Gerda Ratzinger

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.

Targeted drug delivery : Binding and uptake of plant lectins using human 5637 bladder cancer cells

In an effort to detect novel strategies in bladder cancer therapy, the potential and the applicability of different plant lectins was investigated using 5637 cells as a model for human urinary carcinoma. The cell-lectin interaction studies were performed with single cells as well as monolayers using flow cytometry and fluorimetry. As a result, wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA) revealed strongest interaction with single cells demonstrating a high presence of N-acetyl-d-glucosamine, sialic acid and alpha-l-fucose residues on the membrane surface. Considering monolayers, binding of most lectins depended on the culturing period pointing to a change in the glycocalyx composition during cultivation. However, constant binding capacities combined with a high specificity were detected for WGA. Cytoinvasion studies were performed with WGA and revealed a decreased fluorescence intensity at 37 degrees C as compared to 4 degrees C, which points to internalisation of the lectin and accumulation in acidic compartments. Intracellular localization was confirmed by addition of monensin that compensates the pH-gradient between acidic compartments and cytoplasm leading to a full reversal of the decline in fluorescence. According to these findings, some lectins, especially WGA, offer promising features for targeting drugs to bladder cancer cells. This might be interesting for the development of functionalized drug delivery systems for site specific antitumor therapy leading to reduced toxicity, prolonged exposition, and improved efficacy.

An acoustically-driven biochip - impact of flow on the cell-association of targeted drug carriers.

The interaction of targeted drug carriers with epithelial and endothelial barriers in vivo is largely determined by the dynamics of the body fluids. To simulate these conditions in binding assays, a fully biocompatible in vitro model was developed which can accurately mimic a wide range of physiological flow conditions on a thumbnail-format cell-chip. This acoustically-driven microfluidic system was used to study the interaction characteristics of protein-coated particles with cells. Poly(D,L-lactide-co-glycolide) (PLGA) microparticles (2.9 +/- 1 microm) were conjugated with wheat germ agglutinin (WGA-MP, cytoadhesive protein) or bovine serum albumin (BSA-MP, non-specific protein) and their binding to epithelial cell monolayers was investigated under stationary and flow conditions. While mean numbers of 1500 +/- 307 mm(-2) WGA-MP and 94 +/- 64 mm(-2) BSA-MP respectively were detected to be cell-bound in the stationary setup, incubation at increasing flow velocities increasingly antagonized the attachment of both types of surface-modified particles. However, while binding of BSA-MP was totally inhibited by flow, grafting with WGA resulted in a pronounced anchoring effect. This was indicated by a mean number of 747 +/- 241 mm(-2) and 104 +/- 44 mm(-2) attached particles at shear rates of 0.2 s(-1) and 1 s(-1) respectively. Due to the compactness of the fluidic chip which favours parallelization, this setup represents a highly promising approach towards a screening platform for the performance of drug delivery vehicles under physiological flow conditions. In this regard, the flow-chip is expected to provide substantial information for the successful design and development of targeted micro- and nanoparticulate drug carrier systems.

Improving Oral Delivery

It is estimated that 90% of all medicines are oral formulations and their market share is still increasing, due to sound advantages for the patient, the pharmaceutical industry and healthcare systems. Considering biopharmaceutical issues such as physicochemical requirements of the drug and physiological conditions, however, oral delivery is one of the most challenging routes. Recognising solubility, permeability and residence time in the gastrointestinal milieu as key parameters, different characteristics of drugs and their delivery systems such as size, pH, density, diffusion, swelling, adhesion, degradation and permeability can be adjusted to improve oral delivery. Future developments will focus on further improvement in patient compliance as well as the feasibility of administering biotech drugs via the oral route.

Surface modification of PLGA particles: the interplay between stabilizer, ligand size, and hydrophobic interactions.

Therapeutic and diagnostic carriers can be functionalized with active targeters to induce tissue-specific delivery. However, the possible impact of adsorbed steric stabilizer such as the frequently used poloxamers (Pluronics) on surface modification of poly(D,L-lactide-co-glycolide) (PLGA) particles has not been examined so far. Therefore, three model ligands of different molecular weights (653; 36,000; 155,000 g/mol) covering the size range of important targeters were conjugated to the surface of PLGA microparticles in the presence of different concentrations of Pluronic F68 (0.01-5%, w/v). Flow cytometry and fluorimetric quantification revealed for all tested ligands that high Pluronic concentrations decreased the coupling efficiency to a half or even one-third of that achieved in the absence of stabilizer. Moreover, the reduction strongly depends on the ligand size and its propensity for hydrophobic interactions. Apart from that, a high degree of particle aggregation was observed with Pluronic concentrations below 0.1% (w/v). Thus, a compromise has to be found, which combines sufficient stability with the best possible ligand coupling efficiency. For the studied system, 0.1% (w/v) turned out to be the optimum concentration of Pluronic F68.

Targeted PLGA microparticles as a novel concept for treatment of lactose intolerance.

BACKGROUND Peroral β-galactosidase preparations for the management of lactose intolerance need to be administered in large doses (1500 to 6000 U) immediately before or together with a lactose-containing meal. AIM Therefore, this work aimed at developing an innovative long-acting formulation. For this purpose, biodegradable polymeric microcarriers were functionalized with β-galactosidase and targeted with wheat germ agglutinin (WGA) for bioadhesion and thus prolonged residence time in the small intestine. METHODS Spray-dried poly(D,L-lactide-co-glycolide) (PLGA) particles with 2.78±1.05µm in diameter were functionalized with β-galactosidase from Kluyveromyces lactis and WGA using different types of spacers (polyethyleneimine, hexamethylene diamine, 6-aminocaproic acid) and coupling methods (carbodiimide and glutaraldehyde). The particle-bound enzyme activity was determined, and the bioadhesive characteristics were assessed by interaction with mucin coatings and Caco-2 cell monolayers. RESULTS Up to 1470 U β-galactosidase per gram PLGA were immobilized. The best results were obtained with hexamethylene diamine as a spacer applying the carbodiimide method. Thereby, a nearly 6-fold increase in enzyme activity was obtained as compared to particles without spacer. Upon targeting with WGA, binding to artificial human intestinal epithelium was increased considerably. CONCLUSIONS For the delivery of β-galactosidase WGA-targeted PLGA microparticles were prepared, which represent promising candidates for a convenient biomimetic treatment regimen of lactose intolerance.

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.

Characterization of binding and uptake of biomimetic nanoparticles by flow cytometry

To establish a method for characterization of the nanoparticle-cell interaction without labeling, the changes in granularity of Caco-2 cells due to association of biomimetic nanoparticles, consisting of PLGA and wheat germ agglutinin (WGA), were monitored by side scattering using flow cytometry. The reliability was confirmed by a temperature-dependent linear correlation between side scatter data and cell-associated fluorescence intensities of cells loaded with fluorescent biomimetic nanoparticles. Moreover, not only the binding to but also the uptake of the nanoparticles into the cells was detected. Whereas the cell-binding of nanoparticles with lowest WGA-densities was controlled by temperature-dependent diffusional processes, the cytoinvasive properties of the WGA-corona prevailed at higher lectin densities. Due to the multiparametric character of the flow cytometric analysis useful additional information on the viability and agglomeration of cells is provided at the same time.

105 M-Cell Targeting by Neuraminidase Functionalized Microparticles for Future Application in Oral Immunotherapy.

Background Recently, we demonstrated in an experimental mouse study that mucosal M-cell targeting with Aleuria aurantia lectin (AAL) coated Poly(D,L-lactide-co-glycolide) (PLGA) microspheres represents a promising oral treatment approach in IgE mediated allergy. Due to its structural similarities with AAL we aimed to assess Neuraminidase (NA) from Vibrio cholerae as a novel M-cell specific targeters and compared its properties to AAL and wheat germ agglutinin (WGA) representing 2 plant lectins, which target either M-cells or epithelial cells, respectively. Methods The resistance against gastric digestion of NA, AAL and WGA was analyzed in simulated gastric fluid (SGF) experiments. Intestinal epithelial binding was determined using the colon carcinoma cell line Caco2, which represents a well established model for the human intestinal epithelium. Binding specificity was evaluated by inhibition experiments by incubating Caco2 cells with Biotin-labeled NA, AAL or WGA, after preincubation with a-L fucose, monoganglioside (GM1) or N,N′,N″-triacetyl-chitotriose (TCT). The stimulatory effects of the targeting substances on the intestinal microenvironment were investigated by cytokine read-out experiments in real-time PCR. Further, the transeptihelial uptake of NA-, AAL- or WGA-functionalized fluospheres was evaluated in a human M-cell co-culture model. Results All 3 targeters were stable up to 180 minutes in SGF, indicating their suitability for oral application. The binding partners were a-L fucose for AAL and TCT for WGA, whereas NA interacts with intestinal epithelial cells via a-L fucose and additionally GM1. NA skewed the cytokine production by inducing a 2-fold increase of the Th1 cytokine IFNg after 60 minutes, whereas AAL decreased the overall cytokine expression. In a human M-cell co-culture model, a higher transepithelial transport rate of fluospheres coated with NA and AAL was observed as compared to WGA and plain particles. Conclusions NA specifically targets M-cells via a-L fucose and additionally GM1 and, thus, increases the transepithelial transport of NA coated particles. Due to the immunomodulatory capacity on intestinal epithelial cells, NA functionalized microspheres may represent a promising M-cell specific targeting approach for oral immunotherapy.

Targeted PLGA-Microparticles as a Novel Concept for Treatment of Lactose Intolerance

Background: Lactose intolerance is the inability to metabolize lactose because of the absence of the enzyme lactase. It is estimated that 75–90% of birth lactase levels are lost by most people after weaning. [...]