Chitosan-graft-6-mercaptonicotinic Acid: Synthesis, Characterization

Oct 12, 2009 - For this purpose, 6-mercaptonicotinic acid (6-MNA) was covalently .... The media used for the EAhy926 cells consisted of 90% Dulbecco's...
1 downloads 0 Views 150KB Size
Download PDF
Biomacromolecules 2009, 10, 3023–3027

3023

Chitosan-graft-6-mercaptonicotinic Acid: Synthesis, Characterization, and Biocompatibility Gioconda Millotti,† Claudia Samberger,‡ Eleonore Fro¨hlich,‡ and Andreas Bernkop-Schnu¨rch*,† Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University, Innrain 52c, Josef Mo¨ller Haus, A-6020 Innsbruck, Austria, and Medical University Graz, Centre for Medical Research, Stiftingtalstr. 24, A-8010, Graz, Austria Received June 4, 2009; Revised Manuscript Received August 14, 2009

Thiolated chitosans are relatively new thiolated biopolymers exhibiting mucoadhesive, enzyme inhibitory, and permeation enhancing properties. A drawback, however, is their pH dependent reactivity. The aim of this study was therefore to develop a novel thiolated chitosan showing a non pH dependent reactivity of its thiol groups. For this purpose, 6-mercaptonicotinic acid (6-MNA) was covalently attached to chitosan by a carbodiimide mediated reaction. The obtained conjugate was characterized in vitro by quantification of immobilized thiol groups, cytotoxicity, in situ gelling properties, and disulfide bond formation at different pH. The synthesized conjugate displayed up to 973.80 µmol thiol groups per gram of polymer in reduced form. The polymer was nontoxic and showed in situ gelling properties. Furthermore, disulfide bond formation and therefore gelling properties occurred at various pH ranges. The reactivity of thiol groups was in the same range at pH 3 and pH 6.8. According to these results, chitosan-6 mercaptonicotinic acid seems to have some promising features to be used particularly for mucoadhesive formulations.

1. Introduction Thiomers are hydrophilic macromolecules bearing free thiol groups on their backbone. Due to the immobilization of thiol groups on already well-established polymers, their mucoadhesive,1 enzyme inhibitory,2 permeation enhancing,3 and efflux pump inhibiting properties4 are strongly improved. Thiol-bearing ligands can be covalently immobilized on the primary amino groups at the C-2 position of the glucosamine subunits of chitosan. According to this, chitosan-cysteine,5 chitosan-glutathione,6 chitosan-thioethylamidine,7 chitosan-thioglycolic acid,8 chitosan-4-thio-butyl-amidine,9 and chitosan-N-acetyl cysteine conjugates,10 as well as other thiolated chitosans,11,12 have been synthesized. Besides all these advantages, a drawback of the thiomers developed so far is their pH-dependent reactivity. The reactive form of thiomers is the thiolate anion. The pKa or alkyl thiols is in the range of 8-10.13 This means that thiomers will be most reactive in a pH range slightly above the physiological intestinal pH. Consequently, they do not reach their full potential in cases where the pH is lower, like, for example, in the stomach where the pH is very low or the small intestine where the pH is around 5. Furthermore, vaginal pH is in the range of 3.5-5. The most used vaginal formulation is a gel. Therefore, it would be important to have an in situ gelling system that would be active in that pH range. The ideal would be to have a system that would react in a non-pH-dependent manner. It was therefore the aim of this study to synthesize a novel type of thiolated chitosan that offers the benefits of thiomers at a broader spectrum of pH levels. 6-Mercaptonicotinic acid was chosen to develop a novel thiomer with a pH-independent action mechanism. Due to its particular structure, this compound has two * To whom correspondence should be addressed. Tel.: +43 512 507 5383. Fax: +43 512 507 2933. † Leopold-Franzens-University. ‡ Medical University Graz.

tautomeric structures: thiol (SsH) and thione (CdS). In polar solvents such as water, the thione form (CdS) is the most predominant structure. This structure can react with a disulfide bond both as a nucleophile and a proton donor. Therefore, disulfide bonds can be formed even without thiol groups being available on the polymer in the form of thiolate anions. The polymer was characterized in terms of thiol content, disulfide bond formation with a parallel increase in viscosity, and cytotoxicity.

2. Materials and Methods 2.1. Materials. 6-Mercaptonicotinic acid (6-MNA), dioxane, and N-3(dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDAC) were purchased from Sigma-Aldrich. Chitosan medium molecular mass and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were obtained from Fluka. CytoTox-ONE(TM) homogeneous Membrane Integrity Assay, CellTiter-Glo Luminescent Cell Viability Assay, and CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay were purchased from Promega. Surfactant-Free Carboxyl White Polystyrene Latex and Carboxyl White Polystyrene Latex were purchased from Interfacial Dynamics. Pyrogen Ultra (sensitivity ) 0.06 EU/mL (European unit/ mL)) was obtained from Lonza. 2.2. Modification of Chitosan with 6-Mercaptonicotinic Acid. Briefly, 1 g of chitosan (medium molecular mass: 400 kD) was hydrated in 8 mL of 1 M HCl and then dissolved in water, obtaining a 1% (m/v) polymer solution. The pH was adjusted to 3.0 by the addition of 0.5 M NaOH. Afterward, 2.5 g of 6-mercapto nicotinic acid dissolved in 100 mL of a dioxane-water mixture (80 mL + 20 mL) was slowly added under stirring. The pH was adjusted to 5.0 with 0.5 M NaOH. Then EDAC dissolved in 5 mL of demineralized water was added in concentrations as listed in Table 1 under vigorous stirring. The pH was adjusted to 6 and the reaction was allowed to proceed for 7 h at room temperature under permanent and vigorous stirring. Tris(2carboxyethyl)phosphine hydrochloride (TCEP) in a final concentration of 10 mM was added at pH 5 and incubated under vigorous stirring for 30 min. The resulting reduced conjugated polymer was dialyzed in

10.1021/bm9006248 CCC: $40.75  2009 American Chemical Society Published on Web 10/12/2009

3024

Biomacromolecules, Vol. 10, No. 11, 2009

Table 1. Quantification of Thiol Groups on the Conjugates

conjugate A conjugate B

total amt of thiol groups (µmol/g polymer)

reduced thiol groups (µmol/g polymer)

956.41 1858.56

524.27 973.80

tubings (molecular mass cutoff 12 kDa) first against 4.5 L of demineralized water with 7 mM HCl, two times against 7 mM HCl containing 1% NaCl, once against 5 mM HCl, and finally against 1 mM HCl at 10 °C in the dark. Controls were prepared following the same protocol but omitting EDAC. The frozen aqueous solutions of all the conjugates and controls were freeze-dried at -77 °C (Virtis Bench top freeze-drier, Bartelt, Graz, Austria) and stored at 4 °C until use. 2.3. Determination of the Degree of Modification. 6-MNA exhibits an absorption maximum in the range of 260-280 nm in the UV-vis range. The freeze-dried conjugates were dissolved in a 0.1 M acetate buffer pH 3.3-dioxane mixture (1 + 1) in a final concentration of 0.2 mg/mL. The amount of ligand bound to chitosan was calculated from the corresponding calibration curve made with 6-mercaptonicotinic acid (0-0.16 µmol/mL) dissolved in 0.1 M acetate buffer pH 3.3-dioxane mixture (1 + 1) with absorbance values at 268 nm (Beckman DU 650 spectrophotometer) from 0-0.9. 2.4. Determination of Reduced Thiol Groups. The amount of thiol groups in their reduced form was measured via iodometric titration.14 Briefly, different concentrations of both conjugates have been prepared (0.5-0.05 mg/mL) in 0.5 M acetate buffer pH 2.7. Then 1 mL starch aqueous solution (1% m/v) was added to 2 mL of each sample concentration. Afterward, 0.1 M iodine was added until the majority of the samples of each conjugate displayed a blue-violet color. The oxidation reaction was allowed to proceed for 24 h, protected from light. Two calibration curves were elaborated under the same conditions with cysteine-HCl solutions: 20-50 µmol/L for conjugate A and 75-110 µmol/L for conjugate B. The excess iodine that reacts with starch was measured at 560 nm. 2.5. In Situ Gelling Properties. Chitosan and modified chitosan6-MNA were dissolved in acetate buffer pH 5.5, obtaining a solution of 1% (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. In parallel, at each predetermined time point, 200 µL aliquots were withdrawn and 50 µL of 1 M HCl was added to stop any further oxidation. The thiol content was measured by iodometric titration. Briefly, 1 mg of the freeze-dried polymer was dissolved in 1 mL of demineralized water, adjusting the pH to 1-2 with one drop of 1 M HCl. Then, 150 µL of a 2% (m/v) starch solution were added as indicator. The solution was titrated with 1 mM iodine solution until a permanent blue-violet color appeared. The same procedure was followed by incubating conjugate A for 8 h in acetate buffers of pH 3, 4, 5.5 and phosphate buffer at pH 6.8. 2.6. Cell Culture Conditions. For the Caco-2 cell line, the medium was composed of 80% minimum essential medium (MEM) (with Earle’s salts), 20% phosphate buffered saline (PBS) pH 7.2, 1× nonessential amino acids, and 1% penicillin-streptomycin liquid. The media used for the EAhy926 cells consisted of 90% Dulbecco’s modified Eagle’s medium, 10% phosphate buffered saline (PBS), 2 mM L-glutamate, and 1% penicillin-streptomycin liquid. 2.7. LDH Release Assay. A standardized number of cells was seeded in a 96-well plate (100 µL per well) and grown for 24 h in an incubator (37 °C, 5% CO2, 95% relative humidity) prior to stimulation. Medium was removed and 100 µL of the polymer (0-100 µg/mL in medium) were added. Experiments were performed in triplicate. The plate was incubated at 37 °C, 5% CO2, and 95% relative humidity for 4-24 h. The standard assay setup included: blank (culture medium alone), growth control (culture medium with the cells), lysis control

Millotti et al. (untreated cells in medium where 100% lysis was performed), and particulate positive and negative controls (26 nm carboxyl White Polystirene latex as positive control and 160 nm Surfactant-Free Carboxyl white polystyrene Latex as negative control). The CytoToxONE assay kit was performed as indicated by the producer. The average value of the blank was subtracted from every fluorescence value. The percent toxicity was calculated according to the formula:

percent cytotoxicity ) (experimental - culture medium background) 100 × (maximum LDH release - culture medium background) 2.8. MTT Test. Cell seeding and exposure to the polymer was the same as for the LDH release, but no lysis control was included in the setup. For the assay, MTS solution from the kit (tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulpophenyl)-2H-tetrazolium] and PMS solution from the kit (phenazine methosulfate) were thawed. Afterward, 100 µL of the PMS solution was added to 2 mL of MTS solution and gently swirled before addition to the wells. Then, 20 µL of the MTS/PMS solution was pipetted into each well of the 96-well assay plate containing 100 µL, and the plate was incubated for 2 h at 37 °C. The absorbance was recorded at 490 nm with a plate reader. The blank (background from the culture medium) was subtracted from all other absorbance values. 2.9. Quantification of ATP. Cell seeding and exposure to the polymer was the same as for the LDH release, but no lysis control was included in the setup. The assay was performed as indicated by the producer. The blank values were subtracted from all values. 2.10. Quantification of Endotoxins. LAL (Limulus Amebocyte Lysate) was performed using the Pyrogent Ultra kit. This test is a qualitative test for Gram-negative bacterial endotoxin. Gram-negative bacterial endotoxin catalyzes the activation of a proenzyme in the Limulus Amebocyte Lysate. To prepare the Limulus Amebocyte Lysate, the lyophilized lysate was reconstituted by adding 5.2 mL of LAL reagent water (from the kit) to 50 test vial. The solution was gently swirled. For the liquid endotoxin standards, vials containing a liquid preparation of purified endotoxin from E. coli strain (055:B5) were inverted five times to allow mixing of the contents. Afterward, 100 µL of standard (sample or water) were transferred into the reaction tube. Then, 100 µL of the reconstituted LAL were added to each tube beginning with the highest concentration of endotoxin and mixed thoroughly. This procedure was followed for each dilution of the endotoxin. The sample was run in parallel with the endotoxin standards. After the incubation time, each tube was examined for gelation. A positive reaction is characterized by the formation of a firm gel that remains intact when the tube is inverted by a vertical rotation of 180°. 2.11. Statistical Data Analysis. Statistical data analyses were performed using the Student’s t-test with p < 0.05 as the minimum level of significance.

3. Results and Discussion 3.1. Synthesis and Characterization of Chitosan-6-mercaptonicotonic Acid. The covalent attachment of 6-MNA to chitosan has been achieved via the formation of amide bonds between the carboxylic acid group of the 6-MNA and amine groups of chitosan as illustrated in Figure 1. The immobilization of a thiol bearing compound on a well-established biodegradable, cationic hydrophilic polymer like chitosan should offer on the one hand hydrophobic interactions and decrease the swelling behavior of the polymer in aqueous solution, while on the other hand it is advantageous to introduce a sulfhydryl group for crosslinking via the formation of intra- and intermolecular disulfide bonds.10 During the reaction, the solution became more viscous with time, resulting a gel near the end of the incubation time. After

Chitosan-graft-6-mercaptonicotinic Acid

Figure 1. Schematic presentation of the newly synthesized thiolated chitosan.

the addition of the reducing agent TCEP at the end of the reaction, the reaction mixture became liquid again. This behavior was not observed in the control sample. This is an indication of the actual binding of the ligand to the chitosan chains and the formation of intra- and interchain disulfide bonds during the reaction. The reduction of disulfide bonds could be visually monitored by the decrease in viscosity of the solution after the addition of the reducing agent. During the synthesis of other thiolated chitosan conjugates such behavior was not reported. In these studies, however, the degree of thiolation was much lower. The control reaction mixture did not exhibit this behavior since there was no ligand bound to the chitosan chains. The carboxylic acid moieties of 6-mercaptonicotinic acid were activated by EDAC forming an O-acylurea derivative as an intermediate product that reacts with the primary amino groups of chitosan. By changing the EDAC concentration it is possible to covalently attach different quantities of the ligand, as listed in Table 1. The highest quantity of immobilized ligand was obtained with the higher EDAC concentration. However, at EDAC concentrations above 25 mM, the system collapsed and became turbid with precipitate, which was not possible to resuspend. Too much immobilized ligand would make the system too lipophilic causing precipitation. Synthesis of other thiolated chitosans required EDAC concentrations in the range 50-200 mM, while for the synthesis of this novel thiolated chitosan 12.5 or 25 mM EDAC was enough to achieve comparable or higher coupling rates.6,8,10 The much higher EDAC concentrations needed for other carboxylated thiols are probably the result of the occurrence of a side reaction of the nucleophilic thiolate group of these species with EDAC, leading to an intermediate adduct that is subsequently hydrolyzed with the final result the formation of the nonreactive urea product of EDAC. The lyophilized chitosan-6MNA conjugates appeared as a yellowish powder of fibrous structure. The color of the higher coupled conjugate was more intense than that of the less coupled conjugate. The efficacy of the purification method described could be verified by the corresponding controls, which were prepared in exactly the same way but omitting EDAC. The amount of total thiols present in the control was negligible.

Biomacromolecules, Vol. 10, No. 11, 2009

3025

Figure 2. Increase in viscosity of a 1% (m/v) solution of conjugates A (9) and B (2) and unmodified chitosan ([) in acetate buffer, pH 5.5, incubated at 37 °C. Data are means of at least three experiments ( SD (n ) 3).

Figure 3. Decrease in reduced thiol groups on conjugates A (9) and B ([) of a 1% (m/v) polymer solution in acetate buffer, pH 5.5, incubated at 37 °C. Data are means of at least three experiments ( SD (n ) 3).

3.2. Decrease in the Thiol Group Content within the Conjugates and Increase in Viscosity. Thiol groups on the polymer can undergo oxidation to disulfide bonds in aqueous solution both inter- and intrachain. It is quite possible that in this oxidation process 6-MNA, due to its tautomerism, experiences lower energy barriers than common thiol species, but the oxidation mechanism might be much more complicated than that of the thiol-disulfide exchange mechanism. Bagiyan et al., for instance, reported that oxidation of thiol compounds can afford products containing the S atom in different oxidation states including also radicals.15 The results are reported in Figures 2 and 3. For both polymers a rather significant decrease in reduced thiol groups can be noticed. This behavior has already been reported by other authors.7,16 Both conjugates showed an increase in viscosity but not with an expected trend. On the other hand, unmodified chitosan did not show an increase in viscosity; therefore the reason for the increase in viscosity can only be attributed to the formation of disulfide bonds within the polymer. The viscosity of conjugate A increases up to 477fold. At time zero the polymer in solution is a clear liquid, while

3026

Biomacromolecules, Vol. 10, No. 11, 2009

Figure 4. (a) Tautomeric forms of 6-mercaptonicotinic acid. (b) Reaction of a thiolate anion with disulfides. (c) Reaction of the second tautomeric structure of 6-MNA with disulfides.

after 24 h it is in a solid state, maintaining its transparent appearance. Conjugate B showed a little increase in viscosity: 16-fold. Conjugate B did not form a clear solution. No unsolved parts could be detected, but there were some opalescence probably due to the high amount of ligand on the polymer. The unexpectedly lower increase in viscosity of conjugate B, compared to conjugate A, could be explained with the fact that, as other authors already reported,7,16 thiol groups being located closely to each other can form disulfide bonds more rapidly than remaining isolated thiol groups. In case a polymer displays a high amount of thiol groups covalently attached, there are more thiol groups on the same chain close to each other. These thiols will react much faster among each other rather than with thiols allocated on other polymer chains. Therefore, in the case of conjugate B, although there is a decrease in the reduced thiol group content, the viscosity is not increasing at the same rate because there are not many interchain cross-linkings but rather intrachain. Also, the gel appearance provided evidence for this; although there were no unsolved parts, the structure was not homogeneous; it was agglomerate-like. To prove that the newly synthesized polymer reacts in a nonpH-dependent way, through the pyridone-like form and not only via the free thiol groups, the increase in viscosity was measured at different pH values (Figures 4 and 5 of the Supporting Information). There was not a significant difference between the increases in viscosity at various pH levels. This confirms the pHindependent mechanism of chitosan-6MNA. 6-MNA is found in two tautomeric forms (Figure 4a). Indeed, for reaction with disulfides, two mechanisms are possible. The mechanism of the thiol exchange with disulfides is based on nucleophilic attack of a thiol group in the form of a thiolated anion (R-S-) on one of the sulfur atoms of a disulfide bond. A new disulfide bond is formed with the expulsion of a thiolate (Figure 4b). This mechanism has an energy barrier transition state. On the other hand, compounds such as 6-MNA can form disulfide bonds by reacting in their neutral state, acting both as a nucleophile (CdS) and a proton donor (N-H) to the leaving thiolate group. This mechanism avoids high energetic transition states (Figure 4c). Therefore, chitosan 6-MNA could maintain thiomer characteristics at a wider pH range. An increase in viscosity is directly correlated with disulfide bond formation. A series of alkylthiomers reported a much lower formation of disulfide bonds

Millotti et al.

as pH was diminishing. Chitosan-thioglycolic acid did not form disulfide bonds at pH 4 and there was a very pronounced increase of disulfide bond formation at pH 6.5 compared to pH 5.8 Other thiomers reported the same tendency.6,9,17,18 Therefore, the newly synthesized polymer could be used as an in situ gelling formulation for many applications. For example, the main drawback in vaginal drug delivery is the rapid removal of inserted systems.19 The in situ gelling properties of chitosan6-mercaptonicotinic acid might result in promising vaginal drug delivery systems. Vaginal pH is normally acidic. However, after a certain age, vaginal pH reaches more alkaline values. Therefore, due to its pH-independent in situ gelling properties, chitosan-6-MNA could ensure constant gelling properties for a vaginal drug delivery system in different vaginal pH ranges. In situ gel formation of the newly synthesized polymer could also be useful for ocular and nasal delivery. 3.3. LDH Test. This assay permits the investigation of substances that may induce alterations in cell integrity and therefore quantifies the amount of nonviable cells. LDH is a stable enzyme present in the cytosol that is released upon cell lysis.20 The release of lactate dehydrogenase into the culture medium is measured via a coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resofurin product. The amount of fluorescence is proportional to the number of lysed cells. The test was performed both on human colorectal carcinoma cell lines (Caco-2) and on the human endothelial cell line (EAhy926). The tests were performed at an incubation time of 4 and 24 h in different polymer concentrations. The test performed on Caco-2 cells (Figure 7a, b, in the Supporting Information) indicates that both conjugates have the same toxicity as chitosan, which is very low, also after 24 h. There is no correlation between increasing concentration of polymers and % of cell death. The test performed on EAhy926 cells (Figure 7c, d, in the Supporting Information) also indicates that the newly synthesized conjugates exhibit cytotoxicity in the range of unmodified chitosan. Although after 24 h there was a slight increase in LHD release provoked by the conjugates, the value was still very low and it is considered as nontoxic. 3.4. MTT Test. This test is measuring the number of viable cells. Although formazan bioreduction is the classical cytotoxicity screening assay for conventional substances, The MTS (tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulpophenyl)-2H-tetrazolium] is bioreduced by cells into a formazan product that is soluble in tissue culture medium by dehydrogenase enzymes found in metabolically active cells. The tests performed on Caco-2 cell lines (Figure 8a, b, in the Supporting Information), and EAhy926 cell lines (data not shown) showed a cell viability of around 100% for all the concentrations tested after 4 and 24 h, indicating that the polymers are not harmful for the cells. There was no trend in results depending on the concentration used. 3.5. Quantification of ATP. This test measures the number of viable cells by luminescent quantification of the ATP present, an indication of metabolically active cells. The tests performed on both cell lines (data not shown) showed a cell viability of around 100% for all the concentrations tested after 4 and 24 h, indicating that cells maintain their metabolic activity, and therefore, the polymers can be considered as nontoxic. 3.6. Endotoxin Quantification. Endotoxins are natural compounds found inside pathogens such as bacteria. These are not secreted in soluble form by live bacteria, but are a structural component of the bacteria that is released mainly when bacteria

Chitosan-graft-6-mercaptonicotinic Acid

are lysed. The samples in stock solution contained less than 0.06 EU/mL.

4. Conclusion A new thiolated chitosan has been synthesized, named chitosan-6- mercapronicotinic acid. Two different conjugates have been synthesized, differing in the amount of immobilized ligand. The newly synthesized polymer showed excellent in situ gelling properties without the addition of any oxidizing species. Moreover, in contrast to all other thiolated polymers, the gelling properties are pH-independent. This property might be of considerable advantage for drug delivery applications requiring in situ gelling properties where the pH could be different according to age or individual differences, such as the vagina. As the newly synthesized conjugate represents a new class of thiolated chitosans, it was necessary to screen its biodegradability and cytotoxicity. Cytotoxicity tests performed on two human endothelial and gastrointestinal epithelia, measuring both the viability and cell death, proved that the new conjugate is not harmful for the cells. Therefore, this new conjugate has potential to introduce improvements and beneficial features to pharmaceutical formulations, especially for mucoadhesive formulations. Acknowledgment. This work has been supported by the EC. NanoBioPharmaceutics is an Integrated Project funded within the sixth Framework Programme of the European Commission. Supporting Information Available. Figures 4, 5, 7, and 8. This material is available free of charge via the Internet at http:// pubs.acs.org.

References and Notes (1) Bernkop-Schnurch, A.; Schwarz, V.; Steininger, S. Polymers with thiol groups: a new generation of mucoadhesive polymers. Pharm. Res. 1999, 16 (6), 876–81. (2) Bernkop-Schnurch, A.; Zarti, H.; Walker, G. F. Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N. J. Pharm. Sci. 2001, 90 (11), 1907–14. (3) Clausen, A. E.; Kast, C. E.; Bernkop-Schnurch, A. The role of glutathione in the permeation enhancing effect of thiolated polymers. Pharm. Res. 2002, 19 (5), 602–8. (4) Foger, F.; Schmitz, T.; Bernkop-Schnurch, A. In vivo evaluation of an oral delivery system for P-gp substrates based on thiolated chitosan. Biomaterials 2006, 27 (23), 4250–5.

Biomacromolecules, Vol. 10, No. 11, 2009

3027

(5) Bernkop-Schnurch, A.; Brandt, U. M.; Clausen, A. E. Synthesis and in vitro evaluation of chitosan-cysteine conjugates. Sci. Pharm. 1999, (67), 196–208. (6) Kafedjiiski, K.; Foger, F.; Werle, M.; Bernkop-Schnurch, A. Synthesis and in vitro evaluation of a novel chitosan-glutathione conjugate. Pharm. Res. 2005, 22 (9), 1480–8. (7) Kafedjiiski, K.; Krauland, A. H.; Hoffer, M. H.; Bernkop-Schnurch, A. Synthesis and in vitro evaluation of a novel thiolated chitosan. Biomaterials 2005, 26 (7), 819–26. (8) Kast, C. E.; Bernkop-Schnurch, A. Thiolated polymers-thiomers: development and in vitro evaluation of chitosan-thioglycolic acid conjugates. Biomaterials 2001, 22 (17), 2345–52. (9) Bernkop-Schnurch, A.; Hornof, M.; Zoidl, T. Thiolated polymersthiomers: synthesis and in vitro evaluation of chitosan-2-iminothiolane conjugates. Int. J. Pharm. 2003, 260 (2), 229–37. (10) SchmitzT. Synthesis and charachterization of a chitosan-N-acetyl cysteine conjugate. Int. J. Pharm. 2007, doi: 10:1016/ijpharm. 2007.06.040. (11) Jayakumar, R.; Reis, R. L.; Mano, J. F. Synthesis and characterization of pH-sensitive thiol-containing chitosan beads for controlled drug delivery applications. Drug DeliVery 2007, 14 (1), 9–17. (12) Prabaharan, M.; Shaoquin, G. Novel thiolated carboxymethyl chitosang-B-cyclodextrin as mucoadhesive hydrophobic drug delivery carriers. Carbohydr. Polym. 2008, 73, 117–125. (13) Wilson, J. M.; Bayer, R. J.; D, J., H. Structure-reactivity correlations for the thiol-disulfide interchange reaction. J. Am. Chem. Soc. 1977, 99, 7922–7926. (14) Bravo-Osuna, I.; Teutonico, D.; Arpicco, S.; Vauthier, C.; Ponchel, G. Characterization of chitosan thiolation and application to thiol quantification onto nanoparticle surface. Int. J. Pharm. 2007, 340 (12), 173–81. (15) Bagiyan, G. A.; Koroleva, I. K.; Soroka, N. V.; Ufimtsev, A. V. Oxidation of thiol compounds by molecular oxygen in acqueous solutions. Russ. Chem. Bull. 2003, 52 (5), 1135–1141. (16) Dodou, D.; Breedveld, P.; Wieringa, P. A. Mucoadhesives in the gastrointestinal tract: revisiting the literature for novel applications. Eur. J. Pharm. Biopharm. 2005, 60 (1), 1–16. (17) Kafedjiiski, K.; Hoffer, M.; Werle, M.; Bernkop-Schnurch, A. Improved synthesis and in vitro characterization of chitosan-thioethylamidine conjugate. Biomaterials 2006, 27 (1), 127–35. (18) Grabovac, V.; Bernkop-Schnurch, A. Development and in vitro evaluation of surface modified poly(lactide-co-glycolide) nanoparticles with chitosan-4-thiobutylamidine. Drug DeV. Ind. Pharm. 2007, 33, 767–774. (19) Ceschel, G. C.; Maffei, P.; Lombardi Borgia, S.; Ronchi, C.; Rossi, S. Development of a mucoadhesive dosage form for vaginal administration. Drug DeV. Ind. Pharm. 2001, 27, 541–547. (20) Mao, S.; Shuai, X.; Unger, F.; Wittmar, M.; Xie, X.; Kissel, T. Synthesis, characterization and cytotoxicity of poly(ethylene glycol)graft-trimethyl chitosan block copolymers. Biomaterials 2005, 26 (32), 6343–56.

BM9006248