SCI Biobanking and Translational Medicine, Swiss Paraplegic Research, Nottwil, Switzerland.
Institute for Biomechanics, D-HEST, ETH Zurich, Zurich, Switzerland.
Drug Des Devel Ther. 2020 Oct 28;14:4547-4560. doi: 10.2147/DDDT.S258368. eCollection 2020.
The production of nano-erythrosomes (NEs) by extrusion, which is considered the "gold standard", has several disadvantages such as difficult equipment assembly, long procedure time, variable pressure, and problems with sterility. An alternative approach, using ultrasound probe, has been shown to overheat the sample and have suboptimal results compared to the extrusion method. In our study, we propose, develop, and test a new method for the fabrication of NEs based on shear force and then compare it to the "gold standard" extrusion approach.
The new method consists of mechanical shear force disruption of the hemoglobin-depleted erythrocyte ghost membranes, with the aid of a rotor stator based tissue homogenizer. Using the same batches of erythrocyte ghost membranes, we compared NEs produced by shear force to NEs produced by the well-established extrusion approach. NEs were characterized for yield, size, encapsulation efficiency, morphology, and stability by flow cytometry (FC), transmission electron microscopy (TEM), and zeta potential analysis.
The shear force based process was easier to set up, significantly faster, had better sterility control, and decreased variability between batches. The shear force method generated NEs with the desired size distribution (particles diameter ~125 nm), which were morphologically and functionally equivalent to the NEs produced by extrusion. NEs produced by shear force were stable in terms of counts, size, and fluorescence intensity for 3 weeks at +4°C. Moreover, they showed colloidal stability and minimal influence to centrifugal stress, turbulence shock, and hemolytic potential.
The newly proposed shear force method allows faster, easier, and highly reproducible NEs production when compared to the conventional extrusion approach. The new setup allows simultaneous production of sterile batches of NEs, which have homogenous size distribution, good stability, and improved shelf life storage. The ability of the shear force method to process also high concentration samples indicates a future potential development of large-scale NEs production and industrial application, which has been a challenge for the extrusion method.
挤出法被认为是“金标准”，用于生产纳米红细胞（NE），但存在设备组装困难、程序时间长、压力变化以及无菌性问题等缺点。另一种方法是使用超声探头，但与挤出法相比，该方法会使样品过热，效果不理想。在我们的研究中，我们提出、开发并测试了一种基于剪切力的新型 NE 制备方法，并将其与“金标准”挤出方法进行了比较。
新方法包括使用转子定子组织匀浆器机械剪切破坏去血红蛋白红细胞 ghost 膜，然后使用相同批次的红细胞 ghost 膜，我们比较了由剪切力产生的 NE 与由成熟的挤出方法产生的 NE。通过流式细胞术（FC）、透射电子显微镜（TEM）和zeta 电位分析，对 NE 的产率、粒径、包封效率、形态和稳定性进行了表征。
基于剪切力的工艺更容易设置，速度明显更快，具有更好的无菌性控制，批次间变异性降低。剪切力法产生的 NE 具有所需的粒径分布（颗粒直径约 125nm），在形态和功能上与挤出法产生的 NE 相当。在+4°C 下，通过剪切力产生的 NE 在计数、粒径和荧光强度方面稳定 3 周。此外，它们表现出胶体稳定性和最小的离心力、湍流冲击和溶血潜力影响。
与传统的挤出方法相比，新提出的剪切力方法可以更快、更容易、更可重复地生产 NE。新的设置允许同时生产无菌批次的 NE，其具有均匀的粒径分布、良好的稳定性和延长的保质期。剪切力法处理高浓度样品的能力表明，未来可能开发大规模 NE 生产和工业应用，这是挤出法面临的挑战。
