Galuppo Larry D, Kamau Sarah W, Steitz Benedikt, Hassa Paul O, Hilbe Monika, Vaughan Lloyd, Koch Sabine, Fink-Petri Alke, Hofman Margarethe, Hofman Heinrich, Hottiger Michael O, von Rechenberg Brigitte
Musculoskeletal Research Unit, Equine Hospital, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.
J Nanosci Nanotechnol. 2006 Sep-Oct;6(9-10):2841-52. doi: 10.1166/jnn.2006.481.
This study evaluated in vivo gene delivery and subsequent gene expression within cells of the synovium in the presence of static and pulsating magnetic field application following intraarticular injection of superparamagnetic iron oxide nanoparticles linked to plasmids containing reporter genes encoding for fluorescent proteins. Plasmids encoding genes for either green fluorescent protein or red fluorescent protein were bound to superparamagnetic nanoparticles coated with polyethyleneimine. Larger (200-250 nm) and smaller (50 nm) nanoparticles were compared to evaluate the effects of size on transfection efficiency as well as any associated intraarticular reaction. Comparisons between groups were evaluated at 24, 72, and 120 h time periods. Inflammatory response was mild to moderate for all injected particles, but was present in the majority of synovial membrane samples evaluated. Larger particles tended to be associated with more inflammation than smaller ones. Nevertheless, intraarticular application of both experimental and control nanoparticles were well tolerated clinically. Gene expression as determined by observation of either green or red intracellular fluorescence was difficult to assess by both epifluorescent light, and confocal microscopy. An insufficient concentration of nanoparticles in relation to joint volume likely resulted in a limited number of samples with positive evidence of iron staining and with suspected positive evidence of cells expressing fluorescent proteins. Our results indicate that intraarticular administration of functionalized superparamagnetic iron oxide nanoparticles resulted in a mild to moderate synovitis and there was in conclusive evidence of gene expression. Further research is warranted to determine the best and most effective reporter assay for assessment of the in vivo gene delivery into the joints. In addition, the best suited concentration and size of nanoparticles, which will optimize gene delivery and expression, while minimizing intraarticular inflammation, needs to be determined.
本研究评估了在关节腔内注射与含有编码荧光蛋白的报告基因的质粒相连的超顺磁性氧化铁纳米颗粒后，在静态和脉动磁场作用下，体内基因传递以及滑膜细胞内随后的基因表达情况。编码绿色荧光蛋白或红色荧光蛋白的基因的质粒与涂有聚乙烯亚胺的超顺磁性纳米颗粒结合。比较了较大（200 - 250 nm）和较小（50 nm）的纳米颗粒，以评估尺寸对转染效率以及任何相关关节内反应的影响。在24、72和120小时时间段对各组进行比较评估。所有注射颗粒的炎症反应均为轻度至中度，但在所评估的大多数滑膜样本中均存在。较大颗粒比较小颗粒往往与更多炎症相关。然而，实验性和对照纳米颗粒的关节内应用在临床上耐受性良好。通过落射荧光和共聚焦显微镜观察确定的细胞内绿色或红色荧光所反映的基因表达难以评估。纳米颗粒相对于关节体积的浓度不足，可能导致有铁染色阳性证据以及疑似表达荧光蛋白细胞阳性证据的样本数量有限。我们的结果表明，关节腔内给予功能化超顺磁性氧化铁纳米颗粒会导致轻度至中度滑膜炎，且基因表达的证据尚无定论。有必要进一步研究以确定评估体内基因传递至关节的最佳且最有效的报告基因检测方法。此外，需要确定最适合的纳米颗粒浓度和尺寸，以优化基因传递和表达，同时将关节内炎症降至最低。
