Hoya K, Guterman L R, Miskolczi L, Hopkins L N
Department of Neurosurgery, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, USA.
Drug Deliv. 2001 Oct-Dec;8(4):215-22. doi: 10.1080/107175401317245895.
In this study, a novel intravascular drug delivery system was developed in which a drug injected from a catheter was fixed to the vasculature of the targeted tissue. Cellular proteins of viable endothelial cells were first biotinylated directly by biotinylation reagents, and then bound by an avidinated drug or, using avidin as a linker, a biotinylated drug. In the initial experiments, we studied in vitro the biotinylation of cultured bovine aortic endothelial cells (BAECs) by applying biotinylation reagents (NHS-LC-biotin or sulfo-NHS-LC-biotin) onto the washed intact BAEC monolayers and showed that the amount of biotin bound to the cells depended on the concentration of the biotinylation reagents applied. The cell-bound biotin decreased with time after the biotinylation. When fluorescein-labeled avidin (FITC-avidin) was applied to the biotinylated BAEC monolayers, the FITC-avidin readily bound to the cells. An LDH-release assay showed that sulfo-NHS-LC-biotin was only slightly cytotoxic to the BAECs and a colony formation assay showed only slight adverse effects of the reagent. In vivo studies were carried out on the renal arteries of normal rabbits. A solution of NHS-LC-biotin was injected through a catheter to one kidney to biotinylate its vasculature and the vehicle to the other as control, followed by a perfusion with saline. Finally, a solution of FITC-avidin was injected to both kidneys that were then reperfused with the blood flow following the withdrawal of the catheters. In the histological sections, more than 85% of glomeruli was stained with fluorescein in the biotinylated kidney, whereas no glomeruli were stained in the control. In the kidneys harvested 2 days after the same procedure, most glomeruli were still brightly stained. In the final experiment, biotinylated kidneys were injected with a solution of avidin, followed by a solution of fluorescein-biotin. Control kidneys had no prior biotinylation but received the same injections of avidin and fluorescein-biotin as above. More than 80% of glomeruli were stained in the biotinylated kidneys but none in the controls. This indicated that biotinylated drugs can be anchored to the biotinylated vasculature through avidin without being flushed away by blood flows. No apparent adverse effect was found in the functions of biotinylated kidneys. We propose that this drug delivery system is feasible for the treatment of some pathological conditions of blood vessels such as microvascular proliferation in malignant tumors and for continuous drug delivery in certain target organs.
在本研究中，开发了一种新型血管内药物递送系统，其中从导管注入的药物被固定在靶组织的血管系统上。首先用生物素化试剂直接对存活内皮细胞的细胞蛋白进行生物素化，然后用抗生物素蛋白化药物或使用抗生物素蛋白作为连接物，用生物素化药物进行结合。在最初的实验中，我们通过将生物素化试剂（NHS-LC-生物素或磺基-NHS-LC-生物素）应用于洗涤后的完整牛主动脉内皮细胞（BAEC）单层来体外研究培养的BAEC的生物素化，并表明与细胞结合的生物素量取决于所应用的生物素化试剂的浓度。生物素化后，细胞结合的生物素随时间减少。当将荧光素标记的抗生物素蛋白（FITC-抗生物素蛋白）应用于生物素化的BAEC单层时，FITC-抗生物素蛋白很容易与细胞结合。乳酸脱氢酶释放试验表明，磺基-NHS-LC-生物素对BAEC仅有轻微的细胞毒性，集落形成试验表明该试剂仅有轻微的不良反应。在正常兔的肾动脉上进行了体内研究。通过导管将NHS-LC-生物素溶液注入一侧肾脏以对其血管系统进行生物素化，将载体注入另一侧肾脏作为对照，随后用盐水灌注。最后，将FITC-抗生物素蛋白溶液注入两侧肾脏，然后在拔出导管后恢复血流灌注。在组织学切片中，生物素化肾脏中超过85%的肾小球被荧光素染色，而对照中没有肾小球被染色。在相同程序后2天收获的肾脏中，大多数肾小球仍然被明亮地染色。在最终实验中，向生物素化的肾脏注射抗生物素蛋白溶液，然后注射荧光素-生物素溶液。对照肾脏未预先进行生物素化，但接受与上述相同的抗生物素蛋白和荧光素-生物素注射。生物素化肾脏中超过80%的肾小球被染色，而对照中没有。这表明生物素化药物可以通过抗生物素蛋白锚定在生物素化的血管系统上，而不会被血流冲走。在生物素化肾脏的功能中未发现明显的不良反应。我们提出，这种药物递送系统对于治疗某些血管病理状况（如恶性肿瘤中的微血管增殖）以及在某些靶器官中进行持续药物递送是可行的。
