Franz Gabor

Gantrez® AN as a new polymer for the preparation of ligand-nanoparticle conjugates

The aim of this study was to evaluate the feasibility and in vitro activity of ligand-conjugates based on the use of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA or Gantrez AN). Fluorescently labelled PVM/MA nanoparticles were prepared by desolvation and cross-linkage with 1,3-diaminopropane (DP). Conjugates were obtained by incubation between the carriers and Sambucus nigra agglutinin (SNA) for 1 h in an aqueous medium. The lectin binding to the surface of nanoparticles was increased by both increasing the bulk ligand concentration and decreasing the amount of cross-linker. However, a concentration of about 0.3-0.4 mg DP per mg polymer was necessary to obtain maximum agglutination activity. Under optimal conditions, the amount of fixed ligand was 46 microg/mg nanoparticle (binding efficiency of 86%); although the activity of SNA conjugates was 13.3 microg/mg particle. The activity of nanoparticles, measured by the association to Caco-2 monolayers, was higher when SNA was covalently bound onto their surface. The lectin-conjugate interaction was 6-fold higher than conventional nanoparticles. Moreover, energy-dependent mechanisms were only observed in SNA-PVM/MA particles. Finally, the decrease in association in the presence of lactose demonstrates that both SNA- and SNA-conjugate-binding was due to a true lectin-sugar interaction.

Poly(D, L-lactic-co-glycolic acid) microspheres for sustained delivery and stabilization of camptothecin

Camptothecin (CPT) and its water-insoluble derivatives are known as topoisomerase-I inhibitors exhibiting high antitumoral activity against a wide spectrum of human malignancies. Until now clinical application of CPT is restricted by insolubility and instability of the drug in its active lactone form resulting in less antitumor potency and poor bioavailability. For these reasons CPT-loaded-microspheres were prepared by the solvent evaporation method using the H-series of poly(D,L-lactide-co-glycolide) (H-PLGA), which contain more carboxylic acid end chains and hydrate faster than the non-H-series. At 1.2% CPT-payload the drug was molecular dispersed throughout the matrix whereas at higher CPT-payload the amount of crystalline CPT-islets increased with the CPT content. The release pattern of CPT was biphasic comprising a first burst effect delivering 20-35% of the payload and increasing with drug-loading. This phase was followed by sustained delivery of CPT releasing 40-75% of the payload within 160 h. In comparison to PLGA-microspheres, the CPT-release rate from H-PLGA was twofold higher and accelerated. The active CPT-lactone was maintained during preparation, storage and release due to hindered diffusion of acidic oligomers among other mechanisms. Thus stabilization and sustained release of CPT from PLGA-microspheres might reduce local toxicity combined with prolonged efficacy offering new perspectives in CPT chemotherapy.

Lectin-mediated bioadhesion : binding characteristics of plant lectins on the enterocyte-like cell lines Caco-2, HT-29 and HCT-8

In order to take advantage of the biorecognition between lectins and specific carbohydrates for targeted drug delivery, fluorescein-labelled lectins of different carbohydrate specificities were screened for binding to human colorectal carcinoma cell lines by flow cytometry and confocal microscopy. The lectin-binding rate increased as follows: Dolichos biflorus agglutinin, DBA<peanut agglutinin, PNA<Lens culinaris agglutinin, LCA<Solanum tuberosum lectin, STL<Ulex europaeus isoagglutinin I, UEA-I<wheat germ agglutinin, WGA (Caco-2); PNA<UEA-I<WGA (HT-29); DBA<UEA-I<WGA (HCT-8), thus reflecting the glycosylation pattern of the cells. Compared to the BSA-binding capacity of the cells, the extent of nonspecific binding was strongly dependent on the type of cell line and lectin under investigation being lower than 2% in the case of WGA, STL and UEA-I/Caco-2 and HT-29 cells. Whereas 50% of DBA was bound nonspecifically to Caco-2 cells, the interactions DBA/HCT-8 and PNA/HT-29 were due to nonspecific binding. By competitive inhibition of lectin-adhesion to the cells upon addition of the complementary carbohydrate, specificity of lectin-binding was confirmed except for the interaction of DBA/HCT-8 and PNA/HT-29. Following on from this work, we consider WGA-, STL- and UEA-I-mediated drug delivery to be a promising approach for peroral bioadhesive formulations adhering to absorptive enterocytes.

Lectin-mediated drug targeting: preparation, binding characteristics, and antiproliferative activity of wheat germ agglutinin conjugated doxorubicin on Caco-2 cells.

AbstractPurpose. To investigate the usefulness of wheat germ agglutinin as a targeting carrier protein for an acid-labile chemotherapeutic prodrug directed against colon carcinoma cells in vitro. Methods. Cis-aconityl-linked doxorubicin-wheat germ agglutinin was prepared by a two step procedure and the conjugate-binding capacity of target- and non-target cells was assayed by flow cytometry. The antiproliferative activity of the prodrug on Caco-2 and MOLT-4 cells was determined by the XTT- and BrdU-test and compared with that of the parent drug and the lectin alone. Results. At pH 4.0, about 50% of the conjugated doxorubicin were released within 24 h from the water soluble prodrug exhibiting a conjugation number of 24 (mol doxorubicin/mol WGA). The prodrug-binding capacity of colon carcinoma cells exceeded that of human colonocytes and lymphoblastic MOLT-4 cells 4.5-fold. Additionally, the antiproliferative effect of the conjugate on Caco-2 cells was 39% as opposed to 5% in case of MOLT-4 cells. As the unmodified carrier protein inhibited or stimulated Caco-2 cell growth in a concentration-dependent manner, the cytostatic activity of the conjugate was determined at WGA concentrations without an effect on cell-proliferation. Considering 50% release of conjugated drug at the most, the prodrug yielded 160% of the cytostatic activity of free doxorubicin. Conclusions. WGA-prodrug targeting offers new perspectives for site-specific, cytoinvading drug delivery in colon cancer chemotherapy.

Allergen‐loaded biodegradable poly(d,l‐lactic‐co‐glycolic) acid nanoparticles down‐regulate an ongoing Th2 response in the BALB/c mouse model

Background and objective Biocompatible and biodegradable microparticles have gained interest as antigen delivery systems during the recent years. We investigated whether biodegradable poly(d,l‐lactic‐co‐glycolic) acid (PLGA) nanospheres could be used as allergen vehicles for few‐shot therapy of type I allergy.

Stabilisation and determination of the biological activity of l-asparaginase in poly(d,l-lactide-co-glycolide) nanospheres

The preservation of biological activity of protein drugs in formulations is still a major challenge for successful drug delivery. The enzyme L-asparaginase, which exhibits a short in vivo half-life and is only active against leukaemia in its tetrameric form, was encapsulated in poly(D,L-lactide-co-glycolide) nanospheres by the (w/o)/w-emulsion solvent evaporation technique in presence of various potential stabilisers. Elucidation of the preparation steps revealed that the enzyme is denaturated at the aqueous/organic interface and by sonication. The preparation of L-asparaginase nanospheres with trehalose, PEG 400, and glycerol as components of the inner aqueous phase yielded colloidal formulations with increased biological activity as determined by an improved protocol for quantification of the active enzyme encapsulated. After lyophilisation the enzyme activity and particle size distribution were retained only by use of Pluronic F68 as a lyoprotectant. Despite the unaltered particle size and improved biological activity, the release profile of the enzyme was strongly altered by coencapsulation of the stabilisers resulting in increased first bursts. In consequence, the biological activity of L-asparaginase during preparation and storage can be improved by combining appropriate additives but concurrently the release profile is influenced.

Lectin-Mediated Bioadhesion: Preparation, Stability and Caco-2 Binding of Wheat Germ Agglutinin-Functionalized Poly(D,L-lactic-co-glycolic acid)-Microspheres

Abstract To take advantage of the cytoadhesive characteristics of Wheat germ agglutinin (WGA) for improved particulate drug delivery, the interaction between WGA-grafted poly(D,L-lactic-co-glycolic acid)-microspheres and Caco-2 monolayers was investigated using bovine serum albumin (BSA) or glycine coated microspheres as a control. Covalent immobilization of WGA by the carbodiimide/N-hydroxysuccinimide-method on 4 μm microspheres yielded a surface density of 9.67 ± 1.21 × 106 molecules/particle, whereas 0.22 ± 0.04 × 106 WGA-molecules were bound by physical adsorption. After storage for 21 days in HEPES-buffer and treatment of the particles with 5 M urea, 86% of covalently linked lectin was still attached to the particles. At 4°C the Caco-2 binding rate of both, WGA- and BSA-modified particles increased with addition of increasing numbers of particles until saturation was reached at 38150 ± 1740 (WGA) or 12066 ± 1195 (BSA) microspheres bound/mm2 Caco-2 monolayer. Inhibition of Caco-2 binding of WGA-functionalized microspheres by chitotriose indicated for specificity of the interaction. As observed by confocal laser scanning microscopy, the fluorescein-loading of the particles was accumulated intracellularly after incubation of Caco-2 monolayers with WGA-modified microspheres contrary to glycine-grafted microspheres. Additionally, in case of WGA-functionalized microspheres the amount of cell associated fluorescein was 200-fold higher than that of the free solution. In conclusion, WGA-modified microspheres are expected to enhance intestinal transport of incorporated drugs due to cytoadhesion provided by the lectin coating.

Lectin-mediated drug delivery: binding and uptake of BSA-WGA conjugates using the Caco-2 model.

To examine whether the dietary lectin wheat germ agglutinin (WGA) can facilitate binding and uptake of protein drugs due to its cytoadhesive and cytoinvasive properties, conjugates were prepared by covalent coupling of fluorescein-labeled bovine serum albumin (F-BSA) to WGA using divinylsulfone for crosslinking. Increasing the molar ratio of F-BSA/WGA resulted in 2.6-8.7 times higher Caco-2 binding as compared with glycyl-F-BSA. About 75% of F-BSA-WGA were bound specifically to Caco-2 cells according to inhibition studies in presence of the complementary carbohydrate. The Caco-2 association of F-BSA-WGA was temperature-dependent indicating active uptake of membrane bound conjugate, which was confirmed by confocal microscopy. The conjugate accumulated within lysosomal compartments followed by proteolytic degradation of F-BSA-WGA 1-4 h after conjugate loading as observed by equilibrating the intracellular pH with monensin. Finally low molecular weight degradation products of the proteinaceous prodrug appear in the extracellular medium. Contrary to Caco-2 single cells, a minor part of the conjugate is degraded by brush border proteases already 30 min after exposure to Caco-2 monolayers. But most of the conjugate is taken up into differentiated cells and processed as in single cells. Though the enzymic barrier remains to be surmounted, WGA-mediated drug delivery is a promising strategy for peroral delivery of even high molecular weight drugs to overcome the mucosal barrier.

Selection of reliable reference genes for qPCR studies on chondroprotective action

BackgroundChondroprotective agents (CPA) such as glucosamine, curcumin and diacerein represent potential remedies for the management of osteoarthritis and several studies have been performed on their effects in-vitro and in-vivo. For the investigation of chondroprotective action on chondrocyte gene expression, quantitative real-time RT-PCR is the method of choice. However, validation of applied normalization strategies represents a crucial and sometimes neglected step in the analysis process. Therefore, the present study aimed to determine the expression stability of common reference genes (ACTB, Beta actin; GAPDH, Glyceraldehyde-3-phosphate; B2M, Beta-2-microglobulin; HPRT1, Hypoxanthine phosphoribosyl-transferase I; SDHA, Succinate dehydrogenase complex, subunit A; YWHAZ, Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) under the influence of glucosamine, curcumin and diacerein in the IL-1β-stimulated C-28/I2 chondrocyte model, using the geNorm software tool.ResultsCPA treatment of C-28/I2 chondrocytes significantly affected the expression level of many reference genes (p < 0.05). According to their expression stability, geNorm analysis revealed rankings of the 3 most stable genes (from most stable to least stable) as follows: GAPDH, B2M and SDHA in glucosamine treated samples and HPRT1, GAPDH and B2M in curcumin or diacerein treated samples. Interestingly, ACTB was one of the most variably expressed genes throughout all experiments.ConclusionOur study points out the problem of relying on commonly used reference genes without an accurate validation process. For normalization purposes in gene profiling studies on glucosamine action, the genes GAPDH, B2M and SDHA are recommended as single reference genes depending on the expression level of the target gene or more favourably in combination. For experiments with curcumin and diacerein the use of HPRT1, GAPDH and B2M should be considered.

Surface modification of PLGA nanospheres with Gd-DTPA and Gd-DOTA for high-relaxivity MRI contrast agents

The preparation of particulate contrast agents for magnetic resonance imaging (MRI) based on biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanocarriers is reported. By spacer-aided covalent surface-grafting of the prominent chelating ligands diethylenetriaminepentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), respectively, up to 236 μg gadolinium per mg PLGA can be immobilized in a stable manner. Due to the localisation at the particle surface, water protons may effectively interact with the gadolinium chelates and the modified particles exhibit high proton relaxivities as confirmed by T1 relaxivities of up to 17.5 mm(-1)s(-1) (25 °C, 1.41 T) in case of Gd-DOTA-functionalized carriers and also supported by NMRD profiles. The obtained values compare favorably with marketed low-molecular weight contrast agents and thus suggest suitability for in vivo use.