Department of Biomolecular Sciences, University of Urbino Carlo Bo, via Saffi 2, 61029-Urbino, Italy.
J Control Release. 2011 Sep 25;154(3):306-13. doi: 10.1016/j.jconrel.2011.05.024. Epub 2011 May 27.
Cyclosporine A (CsA) and tacrolimus (also known as FK506) are natural compounds with immunosuppressive activity that have improved the outcome of organ transplantation. Unfortunately, both drugs are characterised by high pharmacokinetic variability, poor bioavailability and high toxicity. Until now, no optimal method to deliver immunosuppressant drugs into circulation has been developed. Here we propose the use of engineered erythrocytes as a drug delivery system for the release of immunosuppressants in circulation in order to modify their pharmacokinetic and restrain toxic effects. After administration, FK506 and CsA mainly distribute within erythrocytes owing to the presence into these cells of immunophilins that bind the drugs with very high affinity (FKBP12 for FK506 and cyclophilin A for CsA); therefore, a new strategy aimed to increase the amount of FK506/CsA carried by erythrocytes by increasing the intra-erythrocytic concentration of the respective binding proteins has been developed. We manufactured recombinant forms of human FKBP12 and cyclophilin A to be loaded into RBC through a hypotonic dialysis and isotonic resealing procedure. Erythrocytes loaded with 3.5±1.3, 7.5±3.1 and 15.5±0.4nmol FKBP12 were able to bind 3.5±1.5, 6.0±1.9 and 11.4±2.9μg FK506 per millilitre RBC, respectively, while RBC loaded with 4.0±0.6, 5.0±0.8 and 15.9±2.4nmol of cyclophilin A could bind 8.9±3.4, 12.2±3.5 and 17.0±3.2μg CsA. Thus, both engineered RBC were demonstrated able to bind up to an order of magnitude more drug than corresponding native erythrocytes (1.0±0.3μg FK506 and 3.2±0.3μg CsA). Moreover, FK506 released from FKBP12-RBC is able to be up-taken by T lymphocytes and inhibit IL-2 expression in vitro as free administered drug. In summary, our results indicate that diffusible immunosuppressants could be entrapped into red cells (thanks to the loading of the respective target protein) and suggest that immunophilin-loaded RBC could be employed as potential delivery system for immunosuppressive agents.
环孢素 A（CsA）和他克莫司（也称为 FK506）是具有免疫抑制活性的天然化合物，它们改善了器官移植的结果。不幸的是，这两种药物都具有高药代动力学变异性、生物利用度差和高毒性的特点。到目前为止，还没有开发出将免疫抑制剂药物递送到循环中的最佳方法。在这里，我们提出使用工程化红细胞作为药物递送系统，以在循环中释放免疫抑制剂，从而改变其药代动力学并抑制毒性作用。在给药后，由于免疫细胞中存在与药物具有非常高亲和力结合的免疫亲和素（FK506 结合 FKBP12，CsA 结合 cyclophilin A），FK506 和 CsA 主要分布在红细胞内；因此，开发了一种新的策略，旨在通过增加各自结合蛋白在红细胞内的浓度来增加红细胞携带的 FK506/CsA 量。我们制造了重组形式的人 FKBP12 和 cyclophilin A，通过低渗透析和等渗再封闭过程加载到 RBC 中。负载 3.5±1.3、7.5±3.1 和 15.5±0.4nmol FKBP12 的红细胞分别能够结合 3.5±1.5、6.0±1.9 和 11.4±2.9μg FK506/ml RBC，而负载 4.0±0.6、5.0±0.8 和 15.9±2.4nmol cyclophilin A 的 RBC 可以结合 8.9±3.4、12.2±3.5 和 17.0±3.2μg CsA。因此，与相应的天然红细胞相比，两种工程化 RBC 都能够结合多达一个数量级的药物（1.0±0.3μg FK506 和 3.2±0.3μg CsA）。此外，FK506 从 FKBP12-RBC 中释放出来，可以被 T 淋巴细胞摄取，并在体外抑制 IL-2 的表达，就像自由给予的药物一样。总之，我们的结果表明，可扩散的免疫抑制剂可以被包裹在红细胞中（由于负载相应的靶蛋白），并表明载有免疫亲和素的 RBC 可以用作免疫抑制剂的潜在递送系统。
