Sarah Dünnhaupt

Chitosan-based drug delivery systems

Within the past 20 years, a considerable amount of work has been published on chitosan and its potential use in drug delivery systems. In contrast to all other polysaccharides having a monograph in a pharmacopeia, chitosan has a cationic character because of its primary amino groups. These primary amino groups are responsible for properties such as controlled drug release, mucoadhesion, in situ gellation, transfection, permeation enhancement, and efflux pump inhibitory properties. Due to chemical modifications, most of these properties can even be further improved. Within this review, an overview on the advantages of chitosan for various types of drug delivery systems is provided.

Preactivated thiomers as mucoadhesive polymers for drug delivery

This study was aimed to synthesize polymeric excipients with improved mucoadhesive, cohesive and in situ-gelling properties to assure a prolonged retention time of dosage forms at a given target site, thereby achieving an increased uptake and improved oral bioavailability of certain challenging therapeutic agents such as peptides and proteins. Accordingly, poly(acrylic acid)-cysteine-2-mercaptonicotinic acid (PAA-cys-2MNA) conjugates were synthesized by the oxidative S–S coupling of PAA-cys (100-, 250- and 450 kDa) with 2-mercaptonicotinic acid (2MNA). Unmodified PAAs, PAAs-cys (thiomers) and PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates were compressed into tablets to perform disintegration tests, mucoadhesion studies and rheological measurements. Moreover, cytotoxicty of the polymers was determined using Caco-2 cells. The resulting PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates displayed 113.5 ± 12.7, 122.7 ± 12.2 and 117.3 ± 4.6 μmol/g of 2-mercaptonicotinic acid, respectively. Due to the immobilization of 2MNA, the PAA-cys-2MNA (pre-activated thiomers) conjugates exhibit comparatively higher swelling properties and disintegration time to the corresponding unmodified and thiolated polymers. On the rotating cylinder, tablets based on PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates displayed 5.0-, 5.4- and 960-fold improved mucoadhesion time in comparison to the corresponding unmodified PAAs. Results achieved from tensile studies were found in good agreement with the results obtained by rotating cylinder method. The apparent viscosity of PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates was improved 1.6-, 2.5- and 206.2-fold, respectively, in comparison to the corresponding unmodified PAAs. Moreover, pre-activated thiomers/mucin mixtures showed a time dependent increase in viscosity up to 24 h, leading to 7.0-, 18.9- and 2678-fold increased viscosity in comparison to unmodified PAAs (100-, 250- and 450 kDa), respectively. All polymers were found non-toxic over Caco-2 cells. Thus, on the basis of achieved results the pre-activated thiomers seem to represent a promising generation of mucoadhesive polymers which are safe to use for prolonged residence time of drug delivery systems to target various mucosa.

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.

Development and Evaluation of a Novel Mucus Diffusion Test System Approved by Self-Nanoemulsifying Drug Delivery Systems

The aim of this study was the development of a novel mucus diffusion model and the approval thereof by self-nanoemulsifying drug delivery systems (SNEDDSs). For diffusion experiments, various SNEDD formulations were developed, spiked with fluorescein diacetate, and evaluated for their mucus diffusion behavior through an intestinal mucus layer within the novel setup. In brief, SNEDD formulations resulting in particle sizes of 12.0 nm produced 70.3% of diffused model drug through the mucus layer. In comparison, SNEDDSs with particle sizes of 455.5 nm led to a permeation of 8.3% only. Apart from this size dependence, two SNEDDS excipients namely Cremophor RH 40 and triacetin were identified to strongly affect the permeation through mucus. Hence, it could be demonstrated that particle size and single excipients can positively influence mucus diffusion of SNEDDSs. Furthermore, it could be shown that the developed mucus diffusion model is a promising tool for pharmaceutical research in comparison with already established systems as it allows an easy handling coupled with the possibility to test different kinds of mucus in parallel within one setup.

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.

S-protected thiolated chitosan for oral delivery of hydrophilic macromolecules: evaluation of permeation enhancing and efflux pump inhibitory properties.

The objective of this study was the investigation of permeation enhancing and P-glycoprotein (P-gp) inhibition effects of a novel thiolated chitosan, the so-named S-protected thiolated chitosan. Mediated by a carbodiimide, increasing amounts of thioglycolic acid (TGA) were covalently bound to chitosan (CS) in the first step of modification. In the second step, these thiol groups of thiolated chitosan were protected by disulfide bond formation with the thiolated aromatic residue 6-mercaptonicotinamide (6-MNA). Mucoadhesive properties of all conjugates were evaluated in vitro on porcine intestinal mucosa based on tensile strength investigations. Permeation enhancing effects were evaluated ex vivo using rat intestinal mucosa and in vitro via Caco-2 cells using the hydrophilic macromolecule FD(4) as the model drug. Caco-2 cells were further used to show P-gp inhibition effects by using Rho-123 as P-gp substrate. Apparent permeability coefficients (P(app)) were calculated and compared to values obtained from each buffer control. Three different thiolated chitosans were generated in the first step of modification, which displayed increasing amounts of covalently attached free thiol groups on the polymer backbone. In the second modification step, more than 50% of these free thiol groups were covalently linked with 6-MNA. Within 3 h of permeation studies on excised rat intestine, P(app) values of all S-protected chitosans were at least 1.3-fold higher compared to those of corresponding thiomers and more than twice as high as that of unmodified chitosan. Additional permeation studies on Caco-2 cells confirmed these results. Because of the chemical modification and higher amount of reactive thiol groups, all S-protected thiolated chitosans exhibit at least 1.4-fold pronounced P-gp inhibition effects in contrast to their corresponding thiomers. These features approve S-protected thiolated chitosan as a promising excipient for various drug delivery systems providing improved permeation enhancing and efflux inhibition effects.

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.

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 24 h. The highest reduction in the blood calcium level, to a minimum of 65% of the initial value after 6 h, 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.

S-protected thiolated chitosan: Synthesis and in vitro characterization

Highlights ► Synthesis of a novel mucoadhesive thiolated chitosan with protected thiol groups. ► The novel conjugate exhibited promising mucoadhesive features. ► In vitro cytotoxicity of the new conjugate was evaluated and found to be non-toxic. ► Swelling behavior of the polymer decreased with the increase of protection. ► Enhanced cross linking within the novel conjugate resulted in improved stability.

In vivo evaluation of an oral drug delivery system for peptides based on S-protected thiolated chitosan.

The aim of the present study was the development and evaluation in vitro as well as in vivo of an oral delivery system based on a novel type of thiolated chitosan, so-called S-protected thiolated chitosan, for the peptide drug antide. The sulfhydryl ligand thioglycolic acid (TGA) was covalently attached to chitosan (CS) in the first step of modification. In the second step, these thiol groups of thiolated chitosan were protected by disulfide bond formation with the thiolated aromatic residue 6-mercaptonicotinamide (6-MNA). Absorptive transport studies of antide were evaluated ex vivo using rat intestinal mucosa. Matrix tablets of each polymer sample were prepared and their effect on the absorption of antide evaluated in vivo in male Sprague-Dawley rats. In addition, tablets were examined in terms of their disintegration, swelling and drug release behavior. The resulting S-protected thiomer (TGA-MNA) exhibited 840μmol of covalently linked 6-MNA per gram thiomer. Based on the implementation of this hydrophobic ligand on the thiolated backbone, the disintegration behavior was reduced greatly and a controlled release of the peptide could be achieved. Furthermore, permeation studies with TGA-MNA on rat intestine revealed a 4.5-fold enhanced absorptive transport of the peptide in comparison to antide in solution. Additional in vivo studies confirmed the potential of this novel conjugate. Oral administration of antide in solution led to only very small detectable quantities in plasma with an absolute and relative bioavailability (BA) of 0.003 and 0.03%, only. In contrast, with antide incorporated in TGA-MNA matrix tablets an absolute and relative BA of 1.4 and 10.9% could be reached, resulting in a 421-fold increased area under the plasma concentration time curve (AUC) compared to the antide solution. According to these results, S-protected thiolated chitosan as oral drug delivery system might be a valuable tool for improving the bioavailability of peptides.