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Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome
Acta Biomaterialia xxx (2015) xxx–xxx
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Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome Flavia Laffleur a, Alexander Fischer a, Matthias Schmutzler b, Fabian Hintzen a, Andreas Bernkop-Schnürch a,⇑ a b
Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Austria Institute of Analytical Chemistry & Radiochemistry, CCB – Center for Chemistry and Biomedicine, University of Innsbruck, Austria
a r t i c l e
i n f o
Article history: Received 23 September 2014 Received in revised form 2 April 2015 Accepted 14 April 2015 Available online xxxx Keywords: Thiomers Chitosan–thioglycolic acid Mercaptonicotinamide Preactivated thiomers Dry mouth syndrome
a b s t r a c t Purpose: The objective of this study was to investigate preactivated thiomers for their potential in the treatment of dry mouth syndrome. Methods: Chitosan–thioglycolic–mercaptonicotinamide conjugates (chitosan–TGA–MNA) were synthesized by the oxidative S–S coupling of chitosan–thioglycolic acid (chitosan–TGA) with 6-mercaptonicotinamide (MNA). Test disks were compressed out of unmodified chitosan, chitosan–TGA (thiomers) and chitosan–TGA–MNA conjugates to investigate cohesive properties, cytotoxicity assays and mucoadhesion studies. Results: Immobilizing the MNA achieved higher swelling and cohesive properties of chitosan–TGA–MNA conjugates compared to unmodified chitosan. Rotating cylinder studies displayed a 3.1-fold improvement of mucoadhesiveness of chitosan–TGA–MNA conjugates compared to thiolated polymers. Findings in tensile strength were in good agreement with rotating cylinder ones. Furthermore, preactivated thiomers exhibit higher stability. All conjugates were found non-toxic against Caco-2 cells. Conclusion: Preactivated thiolated chitosan could be a promising system for the treatment of dry mouth syndrome where mucosa requires lubrication and mucoadhesiveness. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Conventional mucoadhesive materials such as carbopol, polycarbophil or sodium carboxymethylcellulose are to a large extent hydrophilic compounds containing numerous hydrogen bond forming groups [1]. Conventional polymers form non-covalent bonds with mucus substructures which result in mainly weak mucoadhesion. However, functionalized polymers bearing sulfhydryl ligands on the polymeric backbone or designed thiomers show the ability to form covalent bonds with cysteine-rich subunits of mucus. By virtue of these features, both benefits of prolonged cohesion and strong mucoadhesion are met in the case of designated thiomers. The interactions of mucus and thiomer are, however, likely to be most intense in the deprotonated state of sulfhydryl groups [2]. Almost all oral degenerative diseases, including tooth decay, are associated with a low pH (acidic) in the body ⇑ Corresponding author at: Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria. Tel.: +43 512 507 58600; fax: +43 512 507 58699. E-mail address: [email protected] (A. Bernkop-Schnürch).
[3,4]. People with a dry mouth have mouth acidity caused by saliva thickening or dying up because of various medications such as anticholinergics, sympathomimetics, anti-HIV drugs as well as bezodiazepines [5] and dehydration [6,7]. Since xerostomia goes hand in hand with a leaky or even lacking mucus gel layer covering intraoral epithelia and consequently reduced properties of the mucosa to keep sufficient moisture on its surface [8,9], one strategy to increase salivary secretion could be using mucoadhesive polymers such as cellulose derivates as lubricants. Thiomers, however, are unstable toward oxidation and aqueous thiomer solutions are unstable. Therefore a novel second generation of thiomers, the so-called preactivated thiomers have been developed exhibiting stability toward oxidation as well as improved mucoadhesion [10]. To overcome these limitations preactivated thiomers bearing an additional ligand with a heteroaromatic structure reacting in a pH independent manner were designed and evaluated within this study. The thiol groups are protected via disulfide bonds due to the additional aromatic ligand 6-mercaptonicotinic amid (MNA). Furthermore chitosan–TGA–MNA is protected against early oxidation. Prevention of an early oxidation before coming into contact with the mucus layer occurs. Hence, more active thiol groups are
http://dx.doi.org/10.1016/j.actbio.2015.04.016 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Laffleur F et al. Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.016
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available for a long intimate contact with mucosal membranes. To date, however, neither thiomers nor preactivated thiomers were investigated regarding their potential for dry mouth syndrome. According to this, it is the aim of this study to evaluate the positive influence of thiomers and especially preactivated thiomers on mucoadhesion for their potential as therapeutic agents for dry mouth syndrome. 2. Material and methods 2.1. Materials Chitosan (low molecular mass 50–100 kDa; degree of deacetylation, 83–85%) was obtained from Fluka Chemie (Buchs, Switzerland). Ellman’s reagent [5,50 dithiobis(2-nitrobenzoic acid) (DTNB), reduced-form glutathione (GSH), tris(2-carboxylethyl)phosphine hydrochloride (TCEP), 6-chlornicotinamide, thiourea and thioglycolic acid, 5,5-dithiobis(2-nitrobenzoic acid) were purchased from Sigma Aldrich, Steinheim, Germany. All other chemicals, reagents and solvents were of analytical grade and were received from commercial sources. 2.2. Synthesis of chitosan thioglycolic acid conjugates Thioglycolic acid was covalently attached to chitosan as previously described by our research group [11]. Thiolated chitosan was synthesized by the covalent attachment of thioglycolic acid to chitosan which formed amide bonds in between the primary amino group of chitosan and the carboxylic acid moieties of the sulfhydryl agent. Briefly, 500 mg of chitosan (low molecular mass 50–100 kDa; degree of deacetylation, 83–85%) was dissolved in 1 M HCl by adding demineralized water in order to obtain a 1% solution of chitosan hydrochloride. In the next step the coupling reaction was mediated by EDAC in a final concentration of 100 mM in order to activate the carboxylic acid moieties of the subsequently added 500 mg of TGA. The reaction mixture was incubated for 3 h at room temperature under constant stirring. Samples prepared by applying the exact same method with the exception of omitting EDAC during the coupling reaction served as controls for the analytical studies. The reaction mixtures were dialyzed in tubings in order to eliminate unbound TGA and to isolate polymer conjugates. The dialysis process is a washing process of the synthesized mixture. For this purpose the synthesized polymer was transferred into dialysis tubing (Cellulose hydrate, cut-off 12.000 Da, Carl Roth, Karlsruhe, Germany). For purifying reasons as well as to get rid of the unbound ligand, the polymer mixture was three times dialyzed against demineralized water containing 1% NaCl and twice against demineralized water. Afterward, samples and controls were lyophilized by drying frozen aqueous polymer solutions at 30 °C and 0.01 mbar (Christ Beta; Germany) (primary drying conditions of 23 h, beginning with 3 h at 30 °C, augmenting to 15 °C for 3 h , up to 10 °C for 2 h, following 17 h at 0 °C; secondary conditions of 8 h, starting with 1 h at +20 °C and 7 h at +25 °C) and finally stored at 4 °C until further use.
was obtained. This was filtered and dried. By adding 2 M NaOH in the second proceeding step the compound was precipitated. Subsequently, it was stirred at room temperature for 45 min. In the following procedure, the pH was adjusted to 4, where the mixture turned into dark yellowish color. By this adjustment, mercaptonicotinamide (MNA) as the synthesized compound was obtained. This was purified by filtration, washed with acetone and dried. 2.3.2. 6,60 -Dithionicotinamide (6,60 -DTNA)- synthesis and purification A suspension of 6-MNA was adjusted to a pH of 7, afterward H2O2 solution was added. This oxidizing reaction was stirred for 1 h at room temperature. During this reaction the yellow colored compound rendered into the white compound 6,6-dithionicotinamide (6,60 -DTNA). 6,60 -DTNA was filtered, washed with water and dried as mentioned above. 2.4. Synthesis of chitosan–thioglycolic acid–mercaptonicotinamide The aromatic ligand was covalently attached by disulfide bond formation. 200 mg of chitosan–TGA was dissolved in a 50 mL mixture of demineralized water/DMSO (3:7) under stirring. Next, 50 mg of 6,60 -DTNA dimer dissolved in 50 mL of DMSO was added and the pH adjusted to 6.2 with 1 M NaOH. The mixture was stirred for 6 h at room temperature. In order to separate the conjugate from the unbound MNA dimer, the reaction solution was dialyzed seven times against 5 L of demineralized water under stirring in the dark at 10 °C. The dialysate was replaced every 12 h. The obtained product was freeze dried for 31 h as mentioned above and stored dry at 4 °C. 2.5. Analyses of thiolated and preactivated chitosan 2.5.1. Spectrophotometric quantification of the thiol groups Immobilization of thiol groups on the chitosan thioglycolic acid backbone was quantified by Ellman0 s reaction which determines the free thiol groups [13]. Polymers and thiomers were hydrated in 0.5 M phosphate buffer pH 8.0. Ellman’s reagent (5,50 -dithiobis (2-nitrobenzoic acid) (DTNB) was dissolved in 0.5 M phosphate buffer pH 8.0) and added to the polymer solutions. After an incubation time of 120 min protected from light, the absorbance of each sample was measured with a microplate reader (Tecan Fluostar; Galaxy BMG, Offenburg, Germany). The quantity of thiol groups being immobilized on the chitosan thioglycolic acid conjugate was determined by referring to cysteine standards [14].
2.3. 6-mercaptonicotinamide (MNA) and 6,60 -dithionicotinamide (6,60 -DTNA) synthesis
2.5.2. Characterization of 6-MNA and 6,60 -DTNA 6-MNA and 6,60 -DTNA were soluble in DMSO. UV-spectrometer measurements were performed with a UV–VIS spectrophotometer (UV mini-1240, Shimadzu, Kyoto, Japan) [15]. 6-MNA revealed a peak at 307 nm and 6,60 -DTNA showed two peaks, one expressive at 297 nm and a second minor one at 253 nm. Furthermore DSC thermograms were evaluated, according to the literature melting points of around 266 °C and 264 °C are expected [16]. For DSC studies, approximately, 2 mg of sample was taken in an aluminum pan, sealed with an aluminum cap and kept under nitrogen purging (atmosphere). Samples were scanned from 0 °C to 300 °C with the scanning rate of 10 °C/min using differential scanning calorimeter (DSC-60, Shimadzu, Japan).
2.3.1. 6-mercaptonicotinamide- synthesis and purification 6-mercaptonicotinamide was synthesized during a two-step synthesis [12]. For this synthesis, a suspension of 6-chlornicotinamide and thiourea in ethanol was generated and refluxed for 6 h at boiling temperature. After the suspension turned yellow, the mixture was stirred for 12 h at room temperature. In this first step the compound S-(5-carbamyl-2-pyridyl) thiuronium chloride
2.5.3. FT-IR spectra of the 6-MNA, dimer 6,60 -DTNA and the polymer conjugates In order to verify the syntheses FT-IR analyses of 6,60 -DTNA, MNA and conjugates were undertaken. Characterization of 6MNA and 6,60 -DTNA was conducted with attenuated-totalreflectance Fourier transform infrared spectroscopy (ATR-FTIR). The spectrum was recorded by a Perkin Elmer Spectrum 100
Please cite this article in press as: Laffleur F et al. Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.016
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ATR-FTIR spectrometer (Perkin Elmer, Waltham, USA) in combination with a Spectrum software version 6.3.1.0134 (Perkin Elmer, Waltham, USA). Samples were measured at a temperature of 22 °C. The testing compounds were split in six sub-samples. Each sub-sample was measured according to eight scans in the range from 4000 cm1 to 600 cm1 and a resolution of 4 cm1. 2.5.4. Quantification of the chitosan–TGA–MNA The amount of conjugated aromatic ligand was quantified with UV- spectrophotometer. For this assay a 0.1% (m/v) thiomer solution was measured at 307 nm. By adding reduced glutathione 0.1% (GSH) the aromatic ligand was exchanged, while quantification of the monomer release was measured at 307 nm. Due to Ellman0 s reaction the remaining free thiol groups immobilized were measured with a microplate reader (Tecan Fluostar; Galaxy BMG, Offenburg, Germany) [17].
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2.7. Evaluation of swelling behavior Test disks of chitosan–TGA–MNA conjugates, thiolated chitosan–TGA and unmodified chitosan were assessed in simulated salivary fluid by a gravimetric method as described previously [20,21]. For the experimental set up, polymer conjugates were mounted on a steel needle. Simulated salivary fluid comprising phosphate buffered saline solution (2.38 g Na2HPO4, 0.19 g KH2PO4 and 8.0 g NaCl per liter of demineralized water) adjusted with phosphoric acid to pH 6.75 at 37 ± 0.5 °C [22]. At predetermined time points, the swollen polymers were weighted in order to monitor the water uptake. The percentage of water uptake was calculated according to the following equation (Wt is the weight of the swollen polymer; W0 is the initial weight):
Water uptake ð%Þ ¼ ððW t W 0 Þ=W t Þ 100
2.6. Cytotoxicity screening
2.8. Evaluation of stability properties
2.6.1. Cell culture conditions Human colorectal carcinoma cell lines (Caco-2) were investigated for viability and cytotoxicity assays and maintained at 37 °C under 5% CO2 and 90% relative humidity in the minimum essential medium (MEM) supplemented with 20% (v/v) heat-inactivated (1 h at 56 °C) fetal calf serum, 2.2 g/L NaHCO3, 200 mM Lglutamine, penicillin (100 U/mL), and streptomycin (100 mg/mL).
Lyophilized unmodified chitosan, chitosan–TGA and chitosan– TGA–MNA were compressed to 30 mg (5.0 mm diameter, 1.2 mm thickness) flat-faced disks with a press (Paul Weber, Remshalden, Germany). During the compaction the force was kept at 10 kN for 20 s. Stability as well as disintegration properties of the disks were evaluated according to the USP 30, volume 1, year 2007. For this purpose the disintegration tester with six tubes was used (ERWEKA ZT 223, Heusenstamm, Germany). Disintegration studies were accomplished with thiolated chitosan, unmodified and preactivated chitosan. The disintegration assay was performed in simulated salivary fluid at 37 ± 0.5 °C with a disintegration test apparatus. Furthermore, oscillating frequency was adjusted to 0.5 s1 [23].
2.6.2. Cytotoxicity assay In order to determine the in vitro cytotoxicity the release of the enzyme lactate dehydrogenase (LDH) from non-viable Caco-2 cells was determined using a commercial test kit (Roche Diagnostics, Meylan, France). 100,000 cells per well were seeded into a 24-well plate. The plate was incubated at 37 °C in 5% CO2 environment. Cells were treated with chitosan, chitosan–TGA and chitosan– TGA–MNA 0.5% (m/v). MEM without phenol red served as negative control and Triton-X 100 2% (m/v) served as positive control, respectively. At time point zero, samples from the supernatant were stored at 4 °C. In the following step cells were incubated for 3 h and 24 h. Afterward samples were centrifuged to remove cell debris. The LDH content of the supernatant was measured using spectrophotometer at 492 nm. The percentage of cytotoxicity was determined by the following equation [18]:
Cytotoxicity ½% ¼
Av arage absorbance v alue of each triplicate negativ e control 100 positiv e control negativ e control
2.6.2. Resazurin assay Resazurin assay determined the cell viability of Caco-2 cells after treatment with testing polymer solutions. For this purpose 1 105 Caco-2 cells were seeded per well in 24-well plates and incubated in a humidified chamber at 37 °C, 5% CO2. Testing solutions were applied in a concentration of 0.5% (w/v). Negative control was minimum essential medium (MEM) without phenol red and 2% (m/v) and positive control was Triton-X 100 solution, respectively [19]. The cells were treated with the testing polymers chitosan–TGA–MNA, chitosan–TGA as well as unmodified chitosan for 3 h and 24 h. In the next step, the solutions were withdrawn and replaced with resazurin solution. After 3 h of incubation, fluorescence was measured using a spectrophotometer at 540 nm (excitation) and 590 nm (emission). The percentage of the cell viability was calculated using the following equation:
Cell v iability ½% ¼
Av erage absorbance v alue of each sample 100 Av erage absorbance v alue of negativ e control
2.9. Mucoadhesive assays 2.9.1. Rotating cylinder method Mucoadhesive properties of chitosan–TGA–MNA, corresponding chitosan–TGA and unmodified chitosan were investigated by working on the rotating cylinder method. The slaughterhouse provided the buccal mucosa, which was cut into pieces. The tissue was cleaned with 0.9% NaCl solution. On a cylinder (diameter: 4.4 cm; height: 5.1 cm; apparatus-cylinder, stainless, steel) the porcine buccal tissue was mounted by using cyanoacrylate adhesive glue (Henkel KGaA, Austria). Disks of chitosan–TGA–MNA, corresponding chitosan–TGA and unmodified chitosan were placed on the mucosa. In the following step the mounted stainless cylinders were steeped into vessels containing 900 mL of simulated salivary fluid. The dissolution apparatus according to the European Pharmacopoeia 7.0, volume 1, year 2011 was set at 37 ± 0.5 °C and rotated with 100 rpm. The mucoadhesive properties and the detachment of the polymers were observed during one week until the complete polymer was detached [24]. 2.9.2. Detachment assay Excised porcine buccal tissue was fixed on a glass platform. The chitosan–TGA–MNA, corresponding chitosan–TGA and unmodified chitosan disks were fixed on a stainless steel flat disk (10 mm in diameter). The flat disk was fixed on a laboratory stand. The mounted glass platform was transferred to a beaker containing simulated salivary fluid. This beaker was transferred to a balance put on a mobile platform. Now, the disk and the mucus were brought in contact. The force which is required to detach the disk from the mucosa was monitored. For this purpose, the mobile platform was lowered at a rate of 0.1 mm/s after 20 min incubation. Data were taken with the computer software (SartoCollect V 1.0;
Please cite this article in press as: Laffleur F et al. Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.016
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Fig. 1. Synthesis of chitosan–thioglycolic acid conjugate. Amide bond formation in between chitosan’s primary amino groups and the carboxylic acid groups of the thiol bearing ligand. (Reaction conditions: 1% chitosan solution, pH 5.8, 100 mM EDAC, TGA in a ratio of 1:1 stirred at room temperature for 3 h.) Synthesis of chitosan–TGA–MNA. By the first reaction of 6-chloronicotinamide with thiourea 6-mercaptonicotinamide (MNA) is resulting. (Reaction conditions: 6-chloronicotinamide with thiourea in the ratio 1:1, suspended in ethanol, stirred for 6 h.) In the second step, 6-MNA is oxidized to 6,60 -dithionicotinamide (6,60 -DTNA). (Reaction conditions: 6-MNA and H2O2; ratio 1:2, pH 7.) The last step was the addition of the compound to the thiomer to obtain preactivated chitosan. (Reaction conditions: pH 6.2, 6 h at room temperature.)
Fig. 2. Attenuated-total-reflectance Fourier transform infrared spectroscopy (ATR-FTIR) of 6-MNA black colored line and 6,60 -DTNA blue colored line, respectively.
Please cite this article in press as: Laffleur F et al. Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.016
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Fig. 3. Attenuated-total-reflectance Fourier transform infrared spectroscopy (ATR-FTIR) of Ch–TGA–MNA.
Sartorius AG, Germany) in connection to the balance with integrated interface. Afterward, calculations of the force versus displacement curves were made in order to obtain the maximum force of detachment (MDF) and the total work of adhesion (TWA). The TWA is AUC in accordance with the trapezoidal rule [17,20]. 2.9.3. Shear strength assay and peel strength assay The parallel detachment force is measured within the shear strength assay. Therefore freshly excised porcine buccal tissue was mounted with testing polymer disks. For the experimental set up the backside of the disks were mounted on a plastic strip with glue whereas the surface of the disk was kept on the buccal mucosa for a period of five min. The weight necessary for the detachment of the disks from the tissue was monitored after the incubation time. By means of the following equations the force of adhesion and the bond strength were calculated.
Force of adhesion ðNÞ ¼
weight ðgÞ 9:81 1000
Bond strength ðN=m2 Þ ¼
force of adhesion ðNÞ surface area of tablet ðm2 Þ
Evaluation of peel strength was performed by detecting the force which is required in order to detach the adhesive disk in a tangential manner from the buccal tissue. Bond strength and force of adhesion were calculated using the equations [25].
Fig. 4. Resazurin assay. Chitosan–TGA–MNA (Ch–TGA–MNA) conjugate, thiolated conjugates (Ch–TGA) and unmodified polymer (Ch) were tested on Caco-2 cells. Cell viability is expressed in percent. White bars and gray bars represent cell viability after 3 and 24 h, respectively. Indicated values are the means of at least five experiments (±SD).
2.10. Statistical data analysis For statistical analysis, analysis of variance and Student’s t test were used where appropriate. A probability of less than 0.05 (P < 0.05) was considered statistically significant. All results are presented as mean ± SD. 3. Results and discussion
primary amino group of chitosan. Synthesis of chitosan–TGA has already been described by our research group [11,26]. The thiol groups immobilized within chitosan–TGA conjugate were determined by Ellman’s test to be 353.78 ± 34 lmol per gram polymer. The lyophilized polymers exhibit white color and were odorless powder of fibrous structure. The polymer was stored at 4 °C until use and was stable toward air oxidation during the course of study. The chemical pathway is presented in Fig. 1.
3.1. Synthesis and characterization of chitosan–TGA 3.2. Synthesis of 6-MNA and 6,60 -DTNA The thiol bearing ligand namely thioglycolic acid was covalently attached to chitosan’s backbone due to amide bond formation between the carboxylic moieties of thioglycolic acid and the
6-mercaptonicotinamide was synthesized based on guidelines reported by Forrest et al. [16]. 6-MNA was a fine yellowish powder.
Please cite this article in press as: Laffleur F et al. Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.016
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Fig. 5. Water uptake studies of preactivated chitosan–TGA–MNA (white square), chitosan–TGA (black triangle) as well as unmodified (open triangle) in artificial saliva pH 6.75 at 37 °C. Indicated values are mean (±SD) of at least five experiments (significant difference between unmodified and thiolated chitosan at p = 0.015, between unmodified and preactivated chitosan at p = 0.015). 0
0
By the addition of H2O2 to 6-MNA, 6,6 -dithionicotinamide (6,6 DTNA) was obtained. 6,60 -DTNA was of white color and in the form of a powder. The percentage according to the initial weight of the compound as well as the purification process was almost 60%.
formation between thiol groups of the polymeric backbone and the aromatic ligand 6,60 -DTNA was achieved as shown in Fig. 1. The concept for these novel thiomers namely preactivated thiomers is based on the reaction scheme for covalent chromatography of resins such as thiopropylsepharose. The tendency of mercapto pyridine substructure toward nucleophilic attack is a further indication of the electron attracting character from free thiol groups. Pyridyl substructure was covalently linked to the thiomer where it can be eluted by the addition of reducing agents or compounds containing thiol groups [28]. The preactivation step of Ch–TGA was conducted in a solution of DMSO and demineralized water. The synthesized 6-MNA dimer is presented in Fig. 1. Disulfide bond formation was achieved between the thiol group of Ch–TGA and the thiol groups of the 6-MNA dimer. The structure of the preactivated polymer is depicted in Fig. 1. Ellman0 s test of preactivated Ch–TGA–MNA was performed in order to prove the grade of preactivation. The thiol group content was 47.38 ± 7.18 lmol per gram of polymer. These results showed that a successful coupling of MNA to the thiol group of chitosan took place. The lesser the amount of the thiol groups the higher was the amount of immobilized MNA. Furthermore, the amount of MNA was determined in the preactivated polymer, showing 60% activation with 212.26 ± 22.38 lmol MNA per gram of Ch–TGA. The proposed mechanism of the oxidative coupling was the covalent coupling of the MNA dimer to the free thiol group of the activated polymer and the removal of free MNA. All lyophilized conjugates were white, of fibrous structure and easily compressible to test disks. 3.5. Cell culture assays The cytotoxicity of chitosan, chitosan–TGA and preactivated chitosan (chitosan–TGA–MNA) was examined on Caco-2 cells at a
3.3. Characterization of 6-MNA and 6,60 -DTNA During UV-analyses two peaks were significant for of 6,60 DTNA, one at 297 nm and a minor one at 253 nm [27]. A peak at 307 nm described 6-MNA. Furthermore, DSC analyses were performed verifying the desired compounds. The literature reported values of 266–268 °C which are in good agreement with taken values of 6-MNA, monitoring a melting point at around 264 °C [16]. Moreover, the attenuated-total-reflectance Fourier transform infrared spectroscopy (ATR-FTIR) detected significant signals. 3161 cm1 and 3159 cm1 showing the C–H stretch of aromatic compounds, 1653 cm1 to 1619 cm1 indicating N–H bend and – C@C– stretch as well as 840 cm1 representing C–H bend, para, aromatic compound could be seen for 6-MNA and 6,60 -DTNA, respectively. The spectrum is presented in Fig. 2. The thiol function of 6-MNA was found as 2515 cm1 weak signal shown in Fig. 2. For the following synthesis 6,60 -DTNA was identified as a preactivation compound for the thiolated chitosan. The IR spectrum shown in Fig. 3 displayed the typical characteristic signals of the Ch–TGA– MNA. 3500–3200 cm1 represents the O–H bend, 1700–1580 expresses the amide bend and the carboxamide bend of the coupling reagent. The aromatic ring (–C@C–) is found at 1550 cm1, 1404 cm1 expresses the –NH– bend, whereas 1100–1000 cm1 indicates the –C–OH as well as the CH2–OH bend. Both spectra of FTIR indicate the succeeding synthesis of chitosan’s preactivation with 6,60 -DTNA . 3.4. Chitosan–TGA–MNA synthesis Chitosan–TGA–MNA was obtained according to a method described previously by our research group [15]. Disulfide bond
Fig. 6. Disintegration behavior study in artificial saliva pH 6.75 at 37 °C of preactivated chitosan, thiolated and unmodified chitosan. The indicated disintegration time represents an average of at least five experiments (±SD) (significant difference between unmodified and thiolated chitosan (p = 0.014), between unmodified and preactivated chitosan (p = 0.024)).
Please cite this article in press as: Laffleur F et al. Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.016
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properties as well as disintegration and drug release. Adhesiveness taking place in between mucus and polymer could be explained by many processes like absorbing, water uptake as well as capillarity processes. The water uptake findings are depicted in Fig. 5, it could be proven that the thiolation step had a high influence on the swelling behavior. By contrast, the water uptake property was diminished when the aromatic ligand was attached compared to the thiolated. Preactivated chitosan shows higher swelling behavior than unmodified chitosan. A steady and fast weight increase could be observed over a time period of 1 h for thiolated chitosan. A maximum of 63 mg was monitored for thiolated chitosan. This is a 2.1fold increase compared to the initial weight. Preactivated chitosan reached continuously a maximum of 66.58 mg over a time period of 2 h. This correlates with a 2.2-fold increase in the initial weight (significant difference between unmodified and thiolated chitosan (p = 0.015), between unmodified and preactivated chitosan (p = 0.015)). The advantage of depressed water uptake profile for prolonged mucoadhesion was discussed by Mortazavi et al. [29]. It was mentioned that overhydration would be the outcome of an exhaustive water uptake, producing a slippery mucilage and resulting in lower mucoadhesiveness [30]. On these grounds, constant and slow water uptake of preactivated chitosan might show advantages in overcoming overhydration and non- adhesion. 3.7. Stability properties
Fig. 7. Adhesion time assay of preactivated chitosan, chitosan–TGA as well as unmodified chitosan with the rotating cylinder method. Indicated values are means (±SD) of at least five experiments. Significant difference between unmodified and thiolated chitosan (p = 0.001), between unmodified and preactivated chitosan (p = 0.001).
concentration of 0.5% (m/v) for 3 h and 24 h. All conjugates displayed more than 80% cell viability as depicted in Fig. 4. Cell viability of more than 80% after 24 h indicated a negligible effect for the cell monolayer. These results were in close correlation with the LDH findings of tested chitosan conjugates. All polymers were tested in a final concentration of 0.5% (m/v). Moreover, the chosen concentration allowed comparing cytotoxicity of the preactivated polymers to that of well-established thiomers. Cell viability of more than 86% and 80% in the presence of chitosan–TGA–MNA after 3 h and 24 h, respectively, indicated that these polymers were not harmful to the cells. 3.6. Evaluation of swelling behavior Water uptake of a mucoadhesive matrix or dosage form is part of a network being responsible for stability, adhesive and cohesive
Stability properties of chitosan–TGA and preactivated chitosan– TGA–MNA were compared with unmodified polymer. The disintegration of unmodified chitosan was more pronounced compared to its derivatives. As a consequence the stability of the polymers matrix of either chitosan–TGA or chitosan–TGA–MNA exhibited a much higher stability in comparison to the unmodified one. By covalent attachment of 6-mercaptonicotinamide (MNA) to the polymeric backbone of thiolated chitosan the stability of these thiomers was significantly enhanced. A 5.2-fold prolonged disintegration times were obtained in the presence of preactivated chitosan conjugates in comparison with the thiolated one (significant difference between unmodified and thiolated chitosan (p = 0.014), between unmodified and preactivated chitosan (p = 0.024)). Explanations for these findings could be given in the particulate nature of MNA together with the higher amount of inter- and/or intramolecular disulfide bonds within the conjugate which are responsible for the improved stability and hardness of preactivated chitosan. The theory of forming covalent bonds disulfide bonds- within the polymer network which resulted in pronounced stability as well as cohesion was in good agreement with the results given from disintegration studies. Stability and cohesion have a great impact on the disintegration time. This gives the reason that when MNA is introduced as thiol protecting group
Fig. 8. In vitro shear strength study of preactivated, thiolated and unmodified chitosan determined. Indicated values are the means (±SD) of at least five experiments (significant difference between preactivated and unmodified chitosan (p = 0.010), between preactivated and thiolated chitosan (p = 0.044)).
Please cite this article in press as: Laffleur F et al. Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.016
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Fig. 9. In vitro peel strength assay preactivated chitosan, thiolated as well as unmodified chitosan. Indicated values are the mean (±SD) of at least five experiments (significant difference between preactivated and unmodified chitosan (p = 0.002), between preactivated and thiolated chitosan (p = 0.004)).
by the particulate nature of MNA. Furthermore, the formed interand/or intramolecular disulfide bonds within the conjugate could be the reason for its enhanced cohesiveness and hardness of chitosan–TGA–MNA. Fig. 6 represents the findings of the stability evaluation.
3.8. Mucoadhesive strength -in vitro evaluation
Fig. 10. Mucoadhesiveness of preactivated chitosan, thiolated as well as unmodified chitosan determined by tensile studies. Black bars represent the TWA and white bars represent the MDF, respectively. Indicated values are the mean (± SD) of at least five experiments. TWA shows significant difference between unmodified and thiolated chitosan (p = 0.003) between thiolated and preactivated chitosan (p = 0.012) and MDF shows significant difference between unmodified and preactivated chitosan (p = 0.001) between thiolated and preactivated chitosan (p = 0.028).
a more prolonged disintegration time occurred. The prolonged disintegration time might be caused by the lipophilic nature of MNA increasing the hydrophobicity of the polymer conjugate combined with an increase in the formation of inter- and/or intramolecular disulfide bonds since this crosslinking shows a great impact on chain mobility and resistance to dissolution. The mucoadhesion process is initiated by two stages, the contact stage and the consolidation stage. The contact stage is described as the art where the polymer comes in contact with the mucosa, therefore it has to show high stability [31]. For the consolidation stage the polymer should be able to interact with the mucosa regarding the formation of mucoadhesive bonds in order to act as a lubricant which is needed in dry mouth syndrome. An explanation could be given
A variety of mucoadhesive strength assays were conducted based on preactivated chitosan conjugate, chitosan–TGA and unmodified one, respectively. Fig. 7 shows the results of the rotating cylinder test. 3.1-fold improvement is gained due to the preactivation procedure of thiolated chitosan in comparison with thiolated chitosan (significant difference between unmodified and thiolated chitosan (p = 0.001), between unmodified and preactivated chitosan (p = 0.001)). Furthermore, peel, shear, and detachment strength assays were performed on freshly excised buccal mucosa. Shear strength results are shown in Fig. 8 and peel strength is depicted in Fig. 9, respectively. Detachment strength and findings such as total work of adhesion (TWA) (significant difference between unmodified and thiolated chitosan (p = 0.003), between thiolated and preactivated chitosan (p = 0.012)) and maximum detachment force (MDF) (significant difference between unmodified and preactivated chitosan (p = 0.001), between thiolated and preactivated chitosan (p = 0.028)) are depicted in Fig. 10. The correlation coefficient for the correlation between MDF and TWA was 0.987. As a result of the detachment study, chitosan–TGA and preactivated chitosan–TGA–MNA demonstrated a higher MDF and the TWA, respectively compared to unmodified chitosan. The improved MDF and TWA were attributed to the more pronounced attraction of the thiolated and preactivated chitosan conjugates on the buccal mucosa. In the case of preactivated chitosan–TGA, TWA was enhanced 20.5-fold in comparison with unmodified chitosan. In contrast, chitosan–TGA exhibited an increase of 14.3-fold of TWA in comparison with unmodified chitosan. Good correlations between MDF and TWA were obtained regarding all tested polymers. Due to these observations, the theory of improved mucoadhesive properties of thiolated polymers, that the thiol groups of the backbone forms disulfide bonds with cysteine substructures of the mucus, could be proven [32,33]. Within this study, buccal porcine tissue was chosen as mucosal platform for the assessment of mucoadhesive strength. The higher the prolonged residence time the lower the interval of drug application leading to a better patient compliance. Benefits associated with the prolonged residence time are increase of drug concentration at the target site. Taking these mucoadhesive findings in consideration, preactivated conjugate is considered as potential representative for a dosage form treating xerostomia.
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4. Conclusion Findings obtained within this present study suggest preactivated thiolated chitosan as a potential matrix for local buccal drug delivery. The synthesis of chitosan–TGA–MNA, thioglycolic acid as thiol bearing ligand was covalently attached through the amide bond formation to the polymeric backbone. The preactivation of the thiolated polymer results in enhanced mucoadhesive features. The augmented mucoadhesion was proven by a 3.1-fold enhancement of remaining on the mucosa due to the preactivation step of thiolated chitosan in comparison with thiolated chitosan as well as by the increase in the total work of adhesion in detachment strength assays. These findings lead to improved mucoadhesiveness which has a great impact on applications where prolonged residence time is required. Water uptake properties as well as stability of preactivated chitosan were enhanced in comparison to unmodified one. Through the preactivation and disulfide bond formation preactivated chitosan exhibited more pronounced cohesive properties as well as beneficial prolonged residence time on buccal mucosae. The preactivated thiolated chitosan synthesized in this present study paves the way for being a promising candidate in the treatment of the dry mouth syndrome due to its mucoadhesive and water uptake properties. Conflict of interest The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. The author reports no declaration of interest. Acknowledgement The work was supported by the University of Innsbruck and the FWF project no. ZFP 235150. Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Fig. 2, are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2015. 04.016. References [1] Asane GS, Nirmal SA, Rasal KB, Naik AA, Mahadik MS, Rao YM. Polymers for mucoadhesive drug delivery system: a current status. Drug Dev Ind Pharm 2008;34(11):1246–66. [2] Sakloetsakun D, Iqbal J, Millotti G, Vetter A, Bernkop-Schnurch A. Thiolated chitosans: influence of various sulfhydryl ligands on permeation-enhancing and P-gp inhibitory properties. Drug Dev Ind Pharm 2011;37(6):648–55. [3] Gannot G, Lancaster HE, Fox PC. Clinical course of primary Sjogren’s syndrome: salivary, oral, and serologic aspects. J Rheumatol 2000;27(8):1905–9. [4] Hearnden V, Sankar V, Hull K, et al. New developments and opportunities in oral mucosal drug delivery for local and systemic disease. Adv Drug Delivery Rev 2012;64(1):16–28. [5] Scully C. Drug effects on salivary glands: dry mouth. Oral Dis 2003; 9(4):165–76.
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[6] Fox RI. Sjogren’s syndrome. Lancet 2005;366(9482):321–31. [7] Garcia-Carrasco M, Ramos-Casals M, Rosas J, et al. Primary sjogren syndrome: clinical and immunologic disease patterns in a cohort of 400 patients. Medicine (Baltimore) 2002;81(4):270–80. [8] Sudhakar Y, Kuotsu K, Bandyopadhyay AK. Buccal bioadhesive drug delivery–a promising option for orally less efficient drugs. J Control Release 2006; 114(1):15–40. [9] Patel VF, Liu F, Brown MB. Advances in oral transmucosal drug delivery. J Control Release 2011;153(2):106–16. [10] Laffleur F, Bernkop-Schnurch A. Thiomers: promising platform for macromolecular drug delivery. Future Med Chem 2012;4(17):2205–16. [11] Laffleur F, Hintzen F, Rahmat D, Shahnaz G, Millotti G, Bernkop-Schnurch A. Enzymatic degradation of thiolated chitosan. Drug Dev Ind Pharm 2012. [12] Dunnhaupt S, Barthelmes J, Thurner CC, Waldner C, Sakloetsakun D, BernkopSchnurch A. S-protected thiolated chitosan: synthesis and in vitro characterization. Carbohydr Polym 2012;90(2):765–72. [13] Shahnaz G, Iqbal J, Rahmat D, et al. Development and in vivo characterization of a novel peptide drug delivery system providing extended plasma half life. J Control Release 2012;157(3):375–82. [14] Rahmat D, Sakloetsakun D, Shahnaz G, Sarti F, Laffleur F, Schnurch AB. HEC-cysteamine conjugates: influence of degree of thiolation on efflux pump inhibitory and permeation enhancing properties. Int J Pharm 2012; 422(1–2):40–6. [15] Dunnhaupt S, Barthelmes J, Rahmat D, et al. S-protected thiolated chitosan for oral delivery of hydrophilic macromolecules: evaluation of permeation enhancing and efflux pump inhibitory properties. Mol Pharm 2012;9(5):1331–41. [16] Forrest HS, Walker J. Chemotherapeutic agents of the sulphone type; 2: 5-disubstituted derivatives of pyridine. J Chem Soc 1948;70:1939–45. [17] Iqbal J, Shahnaz G, Dunnhaupt S, Muller C, Hintzen F, Bernkop-Schnurch A. Preactivated thiomers as mucoadhesive polymers for drug delivery. Biomaterials 2012;33(5):1528–35. [18] Loretz B, Bernkop-Schnurch A. In vitro evaluation of chitosan–EDTA conjugate polyplexes as a nanoparticulate gene delivery system. AAPS J 2006;8(4):E756–64. [19] Rahmat D, Muller C, Shahnaz G, et al. HEC-cysteamine particles: influence of particle size, zeta potential, morphology and sulfhydryl groups on permeation enhancing properties. Drug Dev Ind Pharm 2012. [20] Kast CE, Bernkop-Schnurch A. Thiolated polymers–thiomers: development and in vitro evaluation of chitosan–thioglycolic acid conjugates. Biomaterials 2001;22(17):2345–52. [21] Sarti F, Muller C, Iqbal J, Perera G, Laffleur F, Bernkop-Schnurch A. Development and in vivo evaluation of an oral vitamin B12 delivery system. Eur J Pharm Biopharm 2013;84(1):132–7. [22] Saboktakin MR, Tabatabaie RM, Maharramov A, Ramazanov MA. Development and in vitro evaluation of thiolated chitosan–poly(methacrylic acid) nanoparticles as a local mucoadhesive delivery system. Int J Biol Macromol 2011;48(3):403–7. [23] Laffleur F, Shahnaz G, Islambulchilar Z, Bernkop-Schnurch A. Design and in vitro evaluation of a novel polymeric excipient for buccal applications. Future Med Chem 2013;5(5):511–22. [24] Bernkop-Schnurch A, Steininger S. Synthesis and characterisation of mucoadhesive thiolated polymers. Int J Pharm 2000;194(2):239–47. [25] Metia PK, Bandyopadhyay AK. In vitro evaluation of novel mucoadhesive buccal tablet of oxytocin prepared with diospyros peregrina fruit mucilages. Yakugaku Zasshi 2008;128(4):603–9. [26] Shahnaz G, Vetter A, Barthelmes J, et al. Thiolated chitosan nanoparticles for the nasal administration of leuprolide: bioavailability and pharmacokinetic characterization. Int J Pharm 2012;428(1–2):164–70. [27] Dunnhaupt S, Barthelmes J, Iqbal J, et al. In vivo evaluation of an oral drug delivery system for peptides based on S-protected thiolated chitosan. J Control Release 2012;160(3):477–85. [28] Bernkop-Schnurch A, Krauland AH, Leitner VM, Palmberger T. Thiomers: potential excipients for non-invasive peptide delivery systems. Eur J Pharm Biopharm 2004;58(2):253–63. [29] Mortazavi SA, Smart JD. An investigation into the role of water-movement and mucus gel dehydration in mucoadhesion. J Control Release 1993; 25(3):197–203. [30] Duchene D, Ponchel G. Principle and investigation of the bioadhesion mechanism of solid dosage forms. Biomaterials 1992;13(10):709–14. [31] Boddupalli BM, Mohammed ZN, Nath RA, Banji D. Mucoadhesive drug delivery system: an overview. J Adv Pharm Technol Res 2010;1(4):381–7. [32] Salamat-Miller N, Chittchang M, Johnston TP. The use of mucoadhesive polymers in buccal drug delivery. Adv Drug Deliver Rev 2005;57(11):1666–91. [33] Shinkar DM, Dhake AS, Setty CM. Drug delivery from the oral cavity: a focus on mucoadhesive buccal drug delivery systems. PDA J Pharm Sci Technol 2012;66(5):466–500.
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Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome Flavia Laffleur a, Alexander Fischer a, Matthias Schmutzler b, Fabian Hintzen a, Andreas Bernkop-Schnürch a,⇑ a b
Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Austria Institute of Analytical Chemistry & Radiochemistry, CCB – Center for Chemistry and Biomedicine, University of Innsbruck, Austria
a r t i c l e
i n f o
Article history: Received 23 September 2014 Received in revised form 2 April 2015 Accepted 14 April 2015 Available online xxxx Keywords: Thiomers Chitosan–thioglycolic acid Mercaptonicotinamide Preactivated thiomers Dry mouth syndrome
a b s t r a c t Purpose: The objective of this study was to investigate preactivated thiomers for their potential in the treatment of dry mouth syndrome. Methods: Chitosan–thioglycolic–mercaptonicotinamide conjugates (chitosan–TGA–MNA) were synthesized by the oxidative S–S coupling of chitosan–thioglycolic acid (chitosan–TGA) with 6-mercaptonicotinamide (MNA). Test disks were compressed out of unmodified chitosan, chitosan–TGA (thiomers) and chitosan–TGA–MNA conjugates to investigate cohesive properties, cytotoxicity assays and mucoadhesion studies. Results: Immobilizing the MNA achieved higher swelling and cohesive properties of chitosan–TGA–MNA conjugates compared to unmodified chitosan. Rotating cylinder studies displayed a 3.1-fold improvement of mucoadhesiveness of chitosan–TGA–MNA conjugates compared to thiolated polymers. Findings in tensile strength were in good agreement with rotating cylinder ones. Furthermore, preactivated thiomers exhibit higher stability. All conjugates were found non-toxic against Caco-2 cells. Conclusion: Preactivated thiolated chitosan could be a promising system for the treatment of dry mouth syndrome where mucosa requires lubrication and mucoadhesiveness. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Conventional mucoadhesive materials such as carbopol, polycarbophil or sodium carboxymethylcellulose are to a large extent hydrophilic compounds containing numerous hydrogen bond forming groups [1]. Conventional polymers form non-covalent bonds with mucus substructures which result in mainly weak mucoadhesion. However, functionalized polymers bearing sulfhydryl ligands on the polymeric backbone or designed thiomers show the ability to form covalent bonds with cysteine-rich subunits of mucus. By virtue of these features, both benefits of prolonged cohesion and strong mucoadhesion are met in the case of designated thiomers. The interactions of mucus and thiomer are, however, likely to be most intense in the deprotonated state of sulfhydryl groups [2]. Almost all oral degenerative diseases, including tooth decay, are associated with a low pH (acidic) in the body ⇑ Corresponding author at: Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria. Tel.: +43 512 507 58600; fax: +43 512 507 58699. E-mail address: [email protected] (A. Bernkop-Schnürch).
[3,4]. People with a dry mouth have mouth acidity caused by saliva thickening or dying up because of various medications such as anticholinergics, sympathomimetics, anti-HIV drugs as well as bezodiazepines [5] and dehydration [6,7]. Since xerostomia goes hand in hand with a leaky or even lacking mucus gel layer covering intraoral epithelia and consequently reduced properties of the mucosa to keep sufficient moisture on its surface [8,9], one strategy to increase salivary secretion could be using mucoadhesive polymers such as cellulose derivates as lubricants. Thiomers, however, are unstable toward oxidation and aqueous thiomer solutions are unstable. Therefore a novel second generation of thiomers, the so-called preactivated thiomers have been developed exhibiting stability toward oxidation as well as improved mucoadhesion [10]. To overcome these limitations preactivated thiomers bearing an additional ligand with a heteroaromatic structure reacting in a pH independent manner were designed and evaluated within this study. The thiol groups are protected via disulfide bonds due to the additional aromatic ligand 6-mercaptonicotinic amid (MNA). Furthermore chitosan–TGA–MNA is protected against early oxidation. Prevention of an early oxidation before coming into contact with the mucus layer occurs. Hence, more active thiol groups are
http://dx.doi.org/10.1016/j.actbio.2015.04.016 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Laffleur F et al. Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.016
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available for a long intimate contact with mucosal membranes. To date, however, neither thiomers nor preactivated thiomers were investigated regarding their potential for dry mouth syndrome. According to this, it is the aim of this study to evaluate the positive influence of thiomers and especially preactivated thiomers on mucoadhesion for their potential as therapeutic agents for dry mouth syndrome. 2. Material and methods 2.1. Materials Chitosan (low molecular mass 50–100 kDa; degree of deacetylation, 83–85%) was obtained from Fluka Chemie (Buchs, Switzerland). Ellman’s reagent [5,50 dithiobis(2-nitrobenzoic acid) (DTNB), reduced-form glutathione (GSH), tris(2-carboxylethyl)phosphine hydrochloride (TCEP), 6-chlornicotinamide, thiourea and thioglycolic acid, 5,5-dithiobis(2-nitrobenzoic acid) were purchased from Sigma Aldrich, Steinheim, Germany. All other chemicals, reagents and solvents were of analytical grade and were received from commercial sources. 2.2. Synthesis of chitosan thioglycolic acid conjugates Thioglycolic acid was covalently attached to chitosan as previously described by our research group [11]. Thiolated chitosan was synthesized by the covalent attachment of thioglycolic acid to chitosan which formed amide bonds in between the primary amino group of chitosan and the carboxylic acid moieties of the sulfhydryl agent. Briefly, 500 mg of chitosan (low molecular mass 50–100 kDa; degree of deacetylation, 83–85%) was dissolved in 1 M HCl by adding demineralized water in order to obtain a 1% solution of chitosan hydrochloride. In the next step the coupling reaction was mediated by EDAC in a final concentration of 100 mM in order to activate the carboxylic acid moieties of the subsequently added 500 mg of TGA. The reaction mixture was incubated for 3 h at room temperature under constant stirring. Samples prepared by applying the exact same method with the exception of omitting EDAC during the coupling reaction served as controls for the analytical studies. The reaction mixtures were dialyzed in tubings in order to eliminate unbound TGA and to isolate polymer conjugates. The dialysis process is a washing process of the synthesized mixture. For this purpose the synthesized polymer was transferred into dialysis tubing (Cellulose hydrate, cut-off 12.000 Da, Carl Roth, Karlsruhe, Germany). For purifying reasons as well as to get rid of the unbound ligand, the polymer mixture was three times dialyzed against demineralized water containing 1% NaCl and twice against demineralized water. Afterward, samples and controls were lyophilized by drying frozen aqueous polymer solutions at 30 °C and 0.01 mbar (Christ Beta; Germany) (primary drying conditions of 23 h, beginning with 3 h at 30 °C, augmenting to 15 °C for 3 h , up to 10 °C for 2 h, following 17 h at 0 °C; secondary conditions of 8 h, starting with 1 h at +20 °C and 7 h at +25 °C) and finally stored at 4 °C until further use.
was obtained. This was filtered and dried. By adding 2 M NaOH in the second proceeding step the compound was precipitated. Subsequently, it was stirred at room temperature for 45 min. In the following procedure, the pH was adjusted to 4, where the mixture turned into dark yellowish color. By this adjustment, mercaptonicotinamide (MNA) as the synthesized compound was obtained. This was purified by filtration, washed with acetone and dried. 2.3.2. 6,60 -Dithionicotinamide (6,60 -DTNA)- synthesis and purification A suspension of 6-MNA was adjusted to a pH of 7, afterward H2O2 solution was added. This oxidizing reaction was stirred for 1 h at room temperature. During this reaction the yellow colored compound rendered into the white compound 6,6-dithionicotinamide (6,60 -DTNA). 6,60 -DTNA was filtered, washed with water and dried as mentioned above. 2.4. Synthesis of chitosan–thioglycolic acid–mercaptonicotinamide The aromatic ligand was covalently attached by disulfide bond formation. 200 mg of chitosan–TGA was dissolved in a 50 mL mixture of demineralized water/DMSO (3:7) under stirring. Next, 50 mg of 6,60 -DTNA dimer dissolved in 50 mL of DMSO was added and the pH adjusted to 6.2 with 1 M NaOH. The mixture was stirred for 6 h at room temperature. In order to separate the conjugate from the unbound MNA dimer, the reaction solution was dialyzed seven times against 5 L of demineralized water under stirring in the dark at 10 °C. The dialysate was replaced every 12 h. The obtained product was freeze dried for 31 h as mentioned above and stored dry at 4 °C. 2.5. Analyses of thiolated and preactivated chitosan 2.5.1. Spectrophotometric quantification of the thiol groups Immobilization of thiol groups on the chitosan thioglycolic acid backbone was quantified by Ellman0 s reaction which determines the free thiol groups [13]. Polymers and thiomers were hydrated in 0.5 M phosphate buffer pH 8.0. Ellman’s reagent (5,50 -dithiobis (2-nitrobenzoic acid) (DTNB) was dissolved in 0.5 M phosphate buffer pH 8.0) and added to the polymer solutions. After an incubation time of 120 min protected from light, the absorbance of each sample was measured with a microplate reader (Tecan Fluostar; Galaxy BMG, Offenburg, Germany). The quantity of thiol groups being immobilized on the chitosan thioglycolic acid conjugate was determined by referring to cysteine standards [14].
2.3. 6-mercaptonicotinamide (MNA) and 6,60 -dithionicotinamide (6,60 -DTNA) synthesis
2.5.2. Characterization of 6-MNA and 6,60 -DTNA 6-MNA and 6,60 -DTNA were soluble in DMSO. UV-spectrometer measurements were performed with a UV–VIS spectrophotometer (UV mini-1240, Shimadzu, Kyoto, Japan) [15]. 6-MNA revealed a peak at 307 nm and 6,60 -DTNA showed two peaks, one expressive at 297 nm and a second minor one at 253 nm. Furthermore DSC thermograms were evaluated, according to the literature melting points of around 266 °C and 264 °C are expected [16]. For DSC studies, approximately, 2 mg of sample was taken in an aluminum pan, sealed with an aluminum cap and kept under nitrogen purging (atmosphere). Samples were scanned from 0 °C to 300 °C with the scanning rate of 10 °C/min using differential scanning calorimeter (DSC-60, Shimadzu, Japan).
2.3.1. 6-mercaptonicotinamide- synthesis and purification 6-mercaptonicotinamide was synthesized during a two-step synthesis [12]. For this synthesis, a suspension of 6-chlornicotinamide and thiourea in ethanol was generated and refluxed for 6 h at boiling temperature. After the suspension turned yellow, the mixture was stirred for 12 h at room temperature. In this first step the compound S-(5-carbamyl-2-pyridyl) thiuronium chloride
2.5.3. FT-IR spectra of the 6-MNA, dimer 6,60 -DTNA and the polymer conjugates In order to verify the syntheses FT-IR analyses of 6,60 -DTNA, MNA and conjugates were undertaken. Characterization of 6MNA and 6,60 -DTNA was conducted with attenuated-totalreflectance Fourier transform infrared spectroscopy (ATR-FTIR). The spectrum was recorded by a Perkin Elmer Spectrum 100
Please cite this article in press as: Laffleur F et al. Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.016
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ATR-FTIR spectrometer (Perkin Elmer, Waltham, USA) in combination with a Spectrum software version 6.3.1.0134 (Perkin Elmer, Waltham, USA). Samples were measured at a temperature of 22 °C. The testing compounds were split in six sub-samples. Each sub-sample was measured according to eight scans in the range from 4000 cm1 to 600 cm1 and a resolution of 4 cm1. 2.5.4. Quantification of the chitosan–TGA–MNA The amount of conjugated aromatic ligand was quantified with UV- spectrophotometer. For this assay a 0.1% (m/v) thiomer solution was measured at 307 nm. By adding reduced glutathione 0.1% (GSH) the aromatic ligand was exchanged, while quantification of the monomer release was measured at 307 nm. Due to Ellman0 s reaction the remaining free thiol groups immobilized were measured with a microplate reader (Tecan Fluostar; Galaxy BMG, Offenburg, Germany) [17].
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2.7. Evaluation of swelling behavior Test disks of chitosan–TGA–MNA conjugates, thiolated chitosan–TGA and unmodified chitosan were assessed in simulated salivary fluid by a gravimetric method as described previously [20,21]. For the experimental set up, polymer conjugates were mounted on a steel needle. Simulated salivary fluid comprising phosphate buffered saline solution (2.38 g Na2HPO4, 0.19 g KH2PO4 and 8.0 g NaCl per liter of demineralized water) adjusted with phosphoric acid to pH 6.75 at 37 ± 0.5 °C [22]. At predetermined time points, the swollen polymers were weighted in order to monitor the water uptake. The percentage of water uptake was calculated according to the following equation (Wt is the weight of the swollen polymer; W0 is the initial weight):
Water uptake ð%Þ ¼ ððW t W 0 Þ=W t Þ 100
2.6. Cytotoxicity screening
2.8. Evaluation of stability properties
2.6.1. Cell culture conditions Human colorectal carcinoma cell lines (Caco-2) were investigated for viability and cytotoxicity assays and maintained at 37 °C under 5% CO2 and 90% relative humidity in the minimum essential medium (MEM) supplemented with 20% (v/v) heat-inactivated (1 h at 56 °C) fetal calf serum, 2.2 g/L NaHCO3, 200 mM Lglutamine, penicillin (100 U/mL), and streptomycin (100 mg/mL).
Lyophilized unmodified chitosan, chitosan–TGA and chitosan– TGA–MNA were compressed to 30 mg (5.0 mm diameter, 1.2 mm thickness) flat-faced disks with a press (Paul Weber, Remshalden, Germany). During the compaction the force was kept at 10 kN for 20 s. Stability as well as disintegration properties of the disks were evaluated according to the USP 30, volume 1, year 2007. For this purpose the disintegration tester with six tubes was used (ERWEKA ZT 223, Heusenstamm, Germany). Disintegration studies were accomplished with thiolated chitosan, unmodified and preactivated chitosan. The disintegration assay was performed in simulated salivary fluid at 37 ± 0.5 °C with a disintegration test apparatus. Furthermore, oscillating frequency was adjusted to 0.5 s1 [23].
2.6.2. Cytotoxicity assay In order to determine the in vitro cytotoxicity the release of the enzyme lactate dehydrogenase (LDH) from non-viable Caco-2 cells was determined using a commercial test kit (Roche Diagnostics, Meylan, France). 100,000 cells per well were seeded into a 24-well plate. The plate was incubated at 37 °C in 5% CO2 environment. Cells were treated with chitosan, chitosan–TGA and chitosan– TGA–MNA 0.5% (m/v). MEM without phenol red served as negative control and Triton-X 100 2% (m/v) served as positive control, respectively. At time point zero, samples from the supernatant were stored at 4 °C. In the following step cells were incubated for 3 h and 24 h. Afterward samples were centrifuged to remove cell debris. The LDH content of the supernatant was measured using spectrophotometer at 492 nm. The percentage of cytotoxicity was determined by the following equation [18]:
Cytotoxicity ½% ¼
Av arage absorbance v alue of each triplicate negativ e control 100 positiv e control negativ e control
2.6.2. Resazurin assay Resazurin assay determined the cell viability of Caco-2 cells after treatment with testing polymer solutions. For this purpose 1 105 Caco-2 cells were seeded per well in 24-well plates and incubated in a humidified chamber at 37 °C, 5% CO2. Testing solutions were applied in a concentration of 0.5% (w/v). Negative control was minimum essential medium (MEM) without phenol red and 2% (m/v) and positive control was Triton-X 100 solution, respectively [19]. The cells were treated with the testing polymers chitosan–TGA–MNA, chitosan–TGA as well as unmodified chitosan for 3 h and 24 h. In the next step, the solutions were withdrawn and replaced with resazurin solution. After 3 h of incubation, fluorescence was measured using a spectrophotometer at 540 nm (excitation) and 590 nm (emission). The percentage of the cell viability was calculated using the following equation:
Cell v iability ½% ¼
Av erage absorbance v alue of each sample 100 Av erage absorbance v alue of negativ e control
2.9. Mucoadhesive assays 2.9.1. Rotating cylinder method Mucoadhesive properties of chitosan–TGA–MNA, corresponding chitosan–TGA and unmodified chitosan were investigated by working on the rotating cylinder method. The slaughterhouse provided the buccal mucosa, which was cut into pieces. The tissue was cleaned with 0.9% NaCl solution. On a cylinder (diameter: 4.4 cm; height: 5.1 cm; apparatus-cylinder, stainless, steel) the porcine buccal tissue was mounted by using cyanoacrylate adhesive glue (Henkel KGaA, Austria). Disks of chitosan–TGA–MNA, corresponding chitosan–TGA and unmodified chitosan were placed on the mucosa. In the following step the mounted stainless cylinders were steeped into vessels containing 900 mL of simulated salivary fluid. The dissolution apparatus according to the European Pharmacopoeia 7.0, volume 1, year 2011 was set at 37 ± 0.5 °C and rotated with 100 rpm. The mucoadhesive properties and the detachment of the polymers were observed during one week until the complete polymer was detached [24]. 2.9.2. Detachment assay Excised porcine buccal tissue was fixed on a glass platform. The chitosan–TGA–MNA, corresponding chitosan–TGA and unmodified chitosan disks were fixed on a stainless steel flat disk (10 mm in diameter). The flat disk was fixed on a laboratory stand. The mounted glass platform was transferred to a beaker containing simulated salivary fluid. This beaker was transferred to a balance put on a mobile platform. Now, the disk and the mucus were brought in contact. The force which is required to detach the disk from the mucosa was monitored. For this purpose, the mobile platform was lowered at a rate of 0.1 mm/s after 20 min incubation. Data were taken with the computer software (SartoCollect V 1.0;
Please cite this article in press as: Laffleur F et al. Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.016
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Fig. 1. Synthesis of chitosan–thioglycolic acid conjugate. Amide bond formation in between chitosan’s primary amino groups and the carboxylic acid groups of the thiol bearing ligand. (Reaction conditions: 1% chitosan solution, pH 5.8, 100 mM EDAC, TGA in a ratio of 1:1 stirred at room temperature for 3 h.) Synthesis of chitosan–TGA–MNA. By the first reaction of 6-chloronicotinamide with thiourea 6-mercaptonicotinamide (MNA) is resulting. (Reaction conditions: 6-chloronicotinamide with thiourea in the ratio 1:1, suspended in ethanol, stirred for 6 h.) In the second step, 6-MNA is oxidized to 6,60 -dithionicotinamide (6,60 -DTNA). (Reaction conditions: 6-MNA and H2O2; ratio 1:2, pH 7.) The last step was the addition of the compound to the thiomer to obtain preactivated chitosan. (Reaction conditions: pH 6.2, 6 h at room temperature.)
Fig. 2. Attenuated-total-reflectance Fourier transform infrared spectroscopy (ATR-FTIR) of 6-MNA black colored line and 6,60 -DTNA blue colored line, respectively.
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Fig. 3. Attenuated-total-reflectance Fourier transform infrared spectroscopy (ATR-FTIR) of Ch–TGA–MNA.
Sartorius AG, Germany) in connection to the balance with integrated interface. Afterward, calculations of the force versus displacement curves were made in order to obtain the maximum force of detachment (MDF) and the total work of adhesion (TWA). The TWA is AUC in accordance with the trapezoidal rule [17,20]. 2.9.3. Shear strength assay and peel strength assay The parallel detachment force is measured within the shear strength assay. Therefore freshly excised porcine buccal tissue was mounted with testing polymer disks. For the experimental set up the backside of the disks were mounted on a plastic strip with glue whereas the surface of the disk was kept on the buccal mucosa for a period of five min. The weight necessary for the detachment of the disks from the tissue was monitored after the incubation time. By means of the following equations the force of adhesion and the bond strength were calculated.
Force of adhesion ðNÞ ¼
weight ðgÞ 9:81 1000
Bond strength ðN=m2 Þ ¼
force of adhesion ðNÞ surface area of tablet ðm2 Þ
Evaluation of peel strength was performed by detecting the force which is required in order to detach the adhesive disk in a tangential manner from the buccal tissue. Bond strength and force of adhesion were calculated using the equations [25].
Fig. 4. Resazurin assay. Chitosan–TGA–MNA (Ch–TGA–MNA) conjugate, thiolated conjugates (Ch–TGA) and unmodified polymer (Ch) were tested on Caco-2 cells. Cell viability is expressed in percent. White bars and gray bars represent cell viability after 3 and 24 h, respectively. Indicated values are the means of at least five experiments (±SD).
2.10. Statistical data analysis For statistical analysis, analysis of variance and Student’s t test were used where appropriate. A probability of less than 0.05 (P < 0.05) was considered statistically significant. All results are presented as mean ± SD. 3. Results and discussion
primary amino group of chitosan. Synthesis of chitosan–TGA has already been described by our research group [11,26]. The thiol groups immobilized within chitosan–TGA conjugate were determined by Ellman’s test to be 353.78 ± 34 lmol per gram polymer. The lyophilized polymers exhibit white color and were odorless powder of fibrous structure. The polymer was stored at 4 °C until use and was stable toward air oxidation during the course of study. The chemical pathway is presented in Fig. 1.
3.1. Synthesis and characterization of chitosan–TGA 3.2. Synthesis of 6-MNA and 6,60 -DTNA The thiol bearing ligand namely thioglycolic acid was covalently attached to chitosan’s backbone due to amide bond formation between the carboxylic moieties of thioglycolic acid and the
6-mercaptonicotinamide was synthesized based on guidelines reported by Forrest et al. [16]. 6-MNA was a fine yellowish powder.
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Fig. 5. Water uptake studies of preactivated chitosan–TGA–MNA (white square), chitosan–TGA (black triangle) as well as unmodified (open triangle) in artificial saliva pH 6.75 at 37 °C. Indicated values are mean (±SD) of at least five experiments (significant difference between unmodified and thiolated chitosan at p = 0.015, between unmodified and preactivated chitosan at p = 0.015). 0
0
By the addition of H2O2 to 6-MNA, 6,6 -dithionicotinamide (6,6 DTNA) was obtained. 6,60 -DTNA was of white color and in the form of a powder. The percentage according to the initial weight of the compound as well as the purification process was almost 60%.
formation between thiol groups of the polymeric backbone and the aromatic ligand 6,60 -DTNA was achieved as shown in Fig. 1. The concept for these novel thiomers namely preactivated thiomers is based on the reaction scheme for covalent chromatography of resins such as thiopropylsepharose. The tendency of mercapto pyridine substructure toward nucleophilic attack is a further indication of the electron attracting character from free thiol groups. Pyridyl substructure was covalently linked to the thiomer where it can be eluted by the addition of reducing agents or compounds containing thiol groups [28]. The preactivation step of Ch–TGA was conducted in a solution of DMSO and demineralized water. The synthesized 6-MNA dimer is presented in Fig. 1. Disulfide bond formation was achieved between the thiol group of Ch–TGA and the thiol groups of the 6-MNA dimer. The structure of the preactivated polymer is depicted in Fig. 1. Ellman0 s test of preactivated Ch–TGA–MNA was performed in order to prove the grade of preactivation. The thiol group content was 47.38 ± 7.18 lmol per gram of polymer. These results showed that a successful coupling of MNA to the thiol group of chitosan took place. The lesser the amount of the thiol groups the higher was the amount of immobilized MNA. Furthermore, the amount of MNA was determined in the preactivated polymer, showing 60% activation with 212.26 ± 22.38 lmol MNA per gram of Ch–TGA. The proposed mechanism of the oxidative coupling was the covalent coupling of the MNA dimer to the free thiol group of the activated polymer and the removal of free MNA. All lyophilized conjugates were white, of fibrous structure and easily compressible to test disks. 3.5. Cell culture assays The cytotoxicity of chitosan, chitosan–TGA and preactivated chitosan (chitosan–TGA–MNA) was examined on Caco-2 cells at a
3.3. Characterization of 6-MNA and 6,60 -DTNA During UV-analyses two peaks were significant for of 6,60 DTNA, one at 297 nm and a minor one at 253 nm [27]. A peak at 307 nm described 6-MNA. Furthermore, DSC analyses were performed verifying the desired compounds. The literature reported values of 266–268 °C which are in good agreement with taken values of 6-MNA, monitoring a melting point at around 264 °C [16]. Moreover, the attenuated-total-reflectance Fourier transform infrared spectroscopy (ATR-FTIR) detected significant signals. 3161 cm1 and 3159 cm1 showing the C–H stretch of aromatic compounds, 1653 cm1 to 1619 cm1 indicating N–H bend and – C@C– stretch as well as 840 cm1 representing C–H bend, para, aromatic compound could be seen for 6-MNA and 6,60 -DTNA, respectively. The spectrum is presented in Fig. 2. The thiol function of 6-MNA was found as 2515 cm1 weak signal shown in Fig. 2. For the following synthesis 6,60 -DTNA was identified as a preactivation compound for the thiolated chitosan. The IR spectrum shown in Fig. 3 displayed the typical characteristic signals of the Ch–TGA– MNA. 3500–3200 cm1 represents the O–H bend, 1700–1580 expresses the amide bend and the carboxamide bend of the coupling reagent. The aromatic ring (–C@C–) is found at 1550 cm1, 1404 cm1 expresses the –NH– bend, whereas 1100–1000 cm1 indicates the –C–OH as well as the CH2–OH bend. Both spectra of FTIR indicate the succeeding synthesis of chitosan’s preactivation with 6,60 -DTNA . 3.4. Chitosan–TGA–MNA synthesis Chitosan–TGA–MNA was obtained according to a method described previously by our research group [15]. Disulfide bond
Fig. 6. Disintegration behavior study in artificial saliva pH 6.75 at 37 °C of preactivated chitosan, thiolated and unmodified chitosan. The indicated disintegration time represents an average of at least five experiments (±SD) (significant difference between unmodified and thiolated chitosan (p = 0.014), between unmodified and preactivated chitosan (p = 0.024)).
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properties as well as disintegration and drug release. Adhesiveness taking place in between mucus and polymer could be explained by many processes like absorbing, water uptake as well as capillarity processes. The water uptake findings are depicted in Fig. 5, it could be proven that the thiolation step had a high influence on the swelling behavior. By contrast, the water uptake property was diminished when the aromatic ligand was attached compared to the thiolated. Preactivated chitosan shows higher swelling behavior than unmodified chitosan. A steady and fast weight increase could be observed over a time period of 1 h for thiolated chitosan. A maximum of 63 mg was monitored for thiolated chitosan. This is a 2.1fold increase compared to the initial weight. Preactivated chitosan reached continuously a maximum of 66.58 mg over a time period of 2 h. This correlates with a 2.2-fold increase in the initial weight (significant difference between unmodified and thiolated chitosan (p = 0.015), between unmodified and preactivated chitosan (p = 0.015)). The advantage of depressed water uptake profile for prolonged mucoadhesion was discussed by Mortazavi et al. [29]. It was mentioned that overhydration would be the outcome of an exhaustive water uptake, producing a slippery mucilage and resulting in lower mucoadhesiveness [30]. On these grounds, constant and slow water uptake of preactivated chitosan might show advantages in overcoming overhydration and non- adhesion. 3.7. Stability properties
Fig. 7. Adhesion time assay of preactivated chitosan, chitosan–TGA as well as unmodified chitosan with the rotating cylinder method. Indicated values are means (±SD) of at least five experiments. Significant difference between unmodified and thiolated chitosan (p = 0.001), between unmodified and preactivated chitosan (p = 0.001).
concentration of 0.5% (m/v) for 3 h and 24 h. All conjugates displayed more than 80% cell viability as depicted in Fig. 4. Cell viability of more than 80% after 24 h indicated a negligible effect for the cell monolayer. These results were in close correlation with the LDH findings of tested chitosan conjugates. All polymers were tested in a final concentration of 0.5% (m/v). Moreover, the chosen concentration allowed comparing cytotoxicity of the preactivated polymers to that of well-established thiomers. Cell viability of more than 86% and 80% in the presence of chitosan–TGA–MNA after 3 h and 24 h, respectively, indicated that these polymers were not harmful to the cells. 3.6. Evaluation of swelling behavior Water uptake of a mucoadhesive matrix or dosage form is part of a network being responsible for stability, adhesive and cohesive
Stability properties of chitosan–TGA and preactivated chitosan– TGA–MNA were compared with unmodified polymer. The disintegration of unmodified chitosan was more pronounced compared to its derivatives. As a consequence the stability of the polymers matrix of either chitosan–TGA or chitosan–TGA–MNA exhibited a much higher stability in comparison to the unmodified one. By covalent attachment of 6-mercaptonicotinamide (MNA) to the polymeric backbone of thiolated chitosan the stability of these thiomers was significantly enhanced. A 5.2-fold prolonged disintegration times were obtained in the presence of preactivated chitosan conjugates in comparison with the thiolated one (significant difference between unmodified and thiolated chitosan (p = 0.014), between unmodified and preactivated chitosan (p = 0.024)). Explanations for these findings could be given in the particulate nature of MNA together with the higher amount of inter- and/or intramolecular disulfide bonds within the conjugate which are responsible for the improved stability and hardness of preactivated chitosan. The theory of forming covalent bonds disulfide bonds- within the polymer network which resulted in pronounced stability as well as cohesion was in good agreement with the results given from disintegration studies. Stability and cohesion have a great impact on the disintegration time. This gives the reason that when MNA is introduced as thiol protecting group
Fig. 8. In vitro shear strength study of preactivated, thiolated and unmodified chitosan determined. Indicated values are the means (±SD) of at least five experiments (significant difference between preactivated and unmodified chitosan (p = 0.010), between preactivated and thiolated chitosan (p = 0.044)).
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Fig. 9. In vitro peel strength assay preactivated chitosan, thiolated as well as unmodified chitosan. Indicated values are the mean (±SD) of at least five experiments (significant difference between preactivated and unmodified chitosan (p = 0.002), between preactivated and thiolated chitosan (p = 0.004)).
by the particulate nature of MNA. Furthermore, the formed interand/or intramolecular disulfide bonds within the conjugate could be the reason for its enhanced cohesiveness and hardness of chitosan–TGA–MNA. Fig. 6 represents the findings of the stability evaluation.
3.8. Mucoadhesive strength -in vitro evaluation
Fig. 10. Mucoadhesiveness of preactivated chitosan, thiolated as well as unmodified chitosan determined by tensile studies. Black bars represent the TWA and white bars represent the MDF, respectively. Indicated values are the mean (± SD) of at least five experiments. TWA shows significant difference between unmodified and thiolated chitosan (p = 0.003) between thiolated and preactivated chitosan (p = 0.012) and MDF shows significant difference between unmodified and preactivated chitosan (p = 0.001) between thiolated and preactivated chitosan (p = 0.028).
a more prolonged disintegration time occurred. The prolonged disintegration time might be caused by the lipophilic nature of MNA increasing the hydrophobicity of the polymer conjugate combined with an increase in the formation of inter- and/or intramolecular disulfide bonds since this crosslinking shows a great impact on chain mobility and resistance to dissolution. The mucoadhesion process is initiated by two stages, the contact stage and the consolidation stage. The contact stage is described as the art where the polymer comes in contact with the mucosa, therefore it has to show high stability [31]. For the consolidation stage the polymer should be able to interact with the mucosa regarding the formation of mucoadhesive bonds in order to act as a lubricant which is needed in dry mouth syndrome. An explanation could be given
A variety of mucoadhesive strength assays were conducted based on preactivated chitosan conjugate, chitosan–TGA and unmodified one, respectively. Fig. 7 shows the results of the rotating cylinder test. 3.1-fold improvement is gained due to the preactivation procedure of thiolated chitosan in comparison with thiolated chitosan (significant difference between unmodified and thiolated chitosan (p = 0.001), between unmodified and preactivated chitosan (p = 0.001)). Furthermore, peel, shear, and detachment strength assays were performed on freshly excised buccal mucosa. Shear strength results are shown in Fig. 8 and peel strength is depicted in Fig. 9, respectively. Detachment strength and findings such as total work of adhesion (TWA) (significant difference between unmodified and thiolated chitosan (p = 0.003), between thiolated and preactivated chitosan (p = 0.012)) and maximum detachment force (MDF) (significant difference between unmodified and preactivated chitosan (p = 0.001), between thiolated and preactivated chitosan (p = 0.028)) are depicted in Fig. 10. The correlation coefficient for the correlation between MDF and TWA was 0.987. As a result of the detachment study, chitosan–TGA and preactivated chitosan–TGA–MNA demonstrated a higher MDF and the TWA, respectively compared to unmodified chitosan. The improved MDF and TWA were attributed to the more pronounced attraction of the thiolated and preactivated chitosan conjugates on the buccal mucosa. In the case of preactivated chitosan–TGA, TWA was enhanced 20.5-fold in comparison with unmodified chitosan. In contrast, chitosan–TGA exhibited an increase of 14.3-fold of TWA in comparison with unmodified chitosan. Good correlations between MDF and TWA were obtained regarding all tested polymers. Due to these observations, the theory of improved mucoadhesive properties of thiolated polymers, that the thiol groups of the backbone forms disulfide bonds with cysteine substructures of the mucus, could be proven [32,33]. Within this study, buccal porcine tissue was chosen as mucosal platform for the assessment of mucoadhesive strength. The higher the prolonged residence time the lower the interval of drug application leading to a better patient compliance. Benefits associated with the prolonged residence time are increase of drug concentration at the target site. Taking these mucoadhesive findings in consideration, preactivated conjugate is considered as potential representative for a dosage form treating xerostomia.
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4. Conclusion Findings obtained within this present study suggest preactivated thiolated chitosan as a potential matrix for local buccal drug delivery. The synthesis of chitosan–TGA–MNA, thioglycolic acid as thiol bearing ligand was covalently attached through the amide bond formation to the polymeric backbone. The preactivation of the thiolated polymer results in enhanced mucoadhesive features. The augmented mucoadhesion was proven by a 3.1-fold enhancement of remaining on the mucosa due to the preactivation step of thiolated chitosan in comparison with thiolated chitosan as well as by the increase in the total work of adhesion in detachment strength assays. These findings lead to improved mucoadhesiveness which has a great impact on applications where prolonged residence time is required. Water uptake properties as well as stability of preactivated chitosan were enhanced in comparison to unmodified one. Through the preactivation and disulfide bond formation preactivated chitosan exhibited more pronounced cohesive properties as well as beneficial prolonged residence time on buccal mucosae. The preactivated thiolated chitosan synthesized in this present study paves the way for being a promising candidate in the treatment of the dry mouth syndrome due to its mucoadhesive and water uptake properties. Conflict of interest The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. The author reports no declaration of interest. Acknowledgement The work was supported by the University of Innsbruck and the FWF project no. ZFP 235150. Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Fig. 2, are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2015. 04.016. References [1] Asane GS, Nirmal SA, Rasal KB, Naik AA, Mahadik MS, Rao YM. Polymers for mucoadhesive drug delivery system: a current status. Drug Dev Ind Pharm 2008;34(11):1246–66. [2] Sakloetsakun D, Iqbal J, Millotti G, Vetter A, Bernkop-Schnurch A. Thiolated chitosans: influence of various sulfhydryl ligands on permeation-enhancing and P-gp inhibitory properties. Drug Dev Ind Pharm 2011;37(6):648–55. [3] Gannot G, Lancaster HE, Fox PC. Clinical course of primary Sjogren’s syndrome: salivary, oral, and serologic aspects. J Rheumatol 2000;27(8):1905–9. [4] Hearnden V, Sankar V, Hull K, et al. New developments and opportunities in oral mucosal drug delivery for local and systemic disease. Adv Drug Delivery Rev 2012;64(1):16–28. [5] Scully C. Drug effects on salivary glands: dry mouth. Oral Dis 2003; 9(4):165–76.
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Please cite this article in press as: Laffleur F et al. Evaluation of functional characteristics of preactivated thiolated chitosan as potential therapeutic agent for dry mouth syndrome. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.016