Molecular Vision 2006; 12:606-15 <http://www.molvis.org/molvis/v12/a67/> Received 28 February 2005 | Accepted 19 April 2006 | Published 26 May 2006 The majority of nanoparticle research, to date, has been carried out by materials scientists, but recent trends have brought these tools into the hands of biologists. Nanoparticles have found two broad niches in biology, detection technolo- gies and payload delivery [1,2]. Since the late 1970s, nanoparticles have been used to deliver drugs [2,3]. In fact, the majority of publications concerning biological applications of nanoparticles are focused on the delivery of chemothera- peutic agents with nanoparticles ranging from 2 to 3000 nm. Nanoparticle mediated gene delivery has recently emerged as a promising tool for gene therapy strategies [4-6]. The main problems with using nanoparticles for gene delivery are the construction, cost, and quality control of the nanoparticles themselves. The construction quickly becomes very compli- cated when the number of layers increases. This is due to the interactions between layers and between nanoparticles with incomplete and complete layering. These factors limit the use- fulness of nanotechnology to laboratories that have chemists capable of nanoparticle synthesis or to investigators in col- laboration with chemists. This limits the technology, especially for small laboratories. This study documents the development of a streptavidin nanoparticle system that is simple and quite flexible from a commercially available product intended for other uses. Magnetic nanoparticles have been primarily applied to three fields: magnetic resonance imaging, molecular and cell separation technologies, and drug delivery [7-11]. Many re- searchers use magnetic particles as contrast agents [12-16]. ©2006 Molecular Vision Construction, gene delivery, and expression of DNA tethered nanoparticles Tarl Prow, 1,2,3 Jacob N. Smith, 2 Rhonda Grebe, 3 Jose H. Salazar, 2 Nan Wang, 2 Nicholas Kotov, 4 Gerard Lutty, 3 James Leary 1,2,5 Departments of 1 Pathology and 2 Infectious Diseases, University of Texas Medical Branch, Galveston, TX; 3 The Wilmer Ophthalmo- logic Institute, Department of Ophthalmology, The Johns Hopkins Hospital, Baltimore, MD; 4 Department of Chemical Engineering, University of Michigan, Ann Arbor, MI; 5 Basic Medical Sciences and Biomedical Engineering, Purdue University, West Lafayette, IN Correspondence to: James Leary, Basic Medical Sciences and Bio- medical Engineering, Purdue University, West Lafayette, IN, 47907; Phone: (765) 494-7280; FAX: (765) 496-6443; email: jfleary@purdue.edu Purpose: 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. Methods: 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 expres- sion 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. Results: 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 semi- conductor nanocrystals and MNP had the highest transfection efficiencies. The MNP did not induce ROS formation or necrosis after 48 h of incubation. Conclusions: Once transfected, the MNP had reporter gene expression levels equivalent to TAP. The nanoparticles, how- ever, 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. 606