Construction, gene delivery, and expression of DNA tetherednanoparticles

[摘要] Purpose: Layered nanoparticles have the potential to deliver anynumber of substances to cells both in vitro and in vivo. The purpose ofthis study was to develop and test a relatively simple alternative tocustom synthesized nanoparticles for use in multiple biological systems,with special focus on the eye.Methods: The biotin-labeled transcriptionally active PCR products(TAP) were conjugated to gold, semiconductor nanocrystals, and magneticnanoparticles (MNP) coated with streptavidin. The process ofnanoparticle construction was monitored with gel electrophoresis.Fluorescence microscopy followed by image analysis was used to examinegene expression levels from DNA alone and tethered MNP in human hepatomaderived Huh-7 cells. Adult retinal endothelial cells from both dog(ADREC) and human (HREC) sources were transfected with nanoparticles andreporter gene expression evaluated with confocal and fluorescentmicroscopy. Transmission electron microscopy was used to quantify theconcentration of nanoparticles in a stock solution. Nanoparticles wereevaluated for transfection efficiency, determined by fluorescencemicroscopy cell counts. Cells treated with MNP were evaluated forincreased reactive oxygen species (ROS) and necrosis with flowcytometry.Results: Both 5' and 3' biotin-labeled TAP bound equally to MNP andthere were no differences in functionality between the two tetheringorientations. Free DNA was easily removed by the use of magneticcolumns. These particles were also able to deliver genes to a humanhepatoma cell line, Huh-7, but transfection efficiency was greater thanTAP. The semiconductor nanocrystals and MNP had the highest transfectionefficiencies. The MNP did not induce ROS formation or necrosis after 48h of incubation.Conclusions: Once transfected, the MNP had reporter gene expressionlevels equivalent to TAP. The nanoparticles, however, had bettertransfection efficiencies than TAP. The magnetic nanoparticles were themost easily purified of all the nanoparticles tested. This strategy forbioconjugating TAP to nanoparticles is valuable because nanoparticlecomposition can be changed and the system optimized quickly. Sinceendothelial cells take up MNP, this strategy could be used to targetneovascularization as occurs in proliferative retinopathies. Multiplecell types were used to test this technology and in each thenanoparticles were capable of transfection. In adult endothelial cellsthe MNP appeared innocuous, even at the highest doses tested withrespect to ROS and necrosis. This technology has the potential to beused as more than just a vector for gene transfer, because each layerhas the potential to perform its own unique function and then degrade toexpose the next functional layer.

[学科分类] 生物化学/生物物理