Energetics of ligand-receptor binding affinity on endothelial cells: An in vitro model.
Targeted therapies represent a challenge in modern medicine. In this contest, we propose a rapid and reliable methodology based on Isothermal Titration Calorimetry (ITC) coupled with confluent cell layers cultured around biocompatible templating microparticles to quantify the number of overexpressing receptors on cell membrane and study the energetics of receptor-ligand binding in near-physiological conditions. In the in vitro model here proposed we used the bEnd3 cell line as brain endothelial cells to mimic the blood brain barrier (BBB) cultured on dextran microbeads ranging from 67μm to 80μm in size (Cytodex) and the primary human umbilical vein cells (HUVEC) for comparison. The revealed affinity between transferrin (Tf) and transferrin receptor (TfR) in both systems is very high, Kd values are in the order of nM. Conversely, the value of TfRs/cell reveals a 100-fold increase in the number of TfRs per bEnd3 cells compared to HUVEC cells. The presented methodology can represent a novel and helpful strategy to identify targets, to address drug design and selectively deliver therapeutics that can cross biological barriers such as the blood brain barrier.
Dipartimento di Farmacia, Università di Napoli Federico II, via Domenico Montesano 49, 80131 Naples, Italy.
Iolanda
I
Fotticchia
Istituto Italiano di Tecnologia (IIT)@CRIB, Largo Barsanti e Matteucci 53, 80125 Naples, Italy.
Daniela
D
Guarnieri
Istituto Italiano di Tecnologia (IIT)@CRIB, Largo Barsanti e Matteucci 53, 80125 Naples, Italy.
Teresa
T
Fotticchia
Istituto Italiano di Tecnologia (IIT)@CRIB, Largo Barsanti e Matteucci 53, 80125 Naples, Italy.
Andrea Patrizia
AP
Falanga
Istituto Italiano di Tecnologia (IIT)@CRIB, Largo Barsanti e Matteucci 53, 80125 Naples, Italy; Centro di Ricerca Interdipartimentale sui Biomateriali (CRIB), Università di Napoli Federico II, Piazzale Tecchio 80, 80125 Naples, Italy. Electronic address: raffaele.vecchione@iit.it.
Raffaele
R
Vecchione
Dipartimento di Farmacia, Università di Napoli Federico II, via Domenico Montesano 49, 80131 Naples, Italy; Centro Interuniversitario di Ricerca per le Neuroscienze, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale, Via Sergio Pansini 5, 80131 Naples, Italy. Electronic address: giancola@unina.it.
Concetta
C
Giancola
Istituto Italiano di Tecnologia (IIT)@CRIB, Largo Barsanti e Matteucci 53, 80125 Naples, Italy; Centro di Ricerca Interdipartimentale sui Biomateriali (CRIB), Università di Napoli Federico II, Piazzale Tecchio 80, 80125 Naples, Italy.
Paolo Antonio
PA
Netti
D000818:Animals; D002151:Calorimetry; D042783:Endothelial Cells / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D008863:Microspheres; D008954:Models, Biological; D011485:Protein Binding; D011990:Receptors, Transferrin / Q000378:metabolism*; D012996:Solutions; D013816:Thermodynamics; D014168:Transferrin / Q000378:metabolism*
D008024:Ligands; D011990:Receptors, Transferrin; D012996:Solutions; D014168:Transferrin
Cell adhesion; Isothermal Titration Calorimetry; Receptor-ligand binding; Thermodynamics; blood brain barrier