Role of sonication pre-treatment and cation valence in the sol-gel transition of nano-cellulose suspensions.

Sol-gel transition of carboxylated cellulose nanocrystals has been investigated using rheology, SAXS, NMR and optical spectroscopies to unveil the distinctive roles of ultrasound treatments and addition of various cations. Besides cellulose fiber fragmentation, sonication treatment induces fast gelling of the solution. The gelation is independent of the addition of cations, while the final rheological properties are highly influenced by the type, concentration and sequence of the operations since the cations must be added prior to sonication to produce stiff gels. The gel elastic modulus was found to increase proportionally to the ionic charge rather than the cationic size. In cases where ions were added after sonication, SAXS analysis of the Na+ hydrogel and Ca2+ hydrogel indicated the presence of structurally ordered domains in which water is confined, and 1H-NMR investigation showed the dynamics of water exchange within the hydrogels. Conversely, separated phases containing essentially free water were characteristic of the hydrogels obtained by sonication after Ca2+ addition, confirming that this ion induces irreversible fiber aggregation. The rheological properties of the hydrogels depend on the duration of the ultrasound treatments, enabling the design of programmed materials with tailored energy dissipation response.

Self-emulsifying drug delivery systems: Design of a novel vaginal delivery system for curcumin.

AIM: The aim of this study was to develop a vaginal self-emulsifying delivery system for curcumin being capable of spreading, of permeating the mucus gel layer and of protecting the drug being incorporated in oily nanodroplets towards mucus interactions and immobilization. METHODS: The emulsifying properties of curcumin loaded SEDDS containing 30% Cremophor RH40, 20% Capmul PG-8, 30% Captex 300, 10% DMSO and 10% tetraglycol (SEDD formulation A) as well as 25% PEG 200, 35% Cremophor RH40, 20% Captex 355, 10% Caprylic acid and 10% Tween 80 (SEDD formulation B) after diluting 1+2 with artificial vaginal fluid were characterized regarding droplet size and zeta potential. Collagen swelling test was used to examine the irritation potential of SEDDS. Additionally to mucus binding studies, permeation studies in the mucus were performed. Furthermore, spreading potential of the novel developed formulations was compared with a commercial available o/w cream (non-ionic hydrophilic cream) on vaginal mucosa. RESULTS: SEDDS displayed a mean droplet size between 38 and 141nm and a zeta potential of -0.3 to -1.6mV. The collagen swelling test indicated no significant irritation potential of both formulations over 24h. An immediate interaction of unformulated curcumin with the mucus was determined, whereas both SEDDS facilitated drug permeation through the mucus layer. Formulation B showed a 2.2-fold improved transport ratio of curcumin compared to SEDD formulation A. In comparison to the vaginal cream, SEDD formulation A and B were able to spread over the vaginal mucosa and cover the tissue to a 17.8- and 14.8-fold higher extent, respectively. CONCLUSION: According to these results, SEDDS seems to be a promising tool for vaginal application.

P-glycoprotein inhibitors: synthesis and in vitro evaluation of a preactivated thiomer.

The aim of this study was to synthesize the preactivated thiomer poly(acrylic acid)-cyteine-2-mercaptonicotinic acid (PAA-Cys-2MNA) and to evaluate its P-glycoprotein (P-gp) inhibitory properties. The thiomer (PAA-Cys) was synthesized by covalent immobilization of thiol groups on poly(acrylic acid) (PAA) with a molecular mass of 250 kDa followed by immobilization of 2-mercaptonicotinic acid (2MNA) to thiol groups via disulfide bond formation resulting in PAA-Cys-2MNA. P-gp inhibitory effect of this preactivated thiomer was evaluated on Caco-2 cells. Transports of rhodamine 123 at 37 °C with and without verapamil and at 4 °C were performed to evaluate P-gp function of cells. In total, 1571.81 ± 156.18 µmol thiol groups were immobilized per gram of polymer that were in the next step by 99.88% preactivated. The enhancement ratios of Papp calculated from the ratio between Papp of rhodamine 123 in the presence of P-gp inhibitors and Papp of rhodamine 123 alone were 2.36, 2.09, and 1.84-fold in the presence of PAA-Cys-2MNA, PAA-Cys, and PAA, respectively. Because of its pronounced P-gp inhibitory effect, PAA-Cys-2MNA could be considered as promising macromolecular P-gp inhibitor for various drug delivery systems.

Thiolated silicone oils as adhesive skin protectants for improved barrier function.

OBJECTIVE: The purpose of this study was the evaluation of thiolated silicone oil as novel skin protectant exhibiting prolonged residence time, enhanced barrier function and reinforced occlusivity. METHODS: Two silicone conjugates were synthesized with mercaptopropionic acid (MPA) and thioglycolic acid (TGA) as thiol ligands. Adhesion, protection against artificial urine and water vapour permeability with both a Payne cup set-up and transepidermal water loss (TEWL) measurements on porcine skin were assessed. RESULTS: Silicone thiomers showed pronounced substantivity on skin with 22.1 ± 6.3% and 39.2 ± 6.7% remaining silicone after 8 h for silicone-TGA and silicone-MPA, respectively, whereas unmodified silicone oil and dimethicone were no longer detectable. In particular, silicone-MPA provided a protective shield against artificial urine penetration with less than 25% leakage within 6 h. An up to 2.5-fold improved water vapour impermeability for silicone-MPA in comparison with unmodified control was discovered with the Payne cup model. In addition, for silicone-MPA a reduced TEWL by two-thirds corresponding to non-thiolated control was determined for up to 8 h. CONCLUSION: Thiolation of silicone oil leads to enhanced skin adhesiveness and barrier function, which is a major advantage compared to commonly used silicones and might thus be a promising treatment modality for various topical applications.

P-glycoprotein inhibitors: synthesis and in vitro evaluation of a preactivated thiomer.

The aim of this study was to synthesize the preactivated thiomer poly(acrylic acid)-cyteine-2-mercaptonicotinic acid (PAA-Cys-2MNA) and to evaluate its P-glycoprotein (P-gp) inhibitory properties. The thiomer (PAA-Cys) was synthesized by covalent immobilization of thiol groups on poly(acrylic acid) (PAA) with a molecular mass of 250 kDa followed by immobilization of 2-mercaptonicotinic acid (2MNA) to thiol groups via disulfide bond formation resulting in PAA-Cys-2MNA. P-gp inhibitory effect of this preactivated thiomer was evaluated on Caco-2 cells. Transports of rhodamine 123 at 37 °C with and without verapamil and at 4 °C were performed to evaluate P-gp function of cells. In total, 1571.81 ± 156.18 µmol thiol groups were immobilized per gram of polymer that were in the next step by 99.88% preactivated. The enhancement ratios of Papp calculated from the ratio between Papp of rhodamine 123 in the presence of P-gp inhibitors and Papp of rhodamine 123 alone were 2.36, 2.09, and 1.84-fold in the presence of PAA-Cys-2MNA, PAA-Cys, and PAA, respectively. Because of its pronounced P-gp inhibitory effect, PAA-Cys-2MNA could be considered as promising macromolecular P-gp inhibitor for various drug delivery systems.

Nano-carrier systems: Strategies to overcome the mucus gel barrier.

The present review provides an overview of nanotechnology-based strategies to overcome various mucus gel barriers including the intestinal, nasal, ocular, vaginal, buccal and pulmonary mucus layer without destroying them. It focuses on the one hand on strategies to improve the mucus permeation behavior of particles and on the other hand on systems avoiding the back-diffusion of particles out of the mucus gel layer. Nanocarriers with improved mucus permeation behavior either exhibit a high density of positive and negative charges, bearing mucolytic enzymes such as papain and bromelain on their surface or display a slippery surface due to PEG-ylation. Furthermore, self-nanoemulsifying-drug-delivery-systems (SNEDDS) turned out to exhibit comparatively high mucus permeating properties. Strategies in order to avoid back-diffusion are based on thiolated polymers reacting to a higher extent with cysteine subunits of the mucus at pH 7 in deeper mucus regions than at pH 5 being prevalent in luminal mucus regions of the intestinal and vaginal mucosa. Furthermore, particles changing their zeta potential from negative to positive once they have reached the epithelium seem to be promising carriers. The summarized knowledge should provide a good starting point for further developments in this field.

Mucus permeating thiomer nanoparticles.

The aim of this study was to develop and evaluate a novel mucoadhesive drug delivery system based on thiolated poly(acrylic acid) nanoparticles exhibiting mucolytic properties to enhance particle diffusion into deeper mucus regions before adhesion. Mediated by a carbodiimide, cysteine and the mucolytic enzyme papain were covalently attached to poly(acrylic acid) via amide bond formation. The conjugates were co-precipitated with calcium chloride in order to obtain papain modified (PAA-pap) and thiolated nanoparticles (PAA-cys) as well as particles containing both conjugates (PAA-cys-pap). The nanoparticulate systems were characterized regarding particle size distribution and zeta potential. Particle transport was investigated by diffusion studies across intestinal mucus using two different techniques. Furthermore, mucoadhesive properties of all particles were evaluated via rheological measurements. Results demonstrated that all nanoparticles were in a size range of 158-214 nm and showed negative zeta potentials. Due to the presence of papain, the PAA-cys-pap particles were capable of cleaving mucoglycoprotein substructures and consequently exhibited a 2.0-fold higher penetration into the mucus layer in comparison with PAA-cys particles. Within the rheological studies, an 1.9-fold increase in mucoadhesion could be achieved for the nanoparticulate system based on thiolated PAA compared to papain modified particles (PAA-pap). Therefore, the newly developed particulate system (PAA-cys-pap) is characterized by mucoadhesive as well as mucolytic properties. The combination of both effects - mucus-permeating and mucoadhesive properties - might be a promising strategy for the development of oral drug delivery systems to overcome the mucus barrier and providing a prolonged residence time close to the absorption membrane.

Thiolated nanocarriers for oral delivery of hydrophilic macromolecular drugs.

It was the aim of this study to investigate the effect of unmodified as well as thiolated anionic poly(acrylic acid) (PAA) and cationic chitosan (CS) utilized in free-soluble form and as nanoparticulate system on the absorption of the hydrophilic compound FD4 across intestinal epithelial cell layer with and without a mucus layer. Modifications of these polymers were achieved by conjugation with cysteine to PAA (PAA-Cys) and thioglycolic acid to CS (CS-TGA). Particles were prepared via ionic gelation and characterized based on their amount of thiol groups, particle size and zeta potential. Effects on the cell layer concerning absorption enhancement, transepithelial electrical resistance (TEER) and cytotoxicity were investigated. Permeation enhancement was evaluated with respect to in vitro transport of FD4 across Caco-2 cells, while mucoadhesion was indirectly examined in terms of adsorption behaviour when cells were covered with a mucus layer. Lyophilized particles displayed around 1000 µmol/g of free thiol groups, particle sizes of less than 300 nm and a zeta potential of 18 mV (CS-TGA) and -14 mV (PAA-Cys). Cytotoxicity studies confirmed that all polymer samples were used at nontoxic concentrations (0.5% m/v). Permeation studies revealed that all thiolated formulations had pronounced effects on the paracellular permeability of mucus-free Caco-2 layers and enhanced the permeation of FD4 3.0- to 5.3-fold. Moreover, polymers administered as particles showed a higher permeation enhancement than their corresponding solutions. However, the absorption-enhancing effect of each thiolated formulation was significantly (p<0.05) reduced when cells were covered with mucus layer. In addition, all formulations were able to decrease the TEER of the cell layer significantly (p<0.05). Therefore, both thiolated polymers as nanoparticulate delivery systems represent a promising tool for the oral administration of hydrophilic macromolecules.

Development of a dosage form for accelerated release.

PURPOSE: It was the aim of this study to develop an oral capsule delivery system capable of rapidly ejecting the incorporated payload in the small intestine. METHODS: The capsule consists of four parts: a reaction mixture comprising of a basic and a corresponding acidic component, a plunger necessary to separate the reaction mixture from the inserted ingredients, capsule cap and body (made out of ethylcellulose (EC)), where at the bottom of the body a semipermeable filter membrane is mounted. As soon as water permeates through the membrane, the reaction mixture dissolves and carbon dioxide (CO2) is released resulting in a high speed liberation of inserted compounds onto the intestinal mucosa. Several filter membranes were investigated regarding water influx, capillary force and water retention capacity. CO2 release of sodium hydrogen carbonate (NaHCO3) was examined in presence of several acidic components in different morphological forms (powder, lyophilisate and granule) and the amount of CO2 liberation out of prepared capsules was determined. Furthermore, release of enteric coated capsules was tested within 0.1M HCl and 100mM phosphate buffer pH 6.8. RESULTS: The rank order regarding membrane permeability was determined to be: cellulose acetate with a pore diameter of 12-15 µm>4-12 µm cellulose acetate>8 µm cellulose nitrate>8-12 µm cellulose acetate. NaHCO3 in combination with tartaric acid in form of a granule could be identified as the most promising reaction mixture with the highest amount of released CO2 compared to all other reaction mixture combinations. Stability of enteric coated capsules in HCl and a spontaneous release in phosphate puffer could be demonstrated within in vitro release studies. CONCLUSION: In light of these results, the developed releasing system seems to be a promising tool for an accelerated delivery of several incorporated excipients.

Liposomes coated with thiolated chitosan enhance oral peptide delivery to rats.

The aim of the present study was the in vivo evaluation of thiomer-coated liposomes for an oral application of peptides. For this purpose, salmon calcitonin was chosen as a model drug and encapsulated within liposomes. Subsequently, the drug loaded liposomes were coated with either chitosan-thioglycolic acid (CS-TGA) or an S-protected version of the same polymer (CS-TGA-MNA), leading to an increase in the particle size of about 500 nm and an increase in the zeta potential from approximately -40 mV to a maximum value of about +44 mV, depending on the polymer. Coated liposomes were demonstrated to effectively penetrate the intestinal mucus layer where they came in close contact with the underlying epithelium. To investigate the permeation enhancing properties of the coated liposomes ex vivo, we monitored the transport of fluoresceinisothiocyanate-labeled salmon calcitonin (FITC-sCT) through rat small intestine. Liposomes coated with CS-TGA-MNA showed the highest effect, leading to a 3.8-fold increase in the uptake of FITC-sCT versus the buffer control. In vivo evaluation of the different formulations was carried out by the oral application of 40 µg of sCT per rat, either encapsulated within uncoated liposomes, CS-TGA-coated liposomes or CS-TGA-MNA-coated liposomes, or given as a solution serving as negative control. The blood calcium level was monitored over a time period of 24h. The highest reduction in the blood calcium level, to a minimum of 65% of the initial value after 6h, was achieved for CS-TGA-MNA-coated liposomes. Comparing the areas above curves (AAC) of the blood calcium levels, CS-TGA-MNA-coated liposomes led to an 8.2-fold increase compared to the free sCT solution if applied orally in the same concentration. According to these results, liposomes coated with S-protected thiomers have demonstrated to be highly valuable carriers for enhancing the oral bioavailability of salmon calcitonin.

Thiomer-coated liposomes harbor permeation enhancing and efflux pump inhibitory properties.

An ideal oral drug carrier should facilitate drug delivery to the gastrointestinal tract and its absorption into the systemic circulation. To meet these requirements, we developed a thiomer-coated liposomal delivery system composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and a maleimide-functionalized lipid, to which chitosan-thioglycolic acid (CS-TGA) was covalently coupled. In addition to conventional 77 kDa CS-TGA (CS-TGA77), we tested the 150 kDa homologue (CS-TGA150) as well as an S-protected version of this polymer (CS-TGA150-MNA), in which some of the free SH-groups are conjugated with 6-mercaptonicotinamide to protect them from oxidation. Coupling of CS-TGA to the liposomal surface led to an increase in the particle size of at least 150 nm and an increase in the zeta potential from approximately -33 mV to a maximum of about +36 mV, depending on the polymer. As revealed by fluorescence dequenching the formulations have a storage stability of at least two weeks without releasing any encapsulated compounds. In simulated gastric fluid, the system was shown to be stable over 24 h, while in simulated intestinal fluid, a slow, sustained release of encapsulated compounds was observed. According to our experiments, thiomer-coated liposomes did not induce immunogenic reactions after an oral administration to mice. To evaluate the permeation enhancing and efflux pump inhibiting properties of CS-TGA coated liposomes we monitored the transport of fluoresceinisothiocyanate-dextran (FD(4)) and rhodamine-123 (Rho-123), respectively, through rat small intestine. Permeation studies showed a 2.8-fold higher permeation of FD(4) in the presence of CS-TGA77 coated liposomes and an even 4-fold higher permeation in the presence of CSA-TGA150-MNA coated liposomes. The latter also performed best when we evaluated P-glycoprotein inhibiting properties by monitoring the transport of Rho-123, revealing a 4.2-fold enhancement respective to the buffer control. Taken together, thiomer-coated liposomes were shown to protect encapsulated drugs in the stomach, slowly release them in the small intestine and enhance their absorption through the intestinal tissue by opening tight junctions and inhibiting efflux pumps.

Solubilizing agents in nasal formulations and their effect on ciliary beat frequency.

The purpose of this study was to determine the concentration-dependent effect of selected solubilizers, used in common nasal drug formulations, on ciliary beat frequency (CBF) in human nasal epithelial cell cultures. CBF was measured by a high-speed digital imaging method. Excised ciliated human nasal epithelial cells were incubated for 60min with the solubilizers and determination of the half maximal inhibitory concentration (IC(50)), followed by a reversibility test. LDH test was performed on human nasal epithelial cells with the solubilizing agents. These were applied to nasal epithelial cells in IC(50) values. The following rank order in IC(50) values was obtained for the solubilizers: glycerol>propylene glycol>polyethylene glycol 300>N,N-dimethylacetamide>polyethylene glycol 400>ethanol>ethylendiamindihydrochloride>polyvinylpyrrolidon 25>polyvinylpyrrolidon 90. The highest reversibility of approximately 75% was shown by propylene glycol and polyethylene glycol 300 at a concentration of 30% (v/v). Results from the LDH test showed that N,N-dimethylacetamide displayed the highest cytotoxicity with 5.2% at a concentration of 14.5% (v/v). According to these results, several solubilizers can alter the CBF frequency and thus, have an impact on the nasal mucosa. Therefore, CBF studies with solubilizers used at a concentration relevant for nasal formulations are essential in the design of efficient and most notably safe nasal medicinal products.

Thiolated polyacrylic acid-modified iron oxide nanoparticles for in vitro labeling and MRI of stem cells.

PURPOSE: The purpose of this study was to develop and characterize new surface-modified iron oxide nanoparticles demonstrating the efficiency to be internalized by human endothelial progenitor cells (EPCs) from umbilical cord blood. METHODS: Iron oxide nanoparticles were coated with polyacrylic acid-cysteine (PAA-Cys) by either in situ precipitation or postsynthesis. The nanoparticles were characterized by X-ray powder diffraction. EPCs were labeled with PAA-Cys-modified iron oxide nanoparticles or with uncoated nanoparticles. The relaxivity of uncoated and coated iron oxide nanoparticles as well as EPCs labeled with PAA-Cys-modified iron oxide were determined. RESULTS: Addition of PAA-Cys increased the particle size from 10.4 to 144 and 197 nm, respectively. The X-ray powder diffraction pattern revealed that the particles consist of Fe(3)O(4) with a spinal structure. Postsynthesis coated particles showed a cellular uptake of 85% and 15.26 pg iron/cell. For both types of particles the relaxivity ratio was at least 2-fold higher than that of the gold standard Resovist(®). CONCLUSION: The PAA-Cys coated iron oxide nanoparticles are a promising tool for labeling living cells such as stem cells for diagnostic and therapeutic application in cell-based therapies due to their high relaxivities and their easy uptake by cells.

Development and in vivo bioavailability study of an oral fondaparinux delivery system.

Fondaparinux is an agent of choice for the prevention and initial treatment of venous thromboembolism (VTE) as well as myocardial infarction. Nevertheless, as a negatively charged molecule fondaparinux can pass the intestinal epithelial barrier after oral administration only partially. It was therefore the aim of this study to design a highly efficient small-intestinal-targeted oral delivery system for fondaparinux based on thiolated polycarbophil (PCP-Cys) and glutathione (GSH) combined with sodium decanoate. The formulations were tested in vitro with regard to their release, cytotoxicity profiles and their permeation-enhancing properties across small-intestinal mucosa. For the in vivo study, rats were treated with a single oral dose of fondaparinux gels or mini-tablets (5mg/kg) and the subcutaneous and intravenous groups with a dose of 200µg/kg fondaparinux. The anti-factor Xa activity in the plasma was measured. In the presence of PCP-Cys/GSH/sodium decanoate the uptake of fondaparinux from the intestinal mucosa was 4.1-fold improved. The area under concentration-time curve in rat plasma from 0 to 24h with PCP-Cys/GSH/sodium decanoate gel was 135.3µgmin/ml and 1.3-fold improved with the tablets. C(max) value of mini-tablets was 0.23µg/ml and the absolute bioavailability of 4.4% was 6.2-fold improved, while the control solution was not absorbed orally. PCP-Cys/GSH/sodium decanoate demonstrated potential for increasing the oral bioavailability of the indirect factor Xa inhibitor as an alternative to currently used subcutaneous delivery.

Synthesis, characterization, mucoadhesion and biocompatibility of thiolated carboxymethyl dextran-cysteine conjugate.

This study was aimed at improving the mucoadhesive properties of carboxymethyl dextran by the covalent attachment of cysteine. Mediated by a carbodiimide, l-cysteine was covalently attached to the polymer. The resulting CMD-cysteine conjugate (CMD-(273) conjugate) displayed 273+/-20 micromol thiol groups per gram of polymer (mean+/-S.D.; n=3). Within 2h the viscosity of an aqueous mucus/CMD-(273) conjugate mixture pH 7.4 increased at 37 degrees C by more than 85% compared to a mucus/carboxymethyl dextran mixture indicating enlarged interactions between the mucus and the thiolated polymer. Due to the immobilization of cysteine, the swelling velocity of the polymer was significantly accelerated (p<0.05). In aqueous solutions the CMD-(273) conjugate was capable of forming inter- and/or intramolecular disulfide bonds. Because of this crosslinking process within the polymeric network, the cohesive properties of the conjugate were also improved. Tablets comprising the unmodified polymer disintegrated within 15 min, whereas tablets of the CMD-(273) conjugate remained stable for 160 min (means+/-S.D.; n=3). Results from LDH and MTT assays on Caco-2 cells revealed 4.96+/-0.98% cytotoxicity and 94.1+/-0.9% cell viability for the CMD-(273) conjugate, respectively. Controlled release of model compound from CMD-(273) conjugate tablets was observed over 6h. These findings suggest that CMD-(273) conjugate is a promising novel polymer for drug delivery systems providing improved mucoadhesive and cohesive properties, greater stability and biocompatibility.

Nasal delivery of antisense oligonucleotides: in vitro evaluation of a thiomer/glutathione microparticulate delivery system.

PURPOSE: The aim of this study was to develop a nasal mucoadhesive microparticulate delivery system for phosphorothioate antisense oligonucleotides (PTO-ODNs) utilizing the thiomer technology. METHODS: PTO-ODN microparticles, coated with either the mucoadhesive polymer polycarbophil-cysteine (PCP-Cys) or unmodified PCP and reduced glutathione (GSH) were prepared by the emulsification solvent evaporation technique. Particle size, drug load, decrease in thiol groups on microparticles, swelling properties, release of incorporated PTO-ODN, and mucoadhesive properties were examined. Permeation enhancing effect of the deployed thiomer conjugate was investigated on excised porcine respiratory mucosa of the nasal cavity. RESULTS: Results demonstrated that microparticles were almost of spherical structure displaying particle diameter up to 30 microm. In addition, a controlled drug release of the incorporated PTO-ODN was achieved from these particles. Mucoadhesion studies revealed that thiolated PCP-Cys microparticles display 3-fold higher mucoadhesive properties than the corresponding unthiolated polycarbophil microparticles. The uptake of PTO-ODN, incubated in thiolated polycarbophil and glutathione microparticles, from the nasal mucosa was 2.2-fold improved. CONCLUSIONS: According to these results, the thiolated polycarbophil/reduced GSH microparticles might be a promising formulation for systemic delivery of PTO-ODNs via the nasal route.

In vivo investigation of thiomer-polyvinylpyrrolidon nanoparticles using magnetic resonance imaging.

This study focused on the investigation of the permeation enhancing effects of a stomach targeted, nanoparticulate drug delivery system. The polyacrylic acid-cysteine/polyvinylpyrrolidon nanoparticles were loaded with the magnetic resonance imaging (MRI) contrast agent diethylenetriaminepentaacetic acid gadolinium(III)dihydrogen salt (Gd-DTPA). Average particle size was determined to be 130 nm and the optimum for stability was found to be below a pH of 4.5. In vitro permeation studies were performed on rat gastric mucosa and revealed an eightfold increase in Gd-DTPA uptake when incorporated in the nanoparticles compared to evaluation in the presence of unformulated polyacrylic acid-cysteine. In vivo investigations with rats were performed via the noninvasive MRI method in order to track the nanoparticles way through the gastrointestinal tract. When Gd-DTPA was administered orally as nanoparticulate suspension, an increased MRI signal in the urinary bladder was detected after 34 min, providing evidence for systemic uptake and renal elimination of the contrast agent. As control experiments with Gd-DTPA only or in combination with unformulated polyacrylic acid-cysteine revealed no MRI signal increase at all, the significant permeation enhancing effect could be identified based on the nanoparticulate formulation.

Thiolated polycarbophil as an adjuvant for permeation enhancement in nasal delivery of antisense oligonucleotides.

The purpose of this study was to investigate the effect of thiolated polycarbophil as an adjuvant to enhance the permeation and improve the stability of a phosphorothioate antisense oligonucleotide (PTO-ODN) on the nasal mucosa. Polycarbophil-cysteine (PCP-Cys) was synthesized by the covalent attachment of L-cysteine to the polymeric backbone. Cytotoxicity tests were examined on human nasal epithelial cells from surgery of nasal polyps confirmed by histological studies. Deoxyribonuclease I activity in respiratory region of the porcine nasal cavity was analyzed by an enzymatic assay. The enzymatic degradation of PTO-ODNs on freshly excised porcine nasal mucosa was analyzed and protection of PCP-cysteine toward DNase I degradation was evaluated. Permeation studies were performed in Ussing-type diffusion chambers. PCP-Cys/GSH did not arise a remarkable mortal effect. Porcine respiratory mucosa was shown to possess nuclease activity corresponding to 0.69 Kunitz units/mL. PTO-ODNs were degraded by incubation with nasal mucosa. In the presence of 0.45% thiolated polycarbophil and 0.5% glutathione (GSH), this degradation process could be lowered. In the presence of thiolated polycarbophil and GSH the uptake of PTO-ODNs from the nasal mucosa was 1.7-fold improved. According to these results thiolated polycarbophil/GSH might be a promising excipient for nasal administration of PTO-ODNs.

Characterisation of the barrier caused by luminally secreted gastro-intestinal proteolytic enzymes for two novel cystine-knot microproteins.

It was the aim of this study to evaluate the stability of two novel cystine-knot microproteins (CKM) SE-ET-TP-020 and SE-MC-TR-020 with potential clinical relevance towards luminally secreted proteases of the gastrointestinal tract in order to gain information about their potential for oral administration. Therefore, the stability of the two CKM and the model-drug insulin towards collected porcine gastric and small intestinal juice as well as towards isolated proteolytic enzymes was evaluated under physiological conditions. No intact SE-ET-EP-020 was detected after few seconds of incubation with porcine small intestinal juice. SE-ET-TP-020 was also degraded in porcine gastric juice. Furthermore, SE-ET-TP-020 was extensively degraded by isolated chymotrypsin, trypsin and pepsin. Moreover, it was degraded by elastase. SE-MC-TR-020 was degraded entirely within approximately 2 h when incubated in porcine small intestinal juice, whereas no degradation was observed within a 3 h incubation period with porcine gastric juice. In presence of the isolated proteolytic enzymes, SE-MC-TR-020 was only slightly degraded by trypsin and pepsin, whereas elastase caused no degradation to SE-MC-TR-020 at all. Chymotrypsin was the protease that caused most degradation to SE-MC-TR-020. The model drug insulin was degraded extensively by chymotrypsin, elastase, pepsin and trypsin as well as by porcine gastric and porcine small intestinal juice. In conclusion, a precise characterisation of SE-ET-TP-020 and SE-MC-TR-020 degrading luminally secreted GI enzymes has been made, which is an important and substantial prerequisite for the further optimisation of these CKM.

Correlation of in vitro and in vivo models for the oral absorption of peptide drugs.

The aim of this study was to evaluate two in vitro models, Caco-2 monolayer and rat intestinal mucosa, regarding their linear correlation with in vivo bioavailability data of therapeutic peptide drugs after oral administration in rat and human. Furthermore the impact of molecular mass (Mm) of the according peptides on their permeability was evaluated. Transport experiments with commercially available water soluble peptide drugs were conducted using Caco-2 cell monolayer grown on transwell filter membranes and with freshly excised rat intestinal mucosa mounted in Using type chambers. Apparent permeability coefficients (P (app)) were calculated and compared with in vivo data derived from the literature. It was shown that, besides a few exceptions, the Mm of peptides linearly correlates with permeability across rat intestinal mucosa (R (2) = 0.86; y = -196.22x + 1354.24), with rat oral bioavailability (R (2) = 0.64; y = -401.90x + 1268.86) as well as with human oral bioavailability (R (2) = 0.91; y = -359.43x + 1103.83). Furthermore it was shown that P (app) values of investigated hydrophilic peptides across Caco-2 monolayer displayed lower permeability than across rat intestinal mucosa. A correlation between P (app) values across rat intestinal mucosa and in vivo oral bioavailability in human (R (2) = 0.98; y = 2.11x + 0.34) attests the rat in vitro model to be a very useful prediction model for human oral bioavailability of hydrophilic peptide drugs. Presented correlations encourage the use of the rat in vitro model for the prediction of human oral bioavailabilities of hydrophilic peptide drugs.