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Posted: Mar 01, 2016

 
Researchers involved in a national effort to develop cancer treatments that harness nanotechnology are recommending pivotal changes in the field because experiments with laboratory animals and efforts based on current assumptions about drug delivery have largely failed to translate into successful clinical results.
The assessment was advanced in a perspective piece that (“Targeting the Tumor Microenvironment”; pdf) appeared in the National Cancer Institute’s Cancer Nanotechnology Plan 2015, a 10-year roadmap concerning the use of nanotechnology to attack cancer.

The complex microenvironment of tumors is presenting a challenge in developing effective anticancer treatments


The complex microenvironment of tumors is presenting a challenge in developing effective anticancer treatments that attempt to harness nanotechnology. Researchers are recommending pivotal changes in the field of cancer nanotechnology because experiments with laboratory animals and efforts based on current assumptions about drug delivery have largely failed to translate into successful clinical results. (Image: Bumsoo Han, Kinam Park, Murray Korc) (click on image to enlarge)

Researchers are trying to perfect “targeted delivery” methods using various agents, including an assortment of tiny nanometer-size structures, to selectively attack tumor tissue. However, the current direction of research has brought only limited progress, according to the authors of the article.

“The bottom line is that so far there are only a few successful nanoparticle formulations approved and clinically used, so we need to start thinking out of the box,” said Bumsoo Han, a Purdue University associate professor of mechanical and biomedical engineering.

One approach pursued by researchers has been to design nanoparticles small enough to pass through pores in blood vessels surrounding tumors but too large to pass though the pores of vessels in healthy tissue. The endothelial cells that make up healthy blood vessels are well organized with tight junctions between them. However, the endothelial cells in blood vessels around tumors are irregular and misshapen, with loose gaps between the cells.

“We should realize that having a specific nanosize or functionality alone is not enough to guarantee good drug delivery to target tumors,” said Kinam Park, a professor of pharmaceutics and Purdue’s Showalter Distinguished Professor of Biomedical Engineering. “The tumor microenvironment is just too complex to overcome using this strategy alone.”

The two authored the article with Murray Korc, the Myles Brand Professor of Cancer Research at the Indiana University School of Medicine.

The authors pointed out that research with laboratory mice has rarely translated into successful clinical results in humans, suggesting that a more effective approach might be to concentrate on research using in-vitro experiments that mimic human physiology. For example, one new system under development, called a tumor-microenvironment-on-chip (T-MOC) device, could allow researchers to study the complex environment surrounding tumors and the barriers that prevent the targeted delivery of therapeutic agents.

The approach could help drug makers solve a daunting obstacle: even if drugs are delivered to areas near the target tumor cells, the treatment still is hindered by the complex microenvironment of tumors.

“We used to think that if we just killed the tumor cell it would cure the cancer, but now we know it’s not just the cancer cells alone that we have to deal with,” Korc said. “There are a lot of different cells and blood vessel structure, making for a complex environment that supports the cancer cells.”

An “extracellular matrix” near tumors includes dense collagen bundles and a variety of enzymes, growth factors and cells. For example, surrounding pancreatic tumors is a “stromal compartment” containing a mixture of cells called stromal cells, activated cancer-associated fibroblasts and inflammatory immune cells.

“Particularly for pancreatic cancer, the stromal tissue is much bigger than the tumor itself,” Korc said.

In addition, a compound called hyaluronic acid in this stromal layer increases the toughness of tumor microenvironment tissue, making it difficult for nanoparticles and drugs to penetrate.

“It’s dense, like scar tissue, so it’s more difficult for drugs coming out of the blood vessel to diffuse through this tissue,” Han said.

Another challenge is to develop water-soluble drugs to effectively deliver medicines.

“The cancer drugs need to be aqueous because the body resorbs them better, but a lot of the current chemotherapy drugs have low solubility and usually need different types of solvents to increase their solubility,” Park said.

The T-MOC approach offers some hope of learning how to design more effective cancer treatments.

“Recent advances in tissue engineering and microfluidic technologies present an opportunity to realize in-vitro platforms as alternatives to animal testing,” Park said. “Tumor cells can be grown in 3D matrices with other relevant stromal cells to more closely mirror the complexity of solid tumors in patients. The current ability of forming 3D-perfused tumor tissue needs to be advanced further to create an accurate tumor microenvironment.”

Such a major shift in research focus could play a role in developing personalized medicine, or precision medicine, tailored to a particular type of cancer and specific patients. More effective treatment might require various “priming agents” in combination with several drugs to be administered simultaneously or sequentially.

“This kind of research currently involves a very large number of experiments, and it makes animal testing expensive and time consuming,” Park said. “Moreover, small animal data have not been good predictors of clinical outcome. Thus, it is essential to develop in-vitro test methods that can represent the microenvironment of human tumors.”

Source: By Emil Venere, Purdue University

Cancer Nanoparticle Targets 160210165715_1_540x360In one of the first efforts to date to apply nanotechnology to targeted cancer therapeutics, researchers have created a nanoparticle formulation of a cancer drug that is both effective and nontoxic — qualities harder to achieve with the free drug. Their nanoparticle creation releases the potent but toxic targeted cancer drug directly to tumors, while sparing healthy tissue.

The findings in rodents with human tumors have helped launch clinical trials of the nanoparticle-encapsulated version of the drug, which are currently underway. Aurora kinase inhibitors are molecularly targeted agents that disrupt cancer’s cell cycle.

While effective, the inhibitors have proven highly toxic to patients and have stalled in late-stage trials. Development of several other targeted cancer drugs has been abandoned because of unacceptable toxicity. To improve drug safety and efficacy, Susan Ashton and colleagues designed polymeric nanoparticles called Accurins to deliver an Aurora kinase B inhibitor currently in clinical trials.

The nanoparticle formulation used ion pairing to efficiently encapsulate and control the release of the drug. In colorectal tumor-bearing rats and mice with diffuse large B cell lymphoma, the nanoparticles accumulated specifically in tumors, where they slowly released the drug to cancer cells. Compared to the free drug, the nanoparticle-encapsulated inhibitor blocked tumor growth more effectively at one half the drug dose and caused fewer side effects in the rodents.

Cancer Nanoparticle Targets 160210165715_1_540x360

The polymeric nanoparticle Accurin encapsulates the clinical candidate AZD2811, an Aurora B kinase inhibitor. This material relates to a paper that appeared in the Feb. 10, 2016 issue of Science Translational Medicine, published by AAAS. The paper, by S. Ashton at institution in location, and colleagues was titled, “Aurora kinase inhibitor nanoparticles target tumors with favorable therapeutic index in vivo.”
Credit: Ashton et al., Science Translational Medicine (2016)

A related Focus by David Bearss offers more insights on how Accurin nanoparticles may help enhance the safety and antitumor activity of Aurora kinase inhibitors and other molecularly targeted drugs.


Story Source:

The above post is reprinted from materials provided by American Association for the Advancement of Science. Note: Materials may be edited for content and length.


Journal Reference:

  1. Susan Ashton, Young Ho Song, Jim Nolan, Elaine Cadogan, Jim Murray, Rajesh Odedra, John Foster, Peter A. Hall, Susan Low, Paula Taylor, Rebecca Ellston, Urszula M. Polanska, Joanne Wilson, Colin Howes, Aaron Smith, Richard J. A. Goodwin, John G. Swales, Nicole Strittmatter, Zoltán Takáts, Anna Nilsson, Per Andren, Dawn Trueman, Mike Walker, Corinne L. Reimer, Greg Troiano, Donald Parsons, David De Witt, Marianne Ashford, Jeff Hrkach, Stephen Zale, Philip J. Jewsbury, and Simon T. Barry. Aurora kinase inhibitor nanoparticles target tumors with favorable therapeutic index in vivo. Science Translational Medicine, 2016 DOI: 10.1126/scitranslmed.aad2355

Illumina_illox299The DNA sequencing giant will launch a new company, Grail, to develop blood tests to detect cancer.

The world’s largest DNA sequencing company says it will form a new company to develop blood tests that cost $1,000 or less and can detect many types of cancer before symptoms arise.

Illumina, based in San Diego, said its blood tests should reach the market by 2019, and would be offered through doctors’ offices or possibly a network of testing centers.

The spin-off’s name, Grail, reflects surging expectations around new types of DNA tests that might do more to defeat cancer than the more than $90 billion spent each year by doctors and hospitals on cancer drugs. Illumina CEO Jay Flatley says he hopes the tests could be a “turning point in the war on cancer.”

The startup will be based in San Francisco and has raised more than $100 million from Illumina as well as Bill Gates, Jeff Bezos’s venture fund, Bezos Expeditions, and Arch Venture Partners. Illumina will retain majority control.

The testing concept being pursued by Illumina, sometimes called a “liquid biopsy,” is to use high-speed DNA sequencing machines to scour a person’s blood for fragments of DNA released by cancer cells. If DNA with cancer-causing mutations is present, it often indicates a tumor is already forming, even if it’s too small to cause symptoms or be seen on an imaging machine.

Illumina didn’t invent the idea for the tests, which were first developed by academic centers including at Johns Hopkins University (see “Spotting Cancer in a Vial of Blood”) and in Hong Kong (see “Liquid Biopsy”). But Flatley says only recently has gene-sequencing become inexpensive enough to try to make the cancer screening tests affordable.

Illumina, which is based in San Diego, has established spin-offs in Silicon Valley to address consumer markets for DNA data.

A DNA test able to pick up many kinds of cancer could be revolutionary because tumors caught early can often be cured with surgery or radiation. Since the 1970s, the largest declines in cancer deaths rates are due to either behavioral changes, like declining tobacco use, or because of effective screening tests, principally colonoscopies and pap smear. New drugs have helped, too, though their impact on survival has generally been modest.

Expectations that cancer blood tests will quickly turn into a multibillion-dollar industry has attracted growing interest from investors. For instance, last week, a startup called Guardant, run by former Illumina executives, also said it had raised $100 million.

Guardant’s test isn’t an early detection test, but is instead used to measure tumor DNA in patients already battling cancer and can be prescribed in place of an invasive tissue biopsy (see “The Great Cancer Test Experiment”). Other companies bidding for a share of the testing market include Personal Genome Diagnostics, a spin-off of Johns Hopkins University, as well as Trovagene, Boreal Genomics, and Natera.

In the U.S., the only early-detection liquid biopsy test on the market is from Pathway Genomics, and it costs $699. But since it remains unclear how well these types of tests work, that company received a warning letter from the U.S. Food and Drug Administration questioning its marketing claims (see “Why You Shouldn’t Bother with a $699 Cancer Test”).

Any developer of a presymptomatic screening test for cancer faces daunting obstacles. How often will the test find cancer, and how often will it give a wrong result? What’s more, even tests that do discover cancer early can turn into medical disasters if patients end up getting aggressive and costly treatment for cancers that won’t kill them.

“The hardest part is not only demonstrating the ability to detect cancer early, but being able to say this knowledge is in fact meaningful in terms of patient outcomes,” says J. Leonard Lichtenfeld, deputy chief medical officer of the American Cancer Society. “I can’t tell you how many times we’ve said, ‘Oh, all we have to do is find every cancer early and we would solve the problem.’”

Flatley agrees that most screening tests have failed to help patients and that many companies developing them had suffered reversals as a result. “If you look at this business, it’s littered with failures. With a few exceptions, screening tests have been invariably horrible,” he says. “It’s a big challenge.”

To prove early detection is possible, Flatley says, Grail will spend millions on organizing clinical trials involving as many as 30,000 people. It will test all of them and then see if the tests are able to catch cancer earlier than established methods.

Flatley estimates that the amount of DNA sequencing required for the studies would be the equivalent of decoding the genomes of about 400,000 people at high quality. That makes the project about three times as large as Genomics England, a national effort to study cancer and disease in the U.K.

Flatley says he believes that, right now, Illumina is the only company currently able to implement sequencing technology at a cost that’s low enough to carry out such studies and bring an inexpensive test to market. “In this case, we didn’t think the market could do it fast enough, unless we destroyed our [business] by giving away sequencing,” said Flatley. “We don’t think anyone else can do it at scale. And there are millions of lives at stake.”

Illumina has a price advantage because it makes and sells more than about $2 billion worth of DNA sequencing instruments, chemicals, and test kits annually to university scientists and other labs. But recently it has also sought to jump directly into what it thinks will be key applications for that DNA data. In 2013, it paid almost half a billion dollars to acquire prenatal testing company Verinta (see “Prenatal DNA Sequencing”), and last August said it would partner with investors to create a vast DNA “app store” aimed at consumers. (see “Inside Illumina’s Plans to Lure Consumers with an App Store for Genomes”).

Flatley says Grail’s objective is a “pan-cancer” test able to detect most types of cancer from a single blood draw, but he says early detection of lung and breast cancer could be the first tests to reach the market. Flatley himself says he took an early version of the test, which came back without any problems.

“I was clear,” says Flatley. “But if I have cancer I want to know about it.”

Jay Flatley, the CEO of Illumina, with a DNA sequencing machine. Illumina sequencers account for most of the DNA data generated globally.

Drug Delivery 050815 onereallytin When you take a drug, it travels through your bloodstream, dissolving and dispersing, and eventually reaching its designated target area.

But because the blood containing the drug travels all round your body only a small percentage of the initial dose actually reaches the desired location.

For over-the-counter drugs like paracetamol or ibuprofen, with very few side-effects, this doesn’t matter too much.

But when it comes to cancer drugs, which can affect healthy cells just as much as , this process can cause big problems.

Drug Delivery 050815 onereallytin

Partly because drugs are diluted in their blood, cancer patients need to take these drugs in particularly high doses – and this can cause seriously unpleasant side effects.

But Professor Sonia Trigueros, co-director of the Oxford Martin Programme on Nanotechnology, is inching closer to developing a nano-scale drug delivery system with the aim of specifically targeting cancer cells.

Working with a team of chemists, engineers and physicists, Trigueros has embarked on an ambitious mission to tackle cancer at the ‘nano’ level – less than 100 nanometers wide. For context, this is super-tiny: a nanometre is a thousandth of a thousandth of a millimetre.

There’s still a long way to go, but Trigueros is making decent headway, and has recently tackled a major problem of working at a nano level. And at this year’s Wired Health conference – which looked at the future of health care, wellbeing and genomics – she told us about her recent progress, and her visions for the future.

At the nano level

Some of us will remember the periodic table displayed in our science classrooms which told us about the properties of each element. But working on a nano level everything changes, and elements behave completely differently.

Elements have different properties at the nano level than they do at the micro level, explained Prof Trigueros to the Wired Health 2015 audience.

This poses big problems for researchers trying to make nano-scale devices, which can be made out of a number of different materials, including gold, silver and carbon. All these materials are highly unstable at the nano level.

“After you make the nanostructures you only have minutes to a couple of days to work,” she said. They are really unstable, especially when you put them in water.”

This isn’t ideal, considering our bodies are made up mostly of water.

One (really tiny) step closer to nano-sized cancer drug delivery
Credit: Professor Sonia Trigueros

Trigueros’ recent work has focused on trying to stabilise tiny tubes made of carbon, called carbon nanotubes, which hold drugs inside the tube so they can be delivered into cancer cells.

She has now found a way of keeping them stable for more than two years and in temperatures up to 42ºC.

To do this, she wraps DNA around the structures, like a tortilla wraps around the fillings of a burrito.

While this accomplishes the goal of keeping the nanostructures stable inside the body this doesn’t do much good if the DNA can’t unwrap to deliver the drugs. But, according to Trigueros, she has shown that, once inside a cell, the DNA easily unwinds and releases its payload.

Truly targeted drug delivery

So how does it all work? How do the drugs get into the cancer cells? Trigueros’s nanotubes exploit the differences between cancer cells and – in this case, differences in the membranes that hold them together.

“Cancer cells are more permeable than normal cells so the nanotubes can get through the cell membrane. And once they are in, they unwrap and deliver drug,” explained Trigueros.

Exploiting differences in their permeability is one way to target the cancer cells, but Trigueros explains that there is more than one way to create a truly targeted drug delivery system.

“We can attach whatever we want on DNA,” she said. “So you can attach a protein that recognises cancer cells”.

From theory to reality

While this all sounds great in theory, will it actually work in reality?

One (really tiny) step closer to nano-sized cancer drug delivery
Attaching proteins to DNA could create a truly targeted drug delivery system. Credit: Professor Sonia Trigueros

Trigueros has now started preliminary tests on laboratory grown , she told us during an interview. And this has shown tentative promise, she says, citing unpublished data on their effectiveness at killing these cells in the lab.

Others are cautiously optimistic. “This is a really exciting prospect,” says Professor Duncan Graham, nanotechnology expert and advisor to Cancer Research UK.

“A common concern with carbon nanotubes is toxicity, but when coated with DNA this concern could be removed,” he explains, “and it also addresses a fundamental issue, which is that they collect into clusters that become a solid mass and so are unable to leave the body.”

In theory, once Trigueros’s nanotubes have finished their job they are tiny enough (50 nanometres) to be excreted through urine.

This isn’t the first time carbon nanotubes have been used in cancer research: a US research team has used them, for example, to target and collect images of tumours in mice. But the combination of drug delivery and cancer-specific targeting is what interests Professor Graham.

“Unlike previous work using carbon nanotubes, this approach is set to target the tumour specifically, potentially meaning fewer side effects and a lower dosage. I look forward to seeing this in animal models which is where the real proof of activity lies,” he said.

But he’s cautious, stressing that Trigueros’s work has not yet been peer-reviewed and published.

Next steps

Next Trigueros is aiming towards starting animal trials and, eventually, she wants to begin clinical trials in patients – that is if everything goes well.

She hopes to focus on how nanostructures could be used to cross the blood-brain barrier – the brain’s highly selective ‘bouncer’ that only lets certain molecules across. This has been notoriously difficult to get past, making targeting cancers in the brain more difficult.

But there is a still a long way to go and a lot of problems to tackle. In the shorter term, we’ll be keeping an eager eye on her research, as her ideas continue to develop.

Explore further: Nano packages for anti-cancer drug delivery

 

 

Link to Video Here: http://youtu.be/9ITrc4e8XoM

Published on Jul  9, 2013

What is Nanotechnology? A basic definition: Nanotechnology is the engineering of functional systems at the molecular scale. This covers both current work and concepts that are more advanced. In its original sense, ‘nanotechnology’ refers to the projected ability to construct items from the bottom up, using techniques and tools being developed today to make complete, high performance products.

Nanotechnology (sometimes shortened to “nanotech”) is the manipulation of matter on an atomic and molecular scale. The earliest, widespread description of nanotechnology referred to the particular technological goal of precisely manipulating atoms and molecules for fabrication of macroscale products, also now referred to as molecular nanotechnology. A more generalized description of nanotechnology was subsequently established by the National Nanotechnology Initiative, which defines nanotechnology as the manipulation of matter with at least one dimension sized from 1 to 100 nanometers.

This definition reflects the fact that quantum mechanical effects are important at this quantum-realm scale, and so the definition shifted from a particular technological goal to a research category inclusive of all types of research and technologies that deal with the special properties of matter that occur below the given size threshold. It is therefore common to see the plural form “nanotechnologies” as well as “nanoscale technologies” to refer to the broad range of research and applications whose common trait is size. Because of the variety of potential applications (including industrial and military), governments have invested billions of dollars in nanotechnology research. Through its National Nanotechnology Initiative, the USA has invested 3.7 billion dollars. The European Union has invested 1.2 billion and Japan 750 million dollars

green earth untitledA BBC documentary on nanotechnology advances in Europe “Nano, The Next Dimension”

A very good video to provide “perspective” on how “All Things Nano” have ALREADY impacted our lives and how … the VAST (but tiny!) arena of “Nanotechnologies” (Nano: objects a billionth of a meter in size) will certainly impact ALL of the Sciences, Manufacturing, Communications and Consumer Materials. Impacts such as:

1.  Our abilities to capture and generate abundant renewable sources of energy, (Solar, Hydrogen Fuel Cells)

2. To create abundant sources of CLEAN WATER through vastly improved FILTRATION and WASTE REMEDIATION processes. (Desalination, Oil and Gas Fields)

3. To deliver LIFE SAVING Drug Therapies and provide vastly improved Diagnostics. (Diabetes, Cancer, Alzheimer’s)

4. To create FLEXIBLE SCREENS and PRINTABLE ELECTRONICS that offer vastly improved performance, user experience, with lower energy consumption and with significantly LOWER COSTS. (Flat Panel TV Screens, Smart Phones, Super-Computers, Super-Capacitors, Long-Lived Super Batteries)

5. Completely water, stain proof clothing. Lighter, Stronger Sports Equipment.

6. Coatings and Paints for Buildings, Windows and Highways that capture solar energy. Inks and Sensors that make our everyday life more Secure.

Through the month of January, we will be posting videos, articles and research summaries that focus on the coming accelerated “wave” of nano-supported technologies “that will change the way we innovate everything!”

“Great Things from Small Things!”

 

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(Nanowerk Spotlight) Cancer is one of the leading  causes of death in the world and remains a difficult disease to treat. Current  problems associated with conventional cancer chemotherapies include insolubility  of drugs in aqueous medium; delivery of sub-therapeutic doses to target cells;  lack of bioavailability; and most importantly, non-specific toxicity to normal  tissues. Recent contributions of nanotechnology research address possible  solutions to these conundrums. Nevertheless, challenges remain with respect to  delivery to specific sites, real time tracking of the delivery system, and  control over the release system after the drug has been transported to the  target site.

Nanomedical research on nanoparticles is exploring these issues  and has already been showing potential solutions for cancer diagnosis and  treatment. But a heterogeneous disease like cancer requires smart approaches  where therapeutic and diagnostic platforms are integrated into a theranostic  approach.

Theranostics – a combination of the words therapeutics and diagnostics – describes a treatment platform that combines a  diagnostic test with targeted therapy based on the test results, i.e. a step  towards personalized medicine. Making use of nanotechnology materials and  applications, theranostic nanomedicine can be understood as an integrated  nanotherapeutic system, which can diagnose, deliver targeted therapy and monitor  the response to therapy.

Theranostic nanomedicine has the potential for simultaneous and  real time monitoring of drug delivery, trafficking of drug and therapeutic  responses.

Our Smart Materials and Biodevice group at the Biosensors and Bioelectronics Centre, Linkoping University,  Sweden, has demonstrated for the first time a MRI-visual order-disorder micellar nanostructures for smart  cancer theranostics.

        drug release mechanism via functional outcome of pH response

 

The  drug release mechanism via functional outcome of the pH response illustrated in  the schematic diagram. (Image: Smart Materials and Biodevice group, Linköping  University)   In the report, we fabricated a novel pH-triggered tumour  microenvironment sensitive order-disorder nanomicelle platform for smart  theranostic nanomedicine.           

The real-time monitoring of drug distribution will help  physicians to assess the type and dosage of drug for each patient and thus will  prevent overdose that could result in detrimental side-effects, or suboptimal  dose that could lead to tumour progression.

Additionally, the monitoring of normal healthy tissues by  differentiating with the MRI contrast will help balance the estimation of lethal  dose (for normal tissue) and pharmacologically active doses (for tumour). As a  result, this will help to minimize off-target effects and enhance effective  treatment.

In the present report, the concurrent therapy by doxorubicin and  imaging strategies by superparamagnetic iron oxide nanoparticles with our smart  architecture will provide every detail and thus can enable stratification of  patients into categorized responder (high/medium/low), and has the potential to  enhance the clinical outcome of therapy.

It shows, for the first time, concentration dependent  T2-weighted MRI contrast for a monolayer of clustered cancer cells. The pH  tunable order-disorder transition of the core-shell structure induces the  relative changes in MRI that will be sensitive to tumour microenvironment and  stages.

  MRI visual order-disorder nanostructure for cancer nanomedicine A  novel MRI visual order-disorder nanostructure for cancer nanomedicine explores  pH-trigger mechanism for theranostics of tumour hallmark functions. The pH  tunable order-disorder transition induces the relative changes in MRI contrast.  The outcome elucidates the potential of this material for smart cancer  theranostics by delivering non-invasive real-time diagnosis, targeted therapy  and monitoring the course and response of the action. (Image: Smart Materials  and Biodevice group, Linköping University)  

Our findings illustrate the potential of these biocompatible  smart theranostic micellar nanostructures as a nontoxic, tumour-target specific,  tumour-microenvironment sensitive, pH-responsive drug delivery system with  provision for early stage tumour sensing, tracking and therapy for cells  over-expressed with folate receptors.

The outcomes elucidate the potential of  smart cancer theranostic nanomedicine in non-invasive real-time diagnosis,  targeted therapy and monitoring of the course and response of the action before,  during and after treatment regimen.                     

By Hirak K Patra, Nisar Ul Khali, Thobias Romu, Emilia  Wiechec, Magnus Borga, Anthony PF Turner and Ashutosh Tiwari, Biosensors and Bioelectronics Centre,  Linköping University, Sweden

Read more: http://www.nanowerk.com/spotlight/spotid=33186.php#ixzz2kTi8huZB


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