Multiparametric characterization of red blood cells physiology after hypotonic dialysis based drug encapsulation process.

Red blood cells (RBC) can act as carriers for therapeutic agents and given their biocompatibility and long lifespan in the circulation, they can substantially improve the safety, pharmacokinetics, and pharmacodynamics of many drugs. Maintaining RBC integrity and lifespan is important for the efficacy of RBC as drug carrier and can be a complex challenge of drug encapsulation. We investigated the impact of drug encapsulation by hypotonic dialysis on RBC physiology and integrity. Several parameters were compared between processed RBC loaded with L-asparaginase (“eryaspase�), processed RBC without drug and non-processed RBC. Processed RBC were less hydrated and displayed a reduction of intracellular content, relative to non-processed RBC. This reduction of intracellular content changed the RBC metabolomic profile, as indicated by the activation of the pentose phosphate pathway, but no impact on the global RBC proteomic profile was observed. We found that the encapsulation process caused moderate morphological changes and was accompanied by an increase of microparticles. Despite a decrease in their deformability, processed RBC were not mechanically retained in a spleen-mimicking device. Moreover, processed RBC had increased surface-to-volume ratio and osmotic resistance. Finally, the half-life of processed RBC was not significantly affected in a mouse model and our previous phase 1 clinical study showed that encapsulation of asparaginase in RBC prolonged its in vivo half-life compared to free form. Overall, our study demonstrated that encapsulation by hypotonic dialysis may affect certain  several characteristics of RBC, but does not significantly impact on the in vivo longevity of RBC or their drug carrier function.

红细胞（Red blood cells, RBC）可作为治疗剂的载体，鉴于其良好的生物相容性（biocompatibility）与循环内长寿命特性，能够显著提升诸多药物的安全性、药代动力学（pharmacokinetics）及药效动力学（pharmacodynamics）表现。维持红细胞的结构完整性与存活时长对于其作为药物载体的功效至关重要，而这同时也是药物包封（drug encapsulation）过程中一项颇具挑战性的难题。本研究探究了采用低渗透析（hypotonic dialysis）进行药物包封对红细胞生理状态与结构完整性的影响。我们对三组样本开展了多参数对比分析：负载L-天冬酰胺酶（L-asparaginase，商品名埃里帕酶（eryaspase））的处理红细胞、未负载药物的处理红细胞以及未处理的红细胞。与未处理的红细胞相比，处理后的红细胞水合程度更低，且细胞内内容物含量有所减少。细胞内内容物的这一减少改变了红细胞的代谢组谱（metabolomic profile），表现为磷酸戊糖途径（pentose phosphate pathway）被激活，但未观察到其对红细胞整体蛋白质组谱（proteomic profile）产生影响。研究发现，该包封过程会引发中等程度的形态学变化，并伴随微粒（microparticles）数量的增加。尽管处理后的红细胞变形能力（deformability）有所下降，但它们并未在模拟脾脏装置（spleen-mimicking device）中被机械截留。此外，处理后的红细胞其表面积体积比（surface-to-volume ratio）与渗透压抗性（osmotic resistance）均有所提升。最后，在小鼠模型（mouse model）中，处理后的红细胞半衰期（half-life）未受到显著影响；而我们此前的I期临床研究（phase 1 clinical study）显示，与游离形式（free form）的天冬酰胺酶相比，将其包封于红细胞内可延长其体内半衰期。总体而言，本研究证实，采用低渗透析进行药物包封可能会对红细胞的若干特性产生影响，但并未对红细胞的体内存活时长及其药物载体功能造成显著影响。