Nanoparticles With Honeycomb Cavities Containing Drugs Blast Cancer Cells

ScienceDaily (Apr. 20, 2011) — Melding nanotechnology and medical research, Sandia National Laboratories, the University of New Mexico, and the UNM Cancer Research and Treatment Center have produced an effective strategy that uses nanoparticles to blast cancerous cells with a mélange of killer drugs.

The figure on the left (Hep3B) shows a greenly fluoresced cancerous liver cell penetrated by protocells. The small red dots are lipid bilayer wrappings. Their cargo — drug-filled nanoparticles, their pores here filled with white fluorescent dyes for imaging purposes — penetrate the cancerous cell. (Penetration is more clearly seen in the second image.) The normal cell on the right (hepatocyte) shows no penetration.

In the cover article of the May issue of Nature Materials, available online April 17 , the researchers describe silica nanoparticles about 150 nanometers in diameter as honeycombed with cavities that can store large amounts and varieties of drugs.

"The enormous capacity of the nanoporous core, with its high surface area, combined with the improved targeting of an encapsulating lipid bilayer [called a liposome], permit a single 'protocell' loaded with a drug cocktail to kill a drug-resistant cancer cell," says Sandia researcher and UNM professor Jeff Brinker, the principal investigator. "That's a millionfold increase in efficiency over comparable methods employing liposomes alone -- without nanoparticles -- as drug carriers."

The nanoparticles and the surrounding cell-like membranes formed from liposomes together become the combination referred to as a protocell: the membrane seals in the deadly cargo and is modified with molecules (peptides) that bind specifically to receptors overexpressed on the cancer cell's surface. (Too many receptors is one signal the cell is cancererous.) The nanoparticles provide stability to the supported membrane and contain and release the therapeutic cargo within the cell.

A current Food and Drug Administration-approved nanoparticle delivery strategy is to use liposomes themselves to contain and deliver the cargo. In a head-to-head comparison of targeted liposomes and protocells with identical membrane and peptide compositions, Brinker and colleagues report that the greater cargo capacity, stability and targeting efficacy of protocells leads to many times greater cytotoxicity [destruction] directed specifically toward human liver cancer cells.

Another advantage to protocells over lipsomes alone, says lead author Carlee Ashley, a Harry S. Truman post-doctoral fellow at Sandia's California site in Livermore, is that liposomes used as carriers need specialized loading strategies that make the process more difficult. "We've demonstrated we can just soak nanoparticles to load them with unique drug combinations needed for personalized medicine. They effectively encapsulate toxins as well as siRNA [ribonucleic acid] that silence expressions of proteins."

RNA, the biological messenger that tells cells which proteins to manufacture, in this case is used to silence the cellular factory, a way of causing apoptosis or cell death. "Si" is short for "silence."

The lipids also serve as a shield that restricts toxic chemotherapy drugs from leaking from the nanoparticle until the protocell binds to and takes hold within the cancer cell. This means that few poisons leak into the system of the human host, if the protocells find no cancer cells. This cloaking mitigates toxic side effects expected from conventional chemotherapy.

Instead, the particles -- crafted small enough to float under the radar of the liver and other cleansing organs -- can circulate harmlessly for days or weeks, depending on their engineered size, seeking their prey.

A library of phages -- viruses that attack bacteria -- was created at UNM's nationally accredited cancer center by collaborator David Peabody. This permitted researchers to expose the phages to a group of cancerous cells and normal cells, allowing identification of peptides that bind specifically to cancer cells but not normal cells.

"Proteins modified with a targeting peptide that binds to a particular carcinoma exhibit a 10,000-fold greater affinity for that cancer than for other unrelated cells," Ashley said.

Boosting medicine with nanotechnology strengthens drug cocktail many times over

Brinker adds, "A key feature of our protocell is that its fluid bilayer allows high-affinity binding with just a few of these peptides overall. This reduces nonspecific binding and immune response."

The method is being tested on human cancer cells in vivo, and will shortly be tested on mouse tumors at UNM's cancer center.

The researchers continue to optimize the size of the porous silica particle, which is formed by aerosolizing a precursor solution. The porous nanoparticle fabrication process -- called evaporation-induced self-assembly, and pioneered in the Brinker lab -- produces particles from 50 nanometers to several microns in diameter. Particle sizes between 50 and 150 nanometers in diameter are ideal for maximizing circulation and uptake into cancer cells, so the particles are preselected by size before their formation into protocells.

"Their overall dimensions determine how widely they'll be distributed in the bloodstream," Brinker said. "We're altering our synthesis to favor the smaller sizes."

Also of importance to the circulation time of the particle are its electrical charge and hydrophobicity [avoidance of water], which can improve or detract from its ability to remain free of unwanted molecular or energetic entanglements.

The method may be commercially available in five years, researchers estimate.

Brinker is a Sandia Fellow and UNM Regent's and Distinguished Professor of Chemical and Nuclear Engineering and member of UNM's cancer center.

Other institutions involved in the research include the University of California Davis, and the University of Waterloo in Canada.

Funding was provided by the National Cancer Institute (NCI), the National Science Foundation, the Department of Energy's Basic Energy Sciences program, the Air Force Office of Scientific Research, and Sandia's Laboratory Directed Research and Development office.

The work is the first to show targeted delivery of nanoparticles to cancers that is supported in part by a grant from NCI's Alliance for Nanotechnology in Cancer.

How Peppermint Helps to Relieve Irritable Bowel Syndrome

ScienceDaily (Apr. 20, 2011) — University of Adelaide researchers have shown for the first time how peppermint helps to relieve Irritable Bowel Syndrome, which affects up to 20% of the population.

Peppermint is now clinically proven to be an effective pain reliever for Irritable Bowel Syndrome.

In a paper published in the journalPain, researchers from the University's Nerve-Gut Research Laboratory explain how peppermint activates an "anti-pain" channel in the colon, soothing inflammatory pain in the gastrointestinal tract.

Dr Stuart Brierley says while peppermint has been commonly prescribed by naturopaths for many years, there has been no clinical evidence until now to demonstrate why it is so effective in relieving pain.

"Our research shows that peppermint acts through a specific anti-pain channel called TRPM8 to reduce pain sensing fibres, particularly those activated by mustard and chilli. This is potentially the first step in determining a new type of mainstream clinical treatment for Irritable Bowel Syndrome (IBS)," he says.

IBS is a gastrointestinal disorder, causing abdominal pain, bloating, diarrhea and/or constipation. It affects about 20% of Australians and costs millions of dollars each year in lost productivity, work absenteeism and health care.

"This is a debilitating condition and affects many people on a daily basis, particularly women who are twice as likely to experience Irritable Bowel Syndrome," Dr Brierley says.

"Some people find their symptoms appear after consuming fatty and spicy foods, coffee and alcohol, but it is more complex than that. There appears to be a definite link between IBS and a former bout of gastroenteritis, which leaves nerve pain fibres in a heightened state, altering mechanisms in the gut wall and resulting in ongoing pain."

Dr Brierley says the recent floods in Queensland and Victoria could result in a spike of gastroenteritis cases in Australia due to the contamination of some water supplies in affected regions.

He said case studies in Europe and Canada showed that many people who contracted gastroenteritis from contaminated water supplies went on to experience IBS symptoms that persisted for at least eight years.

There is no cure for IBS and it often comes and goes over a person's lifetime.

Apart from gastroenteritis and food intolerance, IBS can be brought on by food poisoning, stress, a reaction to antibiotics, and in some cases is genetic.

Dr Brierley is one of 25 researchers who work at the University of Adelaide's Nerve-Gut Research Laboratory, hoping to find cures and treatments for a range of intestinal diseases.

Antibiotics Cure Anthrax in Animal Models

ScienceDaily (Apr. 20, 2011) — In the absence of early antibiotic treatment, respiratory anthrax is fatal. The 2001 bioterrorism attacks in the US killed four people, out of 22 infected (10 of them with respiratory anthrax), despite massive antibiotic administration, probably because therapy did not begin until the disease had reached the fulminant stage. But a multi-agent prophylaxis initiated within 24 hours post-infection prevented development of fatal anthrax respiratory disease, and treatment combining antibiotics with immunization with a protective antigen-based vaccine conferred long-term protective immunity against reestablishment of the disease, according to a study in the April 2011 issue of the journal Antimicrobial Agents and Chemotherapy. This study is the first to characterize the severity of respiratory anthrax that can be cured.

The researchers, all from the Israel Institute for Biological Research, Ness-Ziona, tested both the efficiency of different therapeutic approaches in preventing fatal disease from developing in infected animals, and their ability to cure animals in which the disease had developed into a systemic, septic phase. Rescue remains possible with appropriate agents even if initiated two days after infection.

Treatment initiated 24 hours after infection with any of four antibiotics protected the animals during treatment, but many of the animals died of anthrax after treatment was stopped, the antibiotics conferring degrees of protection ranging from 10-90 percent. Combining antibiotic treatment with a protective antigen vaccine left all animals fully protected even after the end of treatment.

Animals whose treatment was delayed beyond 24 hours post-infection developed varying degrees of bacteremia and toxemia. Treatment with doxycycline cured both sick guinea pigs and rabbits exhibiting low to moderate bacteremia; adding protective antigen vaccine to the mix boosted the level of bacteremia that was curable 10-fold in the guinea pigs and 20-fold in the rabbits. But ciprofloxacin plus a monoclonal anti-protective antigen antibody was still more effective.

In all cases, the surviving animals developed immunity against anthrax via subcutaneous challenge.

"Our results suggest that doxycycline and ciprofloxacin are efficient antibiotics to treat anthrax, not only as post-exposure prophylaxis, but also during the systemic phase of the disease," the researchers write. "Treatment with both antibiotics can cure guinea pigs and rabbits in an advanced stage of systemic anthrax"

Ends of Chromosomes Protected by Stacked, Coiled DNA Caps

ScienceDaily (Apr. 20, 2011) — Researchers at the University of Pennsylvania School of Medicine are delving into the details of the complex structure at the ends of chromosomes. Recent work, published online in Nature Structural & Molecular Biology, describes how these structures, called telomeres, can be protected by caps made up of specialized proteins and stacks of DNA called G-quadruplexes, or "G4 DNA." Telomere caps are like a knot at the end of each chromosome "string," with the knot's role preventing the string from unraveling.

This is an open-end view of a G-quadruplex.

"Although G4 DNA has been studied in test tubes for years, we did not know whether it could contribute to telomere protection in actual cells until we performed our studies in yeast cells," stated F. Brad Johnson, MD, PhD, associate professor of Pathology and Laboratory Medicine.

The composition of the particular G4-molecular "knot" studied is complex and unusual, involving a DNA sequence with guanine building blocks that loop back and forth on top of each other to form a four-stranded stack, which is different from the two-stranded arrangement of typical DNA molecules. The stack protects the chromosome from unraveling by specialized enzymes.

The length of telomeres is associated with age. Shortened telomeres are observed in aging cells and in some rare syndromes. There is mounting evidence that loss of telomere capping may contribute to some diseases that become more common with natural aging. An example of extreme aging associated with telomere defects is Werner syndrome, a rare genetic disease in which individuals develop normally until puberty. After this they age rapidly, so that by age 40 or so they often appear several decades older.

The protein missing in people with Werner syndrome but present in healthy people, is a helicase, an enzyme that unzips DNA. A slightly different G4-unwinding helicase is missing in people with a related disease, called Bloom syndrome, which is characterized by chromosome instability and high rates of cancer. It's possible that changes in G4 DNA contribute to the symptoms of these two syndromes.

The normal unwinding of DNA is critical under many particular circumstances, for example during replication. There are hundreds of different types DNA helicases in human cells, and each unwinds DNA under different circumstances. Although it is important to keep the strands of DNA together most of the time, if they can't be unwound when needed, serious problems could occur.

The Penn researchers hope to eventually explore the role of G4 capping in human aging after they know more about the G4 cap in yeast cells, which are easy to study because they can be engineered to make very specific changes in their DNA and proteins.

Recently, Johnson's group found that DNA sequences with the potential to form G4 DNA, which exist not only at telomeres but also at many locations throughout the entire human genome, are closely connected to changes in gene expression in cells from people with Werner or Bloom syndrome. They predict that G4 DNA abnormalities also exist at the telomeres in these human diseases and perhaps those of aging cells.

In their experiments in which telomeres were specifically examined in yeast, both elevated levels of G4 binding protein and inactivation of the yeast helicase that is similar to the one missing in human Werner and Bloom syndrome patients led to increased protection of the telomeres. This suggested that the G4 caps were present on the telomeres and that they protected the telomere from breaking down.

The overall role of G4 DNA is not simple and might seem to be contradictory. For example, work from several other research groups has suggested that G4 DNA can interfere with the replication and capping of telomeres, in contrast to the protective role observed by Johnson's group.

"This points out the complexity of G4 DNA," said Johnson. "On one hand, some G4 DNA may help cap telomeres, but too much G4 DNA or formation of G4 DNA at the wrong times or places may be detrimental. G4 DNA is not a single thing, but is rather a family of related structures, and so it might be possible to target particular types of G4 DNA to, for example, improve telomere capping in normal cells or disrupt the growth of cancer cells. This is a very new field, and it will be fun to see how far it might go."

This study was supported by grants from the National Institute on Aging. The lead author, Jasmine S. Smith, PhD performed this study as part of her Ph.D. dissertation research, which grew from studies begun by the second author, Qijun Chen, PhD. Dr. Liliya Yatsunyk's lab at Swarthmore College performed many of the studies on G4 DNA structure.

How TRIM5 Fights HIV: Scientists Discover Mechanism of Protein That Makes Certain Monkeys Resistant

ScienceDaily (Apr. 20, 2011) — Thanks to a certain protein, certain monkeys are resistant to HIV. Known as TRIM5, the protein prevents the HI virus from multiplying once it has entered the cell. Researchers from the universities of Geneva and Zurich have now discovered the protein's mechanism, as they report in Nature. This also opens up new prospects for fighting HIV in humans.

Structure of the latticed shell of the HI virus. 

Unlike people, certain monkey species, such as rhesus or night monkeys, are resistant to HIV thanks to TRIM5, a cellular protein: In the case of an HIV infection, the protein intercepts the virus as soon as it enters the cell and prevents it from multiplying. We have known about TRIM5 for over six years. However, the mechanism TRIM5 uses to prevent the HI virus from multiplying was still largely unknown.

The majority of the key aspects of TRIM5's defense mechanism against HIV was discovered by the Swiss research teams of Prof. Jeremy Luban, University of Geneva, and Prof. Markus Grütter, University of Zurich, in collaboration with teams from the USA and France. They demonstrated that TRIM5 immediately triggers an immune response if infected with HIV. Consequently, TRIM5 is an HIV sensor in the innate immune system. Unlike the adaptive immune system, which only develops when confronted with a pathogen, the innate immune system is already able to eliminate pathogens as soon as it comes into contact with them.

The HI virus, which penetrates the cell during an infection, has a shell, the components of which are arranged in a lattice, similar to the pattern on a soccer ball. TRIM5 recognizes this lattice structure and specifically attaches itself to it. This stimulates the protein to produce signal molecules known as polyubiquitin chains in the cell. These chains immediately trigger an anti-viral reaction. The "alerted" cell can then start eliminating cells infected with HIV by releasing messenger substances (cytokines).

Humans also have a TRIM5 protein, but it is less effective in fending off HIV. However, the findings in resistant monkeys have opened up new possibilities and ways of fighting HIV in humans. 33 million people are currently infected with HIV worldwide; two million die of AIDS each year. And with 2.7 million people becoming infected every year, HIV remains a major problem.