Xue-Yan Wang

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.

A multichannel acoustically driven microfluidic chip to study particle-cell interactions

Microfluidic devices have emerged as important tools for experimental physiology. They allow to study the effects of hydrodynamic flow on physiological and pathophysiological processes, e.g., in the circulatory system of the body. Such dynamic in vitro test systems are essential in order to address fundamental problems in drug delivery and targeted imaging, such as the binding of particles to cells under flow. In the present work an acoustically driven microfluidic platform is presented in which four miniature flow channels can be operated in parallel at distinct flow velocities with only slight inter-experimental variations. The device can accommodate various channel architectures and is fully compatible with cell culture as well as microscopy. Moreover, the flow channels can be readily separated from the surface acoustic wave pumps and subsequently channel-associated luminescence, absorbance, and/or fluorescence can be determined with a standard microplate reader. In order to create artificial blood vessels, different coatings were evaluated for the cultivation of endothelial cells in the microchannels. It was found that 0.01% fibronectin is the most suitable coating for growth of endothelial monolayers. Finally, the microfluidic system was used to study the binding of 1 μm polystyrene microspheres to three different types of endothelial cell monolayers (HUVEC, HUVECtert, HMEC-1) at different average shear rates. It demonstrated that average shear rates between 0.5 s(-1) and 2.25 s(-1) exert no significant effect on cytoadhesion of particles to all three types of endothelial monolayers. In conclusion, the multichannel microfluidic platform is a promising device to study the impact of hydrodynamic forces on cell physiology and binding of drug carriers to endothelium.

Lectin-coated PLGA microparticles: Thermoresponsive release and in vitro evidence for enhanced cell interaction

PLGA-microparticles with 4.7 μm in diameter were prepared by the double emulsion technique and loaded with 1.7 μg fluorescein/mg PLGA mimicking a hydrophilic API. In an effort to further elucidate the release and bioadhesive characteristics of lectin-grafted formulations in vitro, the particles were coated with wheat germ agglutinin. The microparticles exhibited thermo-responsive release since no free fluorescein was detected at 4 °C or room temperature. At body temperature, however, more than 80% of the payload was released within 48 h. The adhesion of lectin-grafted particles to Caco-2 monolayers, which were applied as a model for the human intestinal epithelium, exceeded that of plain ones 1.5-fold as also observed by fluorescence microscopy. Furthermore, the amount of model drug bound and taken up into the cells was 5.8-fold higher after incubation for 4 h at 37 °C as compared to fluorescein in solution. According to fluorescence imaging a considerable amount of the total fluorescein payload was accumulated intracellularily after incubation for 5 h at 37 °C. These findings not only confirm the utility of bioadhesives of the second generation for improved absorption of low molecular weight hydrophilic compounds but also indicate storage stability of such suspensions at 4 °C and room temperature without any premature loss of API.

A novel cell-based microfluidic multichannel setup, impact of hydrodynamics and surface characteristics on the bioadhesion of polystyrene microspheres

Carboxylated polystyrene microspheres with 1 μm in diameter were surface-modified either by coating with poly(ethyleneimine) (PEI) as cationic polyelectrolyte leading to a conversion of the surface charge from negative to positive, or by covalent immobilization of wheat germ agglutinin (WGA) via a carbodiimide method to obtain a carbohydrate specific biorecognitive surface. To characterize the impact of the binding mechanism on the particle-cell interaction, the binding efficiencies to Caco-2 cells were investigated for both, the biorecognitive WGA-grafted particles and the positively charged PEI-microspheres, and compared to the unmodified negatively charged polystyrene particles. As a result, WGA-grafted particles exhibited the highest binding rates to single cells as well as monolayers as compared to positive and negative particles under stationary conditions. Concerning ionic interactions, PEI-coated particles suffered from a critical agglomeration tendency leading to a high variance in cell binding. Furthermore, in order to elucidate the bioadhesive properties under flow conditions, an acoustically-driven microfluidic multichannel system was applied. Using different setups, it could be demonstrated that the hydrodynamics exerted almost no impact on cell-bound particles with a size of 1 μm at a flow velocity of 2000 μm s(-1). Using this novel microfluidic system, it was thus possible to prove that the omnipresent hydrodynamic drag in vivo is mostly negligible for microparticulate drug delivery systems in the size range of 1 μm or below.

Determination of the glycosylation-pattern of the middle ear mucosa in guinea pigs

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Lectin-Grafted PLGA Microcarriers Loaded with Fluorescent Model Drugs: Characteristics, Release Profiles, and Cytoadhesion Studies.

PLGA microparticles loaded with three different fluorescent model drugs, fluorescein sodium (hydrophilic), sulforhodamine (amphoteric), and boron-dipyrromethene (BODIPY® 493/503, lipophilic), were prepared by the solvent evaporation technique. Due to varying hydrophilicity, the diameters of the microparticles ranged between 4.1 and 4.7 μm. According to fluorimetric analysis, the loading varied from 0.06 to 2.25 μg of the model drug per mg PLGA. In terms of the release profile, the fluorescein sodium-entrapped formulation exhibited thermo-responsive release kinetics. In the case of sulforhodamine- and BODIPY® 493/503-loaded particles, almost no release was observed, neither at 4°C nor 37°C during the first 50 hours. Furthermore, to estimate the bioadhesive properties of such drug delivery systems, the surface of the loaded particles was grafted with wheat germ agglutinin by applying the carbodiimide method. Cytoadhesion studies with Caco-2 monolayers revealed an up to 1.9-fold and 3.6-fold increase in the bioadhesion of the lectin-functionalized, model drug-loaded particles as compared to the albumin- and non-grafted microcarriers, respectively. All in all, the results clearly indicated that the lipophilicity of the polymer matching that of the drug favored entrapment, whereas mismatching impeded loading into the PLGA-microparticles. Even in the case of low loading, these delivery systems might be useful for the fluorescent detections and microscopic imaging of cellular interactions due to their fluorescent properties and lack of dye leakage. Moreover, lectin grafting can mediate bioadhesive properties to such particulate drug carriers which could be a promising approach to improve drug delivery.

Characterization of Vibrio cholerae neuraminidase as an immunomodulator for novel formulation of oral allergy immunotherapy

To improve current mucosal allergen immunotherapy Vibrio cholerae neuraminidase (NA) was evaluated as a novel epithelial targeting molecule for functionalization of allergen-loaded, poly(D,L-lactide-co-glycolide) (PLGA) microparticles (MPs) and compared to the previously described epithelial targeting lectins wheat germ agglutinin (WGA) and Aleuria aurantia lectin (AAL). All targeters revealed binding to Caco-2 cells, but only NA had high binding specificity to α-L fucose and monosialoganglioside-1. An increased transepithelial uptake was found for NA-MPs in a M-cell co-culture model. NA and NA-MPs induced high levels of IFN-γ and IL10 in naive mouse splenocytes and CCL20 expression in Caco-2. Repeated oral gavage of NA-MPs resulted in a modulated, allergen-specific immune response. In conclusion, NA has enhanced M-cell specificity compared to the other targeters. NA functionalized MPs induce a Th1 and T-regulatory driven immune response and avoid allergy effector cell activation. Therefore, it is a promising novel, orally applied formula for allergy therapy.

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. [...]