Characterization of two blood-brain barrier mimicking cell lines: distribution of lectin-binding sites and perspectives for drug delivery.

In the present study plant lectins with distinct sugar specificities were applied to two blood-brain barrier (BBB) mimicking cell lines, namely human ECV304 and porcine brain microvascular endothelial cells PBMEC/C1-2 in order to elucidate their glycosylation pattern and to evaluate the lectin-cell interaction for lectin-mediated targeting. The bioadhesive properties of fluorescein-labeled lectins were investigated with monolayers as well as single cells using fluorimetry and flow cytometry, followed by confirmation of the specificity of binding. For PBMEC/C1-2 layers highest binding capacity was found for wheat germ agglutinin (WGA), followed by Dolichus biflorus agglutinin (DBA) whereas single cell experiments revealed a predominance of DBA only. Analyzing ECV304 monolayers and single cells, WGA yielded the strongest interaction without any changes during cultivation. The binding capacities of the other lectins increased significantly during differentiation. As similar results to primary cells and brain sections were observed, both cell lines seem to be suitable as models for lectin-interaction studies. Thus, an additional focus was set on the mechanisms involved in uptake and intracellular fate of selected lectins. Cytoinvasion studies were performed with WGA for human ECV304 cells and WGA as well as DBA for PBMEC/C1-2 cells. For both lectins, the association rate to the cells was dependent on temperature which indicated cellular uptake.

Alteration of the glycosylation pattern of monocytic THP-1 cells upon differentiation and its impact on lectin-mediated drug delivery.

In the present study, human monocytic THP-1 cells were treated with phorbol-12-myristate-13-acetate (PMA) in order to obtain macrophage-like cells. Before and after treatment, plant lectins with distinct sugar specificities were applied in order to elucidate the glycosylation patterns of both monocytic and macrophage-like cell types and to follow changes during differentiation. As a result of flow-cytometric analyses, for untreated as well as for PMA-differentiated cells WGA yielded the highest binding rate without significant changes in the binding capacity. For the other lectins, divergent results were obtained which point to reorganization of sugar residues on the cell surface during differentiation. Additionally, cytoinvasion being beneficial for enhanced drug absorption was studied with WGA which had displayed a high binding capacity together with a high specificity. For both untreated and PMA-differentiated cells decreased fluorescence intensity at 37 degrees C as compared to 4 degrees C was observable pointing to internalization and accumulation within acidic compartments. Moreover, WGA-functionalized PLGA nanoparticles were prepared, and their uptake evaluated. Uptake rates of 55% in case of PMA-differentiated cells suggested that WGA-grafted drug delivery systems might be an interesting approach for treatment of infectious diseases provoked by parasites, facultative intracellular bacteria, or viruses such as HIV.

Strategies to improve drug delivery in bladder cancer therapy.

Bladder cancer is the ninth most common malignancy in the world featuring very high gender variability in occurrence. Current options for bladder cancer therapy include surgery, immunotherapy, chemotherapy and radiotherapy with a trend towards multimodal treatments. However, successful management remains a challenge for urologists and oncologists because of the high risk for recurrence and progression. Particularly in the field of bladder cancer chemotherapy, efficacy of treatment might be improved by advanced drug delivery strategies aimed at prolonged residence time within the bladder cavity and increased permeability of the bladder wall during intravesical instillation. Moreover, a deeper understanding of the biology of bladder carcinogenesis and malignant progression stimulated the development of a new generation of anticancer drugs for targeted therapies that might result in increased treatment specificity together with lower toxic potential and higher therapeutic indices. This review discusses the available strategies for 'targeted therapy', focusing on molecular targets, and for 'controlled delivery', comprising all other approaches towards improved drug delivery.

Alteration of the glycocalyx of two blood-brain barrier mimicking cell lines is inducible by glioma conditioned media.

Recent studies showed that glioma conditioned medium is able to induce blood-brain barrier properties in in vitro models. In this regard, it was investigated whether glioma conditioned medium can also influence the lectin-binding capacity of blood-brain barrier in vitro models. For the presented study cell lines PBMEC/C1-2 and ECV304 were chosen because it was previously shown that glioma conditioned medium was able to induce specific blood-brain barrier properties in these cell lines. Six different plant lectins (WGA, STL, LCA, UEA-I, DBA, PNA) with distinct sugar specificities were applied in order to elucidate the glycosylation patterns of cell line PBMEC/C1-2 and ECV304. Lectin-binding studies were carried out with monolayers as well as with single cells. In the case of PBMEC/C1-2 monolayers, results showed a significant increase of the binding of lectins WGA, STL, UEA-I, DBA and PNA after application of 25 pmol lectin when cultured in media containing soluble factors derived from glioma cell line C6, whereas the binding capacity for LCA remained similar. For ECV304 monolayers, a significant decrease of WGA, STL and LCA was observable, whereas UEA-I binding increased in comparison to cells grown in the corresponding basal growth medium without soluble C6 factors. Single cell studies showed less significant, but similar changes in the lectin-interactions with the cell surfaces. In conclusion, it was shown that soluble factors derived from glioma cell line C6 can modulate the "glycocalyx" of blood-brain barrier mimicking cell lines.

Lectin binding patterns reflect the phenotypic status of in vitro chondrocyte models.

In vitro studies using chondrocyte cell cultures have increased our understanding of cartilage physiology and the altered chondrocytic cell phenotype in joint diseases. Beside the use of primary cells isolated from cartilage specimens of donors, immortalized chondrocyte cell lines such as C-28/I2 and T/C-28a2 have facilitated reproducible and standardized experiments. Although carbohydrate structures appear of significance for cartilage function, the contribution of the chondrocyte glycocalyx to matrix assembly and alterations of the chondrocyte phenotype is poorly understood. Therefore, the present study aimed to evaluate the glycoprofile of primary human chondrocytes as well as of C-28/I2 and T/C-28a2 cells in culture. First, the chondrocytic phenotype of primary and immortalized cells was assessed using real-time reverse transcriptase polymerase chain reaction, immunofluorescence, and glycosaminoglycans staining. Then, a panel of lectins was selected to probe for a range of oligosaccharide sequences determining specific products of the O-glycosylation and N-glycosylation pathways. We found that differences in the molecular phenotype between primary chondrocytes and the immortalized chondrocyte cell models C-28/I2 and T/C-28a2 are reflected in the glycoprofile of the cells. In this regard, the glycocalyx of immortalized chondrocytes was characterized by reduced levels of high-mannose type and sialic acid-capped N-glycans as well as increased fucosylated O-glycosylation products. In summary, the present report emphasizes the glycophenotype as an integral part of the chondrocyte phenotype and points at a significant role of the glycophenotype in chondrocyte differentiation.

Lectin binding studies on C-28/I2 and T/C-28a2 chondrocytes provide a basis for new tissue engineering and drug delivery perspectives in cartilage research.

The present study was performed to evaluate the applicability of plant lectins as mediators of bioadhesion in cartilage research using human chondrocyte cell lines C-28/I2 and T/C-28a2. The bioadhesive properties of fluorescein-labelled lectins with different carbohydrate specificities were investigated by flow cytometry. Specificity of the lectin-cell interactions was ascertained by competitive inhibition using complementary carbohydrates. As compared to that of other lectins, the interaction between wheat germ agglutinin (WGA) and chondrocytic cells was characterised by remarkable cytoadhesion, adequate binding strength and a high degree of specificity for N-acetyl-glucosamine as contained in hyaluronan chains. We therefore suggest WGA to be a promising candidate for mediating bioadhesion to low-adhesive scaffolds in cartilage tissue engineering. Moreover, the WGA-association rate of C-28/I2 and T/C-28a2 cells was dependent on temperature indicating cellular uptake of membrane-bound WGA. Intracellular enrichment was confirmed by confocal microscopy. Equilibration of intracellular pH gradients with monensin resulted in the reversal of quenching effects indicating accumulation of WGA within acid compartments of chondrocytic cells. Thus, WGA might be internalised into chondrocytes together with hyaluronan via the CD44 receptor-mediated endocytosis pathway and accumulated within lysosomes. This physiological process could represent a feasible pathway to target WGA-functionalised drug delivery devices into chondrocytes.