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

抗生物素蛋白-生物素结合是非常特异性和强的。本课题的研究目的是通过生物素直接修饰内皮细胞并赋予其亲和素分子，将生物素修饰的药物固定在内皮细胞上。在使用兔肾脏的体内研究中，我们从插入肾动脉的血管导管中注射具有NHS-酯的生物素化试剂，其与细胞蛋白质反应。孵育5分钟后，我们应用抗生物素蛋白溶液，随后应用荧光标记的生物素溶液。组织学研究表明，超过80%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上。在体外使用培养的内皮细胞获得了相同的结果。然而，当我们使用浓度低于10 μ M的生物素化药物溶液时<-8>，在细胞中未检测到它们。因此，我们首先将抗生物素蛋白溶液和生物素化药物（r-藻红蛋白-生物素（r-PE-生物素）溶液以100：1（摩尔）的比例混合以制备抗生物素蛋白-生物素化药物复合物。我们将该复合物应用于生物素化的内皮细胞，发现r-PE-生物素被固定在细胞上。基于该结果，我们制备了生物素化的双链（ds）DNA，其具有绿色荧光蛋白（GFP）基因，并将其（10 μ M<-9>和10 μ <-8>M）与过量体积的抗生物素蛋白溶液混合。我们将生物素化的ds-DNA-亲和素复合物应用于对数生长的生物素化细胞。经过两天的培养，我们发现细胞表达GFP。我们相信本方法是一种新的基因转移方法。

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.