Blood Transfusion Research Center, High Institute for Education and Research in Transfusion Medicine, Tehran, Iran.
Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Cardiovasc Hematol Disord Drug Targets. 2020;20(2):131-137. doi: 10.2174/1871529X19666190918141859.
Factor VIII (FVIII) replacement therapy remains a primary treatment for hemophilia A, however, the development of FVIII antibodies (inhibitors) and short half-life of the FVIII products are the major complications. Erythrocytes may prevent rapid removal of drugs from plasma. Erythrocytes are biocompatible and non-immunogenic drug delivery. In this study, in vitro activity of FVIII encapsulated by human erythrocytes was investigated.
FVIII was loaded into erythrocytes using the hypo-osmotic dialysis technique. FVIII activity assay has been analyzed using Activated Partial Thromboplastin Time (APTT). Presence of FVIII on erythrocytes was detected by western blotting and flowcytometry using specific monoclonal antibody (abcam, U.K) against FVIII. Moreover, the osmotic fragility and hematologic parameters of FVIII-loaded carrier erythrocytes were measured.
Our results indicated that FVIII could not cross the membrane, where plenty of FVIII was found on the surface of the carrier erythrocyte. Flow cytometery results showed that 11% of the loaded carrier erythrocytes was positive for FVIII protein on their surface. The greatest activation of FVIII in both groups including lysate and non-lysate FVIII-loaded RBCs was observed on the first day, and the coagulant activity of this factor was gradually reduced on days 3 and 5. In 1:50 dilution of both groups, significant differences in FVIII activity were observed in 1:50 dilution of both groups, especially on the 5th day.
This study aims to introduce erythrocytes as appropriate carriers for FVIII to prolong the dosing intervals in the effective and safe levels for a relatively longer time.
VIII 因子（FVIII）替代疗法仍然是治疗 A 型血友病的主要方法，然而，FVIII 抗体（抑制剂）的产生和 FVIII 产品半衰期短是主要的并发症。红细胞可能会阻止药物从血浆中快速清除。红细胞是生物相容性的和非免疫原性的药物递送载体。在本研究中，研究了包封在人红细胞内的 FVIII 的体外活性。
使用低渗透析技术将 FVIII 载入红细胞。采用活化部分凝血活酶时间（APTT）分析 FVIII 活性测定。使用针对 FVIII 的特异性单克隆抗体（abcam，英国）通过 Western blot 和流式细胞术检测红细胞上 FVIII 的存在。此外，还测量了负载 FVIII 的载体红细胞的渗透脆性和血液学参数。
我们的结果表明，FVIII 不能穿过细胞膜，而大量的 FVIII 被发现存在于载体红细胞的表面。流式细胞术结果显示，在表面上有 11%的负载载体红细胞呈 FVIII 蛋白阳性。在包括裂解物和非裂解物负载 RBC 的两组中，FVIII 的最大激活都在第 1 天观察到，并且该因子的凝血活性在第 3 和第 5 天逐渐降低。在两组的 1:50 稀释度下，在两组的 1:50 稀释度下均观察到 FVIII 活性的显著差异，特别是在第 5 天。
本研究旨在将红细胞作为 FVIII 的合适载体，以在有效和安全的水平上延长给药间隔时间，从而在相对较长的时间内保持相对较长的时间。
