Juliane Hombach

Distribution of thiolated mucoadhesive nanoparticles on intestinal mucosa.

It was the aim of the present study to evaluate and compare the distribution of thiolated mucoadhesive anionic poly(acrylic acid) (PAA) and cationic chitosan (CS) nanoparticles on intestinal mucosa. Modifications of these polymers were achieved by conjugation with cysteine (PAA-Cys) and 2-iminothiolane (CS-TBA). Nanoparticles (NP) were prepared by ionic gelation and labelled with the strong hydrophilic fluorescent dye Alexa Fluor 488 (AF 488) and hydrophobic fluorescein diacetate (FDA). Unmodified and modified CS and PAA NP were examined in vitro in terms of their mucoadhesive and mucus penetrating properties on the mucosa of rat small intestine. To investigate the transport of NP across the mucus layer, their diffusion behaviour through natural porcine intestinal mucus was studied through a new diffusion method developed by our group. Lyophilised particles displayed 526 μmol/g (CS) and 513 μmol/g (PAA) of free thiol groups and a zeta potential of 20 mV (CS) and -14 mV for PAA NP. Nanoparticle distribution on rat intestine suggested that mucoadhesion of thiolated NP is higher than the diffusion into the intestinal mucosa. Modified particles displayed more than a 6-fold increase in mucoadhesion compared to unmodified ones. The rank order with regard to mucoadhesion of all particles was: CS-TBA>PAA-Cys>CS>PAA, whereas CS-TBA showed 2-fold higher mucoadhesive properties compared to PAA-Cys NP. Diffusion through intestinal mucus was much higher for unmodified than for thiolated as well as for anionic compared to cationic particles. Overall, it was shown that thiolated particles of both anionic and cationic polymers have improved mucoadhesive properties and could be promising carriers for mucosal drug delivery.

In situ gelling properties of chitosan-thioglycolic acid conjugate in the presence of oxidizing agents

The rheological behaviour of chitosan-thioglycolic acid conjugate (chitosan-TGA) in the presence of four oxidizing agents was investigated. Chitosan-TGA was synthesized via amide bond formation between the primary amino group of chitosan and the carboxylic acid group of thioglycolic acid. The sol-gel phase transition of the polymer was determined by rheological measurements. Moreover, cytotoxicity of the gel in combination with each oxidizing agent was evaluated utilizing LDH and MTT assay. The modified chitosan displayed 1053+/-44 micromol/g thiol groups. Results of rheological studies showed that 1% (m/v) chitosan-TGA without any oxidizing agents became gel within 40 min. In contrast, when the oxidizing agents hydrogen peroxide, sodium periodate, ammonium persulfate and sodium hypochlorite were added, respectively, gelation took place within a few minutes. Within 20 min, hydrogen peroxide having been added in a final concentration of 25.2 nmol/L increased dynamic viscosity of 1% (m/v) chitosan-TGA up to 16,500-fold. This can be explained by the formation of inter- and/or intramolecular disulfide bonds which were indirectly verified via the decrease in thiol groups. Additionally, evidence of an increase in cross-linking of thiolated chitosan as a function of time was provided by frequency sweep measurements. Furthermore, viability of Caco-2 cells having been incubated with chitosan-TGA/oxidizing agent systems assessed by MTT assay was 70-85% and the percentage of LDH release was only in case of the chitosan-TGA/ammonium persulfate system significantly (p<0.05) raising compared to the negative control. According to these results, chitosan-TGA/oxidizing agent combinations might be a promising novel in situ gelling system for various pharmaceutical applications such as a controlled drug release carrier or for tissue engineering.

Development of a mucoadhesive nanoparticulate drug delivery system for a targeted drug release in the bladder.

PURPOSE Purpose of the present study was the development of a mucoadhesive nanoparticulate drug delivery system for local use in intravesical therapy of interstitial cystitis, since only a small fraction of drug actually reaches the affected site by conventional treatment of bladder diseases via systemic administration. METHODS Chitosan-thioglycolic acid (chitosan-TGA) nanoparticles (NP) and unmodified chitosan NP were formed via ionic gelation with tripolyphosphate (TPP). Trimethoprim (TMP) was incorporated during the preparation process of NP. Thereafter, the mucoadhesive properties of NP were determined in porcine urinary bladders and the release of TMP among simulated conditions with artificial urine was evaluated. RESULTS The particles size ranged from 183nm to 266nm with a positive zeta potential of +7 to +13mV. Under optimized conditions the encapsulation efficiency of TMP was 37%. The adhesion of prehydrated chitosan-TGA NP on the urinary bladder mucosa under continuous urine voiding was 14-fold higher in comparison to unmodified chitosan NP. Release studies indicated a more sustained TMP release from covalently cross linked particles in comparison to unmodified chitosan-TPP NP over a period of 3h in artificial urine at 37°C. CONCLUSION Utilizing the method described here, chitosan-TGA NP might be a useful tool for local intravesical drug delivery in the urinary bladder.

Development and In Vitro Evaluation of a Mucoadhesive Vaginal Delivery System for Nystatin

The purpose of the present study was to design and evaluate a novel vaginal delivery system for nystatin based on mucoadhesive polymers. L-Cysteine and cysteamine, respectively, were covalently attached to poly(acrylic acid), and the two different thiolated polymers were evaluated in vitro regarding their swelling behavior, mucoadhesive properties and release behavior. Tablets comprising these thiolated polymers and nystatin demonstrated a high stability in vaginal fluid simulant pH 4.2 and an increase in weight by swelling whereas control tablets comprising unmodified poly(acrylic acid) disintegrated and dissolved. The mucoadhesion time of tablets on freshly excised bovine vaginal mucosa on a rotating cylinder and the total work of adhesion of gels and tablets increased significantly due to the formation of disulfide bonds between the thiolated polymer and cysteine rich subdomaines of the mucus layer. The drug nystatin was released more slowly out of thiomer tablets and gels than out of PAA control tablets and gels. Therefore these thiolated polymers are promising delivery systems for nystatin providing a prolonged residence time and a sustained drug release in vitro under physiological relevant conditions.

In vitro evaluation of natural and methylated cyclodextrins as buccal permeation enhancing system for omeprazole delivery

In this work the enhancing effect of cyclodextrins on the buccal permeation of a hydrophobic model drug, omeprazole was studied. First, the influence of the complexation with cyclodextrins in the absence and in the presence of an alkali agent, L-arginine, on the drug stability was checked at neutral conditions since omeprazole alone is only stable in basic conditions. In vitro transbuccal permeation of omeprazole non-complexed and complexed with beta- and methyl-beta-cyclodextrin and in presence of L-arginine was examined using freshly obtained porcine buccal mucosa. Tissue viability after incubation with sample solutions was assessed using a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) biochemical assay and histological evaluation. The toxicity of the sample solutions on buccal mucosa was evaluated by measuring lactate dehydrogenase activity. The present results show that complexation with cyclodextrins increases drug stability at neutral conditions; furthermore, L-arginine contributed to higher drug stability. Permeation studies indicate an increase on drug permeation in complexed form of 1.1- and 1.7-fold for beta-cyclodextrin and methyl-beta-cyclodextrin, respectively. The presence of L-arginine increases drug permeation 1.4-fold in omeprazole complexed with beta-cyclodextrin and 2.4-fold in the inclusion complex formed with methyl-beta-cyclodextrin. The cell viability of the buccal mucosa after a 3 h incubation period, with all sample solutions, remained around 70% and lactate dehydrogenase assay showed that studied cyclodextrins, even in the presence of an alkali agent are not cytotoxic for porcine buccal mucosa. Histological evaluation of the tissue demonstrated that the buccal epithelium remains viable after 3 h of incubation with sample solutions.

Design and in vitro evaluation of a novel polymeric P-glycoprotein (P-gp) inhibitor.

The aim of the present study was to improve the inhibitory properties of poly(ethylene glycol) (PEG) as excipient in drug delivery systems by covalent attachment of thiol moieties. This was achieved by grafting PEG to polyethylenimine (PEI) and finally thiolation with γ-thiobutyrolatone. Furthermore, the potential of this novel thiolated PEG-g-PEI co-polymer on the transport of rhodamine-123 (Rho-123) as P-gp substrate across freshly excised rat intestinal mucosa was evaluated in Ussing-type chambers. Apparent permeability coefficients (P(app)) were calculated and compared with values gained from experiments with the well-established P-gp inhibitors verapamil, reduced GSH, 6-mercatopurine and vitamin E-TPGS and the structurally similar compounds, myrj 52 and brij 35. The thiolated co-polymer displayed 145.07 ± 1.64 μmol/g of remaining primary amino groups, 84.30 ± 5.43 μmol/g of immobilized thiol groups and 12.74 ± 1.57 μmol/g of disulfide bonds. The approximate molecular mass of the thiolated co-polymer was 16,000 Da. The (1)H-NMR spectrum of PEG-g-PEI co-polymer was characterized by the presence of signal groups of PEG, hexamethylene diisocyanate (HMDI) and PEI substructures. Studies with Caco-2 cells revealed that the thiolated co-polymer shows 6.69 ± 0.27% of cytotoxicity by LDH assay and 93.33 ± 0.07% of cell viability by MTT assay. The thiolated co-polymer in a concentration of 0.5% (w/v) displayed a more pronounced effect on the absorptive transport of Rho-123 (P(app)=15.2 ± 1.0 × 10(-)(6)cm/s) in comparison to reduced GSH, 6-mercatopurine, vitamin E-TPGS, myrj 52 and brij 35. The thiolated co-polymer increased the transport of Rho-123 up to 3.3-fold in comparison to Rho-123 without any inhibitor used as control (P(app)=4.7 ± 0.1 × 10(-)(6)cm/s). The thiolated co-polymer applied in a concentration of 0.1%, 0.25% and 0. 5% (w/v) did not only enhance the absorption but also decreased the secretory transport of Rho-123 resulting in efflux ratios (secretory P(app)/absorptive P(app)) of 1.0, 1.4 and 2.0, respectively. Because of these features the novel thiolated PEG-g-PEI co-polymer seems to exhibit promising properties as novel P-gp inhibitor.

Chitosan solutions and particles: Evaluation of their permeation enhancing potential on MDCK cells used as blood brain barrier model

It was the aim of the present study to investigate the potential of chitosan of different molecular weight in solution and as particles to enhance the transport into the brain. FITC-dextran 4 (FD4) transport with and without chitosans of different molecular weight across MDCK cell monolayers, a model for the blood brain barrier, was compared. In the following particles of chitosan exhibiting the most appropriate molecular weight were prepared and their particle size and stability were evaluated. Furthermore permeation studies, MDCK cell toxicity test and red blood cell lysis test were performed. The rank order for chitosan for permeation enhancement across MDCK cells was determined to be 20 kDa~150 kDa > 400 kDa~600 kDa. Moreover particles showed a higher permeation enhancement than the corresponding solution and the smaller the particles were the higher the permeation of FD4 was. All particles were stable for 72 h. Particles displayed increased MDCK cell toxicity and red blood cell lysis compared to chitosan in solution. The smaller the particles were, the higher their toxicity was. According to these results chitosan particles are more potent in absorption enhancement than chitosan solutions.

Chitosan-N-acetyl cysteine conjugates: in vitro evaluation of permeation enhancing and P-glycoprotein inhibiting properties.

This study evaluated three chitosan-N-acetyl cysteine (CAC) conjugates of increasing molecular mass as a valuable tool to improve the absorption of drugs by assessing its permeation enhancing effect regarding the active P-gp substrate rhodamine-123 in comparison to the trans- and paracellular marker FD 4 both in rat intestine and Caco 2 monolayers. Additional LDH and MTT cytotoxicity tests have attested a non-toxic profile to CAC, which can consequently be seen as a safe and promising novel drug carrier with the ability to enhance drug absorption and to inhibit P-gp efflux transporters.

Mucoadhesive Drug Delivery Systems

The uptake of drugs is often limited by the short contact time between the formulation and the absorption membrane and by a fast washout. Using mucoadhesive polymers, however, the residence time of the dosage form on the mucosa can be significantly increased. In this chapter the composition of the mucus, the different mucoadhesion theories and binding types between mucus and mucoadhesives, mucoadhesion tests and factors influencing mucoadhesion are introduced. Various mucoadhesive polymers are also described and an overview of various mucoadhesive delivery systems is provided.

In vivo evaluation of thiolated poly(acrylic acid) as a drug absorption modulator for MRP2 efflux pump substrates.

Recently, several polymers have been reported to modulate drug absorption by inhibition of intestinal efflux pumps such as multidrug resistance proteins (MRPs) and P-glycoprotein (P-gp). The aim of the present study was to evaluate the efficiency of thiolated poly(acrylic acid) (PAA-Cys) to act as a drug absorption modulator for MRP2 efflux pump substrates in vivo, using sulforhodamine 101 as representative MRP2 substrate. In vitro, the permeation-enhancing effect of unmodified PAA and PAA(250)-Cys(,) displaying 580 micromol free thiol groups per gram polymer, was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to that of the buffer control, the sulforhodamine 101 transport in the presence of 0.5% unmodified PAA(250) and 0.5% (w/v) PAA(250)-Cys was 1.3- and 4.0-fold improved, respectively. In vivo, sulforhodamine 101 solutions containing 4% (w/v) unmodified PAA(250) or 4% (w/v) thiolated PAA(250) were orally given to rats. The PAA(250)-Cys solution increased the area under the plasma concentration-time curve (AUC(0-12)) of sulforhodamine 101 3.8-fold in comparison to control and 2.2-fold in comparison to unmodified PAA(250). This in vivo study revealed that PAA(250)-Cys significantly increased the oral bioavailability of MRP2 substrate sulforhodamine 101.