Chemical Education Today
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Research Advances by Angela G. King
A Simple Method for Making Lilliputian Cups The technology for making ultrasmall containers—essential in a wide range of modern scientific research—has taken a giant step forward with new research by scientists in India. Researcher G. U. Kulkarni and colleagues from the Jawaharlal Nehru Centre for Advanced Scientific Research report “a simple and straightforward” method for producing metal cups with a capacity measured in femtoliters. A femtoliter may seem uselessly small; however, Kulkarni describes a growing need for ultrasmall containers in scientific research, including dealing with cellular systems and expensive reagents. Uses for such small vials range from holding nanoparticles to serving as nano inkwells for a technology termed “dip pen nanolithography”. The new method of producing the tiny cups is termed pulsed laser ablation, and involves blasting melts (viscous solutions) with a laser beam in a vacuum to produce droplets of molten metal that form into cuplike structures. A YAG laser was focused on a rotating Ag disc in a vacuum chamber, and the resultant plume, consisting of cluster ions, molten droplets, and particulates, was collected on a heated (1173 K) silicon substrate located 4 cm away. Scanning electron microscopy (SEM) revealed several ring-like structures on the silicon. However, the structures had metal deposited across the bottom and thus were not rings, but rather cups. An analysis of the height profile of cup surface reveals that the internal volume of the cups are on a femtoliter scale, ranging from 25 aL to 6 fL (Figure 1). Using this technique, the research team was able to make femtoliter cups from various metals (Au, Cu, Zn, Nb, Cd, Al, In, and Sn) on a variety of substrates (silicon, cover glass, and highly oriented pyrolytic graphite). The metals all exhibit facile cup formation and the morphology appears independent of the exact substrate, given that it is a flat non-reactive surface. To show that the cups work as nanocontainers, Kulkarni’s group filled some with fluorescent biomarkers and metal nanoparticles. The location of the biomarkers and metals was verified to be in the cups by fluorescence imaging and EDX analysis, respectively. Although scientists previously have made even smaller containers, including some with a capacity of a zeptoliter— one million times smaller than a femtoliter—the new method of producing the tiny cups has advantages, including simplicity, over previous methods, according to the researchers.
More Information 1. John, Neena Susan; Selvi, N. R.; Mathur, Manikandan; Govindarajan, Rama; Kulkarni, G. U. A Facile Method of Producing Femtoliter Metal Cups by Pulsed Laser Ablation. J. Phys. Chem. B 2006, 110, 22975–22978.
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Figure 1. 3D projection of an AFM image showing cups of three different sizes. The height profile of the central cup along the dark line is shown. The hashed region gives an estimate of the inner volume. Reprinted with permission from J. Phys. Chem. B 2006, 110, 22975–22978. Copyright 2006 American Chemical Society.
2. More details on laser ablation are available online at http:// www.me.mtu.edu/~microweb/chap4/ch4-2.htm (accessed Mar 2007). 3. More details on Kulkarni’s research can be found at http:// www.jncasr.ac.in/kulkarni/ (accessed Mar 2007).
Uncovering a New Reason Why Patients Respond Differently to the Same Drug Dose Why does the standard dose of certain medications prove dangerously high for some patients and too low to produce beneficial effects in others? Scientists have just added a previously unrecognized factor to the list of explanations (such as age, gender, diet, and genetics) for this common problem of individual variability in response to drugs. Jeffrey P. Krise and Ryan S. Funk, at the University of Kansas, are reporting that variations in the body’s production of hydrogen peroxide—believed to serve as a signaling molecule at low levels—can affect accumulation of drugs inside cells. Oxidative stress refers to a set of conditions that are conducive for the degradation of molecules through a modification that incorporates the chemical addition of molecular oxygen, and it is widely variable among individuals. Increases in oxidative stress levels may be short-lived, as is the case with some inflammation problems, or chronically elevated as caused by both aging and smoking. The research team investigated the effect of M doses of H2O2 on the observed intracellular accumulation of model drugs, specifically the weakly basic anti-cancer drug daunorubicin and the weakly acidic fluorescent dye Oregon Green. The scientists noted, through use of fluorescence microscopy, that pre-treatment with hydrogen peroxide increased the cellular accumulation of both drugs (Figure 2). The scientists postulated that either a decrease in cytosolic pH, compromises to the plasma membrane, or changes
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Reports from Other Journals “The demonstrated correlation between hydrogen peroxide exposure and the concomitant increase in drug accumulation represents a substantial finding,” the study reports. “The results suggest that patients experiencing oxidative stress (or an increase in hydrogen peroxide levels) may have an increased response to a given dosage of a drug relative to a patient with decreased oxidative stress, or those patients chronically taking antioxidants [such as vitamins C or E] with their medications. This could be a very important factor in our continued efforts to provide more individualized dosing of drugs.” Hydrogen peroxide’s effects could be especially important in about two dozen so-called narrow therapeutic index drugs (such as aminophylline, carbamazepine, lithium carbonate, phenytoin, theophylline, and warfarin) for which very small changes in dosage level could cause either subtherapeutic or toxic results, researchers note.
More Information
Figure 2. H2O2 pretreatment increases the cellular accumulation of daunorubicin (DNR) and Oregon Green (OG) in both HL-60 cells (top micrographs) and human skin fibroblasts (bottom micrographs). Designated cells (+H2O2) were incubated in culture media supplemented with 50 M H2O2 for 2 days, washed, and subsequently incubated for 3 h with Oregon Green (1 M for HL60 cells and 2 M for fibroblasts) or daunorubicin (50 nM, both cells) in cell culture media at 37 °C. Cells were subsequently washed and visualized using a fluorescence microscope. Identical microscope settings were employed to allow for meaningful comparisons. Micrographs are representative of at least 5 separate trials. Reprinted with permission from Molecular Pharmaceutics 2007, 4, 154–159. Copyright 2007 American Chemical Society.
in lateral membrane diffusion induced by pre-exposure to H2O2 led to the drug accumulation increases. The team explored these avenues by measuring changes in intracellular pH, membrane integrity, and membrane fluidity. While the intracellular pH and membrane integrity appear to be unaffected by the peroxide pre-treatment dosage, the scientists did observe changes in membrane fluidity. Fluorescence recovery after photobleaching (FRAP) incorporates a dye molecule into a cell plasma membrane. A strong fluorescent beam is focused on a small defined area of the plasma membrane, bleaching the molecules in this area. The rate at which unbleached molecules diffuse into the study area of the membrane is an indicator of membrane fluidity. The researchers routinely observed that untreated cells could recover more rapidly than cells pre-exposed to 50 M H2O2, which is in agreement with previous work showing that oxidative stress reduces membrane fluidity. While it may seem that a more rigid membrane would decrease passive permeability and thus drug accumulation in cells, earlier work shows that an increased level of membrane rigidity results in increased passive permeability of a number of drugs. www.JCE.DivCHED.org
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1. Funk, Ryan S.; Krise, Jeffrey P. Exposure of Cells to Hydrogen Peroxide Can Increase the Intracellular Accumulation of Drugs. Molecular Pharmaceutics 2007, 4, 154–159. 2. Additional research pertaining to oxidative stress and membrane fluidity can be found in Clin. Chim. Acta. 1995, 235, 179– 188; Mol. Chem. Neuropathol. 1997, 31, 53–64; and J. Pharmacol. Exp. Ther. 2004, 313, 104–111. 3. More details on the research projects in Jeffrey Krise’s laboratory can be found at http://www.pharmchem.ku.edu/people-facultyinfo.php?ID=1 (accessed Mar 2007). 4. Additional information on oxidative stress is available online at http://www.portfolio.mvm.ed.ac.uk/studentwebs/session2/group31/ introduc.htm and http://www.sigmaaldrich.com/Area_of_Interest/ Life_Science/Cell_Signaling/Scientific_Resources/ Pathway_Slides___Charts/Oxidative_Stress.html (both sites accessed Apr 2007).
A New Look at Bacterial Infections In an advance in the emerging field of bacterial imaging, scientists are reporting development of a method for identifying specific sites of localized bacterial infections in living animals. Bradley D. Smith at the University of Notre Dame and colleagues developed the in vivo method, which could have applications ranging from food safety to pharmaceuticals. There are multiple strategies for optical imaging of bacteria within living organisms. One relies on genetically-altered fluorescent bacteria. Another avenue uses a molecular probe coupled with a fluorescent reporter group. The molecular probes need two basic components: an affinity ligand to bind them to the study subject and a reporter group that allows detection. The researchers previously discovered fluorescent zinc(II) dipicolylamine (Zn–DPA) zinc molecular probes (Figure 3) could be used as molecular probes to discriminate between common pathogenic bacteria—such as E. coli and Staphylococcus aureus—and mammalian cells. Zn–DPA affinity ligands bind to the anionic surfaces of the bacterial cells. In new research, they report using the probes to pinpoint the sites of
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Reports from Other Journals
N
Zn2+ N
N
N
Zn2+
N
N O
HN H3CO
OCH3
O O N+
–
O3SC4H8
N C4H8SO3 H
Figure 3. The molecular probe designed for fluorescence imaging of bacterial cells. Structure by A. King.
staph infections in living laboratory mice by attaching a nearinfrared (NIR) fluorophore to two Zn–DPA groups. In everyday medicine, physicians may have difficulty distinguishing localized bacterial infections from sites of sterile inflammation. The new bacterial imaging probe allowed scientists to visually locate Staph infections in nude mice. Bacteria were pre-incubated with the designed molecular probe and then injected into the thigh muscles of each mouse. The mice were then anesthetized, placed in an imaging station, and irradiated with 720 ± 35 nm light. During a following 60 s acquisition period, an image of 790 ± 35 nm was collected by a CCD camera. The resulting images document that fluorescence was limited to areas where cells pre-incubated with the molecular probe had been injected, and there was no fluorescence observed if the bacteria cells had been pre-incubated with a control fluorophore. The images also verified that fluorescence from the labeled bacteria can easily penetrate through the skin and muscle of live animals. The research team obtained further confirmation of their conceptual design through in vivo targeting experiments in which unlabeled bacteria were injected in the mouse thigh and incubated for 6 h before the molecular probe was introduced into the bloodstream through a tail vein injection. Images of the mouse show that over time the fluorescent probe is cleared from the bloodstream and accumulates at the site of the injection with maximum signal contrast after approximately 18 h (Figure 4). Using a control fluorophore in place of the molecular probe failed to lead to localized emission.
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Figure 4. Optical images of a mouse with a S. aureus infection in the left rear thigh muscle. Images were obtained before (A), and immediately after (B) intravenous injection of the new molecular probe and at 6 h (C), 12 h (D), 18 h (E), and 21 h (F). Scale represents the same relative fluorescence intensity for all six images in arbitrary units. Reprinted with permission from J. Am. Chem. Soc. 2006, 128, 16476–16477. Copyright 2006 American Chemical Society.
“Bacterial imaging is an emerging technology that has many health and environmental applications,” the researchers note. “For example, there is an obvious need to develop highly sensitive assays that can detect very small numbers of pathogenic bacterial cells in food, drinking water, or biomedical samples. In other situations, the goal is to study in vivo the temporal and spatial distribution of bacteria in live animals.”
More Information 1. Leevy, W. Matthew; Gammon, Seth T.; Jiang, Hua; Johnson, James R.; Maxwell, Dustin J.; Jackson, Erin N.; Marquez, Manuel; Piwnica-Worms, David; Smith, Bradley D. Optical Imaging of Bacterial Infection in Living Mice Using a Fluorescent Near-Infrared Molecular Probe. J. Am. Chem. Soc. 2006, 128, 16476–16477. 2. This Journal has published an article pertaining to NIR in the undergraduate curriculum. See J. Chem. Educ. 1999, 76, 315. 3. For a discussion of transition metal complexes as molecular probes, see Demas, J. N.; DeGraff, B. A. Applications of Luminescent Transition Metal Complexes to Sensor Technology and Molecular Probes. J. Chem. Educ. 1997, 74, 690. 4. More information on Bradley Smith’s research, including optical images of infected mice, can be found online at http:// www.chem.nd.edu/faculty/detail/bsmith3/ (accessed Mar 2007).
Angela G. King is Senior Lecturer in Chemistry at Wake Forest University, P. O. Box 7486, Winston-Salem, NC 27109;
[email protected].
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