Brigitta Loretz

Nanoparticles made from novel starch derivatives for transdermal drug delivery

The goal of this paper was aimed to the formulation of nanoparticles by using two different propyl-starch derivatives - referred to as PS-1 and PS-1.45 - with high degrees of substitution: 1.05 and 1.45 respectively. A simple o/w emulsion diffusion technique, avoiding the use of hazardous solvents such as dichloromethane or dimethyl sulfoxide, was chosen to formulate nanoparticles with both polymers, producing the PS-1 and PS-1.45 nanoparticles. Once the nanoparticles were prepared, a deep physicochemical characterization was carried out, including the evaluation of nanoparticles stability and applicability for lyophilization. Depending on this information, rules on the formation of PS-1 and PS-1.45 nanoparticles could be developed. Encapsulation and release properties of these nanoparticles were studied, showing high encapsulation efficiency for three tested drugs (flufenamic acid, testosterone and caffeine); in addition a close to linear release profile was observed for hydrophobic drugs with a null initial burst effect. Finally, the potential use of these nanoparticles as transdermal drug delivery systems was also tested, displaying a clear enhancer effect for flufenamic acid.

Telomerase as an emerging target to fight cancer: Opportunities and challenges for nanomedicine

Telomerase as an enzyme is responsible for the renewal of the chromosomal ends, the so-called telomeres. By preventing them from shortening with each cell cycle, telomerase is able to inhibit cellular senescence and apoptosis. Telomerase activity, which is detectable in the majority of cancer cells, allows them to maintain their proliferative capacity. The thus obtained immortality of those cells again is a key to their malignancy. Based on these discoveries, it is obvious that telomerase inhibitors would represent an innovative approach to fight cancer, and a variety of such candidate molecules are currently in the pipeline. Telomerase inhibitors largely fall in two classes of compounds: small synthetic molecules and nucleotide-based biologicals. For several candidates, some proof of concept studies have been demonstrated, either on cell cultures or in animal models. But the same studies also revealed that inefficient delivery is largely limiting the translational step into the clinic. The most appealing feature of telomerase inhibitors, which distinguishes them from conventional anticancer drugs, is probably seen in their intrinsic non-toxicity to normal cells. Nevertheless, efficient delivery to the target cells, i.e. to the tumor, is still required. Here, some well-known biopharmaceutical problems such as insufficient solubility, permeability or even metabolic stability are frequently encountered. To address these challenges, there is a clear need for adequate delivery technologies, for example by using nanomedicines, that would allow to overcome their biopharmaceutical shortcomings and to warrant a sufficient bioavailability at the target side. This review first briefly explains the concept of telomerase and telomerase inhibition in cancer therapy. It secondly aims to provide an overview of the different currently known telomerase inhibitors. Finally, the biopharmaceutical limitations of these molecules are discussed as well as the possibilities to overcome those limits by novel drug carrier systems and formulation approaches.

In vitro evaluation of chitosan-EDTA conjugate polyplexes as a nanoparticulate gene delivery system

It was the purpose of this study to evaluate the potential of different molecular-weight chitosan-EDTA conjugates as a carrier matrix for nanoparticulate gene delivery systems. Covalent binding of EDTA to more than one chitosan chain provides a cross-linked polymer that is anticipated to produce stabilized particles. pDNA/chitosan-EDTA particles, generated via coazervation, were characterized in size and zeta potential by electrophoretic light scattering and electron microscopy. Stability was investigated at different pH values by enzymatic degradation and subsequent gel retardation assay. Lactate dehydrogenase assay was performed to determine toxicity. Furthermore, transfection efficiency into Caco-2 cells was assessed using a beta-galactosidase reporter gene. Chitosan-EDTA produced from low-viscous chitosan with 68% amino groups being modified by the covalent attachment of EDTA showed the highest complexing efficacy resulting in nanoparticles of 43 nm mean size and exhibiting a zeta potential of +6.3 mV. These particles were more stable at pH 8 than chitosan control particles. The cytotoxicity of chitosan-EDTA particles was below 1% over a time period of 4 hours. These new nanoplexes showed 35% improved in vitro transfection efficiency compared with unmodified chitosan nanoparticles. According to these results, the chitosan-EDTA conjugate may be a promising polymer for gene transfer.

Oral gene delivery: Strategies to improve stability of pDNA towards intestinal digestion

Purpose: Gastrointestinal (GI) nucleases are responsible for a rapid presystemic degradation of orally administered transgenes. Within the current study, the activity of these degrading enzymes as well as the effect of various nuclease inhibitors on the degradation process were evaluated in order to assess their potential as auxiliary agents in oral gene delivery. Methods: Digestion assays of pDNA with DNaseI and in GI juices were performed in absence and presence of inhibitors. Consequently, a chitosan conjugate with covalently bound ethylendiaminetetraacetic acid disodium salt dihydrat (EDTA) was synthesized and its nuclease inhibitory properties were evaluated. Results: Small intestinal juice was shown to possess a nuclease activity per millilitre corresponding to 0.02 Kunitz units of DNaseI. Inhibition studies revealed that inhibitory activity followed the ranking: EDTA > sodium dodecyl sulfate (SDS) > aurintricarboxylic acid (ATA) > poly (acrylic acid) > cysteine. The chitosan–EDTA conjugate offered good nuclease inhibiting properties. Conclusion: This study determined the nuclease activity of native porcine small intestinal juice as well as enterocytes homogenate. Moreover, several promising strategies to overcome this enzymatic barrier were identified.

In vitro cytotoxicity testing of non-thiolated and thiolated chitosan nanoparticles for oral gene delivery

A comparative cytotoxicity study with various chitosan/pDNA nanoparticles was performed in order to evaluate the influence of thiolation, surface charge and size. In particular, the impact of pH changes as encountered along gastrointestinal tract on zeta potential and subsequently toxicity was investigated. For this purpose chitosan and chitosan-N-acetylcysteine nanoparticles of different polymer:pDNA ratios were prepared and characterised by their physicochemical properties. As endpoints to assess cytotoxicity in Caco-2 cells LDH and MTT assay were utilized. The reduction of transepithelial electrical resistance (TEER) induced by nanoparticle treatment was measured and toxic effects on erythrocytes were evaluated to complete the toxicity profile. Size of particles and amount of bound thiol groups slightly affected toxicity. In contrast a high impact and correlation was found for zeta potential and cytotoxicity. While anionic and neutral nanoparticles causeda minor membrane damage and slightly altered mitochondrial activity, cationic nanoparticles caused severe cytotoxic effects. TEER monitoring indicated sub lethal toxicity for neutral nanoparticles by a reversible resistance reduction of up to a third from initial value depending on the concentration of nanoparticles. Cationic particles evinced also drawbacks in erythrocytes assays by causing agglutination. In conclusion, our results showed evidence that zeta potential is the key feature that contributes most to the toxicity of (thiolated) chitosan/DNA nanoparticles.

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

Summary.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 (Papp) 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 (R2 = 0.86; y = −196.22x + 1354.24), with rat oral bioavailability (R2 = 0.64; y = −401.90x + 1268.86) as well as with human oral bioavailability (R2 = 0.91; y = −359.43x + 1103.83). Furthermore it was shown that Papp values of investigated hydrophilic peptides across Caco-2 monolayer displayed lower permeability than across rat intestinal mucosa. A correlation between Papp values across rat intestinal mucosa and in vivo oral bioavailability in human (R2 = 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.

Design of Starch-graft-PEI Polymers: An Effective and Biodegradable Gene Delivery Platform

Starch and starch derivatives are widely utilized pharmaceutical excipients. The concept of this study was to make use of starch as a biodegradable backbone and to modify it with low-toxic, but poor transfecting low molecular weight polyethylenimine (PEI) in order to achieve better transfection efficacy while maintaining enzymatic degradability. A sufficiently controllable conjugation could be achieved via a water-soluble intermediate of oxidized starch and an optimized reaction protocol. Systematic variation of MW fraction of the starch backbone and the amount of cationic side chains (0.8 kDa bPEI) yielded a series of starch-graft-PEI copolymers. Following purification and chemical characterization, nanoscale complexes with plasmid DNA were generated and studied regarding cytotoxicity and transfection efficacy. The optimal starch-graft-PEI polymers consisted of >100 kDa MW starch and contained 30% (wt) of PEI, showing similar transfection levels as 25 kDa bPEI, and being less cytotoxic and enzymatically biodegradable.

A Hydrophobic Starch Polymer for Nanoparticle‐Mediated Delivery of Docetaxel

A hydrophobic starch derivative is used for safe and enhanced delivery of anticancer agents. The synthesis and characterization of propyl starch with a controlled degree of substitution to modulate the release of the encapsulated hydrophobic drug is reported. The application of this polymer for formulating nanoparticles of docetaxel, an anti-cancer agent effective against numerous types of cancers but possessing intrinsic formulation difficulties is described. The solvent emulsification/diffusion technique is used and the synthesis is optimized with respect to several formulation parameters. Uptake studies with these nanoparticles indicate their enhanced internalization by the cancerous cells and their peri-nuclear localization.

Enhanced transport of P-glycoprotein substrate saquinavir in presence of thiolated chitosan

It was the aim of this study to investigate the effect of chitosan-4-thiobutylamidine (Ch-TBA) and reduced glutathione (GSH) on the absorption of P-glycoprotein (P-gp) and multidrug resistance protein (MRP) substrate saquinavir in vitro and in vivo. Bidirectional transport studies were performed with Caco-2 cell monolayers and additionally with freshly excised rat small intestinal mucosa mounted in Ussing type chambers. Furthermore, a delivery system based on Ch-TBA and GSH was evaluated in vivo in rats. The functional activity of the efflux pumps in Caco-2 cells and rat intestinal mucosa during the experiment was proven by the efflux ratio of saquinavir, which was 6.4 for Caco-2 cells and 2.1 for rat intestinal mucosa, respectively. Ch-TBA and particularly the combination of Ch-TBA with GSH enhanced apical (AP) absorption and decreased the secretory transport of saquinavir. In presence of 0.5% Ch-TBA and 0.5% GSH, the uptake of saquinavir was 1.6-fold improved in Caco-2 monolayer and 2.1-fold improved in rat intestinal mucosa. In vivo, the area under the plasma concentration time curve (AUC) of saquinavir was 1.4-fold and Cmax 1.6-fold increased, in comparison with control. Results of this study showed that Ch-TBA in combination with GSH can be an interesting tool for increasing the oral bioavailability of actively secreted compounds.

Pectin‐cysteine conjugate: synthesis and in‐vitro evaluation of its potential for drug delivery

This study was aimed at improving certain properties of pectin by introduction of thiol moieties on the polymer. Thiolated pectin was synthesized by covalent attachment of cysteine. Pectin‐cysteine conjugate was evaluated for its ability to be degraded by pectinolytic enzyme. The toxicity profile of the thiolated polymer in Caco‐2‐cells, its permeation enhancing effect and its mucoadhesive and swelling properties were studied. Moreover insulin‐loaded hydrogel beads of the new polymer were examined for their stability in simulated gastrointestinal conditions and their drug release profile. The new polymer displayed 892.27 ± 68.68 μmol thiol groups immobilized per g polymer, and proved to have retained its biodegradability, upon addition of Pectinex Ultra SPL in‐vitro, determined by viscosity measurements and titration method. Pectin‐cysteine showed no severe toxicity in Caco‐2 cells, as tested by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Moreover, the synthesized polymer exhibited a relative permeation enhancement ratio of 1.61 for sodium fluorescein, compared to unmodified pectin. Pectin‐cysteine conjugate exhibited approximately 5‐fold increased in in‐vitro adhesion duration and significantly improved cohesive properties. Zinc pectin‐cysteine beads showed improved stability in simulated gastrointestinal media; however, insulin release from these beads followed the same profile as unmodified zinc pectinate beads. Due to favourable safety and biodegradability profile, and improved cohesive and permeation‐enhancing properties, pectin‐cysteine might be a promising excipient in various transmucosal drug delivery systems.