切向流过滤促进了人类红细胞膜碎片的纯化,与离心法相比,切向流过滤更适于从重新封闭的红细胞幽灵中去除未包裹的物质。

IF 2.5 3区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Xiangming Gu, Andre F Palmer
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引用次数: 0

摘要

许多治疗药物的生物分布受到免疫系统的控制。此外,有些分子如果不包裹在较大的细胞结构内,例如包裹在红细胞(RBC)内的血红蛋白(Hb),就会产生细胞毒性。为了抵御免疫系统的识别和细胞毒性,有人提出了基于红细胞膜片段(RBCMFs)的给药系统,作为创造免疫优势疗法的一种策略。然而,将红细胞膜片段用于给药应用需要从裂解的红细胞中大规模纯化不含细胞内成分的红细胞膜片段。在这项研究中,我们成功地利用切向流过滤(TFF)从高浓度(30%-40% v/v)的裂解 RBC 中去除了 >99% 的无细胞 Hb,得到了直径为 2.68 ± 0.17 μm 的 RBCMFs。与之前的研究相比,我们还能对 RBCMFs 进行更全面的表征,包括测量颗粒的 zeta 电位、溶液中无细胞 Hb 浓度的单个 TFF 二周数据和每个二周的时间,以及 RBCMFs 的浓度和大小。除了从裂解的 RBC 中纯化 RBCMFs 外,我们还利用高渗溶液对纯化的 RBCMFs 进行再封闭,使其包裹模型蛋白(Hb),从而得到再封闭的 Hb 包裹 RBC 幽灵(Hb-RBCGs)。然后将 TFF 与离心作为从 Hb-RBCGs 中去除未包被 Hb 的替代方法进行了比较,并评估了每种洗涤方法对 Hb-RBCG 生物物理特性的影响。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Tangential flow filtration-facilitated purification of human red blood cell membrane fragments and its preferential use in removing unencapsulated material from resealed red blood cell ghosts compared to centrifugation.

The biodistribution of many therapeutics is controlled by the immune system. In addition, some molecules are cytotoxic when not encapsulated inside of larger cellular structures, such as hemoglobin (Hb) encapsulation inside of red blood cells (RBCs). To counter immune system recognition and cytotoxicity, drug delivery systems based on red blood cell membrane fragments (RBCMFs) have been proposed as a strategy for creating immunoprivileged therapeutics. However, the use of RBCMFs for drug delivery applications requires purification of RBCMFs at large scale from lysed RBCs free of their intracellular components. In this study, we were able to successfully use tangential flow filtration (TFF) to remove >99% of cell-free Hb from lysed RBCs at high concentrations (30%-40% v/v), producing RBCMFs that were 2.68 ± 0.17 μm in diameter. We were also able to characterize the RBCMFs more thoroughly than prior work, including measurement of particle zeta potential, along with individual TFF diacycle data on the cell-free Hb concentration in solution and time per diacycle, as well as concentration and size of the RBCMFs. In addition to purifying RBCMFs from lysed RBCs, we utilized a hypertonic solution to reseal purified RBCMFs encapsulating a model protein (Hb) to yield resealed Hb-encapsulated RBC ghosts (Hb-RBCGs). TFF was then compared against centrifugation as an alternative method for removing unencapsulated Hb from Hb-RBCGs, and the effects that each washing method on the resulting Hb-RBCG biophysical properties was assessed.

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来源期刊
Biotechnology Progress
Biotechnology Progress 工程技术-生物工程与应用微生物
CiteScore
6.50
自引率
3.40%
发文量
83
审稿时长
4 months
期刊介绍: Biotechnology Progress , an official, bimonthly publication of the American Institute of Chemical Engineers and its technological community, the Society for Biological Engineering, features peer-reviewed research articles, reviews, and descriptions of emerging techniques for the development and design of new processes, products, and devices for the biotechnology, biopharmaceutical and bioprocess industries. Widespread interest includes application of biological and engineering principles in fields such as applied cellular physiology and metabolic engineering, biocatalysis and bioreactor design, bioseparations and downstream processing, cell culture and tissue engineering, biosensors and process control, bioinformatics and systems biology, biomaterials and artificial organs, stem cell biology and genetics, and plant biology and food science. Manuscripts concerning the design of related processes, products, or devices are also encouraged. Four types of manuscripts are printed in the Journal: Research Papers, Topical or Review Papers, Letters to the Editor, and R & D Notes.
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