Nanomedicine Approaches for the Design
and Development of Therapeutic Agents in the
Treatment of Pseudoxanthoma Elasticum (PXE)
and Age Related Macular Degeneration

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By Ravi Shukla
Raghuraman Kannan
Dean Hainsworth
Kavita Katti
Kattesh V. Katti

Editor’s Note: This article reviews research partially funded by NAPE last year and plans for continuing research during the coming year. The presentation at the Salt Lake City conference was introduced with basic information about nanotechnology and its application to medicine. The following article, based on that presentation, does not include the introduction. Instead, readers are encouraged to read “Nanomedicine: Should NAPE Be Interested?” by Kattesh Katti and Raghuraman Kannan (PXE Awareness, November 2006). That article explains how nanomedicine has been applied successfully to the diagnosis and treatment of prostate cancer and suggests its potential for treatment of AMD, PXE and related retinal disorders.

Hypothesis

Dr. Kattesh Katti and his colleagues have built a record of discovery in the diagnosis and treatment of cancers which has brought recognition and support from the National Institutes of Health. Dr. Katti and Dr. Raghuraman Kannan have discovered a library of biocompatible and tumor specific gold nanoparticles that have resulted in recognition by the National Cancer Institute. Among their discoveries is that early stages of various human cancers manifest formation and growth of new blood vessels – a process termed ‘angiogenesis.’ Eye disorders such as PXE and AMD manifest similar blood vessel growth. From this, they hypothesized that nanoparticle-based targeting techniques which can be used for the diagnosis and treatment of cancers should apply to the treatment of various disorders of the eye, including PXE and AMD.

AMD/PXE

Age related macular degeneration (AMD), the leading cause of blindness affecting 90% of human populations across the globe, is characterized as a chronic degenerative disease that can lead to vision loss in either its dry or wet forms. In advanced wet AMD, new leaky blood vessels can cause rapid damage to the retina, typically resulting in central vision loss. The current treatment for wet AMD is repeated needle injections into the eye of Genentech’s Lucentis or Avastin. These drugs target and inhibit vascular endothelial growth factor (VEGF). VEGF is a sub-family of growth factors, specifically, of platelet-derived growth factor or cystine-knot growth factors. They are important signaling proteins involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of leaky blood vessels from pre-existing vasculature).

Research Plan

The similarity of the angiogenesis morphology in the growth of blood vessels in cancer and PXE/AMD led Dr. Katti’s group to work on targeted nanoparticles. Their overall goal is to develop a Super Avastin to achieve superior visual acuity outcomes with fewer intraocular injections. They also are working to develop other new products for easier administration of medications in retinal disorders. This presentation focuses on details of the development of Super Avastin and related animal models to demonstrate retention of Super Avastin within the retina.

Gold nanoparticles have a great capacity for storing diagnostic and therapeutic pharmaceuticals on their surfaces. Because each nanoparticle can carry thousands of atoms on the surface, each atom can be attached with a specific pharmaceutical. For example, if each atom is attached with Avastin, a Super Avastin is created to deliver hundreds/thousands of Avastin molecules in one injection. Traditional Avastin injections deliver only a few molecules of Avastin within the retina. Gold nanoparticle-bound Avastin can deliver hundreds of molecules of Avastin without crossing the toxic threshold.

Nano Sizes

The size relations of gold nanoparticles to proteins, cells and various constituents of the living system is important since such gold nanoparticles can be designed in sizes which penetrate into blood vessels. Thus, VEGF-inhibiting Avastin, coated on gold nanoparticles, can directly penetrate blood vessels in patients with PXE/AMD to improve the therapeutic efficacy far better than traditional injections.

Non-Toxic Gold Nanoparticles

The discovery of the process for forming biocompatible gold nanoparticles using Gum Arabic as a stabilizing agent has opened the door for its use in medical applications. Gum Arabic is widely accepted in the food and pharmaceutical industries. Several tests recently performed at the National Institutes of Health funded Nanocharacterization Laboratory in Frederick, MD, have confirmed the non-toxic features of Gum Arabic stabilized gold nanoparticles. This means they can be used to develop non-toxic applications for treating cancer and PXE/AMD. The discovery, published in 2007, has been cited widely in science and medical media.

It was learned also that the imaging capabilities of biocompatible Gum Arabic protein-coated gold nanoparticles are quite remarkable. Such images provide realistic opportunities for the use of biocompatible gold nanoparticles as diagnostic agents for the detection of early stage cancer, AMD, PXE and related diseases which manifest growth of blood vessels and leaky vasculature.

Targeting Disease Sites

A sense of direction can be attached to gold nanoparticles by tagging them with certain types of chemical molecules called peptides. Specific peptides, such as bombesin peptide, have affinity to cancer cell receptors found in prostate and breast tumors. Attaching gold nanoparticles to tumor targeting peptides allows them to home in on tumor sites when injected in cancer patients. Once gold nanoparticles enter tumor sites, they can be used for both diagnosis and therapy. Similarly released Avastin bound gold nanoparticles should persist within the retina for a longer time than that achieved by traditional injection. Longer retention of Avastin in the retina would mean less frequent need for injections.

Blood Vessel Penetration

Because gold nanoparticles have a high affinity for blood vessels present in angiogenesis in retinal regions of PXE/AMD patients, administration of Avastin-conjugated gold nanoparticles allows penetration into blood vessels. This provides a highly effective delivery mechanism for Avastin at angiogenesis sites. It is most interesting to recognize that gold nanoparticles even without Avastin might be used in reducing/eliminating leaky blood vessels in PXE/AMD.

Animal Studies

Brown Norway rats were chosen for initial study due to their eye pigmentation characteristics. Rats were injected with 5 µL hybrid gold nanoparticles intravitrially and the eyes were visualized with a fundus camera. The gold nanoparticle solution was rapidly dispersed throughout the retina and the blood vessels could not be visualized. When fluorescence angiography was performed seven days post-nanoparticle injection, the presence of gold nanoparticles in the retina was noted. Gold nanoparticle aggregates were found in blood vessels over the retina as well as in laser-induced choroidal neovascular lesions.

In order to quantify the amount of nanoparticles associated with the retina during the course of our studies, rats were sacrificed on day one as well as seven days post-nanoparticle injection. Eyes were enucleated and dissected in two major steps wherein retina and sclera were separated from the rest of the eye including cornea, lens, iris and vitreous. Gold distribution in the tissues was studied by Neutron Activation Analysis. Results confirmed that 95% of the injected gold nanoparticles went to the retina. To further confirm and visualize the presence of gold nanoparticles in the retina, the retinas were detached from sclera seven days post-nanoparticle injection and both sides of the retinas were processed for scanning electron microscopy. Back scatter images of both sides of the retina confirmed the presence and association of gold nanoparticles. Study results clearly demonstrate the selective retinal localization of gold nanoparticles in the eye.

Avastin vs Super Avastin Retention in the Eye

The traditional biological half-life of Avastin in eyes is around 8-10 hours. However, Super Avastin which was produced through the conjugation of Avastin on gold nanoparticles, exhibited the presence of Avastin in the retina and sub-retinal space even after seven days post-nanoparticle injection. Immunohistological findings, one hour post-injection, suggest that gold nanoparticle aggregates are visible beneath the lens in supra-retinal space. Indeed, the clearance of nanoparticles from the supra-retinal regions increased with time providing strong evidence for the interaction of nano aggregates with the retina and associated blood vessels. In sharp contrast to traditional Avastin, seven days post-Super Avastin injection high fluorescence was observed confirming increased biological half-life of Avastin following conjugation to gold nanoparticles.

Summary

In summary, gold nanoparticles conjugated with Avastin are biocompatible. Super Avastin nanoparticles selectively localized and penetrated through the retina and could, therefore, be clinically used to deliver Avastin, related drugs or antibodies into eyes. Our studies also demonstrate that gold nanoparticles under specific concentration ranges do not lead to ocular toxicity, thus presenting a realistic prospect for the creation of a new generation of gold nanoparticulate-based diagnostic and therapeutic agents for the treatment of PXE, AMD and related ophthalmic diseases and disorders. In the coming year our team will continue to investigate and refine these conclusions.

ACKNOWLEDGEMENTS

This work has been supported by funds from the National Association for Pseudoxanthoma Elasticum and the National Cancer Institute through a Cancer Nanotechnology Platform grant.