Chitosan-4-mercaptobenzoic acid: synthesis and characterization of ...

View OnlinePAPERwww.rsc.org/materials | Journal of Materials ChemistryChitosan-4-mercaptobenzoic acid: synthesis and characterization of a novelthiolated chitosan†Gioconda Millotti, a Claudia Samberger, b Eleonore Fr€ohlich, b Duangkamon Sakloetsakun aand Andreas Bernkop-Schn€urch* aReceived 13th August 2009, Accepted 13th January 2010First published as an Advance Article on the web 8th February 2010DOI: 10.1039/b916528bDownloaded by UNIVERSITAETSBIBLIOTHEK INNSBRUCK on 21 October 2010Published on 08 February 2010 on http://pubs.rsc.org | doi:10.1039/B916528BThiomers are thiolated polymers with excellent mucoadhesive properties. The aim of this study was tosynthesize a novel thiolated chitosan with higher reactivity in order to further improve mucoadhesion.For this purpose, 4-mercaptobenzoic acid was chosen to be covalently attached to chitosan. Thearomatic structure of the ligand should exhibit a higher reactivity due to a comparatively low pK a valueof the thiol group, which was determined to be 6.8. Mucoadhesion, in situ gelation, biocompatibilityand toxicity were evaluated. Chitosan-4-mercaptobenzoic acid was proved to be 60-fold moremucoadhesive compared to unmodified chitosan. After 24 hours, the viscosity of the novel thiomersolution increased 2974-fold compared to unmodified chitosan and after 48 hours 4487-fold. Chitosan-4-mercaptobenzoic acid tablets disintegrated twice as slowly as unmodified chitosan. Furthermore, thenovel thiomer proved to be biodegradable and to be non-toxic. Due to the above mentioned properties,chitosan-4-mercaptobenzoic acid is a promising excipient for oral formulations or in situ gellingformulations.IntroductionMucoadhesive delivery systems are in many cases preferred overnon-mucoadhesive delivery systems due to the possibility of druglocalization at the target site, a prolonged residence time at thesite of drug absorption and an increase in drug concentrationgradient on the mucosa. Chitosan is a natural polymer that hasbeen shown to have mucoadhesive properties. These are quiteweak, as they are the result of non-covalent bonds between thepositive charges of the polymer and negative charges of mucusglycoproteins. These mucoadhesive properties can be improvedlog-fold by introducing thiol groups on the polymer structure.Thiomers are hydrophilic polymers whose structure has beenmodified by the covalent attachment of thiol-bearingcompounds. Mucoadhesive properties are improved due todisulfide bond formation between thiol groups present on thepolymer structure and thiols being available on the glycoproteinsubstructures of the mucus. Beside mucoadhesive properties 1thiomers show also enhanced enzyme inhibitory, 2 permeationenhancing 3 and efflux pump inhibiting properties 4 as well as insitu gelling properties. Up to now, a considerable number ofthiolated chitosans have been developed by using alkyl thiolbearing compounds. It is known that aryl thiols (pK a 5–7),however, are far more reactive than alkyl thiols (pK a 8–10). 5 Rayet al., for instance, performed electrochemical studies ofa 6-mercaptonicotinic acid monolayer stating that aromatic oraDepartment of Pharmaceutical Technology, Institute of Pharmacy,Leopold-Franzens-University, Innrain 52c, Josef M€oller Haus, A-6020Innsbruck, Austria. E-mail: andreas.bernkop@uibk.ac.at; Fax: +43 512507 2933; Tel: +43 512 507 5383bMedical University Graz, Centre for Medical Research, Stiftingtalstr. 24,A-8010 Graz, Austria† Electronic supplementary information (ESI) available: FT-IR analysis.See DOI: 10.1039/b916528bheteroaromatic thiols/disulfides are of great interest since theintermolecular interactions are stronger than those between thealkane thiols. 6 Aromatic thiols have already been shown toincrease the folding rate of disulfide containing proteinscompared to aliphatic ones, due to their lower pK a . 7–10 Fora pharmaceutical excipient such as thiolated chitosan theincreased reactivity of the thiol group would further increase itsadvantage over non-thiolated chitosan. This would allowstronger mucoadhesion, resulting in disulfide bond formationbetween the thiols present on the polymer and thiols on mucusglycoproteins. As the pK a range of aromatic thiols is in thephysiological intestinal pH range, they are primarily present inthe form of thiol anions in the intestine with a pH range from 5.5to 7.5, resulting subsequently in much higher mucoadhesion.The aim of this study was to synthesize a novel type of thiolatedchitosan by covalently attaching an aromatic thiol bearingmolecule to the chitosan backbone and to investigate itsmucoadhesion and gelling capacity as well as its biocompatibilityin terms of susceptibility to lysozyme degradation and cytotoxicity.ExperimentalMaterials4-Mercaptobenzoic acid (4-MBA), dioxane and N-3(dimethylaminopropyl)-N-ethylcarbodiimidehydrochloride (EDAC) werepurchased from Sigma-Aldrich. Chitosan low molecular massand tris(2-carboxyethyl)phosphine hydrochloride (TCEP) wereobtained from Fluka. Lysozyme from chicken egg white (185 000U mg 1 ) was purchased from Serva. CytoTox-ONE(TM)homogeneous Membrane Integrity Assay, CellTiter-Glo LuminescentCell Viability Assay and CellTiter 96Ò AQueous Non-Radioactive Cell Proliferation Assay were purchased from2432 | J. Mater. Chem., 2010, 20, 2432–2440 This journal is ª The Royal Society of Chemistry 2010

View OnlineDownloaded by UNIVERSITAETSBIBLIOTHEK INNSBRUCK on 21 October 2010Published on 08 February 2010 on http://pubs.rsc.org | doi:10.1039/B916528Bdisintegration apparatus (Erweka G.m.b.H., Germany) containing1 L of 100 mM phosphate buffer pH 6.8 at 37 C. Theoscillating frequency was set to 0.5 s 1 . Time was recorded whenthe tablets disintegrated completely.Increase in viscosity and decrease in free thiol groups contentChitosan and chitosan-4-MBA were dissolved in 100 mM acetatebuffer pH 5.5 obtaining a solution of 1% (m/v). The samples werekept at 37 C. At predetermined time points the viscosity of 600ml aliquots was measured at 37 C with a plate-plate viscosimeter(Haake MARS) connected to a personal computer for setting theanalysis parameters and for processing and recording the datawith the Haake Rheowin program. In parallel, at each predeterminedtime point, 200 ml aliquots were withdrawn and 50 mLof 1 M HCl was added to stop any further oxidation. The thiolcontent was measured by iodometric titration. Briefly, 1 mg ofthe freeze-dried polymer was dissolved in 1 mL of demineralisedwater, adjusting the pH to 1–2 with one drop of 1 M HCl. Then,150 mL of a 2% (m/v) starch solution were added as indicator.The solution was titrated with 1 mM iodine solution untila permanent blue-violet color appeared.Degradation of chitosan and chitosan-4-MBA by lysozymeFirst, 300 mg of the polymers were hydrated in 8 ml of demineralisedwater. The pH was adjusted to 5.0 by the addition of 1 ml of0.1 M acetic buffer pH 5.0. Afterwards lysozyme previously dissolvedin demineralised water, was added in a final concentrationof 1% (m/v). The final polymer concentration was 3% (m/v). Thesamples were kept at 37 C. At predetermined time points theviscosity of 600 ml aliquots was measured at 37 C with a plateplateviscosimeter (Haake MARS) connected to a personalcomputer for setting the analysis parameters and for processingand recording the data with the Haake Rheowin program. Polymersolutions (3% m/v in demineralised water), without additionof lysozyme, were treated in the same way and served as controls.Cell culture conditionsFor the Caco-2 cell line, the medium was composed of 80%minimum essential medium (MEM) (with Earle’s salts), 20%10 mM phosphate buffered saline (PBS) pH 7.2, 1x non-essentialamino acids, 1% penicillin-streptomycin liquid. The media usedfor the EAhy926 cells consisted of 90% Dulbecco’s modifiedEagle’s medium, 10% 10 mM phosphate buffered saline (PBS),2mML-glutamate and 1% penicillin-streptomycin liquid.LDH release assayA standardized number of cells was seeded in a 96 wells plate(100 ml per well) and grown for 24 h in an incubator (37 C, 5%CO 2 , 95% relative humidity) prior to stimulation. The mediumwas removed and 100 ml of the polymer (0–100 mg ml 1 inmedium) were added. Experiments were performed in triplicate.The plate was incubated at 37 C, 5% CO 2 , 95% relative humidityfor 4 to 24 hours. The standard assay set-up included: blank(culture medium alone), growth control (culture medium withthe cells), lysis control (untreated cells in medium where 100%lysis was performed), particulate positive and negative controls(26 nm carboxyl White Polystyrene latex as positive control and160 nm Surfactant-Free Carboxyl white polystyrene Latex asnegative control). The CytoTox-ONE assay kit was performed asindicated by the producer. The average value of the blank wassubtracted from every fluorescence value. The percent toxicitywas calculated according to the formula:Percent cytotoxicity 100xðExperimental - Culture Medium BackgroundÞ¼ðMaximum LDH Release - Culture Medium BackgroundÞMTT testCell seeding and exposure to the polymer was the same as for theLDH-release but no lysis control was included in the set-up.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. Afterwards, 100 ml of thePMS solution were added to 2 ml of MTS solution and gentlyswirled before addition to the wells. Then, 20 ml of the MTS/PMSsolution were pipetted into each well of the 96 well assay platecontaining 100 ml, and the plate was incubated for 2 hours at 37 C. The absorbance was recorded at 490 nm with a plate reader.The blank (background from the culture medium) was subtractedfrom all other absorbance values.Quantification of ATPCell seeding and exposure to the polymer was the same as for theLDH-release but no lysis control was included in the set-up.Assay was performed as indicated by the producer. The blankvalues were subtracted from all values.Quantification of endotoxinsLAL (Limulus Amebocyte Lysate) was performed using thePyrogent Ultra kit. This test is a qualitative test for Gram-negativebacterial endotoxin. Gram-negative bacterial endotoxin catalysesthe activation of a proenzyme in the Limulus Amebocyte Lysate.To prepare the Limulus Amebocyte Lysate, the lyophilized lysatewas reconstituted by adding 5.2 ml LAL Reagent Water (from thekit) to 50 test vials. The solution was gently swirled. For the liquidendotoxin standards, vials containing a liquid preparation ofpurified endotoxin from E. coli strain (055:B5) were inverted fivetimes to allow mixing of the contents. Afterwards, 100 ml ofstandard (sample or water) were transferred into the reactiontube. Then, 100 ml of the reconstituted LAL were added to eachtube beginning with the highest concentration of endotoxin andmixed thoroughly. This procedure was followed for each dilutionof the endotoxin. The sample was run in parallel with the endotoxinstandards. After the incubation time, each tube was examinedfor gellation. A positive reaction is characterized by theformation of a firm gel that remains intact when the tube isinverted by a vertical rotation of 180 .Statistical data analysisStatistical data analyses were performed using Student’s t-testwith p < 0.05 as the minimum level of significance.2434 | J. Mater. Chem., 2010, 20, 2432–2440 This journal is ª The Royal Society of Chemistry 2010

View OnlineDownloaded by UNIVERSITAETSBIBLIOTHEK INNSBRUCK on 21 October 2010Published on 08 February 2010 on http://pubs.rsc.org | doi:10.1039/B916528BResults and discussionSynthesis and characterization of chitosan-4-mercaptobenzoicacidThe covalent attachment of 4-MBA to chitosan was achieved viathe formation of amide bonds between the carboxylic acid groupof the 4-MBA and amine groups of chitosan, as illustrated inFig. 1. The enhanced reactivity of 4-mercaptobenzoic acid in theintestinal pH range should favour the adhesive interactions withthe mucus, allowing the thiomer incorporated drug to stay incontact with the absorption site for a prolonged period of time,therefore favouring its uptake. Furthermore, the enhanced thiolreactivity is advantageous for in situ gelation. The lyophilizedchitosan-4-MBA conjugates appeared as a white powder offibrous structure.The efficacy of the purification method described could beverified by the corresponding controls which were prepared inexactly the same way but omitting EDAC. The amount of totalthiols present in the control was negligible. The amount ofreduced thiol groups present on the polymer was determined tobe 98.49 mmol g 1 polymer, while the total amount of thiolgroups were 176.27 mmol g 1 polymer. This results in a percent ofsubstitution of the chitosan backbone of 3.8%. After dissolvingthe final product in a 5% NaCl solution and precipitating it inisopropanol, the iodometric titration gave a value of reducedthiol groups of 94.57 mmol g 1 polymer. Therefore, the conjugationof 4-mercaptobenzoic acid to the chitosan backbone haseffectively taken place. The FT-IR spectrum of chitosan-4-mercaptobenzoicacid (ESI†) shows the appearance of new peakswhich are characteristic for an aromatic system in comparison tothe spectra for unmodified chitosan. These are mainly the peak at3100 cm 1 , which is assigned to the C–H bond in aromaticsystems, and the peak at 1650, which is assigned to C]C bond inaromatic systems as well as to C]O in R–CO–NR 2 aromaticsystems. This proves the attachment of 4-mercaptobenzoic acidto chitosan.Theoretical and experimental pK a calculationpK a values are used to express the pH at which acidic and basicgroups are half-ionized in aqueous solutions. Based on a determinedpK a value it is possible to predict the molecules interactionwith charged species or its reactivity.Accordingly, the pK a value was calculated both for the freemolecule and simulated as attached to the chitosan chain, bychanging the free carboxylic group to an amide with a methylending. The pK a value for the thiol group 4-mercaptobenzoicacid non-conjugated to chitosan was calculated to be 6.21 whilethe pK a value for the thiol group simulated as attached to thechitosan chain was calculated to be 6.04. The experimentalresults are presented in Fig. 2, where the distribution of thespecies at different pH values is presented. The curves refer to4-mercaptobenzoic acid non-conjugated to chitosan.The pK a value is determined where the curve representing thedistribution of the species when only one group is deprotonated(AH) and the curve where both groups are deprotonated (A)cross the distribution curve of the protonated molecule (AH2).The experimental value determined for the thiol group is 6.832.This pK a value falls in the range of intestinal pH. Therefore, atintestinal pH values, the thiol group of 4-mercaptobenzoic acidbound to chitosan would be mostly present in the form of a thiolateanion, which is the active form for disulfide bond formation.Mucoadhesive properties would be maximized in this way.This represents a great improvement of aliphatic thiolated chitosanswith a higher pK a , since at intestinal pH the thiol groupwould be only partially present as the thiolate anion. Furthermore,a low pK a value of the thiol group would also have a bigimpact on the in situ gelling properties of thiomers for applicationson targets where the pH is lower, such the vagina. If the pK aFig. 1Schematic structure of the newly synthesized polymer.Fig. 2 Distribution of the species at different pH values and determinationof the pK a value for the thiol group of 4-MBA not bound to thepolymer. AH2 (A) indicates the molecule in its protonated state; AH(-) indicates the molecule in the state where only the carboxylic group isdeprotonated and A (:) indicates the molecule in the state where boththe carboxylic and thiol group are deprotonated.This journal is ª The Royal Society of Chemistry 2010 J. Mater. Chem., 2010, 20, 2432–2440 | 2435

View OnlineDownloaded by UNIVERSITAETSBIBLIOTHEK INNSBRUCK on 21 October 2010Published on 08 February 2010 on http://pubs.rsc.org | doi:10.1039/B916528Bof thiols is high, at physiological pH, the gelling process caneither be too slow or not be significant without the addition ofoxidation agents. Therefore aromatic thiols, by being readilyavailable to react at lower pH in comparison to aliphatic thiols,could improve the in situ gelling properties of various formulations.Swelling behaviourThe swelling behaviour of mucoadhesive polymers is a veryimportant parameter to consider as it determines the adhesiveand cohesive properties. For mucoadhesion to occur, mucoadhesivepolymers need to take up water from the underlyingmucosal tissues by absorbing, swelling and capillary effects. 12 Ifthe delivery system is addressed to the small intestine, however, itwill reach the target in an at least partially hydrated form. Anexcessive water uptake will lead to overhydration with loss ofadhesiveness. Therefore, moderate swelling is necessary to avoidoverhydration and to avoid the loss of adhesive properties beforethe delivery system reaches the target. 13 Water uptake studies, asshown in Fig. 3, demonstrated that the covalent attachment of4-mercaptobenzoic acid to chitosan reduces the swelling behaviourof the polymer.After 360 minutes, water uptake by chitosan-4-MBA wasaround 1.5-fold lower compared to unmodified chitosan. Thisobservation is in contrast to almost all other thiolated chitosansthat did not significantly change their swelling properties incomparison to unmodified chitosan 14,15 or that even increased thewater uptake compared to unmodified chitosan. 13,16 Thisbehaviour is likely based on the lipophilic nature of the ligand.Mucoadhesive propertiesMucoadhesion studies were performed using the rotatingcylinder method. This method is supposed to simulate the in vivosituation better than simple tensile studies, as it imitates theadhesion and cohesiveness of the polymer in physiologicalmedium. 15 The results are shown in Fig. 4. Unmodified chitosandetached from the intestinal mucosa after approximately 3 h,while chitosan-4-MBA remained attached for about 170 hours.Mucoadhesive properties were increased by about 60-fold.Compared with other alkyl-thiolated chitosans exhibitinga similar amount of thiol groups like for instance chitosan-thioethylamidine,exhibiting 139.68 mmol of thiol groups per gramof polymer, chitosan-4-MBA was about 7-fold more mucoadhesive.17 This comparison confirms the higher reactivity of arylthiols. The mechanism of mucoadhesion is explained by theformation of covalent bonds between thiol groups of the polymerand cysteine-rich subdomains of glycoproteins in the mucuslayer. 18 As aromatic thiolated ligands are supposed to be morereactive compared to aliphatic thiols, chitosan-4-MBA shouldexhibit stronger mucoadhesive properties. As the pK a of 4-MBAwas determined to be around 6.8 and the pH of the intestine isaround 6.8, when the thiolated polymer is present in the intestine,its thiol groups will be readily present in the thiol anion form.Therefore, disulfide bond formation with the thiolated glycoproteinsof the mucus would be favoured.Furthermore, due to its structure, 4-MBA should increase thelipophilic character of the polymer. Besides the higher reactivityof the thiol group, mucoadhesion could also be enhanced bylowering the water uptake by the polymer. It is well documentedthat slower swelling rates favor longer duration of adhesion. 19,20Moisture is important as it will plasticize the system allowingmucoadhesive molecules to become more flexible, conform to theshape of the surface, and subsequently to be bound to it.However, the mucoadhesive polymer in an aqueous environmentcan overhydrate to form a slippery mucilage, which is readilyremoved. 21 Controlling the rate and extent of hydration isrequired to produce prolonged adhesion, and strategies such asintroducing hydrophobic entities have been tried to achieve thisgoal. 22 As previously described, the uptake of water of chitosan-4-MBA was 1.5-fold lower compared to unmodified chitosan.Moreover, the attachment of mucin molecules to hydrophobicsurfaces resulted in strong and stable complexes. 23 The inclusionof hydrophobic entities in mucoadhesive systems is thereforea promising strategy to prolong mucoadhesion. 24 Hence, thishydrophobic substructure combined with the disulfide bondFig. 3 Uptake of water by unmodified chitosan (-) and chitosan-4-MBA (A). Tablets of the polymers were fixed on needles and immersedin 100 mM phosphate buffer pH 6.8 and kept at 37 C. The values are theresults of at least 3 experiments SD.Fig. 4 Mucoadhesive properties of unmodified chitosan and chitosan-4-MBA determined using the rotating cylinder method on freshly excisedporcine mucosa in 100 mM phosphate buffer pH 6.8 at 37 C. The resultsare means of at least 3 experiments SD.2436 | J. Mater. Chem., 2010, 20, 2432–2440 This journal is ª The Royal Society of Chemistry 2010

View Onlineformation with mucus glycoproteins seems to be a good strategyand could improve the delivery of many drugs by offering highmucoadhesion.Downloaded by UNIVERSITAETSBIBLIOTHEK INNSBRUCK on 21 October 2010Published on 08 February 2010 on http://pubs.rsc.org | doi:10.1039/B916528BCohesive propertiesUnmodified chitosan tablets were stable for about 6 hours whilechitosan-4-MBA tablets maintained their integrity twice as long(Fig. 5). The increased stability of chitosan-4-MBA is assigned tothe formation of stabilizing disulfide bonds within the polymericnetwork. The cohesiveness of thiomer tablets increases immediatelyin aqueous media due to water interpenetration in thepolymer matrix enabling the crosslinking formation within thepolymer chains. Therefore, disulfide bonds, as an integral part ofthe structure of thiolated polymers, contribute to the cohesivenessof the tablets.Rheological behaviourThe free thiol groups of thiolated polymers are oxidized todisulfide bonds. The extent of the reaction depends on the pK avalue of the thiol group and the pH value of the thiomer solution.Thiomers whose thiol group has a low pK a value will be able toform disulfide bonds at lower pH values, whereas polymerswhose thiol group has a high pK a value will form disulfide bondsto an effective extent only at higher pH values. There is a significantdecrease in thiol groups for chitosan-4-mercaptobenzoicacid although the pH of the experiment was 5.5 (Fig. 6).Other thiomers were quite inefficient to form disulfide bonds atthis pH range. For instance, chitosan-thioglycolic acid showeda substantial decrease of thiol groups only at pH 6.5. At pH 5 theoxidation process was limited. The authors reported that the pK athiol group was 10.6. 16 Moreover, chitosan-2-iminothiolaneshowed very limited disulfide formation at pH 4, 5 and 6. Theauthors estimated the pK a of the thiol group of 2-iminothiolaneattached to the polymer to be 9.94. 25 This comparison allows usto hypothesize that due to the structure of 4-mercaptobenzoicacid and its lower pK a value, the newly synthesised thiomer seemsto have a higher reactivity. Indeed, it was already suggested thatFig. 6 Decrease in reduced thiol groups on chitosan-4-mercaptobenzoicacid (A) of a 1% (m/v) polymer solution in 100 mM acetate buffer pH 5.5incubated at 37 C. Data are means of at least three experiments SD(n ¼ 3).the formation of disulfide bonds could be accelerated bydecreasing the pK a of the thiol groups via electrostatic interactionsor by addition of traces of Cu 2+ . 26 Chitosan-4-MBA offersthe advantage of a lower pK a without the need of further additionalsubstances.The decrease in thiol groups is in good agreement with theincrease in viscosity. There is a very high increase in viscosity ofthe polymer over time, although the coupling rate is rather low(Fig. 7).Beside the high reactivity of the thiol group, such high increasein viscosity could be favoured by the hydrophobic nature of the4-mercaptobenzoic acid. Hydrophobic forces may play animportant role in the gelation process. 27 It has been reported thatthe thickening power of poloxamers in water increased withFig. 5 Cohesive properties of unmodified chitosan and chitosan-4-MBAperformed using the disintegration apparatus in 100 mM phosphatebuffer pH 6.8 at an oscillation frequency of 0.5 s 1 at 37 C. The resultsare means of at least 3 experiments SD.Fig. 7 Increase in viscosity of a 1% (m/v) solutions of chitosan-4-mercaptobenzoicacid (-) and unmodified chitosan (A) in 100 mM acetatebuffer pH 5.5 incubated at 37 C. Data are means of at least threeexperiments SD (n ¼ 3).This journal is ª The Royal Society of Chemistry 2010 J. Mater. Chem., 2010, 20, 2432–2440 | 2437

View OnlineDownloaded by UNIVERSITAETSBIBLIOTHEK INNSBRUCK on 21 October 2010Published on 08 February 2010 on http://pubs.rsc.org | doi:10.1039/B916528Bincreasing hydrophobic molecular mass. 28 Therefore, due to thehigh reactivity and increased hydrophobicity of the ligand, thegelling properties can be greatly improved. The combination ofgelling and mucoadhesive properties renders this novel thiolatedchitosan a useful excipient for various drug delivery systems.Vaginal drug delivery, for instance, has to deal with rapidremoval of inserted systems. 29 Due to its mucoadhesive and insitu gelling properties, chitosan-4-MBA could be a promisingvaginal drug delivery system. Ocular delivery of drugs results inpoor bioavailability. In order to increase the bioavailability ofocular applied drugs, the residence time should be increased. 30Once again, mucoadhesive and in situ gelling properties suchthose exhibited by chitosan-4-MBA could be a solution.Furthermore, another potential application would be the nasaldelivery of drugs as the major drawback is the removal ofsubstances by mucociliary activity. 31Susceptibility towards lysozyme degradationChitosan is a biodegradable polymer which does not accumulatein the body. It is therefore important to evaluate how thiolationwith this new class of thiolated ligands affects biodegradability ofthe original polymer. The loss in viscosity determined by theaddition of lysozyme to the polymer is the result of the degradationof the polymer network in smaller fragments. Some alkylthiolatedchitosans have already been tested regarding lysozymedegradation. For example, chitosan-thioglycolic acid has beenreported to show a slower degree of degradation when comparedto unmodified chitosan. 16 It was reported that chitosan-N-acetylcysteine shows a lower susceptibility towards lysozyme withrespect to unmodified chitosan. Results obtained by studyingchitosan-4-MBA are shown in Fig. 8.Chitosan-4-MBA was degraded to a slightly greater extentcompared to unmodified chitosan. It is probable that thehydrophobic nature of the ligand increases the affinity for theenzyme at the cleavage site. It was observed that by the introductionof hydrophobic entities on the chitosan structure, theblood compatibility properties were improved. 32 Furthermore,aromatic thiols significantly enhanced the folding rate of lysozymerelative to glutathione. The enhanced degradation rateconstant was proposed to be due to the enhanced leaving groupability of aromatic thiols relative to glutathione and theirenhanced nucleophilicity relative to aliphatic thiols with similarthiol pK a values. 10,33Safety aspectsLDH test. This assay permits the investigation of substancesthat may induce alterations in cell integrity and therefore quantifiesthe amount of non-viable cells. LDH is a stable enzymepresent in the cytosol that is released upon cell lysis. 34The release of lactate dehydrogenase into the culture mediumis measured via a coupled enzymatic assay that results in theconversion of resazurin into a fluorescent resofurin product. Theamount of fluorescence is proportional to the number of lysedcells. The test was performed on human colorectal carcinoma cellFig. 8 Rheological studies of unmodified chitosan and chitosan-4-MBA. The viscosity expressed as a percentage of the initial value wasmeasured for both polymers in presence as well as in absence of 1% (m/v)lysozyme over a period of 60 minutes. Legend: (>) unmodified chitosan,(,) chitosan-4-MBA, (A) unmodified chitosan + lysozyme, (-) chitosan-4-MBA+ lysozyme. The results are means of at least 3 experiments SD.Fig. 9 (a) LDH test performed on Caco-2 cell line after 4 hour ofincubation with chitosan-4-MBA (black bars) and unmodified chitosan(grey bars) at indicated polymer concentrations. The results are expressedas % of cell death. The results are means of at least 3 experiments SD.(b) LDH test performed on Caco-2 cell line after 24 hour of incubationwith chitosan-4-MBA (black bars) and unmodified chitosan (grey bars) atindicated polymer concentrations. The results are expressed as % of celldeath. The results are means of at least 3 experiments SD.2438 | J. Mater. Chem., 2010, 20, 2432–2440 This journal is ª The Royal Society of Chemistry 2010

View OnlineDownloaded by UNIVERSITAETSBIBLIOTHEK INNSBRUCK on 21 October 2010Published on 08 February 2010 on http://pubs.rsc.org | doi:10.1039/B916528BFig. 10 (a) Cell viability test performed on Caco-2 cell line for chitosan-4-MBA (black bars) and unmodified chitosan (grey bars) after 4 hours ofincubation. The results are means of at least 3 experiments SD. (b) Cellviability test performed on Caco-2 cell line for chitosan-4-MBA (blackbars) and unmodified chitosan (grey bars) after 24 hours of incubation.The results are means of at least 3 experiments SD.lines (Caco-2). The tests were performed at an incubation time of4 and 24 hours in different polymer concentrations. The test(Fig. 9a,b) indicates that chitosan-4-MBA has the same toxicityas chitosan, which is very low. The values stay constant after24 indicating the lack of toxicity. Therefore, the polymer can beconsidered as relatively safe for in vivo applications.MTT test. This test is measuring the number of viable cells.Although formazan bioreduction is the classical cytotoxicityscreening assay for conventional substances, The MTS (tetrazoliumcompound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]isbioreduced by cells into a formazan product that is soluble intissue culture medium by dehydrogenase enzymes found inmetabolically active cells. The tests performed on Caco-2 celllines showed a cell viability of around 100% for all the concentrationstested after 4 and 24 hours indicating that the polymersare not harmful for the cells. The viability was around 100%.There was no trend in results depending on the concentrationused (data not shown).Quantification of ATP. This test measures the number of viablecells by luminescent quantification of the ATP present, an indicationof metabolically active cells. The tests showed (Fig. 10a,b)a very high cell viability for all the concentration tested after4 and 24 hours, indicating that cells maintain their metabolicactivity, and therefore the polymers can be considered as nontoxic.Endotoxin quantification. Endotoxins are natural compoundsfound inside pathogens such as bacteria. These are not secretedin soluble form by live bacteria but are a structural component ofthe bacteria which is released mainly when bacteria are lysed. Thesamples in stock solution contained less than 0.06 EU ml 1(Endotoxic Unit ml 1 ).ConclusionWithin this study, chitosan-4-mercaptnicotinic acid has beensynthesized and characterized for the first time. The new strategyfor optimizing thiolated chitosans led to a unique type of thiolatedchitosan which exhibits its full reactivity at intestinal pH.The newly synthesized polymer exhibited excellent in situ gellingproperties and improved mucoadhesive properties compared toaliphatic thiolated chitosans with similar coupling rates. Due tothese properties, the newly synthesised polymer is an excellentcandidate as excipient for oral formulations and in situ gellingformulations for various applications, such as vaginal delivery.AcknowledgementsThis work has been supported by the EC. Nano-BioPharmaceutics is an Integrated Project funded within the 6thFramework Programme of the European Commission. Authorsare thankful to Jonny Easmond for help with the pK a calculations.Authors are thankful to Lukas Bittner from the AnalyticalChemistry Department at the University of Innsbruck for theFT-IR analysis.References1 A. Bernkop-Schnurch, V. Schwarz and S. Steininger, Pharm. Res.,1999, 16, 876–881.2 A. Bernkop-Schnurch, H. Zarti and G. F. Walker, J. Pharm. Sci.,2001, 90, 1907–1914.3 A. E. Clausen, C. E. Kast and A. Bernkop-Schnurch, Pharm. Res.,2002, 19, 602–608.4 F. Foger, T. Schmitz and A. Bernkop-Schnurch, Biomaterials, 2006,27, 4250–4255.5 J. M. Wilson, R. J. Bayer and H.D.J., J. Am. Chem. Soc., 1977, 99(24),7922–7926.6 C. R. a. B. Ray and S, J. Electroanal. Chem., 2005, 581, 61–69.7 J. D. Gough, J. M. Gargano, A. E. Donofrio and W. J. Lees,Biochemistry, 2003, 42, 11787–11797.8 J. D. Gough and W. J. Lees, Bioorg. Med. Chem. Lett., 2005, 15, 777–781.9 J. D. Gough and W. J. Lees, J. Biotechnol., 2005, 115, 279–290.10 M. C. Gurbhele-Tupkar, L. R. Perez, Y. Silva and W. J. Lees, Bioorg.Med. Chem., 2008, 16, 2579–2590.11 I. Bravo-Osuna, D. Teutonico, S. Arpicco, C. Vauthier andG. Ponchel, Int. J. Pharm., 2007, 340, 173–181.12 D. Duchene and G. Ponchel, Biomaterials, 1992, 13, 709–714.13 M. Roldo, M. Hornof, P. Caliceti and A. Bernkop-Schnurch, Eur.J. Pharm. Biopharm., 2004, 57, 115–121.14 T. Schmitz, V. Grabovac, T. F. Palmberg, M. H. Hoffer andA. Bernkop-Schnurch, Int. J. Pharm., 2007.This journal is ª The Royal Society of Chemistry 2010 J. Mater. Chem., 2010, 20, 2432–2440 | 2439

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