Newly-established in vitro inner BRB spheroids to elucidate retinal Ang2-linked substance transfer

• Published in: Journal of controlled release 2022-11, Vol.351, p.8-21
• Main Authors: Yamamoto, Yudai, Akanuma, Shin-ichi, Kon, Hideki, Endo, Hiroki, Kubo, Yoshiyuki, Hosoya, Ken-ichi
• Format: Article
• Language: English
• Subjects: Angiopep-2; Green fluorescent protein; Inner blood-retinal barrier; Spheroids; Tight-junctions; Transporter
• ISSN: 0168-3659; 1873-4995
• DOI: 10.1016/j.jconrel.2022.09.019

Conjugation of angiopep-2 (Ang2) with drugs/compounds is known to increase plasma membrane permeability across endothelial barriers. The inner blood-retinal barrier (BRB) regulates retinal drug distribution and is formed by retinal capillary endothelial cells, supported by Müller cells and retinal pericytes. To elucidate the potential of Ang2 conjugation in promoting retinal drug distribution after peripheral administration across the inner BRB, an in vivo administration study and in vitro transport experiments using newly developed multicellular inner BRB spheroids were performed. After intravenous administration of Ang2-linked green fluorescence protein (GFP-Ang2) in mice, GFP-derived signals were observed in the neural retina. In contrast, GFP-derived signals were not observed after intravenous GFP administration, suggesting the promotion of the retinal distribution of substances by Ang2 conjugation. To overcome the limitations of in vitro studies using cells cultured on dishes, inner BRB spheroids were established using conditionally immortalized rat retinal capillary endothelial cells, Müller cells, and retinal pericytes. Immunocytochemistry of marker molecules suggests that the central part of the spheroids is occupied by Müller cells, and encapsulated by retinal pericytes and capillary endothelial cells. Studies on the expression and functions of tight junctions suggest that tight junctions are formed on the surface of the inner BRB spheroids by retinal capillary endothelial cells. The functional expression of drug transporters, such as P-glycoprotein, was observed in the spheroids, implying that the inner side of the spheroids reflects the retinal side of the inner BRB. In the inner BRB spheroids, energy-dependent accumulation of GFP-Ang2 and Ang2-linked 5(6)-carboxyfluorescein (FAM-Ang2) was observed. Moreover, an endocytic inhibition study revealed that clathrin-dependent endocytosis/transcytosis was involved in the transcellular transport of Ang2-conjugated drugs/compounds across the inner BRB. Consequently, it is suggested that the Ang2 linkage is useful for promoting retinal drug distribution via clathrin-dependent transcytosis at the inner BRB. [Display omitted] •Angiopep-2 conjugation promoted retinal protein distribution.•Spheroids were constructed using inner blood-retinal barrier model cells.•Influx transport of the spheroids indicated blood-to-retina transport.•The spheroids appeared to function as tight junctions and transporters.•Angio