Preparation, characterization and application of artificial Caco-2 cell surfaces in the silver nanoparticle enhanced fluorescence technique.
To approach in vivo conditions during real time monitoring of biorecognitive interactions, biomimetic surfaces were prepared by fusion of purified plasma membrane fractions from Caco-2 cells with self-assembled monolayers attached to silver colloid coated standard microplates. Proper orientation and integrity of the membrane coating was confirmed by binding of fluorescein-labelled wheat germ agglutinin (F-WGA) which interacts with certain carbohydrates of the glycocalyx. Additionally, the competition with the complementary carbohydrate decreased the F-WGA binding to 15%. The assay setup offers information about the real time binding kinetics and the affinity of the interaction with the cell membrane excluding interfering events such as internalization and metabolism. As exemplified by F-WGA-binding, the mean velocity of the interaction is 627.07 mFU/s and the working range is 40-240 nM with a detection limit of 1.6 pmol F-WGA. The storage stability of the ready-to-use plates exceeded at least 1 month. The real time monitoring of lectin-prodrug binding to the biomimetic membranes revealed that high conjugation numbers reduces the affinity. The assays are simple and fast one-step reactions without any washing steps as this new technique discriminates between membrane bound and bulk fluorescence. Thus, biomimetic membranes on silver colloid layers represent a versatile tool for high throughput screening at early stages of drug discovery and development.
Lochner
Nina
N
Institute of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstrasse 14, A-1090, Vienna, Austria.
Pittner
Fritz
F
Wirth
Michael
M
Gabor
Franz
F
D018938:Caco-2 Cells; D019793:Fluorescein / Q000138:chemical synthesis / Q000493:pharmacokinetics; D005453:Fluorescence; D006801:Humans; D008567:Membranes, Artificial; D043942:Nanotubes / Q000737:chemistry*; D012834:Silver / Q000737:chemistry* / Q000493:pharmacokinetics*; D014909:Wheat Germ Agglutinins / Q000138:chemical synthesis / Q000493:pharmacokinetics
D008567:Membranes, Artificial; D014909:Wheat Germ Agglutinins; D012834:Silver; D019793:Fluorescein