Dr. Silvia Muro, one of the newest faculty members at UMBI's Center for Biosystems Research (CBR), is finding new ways to deliver lifesaving drugs to the targets in the body where they are most needed. She has focused her research on drug delivery with sub-cellular precision, based on the understanding and utilization of the normal mechanisms by which substances are transported to particular compartments within cells.

The key is to use endocytic pathways, by which cells engulf substances so that they can be brought to the interior of the cells, or in some cases, transported across the cells to other cellular targets. Cells engulf the substances in membrane-bound compartments, called vesicles, which can then be transported within the cellular interior. These vesicles preferentially form when specific substances bind to specific receptors on the cells. Some of these receptors trigger endocytic pathways: the substances bind to the receptors, are bound into vesicles, and then the vesicles containing the substances are transported within the cell. Therefore, "the key to controlled transport is to make use of existing endocytic receptors," says Dr. Muro.

And that is precisely what she does in the laboratory. One major focus of her research has been to rely on naturally-occurring receptors. While Dr. Muro is currently exploring a number of endocytic receptors, in the past, she has mostly focused on ICAM-1 receptors. The density of these receptors increases during a number of pathological conditions, making this an ideal receptor system for controlled transport into diseased cells.

Dr. Muro takes advantage of this opportunity by targeting therapeutics to ICAM-1 receptors. To do this, she couples submicroscopic particles----nanoparticles----to antibodies that bind tightly to ICAM-1 receptors, and then coats these particles with the therapeutics that need to be transported into the cells.

Dr. Muro and colleagues published a paper in the Journal of Cell Science in 2003, describing the pathway by which cells engulf and transport ICAM-1-targeted particles. This was the first description of this pathway. The importance of this paper was marked by an editorial in that same journal. Now, eight years after beginning those studies, the ICAM-1 mediated pathway is recognized as a new route of endocytic transport.

That publication laid the groundwork for all that followed. In 2004, while working at the University of Pennsylvania, Dr. Muro was awarded a prestigious four year grant--for development of promising scientists--from the American Heart Association. This award was continued by a R21 award from NIH, which permitted her to conduct some of the early critical experiments that have led to her current research at UMBI.

These awards focused on one of the applications in which Dr. Muro is most interested: the treatment of lysosomal storage diseases (LySD)----genetic diseases involving enzymes in the compartments of cells----lysosomes----responsible for degradation of defective and toxic substances, as well as certain cell nutrients.

(Fluorescently-labeled particles (green color), internalized by endothelial cells, are contained in vesicles surrounding the cell nucleus (red color))

LySD lead to toxic accumulation of substances in cells, leading to cellular dysfunction and multiple organ failure, and are often fatal in the first months or years of life. There is no current cure. Gaucher syndrome was the first LySD that was described in 1882, and occurs in about 1 in 25,000 live births. This was followed by Fabry disease in 1898. Identification of the lysosomal compartment of cells in the 1950s led to an understanding of the physiological mechanism of LySD, and numerous other LySD have since been described.

An interesting approach to treatment of LySD, known as enzyme replacement therapy, was approved for clinical use about 10 to 12 years ago. In this approach, the lysosomal enzyme that is missing in the patient is infused intravenously. This approach is not very effective, however, since the enzyme is removed from the circulation by the liver and spleen. The other critical organs that need the missing enzyme----such as lungs, heart, kidneys, and brain----do not take up significant amounts, and consequently fail.

By targeting enzymes using particles addressed to endocytic receptors in these organs, Dr. Muro hopes to improve treatment for these diseases. In her earlier experiments on this application, she used polystyrene latex particles, which made a great model system for study, but are not ideal for medical use. However, in more recent experiments, she has switched to PLGA particles (poly-lactic-glycolic-acid)----a safe, biodegradable material that was already approved by FDA for use in surgical sutures. The delivery system consists of the biodegradable PLGA particles which are coupled to ICAM-1 antibodies, and coated in therapeutic enzymes. The antibodies bind to the ICAM-1 receptors and deliver the enzymes to the lysosomes via endocytosis, where they are needed. The nanoparticles made from PLGA are naturally degraded by the cells of the target organs. As they are degraded, the particles release their payload of lifesaving enzymes inside of the organs that need them.

Results from this new delivery system are very promising, both in cell cultures and in experiments involving mice. There is clear evidence, using tools such as radioisotope tracing, fluorescence and electron microscopy, and enzymatic activity, that large amounts of the enzymes are delivered to target organs such as heart and lungs. Unlike enzyme replacement therapy----in which most of the enzymes remain in the blood, and are cleared by organs such as the liver----Dr. Muro's ICAM-1 targeted nanoparticles deliver much larger amounts of enzymes to the cells, and a much smaller proportion of the enzymes remain in the blood. With the ICAM-1 targeted particles, as much as 80% of the injected enzymes can actually get into the cells of organs that need them, such as the lung which received 20% of the injected enzyme.

This promising work has resulted in numerous publications over the years, including papers in Current Vascular Pharmacology in 2004, Blood in 2005 and 2008, the American Journal of Physiology in 2006, Molecular Therapies in 2006 and 2008, the Journal of Pharmacology and Experimental Therapeutics in 2008, Journal of Controlled Release in 2008, and others.

Recently, Dr. Muro has found that the size and shape of the particles for these targeted therapeutics plays an important role in the effectiveness of delivery. In addition to lysosomes, another cellular compartment----the endosome----is also of interest in targeted therapeutics. Dr. Muro's team found that an oblong shape of microscopic dimensions proved most effective for delivery to endosomal compartments, while sub-microscopic spheres were more efficient for lysosomal delivery. This work was featured on the cover of the journal of Molecular Therapy in 2008.

Now Dr. Muro is also exploring the ability of these delivery methods to cross the blood-brain barrier. This research is based on an understanding and utilization of the normal mechanisms by which substances are transported from the circulation into the cells of target organs. Much of this transport takes place in capillaries: the finely branched blood vessels that connect tissues to the blood supply.

(Fluorescently-labeled particles (green color), internalized by endothelial cells, traffic within the cell body helped by the cell actin cytoskeleton (red color))

Capillaries have a single layer of endothelial cells that separate cells of various organs from the bloodstream. These endothelial cells control the transport of substances to organs across the capillaries. Transport of substances other than small ions and molecules is controlled by two major transport routes. The first route, the para-cellular route, is accomplished by means of opening of gaps, and leakage of substances----or even whole cells, such as white blood cells----between the endothelial layer. This is the route that is often used by the body during physiological emergencies, such as infections. This pathway, since it involves gaps and leaks, does not permit much control over the substances that pass from the bloodstream to the organs.

The second major route is the trans-cellular route, in which substances are transported through the endothelial cells in a controlled fashion. In this pathway, cells engulf material from blood in membrane-bound compartments, called vesicles, which then cross the endothelial cells in a controlled manner, and are released when the vesicles open on the other side of the cells, a process known as transcytosis. This is a safer, more controlled method for transporting substances than the leaky para-cellular route.

So to lay the groundwork of research that may someday lead to clinical treatment of LySD (and other diseases requiring targeted delivery of lifesaving drugs), Dr. Muro asked the following question: Is it possible to deliver therapeutic enzymes to their target organs by making use of the endothelial transport pathways?

"From the perspective of transporting therapeutics in a safe manner, the trans-cellular pathway would be the goal," says Dr. Muro. This specific, controlled mechanism prevents leakage of unwanted substances or cells.

Now, Dr. Muro is looking more closely at the various cellular mechanisms by which the particles can be transported across endothelial cells. She is looking forward to establishing a colony of experimental mice at UMBI, deficient in key enzymes, which will allow her to do more detailed whole-organism (in vivo) studies.

Dr. Carmen Garnacho, a postdoctoral researcher working with Dr. Muro, assisted in the work that was performed both at the University of Pennsylvania and at UMBI. She recently moved back to Spain. Currently, in her laboratory at UMBI, Dr. Muro has been mentoring graduate students from three different programs who have been doing rotations in the lab. Two of these rotating students were able to submit abstracts for international release.

One student, Daniel Serrano, has recently joined Dr. Muro's lab, and will be pursuing his Masters and PhD. degrees there. Dr. Muro is also currently mentoring a postdoctoral researcher, Dr. Tridib Bhowmick, an undergraduate student from the Biology Honors program, Rasa Ghaffarian, and a senior student from the Eleanor Roosevelt High School Science&Technology Program, Ashlee Greene. Looking to the future, Dr. Muro has submitted several grant applications that would enable her to further expand the scientific talent in her lab.

Dr. Muro has tremendous enthusiasm and energy for her research----both for the science, and for the future clinical applications. "I am very passionate about my work----I really love it," she says.