Kobayashi Naoki, Hirata Kazuhiro, Chen Shi, Kawase Atsushi, Nishikawa Makiya, Takakura Yoshinobu
Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
J Gene Med. 2004 Apr;6(4):455-63. doi: 10.1002/jgm.531.
A large-volume intravenous (i.v.) injection of DNA, i.e. a hydrodynamics-based transfection procedure, is known to be an efficient and liver-specific method of in vivo gene delivery. However, little is available on an applicable particle size in the procedure.
We examined the effect of particle size on the hepatic delivery by the hydrodynamics-based procedure, using fluorescein isothiocyanate labeled polystyrene microspheres (MS) of 50, 200 or 500 nm in diameter. MS were injected intravenously to mice by a conventional (normal) or the hydrodynamics-based procedure and their degree of hepatic uptake was determined fluorometrically.
For all sizes tested, the two procedures were similar in terms of the apparent degree of hepatic uptake, whereas the intrahepatic localization of MS was apparently different between the procedures as shown by an examination of frozen tissue sections. In mice with gadolinium chloride induced Kupffer cell blockade, the hepatic uptake of MS following the normal procedure was decreased while that of the hydrodynamics-based procedure was less affected. This phenomenon of enhanced hepatic delivery seemed to be more effective for larger particles. Confocal microscopic observation of hepatocyte suspensions indicated that part of the injected MS-50 was delivered intracellularly following the hydrodynamics-based procedure, whereas almost all the observed MS-200 and MS-500 were detected in the extracellular compartment or on the surface of the cells. This was supported by the fact that most of the injected MS existed pericellularly around the transgene-expressing cells.
The hydrodynamics-based procedure facilitated extravasation and hepatic delivery of MS. Larger MS were more efficiently extravasated and trapped by the liver, whereas intracellular delivery hardly occurred with them.
大量静脉注射DNA，即基于流体动力学的转染程序，是一种高效且肝脏特异性的体内基因递送方法。然而，关于该程序中适用的粒径的信息很少。
我们使用直径为50、200或500nm的异硫氰酸荧光素标记的聚苯乙烯微球（MS），通过基于流体动力学的程序研究了粒径对肝脏递送的影响。通过常规（正常）或基于流体动力学的程序将MS静脉注射到小鼠体内，并通过荧光法测定其肝脏摄取程度。
对于所有测试的粒径，两种程序在肝脏摄取的表观程度方面相似，而通过冷冻组织切片检查显示，两种程序之间MS的肝内定位明显不同。在氯化钆诱导的库普弗细胞阻滞的小鼠中，正常程序后MS的肝脏摄取减少，而基于流体动力学的程序的摄取受影响较小。这种肝脏递送增强的现象似乎对较大颗粒更有效。对肝细胞悬液的共聚焦显微镜观察表明，基于流体动力学的程序后，部分注射的MS-50被递送至细胞内，而几乎所有观察到的MS-200和MS-500都在细胞外区室或细胞表面被检测到。这得到以下事实的支持：大多数注射的MS存在于转基因表达细胞周围的细胞外。
基于流体动力学的程序促进了MS的渗出和肝脏递送。较大的MS更有效地渗出并被肝脏捕获，而它们几乎不发生细胞内递送。
