A novel drug delivery method using endothelial biotinylation and avidin-biotin binding.

一种利用内皮生物素化和亲和素-生物素结合的新型药物递送方法。

• 批准号: 11671384
• 负责人: HOYA Katsumi
• 金额: $ 2.05万
• 依托单位: Dokkyo Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671384/
• 关键词: biotinylation; avidin; endothelium; gene therapy; drug targeting; biotin; drug delivery; drug deliovery

The avidin-biotin binding is very specific and strong. The objects of this project are to fix biotinylated drugs on the endothelia by biotinylating directly the cells and giving them avidin molecules. In vivo study using rabbit kidneys, we injected a biotinylating reagent with NHS-ester, which react with cellular proteins, from a vascular catheter inserted into a renal artery. After five-minute incubation, we applied avidin solution and, subsequently, fluorescence-labeled biotin solution. A histological study showed that more than 80% of glomeruli were stained with fluorescence, which meant that biotinylated materials can be fixed on the endothelia in vivo by the present procedure. The same result was obtained in vitro using cultured endothelial cells. However, when we used biotinylated drug solutions whose concentrations were lower than 10^<-8>M, they were not detected in the cells. So we first mixed an avidin solution and a biotinylated drug (r-phycoerythrin-biotin (r-PE-biotin) solution in the ratio 100:1 (in mole) to make avidin-biotinylated drug complex. We applied the complex to the biotinylated endothelia, and found that r-PE-biotin was fixed on the cells. Based on the result, we made biotinylated double strand (ds) DNA, which had the gene of green fluorescence protein (GFP), and mixed it (10^<-9> and 10^<-8>M) with excessive volume of avidin solution. We applied the biotinylated ds-DNA- avidin complex to the biotinylated cells in logarithmic growth. After two-day incubation, we found the cells expressing GFP. We believe the present procedure is a novel method for gene transfer.

亲和素-生物素结合非常特异和强烈。这个项目的目标是通过直接生物素化细胞并给它们提供亲和素分子来将生物素化药物固定在内皮细胞上。在兔肾脏的活体研究中，我们从插入肾动脉的血管导管向生物素试剂注入NHS-酯，它与细胞蛋白发生反应。孵育五分钟后，我们应用亲和素溶液，随后应用荧光标记的生物素溶液。组织学研究表明，80%以上的肾小球被荧光染色，这意味着生物素化材料可以通过本方法在体内固定在内皮细胞上。在体外使用培养的内皮细胞也得到了同样的结果。然而，当我们使用浓度低于10^&lt；-8&gt；M的生物素化药物溶液时，在细胞中没有检测到它们。因此，我们首先将亲和素溶液与生物素化药物(r-藻红蛋白-生物素(r-PE-Biotin)溶液)以100：1的摩尔比混合制成亲和素-生物素化药物复合体。我们将该络合物应用于生物素标记的内皮细胞，发现r-PE-生物素固定在细胞上。在此基础上，我们制备了含有绿色荧光蛋白(GFP)基因的生物素化双链DNA，并将其与过量的亲和素溶液混合。我们将生物素标记的ds-DNA-亲和素复合体应用于生物素化细胞的对数生长。培养两天后，我们发现了表达GFP的细胞。我们相信，本方法是一种新的基因转移方法。