Bocsik Alexandra, Walter Fruzsina R, Gyebrovszki Andrea, Fülöp Lívia, Blasig Ingolf, Dabrowski Sebastian, Ötvös Ferenc, Tóth András, Rákhely Gábor, Veszelka Szilvia, Vastag Monika, Szabó-Révész Piroska, Deli Mária A
Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, H-6726 Szeged, Hungary; Department of Pharmaceutical Technology, University of Szeged, H-6720 Szeged, Hungary.
Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, H-6726 Szeged, Hungary.
J Pharm Sci. 2016 Feb;105(2):754-765. doi: 10.1016/j.xphs.2015.11.018.
The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, β-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers.
细胞间连接限制亲水性化合物通过细胞旁间隙的自由通行。生物屏障紧密连接的可逆开放作为增加药物向体循环或中枢神经系统递送的方法之一进行了研究。对作用于不同整合膜蛋白和连接蛋白的六种肽（ADT-6、HAV-6、C-CPE、七肽（FDFWITP、PN-78）、AT-1002和PN-159）在Caco-2肠上皮细胞系和血脑屏障共培养模型上进行了测试。所有在无毒浓度下测试的肽均显示出可逆的紧密连接调节作用，并且有效地为标记分子荧光素和白蛋白打开了细胞旁通道。通过对闭合蛋白、紧密连接蛋白-4、-5、闭锁小带蛋白-1、β-连环蛋白和E-钙黏蛋白进行免疫染色，验证了细胞间连接结构的变化。在两种模型中均测量了闭合蛋白和紧密连接蛋白的表达水平。我们能够证明C-CPE、ADT-6和HAV-6肽对上皮细胞具有选择性，而七肽和AT-1002肽对脑内皮细胞具有选择性。PN-159可能通过紧密连接蛋白-1和-5发挥作用，是两种模型中最有效的连接通透性调节剂。我们的结果表明，这些肽可以有效地、选择性地用作潜在的药物辅料，以改善药物跨生物屏障的递送。
