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Preactivated thiomers: permeation enhancing properties.
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MedLine Citation:
PMID:  22960503     Owner:  NLM     Status:  MEDLINE    
The study was aimed to prepare a series of poly(acrylic acid)-cysteine-2-mercaptonicotinic acid conjugates (preactivated thiomers) and to evaluate the influence of molecular mass or degree of preactivation with 2-mercaptonicotinic acid (2MNA) on their permeation enhancing properties. Preactivated thiomers with different molecular mass and different degree of preactivation were synthesized and categorized on the basis of their molecular mass and degree of preactivation as PAA(100)-Cys-2MNA (h), PAA(250)-Cys-2MNA (h), PAA(450)-Cys-2MNA (h), PAA(450)-Cys-2MNA (m) and PAA(450)-Cys-2MNA (l). In vitro permeation studies, the permeation enhancement ability for preactivated thiomers was ranked as PAA(450)-Cys-2MNA (h)>PAA(250)-Cys-2MNA (h)>PAA(100)-Cys-2MNA (h) on both Caco-2 cell monolayers and rat intestinal mucosa. Comparing the influence of degree of preactivation with 2MNA on permeation enhancement, the following order PAA(450)-Cys-2MNA (h)>PAA(450)-Cys-2MNA (m)≈PAA(450)-Cys-2MNA (l) on Caco-2 cell monolayers and PAA(450)-Cys-2MNA (m)>PAA(450)-Cys-2MNA (h)>PAA(450)-Cys-2MNA (l) on intestinal mucosa was observed. The P(app) of sodium fluorescein was 5.08-fold improved on Caco-2 cell monolayers for PAA(450)-Cys-2MNA (h) and 2.46-fold improved on intestinal mucosa for PAA(450)-Cys-2MNA (m), respectively, in comparison to sodium fluorescein in buffer only. These results indicated that preactivated thiomers could be considered as a promising macromolecular permeation enhancing polymer for non-invasive drug administration.
Xueqing Wang; Javed Iqbal; Deni Rahmat; Andreas Bernkop-Schnürch
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Publication Detail:
Type:  In Vitro; Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-08-31
Journal Detail:
Title:  International journal of pharmaceutics     Volume:  438     ISSN:  1873-3476     ISO Abbreviation:  Int J Pharm     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-10-12     Completed Date:  2013-03-19     Revised Date:  2014-03-27    
Medline Journal Info:
Nlm Unique ID:  7804127     Medline TA:  Int J Pharm     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  217-24     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 Elsevier B.V. All rights reserved.
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MeSH Terms
Acrylic Resins / chemistry,  metabolism*
Biological Transport
Caco-2 Cells
Cysteine / chemistry,  metabolism*
Drug Delivery Systems*
Intestinal Absorption
Intestinal Mucosa / metabolism
Nicotinic Acids / chemistry,  metabolism*
Rats, Sprague-Dawley
Sulfhydryl Compounds / analysis,  chemistry,  metabolism*
Grant Support
P 23515-B11//Austrian Science Fund FWF
Reg. No./Substance:
0/Acrylic Resins; 0/Nicotinic Acids; 0/Sulfhydryl Compounds; 9003-01-4/carbopol 940; EU7D859ABZ/2-mercaptonicotinic acid; K848JZ4886/Cysteine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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Journal Information
Journal ID (nlm-ta): Int J Pharm
Journal ID (iso-abbrev): Int J Pharm
ISSN: 0378-5173
ISSN: 1873-3476
Publisher: Elsevier/North-Holland Biomedical Press
Article Information
© 2012 Elsevier B.V.
Received Day: 20 Month: 6 Year: 2012
Revision Received Day: 21 Month: 8 Year: 2012
Accepted Day: 24 Month: 8 Year: 2012
pmc-release publication date: Day: 15 Month: 11 Year: 2012
Print publication date: Day: 15 Month: 11 Year: 2012
Volume: 438 Issue: 1-2
First Page: 217 Last Page: 224
PubMed Id: 22960503
ID: 3484403
Publisher Id: IJP12837
DOI: 10.1016/j.ijpharm.2012.08.045

Preactivated thiomers: Permeation enhancing properties
Xueqing Wangab
Javed Iqbalbc
Deni Rahmatbd
Andreas Bernkop-Schnürchb Email:
aDepartment of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing, China
bDepartment of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Austria
cPCSIR Laboratories Complex, Karachi, Off University Road, Karachi-75280, Pakistan
dFaculty of Pharmacy, Pancasila University, Srengseng Sawah, Jagakarsa, Jakarta Selatan, Indonesia
Corresponding author at: Institute of Pharmacy, Leopold-Franzens-University Innsbruck, Josef-Moeller-Haus, Innrain 52c, 6020 Innsbruck, Austria/Europe. Tel.: +43 512 507 5383; fax: +43 512 507 2933.


The oral bioavailability of protein and peptide drugs is strongly limited by an insufficient uptake from the mucosa. In order to overcome the absorption barrier, permeation enhancers are used as auxiliary agents in oral drug delivery systems. State of the art permeation enhancers are mostly low molecular mass agents. They are easily absorbed through the gastrointestinal mucosa into the circulation and systemic toxic side-effects of these auxiliary agents consequently cannot be excluded (Bernkop-Schnürch et al., 2003). In contrast, macromolecular permeation enhancers such as poly(acrylic acid) derivatives, chitosan derivatives and thiolated polymers are too big in size to be taken up into the systemic circulation (Salamat-Miller and Johnston, 2005). In addition, they remain at the site where drug absorption shall take place for a comparatively longer time period.

In recent years, thiolated polymers have gained considerable attention (Shen et al., 2009; Davidovich-Pinhas et al., 2009; Martínez et al., 2012). Thiolated polymers or thiomers are a group of polymers bearing thiol substructures. Due to the immobilization of thiol groups on already well-established polymeric excipients such as polyacrylates (Leitner et al., 2003; Hombach et al., 2009; Bernkop-Schnürch and Thaler, 2000a; Kast et al., 2003) or chitosans (Sakloetsakun et al., 2009; Hombach et al., 2009; Jin et al., 2011; Li et al., 2011), features such as mucoadhesive (Hombach et al., 2009; Bernkop-Schnürch et al., 1999), in situ gelling (Sakloetsakun et al., 2009), efflux pump inhibiting (Greindl et al., 2009) and permeation enhancing properties (Kast et al., 2003; Hombach et al., 2008) are strongly improved. However, there are also shortcomings of thiomers, in particular regarding storage stability. Thiomers in solutions and semisolid formulations are subject to thiol oxidation at pH above 5, unless sealed under inert conditions. At pH 6–7, for instance, the amount of remaining thiol groups on chitosan-thioglycolic acid (Chi-TGA) and polycarbophil-cysteine (PCP-Cys) conjugates decreased by 70% within 3 h (Kast and Bernkop-Schnürch, 2001) and 45% within 2 h, respectively (Bernkop-Schnürch et al., 1999).

To overcome this drawback, novel thiomers – designated preactivated thiomers – were synthesized in this study. The concept for these novel thiomers is based on the reaction scheme for covalent chromatography of resins such as thiopropyl sepharose 6B (Fig. 1A). The thiolated resin being activated by a mercaptopyridine group is stable toward oxidation and can react with solutes containing thiol groups under mild conditions to form mixed disulfides (from insructions for thiopropyl sepharose 6B from Amersham Biosciences). In analogy the reaction can also take place when such polymers come into contact with the mucosa where thiol-rich substructures are available (Bernkop-Schnürch et al., 2004).

Following this strategy very recently thiolated polyacrylates were preactivated with 2-mercaptonicotinic acid and strongly improved mucoadhesive properties in comparison to just thiolated polyacrylates were demonstrated (Iqbal et al., 2012). In contrast to these improved mucoadhesive properties which could be anticipated because of the chemistry behind, the impact of preactivation on the permeation enhancing properties of thiomers cannot be foreseen, as the mechanisms involved in permeation enhancement of polymeric excipients is comparatively complex and only partially understood. Therefore the aim of this study was to prepare preactivated thiomers and evaluate their permeation-enhancing properties. Poly(acrylic acid) (PAA) was chosen as backbone because it has been previously successfully modified by cysteine. Sodium fluorescein was used as a paracellular marker (Clausen et al., 2002). Preactivated thiomers of different molecular mass and different degree of preactivation with 2-mercaptonicotinic acid (2MNA) were synthesized and their influence on the permeation enhancing properties were investigated on Caco-2 cell monolayers and freshly excised rat intestinal mucosa.

Materials and methods
2.1  Materials

Poly(acrylic acid) (PAA) (100, 250 and 450 kDa), 2-mercaptonicotinic acid (2MNA), L-cysteine hydrochloride (Cys), reduced glutathione (GSH), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) and sodium fluorescein (Na-Flu) were purchased from Sigma–Aldrich. All other reagents used were of analytical grade.

2.2  Synthesis of poly(acrylic acid)-cysteine conjugates

PAA-cysteine conjugates (PAA100-Cys, PAA250-Cys and PAA450-Cys) were synthesized by the covalent attachment of cysteine to poly(acrylic acid) according to a method described previously (Bernkop-Schnürch and Steininger, 2000b) (Fig. 1B). Briefly, 1 g each of PAA (with the molecular mass 100, 250 and 450 kDa) was hydrated separately in demineralized water and the pH value of the solutions was adjusted to 4.5 by the addition of 5 M NaOH. Then, EDAC in the final concentration of 200 mM was slowly added in order to activate the carboxylic acid moieties of each of the hydrated polymers. After 20 min of incubation under stirring at room temperature, 1 g of L-cysteine hydrochloride (pH adjust to 4.5) was added to each of the hydrated PAA solutions and the pH maintained at 4.5. Reaction mixtures were incubated for 3 h at room temperature under stirring. Neutralized polymers (PAA100, PAA250 and PAA450) prepared in the same way as the PAA-Cys conjugates but omitting EDAC during coupling reaction served as references. In order to eliminate unbound reacting species from the polymers, each of the above mentioned reaction mixtures was dialyzed five times using Spectra/Por® 3 membrane (MWCO: 1200) at (low acidic) ∼3 for 3 days in total at 10 °C in the dark, two times against 1 mM HCl, two times against the same medium but containing 1% NaCl and one time against 0.2 mM HCl. Thereafter, the dialyzed products were freeze-dried for 3 days at −80 °C under reduce pressure and stored at 4 °C until use.

2.3  Synthesis of poly(acrylic acid)-cysteine-2-mercaptonicotinic acid

Poly(acrylic acid)-cysteine-2-mercaptonicotinic acid (PAA-Cys-2MNA) of increasing molecular mass named PAA100-Cys-2MNA, PAA250-Cys-2MNA, PAA450-Cys-2MNA and PAA450-Cys-2MNA of increasing degree of preactivation with 2MNA were synthesized. 2MNA dimer (2,2′-dithiodinicotinic acid) was first prepared by oxidation of 2MNA with hydrogen peroxide under neutral pH conditions. Briefly, 4 g of 2MNA were dispersed in 100 ml of demineralized water by ultrasonicating for half an hour. Then the pH was adjusted to 7 and a clear solution was obtained. 5.3 ml of hydrogen peroxide (30%, v/v) was added and the pH maintained at 7. The resulting clear solution was 2MNA dimer. The formation of 2MNA dimer was proved by the measurment and comparison of UV absorption spectrums of monomer and dimer solution over a wavelength range between 200 nm and 400 nm using a UV–Vis spectrophotomer (UVmini-1240, Shimadzu Corp., Kyoto, Japan). Then, 0.2 g each of PAA-Cys (PAA100-Cys, PAA250-Cys, PAA450-Cys) was hydrated separately in 25 ml of demineralized water under permanent stirring. To prepare PAA-Cys-2MNA with high degree of preactivation (PAA100-Cys-2MNA (h), PAA250-Cys-2MNA (h) and PAA450-Cys-2MNA (h)), the freshly prepared 2MNA dimer solution containing 0.6 g of dimer mixed with 20 ml of DMSO was added into the PAA-Cys solution. The pH value of the mixture was adjusted to 6 under continuous stirring for 6 h at room temperature (Fig. 1C). The preparation of PAA450-Cys-2MNA with medium degree of preactivation (PAA450-Cys-2MNA (m)) was the same as that of PAA450-Cys-2MNA (h) except DMSO was omitted and the pH was adjusted to 7–8 (Fig. 1C). PAA450-Cys-2MNA with low degree of preactivation (PAA450-Cys-2MNA (l)) was prepared as outlined in Fig. 1D. In brief, 10 ml (25 mg/ml, pH 7–8) of the 2MNA solution was added into the PAA450-Cys solution and the pH value of the mixture was adjusted to 7–8. Then, 1 ml of hydrogen peroxide (H2O2, 30%, v/v) was added under continuous stirring for 6 h at room temperature.

Each of the above reaction mixtures were dialyzed five times using Spectra/Por® 3 membrane (MWCO: 1200) in 5 L of demineralized water for 3 days in total at 10 °C in the dark. Thereafter, the dialyzed products were freeze-dried for 3 days at −80 °C under reduced pressure and stored at 4 °C until use.

2.4  Determination of the thiol group content

The total amount of thiol groups and free thiol groups immobilized on the polymer conjugates was determined photometrically using Ellman's reagent as described previously. L-Cysteine hydrochloride was employed to establish calibration curve for all polymer conjugates (Bernkop-Schnürch et al., 1999).

2.5  Quantification of conjugated 2-mercaptonicotinic acid

The amount of conjugated 2MNA was determined photometrically. Briefly, 0.5 mg of each of the PAA-Cys-2MNA (100, 250 and 450 kDa) was hydrated in 0.5 M phosphate buffer (pH 6.8) with 2% reduced glutathione. After 60 min of incubation at room temperature, absorbance of 300 μl solution was measured at 354 nm. 2MNA was employed to establish calibration curve for all polymer conjugates.

2.6  In vitro transport studies across Caco-2 cell monolayers

Caco-2 cells (passage number 11–15) were seeded onto 12 well Transwell polyester®. The cells were cultured in MEM medium supplemented with 20% FCS and stored in a 5% CO2 environment in an incubator maintained at 37 °C. The culture medium was exchanged every 48 h. The cells were allowed to grow and differentiate for 21 days. Transepithelial electrical resistance (TEER) of the monolayers was measured with EVOM instrument (World Precision Instrument, Sarasota, FL). Prior to all experiments, each well (monolayers) was washed with 1 ml of 100 mM phosphate buffered saline (PBS). Then, 1 ml of transport medium (MEM, without FCS) was added to apical (AP) and 1 ml to basal (BL) chambers. After an equilibration period of 30 min in 5% CO2 incubator, the media of the donor compartment (AP chamber) was substituted by 0.5% (m/v) preactivated thiomers PAA100-Cys-2MNA, PAA250-Cys-2MNA and PAA450-Cys-2MNA of different degree of preactivation. Sodium fluorescein was used as model drug with a final concentration of 0.001% (m/v) and its MEM solution was used as control. At 0, 60, 120 and 180 min, 100 μl samples were taken out from the acceptor chambers and were replaced by the same amount of fresh transport medium. The amount of permeated sodium fluorescein was determined by fluorimetric detection by a microplate reader (Tecan Austria GmbH, Austria. 485 nm for extinction wavelength and 535 nm for emission wavelength) and was calculated by interpolation from an according standard curve. Cumulative corrections were made for previously removed samples. Apparent permeability coefficients (Papp) for sodium fluorescein were calculated as follows: Papp = Q/Act, where Q is the total amount permeated throughout the incubation time (μg), A is the the area of the transwell inserts (1.13 cm2), c is the initial concentration of sodium fluorescein in the donor chamber (μg/cm3), and t is the time of the permeation study (s). Each experiment was performed in triplicate. Transport enhancement ratios (R) were calculated from Papp values by: R = Papp(sample)/Papp(control).

2.7  In vitro transport studies across freshly excised rat intestinal mucosa

For in vitro permeation studies, non-fasting male Sprague–Dawley rats weighting between 240 and 250 g were used. After sacrificing the rats, the lower jejunum and ileum of the small intestine were immediately removed. The excised intestine was cut into strips of 1.5 cm, rinsed free of luminal contents and mounted in Ussing-type chambers (0.64 cm2 surface area) without stripping off the underlying muscle layer. For the apical chamber a buffer comprising 138 mM NaCl, 5 mM KCl, 10 mM glucose and 10 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) was prepared at pH 6.8. The buffer for the basolateral chamber included the same components plus 2 mM CaCl2 and 1 mM MgCl2 at pH 6.8. Ca2+ and Mg2+ were omitted in the apical chamber in order to prevent PAA-Cys complexes with Ca2+/Mg2+. The intestinal membrane was pre-incubated for 30 min with these artificial fluids. The model compound for permeation studies was sodium fluorescein. After pre-incubation, polymer solutions with sodium fluorescein (final concentration was 0.001% (m/v)) were added to the apical chamber for absorptive (AP to BL) transport. The polymers used in the transport study are shown in Table 1, including 0.5% (m/v) unmodified PAA100, PAA250, PAA450; 0.5% (m/v) PAA100-Cys, PAA250-Cys, PAA450-Cys or 0.5% (m/v) PAA100-Cys-2MNA (h), PAA250-Cys-2MNA (h), PAA450-Cys-2MNA (h), PAA450-Cys-2MNA (m) and PAA450-Cys-2MNA (l). To investigate the influence of GSH on the permeation enhancing properties for the preactivated thiomers, GSH was added at a final concentration of 0.5% (m/v). After 0, 30, 60, 90, 120, 150 and 180 min, 100 μl samples were withdrawn from the acceptor chambers and were replaced by the same amount of fresh transport medium. The following steps were the same as those described in Section 2.6 with the exception that for calculations the diffusion area of the Ussing-type chambers 0.64 cm2 was used.

2.8  Statistical data analysis

Statistical data analysis was performed using the ANOVA with P < 0.05 as the minimal level of significance.

Results and discussion
3.1  Synthesis and characterization of thiolated polymers

Synthesis of PAA-Cys has already been described by our research group previously (Bernkop-Schnürch and Steininger, 2000b). In general, the primary amino groups of L-cysteine were covalently attached to activated carboxylic acid groups of PAA by forming amide bonds. The chemical substructure of PAA-Cys is shown in Fig. 1B. The obtained PAA-Cys conjugates appeared as white, odorless powder of fibrous structure. The amounts of thiol groups attached to PAA determined by the Ellman's test are shown in Table 1. All the PAA-Cys exhibited a similar degree of modification with reduced thiol groups in the range of 700 μmol SH per gram polymer.

To study the influence of the molecular mass of PAA-Cys-2MNA and the degree of preactivation with 2MNA on the permeation enhancement property, a series of preactivated thiomers were synthesized. The conjugates were generated by the exchange of disulfide bonds between thiol functions of PAA-Cys and disulfide bonds of 2MNA dimer. The UV absorption spectrums of 2MNA monomer and dimer solution are shown in Fig. 2. Significant peaks were observed at 340.8 nm and 270 nm for the monomer and at 241 nm for the dimer. In the preparation of PAA-Cys-2MNA with high degree of preactivation, DMSO was used as a reaction medium due to the poor solubility of 2MNA and 2,2′-dithiodinicotinic acid in water. Moreover, addition of DMSO into the medium also avoids the precipitates formation during the reaction process. At the end of the reaction, DMSO was removed by dialysis against demineralized water. Moreover, the demineralized water used for dialysis was measured at 265 nm in order to verify the complete removal of DMSO form the dialysed products.

The chemical substructure of PAA-Cys-2MNA conjugates is illustrated in Fig. 1C. The lyophilized PAA-Cys-2MNA conjugates appeared as soft cotton-like fiber. With increasing degree of preactivation, the color turned from off-white to light yellow. The degree of preactivation with 2MNA of each preactivated thiomer is shown in Table 1. Generally, thiomers were divided into two groups: one group with high degree of preactivation (PAA100-Cys-2MNA (h), PAA250-Cys-2MNA (h) and PAA450-Cys-2MNA (h), ≥700 μmol 2MNA per gram polymer) and different molecular mass (100, 250 and 450 kDa) and one group with the same molecular mass (450 kDa) but with different degree of preactivation (high, ≥700 μmol; middle, 300–400 μmol and low, ≤200 μmol 2MNA per gram polymer).

3.2  Influence of molecular mass of PAA on permeation across freshly excised rat intestinal mucosa

The influence of molecular mass of preactivated thiomers on the permeation across freshly excised rat intestinal mucosa is shown in Fig. 3. When PAA-Cys-2MNAs displayed a similar degree of preactivation, the permeation ability increased with the increase in molecular mass of PAA (Fig. 3A). The Papp of the three preactivated thiomers are shown in Table 2. The rank order was: PAA450-Cys-2MNA (h) > PAA250-Cys-2MNA (h) > PAA100-Cys-2MNA (h). Using GSH as permeation mediator, the amount of permeated Na-Flu was to some extent improved for PAA250-Cys-2MNA (h) and PAA450-Cys-2MNA (h) (Fig. 3B and Table 2). The permeation ability rank order was: PAA450-Cys-2MNA (h) > PAA250-Cys-2MNA (h) > PAA100-Cys-2MNA (h), same as that without GSH. These results demonstrated the same size dependent effect for preactivated thiomer as shown for conventional thiomers, i.e. the larger the molecular mass, the higher the permeation enhancement ability (Kast and Bernkop-Schnürch, 2002). PAA100-Cys-2MNA (h), PAA250-Cys-2MNA (h) and PAA100-Cys-2MNA (h) with GSH had no enhancement effect compared to the control. The reason might be explained from two aspects. On freshly excised rat intestine model, PAA-Cys-2MNAs with high degree of preactivation showed poor permeation enhancement, while PAA450-Cys-2MNA (m) showed the most pronounced enhancement properties (reasons discussed in Section 3.4). The other reason might be the relative low molecular mass for PAA100-Cys-2MNA and PAA250-Cys-2MNA. As Kast (Kast and Bernkop-Schnürch, 2002) reported, the permeation enhancement ability from high to low was PCP > PAA450 > PAA100 > PAA30. PAA450 and PAA100 have no significant increase in permeation of Na-Flu. In fact, some polymer with low molecular mass, such as PAA30, decreased the permeation. There was not a well-founded explanation for this phenomenon up to now.

3.3  Permeation enhancing properties of PAA, PAA-Cys, PAA-Cys-2MNA on freshly excised rat intestinal mucosa

The permeation enhancing properties of unmodified PAAs, PAA-Cyses and PAA-Cys-2MNAs on the transport of Na-Flu were evaluated in vitro. Freshly excised rat intestine mounted in Ussing chambers served as model membrane. The unmodified PAA100, PAA100-Cys, PAA250, PAA250-Cys showed no significant enhancement effect on the permeation of Na-Flu (data not shown). The permeation studies with the unmodified PAA450, PAA450-Cys, PAA450-Cys-2MNA (h) and PAA450-Cys-2MNA (m) on mucosal uptake of Na-Flu are shown in Fig. 4 and their Papp values are listed in Table 2. PAA450-Cys-2MNA (h) showed some increase in the permeation but without a significant difference (1.22-fold, P = 0.2480) when compared with the control Na-Flu in buffer only. PAA450-Cys-2MNA (m) had obviously enhancement properties (2.46-fold, P = 0.0087). When the enhancement ability of PAA450, PAA450-Cys and PAA450-Cys-2MNA was compared, PAA450-Cys-2MNA showed the highest and PAA450 showed the lowest effect. These observations indicated that PAA450-Cys-2MNA might be a more effective macromolecular permeation enhancer. The underlying mechanism for the permeation enhancing effect of the preactivated thiomers is still not satisfactorily explained. We tried to explain it on two aspects: on the one hand, PAA450-Cys-2MNA was more active than PAA450-Cys because of the introduction of mercaptopyridine group into the molecule. The preactivated thiomer could therefore interact with thiol groups on the mucosal surface to a higher extent. These improved mucoadhesive properties of preactivated thiomers have already been demonstrated by Iqbal et al. (2012). Due to the immobilization of 2MNA, tablets based on PAA450-Cys-2MNA displayed 960-fold improved mucoadhesion in comparison to the corresponding unmodified PAA450 and the apparent viscosity was 206.2-fold improved. These properties led to a prolonged residence time of the preactivated thiomers on the mucosa. Similar as conventional thiomers, the preactivated thiomers would provide a comparatively high concentration of GSH on the membrane and lead to the opening of tight junctions (Bernkop-Schnürch et al., 2003). On the other hand, preactivated thiomers might directly interact with thiol groups on the tight junction proteins, which would affect the conformation of proteins and would change the membrane structure resulting in an improved drug uptake.

3.4  Influence of the degree of preactivation on permeation across freshly excised rat intestinal mucosa

This influence of the degree of preactivation of thiomers on the permeation enhancing effect on freshly excised rat intestinal mucosa is shown in Fig. 5. All the PAA450-Cys-2MNAs were of constant molecular mass (450 kDa). As illustrated in Fig 5A, the Papp order of the three polymers was PAA450-Cys-2MNA (m) > PAA450-Cys-2MNA (h) > PAA450-Cys-2MNA (l) and the Papp of Na-Flu in 0.5% PAA450-Cys-2MNA (m) was 2.46- times higher than that in buffer (Table 2). Not the thiomer exhibiting the highest degree of preactivation but PAA450-Cys-2MNA (m) showed the most pronounced enhancement properties. The results were different from that of conventional thiomers, which was general reported as the higher the degree of thiolation, the higher was the enhancement effect (Kast and Bernkop-Schnürch, 2002; Clausen and Bernkop-Schnürch, 2001).

This phenomenon might be explained from two aspects. First, free thiol groups immobilized within conventional thiomers play an important role in the permeation enhancing effect. Because the —SH in thiomers were prone to be oxidized to disulfides (S—S) at pH above 5, a large amount of free thiol groups in thiomers was necessary to maintain enough —SH for the permeation enhancement function. As for the preactivated thiomers, the active groups were disulfides in the molecules. They functioned as permeation enhancers due to disulfide exchange with oxidized glutathione (GSSG) or thiol groups on the mucosal surface. Preactivated thiomers were stable in a broad pH range. So a moderate amount of preactivated thiomers was sufficient for the reaction with oxidized glutathione (GSSG) or thiol groups on the mucosal surface.

Second, why PAA450-Cys-2MNA (h) showed poor permeation enhancement ability compared to PAA450-Cys-2MNA (m)? It was supposed as the influence of the hydrophobic ligand of 2MNA. When 2MNA was conjugated to PAA450-Cys, the hydrophilic character of the thiomer decreased. The higher the content of 2MNA, the more hydrophobic was the resulting preactivated thiomer. As it is well known, polymers exhibiting a similar solubility to mucin were able to penetrate the mucus layer easily (Huang et al., 2000). That was to say, hydrophilic character was critical for polymers to deliver drugs across mucus layer. Due to the high hydrophobic character of PAA450-Cys-2MNA (h), not all the preactivated substructures might be available. As for PAA450-Cys-2MNA (m) displays higher permeation ability than PAA450-Cys-2MNA (l), it might be the result of more disulfides from mercaptopyridine group.

The permeation enhancing effect was slightly increased in the presence of GSH of PAA450-Cys-2MNA (h) and PAA450-Cys-2MNA (m) (Fig. 5B and Table 2) without changing the enhancement rank order having been described above.

3.5  Influence of molecular mass and degree of preactivation on the permeation in Caco-2 cell monolayers

Caco-2 cell monolayers with a TEER value more than 700 Ω/cm2 were selected for the permeation experiments. The influence of molecular mass on the permeation enhancement of preactivated thiomers on Caco-2 cell monolayers is illustrated in Fig. 6. When PAA-Cys-2MNA showed a similar degree of modification with 2MNA, the enhancement increased with increasing molecular mass of PAA. The Papp values of the three preactivated thiomers are listed in Table 3. They ranked as PAA450-Cys-2MNA (h) > PAA250-Cys-2MNA (h) > PAA100-Cys-2MNA (h) being in good agreement with the rank order having been obtained on freshly excised intestinal mucosa.

The influence of the degree of preactivation on the permeation is shown in Fig. 7. Among PAA-Cys-2MNAs of the same molecular mass (450 kDa), PAA450-Cys-2MNA (h) exhibited the most pronounced enhancement effect. The Papp of the three preactivated thiomers ranked as PAA450-Cys-2MNA (h) > PAA450-Cys-2MNA (m) ≈ PAA450-Cys-2MNA (l) (Table 3). The Papp of Na-Flu was 5.08-fold increased in the presence of PAA450-Cys-2MNA (h) in comparison to that in buffer only. The result of the influence of the degree of preactivation on the permeation on Caco-2 cell monolayers was different from that on freshly excised rat intestine. This might have resulted from the structure difference between rat intestinal mucosa and Caco-2 cell monolayers. The mucus layer covering intestinal epithelial cells may restrict the access of higher molecular weight compounds (such as peptide and pharmaceutical excipients) to the absorption membrane (Schipper et al., 1999). However, the Caco-2 cell monolayers are mucus-free drug absorption model (Meaney and O’Driscoll, 1999). When polymer solution containing model compound was added to the apical chambers of the transwell, the polymer would get into intimate contact with the Caco-2 cells providing comparatively more pronounced interactions. The phenomenon had been proven by the fact that chitosans had pronounced effect on the permeability of mucus-free Caco-2 monolayers. In contrast, enhancement through rat ileum was modest (Meaney and O’Driscoll, 1999). In this study, when the preactivated thiomers were incubated with the Caco-2 cell monolayers, they took part directly in interactions with the epithelial cells without having to diffuse through the mucus layer. So solubility of preactivated thiomers was not so crucial as on the intestinal mucosa. Furthermore, thiomers with high degree of preactivation could show high activity on opening the tight junctions of Caco-2 cell monolayers.


During this study, novel designed preactivated thiomers were prepared by immobilizing 2MNA on the polymeric backbone polyacrylates. The influence of molecular mass and degree of preactivation with 2MNA on permeation enhancing properties of the thiolated polyacrylates was evaluated. Some preactivated thiomers could improve the permeation of sodium fluorescein across freshly excised rat intestinal mucosa and Caco-2 cell monolayer models significantly. On rat intestinal mucosa, the polymer with the highest molecular mass and medium degree of preactivation (PAA450-Cys-2MNA (m)) showed the highest permeation enhancing effect (2.89-fold) compared to Na-Flu in buffer only. In Caco-2 cell monolayers, the polymer with high molecular mass and high degree of preactivation (PAA450-Cys-2MNA (h)) displayed the highest permeation enhancing effect (5.08-fold). According to these results preactivated thiomers could be considered as a promising macromolecular permeation enhancing polymer for oral drug administration and their enhancement mechanism needs further investigation.

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This study was supported by the National Basic Research Program of China (No. 2009CB930300), National Natural Science Foundation of China (No. 81273456), Austrian Agency for International Cooperation in Education and Research (ÖAD), Higher Education Commission Pakistan (HEC) and the FWF (Fonds zur Förderung der wissenschaftlichen Forschung) project No. ZFP 235150.

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Keywords: Keywords Thiomers, Poly(acrylic acid)-cysteine, Preactivated thiomers, Permeation enhancer.

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