Comparison of mucoadhesive and cohesive features of poly(acrylic acid)-conjugates respective their molecular mass

European Journal of Pharmaceutics and Biopharmaceutics 113 (2017) 149–156

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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

Research paper

Comparison of mucoadhesive and cohesive features of poly(acrylic acid)-conjugates respective their molecular mass Flavia Laffleur a,⇑, Kathrin Knapp a, Wongsakorn Suchaoin a, Alexandra Partenhauser a, Kesinee Netsomboon a,b, Andreas Bernkop-Schnürch a a b

Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Austria Faculty of Pharmacy, Thammasat University, Rungsit Campus, Phahonyothin Road, Khlong Luang, Pathumthani 12120, Thailand

a r t i c l e

i n f o

Article history: Received 5 October 2016 Revised 29 November 2016 Accepted in revised form 6 December 2016 Available online 12 January 2017 Keywords: Buccal delivery Mucoadhesive Poly(acrylic acid) Resazurin assay Rheology

a b s t r a c t Objectives: This study aimed to assess the impact of molecular mass as well as the differences between poly(acrylic acid)-thiol-conjugates (PAA100,250,450 KDa) on their mucoadhesive and cohesive qualities. Methods: Covalent attachment of cysteine (CYS), cysteamine (CYSM) and L-gluthathione (GSH) to poly (acrylic acid) was achieved by formation of amide bonds between primary amino group of the amino acid (in the case of cysteine and glutathione), respectively the amino group of the aminothiol cysteamine and carboxylic acid group of the polymer. Obtained polymer conjugates were evaluated in regard to their safety profile, mucoadhesive properties on the buccal mucosa by rotating cylinder, tensile strength and rheological investigations, respectively. Furthermore, stability, cohesive and water uptake studies were performed. Key findings: Mucoadhesive studies revealed that maximum detachment force of PAACYS450 was 24.3fold higher in comparison to the respective controls. Stability studies revealed for PAACYS450 a 50.2-fold higher stability compared to controls. Conclusion: Taken together, among all polymers tested, PAACYS450 evinced the most favorable qualities regarding mucoadhesion and cohesion, followed by PAACYSM450 and PAACYS250. Ó 2017 Elsevier B.V. All rights reserved.

1. Introduction A current key ambition in pharmaceutical technology is finding alternative routes to the parental one. By this approach, it is aimed to make drugs systemically available, without pain, fear and risks. Amidst the divergent routes of drug delivery, oral pathway is one of the most pleasant one for patients [1]. Nevertheless, being orally administrated several detriments such as first pass metabolism and enzymatic degradation inside the gastrointestinal tract, on the basis of this disadvantages oral administration of several classes of drugs are impeded. For that reason, buccal mucosa is on a variety of grounds a very auspicious and promising target area [2]. Comprising rich blood supply, the fact of being relatively permeable and robust, as well as a short recovery time after stress or damage renders the oral mucosa distinguished for the systemic as well as the local application of drugs [3]. A crucial pro of buccal drug delivery systems is an emended patients’ compliance favoring ⇑ Corresponding author at: Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria. E-mail address: [email protected] (F. Laffleur). http://dx.doi.org/10.1016/j.ejpb.2016.12.022 0939-6411/Ó 2017 Elsevier B.V. All rights reserved.

the ease of administration, but also circumventing first-pass effect and avoiding acid hydrolysis in the gastrointestinal tract [4]. However, buccal delivery systems have to boast specific traits to overcome diffusion barrier, enzymatic barrier and absorption barrier [5,6]. Apart from this, the barrier represented by efflux pumps limiting the bioavailability of different drugs needs to be surmounted. One of the most encouraging attempts of drug carrier-systems for multifunctional buccal delivery are application systems comprising thiolated polymers or so called thiomers [7]. By further functionalization of well-established polymers, such as poly(acrylic acid) via implementation of thiol-bearing ligands into polymeric backbone, mucoadhesive qualities are substantially amended guaranteeing a sustained and also intimate contact among polymeric delivery system and mucosal membrane [8]. Thiomers offer their mucoadhesive potential by building covalent bonds by formation of disulfide bridges between their sulfhydryl residues and cysteine-rich subdomains of the mucus, due to thiol/disulfide exchange reactions or plain oxidation processes. In this manner, mucoadhesion is immensely fostered compared with carriers absent from thiol bearing residues possessing solely noncovalent bonds [9].

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Therefore, this study aimed to appraise the mucoadhesive and cohesive features of poly(acrylic acid)-conjugates respective unmodified poly(acrylic acid) with regard to their molecular mass. 2. Materials and methods 2.1. Materials L-Cysteine, cysteamine, 5,50 -Dithiobis-(2-nitrobenzoic acid) DTNB, 1-Ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDAC), l-glutathione, hydrochloric acid, potassium phosphate, sodium borohydride, di-sodium hydrogen phosphate, poly(acrylic acid) 100 kDa, poly(acrylic acid) 250 kDa, poly(acrylic acid) 450 kDa, tris (hydroxymethyl) aminomethane hydrochloride, Resazurin salt, Minimum Essential Medium (MEM) and TritonÒX-100 were obtained from Sigma-Aldrich (Vienna, Austria). All other reagents used were of analytical grade. Cell culture supplements were purchased from Biochrom AG, Berlin, Germany. Multiwell plates and tissue culture flasks were received from Greiner bio-one, Kremsmünster, Austria. Caco-2 cells were purchased from the European Collection of Cell Culture (ECACC), Salisbury, England. 2.2. Methods 2.2.1. Synthesis of modified Poly(acrylic acid) conjugates The covalent attachment of cysteine, cysteamine and lglutathione to poly(acrylic acid) was achieved by the formation of amide bonds between the primary amino group of the amino acid (in the case of cysteine and glutathione), respectively the amino group of the aminothiol cysteamine and the carboxylic acid group of the polymer [10–12]. Briefly, polymer was hydrated in demineralized water and the pH was adjusted to pH 6 by addition of 5 M NaOH. EDAC was added for the purpose of activate the carboxylic acid moieties of the hydrated poly(acrylic acid). After 20 min incubation at room temperature, thiol-constituents cysteine, cysteamine and l-glutathione were added as shown in Table 1 and pH was readjusted to pH 6. Afterwards, resulting conjugates were isolated by dialysing at 10 °C in the dark against 1 mM HCl and 1 mM HCl containing 1% NaCl. Control polymers were prepared in the same way but omitting carbodiimide during the coupling reaction. After dialysis, pH of the solutions were readjusted and mixtures were lyophilized at 30 °C and 0.01 mbar (Christ Gamma 1-16 LSC Freeze dryer) [13]. Afterwards, 30 mg of the lyophilized polymer conjugates were compressed into flat-faced discs. To obtain discs with a diameter of 5.0 mm a single-punch eccentric press (Paul Weber, Germany) was used keeping constant compaction pressure of 11 kN during. The lyophilized polymers were stored at 8 °C until further use [9]. 2.2.2. Characterization of obtained conjugates The amount of thiol groups immobilized on the poly(acrylic acid) backbone were determined photometrically applying Ell-

man’s reagent as described previously by the research group of Bernkop-Schnürch et al. [14] First of all 0.5–1 mg of samples and controls was dissolved in 500 lL Ellman’s buffer consisting of 0.5 M phosphate buffer pH 8 and subsequently 500 lL fresh prepared Ellman’s reagent (consisting of 3 mg DTNB dissolved in 1 mL Ellman’s buffer) was added to the mixtures incubating at room temperature for 90 min in the dark [15]. A blank-test and a serial dilution were prepared equal to the samples and controls. Upon completion of incubation aliquots of 100 lL of each solutions were transferred 96 microtiter wellplate and measured at 450 nm using Tecan infinite M200 (Grödig, Austria). Furthermore, disulfide bond test was performed to quantify disulfide bonds formed due to oxidation during the reaction. Determination of disulfide content occurred in a resembling way to thiol group content determination. Initially, samples were dissolved in Tris-buffer (0788 g Tris-HCl in 100 mL demineralized water, adjusted to pH 7.6) and reduced with NaBH4-solution (400 mg NaBH4 solved in 10 mL H2O). After 2 h of incubation at 37 °C in a shaking bath the reaction was stopped with 250 lL 5 N HCl. Afterwards 1 mL phosphate buffer pH 8 and 100 lL freshly prepared Elman’s reagent was added and after 90 min incubating at room temperature in the dark the measurement was accomplished at 450 nm using Tecan infinite M200 (Grödig, Austria). 2.2.3. Cell viability study according to Resazurin assay Cell viability studies were carried by Resazurin assay. To assess the toxicity of both thiolated and non- thiolated polymers on Caco2 cells, 1x 105 Caco-2 cells were seeded per well in 24-well plates and consecutively incubated in a moisturized chamber at a retained temperature of 37 °C, and 5% CO2. At a concentration of 0.5% (w/V) the cytotoxicity of tested thiomers as well as controls was examined. As a control conduced minimum essential medium (MEM, pH 7.1–7.4) without phenol red. After an incubation time of 24 h, testing samples were removed and 0.5 mL of Resazurin solution was added. Cells were incubated for further 3 h and subsequently fluorescence of the supernatants was measured at 540 nm excitation and 590 nm emission using Tecan infinite M200 (Grödig, Austria) and cell viability was calculated according following equation [16].

Cell viability ð%Þ ¼ ðAverage fluorescence value of each sample= Average fluorescence of low controlÞ  100 2.2.4. Evaluation of the swelling behavior The water-absorbing capacity was determined by a gravimetric method. Test discs of all examined polymers, thiolated as well as non-thiolated were fixed on a needle and submersed in a test tube comprising simulated saliva fluid (2.38 g Na2HPO4, 0.19 g KH2PO4 and 8.0 g NaCl dissolved in 1 L demineralized water adjusted to pH 6.75 [13]) at 37 °C.

Table 1 Composition of used reagents for reaction mixtures. Conjugate

Polymer (g/80 mL)

EDAC (mM)

Cysteine (g)

PAA100-Cysteine PAACYS100 PAA250-Cysteine PAACYS250 PAA450-Cysteine PAACYS450 PAA100-Cysteamine PAACYSM100 PAA250-Cysteamine PAACYSM250 PAA450-Cysteamine PAACYSM450 PAA100-Glutathione PAAGSH100 PAA250-Glutathione PAAGSH250 PAA450-Glutathione PAAGSH450

1.0 1.0 1.0 0.8 0.8 0.8 1.0 1.0 1.0

200 200 200 100 100 100 50 50 50

1.0 1.0 1.0

Cysteamine-HCl (g)

Glutathione (g)

0.75 0.75 0.75 2.0 2.0 2.0

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Increase in weight of each particular disc was determined at prescheduled time intervals. For this reason, hydrated test discs were taken out of incubation medium, excess water was removed and discs were reweighed. This measurements were repeated until a constant or declining weight was observed. The amount of absorbed water was determined by subtracting the initial weight (W0) of each disc and the weight of each disc (Wt) taken at defined time points [17].

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combination (Haake Mars Rheometer, 379–0200, Thermo Electron Gmbh, Karlsruhe, Germany; Rotor: C35/1°; D = 35 mm). The apparent viscosity (g) was determined at a shear rate of 50 s1 and the temperature fixed at 37 °C [23,24]. 1% (w/w) gels of thiolated polymers and controls, respectively, were prepared in a ratio 1:1 (polymer:mucus). The test samples were measured at two predetermined time points, immediately after mixing and after 4 h of incubation.

Swelling ð%Þ ¼ W0  Wt=  100 3. Results 2.2.5. Erosion study For this study, test discs were fixed on needles and submerged in test tubes containing simulated saliva fluid at 37 °C ± 0.5 °C. Ensuing from the evaluated maximum time point of wateruptake test discs were taken out of the medium, excess water was removed and transferred in a compartment drier and desiccated at 30 °C for 24 h [18]. Afterwards, discs were kept in an desiccator until an equilibrium weight was attained. The percentage of erosion was computed in accordance with the pursuant equation:

Erosion ð%Þ ¼ ðW0  WtÞ=Wt  100 2.2.6. Disintegration study The disintegration behavior of discs in stimulated saliva fluid pH 6.75 was analyzed with a disintegration test apparatus according to the European Pharmacopeia. The oscillating frequency was adjusted to 0.5 s1 and the temperature was kept at 37 °C. The time of degradation was assessed visually [19]. 2.2.7. Mucoadhesive investigations by rotating cylinder assay The rotating cylinder assay is an appropriate visual test method to evaluate the capability of mucoadhesive formulations to retain contact with a mucosal surface area in a wet environment while being subjected to shear forces [20]. For the implementation of this method, test discs were attached to freshly excised buccal porcine mucosa being fixed on a stainless steel cylinder (diameter 4.4 cm; height: 5.1 cm; apparatus four-cylinder, USP XXIII). Thereupon cylinders of dissolution test apparatus (Erweka DT 700, Heusenstamm, Germany) were submerged in saliva fluid and 100 rev/ min speed of rotation and a temperature of 37 °C were kept. The time needed for detachment or disintegration has been ascertained visually. 2.2.8. Mucoadhesive assessment according to tensile study Tensile studies were carried out on freshly excised native porcine buccal mucosa. The polymer discs were glued to a stainless steel flat disc (5 mm in diameter) which was hung from a laboratory stand with a nylon thread. The porcine mucosa was fixed and placed on a balance on a mobile platform and subsequently hydrated with simulated saliva fluid to mimic physiological conditions. Thereafter test discs (attached at stainless steel flat discs) were brought into contact with mucosa for 20 min by raising carefully the mobile platform. After 20 min of incubation mucosa was pulled down from discs at a rate of 0.1 mm/s by regulation knob provided by the mobile platform [21]. Data points were collected every second by computer software (SartoCollect V 1.0; Satorius AG, Germany) linked to a balance with integrated interface. To calculate maximum force of detachment (MDF) and total work of adhesion data was transferred to EXCEL and the force versus displacement curves were analyzed [22]. 2.2.9. Rheological investigations evaluating polymer/mucus interactions The viscoelastic properties of thiolated as well as non-thiolated manufactured PAA-conjugates were determined with a cone-plate

3.1. Synthesis and characterization of obtained polymer-conjugates Syntheses of poly(acrylic acid)-conjugates was achieved by the formation of amide bonds between carboxylic acid groups of respective PAAs (100-, 250-, and 450 kDa) and primary amino groups of l-cysteine, cysteamine and glutathione as displayed in Fig. 1. For this reaction, the carboxylic acid moieties of PAA were activated by EDAC. The obtained polymers appeared as a fibrous structured, nearly white powder without any scent, being easily soluble at physiological conditions in aqueous solution and no further comminution or pulverization was carried out for the experiments. Characterization and determination of the thiol groups by Elman’s test is shown in Table 2. 3.2. Safety screening – Resazurin assay Resazurin assay was performed on Caco-2 cell line to determine the impact of polymer conjugates on the cell viability. Cells were incubated with both thiolated and non-thiolated polymers. Cell viability of more than 84%(PAACYS100), 94% (PAAGSH100), 87% (PAACYSM100), 91% (PAACYS250), 87% (PAAGSH250), 91% (PAACYSM250), 90% (PAACYS450), 90% (PAAGSH450) and 90% (PAACYSM450) indicated these polymers being not at all harmful for cells as shown in Fig. 2. All polymers were tested in a final concentration of 0.5% (w/V). All findings were in good correlation with previous published results from MTT and LDH assays [25]. 3.3. Swelling evaluation Swelling behavior of mucoadhesive polymers has a great impact on adhesiveness, cohesiveness and stability. Mucoadesive polymers are able to absorb water from the underlying mucosal tissue via capillary action and diffusion [26]. In order to assess the correlation between the swelling behavior and mucoadhesion, water uptake studies were implemented using simulated salvia fluid as the incubation medium to simulate physiological conditions. Results of this study are shown in Fig. 3. Non-thiolated polymers (PAA100, PAA250, PAA450) dissolved in the salvia fluid within 30 min whereas modified test discs showed higher water uptake levels over 200 min: period of water uptake was in the case of PAACYS100 3.0-fold, PAACYS250 2.0-fold, PAACYS450 7.0-fold and in case of PAACYSM450 6.0-fold, respectively, prolonged. PAACYS450 and PAACYSM450 exhibited an almost constant water uptake up to a period of 180–210 min. By means of the swelling properties, it could be shown that in virtue of the formation of disulfide bonds within thiolated conjugates, stability of the polymeric matrix was substantially enhanced. 3.4. Erosion study evaluating the stability The execution of erosion studies is an important tool for measuring weight loss from matrix discs immersed in simulated salvia fluid as a function of time. In hydrophilic polymeric matrix systems, carrier on the interface of the matrix primarily hydrates dur-

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Fig. 1. Synopsis of syntheses.

Table 2 Characterization of reaction mixtures by Elman’s assay. Evaluated polymers

Thiol group content

PAA100 PAA250 PAA450 PAA100CYS PAA250CYS PAA450CYS PAA100CYSM PAA250CYSM PAA450CYSM PAA100 GSH PAA250 GSH PAA450 GSH

10.27 ± 2.21 lmol/g 10.03 ± 1.90 lmol/g 13.33 ± 0.24 lmol/g 1082.07 ± 24.06 lmol/g 1144.99 ± 214.27 lmol/g 956.22 ± 102.23 lmol/g 462.73 ± 67.21 lmol/g 397.38 ± 39.09 lmol/g 332.79 ± 76.13 lmol/g 69.20 ± 4.32 lmol/g 75.43 ± 16.10 lmol/g 212.47 ± 60.20 lmol/g

ing dissolution to constitute an exterior viscous gel layer, followed by ensuing matrix bulk hydration, swelling and erosion. Drug availability and the entire dissolution rate is eventually defined by the rate of swelling, matrix erosion and drug diffusion across the gel layer. The percentage remaining of the test discs reflects the amount of dissolved polymer and the erosion of discs during the maximum time of swelling process. The results of erosion studies as shown in Fig. 4 reveal the extent of polymer dissolved and the erosion at the time of maximal swelling process. The percent weight loss of the various thiolated and unmodified polymeric matrix was diverging, but there is a distinct tendency in every weight category (PAA100, PAA250, PAA450) of less erosion of thiolated polymers in relation to unmodified polyacrylic acid. In comparison to unthiolated polyacrylic acid discs with a molecular weight of 100, the percent weight loss of the cysteine-conjugate was 1.4-fold, of the glutathione-conjugate 1.1-fold and of the

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Fig. 2. Cell viability after 24 h was tested on Caco-2 cells by Resazurin assay. Indicated values are the means of at least three experiments (±SD).

Fig. 3. Swelling behavior of PAA-conjugates in artificial saliva pH 6.75 at 37 °C. Indicated values are means (±SD) of at least three experiments.

Fig. 4. Erosion study of unmodified PAA and modified PAA-conjugates. Indicated values are means (±SD) of at least three experiments.

Fig. 5. Overview of the disintegration profile. Disintegration of PAA-conjugates was evaluated according to the USP. All indicated values represent an average of at least three experiments (±S.D.).Arrows in the graphs indicate the long lasting disintegration.

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early. Consequently, disintegration studies comprising thiolated and unmodified PAA-polymers with respectively various molecular masses were executed and findings were shown in Fig. 5. Results demonstrated a significantly higher stability of the thiolated test discs, moreover the disintegration time overall increased with increasing molecular mass of the samples with the solitary exception of the higher values for PAACYSM100-conjugates in comparison to PACYSM250-conjugates. Studies revealed for PAACYS100-conjugate a 18.8-fold, PAA100GSH-conjugate a 1.5-fold, PAACYSM100-conjugate a 7.8fold, PAACYS250 -conjugate a 52.4-fold, PAAGSH250-conjugate a 1.6-fold, PAACYSM250-conjugate a 2.9-fold, PAACYS450conjugate a 50.2-fold, PAAGSH450-conjugate a 3.2-fold and PAACYSM450-conjugate a 50.2-fold higher stability, respectively, compared to unmodified ones. Fig. 6. Comparison of the adhesion time of unmodified and thiolated polyacrylic acid polymer discs (30 mg) on freshly excised porcine buccal mucosa in synthetic saliva fluid (pH 6.75) at 37 °C. The denoted values represent an average of at least three experiments (±SD). Arrows in the graphs indicate higher mucoadhesion than 720 min.

cysteamine-conjugate even 2.0-fold lower. Referring to the thiolated PAA250 conjugates, the results show a 1.5-fold for glutathione-, a 1.0-fold for cysteamine-conjugate and a 75-fold lower weight loss. Almost equal convincing results revealed conjugates of PAA450 with an 8.1-fold (PAA450CYS), a 1.4-fold (PAAGSH450) and a 2.4-fold (PACYSM450) lower percent weight loss.

3.5. Disintegration assessment The disintegration behavior has a substantive influence on mucoadhesive attributes of polymers. If the adhesive bond within the delivery system itself is insufficient, even a strong adhesion of the delivery system to the mucosa is less effective. Furthermore, it is not possible to ensure controlled drug release, if the disintegration of the polymeric network of mucoadhesive systems occurs too

3.6. Mucoadhesive investigations by rotating cylinder assay Rotating cylinder assay is a suitable method to evaluate mucoadhesiveness of testing polymers being exposed to shear forces. A number of physiological aspects of the oral cavity play a substantive role, including pH, enzyme activity, fluid volume, and the permeability of the oral mucosa. Furthermore structure and turnover of mucosal area are determinants of mucoadhesivness [27]. This approach emulates adhesiveness and cohesiveness of the test discs in the physiological environment and is hence assumed to be similar to in vivo conditions. As depicted in Fig. 6, mucoadhesive properties of thiolated polyacrylic acid polymers overtake the mucoadhesive properties of unmodified polyacrylic acid polymers with 1.2-fold (PAACYS100), 1.5-fold (PAGSH100), 15.9-fold (PAACYSM100), 2.0-fold (PAACYS250), 1.7-fold (PAAGSH250), 2.1-fold (PAACYSM250), 1.2-fold (PAACYS450), 3.2-fold (PAAGSH450) and 7.7-fold (PAACYSM450) improvement, respectively. Whereas the simple poly(acrylic acid) polymer just kept sticking onto the mucosal surface area for almost 22, 31 and 47 min, certain thiomers (PAACYSM100 and PAACYSM450) reached an adhesive maximum of over 360 min (by this point the examination was terminated). On account of this, thiolated

Fig. 7. Comparison of mucoadhesive strength by evaluation of total work of adhesion (TWA) (black bars) and maximum detachment force (MDF) of PAA-conjugates. Indicated values are means (±SD) of at least three experiments.

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polymers are able to imitate naturally-occurring mechanism in the manner of mucus glycoproteins are immobilized in the mucosal layer. 3.7. Tensile strength evaluation To corroborate the results of the rotating cylinder assay, a further mucoadhesion test was carried out. The force required to segregate test discs from mucosal surface area by means of an external force was investigated in order to determine the adhesive tensile strength among buccal mucosa and thiolated as well as respective unmodified polymers. The results as TWA (total work of adhesion) and MDF (maximum detachment force) were displayed in Fig. 7. Thiolated polymers bear active thiol groups being able to interact with mucus. Evident, with exception of the MDF resulting from PAA100-glutathione-conjugate, TWA (in descending order) of the discs of PAACYS450 was 26.5-fold, PAACYSM450 20.1-fold, PAAGSH450 19.2-fold, PAACYS250 7.0-fold, PAACYS100 was 4.6fold, PAAGSH250 4.2-fold, PAACYSM250 4.0-fold, PAACYSM100 3.5-fold, and PAAGSH100 1.5-fold higher in comparison to the respective controls. The MDF (in descending order) of the discs of PAACYS450 was 24.3-fold, PAACYSM450 12.6-fold, PAACYS250

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9.5-fold, PAAGSH450 9.0-fold, PAACYS100 4.2-fold, PAAGSH250 3.9-fold, PAACYSM250 2.0-fold and PAACYSM100 1.7-fold higher in comparison to the respective controls. This findings are in good correlation with previous studies. The molecular mass better the molecular chain length defines the strength of bonds between polymer and mucosal area. This can be explained by the ability of polymer and mucus to form entanglements being affected by molecular mobility and flexibility. It is assumed, that an ideally high molecular mass concurs to maximum adhesion [28]. Among all evaluated polymers, poly(acrylic acid)-cysteine-conjugates indicated the strongest mucoadhesive properties. In regard to the molecular mass, poly(acrylic acid)-conjugates with a mass of 250 kDa as well as conjugates with a molecular mass of 450 kDa reached the most promising results. 3.8. Rheological investigations Rheological measurements exhibit paramount importance as far as mucoadhesive polymers are involved. It is well known, that the inner cohesiveness is even-handedly crucial in common with a high adhesiveness. If the adhesive bond falls through within the polymer itself, a substantial rate will be washed away from muco-

Fig. 8. Comparison of viscosity of thiolated and unmodified polyacrylic acid mixtures with native porcine mucin, on the one hand immediately after preparing (black bars) and on the otherhand after incubating for 4 h (white bars) at room temperature. The denoted values represents an average of at least three experiments (±SD); measurements were worked out at room temperature at 6.283 rad/s (=1 Hz).

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sal surface area already well before an adequate percentage of polymer’s surface area effectively reached contact with the mucosa. An ideal bioadhesive polymer is capable to attach to the mucosal surface area in manner of forming reliable inner cohesion. Ascending viscoelastic properties are alleged to proof inter- and intramolecular disulfide crosslinking [29]. Results illustrated in Fig. 8 showed higher viscosity of the thiolated polymers compared to unmodified polymers. In particular, thiolated samples of poly(acrylic acid) with a molecular mass of 450 kDa as well as samples poly(acrylic acid) with a molecular mass of 250 kDa revealed exceptional high outcomes. Immediately after preparing PAA450-cyteine-conjugate (PAACYS450) reached the highest values with 295.0-fold in comparison to the obtained derivates. After incubating for 4 h the viscosity PAA250glutathione-conjugate (PAAGSH250) exhibited with 319.1-fold the highest viscosity in respect to controls.

sideration, this study provides a comprehensive characterization of a broad range of thiomers simplifying the design and development of novel drug delivery systems appropriating prolonged residence time on mucosal surface area.

4. Discussion

The authors thank the slaughterhouse Josef Mayr in Natters for the porcine mucosa.

According to the theory, that the molecular mass-pertaining to the molecular chain length- defines the strength of mucoadhesive bonds as molecular mobility and flexibility influence the capability of polymer and mucus to form implications, it is believed that an ideally high molecular mass complies with uppermost adhesiveness [28]. An crucial factor for both mucoadhesion and cohesion of thiomers is their aptitude to form covalent bonds between the polymer and the mucus layer as well as within the polymer itself [30]. The smaller the molecules the fewer thiol groups and thus they are not capable to build highly cohesive three-dimensional networks based on the formation of intermolecular disulfide bonds [31]. Nevertheless, the accomplishment to diffuse into the mucosal gel layer is restricted by too long polymer chains [32]. In addition to the mechanism of adhesion, equally the cohesiveness of dosage forms has a substantive influence on mucoadhesion. If binding miscarry within the delivery system itself, the strongest adhesive attributes of a dosage form to the mucus layer is immaterial. Results of investigations regarding disintegration behavior showed conjugates based on unmodified poly(acrylic acid) but also certain modified ones with a low molecular mass not being stable. Discs based on thiolated poly(acrylic acid) especially those coupled with cysteine of molecular mass 250 kDa as well as 450 kDa and poly (acrylic acid) of molecular mass of 450 kDa coupled with cysteamine displayed a remarkable stability in comparison with the unmodified ones. This high stability can be expounded by the formation of both inter- as well as intramolecular disulfide bonds within these polymers. The functional interaction of strong mucoadhesive attributes and high cohesiveness - as shown by thiomers - seems hence to be highly advantageous for adhesive dosage forms. 5. Conclusion Within the present study mucoadhesive and cohesive attributes of nine different poly(acrylic acid)-conjugates with molecular masses in the range of 100 kDa-450 kDa were compared and evaluated on buccal porcine mucosa. Comparatists between thiolated and unmodified polymers were investigated in terms of stability (swelling behavior, erosion studies, disintegration behavior) and mucoadhesion (tensile studies, rotating cylinder assay, rheological studies). Among all polymers tested, poly(acrylic acid)-cysteineconjugate (PAACYS450) evinced the most favorable and auspicious qualities regarding mucoadhesion and cohesion, followed by poly (acrylic acid)-cysteamine-conjugate (PAACYSM450) and poly (acrylic acid)-cysteine (PAACYS250). Taking these findings in con-

Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. Acknowledgement

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