Scientists Engineer Nanoscale Vaults to Encapsulate 'Nanodisks' for Drug Delivery

ScienceDaily (Apr. 21, 2011) — There's no question, drugs work in treating disease. But can they work better, and safer? In recent years, researchers have grappled with the challenge of administering therapeutics in a way that boosts their effectiveness by targeting specific cells in the body while minimizing their potential damage to healthy tissue.

Vault containing nanodisk. The image shows a single-particle electron microscope tomography reconstruction, which reveals that a fully assembled drug-loaded nanodisk (red) can be packaged into the vault lumen (green) as a viable method for vault-mediated drug delivery. The electron micrograph in the background shows negatively stained vaults containing nanodisks.

The development of new methods that use engineered nanomaterials to transport drugs and release them directly into cells holds great potential in this area. And while several such drug-delivery systems -- including some that use dendrimers, liposomes or polyethylene glycol -- have won approval for clinical use, they have been hampered by size limitations and ineffectiveness in accurately targeting tissues.

Now, researchers at UCLA have developed a new and potentially far more effective means of targeted drug delivery using nanotechnology.

In a study to be published in the May 23 print issue of the journal Small, they demonstrate the ability to package drug-loaded "nanodisks" into vault nanoparticles, naturally occurring nanoscale capsules that have been engineered for therapeutic drug delivery. The study represents the first example of using vaults toward this goal.

The UCLA research team was led by Leonard H. Rome and included his colleagues Daniel C. Buehler and Valerie Kickhoefer from the UCLA Department of Biological Chemistry; Daniel B. Toso and Z. Hong Zhou from the UCLA Department of Microbiology, Immunology and Molecular Genetics; and the California NanoSystems Institute (CNSI) at UCLA.

Vault nanoparticles are found in the cytoplasm of all mammalian cells and are one of the largest known ribonucleoprotein complexes in the sub-100-nanometer range. A vault is essentially barrel-shaped nanocapsule with a large, hollow interior -- properties that make them ripe for engineering into a drug-delivery vehicles. The ability to encapsulate small-molecule therapeutic compounds into vaults is critical to their development for drug delivery.

Recombinant vaults are nonimmunogenic and have undergone significant engineering, including cell-surface receptor targeting and the encapsulation of a wide variety of proteins.

"A vault is a naturally occurring protein particle and so it causes no harm to the body," said Rome, CNSI associate director and a professor of biological chemistry. "These vaults release therapeutics slowly, like a strainer, through tiny, tiny holes, which provides great flexibility for drug delivery."

The internal cavity of the recombinant vault nanoparticle is large enough to hold hundreds of drugs, and because vaults are the size of small microbes, a vault particle containing drugs can easily be taken up into targeted cells.

With the goal of creating a vault capable of encapsulating therapeutic compounds for drug delivery, UCLA doctoral student Daniel Buhler designed a strategy to package another nanoparticle, known as a nanodisk (ND), into the vault's inner cavity, or lumen.

"By packaging drug-loaded NDs into the vault lumen, the ND and its contents would be shielded from the external medium," Buehler said. "Moreover, given the large vault interior, it is conceivable that multiple NDs could be packaged, which would considerably increase the localized drug concentration."

According to researcher Zhou, a professor of microbiology, immunology and molecular genetics and director of the CNSI's Electron Imaging Center for NanoMachines, electron microscopy and X-ray crystallography studies have revealed that both endogenous and recombinant vaults have a thin protein shell enclosing a large internal volume of about 100,000 cubic nanometers, which could potentially hold hundreds to thousands of small-molecular-weight compounds.

"These features make recombinant vaults an attractive target for engineering as a platform for drug delivery," Zhou said. "Our study represents the first example of using vaults toward this goal."

"Vaults can have a broad nanosystems application as malleable nanocapsules," Rome added.

The recombinant vaults are engineered to encapsulate the highly insoluble and toxic hydrophobic compound all-trans retinoic acid (ATRA) using a vault-binding lipoprotein complex that forms a lipid bilayer nanodisk.

The research was supported by the UC Discovery Grant Program, in collaboration with the research team's corporate sponsor, Abraxis Biosciences Inc., and by the Mather's Charitable Foundation and an NIH/NIBIB Award.

Molecule That Can Increase Blood Flow in Vascular Disease Identified

ScienceDaily (Apr. 21, 2011) — Circulating through the bloodstream of every human being is a rare and powerful type of cell, one that can actually create new blood vessels to bypass blockages that cause heart attacks and peripheral artery disease. Though everyone has these cells -- called endothelial progenitor cells -- they are often dysfunctional in people prone to vascular disease.

Human endothelial progenitor cells grown in the lab (left) and forming capillary tube like structures (right).

Now researchers at the University of North Carolina at Chapel Hill have discovered that a molecule -- called Wnt1 -- can improve the function of endothelial progenitor cells, increasing the blood flow to organs that previously had been cut off from the circulation. The finding could enhance clinical trials already testing these powerful cells in patients hospitalized with cardiac arrest.

"The premise of these trials is that these cells will supply the ischemic organ with new blood vessels and allow the damaged organ to function better," said senior study author Arjun Deb, MD, assistant professor of medicine in the UNC School of Medicine. "But because you are isolating these cells from the patients themselves, you know that the cells are dysfunctional -- so the approach is almost flawed from the very beginning. We want to see how we can improve the function of these cells so they can do their job better."

The study, published online Feb. 14, 2011, in the FASEB (Federation of American Societies for Experimental Biology) Journal, is the first to show that the Wnt1 protein, one of a family of 19 such molecules, can stimulate blood vessel formation.

A number of studies in the past few years have suggested that genes that play an important role during early development and get "turned off" during adulthood may also get "turned on" or expressed again in response to injury, such as heart attack.

Deb, who studies the Wnt family of developmental genes, looked to see if any of its members follow this same pattern. He found that one gene in particular, Wnt1, was expressed during development of blood vessels, shut off during adulthood and then re-expressed in angiosarcoma, a cancer of endothelial cells.

Deb wanted to see what would happen if he put the Wnt1 protein on human endothelial progenitor cells. He found that treating these special cells with Wnt1 not only greatly increased their function but also their number. Next, Deb and his colleagues investigated what effect the protein would have on a mouse model of peripheral artery disease, an illness in humans caused by decreased blood flow to the extremities. They found that treating these animals with a single injection of the Wnt1 protein resulted in almost three fold increase in blood flow in the affected areas.

"We found that Wnt1 is a novel proangiogenic molecule, something that has never been shown before," said Deb. "It gives us hope that injecting the Wnt1 protein -- or molecules that stimulate the Wnt1 signaling pathway -- into ischemic tissues in humans could improve blood flow and assert a therapeutic effect."

Approximately 1 in 4 deaths in adults in the US are secondary to heart disease and as many as 15 percent of Americans age 65 and older have peripheral artery disease. In the future, Deb plans to use his findings to identify such small molecules or drug candidates that could reverse the endothelial progenitor cell dysfunction observed in so many patients with vascular disease.

The research was funded by the National Institutes of Health and Ellison Medical Foundation. Study co-authors were Costin M. Gherghe, MD, PhD, postdoctoral fellow in Deb's lab; Jinzhu Duan, PhD, postdoctoral fellow in Deb's lab; Jucheng Gong, lab manager in Deb's lab; Mauricio Rojas, MD, MPH, director of mouse cardiovascular models core lab; Nancy Klauber-Demore, MD, associate professor of surgery; and Mark Majesky, PhD, Professor of Pediatrics, University of Washington, Seattle.

Genetic Discovery Offers New Hope in Fight Against Deadly Pulmonary Fibrosis

ScienceDaily (Apr. 21, 2011) — A team led by researchers at National Jewish Health has discovered a new genetic variation that increases the risk of developing pulmonary fibrosis by 7 to 22 times. The researchers report in the April 21, 2011, issue of The New England Journal of Medicine that nearly two-thirds of patients with idiopathic pulmonary fibrosis or familial interstitial pneumonia carry the genetic variation. It is associated with the MUC5B gene, which codes for a mucus-forming protein.

"This discovery not only identifies a major risk factor for pulmonary fibrosis, but also points us in an entirely new direction for research into the causes and potential treatments for this difficult disease," said Max Seibold, PhD, first author and research instructor at National Jewish Health and the Center for Genes, Environment and Health. "The research also demonstrates how a genetic approach to disease can uncover a previously unknown and unsuspected association disease."

Idiopathic pulmonary fibrosis (IPF) and familial interstitial pneumonia (FIP) are similar, invariably fatal diseases that involve progressive scarring of the lungs. The scarring prevents oxygen transport to the tissues, and most people die of respiratory failure within a few years of diagnosis. The diseases are relatively rare, but account for approximately 40,000 deaths each year, the same number as die of breast cancer. There is no approved treatment for the diseases.

Research into pulmonary fibrosis has been quite difficult. Little is understood about the biological roots of the diseases, and recent clinical trials of several experimental medications have failed to effectively treat them. Previous research has focused primarily on the scarring and inflammatory processes evident in the disease.

In the study funded by the National Heart, Lung and Blood Institute, National Jewish Health researchers and their colleagues took an "agnostic" approach, statistically analyzing the entire genome of 82 afflicted families. They found an association with the diseases in a region of chromosome 11 that contains four mucin genes involved in the production of mucus. Narrowing their search with fine mapping, then sequencing, they eventually found a common variation near the MUC5B gene, presumably in a regulatory element, that is strongly associated with the disease.

"This research suggests that mucus production where the small airways and the air sacs converge may play a significant role in pulmonary fibrosis," said senior author David Schwartz, MD, Chair of the Department of Medicine at the University of Colorado School of Medicine and Director of the Center for Genes, Environment and Health at National Jewish Health.

The variation exists in 19 percent of healthy controls, 59 percent of FIP patients, and 67 percent of IPF patients. Carrying one copy of the gene increases the risk of developing FPF by 6.8 times, and IPF by 9.0 times. Carrying two copies of the variation increases risk 20.8 times and 21.8 times, respectively.

The researchers discovered that the genetic variant increases production of MUC5B more than thirtyfold in unaffected patients. They also found that MUC5B production is elevated in pulmonary fibrosis patients both with and without the gene.

"There are several biologically plausible ways in which excess mucus could cause disease," said Dr. Schwartz. "We are currently investigating all of these mechanisms as potential causes of disease."

Mucus is a vital part of lung biology, protecting delicate cells from direct exposure to inhaled irritants and toxins, and helping to clear them from the lungs. The researchers hypothesize that excess mucus production caused by the MUC5B variant could slow clearance of mucus contaminated with irritants and toxins. Excess mucus might also interfere with repair of air sacs damaged by these contaminants. Another scenario suggests that the genetic variation could trigger the production of mucus in areas where it is not normally present. In addition to National Jewish Health, other institutes contributing to this study were the University of Colorado School of Medicine, Colorado School of Public Health, Duke University Medical Center, North Carolina State University, Vanderbilt University School of Medicine, Landspitali University Hospital, in Reykjavik, Iceland, the University of Texas M. D. Anderson Cancer Center, the University of Miami, and the National Institute of Environmental Health Sciences.

What's Your Gut Type? Gut Bacteria Could Help With Diagnostics and Influence Treatments

ScienceDaily (Apr. 21, 2011) — In the future, when you walk into a doctor's surgery or hospital, you could be asked not just about your allergies and blood group, but also about your gut type. Scientists at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, and collaborators in the international MetaHIT consortium, have found that humans have three different gut types.

This is an artistic impression of the three human gut types.

The study, published in Nature, also uncovers microbial genetic markers that are related to traits like age, gender and body-mass index. These bacterial genes could one day be used to help diagnose and predict outcomes for diseases like colo-rectal cancer, while information about a person's gut type could help inform treatment.

We all have bacteria in our gut that help digest food, break down toxins, produce some vitamins and essential amino acids, and form a barrier against invaders. But the composition of that microbial community -- the relative numbers of different kinds of bacteria -- varies from person to person.

"We found that the combination of microbes in the human intestine isn't random," says Peer Bork, who led the study at EMBL: "our gut flora can settle into three different types of community -- three different ecosystems, if you like."

Bork and colleagues first used stool samples to analyse the gut bacteria of 39 individuals from three different continents (Europe, Asia and America), and later extended the study to an extra 85 people from Denmark and 154 from America. They found that all these cases could be divided into three groups, based on which species of bacteria occurred in high numbers in their gut: each person could be said to have one of three gut types, or enterotypes.

The scientists don't yet know why people have these different gut types, but speculate that they could be related to differences in how their immune systems distinguish between 'friendly' and harmful bacteria, or to different ways of releasing hydrogen waste from cells.

Like blood groups, these gut types are independent of traits like age, gender, nationality and body-mass index. But the scientists did find for example, that the guts of older people appear to have more microbial genes involved in breaking down carbohydrates than those of youngsters, possibly because as we age we become less efficient at processing those nutrients, so in order to survive in the human gut, bacteria have to take up the task.

"The fact that there are bacterial genes associated with traits like age and weight indicates that there may also be markers for traits like obesity or diseases like colo-rectal cancer," Bork says, "which could have implications for diagnosis and prognosis."

If this proves to be the case, when diagnosing or assessing the likelihood of a patient contracting a particular disease, doctors could look for clues not only in the patient's body but also in the bacteria that live in it. And after diagnosis, treatment could be adapted to the patient's gut type to ensure the best results.

Genes Causing Antimalarial Drug Resistance Identified

ScienceDaily (Apr. 21, 2011) — Using a pair of powerful genome-search techniques, researchers from the Harvard School of Public Health (HSPH), Harvard University, and the Broad Institute have identified several genes that may be implicated in the malaria parasite's notorious ability to rapidly evade drug treatments. Further testing revealed that one of the genes, when inserted into drug-sensitive parasites, rendered them less vulnerable to three antimalarial drugs.

The successful experiments suggest that the genomic methods are useful tools for probing the genetic mechanisms underlying drug resistance in the Plasmodium falciparum malaria parasite and potentially other types of disease-causing parasites as well.

The study appears online April 21, 2011, in PLoS Genetics, and is timed to coincide with World Malaria Day on April 25.

"Identification of mutations associated with drug resistance helps us understand how the parasite evades the effects of the drug," said Sarah Volkman, senior research scientist at HSPH and a co-senior author of the paper. "Once we understand the processes used by the parasite to avoid the effects of the antimalarial treatment, scientists can develop new drugs that circumvent the strategies employed by the drug-resistant malaria parasite."

In addition, said Volkman, knowing the mutations that signal that a parasite has become resistant to an antimalarial compound allows researchers to develop tools that can be used for monitoring and surveillance of drug-resistant parasites.

Reducing the toll of malaria, which kills nearly a million people a year--mainly young children in sub-Sahara Africa--is a major challenge because of the parasite's talent for swiftly developing resistance to multiple drugs. To counter the shape-shifting parasite's defenses, scientists say they must improve on their meager understanding of the molecular and genetic mechanisms of resistance.

Genetically diverse populations of the blood-borne malaria parasite are endemic in Africa, Asia, and South America. When exposed to antimalarial drugs and the human immune system, Plasmodium falciparum has a remarkable ability to quickly generate resistant clones of parasites, a major obstacle to successful treatment.

For the study, the scientists, including Volkman, Dyann Wirth, and co-first author Daria Van Tyne of HSPH and the Broad, co-first author Danny Park and Pardis Sabeti of the Broad and Harvard University, and Daniel Neafsey and Stephen Schaffner of the Broad, analyzed the DNA of 57 parasites from the three continents, using a high-density genome-wide array that examines more than 17,000 mutations. They also measured the parasites' responses to 13 antimalarial drugs.

The scientists examined diversity of the parasite to identify 20 rapidly evolving loci in the genome, and then carried out a genome-wide association study (GWAS) to identify genetic variants that correlated with or are associated with the drug-resistance trait. These genetic variants are necessarily enriched in the drug-resistant, but not drug-sensitive parasites, allowing the researchers to home in on the candidate genes that are involved in modulating drug responses. That search netted 11 genes implicated in drug resistance -- one previously known and others discovered for the first time.

Van Tyne pursued one of the novel genes, PF10_0355, for follow-up functional testing. She used an experimental technique that introduced extra copies of the gene from a resistant parasite into a drug-sensitive one, and found that the formerly sensitive parasite was now rendered more resistant to three standard antimalarial agents.

"This demonstration suggests that the gene is involved in modifying parasite drug response," said Van Tyne, a graduate student in the laboratory of Wirth, chair of the Department of Immunology and Infectious Diseases at HSPH and a co-director of the Infectious Disease Initiative at the Broad. "We feel that this is one gene of potentially many that affect drug-resistance mechanisms. We're now working to follow up and understand how these and the other genes identified work."

Drug resistance is a major concern that threatens to undermine global efforts to control or eradicate malaria. Understanding how the parasite is changing before clinical drug resistance is apparent offers some hope that we might be able to extend the useful life of available drugs and identify new effective antimalarials, said Volkman.

The study was funded by the Bill and Melinda Gates Foundation, the Ellison Medical Foundation, the ExxonMobil Foundation, the Fogarty International Center at the NIH and the National Institute of Allergy and Infectious Diseases.