Fluorescence Impacting Pharmaceuticals - Essay Example

How can the Phenomenon of Fluorescence be Used to Study the Effect of Pharmaceuticals on Cell Systems Introduction: Fluorescence illumination and observation is one of the most rapidly expanding technologies of our time when it is concerned with the staggering achievements of science and technology in the fields of Medicine, Biochemistry, Biotechnology, and Pharmaceuticals (Young M.R., 1961). The observation of fluorescence and analysis of its implications as applied to the domain of Biology has lead to development of more sophisticated technologies and numerous technological accessories dependent on fluorescence (Tobie, J.E., 1958). Physicochemical Principles: Fluorescence can be called a family of processes in which specific susceptible molecules emit light. This emission of light happens as a result of electronic excitation of the target. The electronic excitation can be achieved by several ways, physical-by light absorption, mechanical-friction, or chemical mechanisms(Huang et al, 2002). Some molecules are capable of being excited via absorption of light energy where photons or ultraviolet ray is used to excite them to a higher energy state, also called an excited state. The energy of the excited state, which cannot be sustained for long, "decays" or decreases, resulting in the emission of light energy. This process is called fluorescence. To "fluoresce" means to emit light via this process, but this is substance specific, meaning some molecules absorb light only at a particular wavelength to get excited, only to emit light of their own at a longer wavelength, which is characteristic of that substance. The question is does this phenomenon happen to everything The answer is yes. Only the emitted light may be of such a short wavelength that naked eye visibility is nil. The present day fluorescent microscopy with electron microscopy enhancements can detect even a subliminal threat of illumination and can enhance that. With appropriate tagging, fluorescence microscopy and techniques can now image single molecular species based solely on the properties of fluorescence emission. In short, the physical events that occur with fluorescence can by pin-pointed by the following. Excitation of a fluorophore through the absorption of light energy, a transient excited lifetime with some loss of energy, and return of the fluorophore to its ground state accompanied by the emission of light. Due to the energy lost during the transient excited lifetime, the light energy emitted is always of a longer wavelength than the light energy absorbed, and that is used to study different life processes (Molecular expressions). Today, there is an increased use of these techniques encouraged mainly by labeled antibody techniques (Coons and Kaplan, 1950) and by application of fluorescent dyes as tracers in histochemical techniques. Aminoacridine compounds have special affinity for nucleic acids; a sensitive fluorescence technique in which acridine orange is used for the identification of DNA and RNA in mammalian cells (Anderson, Armstrong, and Niven, 1959). Thus using fluorescence techniques and microscopy, the precise location and dynamics of intracellular components labeled with specific fluorophore designed for the cell system and the targeted interaction as applied to a pharmaceutical agent. This domain also, as a result, includes the study of other physicochemical properties of the concerned molecule, diffusion coefficient, transport characteristics, and above all the interaction with other biomolecules present. When applied to the field of study of pharmaceuticals and their effect on cell systems, this can allow one to study the phenomenal response in fluorescence to localized cellular environmental variables, such as, variation in pH, viscosity, refractive index, ionic concentrations, membrane potentials, and solvent polarity in living cell systems and tissue preparations with extraordinarysensitivity and selectivity. Dyes and stains have long been used to detect and visualize structures and processes in cell systems. Many dyes and stains have a fluorescent component. Examples of some widely used fluorescent dyes are ethidium bromide, Alexa Fluor dyes, Cy dyes, and fluorescein (K Schauenstein, E Schauenstein, and G Wick, 1978). Instruments: Apart from the minute biomeolecules in cell organelles, while studying the effect of pharmaceuticals in the cell systems, the effect is always evident on the molecular level. The action of a drug happens on the cells by several known processes, but cellular molecular architecture, configuration, polarity, ion channels, and electronic, atomic, or molecular exchanges either by mediation of radicles, receptors, cell surface proteins, and ultimately mediated by cellular protein synthesis, that is, RNA and DNA actions. One this is common about these. These are minute molecules. Even a little change in their configurations induced by pharmaceuticals would lead to remarkable changes in the cellular metabolic determinants that will be translated into therapeutic effects or side effects or adverse reaction of the pharmaceutical. To address these minute quantities, very sensitive techniques are required. Fluorescence correlation spectroscopy: Among those that allow even single molecule measurements, is fluorescence spectroscopy. It is comparatively noninvasive, hence is perfectly suitable for measurements or visualization inside a living cells. Fluorescence correlation spectroscopy (FCS) is one of the many different methods of high-resolution spatial and temporal analysis of extreme low concentration of biomolecules(Elson, E. L., and D. Madge. 1974). The focus in this method is not the intensity of emission, rather spontaneous intensity fluctuations caused by minute deviations in the thermal equilibrium of the small cell system. As all physical parameters in this kind of microenvironment give (Berland, K. M. et al, 1996) rise to fluctuations in the fluorescence signal, it determines directly local concentrations of drugs, mobility coefficients or characteristic rate constants of inter- or intramolecular reactions of the drug, of fluorescently labeled biomolecules in concentrations that are in nanomoles (Berland, K. M. et al, 1995). Widefield fluorescence and laser scanning confocal microscopy: This instrumentation technique relies solely on secondary fluorescence emission as an imaging mode. This is because, this is a sensitive technique with ability to target structural components and dynamic processes in chemically fixed as well as living cells and tissues. Many fluorescent probes are constructed with synthetic aromatic organic chemicals that are designed to bind to a biological macromolecule (Shotton, D, 1989). This hence is useful to monitor cellular integrity, live versus dead or apoptosis. This can also track the pharamokinetics of given drug by diagnosing endocytosis, exocytosis, membrane fluidity, protein trafficking, signal transduction, and enzymatic activity. Thus laser-induced fluorescence had been used to quantify different varieties of prostaglandins (Ramwell, P. W. 1973, 1974, 1975), which have different classes and subclasses and entirely different functionalities, in femtomolar levels (McGuffin, V. L. & Zare, R. N. 1985). Two-Photon Fluorescence Spectroscopy and Microscopy: This method employes redox fluorometry based on intrinsic fluorescence of the reduced pyridine nucleotides, NADH and NADPH and oxidized flavoproteins. This has been a very useful tool for studying cellular energy metabolism. (reviewed by Balaban and Mandel, 1990; Chance, 1991; Masters, 1984; Masters and Chance, 1993). Previously, this involved one-photon excitation at near-UV and visible wavelengths for NADPH and flavoproteins. Technical difficulties of poor resolution, sample turbidity, and phtobleaching inherent with this technique has been avoided by multiphoton microscopy coupled with near-infrared excitation (Denk et al., 1990; reviewed by Denk et al., 1995; Xu and Webb, 1997). Consequently, two-photon (2P) NAD(P)H fluorescence has been and applied to several biological studies (Piston et al., 1995; Konig et al., 1996; Masters et al., 1998). Fluorescence-based technique (High-Throughput): We shall use this technique for our discussion. This method has been widely used for antimalarial dug screening, like quinines. As opposed to usual radioisotope assays, in high-throughput drug testing as applicable to antimalarial quinine, which is a quinine derivative, can be accomplished with simple fluorescence assay. Principles: Quinine is a strongly fluorescent compound in dilute acidic solution. In 0.05 M sulfuric acid, this drug has two absorption bands that are used for excitation, at 250 and 350 nm with the peak fluorescence occurring at 450 nm. The excitation spectrum of quinine is measured, and the best excitation spectrum is selected to measure the fluorescent signal for a wide range of quinine concentration to determine the range of linear response. This can be used for a fluorescence assay for antimalarial drug screening. The parasite growth is determined by SYBR Green I. This is a dye which has marked fluorescence enhancement in contact with Plasomodium DNA. This assay method is used to determine effective concentration of the drug that is lethal for 50% of the parasites (Smilkstein et al, 2004). The prevalence of malaria and spread of drug-resistant malaria make the need for improved therapy a global demand. Assessment of both existing drugs and new therapeutic agents, alone or in combination requires dependable and reliable methods of testing these drugs in vivo. This assay is based on the scientific fact that host erythrocytes lack both DNA and RNA. The malaria parasites in contrast have both DNA and RNA, hence are readily stained with dyes that show enhanced fluorescence in presence of nucleic acids (Davis, W., C. Wyatt, M. Hamilton, and W. Goff. 1992). The fluorescence assay can be done after 48 hours of growth of the protozoa, the SYBR green in lysis buffer would be added to the wells containing the parasites. The Contents should be mixed until all the red cells in the media become invisible. This would need to be incubated in the dark at room temperature, the fluorescence would be measured in Cytofluor II fluorescence multiwell plate reader. The excitation and emission wavelengths at 485 and 530 nm with gain setting at 50. The accompanying Cytofluor software could be used to get a background reading of the empty well to yield fluorescent count analysis ( Martin Smilkstein et al., 2004). TABLE 1. EC50s of the test drug by fluorescence assays Test drug EC50 (nM)a Fluorescence method [3H]ethanolamine a Values are means _ standard errors of the means of a single parallel determination run in triplicate. At 1 h After freezing- method thawing Chloroquine 21 _ 1.7 20 _ 3.0 21 _ 1.7 Quinine 62 _ 12.9 41 _ 3.7 44 _ 3.1 Mefloquine 61 _ 3.0 32 _ 5.0 47 _ 3.0 Artemisinin 16 _ 0.4 15 _ 1.8 13 _ 0.8 C5 137 _ 3.6 118 _ 4.9 134 _ 5.8 (Adapted from Martin Smilkstein et al, 2004) . (Adapted from Martin Smilkstein et al, 2004) This data clearly shows the results of fluorescence based assays of high-throughput screening for quinine antimalarials. These table and plotting of the data show a direct linear correlation with the level of the parasite load. The drugs tested vary in their potencies, structures, and actions, yet they show a linearity in response against the plasmodia. It must be mentioned that this method may be applicable to study of other drugs like atropine, but the advantages and limitations are based on the technology of the assay. Even subtle changes in the hematocrit, parasitemia, parasite development stage will affect the outcome of this technique, but these variables are present in any other method of this kind of drug assay. One possible limitation is drug interference with fluorescence excitation and emission over a range of fluorescence excitation and emission similar to that of CYBR Greeen. The drugs that interact with nucleic acid may not be assayed by this method. Reference List Albota, M. A., C. Xu, And W. W. Webb. 1998. Two-Photon Fluorescence Excitation Cross Sections Of Biomolecular Probes From 690 To 960 Nm. Appl. Opt. 37:7352-7356. Anderson, E. S., Armstrong, J. A., And Niven, J. S. F., 1959. 'Observation Of Virus GrowthsWith Aminoacridines.' 9th Symposium Of The Society For General Microbiology, April,1959. Cambridge (University Press). Balaban, R. S., And L. J. Mandel. 1990. Optical Methods For The Study Of Metabolism In Intact Cells. In Noninvasive Techniques In Cell Biology. Berland, K. M., P. T. So, Y. Chen, W. W. Mantulin, And E. Gratton. 1996. Scanning Two-Photon Fluctuation Correlation Spectroscopy: Particle Counting Measurements For Detection Of Molecular Aggregation. Biophys. J. 71:410-420. Berland, K. M., P. T. So, And E. Gratton. 1995. Two-Photon Fluorescence Correlation Spectroscopy: Method And Application To The Intracellular Environment. Biophys. J. 68:694-701. Brian Herman And Victoria E. Centonze Frohlich - Department Of Cellular And Structural Biology, University Of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229. Chance, B. 1991. Optical Method. Annu. Rev. Biophys. Biophys. Chem. 20:1-28. Coons, A. H., And Kaplan, M. H., 1950. 'Localization Of Antigen In Tissue Cells.' J. Exp.Med., 91, 1. Daniel Axelrod - Department Of Biophysics, 930 North University Ave., University Of Michigan, Ann Arbor, Michigan 48109. David M. Shotton,Confocal Scanning Optical Microscopy And Its Applications For Biological Specimens,J. Cell Sci., Oct 1989; 94: 175 - 206. Davis, W., C. Wyatt, M. Hamilton, And W. Goff. 1992. A Rapid, Reliable Method Of Evaluating Growth And Viability Of Intraerythrocytic Protozoan Hemoparasites Using Fluorescence Flow Cytometry. Mem. Inst. Oswaldo Cruz 87:235-239. Reference List Denk, W., D. W. Piston, And W. W. Webb. 1995. Two-Photon Molecular Excitation In Laser-Scanning Microscopy. In Handbook Of Biological Confocal Microscopy. J. B. Pawley, Editor. Plenum Publishing, New York. 445-458. Denk, W., J. H. Strickler, And W. W. Webb. 1990. Two-Photon Laser Scanning Fluorescence Microscopy. Science. 248:73-76. Elson, E. L., And D. Madge. 1974. Fluorescence Correlation Spectroscopy. I. Conceptual Basis And Theory. Biopolymers. 12:1-27. J. K. Foskett And S. Grinstein, Editors. Wiley-Liss, New York. 213-236. John E. Tobie, Certain Technical Aspects Of Fluorescence Microscopy And The Coons Fluorescent Antibody Technique, J. Histochem. Cytochem., Jul 1958; 6: 271 - 277. Konig, K., P. T. So, W. W. Mantulin, B. J. Tromberg, And E. Gratton. 1996. Two-Photon Excited Lifetime Imaging Of Autofluorescence In Cells During UVA And NIR Photostress. J. Microsc. 183:197-204. K Schauenstein, E Schauenstein, And G Wick,Fluorescence Properties Of Free And Protein Bound Fluorescein Dyes. I. Macrospectrofluorometric Measurements J. Histochem. Cytochem., Apr 1978; 26: 277. Masters, B. R., And B. Chance. 1993. Redox Confocal Imaging: Intrinsic Fluorescent Probes Of Cellular Metabolism. In Fluorescent And Luminescent Probes For Biological Activity. W. T. Mason, Editor. Academic Press, New York. 44-57 Martin Smilkstein,1,2* Nongluk Sriwilaijaroen,3 Jane Xu Kelly,1 Prapon Wilairat,3 And Michael Riscoe1,2,Simple And Inexpensive Fluorescence-Based Technique For High-Throughput Antimalarial Drug Screening Medical Research Service, Department Of Veterans Affairs Medical Center,1 And Oregon Health And Science University,2 Portland, Oregon, And Department Of Biochemistry, Mahidol University,Bangkok, Thailand3 Mcguffin, V. L. & Zare, R. N. (1985) Appl. Spectrosc. 39, 847-853. Piston, D. W., S. M. Knobel, C. Postic, K. D. Shelton, And M. A. Reference List Magnuson. 1999. Adenovirus-Mediated Knockout Of A Conditional Glucokinase Gene In Isolated Pancreatic Islets Reveals An Essential Role For Proximal Metabolic Coupling Events In Glucose-Stimulated Insulin Secretion. J. Biol. Chem. 274:1000-1004. Ramwell, P. W. (1973, 1974, 1975) The Prostaglandins (Plenum,New York), Vol. 1, 2, 3. Shaohui Huang, Ahmed A. Heikal, And Watt W. Webb,Two-Photon Fluorescence Spectroscopy And Microscopy Of NAD(P)H And Flavoprotein,Biophys. J., May 2002; 82: 2811 - 2825. (Http://Micro.Magnet.Fsu.Edu/Primer/Techniques/Fluorescence/Fluorhome.Html.) Molecular Expressions Young. M. R,Principles And Technique Of Fluorescence Microscopy, J. Cell Sci., Dec 1961; S3-102: 419 - 449. Read More