Summary
It is increasingly apparent that bacteriophages, viruses that infect bacteria and more commonly referred to as simply phages, have tropisms outside their bacterial hosts. Using live tissue culture cell imaging, we demonstrate that cell type, phage size, and morphology play a major role in phage internalization. Uptake was validated under physiological conditions using a microfluidic device. Phages adhered to mammalian tissues, with adherent phages being subsequently internalized by macropinocytosis, with functional phages accumulating intracellularly. We incorporated these results into a pharmacokinetic model demonstrating the potential impact of phage accumulation by cell layers, which represents a potential sink for circulating phages in the body. During phage therapy, high doses of phages are directly administered to a patient in order to treat a bacterial infection, thereby facilitating broad interactions between phages and mammalian cells. Understanding these interactions will have important implications on innate immune responses, phage pharmacokinetics, and the efficacy of phage therapy.
Here,
we have developed and evaluated a microfluidic-based human
blood–brain-barrier (μBBB) platform that models and predicts
brain tissue uptake of small molecule drugs and nanoparticles (NPs)
targeting the central nervous system. By using a photocrosslinkable
copolymer that was prepared from monomers containing benzophenone
and N-hydroxysuccinimide ester functional groups,
we were able to evenly coat and functionalize μBBB chip channels in situ, providing a covalently attached homogenous layer
of extracellular matrix proteins. This novel approach allowed the
coculture of human endothelial cells, pericytes, and astrocytes and
resulted in the formation of a mimic of cerebral endothelium expressing
tight junction markers and efflux proteins, resembling the native
BBB. The permeability coefficients of a number of compounds, including
caffeine, nitrofurantoin, dextran, sucrose, glucose, and alanine,
were measured on our μBBB platform and were found to agree with
reported values. In addition, we successfully visualized the receptor-mediated
uptake and transcytosis of transferrin-functionalized NPs. The BBB-penetrating
NPs were able to target glioma cells cultured in 3D in the brain compartment
of our μBBB. In conclusion, our μBBB was able to accurately
predict the BBB permeability of both small molecule pharmaceuticals
and nanovectors and allowed time-resolved visualization of transcytosis.
Our versatile chip design accommodates different brain disease models
and is expected to be exploited in further BBB studies, aiming at
replacing animal experiments.
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