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. 2017 Sep 4;7:10396. doi: 10.1038/s41598-017-11220-1

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Flow cytometry evaluation of the association between immunoliposomes and BCECs in vitro. (A) BCECs were incubated with either stealth liposomes, isotype IgG, or OX26 immunoliposomes labelled with a fluorophore in the lipid membrane, and the treated cells were analyzed by flow cytometry to evaluate the association. OX26 immunoliposomes had a five-fold higher association compared to the isotype IgG immunoliposomes, whereas stealth liposomes had no association above the background of untreated cells (p < 0.0001). Due to the large difference in association between the two control liposomes, isotype IgG immunoliposomes was chosen as the most relevant control for subsequent experiments. (B) The association between BCECs and OX26 immunoliposomes was further characterized by co-incubation with free OX26 antibodies, which decreased the association significantly. Furthermore, incubation at 4°C also reduced the association, indicating an energy-demanding uptake mechanism (p < 0.0001). Data are presented as mean + SEM (n = 4–8), and the p-values depicted were derived from a one-way ANOVA with Tukey’s multiple comparisons post hoc test. MFI: Median fluorescence intensity.

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