Prow Tarl, Smith Jacob N, Grebe Rhonda, Salazar Jose H, Wang Nan, Kotov Nicholas, Lutty Gerard, Leary James
Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
Mol Vis. 2006 May 26;12:606-15.
Layered nanoparticles have the potential to deliver any number of substances to cells both in vitro and in vivo. The purpose of this study was to develop and test a relatively simple alternative to custom synthesized nanoparticles for use in multiple biological systems, with special focus on the eye.
The biotin-labeled transcriptionally active PCR products (TAP) were conjugated to gold, semiconductor nanocrystals, and magnetic nanoparticles (MNP) coated with streptavidin. The process of nanoparticle construction was monitored with gel electrophoresis. Fluorescence microscopy followed by image analysis was used to examine gene expression levels from DNA alone and tethered MNP in human hepatoma derived Huh-7 cells. Adult retinal endothelial cells from both dog (ADREC) and human (HREC) sources were transfected with nanoparticles and reporter gene expression evaluated with confocal and fluorescent microscopy. Transmission electron microscopy was used to quantify the concentration of nanoparticles in a stock solution. Nanoparticles were evaluated for transfection efficiency, determined by fluorescence microscopy cell counts. Cells treated with MNP were evaluated for increased reactive oxygen species (ROS) and necrosis with flow cytometry.
Both 5' and 3' biotin-labeled TAP bound equally to MNP and there were no differences in functionality between the two tethering orientations. Free DNA was easily removed by the use of magnetic columns. These particles were also able to deliver genes to a human hepatoma cell line, Huh-7, but transfection efficiency was greater than TAP. The semiconductor nanocrystals and MNP had the highest transfection efficiencies. The MNP did not induce ROS formation or necrosis after 48 h of incubation.
Once transfected, the MNP had reporter gene expression levels equivalent to TAP. The nanoparticles, however, had better transfection efficiencies than TAP. The magnetic nanoparticles were the most easily purified of all the nanoparticles tested. This strategy for bioconjugating TAP to nanoparticles is valuable because nanoparticle composition can be changed and the system optimized quickly. Since endothelial cells take up MNP, this strategy could be used to target neovascularization as occurs in proliferative retinopathies. Multiple cell types were used to test this technology and in each the nanoparticles were capable of transfection. In adult endothelial cells the MNP appeared innocuous, even at the highest doses tested with respect to ROS and necrosis. This technology has the potential to be used as more than just a vector for gene transfer, because each layer has the potential to perform its own unique function and then degrade to expose the next functional layer.
层状纳米颗粒有潜力在体外和体内将多种物质递送至细胞。本研究的目的是开发并测试一种相对简单的替代定制合成纳米颗粒的方法，用于多种生物系统，特别关注眼部。
将生物素标记的转录活性PCR产物（TAP）与金、半导体纳米晶体以及包被有链霉亲和素的磁性纳米颗粒（MNP）偶联。用凝胶电泳监测纳米颗粒构建过程。采用荧光显微镜结合图像分析来检测人肝癌来源的Huh-7细胞中单独DNA和连接MNP后的基因表达水平。用纳米颗粒转染犬源（ADREC）和人源（HREC）的成年视网膜内皮细胞，并用共聚焦显微镜和荧光显微镜评估报告基因表达。用透射电子显微镜定量储备溶液中纳米颗粒的浓度。通过荧光显微镜细胞计数确定纳米颗粒的转染效率。用流式细胞术评估用MNP处理的细胞中活性氧（ROS）增加情况和坏死情况。
5'和3'生物素标记的TAP与MNP的结合程度相同，两种连接方向在功能上无差异。使用磁柱可轻松去除游离DNA。这些颗粒也能够将基因递送至人肝癌细胞系Huh-7，但转染效率高于TAP。半导体纳米晶体和MNP的转染效率最高。孵育48小时后，MNP未诱导ROS形成或坏死。
一旦转染，MNP的报告基因表达水平与TAP相当。然而，纳米颗粒的转染效率比TAP更高。磁性纳米颗粒是所有测试纳米颗粒中最易于纯化的。将TAP生物偶联至纳米颗粒的这种策略很有价值，因为可以改变纳米颗粒组成并快速优化该系统。由于内皮细胞会摄取MNP，这种策略可用于靶向增殖性视网膜病变中出现的新生血管形成。使用多种细胞类型测试了该技术，在每种细胞中纳米颗粒都能够进行转染。在成年内皮细胞中，即使在测试的最高剂量下，MNP相对于ROS和坏死而言似乎也是无害的。这项技术有潜力不仅用作基因转移载体，因为每一层都有潜力执行其自身独特的功能，然后降解以暴露下一个功能层。
