Biomimickry of UPEC Cytoinvasion: A Novel Concept for Improved Drug Delivery in UTI.

Urinary tract infections (UTIs) are among the most common bacterial infections. In an increasing number of cases, pathogen (multi-)resistance hampers durable treatment success via the standard therapies. On the functional level, the activity of urinary excreted antibiotics is compromized by the efficient tissue colonization mechanism of uropathogenic Escherichia coli (UPEC). Advanced drug delivery systems aim at exploiting a glycan-mediated targeting mechanism, similar to the UPEC invasion pathway, to increase bioavailability. This may be realized by conjugation of intravesically applied drugs or drug carriers to chosen plant lectins. Higher local drug concentrations in or nearby bacterial reservoirs may be gained, with higher chances for complete eradication. In this study, preliminary parameters to clarify the potential of this biorecognitive approach were evaluated. Glycan-triggered interaction cascades and uptake processes of several plant lectins with distinct carbohydrate specificities were characterized, and wheat germ agglutinin (WGA) could be identified as the most promising targeter for crossing the urothelial membrane barrier. In partially differentiated primary cells, intracellular accumulation sites were largely identical for GlcNAc- and Mannose-specific lectins. This indicates that WGA-mediated delivery may also enter host cells via the FimH-dependent uptake pathway.

Glycan-mediated uptake in urothelial primary cells: Perspectives for improved intravesical drug delivery in urinary tract infections.

Urinary tract infections (UTIs) are among the most common bacterial infections. Despite a wide range of therapeutic options, treatment success is compromised by multiresistance and the efficient mechanism of tissue colonization of uropathogenic Escherichia coli (UPEC). In advanced drug delivery systems, a similar, glycan-mediated targeting mechanism may be realized by conjugating the drug to a plant lectin. This may lead to the drug being more efficiently accumulated at the desired site of action, the bacterial reservoirs. In this study, we aimed at elucidating the potential of this biorecognitive approach. Glycan-triggered interaction cascades and uptake processes of several plant lectins with distinct carbohydrate specificities were characterized using single cells and monolayer culture. Due to pronounced cytoadhesive and cytoinvasive properties, wheat germ agglutinin (WGA) emerged as a promising targeter in porcine urothelial primary cells. The lectin-cell interaction proved highly stabile in artificial urine, simulating the conditions in actual application. Colocalisation studies with internalized WGA and lens culinaris agglutinin (LCA) revealed that intracellular accumulation sites were largely identical for GlcNAc- and Mannose-specific lectins. This indicates that WGA-mediated delivery may indeed constitute a potent tool to reach bacteria taken up via a FimH-triggered invasion process. Existing pitfalls in intravesical treatment schedules may soon be overcome.

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.

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.