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Medical Science News While pondering the challenges of distinguishing one nanosize probe image from another in a mass of hundreds or thousands of nanoprobes, two investigators at Emory University and the Georgia Institute of Technology made an interesting observation. The tiny, clustered dots of light looked a lot like a starry sky on a clear night.

The biomedical researchers realized that astronomers had already made great strides in solving a problem very similar to their own-isolating and analyzing one dot (in this case, a star) in a crowded field of light. They hypothesized that a computer system designed for stellar photometry, a branch of astronomy focused on measuring the brightness of stars, could hold the solution to their problem.

Now, May Wang, Ph.D., at Georgia Tech, and Shuming Nie, Ph.D., and their collaborators have created a technology based on stellar photometry software that provides more precise images of single molecules tagged with nanoprobes, particles specially designed to bind with a certain type of cell or molecule and illuminate when the target is found. The clearer images allow researchers to collect more detailed information about a single molecule, such as how the molecule is binding in a gene sequence, taking scientists a few steps closer to truly personalized and predictive medicine as well as more complex biomolecular structural mapping. In addition to biomedical applications, the system could be used to clarify other types of nanoparticle probes, including tagged particles or molecules. Their research appears in the Proceedings of the National Academy of Sciences of the United States of America (PNAS).

"As more powerful imaging technologies are developed, scientists face a real challenge to quantitatively analyze and interpret these new mountains of data," said Wang. "This paper is only a start, but I expect that innovative computing and data processing will be increasingly used to reveal detailed and quantitative features not currently available to biomedical researchers."

"This work is pointing to a new era in light microscopy in which single-molecule detection is achieved at nanometer resolution," added Nie. "This is also an example of interdisciplinary research in which advanced computing meets nanotechnology. I envision major applications not only for single-molecule imaging but also for ultrasensitive medical diagnostics."

Because scientists frequently use several different colors of nanoprobes to color-code genes and proteins, a blended color dot is a common challenge when analyzing images. For every few green or red dots in an image, there could be a few yellow dots as well, indicating that at least two dots are clustering to create the appearance of a new color. Although less than precise nanoprobe images yield valuable information, the Georgia Tech and Emory research team knew that better technology was needed to pinpoint the exact distance in nanometers between probes to reveal important information about the size and binding geometry of targeted molecules.

"We had no way of knowing for sure if we were looking at one molecule or two or three molecules very near one another," said Wang. "The fuzzy dot images were not precise enough on the nanometer level to truly tell us how these markers reflect DNA, but this system allows us to collect quantitative data and prove-not hypothesize-how genes are behaving."

Instead of starting from scratch to create a system to isolate the clumped nanoprobe images, the Georgia Tech and Emory researchers pursued their stellar photometry idea by adapting DAOPHOT, a program written by Peter Stetson, Ph.D., at the Dominion Astrophysical Observatory, which was designed to handle crowded fields of stars. After adapting DAOPHOT, the research team used color-coded nanoparticles to beat the traditional diffraction limit by nearly two orders of magnitude, allowing routine superresolution imaging at 1-nanometer resolution. And by using DNA molecules, two color-coded nanoparticles are designed to recognize two binding sites on a single target. Then the particles are brought together within nanometer distances after target binding.

These distances are sorted out by highly efficient image processing technology, leading to the detection and identification of individual molecules based on the target's geometric size. Compared with other single-molecule imaging methods, the Georgia Tech and Emory system allows for higher speed detection involving much larger sample volumes (microliter to milliliters).

This work, which was supported in part by the NCI's Alliance for Nanotechnology in Cancer, is detailed in the paper "Nanometer-scale mapping and single-molecule detection with color-coded nanoparticle probes." An investigator from Georgia State University also participated in this study. This paper is available through open access at the journal's Web site.

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http://nano.cancer.gov

Medical Science News Small pieces of nucleic acid, known as siRNAs (short interfering RNAs), can turn off the production of specific proteins, a property that makes them one of the more promising new classes of anticancer drugs in development. Indeed, at least two siRNA-based anticancer therapies, both delivered to tumors in nanoparticles, have begun human clinical trials. Now, three new reports highlight the progress that researchers are making in developing broadly applicable, nanoparticle-enabled siRNA anticancer therapeutics.

In the first report, Mark E. Davis, Ph.D., an investigator in the Nanosystems Biology Cancer Center at the California Institute of Technology, and former graduate student Derek Bartlett, Ph.D., now at the City of Hope, used mathematical modeling and results from dosing experiments in a mouse model of human cancer to explain therapeutic response with various dosing regimes for both targeted and untargeted siRNA-containing nanoparticles. The results of this work, published in the journal Biotechnology and Bioengineering, provide guidelines for optimizing the design of siRNA-based anticancer therapies.

In their experiments, the investigators used a cyclodextrin-based nanoparticle to deliver an siRNA agent designed to reduce production of ribonucleotide reductase subunit M2 (RRM2), which plays an important role in tumor growth. The investigators created two versions of their nanoparticle formulation, one targeted to transferrin, a protein overexpressed by many tumors, and the other untargeted. They also used two different dosing regimens, one consisting of three consecutive daily injections, the other consisting of three injections spaced 3 days apart.

Data from these experiments showed that targeted nanoparticles were far more effective than untargeted nanoparticles at reducing tumor growth. Dosing regimen, however, had no statistically significant impact on the outcome for either nanoparticle formulation. Closer examination of tumors removed from the animals following treatment showed that the targeted nanoparticles were able to deliver siRNA into the tumors, although the final distribution of siRNA throughout the tumors was not uniform. The investigators then modeled the observed responses; the results of these simulations led them to conclude that it is not necessary to persistently shut down protein production in order to achieve a therapeutic response using siRNA. Instead, they concluded, it is more important to maximize the number of cells reached with a sufficient dose of siRNA agent.

In a second report, Leaf Huang, Ph.D., and his colleagues at The University of North Carolina at Chapel Hill, describe their development of a self-assembling siRNA-liposomal formulation that they can then coat with poly(ethylene glycol) (PEG) linked to a targeting agent. This targeted liposome was fourfold more effective than an untargeted, but otherwise identical, liposome at delivering siRNA into tumors. Gene silencing activity was also higher for the targeted version, with the therapeutic effect lasting 4 days. The investigators also found that although the targeted nanoparticle effectively penetrated lung metastases, it did not enter liver cells. In addition, the targeted nanoparticle showed little immunotoxicity. These results appear in the Journal of Controlled Release.

Another paper published in the same journal, this one from Stefaan De Smedt, Ph.D., and his collaborators at Ghent University in Belgium, describes a method that could prove useful in both preclinical and clinical studies of nanoparticle-enabled siRNA therapeutics. Their new technique uses fluorescence fluctuation spectroscopy to measure the stability of these formulations, even at low concentrations, in human serum in less than 1 minute. Serum stability of siRNA-containing nanoparticles is essential to therapeutic efficacy, given that most studies have shown that naked siRNA has little effect on tumors. Using this method, the investigators were able to show that even PEGylated siRNA-containing liposomes were releasing the bulk of their cargo in serum.

The work from Drs. Davis and Bartlett, supported by the NCI's Alliance for Nanotechnology in Cancer, is detailed in the paper "Impact of tumor-specific targeting and dosing schedule on tumor growth inhibition after intravenous administration of siRNA-containing nanoparticles." An abstract of this paper is available through PubMed.

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The work from Dr. Huang's group is detailed in the paper "Efficient gene silencing in metastatic tumor by siRNA formulated in surface-modified nanoparticles." An investigator from Hokkaido Pharmaceutical University also participated in this study. An abstract of this paper is available through PubMed.

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The work from Dr. De Smedt and colleagues is detailed in the paper "A fast and sensitive method for measuring the integrity of siRNA-carrier complexes in full human serum." Investigators from the University of Leuven (Belgium) also participated in this study. An abstract of this paper is available through PubMed.

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Medical Science News As a wide variety of nanoparticles continue to demonstrate their ability to improve the delivery of imaging agents and drugs to tumors, nanoparticle researchers have turned their attention to the challenge of systematically determining how a given nanoparticle's physical and chemical characteristics affect its ability to target tumors. Such data could provide drug developers with guidelines to help them select the most effective type of nanoparticle for a given therapeutic or imaging application.

In a paper published in the PNAS, a team of investigators at the MIT-Harvard Center for Cancer Nanotechnology Excellence, led by Omid Farokhzad, Ph.D., at the Harvard Medical School, and Robert Langer, Ph.D., at MIT, describe one such approach to systematizing nanoparticle development. In their research, the investigators created used two self-assembling polymer families to create series of tumor-targeted nanoparticles that varied slightly from one another in terms of their physical characteristics and their biopharmaceutical properties.

By changing the exact composition of each of the two polymers, as well as the ratio of the two polymers, the investigators found that they could fine-tune both the size and drug-releasing properties of the nanoparticles, which were targeted to the prostate-specific membrane antigen found on the surface of prostate cancer cells. The researchers also were able to vary the amount of targeting agent on the nanoparticle surface, as well as the "stealth" characteristics of the nanoparticle, that is, the ability to evade the immune system. By studying the effect of each change on nanoparticle uptake by prostate cancer cells growing in tissue culture, the investigators were able to identify the specific formulation that optimized tumor uptake in vivo.

Investigators at the University of California, Berkeley, and the University of California, San Francisco, achieved similar results with a different class of polymer nanoparticles known as dendrimers. Jean Fréchet, Ph.D., at UC-Berkeley, and Francis Szoka, at UCSF and a member of the Carolina Center of Cancer Nanotechnology Excellence, led the team of collaborators that created libraries of dendrimers containing a variety of functional groups on their surfaces. These functional groups enable the investigators to attach both PEG and any number of targeting, imaging, and therapeutic agents to the dendrimer surface in a systematic manner.

Experiments using radiolabeled dendrimers demonstrated that these nanoparticle were able to circulate in blood for long periods of time. Subsequent experiments using a dendrimer linked to the antitumor agent doxorubicin showed that drug-loaded carrier accumulated in tumors but far less in healthy tissue compared with liposomal doxorubicin, the first nanoparticle-based drug approved to treat cancer.

The work from Drs. Farokhzad and Langer, which was supported in part by the NCI's Alliance for Nanotechnology in Cancer, is detailed in the paper "Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers." An abstract of this paper is available through PubMed.

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The work from Drs. Fréchet and Szoka is detailed in the paper "PEGylated dendrimers with core functionality for biological applications." An abstract of this paper is available through PubMed.

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http://nano.cancer.gov

Medical Research News In an article featured on the cover of the March issue of Nature Nanotechnology, Mauro Ferrari, Ph.D., of The University of Texas Health Science Center at Houston, presented a proof-of-concept study on a new multistage delivery system (MDS) for imaging and therapeutic applications. This discovery could go a long way toward making injectable drugs more effective.

"This is next-generation nanomedicine," said Ferrari, who played a critical role in the development of the National Cancer Institute's (NCI) Alliance for Nanotechnology in Cancer. "Now we're engineering sophisticated nanostructures to elude the body's natural defenses, locate tumors and other diseased cells, and release a payload of therapeutics, contrasting agents, or both over a controlled period."

Nanotechnology offers new and powerful tools to design and engineer novel drug delivery systems and to predict how they will work once inside the body. "The field of therapeutic nanoparticles began with tiny drug-encapsulated fat bubbles called liposomes, now commonly used in cancer clinics worldwide. Targeting molecules were later added to liposomes and other nanovectors to assist in directing them to diseased cells," Ferrari said.

Getting intravenous agents to their intended targets is no easy task. It is estimated that approximately 1 of every 100,000 molecules of agent reaches its desired destination. Physicians are faced with the quandary of increasing the dosage, which can lead to side effects, or reducing the dosage, which can limit the therapeutic benefits.

The multistage approach, according to Ferrari, is needed to circumvent the body's natural defenses or biobarriers, which act as obstacles to foreign objects injected in the bloodstream. "To overcome this problem, we hypothesized and developed a multifunctional MDS comprising stage 1 mesoporous particles loaded with one or more types of stage 2 nanoparticles, which in turn can carry either active agents or higher stage particles. We have demonstrated the loading, controlled release, and simultaneous in vitro delivery of quantum dots and carbon nanotubes to human vascular cells," said Ferrari.

In addition to circumventing biobarriers, Ferrari's team is working on the biochemical modifications required to efficiently deliver the MDS to a specific cancer lesion. "We have preliminary data that show that we can localize a payload of diagnostic agents, therapeutic agents, or combination of both to target cells. Once on site, the molecules can be released in a controlled way, and then the MDS will degrade in 24 to 48 hours, be transformed into orthosilicic acid, and leave no trace in the body," Ferrari said.

One of Ferrari's coauthors, Ennio Tasciotti, Ph.D., said the proof-of-concept study would not have been possible without a multidisciplinary effort that included contributions from mathematicians, physicists, engineers, chemists, and biologists. "We are dealing with objects that are in the billionth of a meter size range, and to study such objects we used cutting-edge technologies," Tasciotti said. "The characterization of the particles was performed using scanning electron and atomic force microscopy, dynamic light scattering, fluorimetry, and flow cytometry. The interaction of particles with cells was studied using fluorescence and confocal microscopy as well as a series of assays intended to determine cell viability and internalization rate of the nanoparticles."

This work, which was supported in part by the NCI, is detailed the paper "Mesoporous silicon particles as a multistage delivery system for imaging and therapeutic applications." Investigators from The University of Texas M. D. Anderson Cancer Center and Rice University also participated in this study. An abstract of this paper is available at the journal's Web site.

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http://nano.cancer.gov

Medical Studies/Trials A study has found only 16 per cent of the 352,082 Australians who filled a prescription for asthma preventer medications for the first time during the period July 2004 to June 2005, went on to use them regularly.

Most (61 per cent) 'first time' users did not fill another prescription in the next two years while 22 per cent did so sporadically.

The research will be presented at the Thoracic Society of Australia and New Zealand (TSANZ) Conference which starts this Sunday in Melbourne.

The study was conducted by the Australian Centre for Asthma Monitoring, a collaborating unit of the Australian Institute of Health and Welfare based at the University's Woolcock Institute of Medical Research in Sydney. It analysed the anonymous Pharmaceutical Benefits Scheme (PBS) records of individuals who filled a prescription for preventer medications for asthma for the first time between July 2004 and June 2005, and their subsequent prescription activity over a period of two years. The most commonly used form of preventer medication is inhaled corticosteroids.

Professor Guy Marks, Head of Epidemiology Research at the Woolcock Institute, said the results indicated that the prevalence of one-time and sporadic use was highest in young adults (age 15-34 years) with regular use most common in adults aged 65 years and over.

"The PBS dataset is a valuable tool for assessing patterns of asthma medication use," he said.

"Importantly this study shows that while guidelines recommend regular use of preventer medication, this certainly isn't happening in the community. At least some of those people who are now using preventer medication sporadically, could be expected to benefit from regular use of this class of medications.

The results of the study will be presented at the upcoming Thoracic Society of Australia and New Zealand (TSANZ) Conference being held in Melbourne from March 30 to April 2.

Professor Marks will present the abstract titled Patterns of Asthma Medication Use: An Australian Population-Based Longitudinal Cohort Study on the afternoon of Monday 31 March.

http://www.usyd.edu.au/