Casagrande Giustina, Arienti Flavio, Mazzocchi Arabella, Taverna Francesca, Ravagnani Fernando, Costantino MariaLaura
Department of Chemistry, Materials and Chemical Engineering "Giulio Natta,", Politecnico di Milano, Milan, Italy.
Service of Immunohematology & Transfusion Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.
Artif Organs. 2016 Oct;40(10):959-970. doi: 10.1111/aor.12662. Epub 2016 Jan 27.
Human red blood cells (RBCs) have a remarkable capacity to undergo reversible membrane swelling. Resealed erythrocytes have been proposed as carriers and bioreactors to be used in the treatment of various diseases. This work is aimed at developing a setup allowing the encapsulation of test molecules into erythrocytes by inducing reversible pore formation on the RBC membrane through the application of controlled mechanical shear stresses. The designed setup consists of two reservoirs connected by a glass capillary. Each reservoir is connected to a compressor; during the tests, the reservoirs were in turn pressurized to promote erythrocyte flow through the capillary. The setup was filled with a suspension of erythrocytes, phosphate buffer, and FITC-dextran. Dextran was chosen as the diffusive molecule to check membrane pore dimensions. Samples of the suspension were withdrawn at scheduled times while the setup was operating. Flow cytometry and stereo-optical microscopy analyses were used to evaluate the erythrocyte dextran uptake. The setup was shown to be safe, well controlled, and adjustable. The outcomes of the experimental tests showed significant dextran uptake by RBCs up to 8%. Microscopy observations highlighted the formation of echinocytes in the analyzed samples. Erythrocytes from different donors showed different reactions to mechanical stresses. The experimental outcomes proved the possibility to encapsulate test molecules into erythrocytes by applying controlled mechanical shear stresses on the RBC membrane, encouraging further studies.
人类红细胞（RBCs）具有显著的可逆性膜肿胀能力。重新密封的红细胞已被提议用作载体和生物反应器，用于治疗各种疾病。这项工作旨在开发一种装置，通过施加可控的机械剪切应力在红细胞膜上诱导可逆孔形成，从而将测试分子封装到红细胞中。设计的装置由两个通过玻璃毛细管连接的储液器组成。每个储液器都连接到一台压缩机；在测试过程中，轮流对储液器加压以促进红细胞通过毛细管流动。装置中充满了红细胞、磷酸盐缓冲液和异硫氰酸荧光素标记的葡聚糖（FITC - dextran）的悬浮液。选择葡聚糖作为扩散分子来检查膜孔尺寸。在装置运行期间，按预定时间抽取悬浮液样本。使用流式细胞术和立体光学显微镜分析来评估红细胞对葡聚糖的摄取。结果表明该装置安全、易于控制且可调节。实验测试结果显示红细胞对葡聚糖的摄取量高达8%，具有显著意义。显微镜观察突出了分析样本中棘状红细胞的形成。来自不同供体的红细胞对机械应力表现出不同的反应。实验结果证明了通过对红细胞膜施加可控的机械剪切应力将测试分子封装到红细胞中的可能性，这鼓励了进一步的研究。
