Several methods for the preparation of sterically stabilized immunoliposomes (SIL) have recently been described. This report examines an established method for coupling anti-CD34 My10 mAb to poly(ethylene glycol)-liposomes (PEG-liposomes) containing the anchor pyridyldithiopropionylamino-PEG-phosphatidylethanolamine (PDP-PEG-PE) via a cleavable disulfide bond. Efficient attachment of pyridyldithio-derivatized mAb took place (equivalent to coupling ca. 70% of total input protein) at 2 mol percent of the functionalized PEG-lipid. The My10-SIL bound specifically to CD34+ cells (human leukemic KG-1a and hematopoietic progenitor cells) and the extent of binding was a function of liposomal lipid concentration, the mAb density in the liposome surface and the CD34 cell expression. In mixtures with CD34- cells (CHO or Jurkat), CD34+KG-1a cells were determined by flow cytometry at percentages (1-4%) similar to those reported in clinical samples (such as cord blood, mobilized peripheral blood and bone marrow) using a direct immunostaining with My10-SIL. The disulfide bond was stable in cell culture medium (10% of fetal calf serum) during 8 h and cell-bound SIL can be released from cells by treatment with dithiothreitol as reducing agent under mild conditions (1 h of incubation with 50 mM DTT at 20 degrees C). SIL binding and subsequent dithiothreitol treatment did not influence the cell viability. Our approach should contribute to the development of targetable liposomal vehicles to CD34+ cells for use in ex vivo conditions as sorting of hematopoietic stem cells.

译文

最近已经描述了几种制备空间稳定的免疫脂质体 (SIL) 的方法。本报告研究了一种通过可切割的二硫键将anti-CD34 My10 mAb与含有锚定吡啶基二硫代丙酰氨基-PEG-磷脂酰乙醇胺 (PDP-PEG-PE) 的聚 (乙二醇)-脂质体 (PEG-脂质体) 偶联的方法。在2摩尔 % 的官能化PEG-脂质处发生吡啶基二硫基衍生的mAb的有效附着 (相当于总输入蛋白的约70% 偶联)。My10-SIL与CD34细胞 (人白血病KG-1a和造血祖细胞) 特异性结合,结合程度是脂质体脂质浓度,脂质体表面mAb密度和CD34细胞表达的功能。在与CD34-细胞 (CHO或Jurkat) 的混合物中,通过流式细胞术以与临床样品 (如脐带血、动员的外周血和骨髓) 中报道的相似的百分比 (1-4%) 用My10-SIL直接免疫染色测定CD34 + KG-1a细胞。在8 h期间,二硫键在细胞培养基 (胎牛血清的10%) 中是稳定的,并且可以通过在温和条件下用二硫苏糖醇作为还原剂处理 (在20 ℃ 下与50 mmdtt孵育1小时) 从细胞释放细胞结合的SIL。SIL结合和随后的二硫苏糖醇处理不会影响细胞活力。我们的方法应有助于开发针对CD34细胞的可靶向脂质体载体,以在离体条件下用作造血干细胞的分选。

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