International Journal of Pharmaceutics 478 (2015) 383–389
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Personalised medicine
Preactivated hyaluronic acid: A potential mucoadhesive polymer for vaginal delivery Jessika Nowak, Flavia Laffleur, Andreas Bernkop-Schnürch * Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Austria
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 July 2014 Received in revised form 20 November 2014 Accepted 21 November 2014 Available online 24 November 2014
The objective of this study was to develop mucoadhesive polymeric excipients for vaginal drug delivery systems. Hyaluronic acid was thiolated and subsequently preactivated with 6-mercaptonicotinamide (HA-CYS–MNA) to enhance stability and mucoadhesive properties on vaginal mucosa. After determination of the thiol group content, disintegration studies and in vitro mucoadhesion studies (rotating cylinder and tensile) were performed. Furthermore, swelling behavior and cytotoxicity studies were performed in comparison with corresponding polymers. Both, disintegration and in vitro mucoadhesive studies revealed that modifying HA-CYS with MNA resulted in higher stability (3.6-fold prolonged disintegration time compared to unmodified hyaluronic acid) and prolonged mucoadhesion time. MTT assay and LDH revealed no toxicity for the polymeric excipients and safe for their use. Disintegration and swelling results conducted more pronounced stability of the preactivated thiomers compared to corresponding unmodified ones. According to these results preactivated hyaluronic acid might be a useful tool for vaginal delivery systems. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Mucoadhesion Hyaluronic acid Preactivated thiomers Thiomers Vaginal delivery
1. Introduction Mucoadhesive polymers are of pharmaceutical interest since the introduction of mucoadhesion in the 1980s. Belonging to the group of mucoadhesive polymers, thiomers or so called thiolated polymers swell rapidly in an aqueous environment attached to the mucus through formation of disulfide bonds (Bernkop-Schnurch, 2005; Bernkop-Schnurch et al., 2004). Thus, the residence time is prolonged and the drug uptake is enhanced. Thiolated polymers belong to a unique and new generation of mucoadhesive polymers. They form disulfide bonds with cysteine bearing subdomains of the mucus glycoproteins which are stronger than non-covalent interactions and therefore provide stronger adhesion. Thiomers have high in-situ gelling properties, are mucoadhesive, offer the possibility to apply drugs in a controlled release form (Wang et al., 2012). This could be shown lately for hyaluronic acid (Li et al., 2012). But one disadvantage of thiomers is the poor stability in solutions and the high ability of thiol oxidation at a pH above 5
* Corresponding author at: Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, Innrain 80-82, A-6020 Innsbruck, Austria. Tel.: +43 51250758600; fax: +43 51250758699. E-mail address: [email protected] (A. Bernkop-Schnürch). http://dx.doi.org/10.1016/j.ijpharm.2014.11.048 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
(Bernkop-Schnurch et al., 2003). To overcome this drawback new preactivated thiomers are synthesized to enhance stability, mucoadhesion and cohesive properties of the thiomers. Hyaluronic acid (HA), which is also known as hyaluronan or hyaluronate, is a viscoelastic linear polysaccharide which consists of alternating disaccharide units of D-glucuronic acid and N-acetylD-glucosamine linked by b-1–3 and b-1–4 glycosides bonds (Kafedjiiski et al., 2007). HA was investigated as a possible drug delivery agent using various routes of administration such as ophthalmic, nasal, pulmonary, oral, parenteral and topical (Yadav et al., 2008). High vascularization of the vagina turns this mucosa to a possible and promising administration site for not only locally efficacious drugs but also for systemic drug delivery. By using the vagina as a possible drug delivery route several advantages such as a great permeation area because of the high vascularization (vaginal cavity approximately 60 cm2), the avoidance of hepatic first-pass metabolism and therefore less gastrointestinal side effects are offered (Hussain and Ahsan, 2005; Valenta, 2005; Baloglu et al., 2009). In this study HA-CYS was preactivated with 6-mercaptonicotinamide to prevent oxidation and therefore enhance the stability in an aqueous environment and investigated as a possible mucoadhesive polymer (Laffleur and Bernkop-Schnurch, 2012).
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2. Material and methods
with an increasing amount of L-cysteine hydrochloride were used (Rahmat et al., 2012).
2.1. Materials 2.6. Disulfide bond test Hyaluronic acid sodium salt from Streptococcus equi (1.5– 1.8 106 Da), 6-chloronicotinamide, thiourea, hydrogen peroxide, L-cysteine ethyl ester hydrochloride, reduced glutathione, Nhydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma–Aldrich. All other reagents used were of analytical grade.
The disulfide bond test was performed to measure the amount of disulfide bonds as an indicator for successful activation and preactivation, respectively. Thereby NaBH4 was used to reduce the disulfide bonds and after that the thiol group content was measured as mentioned above (Bernkop-Schnurch et al., 1999). 2.7. Quantification of conjugated 6-mercaptonicotinamide
2.2. Synthesis of hyaluronic acid-L-cysteine ethyl ester hydrochloride conjugates Our research group was the first who synthesized hyaluronic acid-L-cysteine ethyl ester (HA-CYS) (Kafedjiiski et al., 2007). In brief, 0.200 g of hyaluronic acid was dissolved in 50 mL demineralized water to gain a 0.4% (w/v) solution. The pH was adjusted to 5.5 with 0.1 M hydrochloric acid. Afterwards 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and Nhydroxysuccinimide (NHS) were added in a molar ratio of 1:1. Again, the pH was adjusted to 5.5. After stirring at room temperature for 15 min 0.250 g of L-cysteine ethyl ester hydrochloride was added resulting in pH 6. The solution was incubated under further stirring for 4 h and then filled into tubings with a molecular weight cut off (MWCO) of 12,000–20,000 Da. The tubings were dialyzed thrice against demineralized water containing 1% of NaCl and once against demineralized water in the dark at 10 C. Finally the obtained solution was frozen; freeze dried and kept in the refrigerator at 4 C until further use.
The amount of MNA was measured with a photometer. 0.5 mg of the HA-CYS–MNA was dissolved in 0.5 M phosphate buffer pH 6.8. After adding a 2% reduced glutathione solution, the samples were incubated for 1 h at 37 C. The absorbance of 100 mL of this solution was measured at 317 nm (MNA peak). MNA was employed to establish the calibration curve. 2.8. FT-IR characterization of hyaluronic acid-L-cysteinemercaptonicotinamide The dimer was characterized by FT-IR analysis. The characterization was conducted by attenuated-total-reflectance Fourier transform infrared spectroscopy (ATR–FTIR). The spectra were obtained by a PerkinElmer Spectrum 100 ATR–FTIR spectrometer (PerkinElmer, Waltham, USA) in combination with a Spectrum software version 6.3.1.0134 (PerkinElmer, Waltham, USA). Samples were measured at 22 C and were divided in six subsamples. 8 scans in a range from 4000 cm1 to 600 cm1 and a resolution of 4 cm1 were taken.
2.3. Synthesis of 6-mercaptonicotinamide 2.9. Cell viability screening-MTT assay 6-Mercaptonicotinamide was synthesized using 6-chloronicotinamide and thiourea. 4.4 g of thiourea and 9.0 g of 6-chloronicotinamide were dissolved in 79.4 mL of ethanol. The solution was stirred for 6 h at 90 C at reflux, and overnight without any heating. Afterwards the solvent was removed under vacuum. The product was dissolved in demineralized water and 5 M sodium hydroxide solution and stirred for another 45 min. Afterwards the pH was adjusted to 4.2 with glacial acetic acid and again stirred for 15 min. The solvent was removed with a suction filter and dried at 40 C. In the second step the product was dimerised. The MNA monomer was suspended in water and the pH was adjusted to 7–8 with sodium hydroxide solution. After adding hydrogen peroxide (30% w/v) drop wisely the product was filtered, washed with demineralized water and dried at 40 C. It was kept at room temperature for further use. 2.4. Synthesis of hyaluronic acid-L-cysteine-mercaptonicotinamide For preactivation HA-CYS solution of 0.1% (w/v) was prepared in a DMSO/water mixture with a ratio of 7:3. The pH was adjusted to 6.0. The MNA dimer was re-suspended in DMSO. For the preactivation HA-CYS and MNA were used in a molecular ratio of 1:1. After addition of MNA aliquots to the thiomer mixture, a 24 h stirring process followed. As following step, the dialysation took place in tubings (MWCO of 12,000–20,000 Da) at 10 C under light protection. After lyophilization, the obtained hyaluronic acid-Lcysteine-mercaptonicotinamide was kept at 4 C until further use. 2.5. Determination of the thiol group content The amount of thiol groups was quantified spectrophometrically using ellman’s reagent. For the calibration curve solutions
To determine cell viability the MTT assay was performed on Caco-2 cells (Mosmann, 1983). Therefore 10 104 cells (Caco2) per mL were supplemented with 5% FBS (fetal bovine serum), antibiotics, non essential amino acid (NEAA) and sodium pyruvate. 100 mL of the cells were cultured in each well from the 96 well plates. The cells were cultivated for 24 h at 37 C in a humidified atmosphere with 5% CO2. The next day the polymers were dissolved in MEM containing 5% FBS, antibiotics, non essential amino acid (NEAA) and sodium pyruvate in a concentration of 0.5% m/v. In the following the test solutions, the negative control (MEM containing 5% FBS, antibiotics, NEAA and sodium pyruvate) and the positive control (Triton-X100, 1 mM) were added in triplicate to the cells in 100 mL quantity after being washed twice with PBS. The plates were cultivated for 3 and 24 h at 37 C in a humidified atmosphere with 5% CO2, respectively. The test solutions were then removed and fresh medium containing 10% MTT was added to each plate in a quantity of 100 mL. After incubating for 3 h at 37 C in a humidified atmosphere with 5% CO2 the MTT solution was removed and 100 mL of DMSO was added into each plate to dissolve the obtained formazan crystals. The plate was put on an orbital shaker and mixed for 2 min before measuring the absorbance at 570 nm. 2.10. Cytotoxicity LDH (lactate dehydrogenase) Lactate dehydrogenase (LDH) is a cytosolic enzyme available in cells. By damaging the cell membrane LDH is released in the culture medium. By working on LDH assay, the cytotoxicity effect of testing substrates can be detected. For this purpose, cells were seeded in 24 well plates in a density of 100,000 cells/well. Cells were treated for 3 h and 24 h with HA 0.5% (w/v), HA-CYS
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0.5% (w/v), minimum essential medium as negative control and Triton X-100 2% (v/v) as positive control, respectively. The released amount of LDH was detected via a commercial test kit (Roche Diagnostics, Meylan, France) by measuring at 492 nm with Tecan infinite M200 spectrophotometer (Tecan, Grödig, Austria). 2.11. Preparation of tablets Lyophilized HA, HA-CYS and HA-CYS–MNA were compressed to 30 mg (5.0 mm diameter, 1.2 mm thickness) flat-faced tablets with a tablet press (Paul Weber, Maschinen- und Apparatebau, Remshalden, Germany). During the compaction the force was kept at 10 kN for 20 s.
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mobile platform until the tablet attached to the mucosa. After 20 min of incubation at room temperature the balance was pulled down manually with a rate of 0.1 mm/s using a knob, which was provided on the platform. To ensure the precise displacement of 0.1 mm/s the regulating knob was marked and validated via length measurements. The data was collected every second with appropriate software (SartoCollect V 1.0; Satorius AG, Germany) and transferred to Excel 2010. Force versus displacement curves was analyzed to calculate the total work of adhesion as the area under the curve in accordance with the trapezoidal rule. 3. Results and discussion 3.1. Synthesis and characterization of preactivated thiolated polymers
2.12. Disintegration studies 2.12.1. Evaluation of the swelling behavior The water absorbing capacity of HA, HA-CYS and HA-CYS–MNA was measured in simulated vaginal fluid [NaCl 3.51 g, KOH 1.40 g, Ca(OH)2 0.222 g, albumin 0.018 g, acetic acid 1.00 g, lactic acid 2.0 g, glycerol 0.16 g, urea 0.4 g, glucose 5.0 g, aqua dest. ad 1000 mL (pH was adjusted to 4.2 with HCl)] (Baloglu et al., 2011) with a gravimetric method (Laffleur et al., 2014). The 30 mg tablets were weighed and fixed on the tip of a needle. The tablets were put in 15 mL falcon tubes filled with the simulated vaginal fluid which was preheated at 37 C. During the whole experiment the tubes were kept in a water bath at 37 C. After certain time points the tablets were carefully taken out of the medium, excess water was removed with a tissue and then the tablets were weighed again and the water uptake (%) was determined using the following equation: water uptakeð%Þ ¼
Wt W0 W0 100
Wt is the weight of the polymer at a given time and W0 is the polymer in the dry form. 2.12.2. Disintegration test Disintegration behavior of the tablets comprising hyaluronic acid, HA-CYS and HA-CYS–MNA were evaluated in simulated vaginal fluid pH 4.2 at 37 0.5 C with a disintegration test apparatus according to the European Pharmacopoeia. The oscillating frequency was adjusted to 0.5 s1 (Kafedjiiski et al., 2005).
Hyaluronic acid served as polymeric backbone for the immobilization of thiol moieties of the thiol bearing compound L-cysteine ethyl ester where the covalent attachment occurs due to an amide bond formation (Bernkop-Schnurch et al., 1999). Via ellman’s reagent the amount of thiol groups were determined exhibiting 218.87 9.19 mmol per gram of hyaluronic acid. The amount of disulfide bonds were 173.80 63.5 mmol per gram of hyaluronic acid as presented in Table 1. HA-CYS showed to be a white fibrous powder. The preactivation of HA-CYS was performed in a solution of DMSO and demineralized water. The synthesis of the 6-MNA dimer is shown in Fig. 1. A disulfide bond was formed between the thiol group of HA-CYS and the thiol groups of the 6-MNA dimer. The structure of the obtained polymer is shown in Fig. 2. The lyophilized HA-CYS–MNA is an odorless powder of fibrous structure and of yellow color. The powder is poorly soluble in water. Ellman’s test and disulfide bond test were performed to show the grade of preactivation. The thiol group content was 20.46 2.05 mmol per gram of polymer and the disulfide bonds were 163.97 59.83 mmol per gram of polymer. These results show that a successful coupling of MNA to the thiol group of hyaluronic acid took place. The higher the amount of the disulfide bonds the higher is the amount of immobilized MNA. In addition the amount of MNA was determined in the preactivated polymer, Table 1 The amount of thiol/disulfide groups and MNA for HA-CYS and HA-CYS–MNA. Conjugates
Thiol groups (mmol/g polymer SD; n = 3)
Disulfide groups (mmol/g polymer SD; n = 3)
HA-CYS HA-CYS– MNA
218.87 9.19 20.46 2.05
173.80 63.5 163.97 59.83
2.13. In vitro mucoadhesion studies 2.13.1. Rotating cylinder method Mucoadhesion was measured with a disintegration tester (Erweka DT 700, Germany). Freshly excised bovine vaginal mucosa was used, which was obtained from the local slaughterhouse. The vaginal mucosa was fixed with glue on the cylinders and the tablets of HA, HA-CYS and HA-CYS–MNA were attached to the mucosa with soft pressure. The baskets were filled with 900 mL simulated vaginal fluid pH 4.2 and preheated to 37 C. This temperature was kept constant and the cylinders rotated with 100 rotations/min during the whole experiment. The time when the tablets detached from the mucosa was measured (Iqbal et al., 2012). 2.13.2. Tensile studies Tensile strength assays were conducted on freshly excised bovine vaginal mucosa according to a method described previously (Bernkop-Schnurch et al., 2001). Briefly, the mucosa was fixed on a glass platform with cyanoacrylate glue. The tablet was glued to a stainless steel flat disc (10 mm in diameter), which was hung from a laboratory with a nylon thread (15 cm). The glass platform with the mucosa was placed on a balance and carefully raised by a
Fig. 1. Synthesis of 6-MNA.
MNA (mmol/g polymer SD; n = 3)
186.60 48.29
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Fig. 2. Preactivation of hyaluronic acid with L-cysteine ethyl ester hydrochloride and coupling with MNA.
showing 85.3% activation with 186.60 48.29 mmol MNA per gram of HA-CYS. The proposed mechanism of the oxidative coupling is the covalent coupling of MNA dimer to the free thiol group of the activated polymer and the removal of free MNA. Furthermore, the IR spectra were in good agreement with the synthesized thiomer. The typical signals such as 3161 cm1 and 3159 cm1 representing the C—H stretch as well as the N—H bend and —C¼C— were shown at 1653–1619 cm1. Moreover, the
proving amide bond formation was found in HA-CYS–MNA with 1735–1700 cm1, where as it could not be detected for unmodified HA as shown in Fig. 3. 3.2. MTT assay Caco-2 cells were incubated with hyaluronic acid, HA-CYS and the HA-CYS–MNA for either 3 or 24 h to determine their effect on
Fig. 3. Comparison of FT-IR spectra of unmodified hyaluronic acid (black line) and preactivated HA-CYS–MNA (red line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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cell viability. The results are shown in Fig. 4. After 3 h incubation HA, HA-CYS and HA-CYS–MNA showed cell viability of 95.52 7.04%, 91.20 18.49% and 99.90 17.6%. The results for HA, HA-CYS and HA-CYS–MNA after the 24 h incubation exhibited were 70.27 10.99%, 71.38 4.13% and 66.94 8.03% cell viability, respectively. This showed that these polymeric excipients are not cytotoxic and not harmful to the cells. The polymers were all tested in a concentration of 0.5% (w/v) because a higher concentration would have led to a higher viscosity and therefore the measurement would have been impossible. 3.3. LDH cell toxicity test In vitro cytotoxicity of the polymer was determined by measuring the release of the enzyme lactate dehydrogenase (LDH) from damaged Caco-2 cells. In vitro cytotoxicity results are presented in Fig. 5. These results showed that negative control (MEM without FCS and phenol red) and positive control (Triton-X) indicate 0.20 0.06% and 100 0.63% cytotoxicity, respectively. The resulting thiolated HA (0.5% w/v) exhibited only 7.50 0.18% and 25.27 0.20% of toxicity after 3 and 24 h, respectively. Unmodified HA was found 5.43 0.06% and 17.14 0.24% toxic after 3 and 24 h, respectively. Preactivated HA showed 8.55 0.05% and 27.28 0.84% cytotoxicity which might be due to the presence of mercaptonicotinic groups present on the activated adduct. 3.4. Swelling behavior The swelling behavior of mucoadhesive polymers is an important parameter as it correlates with the stability, adhesive properties and cohesiveness. After attaching to the mucus the tablet swelled up by initiating water absorption from the underlying mucosal surface via capillary action and diffusion process leading to adhesion. In contrary, overhydration results in less cohesiveness and less mucoadhesion. Therefore moderate swelling is necessary to minimize the risk of detachment of the polymer from the mucus and to maximize the time of adhesion. The water uptake studies were performed in simulated vaginal fluid with a pH of 4.2 at 37 C with 30 mg flat faced tablets based on the unmodified polymer (HA), HA-CYS and HA-CYS–MNA. The results are shown in Fig. 6. The unmodified polymer showed a strong and linear water uptake. At 120 min hyaluronic acid disintegrated and no further water uptake could be measured. It was observed that the covalent attachment of L-cysteine ethyl ester to hyaluronic acid significantly
Fig. 5. Cytotoxicity assay working on LDH. Caco-2 cells were treated with HA 0.5%, HA-CYS 0.5% and HA-CYS–MNA 0.5% for 3 h (black bars) and 24 h (white bars), respectively. Results are expressed as means SD (n = 3).
enhanced the swelling capacity in the first 30 min. After this time period no further significant water uptake could be demonstrated. At 150 min the thiomer disintegrated. In comparison to the above mentioned polymers HA-CYS–MNA showed the slowest increase in water uptake, therefore a constant linear rise. At 150 min the polymer still swelled up, but after 180 min it disintegrated. This might be explained by the lipophilic nature of MNA together with the higher amount of inter and intramolecular disulfide bonds within the polymer resulting in an increased stability and higher swelling capacity than HA-CYS. 3.5. Disintegration Disintegration studies revealed that disintegration time increased with degree of chemical modification as the difference in
Fig. 4. MTT cell viability of HA, HA-CYS, HA-CYS–MNA obtained by 3 h (grey bars) and 24 h (black bars), respectively. Data are expressed as means SD (n = 3).
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Fig. 6. Swelling behavior of HA, HA-CYS and HA-CYS–MNA; data are expressed as means SD (n = 3).
disintegration time between unmodified, thiolated and pre-activated polymers become more pronounced regarding the thiol group amounts. Results are shown in Fig. 7. As expected, tablets comprising unmodified hyaluronic acid completely disintegrated within 45 min. It is clear that thiolation process greatly improves the crosslinking properties of polymers. Tablets comprising polymers with L-cysteine ethyl ester in comparison to the tablets comprising corresponding unmodified polymers displayed 3.1-fold prolonged disintegration time. Moreover, the covalent attachment of 6-mercaptonicotinamide (MNA) to HA-CYS significantly enhanced the stability of these thiomers and 3.6-fold prolonged disintegration times were achieved in the presence of HA-CYS–MNA conjugates compared to unmodified hyaluronic acid. These observations might be explained by the particulate nature of MNA together with the higher amount of interand/or intramolecular disulfide bonds within the conjugate being responsible for increased stability and hardness of HA-CYS–MNA. The results obtained from disintegration studies are in accordance with the theory that formation of covalent bonds (disulfide bonds)
Fig. 7. Disintegration time for HA, HA-CYS and HA-CYS–MNA, data are expressed as means SD (n = 3).
within polymer structure resulted in enhanced stability and cohesion. The formation of covalent bonds (disulfide bonds) within the polymer leads to an enhanced stability and cohesion that is responsible for the disintegration time. In accordance, the introduction of MNA as S-protective leaving group further prolonged the disintegration of disks in both media. This might be caused by the lipophilic nature of MNA that increases hydrophobicity of the conjugate, in combination with an increased formation of inter- and/ or intramolecular disulfide bonds since crosslinking influences chain mobility and resistance to dissolution. 3.6. In vitro mucoadhesion studies In vitro mucoadhesion studies with the rotating cylinder showed the time when the unmodified polymer, HA-CYS and HA-CYS–MNA detached from the mucosa in a time period of 48 h. As expected, HA firstly detached after a time period of 2.8 h as shown in Fig. 8. This can be explained by the strong hydration and by the lack of disulfide bond formation – only non-covalent bonds are formed to stay on the mucus. HA-CYS detached after 11.5 h. This correlates with the theory that disulfide bonds between thiolated polymers and glycoproteins from the mucus are formed and therefore mucoadhesion is enhanced. In contrast to HA and HACYS, HA-CYS–MNA did not detach from the mucosa during the whole experiment. This can be explained by the moderate swelling behavior and by preactivation with MNA which led to higher stability because of less oxidation of thiol groups and higher attendance to form disulfide bonds with the mucus. Tensile strength studies which were performed with freshly excised bovine vaginal mucosa confirm the results obtained in the rotating cylinder study. The results are shown in Fig. 9. HA-CYS– MNA showed the highest adhesive force. This can be explained by the presence of disulfide bond which prevent early oxidation of thiol groups before attaching to the mucus layer. MNA as a hydrophobic agent decreases the dispersive properties of HA-CYS resulting in higher stability and mucoadhesion. The in vitro studies are in accordance with the theory that a thiolation leads to an enhancement of mucoadhesion by forming disulfide bonds with the mucus layer and that preactivation minimizes the risk of oxidation.
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mucoadhesive drug delivery systems and leads to additional possibilities in the field of vaginal formulation development. Acknowledgement The authors wish to thank the local slaughterhouse Piegger in Sistrans for the vaginal mucosa and the FWF (Fonds zur Förderung der wissenschaftlichen Forschung) project no P23515 – B11. References
Fig. 8. Rotating cylinder study with HA, HA-CYS, HA-CYS–MNA; data are expressed as means SD (n = 3).
Fig. 9. Total work of adhesion (TWA) in mJ of HA, HA-CYS, HA-CYS–MNA; data are expressed as means SD (n = 3).
4. Conclusion Results obtained within the present study suggest that preactivated HA-CYS–MNA can influence bioadhesion of vaginal drug delivery positively, mostly due to the improved mucoadhesive properties of this second class of thiomers. Covalent attachments could be achieved via disulfide bond formation, resulting in an increased retention time of vaginally administered formulations. Consequently, HA-CYS–MNA seems to be a promising tool for
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Personalised medicine
Preactivated hyaluronic acid: A potential mucoadhesive polymer for vaginal delivery Jessika Nowak, Flavia Laffleur, Andreas Bernkop-Schnürch * Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Austria
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 July 2014 Received in revised form 20 November 2014 Accepted 21 November 2014 Available online 24 November 2014
The objective of this study was to develop mucoadhesive polymeric excipients for vaginal drug delivery systems. Hyaluronic acid was thiolated and subsequently preactivated with 6-mercaptonicotinamide (HA-CYS–MNA) to enhance stability and mucoadhesive properties on vaginal mucosa. After determination of the thiol group content, disintegration studies and in vitro mucoadhesion studies (rotating cylinder and tensile) were performed. Furthermore, swelling behavior and cytotoxicity studies were performed in comparison with corresponding polymers. Both, disintegration and in vitro mucoadhesive studies revealed that modifying HA-CYS with MNA resulted in higher stability (3.6-fold prolonged disintegration time compared to unmodified hyaluronic acid) and prolonged mucoadhesion time. MTT assay and LDH revealed no toxicity for the polymeric excipients and safe for their use. Disintegration and swelling results conducted more pronounced stability of the preactivated thiomers compared to corresponding unmodified ones. According to these results preactivated hyaluronic acid might be a useful tool for vaginal delivery systems. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Mucoadhesion Hyaluronic acid Preactivated thiomers Thiomers Vaginal delivery
1. Introduction Mucoadhesive polymers are of pharmaceutical interest since the introduction of mucoadhesion in the 1980s. Belonging to the group of mucoadhesive polymers, thiomers or so called thiolated polymers swell rapidly in an aqueous environment attached to the mucus through formation of disulfide bonds (Bernkop-Schnurch, 2005; Bernkop-Schnurch et al., 2004). Thus, the residence time is prolonged and the drug uptake is enhanced. Thiolated polymers belong to a unique and new generation of mucoadhesive polymers. They form disulfide bonds with cysteine bearing subdomains of the mucus glycoproteins which are stronger than non-covalent interactions and therefore provide stronger adhesion. Thiomers have high in-situ gelling properties, are mucoadhesive, offer the possibility to apply drugs in a controlled release form (Wang et al., 2012). This could be shown lately for hyaluronic acid (Li et al., 2012). But one disadvantage of thiomers is the poor stability in solutions and the high ability of thiol oxidation at a pH above 5
* Corresponding author at: Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, Innrain 80-82, A-6020 Innsbruck, Austria. Tel.: +43 51250758600; fax: +43 51250758699. E-mail address: [email protected] (A. Bernkop-Schnürch). http://dx.doi.org/10.1016/j.ijpharm.2014.11.048 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
(Bernkop-Schnurch et al., 2003). To overcome this drawback new preactivated thiomers are synthesized to enhance stability, mucoadhesion and cohesive properties of the thiomers. Hyaluronic acid (HA), which is also known as hyaluronan or hyaluronate, is a viscoelastic linear polysaccharide which consists of alternating disaccharide units of D-glucuronic acid and N-acetylD-glucosamine linked by b-1–3 and b-1–4 glycosides bonds (Kafedjiiski et al., 2007). HA was investigated as a possible drug delivery agent using various routes of administration such as ophthalmic, nasal, pulmonary, oral, parenteral and topical (Yadav et al., 2008). High vascularization of the vagina turns this mucosa to a possible and promising administration site for not only locally efficacious drugs but also for systemic drug delivery. By using the vagina as a possible drug delivery route several advantages such as a great permeation area because of the high vascularization (vaginal cavity approximately 60 cm2), the avoidance of hepatic first-pass metabolism and therefore less gastrointestinal side effects are offered (Hussain and Ahsan, 2005; Valenta, 2005; Baloglu et al., 2009). In this study HA-CYS was preactivated with 6-mercaptonicotinamide to prevent oxidation and therefore enhance the stability in an aqueous environment and investigated as a possible mucoadhesive polymer (Laffleur and Bernkop-Schnurch, 2012).
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2. Material and methods
with an increasing amount of L-cysteine hydrochloride were used (Rahmat et al., 2012).
2.1. Materials 2.6. Disulfide bond test Hyaluronic acid sodium salt from Streptococcus equi (1.5– 1.8 106 Da), 6-chloronicotinamide, thiourea, hydrogen peroxide, L-cysteine ethyl ester hydrochloride, reduced glutathione, Nhydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma–Aldrich. All other reagents used were of analytical grade.
The disulfide bond test was performed to measure the amount of disulfide bonds as an indicator for successful activation and preactivation, respectively. Thereby NaBH4 was used to reduce the disulfide bonds and after that the thiol group content was measured as mentioned above (Bernkop-Schnurch et al., 1999). 2.7. Quantification of conjugated 6-mercaptonicotinamide
2.2. Synthesis of hyaluronic acid-L-cysteine ethyl ester hydrochloride conjugates Our research group was the first who synthesized hyaluronic acid-L-cysteine ethyl ester (HA-CYS) (Kafedjiiski et al., 2007). In brief, 0.200 g of hyaluronic acid was dissolved in 50 mL demineralized water to gain a 0.4% (w/v) solution. The pH was adjusted to 5.5 with 0.1 M hydrochloric acid. Afterwards 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and Nhydroxysuccinimide (NHS) were added in a molar ratio of 1:1. Again, the pH was adjusted to 5.5. After stirring at room temperature for 15 min 0.250 g of L-cysteine ethyl ester hydrochloride was added resulting in pH 6. The solution was incubated under further stirring for 4 h and then filled into tubings with a molecular weight cut off (MWCO) of 12,000–20,000 Da. The tubings were dialyzed thrice against demineralized water containing 1% of NaCl and once against demineralized water in the dark at 10 C. Finally the obtained solution was frozen; freeze dried and kept in the refrigerator at 4 C until further use.
The amount of MNA was measured with a photometer. 0.5 mg of the HA-CYS–MNA was dissolved in 0.5 M phosphate buffer pH 6.8. After adding a 2% reduced glutathione solution, the samples were incubated for 1 h at 37 C. The absorbance of 100 mL of this solution was measured at 317 nm (MNA peak). MNA was employed to establish the calibration curve. 2.8. FT-IR characterization of hyaluronic acid-L-cysteinemercaptonicotinamide The dimer was characterized by FT-IR analysis. The characterization was conducted by attenuated-total-reflectance Fourier transform infrared spectroscopy (ATR–FTIR). The spectra were obtained by a PerkinElmer Spectrum 100 ATR–FTIR spectrometer (PerkinElmer, Waltham, USA) in combination with a Spectrum software version 6.3.1.0134 (PerkinElmer, Waltham, USA). Samples were measured at 22 C and were divided in six subsamples. 8 scans in a range from 4000 cm1 to 600 cm1 and a resolution of 4 cm1 were taken.
2.3. Synthesis of 6-mercaptonicotinamide 2.9. Cell viability screening-MTT assay 6-Mercaptonicotinamide was synthesized using 6-chloronicotinamide and thiourea. 4.4 g of thiourea and 9.0 g of 6-chloronicotinamide were dissolved in 79.4 mL of ethanol. The solution was stirred for 6 h at 90 C at reflux, and overnight without any heating. Afterwards the solvent was removed under vacuum. The product was dissolved in demineralized water and 5 M sodium hydroxide solution and stirred for another 45 min. Afterwards the pH was adjusted to 4.2 with glacial acetic acid and again stirred for 15 min. The solvent was removed with a suction filter and dried at 40 C. In the second step the product was dimerised. The MNA monomer was suspended in water and the pH was adjusted to 7–8 with sodium hydroxide solution. After adding hydrogen peroxide (30% w/v) drop wisely the product was filtered, washed with demineralized water and dried at 40 C. It was kept at room temperature for further use. 2.4. Synthesis of hyaluronic acid-L-cysteine-mercaptonicotinamide For preactivation HA-CYS solution of 0.1% (w/v) was prepared in a DMSO/water mixture with a ratio of 7:3. The pH was adjusted to 6.0. The MNA dimer was re-suspended in DMSO. For the preactivation HA-CYS and MNA were used in a molecular ratio of 1:1. After addition of MNA aliquots to the thiomer mixture, a 24 h stirring process followed. As following step, the dialysation took place in tubings (MWCO of 12,000–20,000 Da) at 10 C under light protection. After lyophilization, the obtained hyaluronic acid-Lcysteine-mercaptonicotinamide was kept at 4 C until further use. 2.5. Determination of the thiol group content The amount of thiol groups was quantified spectrophometrically using ellman’s reagent. For the calibration curve solutions
To determine cell viability the MTT assay was performed on Caco-2 cells (Mosmann, 1983). Therefore 10 104 cells (Caco2) per mL were supplemented with 5% FBS (fetal bovine serum), antibiotics, non essential amino acid (NEAA) and sodium pyruvate. 100 mL of the cells were cultured in each well from the 96 well plates. The cells were cultivated for 24 h at 37 C in a humidified atmosphere with 5% CO2. The next day the polymers were dissolved in MEM containing 5% FBS, antibiotics, non essential amino acid (NEAA) and sodium pyruvate in a concentration of 0.5% m/v. In the following the test solutions, the negative control (MEM containing 5% FBS, antibiotics, NEAA and sodium pyruvate) and the positive control (Triton-X100, 1 mM) were added in triplicate to the cells in 100 mL quantity after being washed twice with PBS. The plates were cultivated for 3 and 24 h at 37 C in a humidified atmosphere with 5% CO2, respectively. The test solutions were then removed and fresh medium containing 10% MTT was added to each plate in a quantity of 100 mL. After incubating for 3 h at 37 C in a humidified atmosphere with 5% CO2 the MTT solution was removed and 100 mL of DMSO was added into each plate to dissolve the obtained formazan crystals. The plate was put on an orbital shaker and mixed for 2 min before measuring the absorbance at 570 nm. 2.10. Cytotoxicity LDH (lactate dehydrogenase) Lactate dehydrogenase (LDH) is a cytosolic enzyme available in cells. By damaging the cell membrane LDH is released in the culture medium. By working on LDH assay, the cytotoxicity effect of testing substrates can be detected. For this purpose, cells were seeded in 24 well plates in a density of 100,000 cells/well. Cells were treated for 3 h and 24 h with HA 0.5% (w/v), HA-CYS
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0.5% (w/v), minimum essential medium as negative control and Triton X-100 2% (v/v) as positive control, respectively. The released amount of LDH was detected via a commercial test kit (Roche Diagnostics, Meylan, France) by measuring at 492 nm with Tecan infinite M200 spectrophotometer (Tecan, Grödig, Austria). 2.11. Preparation of tablets Lyophilized HA, HA-CYS and HA-CYS–MNA were compressed to 30 mg (5.0 mm diameter, 1.2 mm thickness) flat-faced tablets with a tablet press (Paul Weber, Maschinen- und Apparatebau, Remshalden, Germany). During the compaction the force was kept at 10 kN for 20 s.
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mobile platform until the tablet attached to the mucosa. After 20 min of incubation at room temperature the balance was pulled down manually with a rate of 0.1 mm/s using a knob, which was provided on the platform. To ensure the precise displacement of 0.1 mm/s the regulating knob was marked and validated via length measurements. The data was collected every second with appropriate software (SartoCollect V 1.0; Satorius AG, Germany) and transferred to Excel 2010. Force versus displacement curves was analyzed to calculate the total work of adhesion as the area under the curve in accordance with the trapezoidal rule. 3. Results and discussion 3.1. Synthesis and characterization of preactivated thiolated polymers
2.12. Disintegration studies 2.12.1. Evaluation of the swelling behavior The water absorbing capacity of HA, HA-CYS and HA-CYS–MNA was measured in simulated vaginal fluid [NaCl 3.51 g, KOH 1.40 g, Ca(OH)2 0.222 g, albumin 0.018 g, acetic acid 1.00 g, lactic acid 2.0 g, glycerol 0.16 g, urea 0.4 g, glucose 5.0 g, aqua dest. ad 1000 mL (pH was adjusted to 4.2 with HCl)] (Baloglu et al., 2011) with a gravimetric method (Laffleur et al., 2014). The 30 mg tablets were weighed and fixed on the tip of a needle. The tablets were put in 15 mL falcon tubes filled with the simulated vaginal fluid which was preheated at 37 C. During the whole experiment the tubes were kept in a water bath at 37 C. After certain time points the tablets were carefully taken out of the medium, excess water was removed with a tissue and then the tablets were weighed again and the water uptake (%) was determined using the following equation: water uptakeð%Þ ¼
Wt W0 W0 100
Wt is the weight of the polymer at a given time and W0 is the polymer in the dry form. 2.12.2. Disintegration test Disintegration behavior of the tablets comprising hyaluronic acid, HA-CYS and HA-CYS–MNA were evaluated in simulated vaginal fluid pH 4.2 at 37 0.5 C with a disintegration test apparatus according to the European Pharmacopoeia. The oscillating frequency was adjusted to 0.5 s1 (Kafedjiiski et al., 2005).
Hyaluronic acid served as polymeric backbone for the immobilization of thiol moieties of the thiol bearing compound L-cysteine ethyl ester where the covalent attachment occurs due to an amide bond formation (Bernkop-Schnurch et al., 1999). Via ellman’s reagent the amount of thiol groups were determined exhibiting 218.87 9.19 mmol per gram of hyaluronic acid. The amount of disulfide bonds were 173.80 63.5 mmol per gram of hyaluronic acid as presented in Table 1. HA-CYS showed to be a white fibrous powder. The preactivation of HA-CYS was performed in a solution of DMSO and demineralized water. The synthesis of the 6-MNA dimer is shown in Fig. 1. A disulfide bond was formed between the thiol group of HA-CYS and the thiol groups of the 6-MNA dimer. The structure of the obtained polymer is shown in Fig. 2. The lyophilized HA-CYS–MNA is an odorless powder of fibrous structure and of yellow color. The powder is poorly soluble in water. Ellman’s test and disulfide bond test were performed to show the grade of preactivation. The thiol group content was 20.46 2.05 mmol per gram of polymer and the disulfide bonds were 163.97 59.83 mmol per gram of polymer. These results show that a successful coupling of MNA to the thiol group of hyaluronic acid took place. The higher the amount of the disulfide bonds the higher is the amount of immobilized MNA. In addition the amount of MNA was determined in the preactivated polymer, Table 1 The amount of thiol/disulfide groups and MNA for HA-CYS and HA-CYS–MNA. Conjugates
Thiol groups (mmol/g polymer SD; n = 3)
Disulfide groups (mmol/g polymer SD; n = 3)
HA-CYS HA-CYS– MNA
218.87 9.19 20.46 2.05
173.80 63.5 163.97 59.83
2.13. In vitro mucoadhesion studies 2.13.1. Rotating cylinder method Mucoadhesion was measured with a disintegration tester (Erweka DT 700, Germany). Freshly excised bovine vaginal mucosa was used, which was obtained from the local slaughterhouse. The vaginal mucosa was fixed with glue on the cylinders and the tablets of HA, HA-CYS and HA-CYS–MNA were attached to the mucosa with soft pressure. The baskets were filled with 900 mL simulated vaginal fluid pH 4.2 and preheated to 37 C. This temperature was kept constant and the cylinders rotated with 100 rotations/min during the whole experiment. The time when the tablets detached from the mucosa was measured (Iqbal et al., 2012). 2.13.2. Tensile studies Tensile strength assays were conducted on freshly excised bovine vaginal mucosa according to a method described previously (Bernkop-Schnurch et al., 2001). Briefly, the mucosa was fixed on a glass platform with cyanoacrylate glue. The tablet was glued to a stainless steel flat disc (10 mm in diameter), which was hung from a laboratory with a nylon thread (15 cm). The glass platform with the mucosa was placed on a balance and carefully raised by a
Fig. 1. Synthesis of 6-MNA.
MNA (mmol/g polymer SD; n = 3)
186.60 48.29
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Fig. 2. Preactivation of hyaluronic acid with L-cysteine ethyl ester hydrochloride and coupling with MNA.
showing 85.3% activation with 186.60 48.29 mmol MNA per gram of HA-CYS. The proposed mechanism of the oxidative coupling is the covalent coupling of MNA dimer to the free thiol group of the activated polymer and the removal of free MNA. Furthermore, the IR spectra were in good agreement with the synthesized thiomer. The typical signals such as 3161 cm1 and 3159 cm1 representing the C—H stretch as well as the N—H bend and —C¼C— were shown at 1653–1619 cm1. Moreover, the
proving amide bond formation was found in HA-CYS–MNA with 1735–1700 cm1, where as it could not be detected for unmodified HA as shown in Fig. 3. 3.2. MTT assay Caco-2 cells were incubated with hyaluronic acid, HA-CYS and the HA-CYS–MNA for either 3 or 24 h to determine their effect on
Fig. 3. Comparison of FT-IR spectra of unmodified hyaluronic acid (black line) and preactivated HA-CYS–MNA (red line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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cell viability. The results are shown in Fig. 4. After 3 h incubation HA, HA-CYS and HA-CYS–MNA showed cell viability of 95.52 7.04%, 91.20 18.49% and 99.90 17.6%. The results for HA, HA-CYS and HA-CYS–MNA after the 24 h incubation exhibited were 70.27 10.99%, 71.38 4.13% and 66.94 8.03% cell viability, respectively. This showed that these polymeric excipients are not cytotoxic and not harmful to the cells. The polymers were all tested in a concentration of 0.5% (w/v) because a higher concentration would have led to a higher viscosity and therefore the measurement would have been impossible. 3.3. LDH cell toxicity test In vitro cytotoxicity of the polymer was determined by measuring the release of the enzyme lactate dehydrogenase (LDH) from damaged Caco-2 cells. In vitro cytotoxicity results are presented in Fig. 5. These results showed that negative control (MEM without FCS and phenol red) and positive control (Triton-X) indicate 0.20 0.06% and 100 0.63% cytotoxicity, respectively. The resulting thiolated HA (0.5% w/v) exhibited only 7.50 0.18% and 25.27 0.20% of toxicity after 3 and 24 h, respectively. Unmodified HA was found 5.43 0.06% and 17.14 0.24% toxic after 3 and 24 h, respectively. Preactivated HA showed 8.55 0.05% and 27.28 0.84% cytotoxicity which might be due to the presence of mercaptonicotinic groups present on the activated adduct. 3.4. Swelling behavior The swelling behavior of mucoadhesive polymers is an important parameter as it correlates with the stability, adhesive properties and cohesiveness. After attaching to the mucus the tablet swelled up by initiating water absorption from the underlying mucosal surface via capillary action and diffusion process leading to adhesion. In contrary, overhydration results in less cohesiveness and less mucoadhesion. Therefore moderate swelling is necessary to minimize the risk of detachment of the polymer from the mucus and to maximize the time of adhesion. The water uptake studies were performed in simulated vaginal fluid with a pH of 4.2 at 37 C with 30 mg flat faced tablets based on the unmodified polymer (HA), HA-CYS and HA-CYS–MNA. The results are shown in Fig. 6. The unmodified polymer showed a strong and linear water uptake. At 120 min hyaluronic acid disintegrated and no further water uptake could be measured. It was observed that the covalent attachment of L-cysteine ethyl ester to hyaluronic acid significantly
Fig. 5. Cytotoxicity assay working on LDH. Caco-2 cells were treated with HA 0.5%, HA-CYS 0.5% and HA-CYS–MNA 0.5% for 3 h (black bars) and 24 h (white bars), respectively. Results are expressed as means SD (n = 3).
enhanced the swelling capacity in the first 30 min. After this time period no further significant water uptake could be demonstrated. At 150 min the thiomer disintegrated. In comparison to the above mentioned polymers HA-CYS–MNA showed the slowest increase in water uptake, therefore a constant linear rise. At 150 min the polymer still swelled up, but after 180 min it disintegrated. This might be explained by the lipophilic nature of MNA together with the higher amount of inter and intramolecular disulfide bonds within the polymer resulting in an increased stability and higher swelling capacity than HA-CYS. 3.5. Disintegration Disintegration studies revealed that disintegration time increased with degree of chemical modification as the difference in
Fig. 4. MTT cell viability of HA, HA-CYS, HA-CYS–MNA obtained by 3 h (grey bars) and 24 h (black bars), respectively. Data are expressed as means SD (n = 3).
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Fig. 6. Swelling behavior of HA, HA-CYS and HA-CYS–MNA; data are expressed as means SD (n = 3).
disintegration time between unmodified, thiolated and pre-activated polymers become more pronounced regarding the thiol group amounts. Results are shown in Fig. 7. As expected, tablets comprising unmodified hyaluronic acid completely disintegrated within 45 min. It is clear that thiolation process greatly improves the crosslinking properties of polymers. Tablets comprising polymers with L-cysteine ethyl ester in comparison to the tablets comprising corresponding unmodified polymers displayed 3.1-fold prolonged disintegration time. Moreover, the covalent attachment of 6-mercaptonicotinamide (MNA) to HA-CYS significantly enhanced the stability of these thiomers and 3.6-fold prolonged disintegration times were achieved in the presence of HA-CYS–MNA conjugates compared to unmodified hyaluronic acid. These observations might be explained by the particulate nature of MNA together with the higher amount of interand/or intramolecular disulfide bonds within the conjugate being responsible for increased stability and hardness of HA-CYS–MNA. The results obtained from disintegration studies are in accordance with the theory that formation of covalent bonds (disulfide bonds)
Fig. 7. Disintegration time for HA, HA-CYS and HA-CYS–MNA, data are expressed as means SD (n = 3).
within polymer structure resulted in enhanced stability and cohesion. The formation of covalent bonds (disulfide bonds) within the polymer leads to an enhanced stability and cohesion that is responsible for the disintegration time. In accordance, the introduction of MNA as S-protective leaving group further prolonged the disintegration of disks in both media. This might be caused by the lipophilic nature of MNA that increases hydrophobicity of the conjugate, in combination with an increased formation of inter- and/ or intramolecular disulfide bonds since crosslinking influences chain mobility and resistance to dissolution. 3.6. In vitro mucoadhesion studies In vitro mucoadhesion studies with the rotating cylinder showed the time when the unmodified polymer, HA-CYS and HA-CYS–MNA detached from the mucosa in a time period of 48 h. As expected, HA firstly detached after a time period of 2.8 h as shown in Fig. 8. This can be explained by the strong hydration and by the lack of disulfide bond formation – only non-covalent bonds are formed to stay on the mucus. HA-CYS detached after 11.5 h. This correlates with the theory that disulfide bonds between thiolated polymers and glycoproteins from the mucus are formed and therefore mucoadhesion is enhanced. In contrast to HA and HACYS, HA-CYS–MNA did not detach from the mucosa during the whole experiment. This can be explained by the moderate swelling behavior and by preactivation with MNA which led to higher stability because of less oxidation of thiol groups and higher attendance to form disulfide bonds with the mucus. Tensile strength studies which were performed with freshly excised bovine vaginal mucosa confirm the results obtained in the rotating cylinder study. The results are shown in Fig. 9. HA-CYS– MNA showed the highest adhesive force. This can be explained by the presence of disulfide bond which prevent early oxidation of thiol groups before attaching to the mucus layer. MNA as a hydrophobic agent decreases the dispersive properties of HA-CYS resulting in higher stability and mucoadhesion. The in vitro studies are in accordance with the theory that a thiolation leads to an enhancement of mucoadhesion by forming disulfide bonds with the mucus layer and that preactivation minimizes the risk of oxidation.
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mucoadhesive drug delivery systems and leads to additional possibilities in the field of vaginal formulation development. Acknowledgement The authors wish to thank the local slaughterhouse Piegger in Sistrans for the vaginal mucosa and the FWF (Fonds zur Förderung der wissenschaftlichen Forschung) project no P23515 – B11. References
Fig. 8. Rotating cylinder study with HA, HA-CYS, HA-CYS–MNA; data are expressed as means SD (n = 3).
Fig. 9. Total work of adhesion (TWA) in mJ of HA, HA-CYS, HA-CYS–MNA; data are expressed as means SD (n = 3).
4. Conclusion Results obtained within the present study suggest that preactivated HA-CYS–MNA can influence bioadhesion of vaginal drug delivery positively, mostly due to the improved mucoadhesive properties of this second class of thiomers. Covalent attachments could be achieved via disulfide bond formation, resulting in an increased retention time of vaginally administered formulations. Consequently, HA-CYS–MNA seems to be a promising tool for
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