October 9, 2008
Gold Bullets Take Aim at Parasites
By Valenzuela, Stella Cortie, Michael
Gold nanoparticles can target and kill the parasite Toxoplasma gondii, opening the way for a new treatment for parasitic infections in humans. Over the ages, people worldwide have revered gold as a precious metal, with its uses being many and varied. In cultures where the Sun was worshiped as a deity, gold was viewed as its representative form on Earth. It has also commonly been used for decorative purposes in art, food and jewellery. Other uses include wiring in electronics, as a medium of monetary exchange and for coinage.
In addition, gold alloys and salts have been used in both dentistry and medicine, respectively. Medical uses of gold can be traced back as far as the ancient Egyptians and to early Indian and Chinese cultures, where gold preparations were considered useful in the treatment of cardiac and liver ailments, as a cure for smallpox and as an aphrodisiac. In medieval times it was also used as the basis of an "elixir of life" that could hopefully restore youthfulness. Since the early 1900s gold has been used in the treatment of rheumatoid arthritis but, due to numerous side-effects and limited efficacy, its use in this way has been generally superseded by more effective drug treatments.
With the emergence of nanotechnologies, gold has again come to the fore as a potential therapeutic agent. Today, however, most of the interest is focused on nanoparticles of metallic gold rather than on its salts. The useful features of gold in this form include resistance to oxide formation, a readiness to form uniform nanosized particles of different sizes and shapes, a surface chemistry that makes the attachment of biological molecules relatively straightforward, and special optical properties.
A nanoparticle itself is nothing too sophisticated: it is merely a little piece of solid matter measuring 1-100 nm in size. Although these nanoparticles have more in common with ordinary molecules than the high-tech nanobots promoted in popular culture, the small size imbues the gold particle with the additional useful properties of an enormous surface area and colloidal stability - and especially the ability to resonate with, and absorb, particular wavelengths of light. The absorbed light is then released as heat, and this phenomenon can be exploited to generate heat with pinpoint precision in a microstructure.
The point to note, however, is that under normal circumstances solitary, unbound nanoparticles are exceedingly rare in living systems. This is because a conspiracy of natural laws ensures that nanoparticles tend to aggregate in our bodies, or in the environment, so that they soon become quite ordinary microparticles. However, if you can stabilise them then discrete nanoparticles of gold will reveal the unique and interesting chemical and physical properties mentioned above.
The trick is to "functionalise" the nanoparticles by coating them with some useful molecule to stop them from aggregating or reacting prematurely. In the case of gold, this is usually achieved by applying a monolayer of an organic molecule or an adsorbed ion around the particle, and then using this layer as a foundation onto which additional molecules, such as antibodies, can be attached. The resulting composite particle in this case is often termed a "gold- antibody conjugate".
Conjugated gold nanoparticles are now being intensely investigated in the area of nanomedicine. In particular there is interest in using gold nanoparticles as the vector to deliver a thermal, chemical or genetic payload to a target cell type.
Here we will discuss only the exploitation of the thermal mode of operation. Much of the pioneering work in this area was done in the USA by Prof. Naomi Halas, who realised about 8 years ago that a particular type of gold nanoparticle, the gold "nanoshell", had an optical resonance that could be tuned to the so-called "tissue window". This is the region of the electromagnetic spectrum (in the near-infrared) in which our body's tissues and cells are most transparent to light.
The work being pursued by Halas is generally directed towards various types of cancer. A major challenge is to find a way to concentrate gold nanoshells or nanorods in the desired target region.
One scheme, known as "active targeting", relies upon an antibody bound to the gold nanoparticle. Depending on the antibody used, the functionalised gold particles will recognise and bind to the target cell of interest.
The next phase of the treatment is to illuminate the region containing the target tissue with a laser light of appropriate wavelength and intensity. This causes the gold particles to resonate and emit energy in the form of heat. It is the very localised rise in temperature that kills the target cells selectively.
Our work differs from that of the American group, and others in the field, because we have sought to target the infectious parasite Toxoplasma gondii rather than cancer cells. This parasite is responsible for the intracellular infection known as toxoplasmosis.
T. gondii infects a number of warm-blooded animals, including humans, birds and livestock. It has a complex life cycle that includes both sexual and asexual reproduction.
Cats, both domesticated and wild, are the definitive hosts of T. gondii. This means that the parasite is only able to sexually reproduce in cats, but it can reproduce a sexually in many other mammals.
Almost half of all humans have been exposed to T. gondii, and therefore infection and the risk of disease is relatively high. Usually infection in healthy human adults results in no symptoms, and therefore most people don't even know they have been exposed.
Unfortunately, this is not the case for pregnant women, as toxoplasmosis can cause abortion and congenital defects to the unborn child. Similarly, immunocompromised individuals such as people infected with HIV, organ transplant recipients and patients undergoing certain types of chemotherapy are at a higher risk, with severe toxoplasmosis potentially causing damage to the eyes, brain and other organs.
To date, most pharmaceutical compounds are not entirely specific in their action, and they may cause undesired effects on healthy body cells. The advantage of using gold nanoparticles, functionalised with antibodies and laser treatment, over conventional drug therapy is that it offers opportunities to improve the specificity of targeting in therapeutic technologies. Not only can the gold particles be tailored to bind to specific targets but, in combination with controlled laser beam exposure, the area of the body to be treated can be specifically defined.
In addition, side-effects are minimised by the fact that the wavelength of light needed to activate and cause heating of the gold particles coincides with the region of maximum transparency of the body's tissues. Therefore, non-target tissues are not subject to adverse effects by the laser.
Our project at University of Technology Sydney (UTS) has been carried out by PhD student Ms Dakrong Pissuwan, with assistance from the UTS Institute for the Biology of Infectious Diseases. In this work we functionalised both spherical and rod-shaped gold nanoparticles with an antibody that specifically binds to the tachyzoite phase of T. gondii's life cycle. In this way we make the gold nanoparticles target and bind to the tachyzoites but not to the host body cells.
The gold-antibody conjugate particles are then incubated with a culture containing a mixture of live Toxoplasma organisms and mammalian host cells. The gold nanoparticles stick selectively to the tachyzoites, which can then be killed by illuminating the culture with low power laser light tuned to the particular optical extinction peak of the nanoparticle.
So far Pissuwan has shown that selective targeting and destruction is readily achieved in vitro. Exposure to a single laser treatment of 10 minutes resulted in the killing of four times as many tachyzoites that were treated with the gold-antibody conjugate particles compared with controls. Similarly, host cells that did not have gold-antibody conjugate particles attached to them were also shown to have no significant cell death after exposure of the laser light alone.
These results are extremely promising as we now have demonstrated both an effective way of targeting and killing parasitic cells without causing damage to host cells. The challenge that now lies ahead is to translate and apply this approach to an in vivo system, where we would need to show significant mortality of an infecting parasite within a live host.
These are exciting times, where the coming together of discipline areas such as medicine, biology and physics is allowing us to exploit new technologies to treat the growing list of diseases for which purely chemical treatments are now less effective. Treatments built on one or more of the unique physical properties of nanoparticles can target diseases in ways that conventional treatments cannot.
Small yet significant steps, such as the ones we have described here, are providing the impetus for the next breakthrough in the biomedical sciences.
Target cells (in this case mouse macrophage cells, a type of white blood cell) to which antibody-functionalised gold nanoparticles have been attached. The particles are made visible by the secondary application of a fluorescent dye. Photo: Ms Dakrong Pissuwan]. Dr Stella Valenzuela is Senior Lecturer at the Department of Medical and Molecular Biosciences, University of Technology, Sydney. Prof Michael Cortie is Director of the Institute for Nanoscale Technology, University of Technology, Sydney.
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