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Diphtheria Toxin Mutant CRM197-Mediated Transcytosis across Blood–Brain Barrier In Vitro

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Abstract

Blood–brain barrier (BBB) is specialized to limit brain drug delivery. Cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin, could act as a diphtheria toxin receptor-specific carrier protein and deliver drugs across the BBB. CRM197 has previously been shown to inhibit phospatidyl-3-inositol-kinase (PI3K)/Akt signaling. Other studies have demonstrated a link between PI3K/Akt signaling and forkhead transcription factors in endothelial cells. We therefore investigated the effects and mechanisms underlying the potential of CRM197, not only as a carrier protein for targeted drug delivery to the brain, but also for inducing signaling to affect endocytosis in endothelial cells. The hCMEC/D3 cell line had been used to establish a BBB in vitro model; the transport efficiency of CRM197 was analyzed both by association and transcytosis experiments. CRM197 was shown to prefer apical-to-basal transcytosis, which involved the caveolae-mediated pathway. The uptake of CRM197 conjugates by endothelial cells reached equilibrium after 60 min of treatment. The caveolin-1 mRNA and protein expression levels were significantly increased by CRM197. The up-regulation of caveolin-1 may be mediated by CRM197 via a PI3K/Akt dependent pathway and reduction of the phospho-FOXO1A (forkhead box O) transcription factor. Our results indicate that carrier protein CRM197-mediated delivery across the BBB is involved in the induction of FOXO1A transcriptional activity and upregulation of caveolin-1 expression.

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Acknowledgments

hCMEC/D3 cells were kindly provided by Prof. Dr. Pierre-Olivier Couraud (INSERM U567, Paris, France). This work is supported by grants from the Natural Science Foundation of China (No. 30670723, 30700861, 30800451, 30872656, 30973079), Natural Science Foundation of Liaoning Province in China (No. 20052102, 20082102), Science and Technology Plan Project of Educational Department of Liaoning Province (No. 2008850), and Shenyang Science and Technology Plan Projects (No. 1091175-1-01, 1081266-9-00).

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Authors and Affiliations

  1. Department of Neurobiology, China Medical University, Shenyang, 110001, China

    Ping Wang & Yixue Xue

  2. Department of Neurology, First Affiliated Hospital of China Medical University, Shenyang, 110001, China

    Xiuli Shang

  3. Department of Neurosurgery, Shengjing Affiliated Hospital of China Medical University, Shenyang, 110004, China

    Yunhui Liu

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  1. Ping Wang
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Correspondence to Yixue Xue.

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Ping Wang and Yixue Xue have contributed equally to this work.

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Wang, P., Xue, Y., Shang, X. et al. Diphtheria Toxin Mutant CRM197-Mediated Transcytosis across Blood–Brain Barrier In Vitro. Cell Mol Neurobiol 30, 717–725 (2010). https://doi.org/10.1007/s10571-010-9496-x

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