg over a time period of 420 min. Data are means of at least three experiments ± SD. *Differ significantly with p < 0.001.
3. Swelling behavior
The adhesive properties, cohesiveness and drug release profile of polymers are generally affected by their swelling behavior [7]. Mucoadhesive materials need to take up water from the underlying mucosal tissues by absorbing, swelling and capillary effects leading to a considerably stronger adhesion [17]. However, excessive water uptake will lead to overhydration forming slippery mucilage, which results in loss of adhesiveness. Therefore, slow swelling is necessary
to avoid overhydration and loss of adhesiveness [5]. Moreover, overhydration or very rapid hydration can also lead to a burst release of the incorporated drug. Water uptake studies as shown in Figure 5 demonstrated that the covalent attachment of 6-mercaptonicotinic acid to chitosan 183
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6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems G. Millotti, H. Hoyer, J.F.J. Engbersen, A. Bernkop-Schnürch
significantly influenced the swelling behavior of the polymer. This observation is in contrast to almost all other thiolated chitosans that did not significantly alter their swelling properties in comparison to unmodified chitosan [4, 18] or that even increased the water uptake compared to unmodified chitosan [5, 19]. Conjugates A and B were almost 50 and 75 % less swollen than unmodified chitosan, respectively. This behavior is probably based on the lipophilic nature of the ligand. Eventually, lower water uptake could also be useful to protect the incorporated drug from an enzymatic degradation.
4. Disintegration studies
At pH of 6.8 both the conjugates and unmodified chitosan tablets did not disintegrate completely within a period of 3 days. At pH 5.8 the situation was different (Figure 6). Indeed unmodified chitosan tablets disintegrated in about 40 min, conjugate A tablets dissolved after about 2 h and conjugate B tablets proved stable for a period of approximately 50 h. Although conjugate A solutions showed much higher viscosity than unmodified chitosan, meaning a highly cross-linked structure, the disintegration occurred relatively quickly. However, it should be noted that the conditions of the viscosity and disintegration experiments were quite different as in this case the conjugates were tested in a solid tablet form and were agitated in a liquid medium, while the increase in viscosity was measured under non-agitated conditions and the polymer was initially in a solution. Nevertheless, the disintegration time of conjugate A is still 2-3 fold increased compared to unmodified chitosan. Conjugate B tablets were much more stable compared to unmodified chitosan and conjugate A. In addition to the oxidation process taking place in the conjugate network, an important factor is the lower swelling behavior and the lipophilicity of the ligand that will prevent the tablet from dissolving too fast. However, both conjugates stabilized the matrix tablet compared to the unmodified chitosan tablet.
Figure 6 - Disintegration times for tablets consisting of unmodified chitosan, conjugate A and conjugate B, in a 0.1 M phosphate buffer pH 5.8. The results are means of at least 3 experiments ± SD. *Differ significantly with p < 0.001. **Differ significantly with p < 0.05.
5. Drug release studies
It has been demonstrated that unmodified chitosan, in a phosphate buffer of pH 6.8, releases the total amount of drug very quickly, and the plateau is reached in less than 1 h [20]. Indeed, chitosan is a biopolymer with a limited solubility imposed by pH, as it is insoluble at pH > 6.5 [21]. Both conjugates A and B exhibited a significant delay in the release of the model drug FD4, making the release profile more linear (Figure 7). Reasons for the slower release profile can be found both in the presence of disulfide bonds within the matrix tablet, making it more compact, as well as the lipophilic character of the ligand that prevents extensive hydration, therefore lowering the velocity of drug molecule diffusion.
Figure 7 - Release profile of FD4 from unmodified chitosan (◊), Conjugate A (p) and conjugate B (D) tablets in phosphate buffer pH 6.8. The results are the mean of at least five experiments ± SD.
6. Susceptibility towards lysozyme degradation
It is known that chitosan is a biodegradable polymer which does not accumulate in the body. Therefore this test aimed to study how the thiolation with this new class of thiol ligands affects the biodegradability of the original polymer. Different studies have been undertaken to study the degradation of chitosan by lysozyme and the influence of different degrees of deacetylation, reacetylation and acylation [22, 23]. However, the results are not in full agreement with each other, most likely due to the different origin of the enzyme, the different degrees of substitution and the physical form of the samples [24]. Chitosanthioglycolic acid has been reported to have a much slower rate of degradation compared to unmodified chitosan in proportion to the coupling degree [19]. Chitosan –N-acetyl cysteine was also reported to exhibit a lower susceptibility towards lysozyme with respect to unmodified chitosan. Surprisingly, for the chitosan-6-MNA derivatives an opposite behavior was found as a significant decrease in viscosity was observed upon exposure of the newly synthesized polymers to lysozyme solution (Figure 8). The decrease in viscosity of the newly synthesized polymers is much higher in the conjugated chitosan than
Figure 8 - Susceptibility towards lysozyme degradation expressed as percent of loss in viscosity. Indicated values are means ± SD (n = 4). The polymers were used in a concentration of 3 % (m/v) while lysozyme in final concentration of 1 % (m/v). The experiment was performed at pH of 5.0. Unmodified chitosan (u), conjugate A (n), conjugate B (s), unmodified chitosan + lysozyme (◊), conjugate A + lysozyme (p) conjugate B + lysozyme (D).
in the unmodified chitosan. A correlation was observed between an amount of 6-mercaptonicotinamide groups present in the polymer and the susceptibility towards lysozyme. A number of effects may play a 184
6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems G. Millotti, H. Hoyer, J.F.J. Engbersen, A. Bernkop-Schnürch
J. DRUG DEL. SCI. TECH., 20 (3) 181-186 2010
4. 5. Figure 9 - Possible catalysis by 6-mercaptopyridine groups in the hydrolytic cleavage of the glycoside bonds in chitosan.
role in this behavior. Firstly, it is possible that the acylation of part of the amino groups in chitosan by 6-MNA and the concomitant increase in hydrophobic nature of the chitosan derivatives resulted in a higher affinity of the enzyme for the site of cleavage. It has been reported that N-acylation of chitosan leads to higher lysozyme susceptibility [24, 25]. It was suggested that acylation of primary amines in a subdomain of chitosan increases the accessibility of the active site of lysozyme to the glycosidic moieties present in this subdomain [25]. Other authors explained that by altering the hydrophilic nature of chitosan by introducing hydrophobic entities, the balance in the hydrophobic and hydrophilic properties thus improves the blood compatible properties [25]. Moreover, due to the presence of disulfide bonds in lysozyme, the three dimensional structure of lysozyme, and hence its catalytic activity, is sensitive to the concentration and nature of redox species in solution. Thus, the redox active groups in the chitosan derivatives may affect the lysozyme activity. Furthermore, as explained above, 6-mercaptopyridine (6-thiopyridone) is a potential bifunctional catalyst in processes in which simultaneous proton exchange can avoid highly energetic (charged) intermediates or transition stated. Thus, the tautomeric properties of 6-mercaptopyrydine by providing a proton and accepting a proton at different sites in the molecule may also be involved in hydrolysis of the glycoside group and takes into consideration as an additional effect in order to explain the enhanced degradation of the conjugate in comparison to unmodified chitosan (Figure 9).
6.
*
16.
In this work, two novel derivatives of chitosan, functionalized with different degrees of 6-mercapronicotinamide groups have been synthesized and characterized in terms of mucoadhesion, swelling behavior, controlled release, tablet disintegration behavior and biocompatibility. In vitro mucoadhesion was very high placing these new polymers among the most effective mucoadhesive thiomers. The lower swelling capacity compared to unmodified chitosan is advantageous for both the adhesion properties as well as a sustained drug release. Matrix tablets comprising this novel thiolated chitosan exhibited a prolonged disintegration time. Furthermore, the modification of chitosan with 6-mercaptonicotinic acid improved chitosan biodegradability. These properties could be useful in order to prolong the stability and adhesion time of various dosage forms on various mucosal tissues. Therefore this new conjugate has a potential to contribute to the development of more beneficial and specific pharmaceutical formulations.
17.
7. 8. 9. 10.
11. 12.
13. 14. 15.
18. 19. 20.
21.
22. 23.
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J. DRUG DEL. SCI. TECH., 20 (3) 181-186 2010
6-mercaptonicotinamide-functionalized chitosan: a potential excipient for mucoadhesive drug delivery systems G. Millotti, H. Hoyer, J.F.J. Engbersen, A. Bernkop-Schnürch
Acknowledgement
Manuscript
This work has been supported by the EC. NanoBioPharmaceutics is an Integrated Project funded within the 6th Framework Programme of the European Commission.
Received 4 February 2010, accepted for publication 15 March 2010.
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