Applications in ophthalomology include gene delivery, cell delivery and nanosurgery.
Nanotechnology is defined as the creation and use of materials and devices on the mi-nute scale of intracellular molecules and structures; these systems are generally less than 100 nanometers in size, said Dr Zarbin, the Alfonse A. Cinotti, MD/Lions Eye Research Professor, chairman of the Institute of Ophthalmology and Visual Science, and professor of neuroscience at the UMDNJ-New Jersey Medical School, Newark.
To put the size into perspective, he contrasted the height of an average man (2 billion nm) and the width of a red cell (7,000 nm) with the width of a strand of DNA (about 2 nm).
Polypexes are nanoparticles, according to Dr Zarbin, that are complexes of cationic polymers and DNA, which is negatively charged.
"They can have a transfection efficiency comparable to that of viral vectors," Dr Zarbin said. "The advantages over the traditional nonviral approaches, such as lipofection, are that polypexes are small and easy to prepare, they have a large vector capacity, are stable in nuclease-rich environments, and they deliver genes to dividing and nondividing cells. To the best of our knowledge, they are nontoxic in the eye."
However, some polypexes have low transfection efficiency, and the duration of gene expression can be short.
Xue Cai and associates exploited the use of polypexes to treat a form of retinitis pigmentosa in the retinal degeneration slow (Rds) mouse model. The mouse has a mutation in a photoreceptor protein, peripherin 2. The investigators developed a nanoparticle (~100 nm long and ~8 nm wide) to deliver normal mouse peripherin 2 DNA to the photoreceptors.
Electrophysiological and histological studies performed ~120 days after treatment showed that in the animals with the nanoparticles injected, photopic b-wave amplitudes and outer nuclear layer thickness and architecture were greatly improved. The findings indicated that cone degeneration was attenuated in the treated animals.
"This very simple idea of a nanoparticle can be made more complicated and better," Dr Zarbin said.
He described the work of James Leary, PhD, and colleagues, who developed a multilayered magnetic nanoparticle.
Using this technology, the outer layer targets the nanoparticle to the desired cell, and an inner second layer targets the nanoparticle to the desired subcellular organelle; a third layer has a biosensor that turns therapeutic gene expression on or off depending on the microenvironment that is present. The innermost layer is the superparamagnetic iron oxide core, which can be imaged.
"This particle operates like a nanomachine that only delivers a treatment if there is a problem present," Dr Zarbin said. "This device can be both diagnostic and therapeutic."
Prow, Lutty, and coworkers have provided an example of the use of nanotechnology to create biosensors for health maintenance. This team developed a biosensor DNA tethered to a magnetic nanoparticle.
The biosensor is based on an enhanced green fluorescent protein (EGFP) reporter gene driven by an antioxidant response element (ARE), which normally is activated in the setting of oxidative stress and enhances the expression of genes downstream in its sequence. This engineered nanoparticle penetrates endothelial cells, and exposure of the cells to hyperoxia drives the expression of EGFP. After subretinal injection, these biosensor nanoparticles reported the activation of the ARE in diabetic rat retinal pigment epithelium cells.
The antioxidant biosensor could provide a means for clinicians to identify patients likely to need therapy at a time before clinical manifestations of severe disease are evident. By coupling a therapeutic gene to the ARE (instead of a reporter gene such as EGFP), one can create a combined diagnostic-therapeutic device that enables endothelial cells (or any cell that takes up the nanoparticle) to "treat themselves" in the setting of oxidative damage.
Polymer scaffolds can increase retinal progenitor cell survival and differentiation. Surgically, use of such scaffolds enables one to target cell delivery spatially, Dr Zarbin said.
"In principle, these nanostructures can be used not only to deliver cells but also to deliver growth factors and other needed substances to enhance integration with the host and proper differentiation, thus improving transplant success," Dr Zarbin said.
Sarah Tao, PhD, and colleagues used microelectromechanical systems (MEMS)-based technology to build polymethyl methacrylate sheets with highly specific, reproducible topography (6 µm thick, 11 µm diameter pores, 63 µm pore separation). When implanted into rats, retinal progenitor cell survival and migration into the host was enhanced greatly (150%) if the transplants were delivered on a porous (versus non-porous) scaffold.
"The surface chemistry can be changed on these scaffolds with the goal of further improving progenitor cell survival, differentiation, and integration with the host retina," Dr Zarbin said.
Nanosurgery currently is science fiction, but it is on the verge of becoming a clinical reality, according to Dr Zarbin.
The first step in progress toward nanosurgery has been shrinking the size of the surgical instrumentation, he said.
Additional steps will require providing a very high magnification operating microscope and developing an image stabilization system that keeps the instruments and target tissue in focus as they move during respiration or in association with the pulsation of the ocular arteries.
David Sretavan, MD, PhD, and associates are working on axonal surgery and have created a 1-mm3 device to facilitate axonal repair. The cutting device consists of a silicon nitride knife with an ultra-sharp knife-edge mounted onto a silicon-based compliant knife suspension. The knife-edge's radius of curvature (~20 nm) is similar to the diameter of a single microtubule. The device comprises 16 pieces that can fit on a penny and are assembled by microforceps under a microscope.
"The concept of axonal surgery is the cutting out of a damaged axon segment that is then replaced with a healthy axon segment; the ends of the donor and recipient axon are fused, and axonal transport and other neuronal functions resume without interruption. The injured axons are severed using the 1-mm3 cutting platform developed by Sretavan and co-workers, and the severed tissue is removed by dielectrophoresis (force is exerted on a dielectric particle that is exposed to an electric field). (Dielectrophoresis is used currently in cell sorting technology.) The axonal ends can be fused by electrofusion, a technique that is used currently to make hybridoma cells," he said.
Nanotweezers also have been developed by Drs Kim and Lieber. A conductive material such as gold is put on a glass electrode to which are attached two rigid multiwalled nanotubes (50 nm diameter, 4 µm length). Each nanotube serves as one arm of the tweezer. When voltage is applied across the nanotubes (which conduct electricity), an electrostatic force brings the two arms of the tweezers together. These nanotweezers might not work well in the eye, however, due to its water-filled environment and also the possibility that the applied current might coagulate the target tissue.
"Nanotechnology is going to have a huge impact on the way we treat patients," Dr Zarbin concluded. "The earliest advances will be in drug delivery and cell delivery."