The blood-brain barrier (BBB) hinders the brain delivery of therapeutic immunoglobulin γ (igG) antibodies. Evidence suggests that IgG-specific processing occurs within the endothelium of the BBB, but any influence on transcytosis remains unclear. Here, involvement of the neonatal Fc receptor (FcRn), which mediates IgG recycling and transcytosis in peripheral endothelium, was investigated by evaluating the transcytosis of IgGs with native or reduced FcRn engagement across human induced pluripotent stem cell-derived brain endothelial-like cells. Despite differential trafficking, the permeability of all tested IgGs were comparable and remained constant irrespective of concentration or competition with excess IgG, suggesting IgG transcytosis occurs nonspecifically and originates from fluid-phase endocytosis. Comparison with the receptor-enhanced permeability of transferrin indicates that the phenomena observed for IgG is ubiquitous for most macromolecules. However, increased permeability was observed for macromolecules with biophysical properties known to engage alternative endocytosis mechanisms, highlighting the importance of biophysical characterizations in assessing transcytosis mechanisms.The brain endothelial cells (BECs) that form the main structural component of the blood-brain barrier (BBB) are central to the protection of brain parenchyma. Unique from peripheral endothelium, BECs exhibit substantially reduced permeability of most bloodborne molecules 1 . Accordingly, this restrictive physiology also poses a formidable obstacle in the brain delivery of therapeutic molecules 2 . In particular, conventional immunoglobulin γ (IgG) antibody-based passive immunotherapies, which are structurally and functionally similar to endogenous IgGs, only demonstrate brain uptake of 0.1-0.3% of the injected dose 3,4 . Despite the need to improve the brain delivery of conventional therapeutic IgGs 5 , progress is hindered by the current lack of understanding regarding their interactions with BECs.IgG-endothelium interactions in the periphery are dominated by the neonatal fragment crystallizable (Fc) receptor (FcRn). Following internalization of circulating IgG, the acidic microenvironment of endosomal compartments enables FcRn to bind and recycle IgG back to the lumen in a pH-dependent manner 6 . FcRn-mediated recycling therefore limits lysosomal degradation and contributes to the extended serum half-life of IgGs. However, FcRn can also mediate the abluminal transcytosis of IgG, which is exemplified in the transfer of maternal IgG across the placental endothelium. In this regard, FcRn is a unique transcytosis receptor, e.g. compared to the transferrin receptor (TfR) 7 , as it can shuttle its ligand bidirectionally to either cell surface (i.e. luminal recycling or abluminal transcytosis) 8 . Traditional transcytosis pathways are categorized as fluid-phase (e.g. macropinocytosis) or adsorptive-mediated, which occur via specific (e.g. receptor-mediated transcytosis (RMT)) or nonspecific (e.g. electrostatic adsorption) processes....
Representative in vitro blood-brain barrier (BBB) models can support the development of strategies to efficiently deliver therapeutic drugs to the brain by aiding the characterization of their internalization, trafficking, and subsequent transport across the BBB. A collagen type I (COL1) hydrogel-based in vitro BBB model was developed to enable the simultaneous characterization of drug transport and intracellular processing using confocal microscopy, in a way that traditional insert-based in vitro BBB models cannot. Human induced pluripotent stem cells (hiPSCs) were differentiated into cells that exhibited a BBB-like phenotype on COL1 hydrogels, which included the expression of key BBB-specific proteins and low permeability of representative small and large molecule therapeutics. Furthermore, the BBB phenotype observed on the COL1 hydrogel was similar to that previously reported on porous inserts. The intracellular visualization of two small molecule efflux pump substrates within the hiPSC-derived BBB-like cells demonstrated a difference in cytosolic and vesicular accumulation, which complemented permeability measurements demonstrating a difference in transport rate. The easy-to-construct COL1-based hiPSC-derived BBB model presented here is the first in vitro two-dimensional BBB experimental system that enables the simultaneous quantification of cellular permeability and visualization of intracellular processes by utilizing confocal microscopy, which can provide insights regarding the relationship between transport and intracellular trafficking of therapeutic drugs.
Brain endothelial cells (BECs) hinder macromolecules from reaching brain parenchyma, necessitating the evaluation and engineering of therapeutic immunoglobulin γ (IgG) for improved brain delivery. Emerging fluorescent-based approaches to assess IgG brain exposure can expedite and complement current methods; however, alterations in IgG pharmacokinetics following fluorophore conjugation, which remain unexplained, indicate that conjugation may confound analysis of native IgG processing. Here, changes in transcytosis and intracellular processing of IgG conjugates (with sulfonated cyanine 5) were examined using human induced pluripotent stem cell-derived BECs (iBECs). Above a critical degree of labeling, transcytosis rates increased significantly but could be attenuated by nonspecific protein competition. Concurrent increases in intracellular accumulation, which was not attributable to disrupted binding by the neonatal Fc receptor (FcRn), are indicative of indirect reduction of FcRn-mediated recycling that agrees with reported aberrations in the pharmacokinetics of certain unconjugated IgGs. Overall, these findings support the notion that certain fluorophore−IgG conjugates can engage in adsorptive interactions with cell surface moieties, reminiscent of phenomena exhibited by cationized IgG, and provide in vitro criteria to identify changes in IgG processing following fluorophore conjugation.
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