New perspectives of starch: Synthesis and in vitro assessment of novel thiolated mucoadhesive derivatives.

AIM: The purpose of this study was to develop a novel thiolated starch polymer with improved mucoadhesive properties by conjugation of cysteamine to starch as a natural polymer of restricted mucoadhesive properties. METHODS: Aldehyde substructures were integrated into starch via oxidative cleavage of vicinal diols by increasing amounts of sodium periodate followed by covalent attachment of cysteamine to oxidized starch via reductive amination. Thiol groups were quantified via Ellman's reaction and their impact on mucoadhesion was analyzed by rheological investigations, the rotating cylinder method and tensile studies on porcine mucosa. RESULTS: The total amount of immobilized thiol groups revealed a correlation between degree of oxidation and thiolation. Modified starch demonstrated an up to 1.66-fold increase in water uptake in comparison to native starch. Modification of starch resulted in greatly improved cohesive properties and improvement in mucoadhesion. Rheological investigations revealed a 2- to 4-fold rise in viscosity of mucus. Tensile studies revealed a linear correlation between degree of oxidation/thiolation and enhancement of maximum detachment force and total work adhesion. CONCLUSION: In terms of these results, thiolated starch is a new, promising, polymer in the field of mucoadhesive drug delivery systems.

Impact of different hydrophobic ion pairs of octreotide on its oral bioavailability in pigs.

The objective of this study was to investigate the impact of different hydrophobic ion pairs (HIP) on the oral bioavailability of the model drug octreotide in pigs. Octreotide was ion paired with the anionic surfactants deoxycholate, decanoate and docusate differing in lipophilicity. These hydrophobic ion pairs were incorporated in self-emulsifying drug delivery systems (SEDDS) based on BrijO10, octyldodecanol, propylene glycol and ethanol in a concentration of 5mg/ml. SEDDS were characterized regarding size distribution, zeta potential, stability towards lipase, log DSEDDS/release medium and mucus diffusion behavior. The oral bioavailability of octreotide was evaluated in pigs via LC-MS/MS analyses. Most efficient ion pairing was achieved at a molar ratio of 1:3 (peptide: surfactant). SEDDS containing the octreotide-deoxycholate, -decanoate and -docusate ion pair exhibited a mean droplet size of 152nm, 112nm and 191nm and a zeta potential of -3.7, -4.6 and -5.7mV, respectively. They were completely stable towards degradation by lipase and showed a log DSEDDS/release medium of 1.7, 1.8 and 2.7, respectively. The diffusion coefficient of these SEDDS was in the range of 0.03, 0.11 and 0.17×10-9cm2/s, respectively. In vivo studies with these HIPs showed no improvement in the oral bioavailability in case of octreotide-decanoate. In contrast, octreotide-deoxycholate and octreotide-docusate SEDDS resulted in a 17.9-fold and 4.2-fold higher bioavailability vs. CONTROL: According to these results, hydrophobic ion pairing could be identified as a key parameter for SEDDS to achieve high oral bioavailability.

ζ potential changing nanoparticles as cystic fibrosis transmembrane conductance regulator gene delivery system: an in vitro evaluation.

AIM: Aim of the study was the development of ζ potential changing nanoparticles as gene delivery system for the cystic fibrosis transmembrane conductance regulator gene. METHODS: Chitosan and carboxymethyl cellulose were modified with phosphotyrosine, a substrate for the brush border enzyme alkaline phosphatase. With these synthesized derivatives, different nanoparticle formulations, including the cystic fibrosis transmembrane conductance regulator gene were prepared by ionic gelation. RESULTS: A change from negative to positive ζ potential after enzymatic cleavage could be observed. Transfection studies with HEK-293 and Caco-2 cells showed transfection rates comparable to Lipofectamine 2000. Transfection efficiencies were significantly decreased when phosphate cleavage and thus ζ potential change was inhibited by phosphatase inhibitor. CONCLUSION: The developed nanoparticles represent a promising gene delivery system.

Enhancing the efficiency of thiomers: Utilizing a highly mucoadhesive polymer as backbone for thiolation and preactivation.

The objective of this study was to develop a novel thiomer with enhanced mucoadhesive properties using a highly mucoadhesive polymeric backbone. Fixomer™ A-30 (poly(methacrylic acid-co-sodium acrylamidomethyl propane sulfonate)), exhibiting a mucoadhesive strength superior to that of all other polymers, was thiolated by conjugation with l-cysteine and furthermore preactivated with 2-mercaptonicotinic acid (MNA). The resulting derivatives Fix-SH and Fix-S-MNA exhibited coupling rates of 755µmol thiol groups and 304µmol MNA per gram polymer, respectively. The mucoadhesive profile was evaluated with three different methods: tensile studies, rotating cylinder and rheological synergism. In tensile studies, a total work of adhesion of above 500µJ was determined for the unmodified polymer that increased to around 750µJ after thiolation and around 1500µJ after preactivation. The adhesion time of Fix-SH on the rotating cylinder was 3.7-fold and that of Fix-S-MNA 6.8-fold longer compared to the unmodified polymer. A rheological synergism was observed for the unmodified polymer as well as the derivatives with a non-significant difference for Fix-SH but a 5.44-fold improvement for Fix-S-MNA. Fix-S-MNA showed a significantly improved swelling behavior with a water-uptake up to the 30-fold of its initial weight over >50h whereas thiolation showed only slight improvements. Derivatization had no significant influence on cell viability. According to the results, Fix-S-MNA seems to be a suitable polymer for mucoadhesive drug delivery systems.

Thiolated polymers: evaluation of their potential as dermoadhesive excipients.

The objective of this study was to evaluate and compare four different thiolated polymers regarding their dermoadhesive potential. Therefore, three hydrophilic polymers (poly(acrylic acid), Carbopol 971 and carboxymethylcellulose) and a lipophilic polymer (silicone oil) were chosen to generate thiolated polymers followed by characterization. The total work of adhesion (TWA) and the maximum detachment force (MDF) of formulations containing modified and unmodified polymers were investigated on skin obtained from pig ears using a tensile sandwich technique. The synthesis of thiolated polymers provided 564 µmol, 1079 µmol, 482 µmol and 217 µmol thiol groups per gram poly(acrylic acid), Carbopol 971, carboxymethylcellulose and silicone oil, respectively. Hydrogels containing poly(acrylic acid)-cysteine, Carbopol 971-cysteine, and carboxymethylcellulose-cysteamine exhibited a 6-fold, 25-fold and 9-fold prolonged adhesion on porcine skin than the hydrogel formulations prepared from the corresponding unmodified polymers, respectively. Furthermore, thiolation of silicone oil with thioglycolic acid led to a 5-fold improvement in adhesion compared to the unmodified silicone oil. A comparison between the four thiolated polymer formulations showed a clear correlation between the amount of coupled thiol groups and the TWA. According to these results thiomers might also be useful excipients to provide a prolonged dermal resistance time of various formulations.

Charge changing phosphorylated polymers: Proof of in situ mucoadhesive properties.

The objective of this study was to design a novel polyethylene glycol (PEG) derivative exhibiting mucus permeating and mucoadhesive properties. Therefore, the enzymatically degradable phosphate ester, phosphotyrosine (Ptyr) was covalently attached to PEG-diamine. The synthesized PEG-Ptyr was studied in terms of enzymatic degradability on Caco 2 cells and by isolated intestinal alkaline phosphatase (IAP). Furthermore, the influence of enzymatic degradation on charge distribution of the polymer as well as on mucus diffusion and mucoadhesion was investigated. Within this study, the phosphate ester in PEG-Ptyr could be cleaved on the cell monolayer and by the isolated IAP, whereby the degradation rate was 10-fold higher utilizing the isolated enzyme. Implementation of negative charges on PEG due to modification with Ptyr led to an increased electrophoretic mobility, which was reduced after enzymatic degradation of the phosphate ester, most likely due to the alterations in charge distribution on the polymeric backbone. Interactions with mucus components were determined within mucus diffusion studies and rheological investigations. Herein, PEG-Ptyr showed a 3-fold lower mucus diffusion, after incubation with IAP. Within rheological investigations, dynamic viscosities increased by the factor of 3, after the phosphate ester in PEG-Ptyr was degraded by IAP. Results obtained within these experiments provided evidence for the in situ mucoadhesive properties of charge changing phosphorylated polymers. The combination of mucus permeating and mucoadhesive features of phosphorylated PEGs could be a highly interesting tool for future applications, such as for coating nanoparticles.

S-protected thiolated hydroxyethyl cellulose (HEC): Novel mucoadhesive excipient with improved stability.

The aim of this study was the design of novel S-protected thiolated hydroxyethyl cellulose (HEC) and the assessment of its mucoadhesive properties and biodegradability compared to the corresponding unmodified polymer. Thiolated HEC was S-protected via disulfide bond formation between 6-mercaptonicotinamide (6-MNA) and the thiol substructures of the polymer. In vitro screening of mucoadhesive properties was accomplished using two different methods: rotating cylinder studies and viscosity measurements. Moreover, biodegradability of these polymers by cellulase, xylanase and lysozyme was evaluated. MTT and LDH assays were performed on Caco-2 cells to determine the cytotoxicity of S-protected thiolated HEC. Thiolated HEC displayed 280.09±1.70µmol of free thiol groups per gram polymer. S-protected thiolated HEC exhibiting 270.8±21.11µmol immobilized 6-MNA ligands per gram of polymer was shown being 2.4-fold more mucoadhesive compared to thiolated HEC. No mucoadhesion was observed in case of unmodified HEC. Results were in a good agreement with rheological studies. The presence of free thiol moieties likely caused lower degree of hydrolysis by xylanase, whereas the degradation by both enzymes cellulase and xylanase was more hampered when 6-MNA was introduced as ligand for thiol group's protection. Findings in cell viability revealed that all three conjugates were non-toxic. S-protection of thiolated hydroxyethyl cellulose improved mucoadhesive properties and provided pronounced stability towards enzymatic attack, that makes this excipient superior for non-invasive drug administration over thiolated and unmodified forms.

Development of oral self nano-emulsifying delivery system(s) of lanreotide with improved stability against presystemic thiol-disulfide exchange reactions.

OBJECTIVES: To develop a self nano-emulsifying delivery system (SNEDS) for model peptide lanreotide providing a protective effect towards thiol-disulfide exchange reactions. METHODS: Ion-paired complexes of lanreotide with surfactants were prepared. In the following, Log P (octanol/water) of these complexes was determined. Lanreotide-loaded SNEDS (Lan/Deo-SN2 and Lan/Deo-SN3) were characterized for payload, droplet size and zeta potential. Lan/Deo-SN2 and Lan/Deo-SN3 were incubated with reduced glutathione (GSH) and thiol-enriched casein peptones for the assessment of thiol-disulfide exchange reactions. Ultra-centrifugation was used for separation of lanreotide released from SNEDS. RESULTS: A maximum payload of 6.4% was achieved for Lan/Deo-SN2. Mean droplet size of Lan/Deo-SN2 and Lan/Deo-SN3 was 45 ± 0.20 nm and 37 ± 0.02 nm, respectively. Both formulations showed significant protection towards thiol-disulfide exchange reactions. After 3 h of incubation with GSH, 48% and 80% of lanreotide remained intact when incorporated in Lan/Deo-SN2 and Lan/Deo-SN3, respectively. Furthermore, Lan/Deo-SN2 and Lan/Deo-SN3 showed 47% and 51% protection against thiol enriched casein peptones, respectively. Both formulations showed sustained lanreotide release over a period of 3 h. CONCLUSION: Owing to the results, the above-mentioned approach might be a useful tool to overcome the sulfhydryl barrier of the GI-tract.

Novel bioadhesive polymers as intra-articular agents: Chondroitin sulfate-cysteine conjugates.

The aim of this study was to generate and characterize a chondroitin sulfate-cysteine conjugate (CS-cys) as a novel bioadhesive agent for intra-articular use. Mucoadhesive properties of synthesized CS-cys were investigated by rheological measurement of polymer-mucus mixture and rotating cylinder method, while bioadhesive features of CS-cys on porcine articular cartilage were evaluated via tensile studies. Thiolation was achieved by attachment of l-cysteine to CS via amide bond formation mediated by carbodiimide as a coupling reagent. The conjugate exhibited 421.17±35.14 µmol free thiol groups per gram polymer. The reduced CS-cys displayed 675.09±39.67 µmol free thiol groups per gram polymer after disulfide bonds reduction using tris(2-carboxyethyl)phosphine hydrochloride. The increase in dynamic viscosity of thiolated CS due to oxidative disulfide bond formation was demonstrated using capillary viscometer. The combination of CS-cys and mucus led to 4.57-fold increase in dynamic viscosity in comparison with mucus control. Furthermore, adhesion time to porcine mucosa of CS-cys-based test disk was enhanced by 2.48-fold compared to unmodified CS as measured by rotating cylinder method suggesting the interaction between thiomers and mucus gel layer via disulfide bonds formation. Tensile studies of thiolated CS on porcine articular cartilage showed 5.37- and 1.76-fold increase in the total work of adhesion and the maximum detachment force, respectively, in comparison with unmodified CS indicating bioadhesive features of CS-cys. Cytotoxicity of CS-cys was assessed in Caco-2 cells and rat primary articular chondrocytes using MTT and LDH release assay, thereby showing the safety of CS-cys at a concentration of 0.25% (w/v) in Caco-2 cells. Furthermore, 0.1% of CS-cys was found non-toxic to rat primary articular chondrocytes. According to these results, CS-cys provides improved bioadhesive properties that might be useful as an intra-articular agent for treatment of osteoarthritis.

Preactivated thiolated nanoparticles: A novel mucoadhesive dosage form.

Within this study a novel form of mucoadhesive nanoparticles (NPs) exhibiting a prolonged residence time on mucosal tissues was developed. In order to achieve this goal a new thiomer was synthesized by the covalent attachment of the amino acid l-cysteine ethyl ester to poly(acrylic acid) (100 kDa). The free thiol groups were in the following preactivated with the aromatic thiol bearing ligand 2-mercaptonicotinic acid (2-MNA) and the amount of coupled l-cysteine ethyl ester as well as the amount of attached 2-MNA was determined. Based on this, preactivated thiomer NPs were prepared by ionic gelation with polyethylenimine (PEI). The resulting NPs were characterized regarding size and zeta potential. Furthermore their mucoadhesive properties were investigated via rheological measurements with porcine intestinal mucus and via determination of the particles' mucosal residence time. Results showed that 1666.74 µmol l-cysteine ethyl ester and 603.07 µmol 2-MNA could be attached per gram polymer. NPs were in a size range of 112.67-252.84 nm exhibiting a zeta potential of -29 mV. Thiolated NPs only led to a 2-fold increase in mucus viscosity whereas preactivated NPs showed a 6-fold higher mucus viscosity than unmodified NPs. The mucosal residence time of thiolated NPs was 1.6-fold prolonged and that of preactivated NPs even 4.4-fold higher compared to unmodified particles. Accordingly, preactivated thiolated NPs providing a prolonged residence time on mucosal membranes could be a promising dosage form for various applications.

Zeta potential changing phosphorylated nanocomplexes for pDNA delivery.

The objective of this study was to evaluate the suitability of a zeta potential changing system as gene delivery system. The phosphate ester bearing ligand 6-phosphogluconic acid (6-PGA) was attached to linear and branched polyethyleneimine (PEI) via a carbodiimide-mediated reaction whereby 287 µmol and 413 µmol 6-PGA could be coupled per gram polymer. Nanocomplexes of these modified polymers with pDNA showed a zeta potential of +12 mV for nanocomplexes with the linear PEI-6PGA and +16 mV in case of the branched derivative. By the addition of carboxymethylcellulose (CMC), zeta potentials of the complexes were reduced to +2.86 and +3.25, respectively. Phosphate release studies on Caco 2 cells and HEK-293 cells demonstrated the ability to cleave the phosphate ester. Compared to HEK-293 cells, enzymatic degradation of the phosphate ester in Caco 2 cells was 2.3-fold higher from nanocomplexes comprising linear PEI and 4.3-fold higher from those with branched PEI. Furthermore, incubation with alkaline phosphatase led to an increase in the zeta potential of nanocomplexes based on linear PEI-6PGA to +6.96mV and +8.26 mV in nanocomplexes comprising branched PEI-6PGA. Studying transfection efficiency in Caco 2 cells and HEK-293 cells, a higher expression of the green fluorescent protein (GFP) could be detected in HEK-293 cells. In presence of a phosphate inhibitor, transfection efficiencies were decreased in both cells lines, due to a lacking shift of the zeta potential of the tested pDNA complexes. According to these results, zeta potential changing systems seem to be a promising strategy for future gene delivery systems, as this concept allows the in situ generation of positive charges in close proximity to the cellular surface.

Can thiolation render a low molecular weight polymer of just 20-kDa mucoadhesive?

The objective was to investigate whether even low-molecular weight polymers (LMWPs) can be rendered mucoadhesive due to thiolation. Interceded by the double catalytic system carbodiimide/N-hydroxysuccinimide, cysteamine was covalently attached to a copolymer, poly(4-styrenesulfonic acid-co-maleic acid) (PSSA-MA) exhibiting a molecular weight of just 20 kDa. Depending on the amount of added N-hydroxysuccinimide and cysteamine, the resulting PSSA-MA-cysteamine (PC) conjugates exhibited increasing degree of thiolation, highest being "PC 2300" exhibiting 2300.16 ± 149.86 µmol thiol groups per gram of polymer (mean ± SD; n = 3). This newly developed thiolated polymer was evaluated regarding mucoadhesive, rheological and drug release properties as well from the toxicological point of view. Swelling behavior in 100 mM phosphate buffer pH 6.8 was improved up to 180-fold. Furthermore, due to thiolation, the mucoadhesive properties of the polymer were 240-fold improved. Rheological measurements of polymer/mucus mixtures confirmed results obtained by mucoadhesion studies. In comparison to unmodified polymer, PC 2300 showed 2.3-, 2.3- and 2.4-fold increase in dynamic viscosity, elastic modulus and viscous modulus, respectively. Sustained release of the model drug codeine HCl out of the thiomer was provided for 2.5 h (p < 0.05), whereas the drug was immediately released from the unmodified polymer. Moreover, the thiomer was found non-toxic over Caco-2 cells for a period of 6- and 24-h exposure. Findings of the present study provide evidence that due to thiolation LMWPs can be rendered highly mucoadhesive as well as cohesive and that a controlled drug release out of such polymers can be provided.

An in-vitro exploration of permeation enhancement by novel polysulfonate thiomers.

The study was aimed to synthesize preactivated polysulfonate thiomers using poly(4-styrenesulfonic acid-co-maleic acid) (PSSA-MA) and to evaluate their permeation enhancing properties. PSSA-MA-cysteamine (PC) conjugates with 2-mercaptonicotinic acid (2MNA) having different degree of preactivation (PC1608-2MNA, PC2300-2MNA, PC3100-2MNA) were synthesized from the subsequent PSSA-MA-cysteamine thiomers (PC1608, PC2300 and PC3100). The permeation-enhancing features were evaluated by in-vitro models using low-molecular size marker sodium fluorescein (Na-Flu) and with high-molecular size marker fluorescein isothiocyanate-dextran (FD4). Associating the influence of degree of preactivation on permeation enhancement, following rank order PC3100-2MNA>PC3100>PC2300-2MNA>PC2300>PC1608-2MNA>PC1608>PSSA-MA>control was observed on Caco-2 cell monolayers and with little change in sequence on freshly excised rat intestine. The apparent permeability (Papp) was improved 3.16-fold for Na-Flu and 3.51-fold for FD4 on Caco-2 cell monolayers. Similarly, 4.17- and 3.60-fold improved Papp values were observed on freshly excised rat intestine for Na-Flu and FD4, respectively. More pronounced permeation effects on rat intestine compared to Caco-2 cell monolayer by the thiomer/preactivated conjugates indicated their mucus-interpenetration capability in addition to mucoadhesion. Thus relatively low molecular weight (LMW) preactivated polysulfonate thiomers could to a higher extent enhance permeation on membranes covered by mucus layer compared to high molecular weight thiomers/preactivated conjugates that usually exhibit higher permeation enhancing effects on Caco-2 cell monolayer.

Synthesis and characterization of thiolated ß-cyclodextrin as a novel mucoadhesive excipient for intra-oral drug delivery.

The objective of the present study was to synthesize and characterize cysteamine conjugated ß-cyclodextrin (ß-CD-Cys) as a novel mucoadhesive oligomeric excipient for intra-oral drug delivery. ß-CD-Cys conjugates were obtained in a two-step synthetic pathway, whereby, vicinal diol groups of the oligomer were oxidized using increasing concentrations of sodium-per-iodate (NaIO4), prior to the covalent coupling of cysteamine via reductive amination. Quantification of immobilized thiol groups through Ellman's test revealed 561.56 ± 81 µmol/g, 1054.26 ± 131 µmol/g and 1783.92 ± 201 µmol/g of free thiol groups attached to the oligomer backbone depending on the extent of oxidation. ß-CD-Cys conjugates at concentrations of 0.5% (m/v) showed no toxic effects on Caco-2 cells within 72 h. Furthermore, ß-CD-Cys conjugates displayed a 4-fold improved water solubility compared to the parent oligomer. ß-CD-Cys conjugates (ß-CD-Cys561, ß-CD-Cys1054 and ß-CD-Cys1783) showed 2.86-, 15.09- and 49.08-fold improved retention time on porcine intestinal mucosa and 9.66-, 16.43- and 34.51-fold improved on the porcine buccal mucosa, respectively. Formation of inclusion complexes of miconazole nitrate and ß-CD-Cys1054 resulted in 150-fold increased solubility of miconazole nitrate. According to these results, it seems that ß-CD-Cys conjugates might provide a new promising tool for delivery of poorly water soluble therapeutic agents, such as miconazole nitrate for intra-oral delivery.

Synthesis and In Vitro Evaluation of Thiolated Carrageenan.

The aim of this study was to generate and characterize a thiolated carrageenan. Thiolated carrageenan (carrageenan-SH) was synthesized from kappa (κ)- and iota (ι)-carrageenan by bromine replacement of the hydroxyl moieties followed by substitution to thiol groups using thiourea. Thiolated κ- and ι-carrageenan exhibited 176.57 ± 20.11 and 109.51 ± 18.26 µmol thiol groups per gram polymer, respectively. The resazurin test in Caco-2 cells revealed no toxic effect of both thiolated carrageenans at a concentration below 0.1% (w/v). Regarding efflux pump inhibitory effect, cellular accumulation of multidrug-resistance protein 2 substrate, sulforhodamine 101, was 1.38- and 1.35-fold increased in cells treated with thiolated κ- and ι-carrageenan, respectively. Modification of κ- and ι-carrageenan led to 3.9- and 2.0-fold increase in dynamic viscosity of mucus-thiolated carrageenan mixture within 4 h. Furthermore, residence time of κ- and ι-carrageenan-SH on porcine intestinal mucosa was 6.4- and 1.8-fold prolonged, respectively, as demonstrated by rotating cylinder method, indicating improved mucoadhesive properties. Hence, thiolation of carrageenans led to novel pharmaceutical excipients for various applications.

Thiolated alkyl-modified carbomers: Novel excipients for mucoadhesive emulsions.

The aim of this study was the design and evaluation of mucoadhesive emulsifying polymeric excipients. Three thiol bearing ligands with increasing pKa values of their sulfhydryl group, namely 4-aminothiophenol (pKa=6.86), l-cysteine (pKa=8.4) and d/l-homocysteine (pKa=10.0) were coupled to the polymeric backbone of alkyl-modified carbomer (PA1030). Resulting conjugates displayed 818.5µmol 4-aminothiophenol, 698.5µmol cysteine and 651.5µmol homocysteine per gram polymer and were evaluated regarding the reactivity of thiol groups, emulsifying and mucoadhesive properties. In general, the synthesized conjugates showed a pH dependent reactivity, whereby the fastest oxidation occurred in PA1030-cysteine, as almost no free thiol groups could be detected after 120min. Emulsification of medium chain triglycerides was feasible with all synthesized conjugates leading to oil-in-water-emulsions. Emulsions with PA1030-cysteine displayed the highest stability and the smallest droplet size among the tested formulations. Oxidation and consequently cross-linking of the thiomers prior to the emulsification process led to an overall decreased emulsion stability. Evaluating mucosal residence time of thiomer emulsions on porcine buccal mucosa, a 9.2-fold higher amount of formulation based on PA1030-cysteine remained on the mucosal tissue within 3h compared to the unmodified polymer. According to these results, the highest reactive ligand l-cysteine seems to be most promising in order to obtain thiolated polymers for the preparation of mucoadhesive o/w-emulsions.

Development of phosphorylated nanoparticles as zeta potential inverting systems.

The objective of this study was to generate nanoparticles with a slightly negative zeta potential which switches to positive values under the influence of intestinal alkaline phosphatase in order to address two major physiological barriers (mucus and membrane barrier). Carboxymethyl cellulose and chitosan were modified with phosphotyrosine by means of a water-soluble carbodiimide and polyelectrolyte complexes were formed by mixing two polymer solutions in an appropriate ratio. Due to this modification, phosphate ions could potentially be released which would lead to a change in zeta potential. Their sizes were found to be between 200 and 300 nm while their zeta potentials ranged from -8 mV to -5 mV prior to incubation with the enzyme. It could be shown that phosphate ions are released from the modified polymers and nanoparticles by isolated phosphatase and in a Caco-2 cell model. Incubation with phosphatase led to a change in zeta potential of the nanoparticles up to +8 mV. As neither polymers nor particles display toxic properties within the resazurin assay, these nanoparticles appear to be useful tools in future drug delivery systems as they have appropriate properties regarding particle size and surface charge in order to overcome the mucus and the membrane barrier.

Polyethylene imine-6-phosphogluconic acid nanoparticles--a novel zeta potential changing system.

The aim of the study was to develop nanoparticles with the ability to change their zeta potential. By covalent attachment of 6-phosphogluconic acid to polyethylene imine, a charged, enzymatically removable moiety was introduced into the polymer. The novel conjugate displayed 400 µmol phosphate per gram polymer, as determined by malachite green assay. Studies evaluating the cleavage by intestinal alkaline phosphatase revealed that 69 % of the coupled phosphate could be released from the polymer. Furthermore, nanoparticles generated by polyelectrolyte complexation technique using carboxymethyl cellulose as negatively charged component exhibited a zeta potential of -6 mV and an average particle size of 300 nm. Enzymatic cleavage of the phosphate ester moiety by isolated intestinal alkaline phosphatase on these nanoparticles caused shift of the zeta potential from negative to positive value of +3 mV whereby 58% of the total amount of phosphate were released. Studies on Caco-2 cells revealed the capability of a living system to hydrolyze the phosphate ester in the novel conjugate as well as on the nanoparticles via their intestinal alkaline phosphatase. Based on these results, polymeric nanoparticles comprising an enzymatically degradable phosphate ester moiety can provide a promising strategy for zeta potential changing systems.

Preactivated thiolated poly(methacrylic acid-co-ethyl acrylate): synthesis and evaluation of mucoadhesive potential.

The study was aimed to developed and investigate a novel polymer for intestinal drug delivery with improved mucoadhesive properties. Therefore Eudragit® L 100-55 (poly(methacrylic acid-co-ethyl acrylate)) was thiolated by covalent attachment of L-cysteine. The immobilized thiol groups were preactivated by disulfide bond formation with 2-mercaptonicotinic acid. Resulting derivative (Eu-S-MNA) was investigated in terms of mucoadhesion via three different methods: tensile studies, rotating cylinder studies and rheological synergism method, as well as water-uptake capacity and cytotoxicity. Different derivatives were obtained with increasing amount of bound L-cysteine (60, 140 and 266 µmol/g polymer) and degree of preactivation (33, 45 and 51 µmol/g polymer). Tensile studies revealed a 30.5-, 35.3- and 52.2-fold rise of total work of adhesion for the preactivated polymers compared to the unmodified Eudragit. The adhesion time on the rotating cylinder was prolonged up to 17-fold in case of thiolated polymer and up to 34-fold prolonged in case of the preactivated polymer. Rheological synergism revealed remarkable interaction of all investigated modified derivatives with mucus. Further, water-uptake studies showed an over 7h continuing weight gain for the modified polymers whereat disintegration took place for the unmodified polymer within the first hour. Cell viability studies revealed no impact of modification. Accordingly, the novel preactivated thiolated Eudragit-derivative seems to be a promising excipient for intestinal drug delivery.

Thiomers--from bench to market.

Thiolated polymers or designated thiomers are obtained by immobilization of sulhydryl bearing ligands on the polymeric backbone of well-established polymers such as poly(acrylates) or chitosans. This functionalization leads to significantly improved mucoadhesive properties compared to the corresponding unmodified polymers, as disulfide bonds between thiol groups of thiomers and cysteine-rich glycoproteins of the mucus gel layer are formed. Furthermore, enzyme- and efflux-pump inhibiting as well as improved permeation-enhancing properties are advantages of thiolization. By the covalent attachment of mercaptonicotinamide substructures via disulfide bonds to thiolated polymers these properties are even substantially further improved and stability towards oxidation even in aqueous media can be provided. Meanwhile, more than 50 research groups worldwide are working on thiolated polymers. For certain thiomers the scale up process for industrial production has already been done and GMP material is available. Furthermore, safety of thiolated poly(acrylic acid), thiolated chitosan and thiolated hyaluronic acid could be demonstrated via orientating studies in human volunteers and via various clinical trials. The first product (Lacrimera® eye drops, Croma-Pharma) containing a chitosan-N-acetylcysteine conjugate for treatment of dry eye syndrome will enter the European market this year. It is the only product providing a sustained protective effect on the ocular surface due to its comparatively much more prolonged residence time worldwide. Various further products utilizing, for instance, thiolated hyaluronic acid in ocular surgery are in the pipeline.