Researchers are using nanotech tools to study areas such as neural regeneration of the optic nerve, as well as into ophthalmic genetics.
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The anatomy of the human eye (shown here) is very complex, and researchers trying to find ways to treat or cure ophthalmic diseases must deal with this complexity. (Source: The National Health Museum).
In the minds of many people, the word “nanotechnology” conjures up images of tiny robots that burrow deep into cells to perform surgery on diseased molecules, which, if not managed properly, could run out of control and cover the earth with a thick layer of grey goo.
Although these images are the stuff of science fiction, the development of nanotechnology for medical applications is a serious endeavour that is beginning to bear fruit. Last year, the National Institutes of Health (NIH) launched four Nanomedicine Development Centers and anticipates the addition of another this year. Nanotechnology was on the agenda at the 2006 meeting of the Association for Research in Vision and Ophthalmology in Fort Lauderdale, Fla., where researchers convened to discuss its potential in eye research and clinical development.
“The NIH Nanotechnology Initiative is to create conceptual and literal interfaces between biology and medical application at the scale of biomolecular processes,” Paul Sieving, MD, PhD, director of the National Eye Institute and chair of its Nanomedicine Initiative, told meeting delegates. “The goals are to: one, characterize quantitatively the physical and chemical properties of molecules and nanomachinery in living cells; two, to gain an understanding of the engineering principles used in living cells to ‘build’ molecules, molecular complexes, organelles, cells, and tissues; and three, to use knowledge of properties and design principles to develop new technologies, and engineer devices and hybrid structures for repairing tissues as well as preventing and curing disease.”
Sieving said some of the areas most suited for nanotechnology in ophthalmology are in hydraulics and biomechanics (e.g., to assist in glaucoma treatment and retinal vascular surgery), drug delivery (e.g., to better target the retina), neural prosthetics (e.g., neuron-sized electrodes to improve current flow and image resolution), as well as imaging, diagnostics, immuno-modulation, lens and retina repair, and corneal replacement.
One such application could be neural regeneration of the optic nerve. At the Massachusetts Institute of Technology, Cambridge, Mass., Rutledge Ellis-Behnke, PhD, and colleagues have used a self-assembling peptide nanofiber scaffold (SAPNS) to “nano neuro knit” damaged tissue deep within the mammalian brain. After transecting the optic tract in hamsters at the superior colliculus, thus depriving them of vision, the investigators injected a solution containing SAPNS into the area. The self-assembling scaffold allowed the axons to regenerate and “knit” the surrounding brain tissue together to the point where functional vision returned in the animals. The technology is far from being tested in humans, but the results are promising.
Multiple uses for dendrimers
Dendrimers are three-dimensional, hyper-branched polymers constructed by adding branch-upon-branch units onto a core moiety.
At the Imperial College at Hammersmith Hospital in London, Sunil Shaunak, MD, PhD, and his colleagues are examining the use of anionic dendrimers for ophthalmic applications. One of the key motivations for looking at these compounds is to overcome the limitations of agents targeted to a single molecule or receptor. “We became concerned that we have been working with only small-molecule monovalent medicines for a long time. . . . So we started thinking about polyvalent medicines, larger molecules where you have several ligands binding to several receptors in order to get fundamentally new biological responses.”
Shaunak says surgery causes the release of tissue enzymes, which in turn causes the release of small molecular weight molecules of heparan sulfate, triggers for Toll-like receptor 4 (TLR4). “This is almost certainly why we see patients who have a septic shock after surgery and don’t respond to antibiotics, because you actually have two ligands—bacterial LPS and small soluble molecular weight heparan sulphate—stimulating the receptor,” he said.
Shaunak decided to use this to find a solution for a common problem in ophthalmology—scar formation after glaucoma surgery. He and his group constructed a dendrimer that interfered with the ability of heparan sulfate to bind to TRL4, thus interrupting the inflammation cascade close to its source. They also made a sulfated dendrimer that competes directly with fibroblast growth factor at its receptor, thus disrupting angiogenesis close to its source. Both pathways are important components of scar formation. Shaunak says dendrimers could also be used to disrupt inflammation and angiogenesis at the back of the eye for various diseases.
Ophthalmic genetics, genomics
Dendrimers and other nanotechnology devices are also finding applications in the area of ophthalmic genetics and genomics research. Julia Richards, PhD, associate professor at the University of Michigan, finds the field filled with potential for glaucoma research. Three items on her “ophthalmo-geneticists nanotechnology wish list” are devices that will enable genotyping in real time in clinical or field situations; nanoscale devices for assaying levels of gene expression in situ to enable evaluation of effects during attempted interventions; and methods to deliver gene therapy to specific cell types without the hazards of viral gene therapy vectors.
At the University of Michigan, David Burke, PhD, and colleagues are developing a handheld “lab on a chip” device for on-the-spot genotyping. While it is a microscale device—huge in comparison to nanodevices—it deals with nanoliter volumes. The device is capable of measuring reagents and DNA-containing solutions, mixing the solutions, amplifying or digesting the DNA to form discrete products, and separating and detecting those products.
Richards and Burke are applying this technology to large field studies, where blood samples from many individuals need to be collected and analyzed quickly to determine the direction of the research. One is a recent study involving a family with 857 individuals. “In trying to study a family like that, especially when they are spread around the world, we’d like to be able to trace particular branches of family on the spot without having to actually have every single person in this family go in and be seen by ophthalmologists for extensive testing,” Richards said. “If we can follow them in the field and trace down the paths we want to deal with, we can carry out these studies with much greater efficiency.”
Richards noted that one of the problems in glaucoma treatment is getting the drug into the cell rather than in the cell surface or surrounding spaces. Trabecular meshwork cells have phagocytic properties and could be induced to take up a variety of different carrier particles. “So in this case, I hypothesized the use of a latrotoxin analog to direct the dendrimer to the latrotoxin receptor on the surface of the trabecular meshwork cells,” says Richards. “We may want to release the molecule once internalized, so the presence of the relatively specific protease, PCSK1, in TM cells offers the possibility of genetically engineering the protein to be attached to the particle.” This will be done via a cleavable peptide attachment that will result in its release from the dendrimer once it is inside the cell in which the protease is located.
At the University of Wisconsin’s School of Veterinary Medicine in Madison, Christopher Murphy, DVM, PhD, is developing cell culture surfaces which modulate
fundamental cell behaviors such as morphology, alignment, adhesion, migration, and many others. Although “topographic cueing” is not a new concept, Murphy is one of the first to explore this phenomenon at the nanoscale level.
Most epithelial cells must adhere to and spread on a surface to survive. Normally, they adhere to basement membranes composed mainly of type IV collagen and laminin. Under the microscope, basement membranes have a felt-like appearance with pores and fibers of nanoscale dimensions. In other words, throughout its life, each epithelial cell remains in contact with thousands of topographic features that influence its function. Murphy’s aim is to fabricate well-defined biomimetic nanostructured surfaces to modulate cell behaviors by nanotopography.
Using advanced lithography, etching, and other techniques, he and members of his lab have manufactured a range of substrata with parallel nanogrooves and ridges ranging from 400 nm to 4,000 nm in width. Although these are meant to mimic the basement membrane of the human cornea, the ordered configurations of the surfaces are designed to manipulate cell behavior.
The investigators found that cells grown on these surfaces became elongated and aligned along patterns of grooves and ridges with feature dimensions as small as 70 nm, whereas cells grown on smooth substrates remained mostly round. In fact, cells were found to adhere best to smaller scale features, an interesting observation considering that the cell substrate contact surface was lowest in these examples.
The orientation of focal adhesions and stress fibers depend upon the pattern pitch (i.e., width of one groove and ridge). Cells grown on a surface with a 400 nm or 4,000 nm pitch become obliquely oriented, whereas those grown on a surface with a 1,200 nm pitch become parallel-aligned. This transition phenomenon was found to be true for several types of cell behavior. Once a pitch of 1,200 nm to 1,600 nm is reached, orientation, adhesion, cell migration, and proliferation show sudden shifts. Murphy calls this the “biomimetic zone.”
“Once you reach the biomimetic zone, it changes the anatomy and the orientation of the focal adhesions,” he says. “It even changes the orientation of the stress fibers to the underlying focal adhesions. We don’t know what that means but we do know that cytoskeletal changes translate in many cases to changes in intracellular signalling.” Murphy is using this nanotechnology to develop an artificial cornea. By using the appropriate topographic feature, he has been able to determine the direction of corneal epithelial cell migration and their level of proliferation.
Admittedly, nanotechnology has a long way to go before it yields practical treatments in mainstream ophthalmology practice. But the implications and possibilities fire the imagination like nothing else in the rapidly developing field of vision research, says Robert Ritch, MD, professor of clinical ophthalmology at the New York Medical College and chair of the symposium. “This is something that is already beginning to revolutionize our life and our society, and we can’t even imagine the scientific outgrowth that is going to come out of this.”
About the Author
Wilson is a freelance writer based in Montreal.
This article was published in Drug Discovery & Development magazine: Vol. 9, No. 9, September, 2007, pp. 56-60.
Filed Under: Drug Discovery