Fluorescence anisotropy based single liposome assay to measure molecule-membrane interactions.

Nanometer-scaled liposomes are used frequently for research, therapeutic, and analytical applications as carriers for water-soluble molecules. Recent technical advances allow the monitoring of single liposomes, which provides information on heterogeneous properties that were otherwise hidden due to ensemble averaging. Recent observations demonstrated that the efficiency of entrapping water-soluble molecules increases with decreasing vesicle size. The molecular mechanism behind this observation is not clear, but enhanced molecule-membrane interactions due to the increase of the surface area-to-volume ratio could play an important role. To investigate this hypothesis, we extended our single liposome assay based on confocal fluorescence imaging by implementation of fluorescence anisotropy. This combination has not been widely exploited, and confocal fluorescence anisotropy imaging in particular has seldom been used. We investigated different small dye molecules and were able to determine if these molecules interact or not with the liposome membrane. We confirm the liposome size-dependent entrapment of molecules whereas the molecule-membrane interactions appear to be independent of liposome size. Our fluorescence anisotropy assay can be used as a general method to investigate molecule-membrane interactions or molecule-molecule interactions in a high-throughput manner in nanometer-scaled containers like liposomes.

[1]  G. Gregoriadis,et al.  Lysosomal localization of -fructofuranosidase-containing liposomes injected into rats. , 1972, The Biochemical journal.

[2]  F. Szoka,et al.  Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. , 1979, Biochimica et biophysica acta.

[3]  D. Axelrod.  Fluorescence polarization microscopy. , 1989, Methods in cell biology.

[4]  L. Runnels,et al.  Theory and application of fluorescence homotransfer to melittin oligomerization. , 1995, Biophysical journal.

[5]  D. Piston,et al.  The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy. , 1996, Biophysical journal.

[6]  Y. Wu,et al.  Changes in membrane properties during energy depletion-induced cell injury studied with fluorescence microscopy. , 1996, Biophysical journal.

[7]  Andrew D. Miller.  Cationic Liposomes for Gene Therapy , 1998 .

[8]  C. Ropert.  Liposomes as a gene delivery system. , 1999, Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas.

[9]  S. Hell,et al.  Depolarization by high aperture focusing , 2000 .

[10]  D. Lilley,et al.  Location of cyanine-3 on double-stranded DNA: importance for fluorescence resonance energy transfer studies. , 2000, Biochemistry.

[11]  J. Nicolas,et al.  Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins. , 2001, Biophysical journal.

[12]  G. Haran,et al.  Immobilization in Surface-Tethered Lipid Vesicles as a New Tool for Single Biomolecule Spectroscopy , 2001 .

[13]  G. Danuser,et al.  A novel approach to produce biologically relevant chemical patterns at the nanometer scale: Selective molecular assembly patterning combined with colloidal lithography , 2002 .

[14]  V. Subramaniam,et al.  Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM). , 2002, Biophysical journal.

[15]  F. Höök,et al.  Patterns of DNA‐Labeled and scFv‐Antibody‐Carrying Lipid Vesicles Directed by Material‐Specific Immobilization of DNA and Supported Lipid Bilayer Formation on an Au/SiO2 Template , 2003, Chembiochem : a European journal of chemical biology.

[16]  V. Subramaniam,et al.  Photophysics of green and red fluorescent proteins: implications for quantitative microscopy. , 2003, Methods in enzymology.

[17]  E. Delamarche,et al.  Self-assembled microarrays of attoliter molecular vessels. , 2003, Angewandte Chemie.

[18]  D. Piston,et al.  Intrasequence GFP in class I MHC molecules, a rigid probe for fluorescence anisotropy measurements of the membrane environment. , 2003, Biophysical journal.

[19]  T. Foster,et al.  Imaging enzyme activity with polarization‐sensitive confocal fluorescence microscopy , 2004, Journal of microscopy.

[20]  P. Verveer,et al.  Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells. , 2004, Journal of structural biology.

[21]  H. Vogel,et al.  Integrated nanoreactor systems: triggering the release and mixing of compounds inside single vesicles. , 2004, Journal of the American Chemical Society.

[22]  D. Chiu,et al.  Determination of the encapsulation efficiency of individual vesicles using single-vesicle photolysis and confocal single-molecule detection. , 2005, Analytical chemistry.

[23]  Keith A. Lidke,et al.  The role of photon statistics in fluorescence anisotropy imaging , 2005, IEEE Transactions on Image Processing.

[24]  D. Chiu,et al.  Proton permeation into single vesicles occurs via a sequential two-step mechanism and is heterogeneous. , 2006, Journal of the American Chemical Society.

[25]  Confocal fluorescence polarization microscopy in turbid media: effects of scattering-induced depolarization. , 2006, Journal of the Optical Society of America. A, Optics, image science, and vision.

[26]  T. Ha,et al.  Multiple intermediates in SNARE-induced membrane fusion , 2006, Proceedings of the National Academy of Sciences.

[27]  Perry G. Schiro,et al.  Accurate sizing of nanoparticles using confocal correlation spectroscopy. , 2006, Journal of Physical Chemistry B.

[28]  Sune M. Christensen,et al.  Surface-based lipid vesicle reactor systems: fabrication and applications. , 2007, Soft matter.

[29]  S. Boxer,et al.  Model membrane systems and their applications. , 2007, Current opinion in chemical biology.

[30]  S. Boxer,et al.  Kinetics of DNA-mediated docking reactions between vesicles tethered to supported lipid bilayers , 2007, Proceedings of the National Academy of Sciences.

[31]  A. Bader,et al.  Imaging of protein cluster sizes by means of confocal time-gated fluorescence anisotropy microscopy. , 2007, Optics express.

[32]  M. Sanborn,et al.  Fluorescence properties and photophysics of the sulfoindocyanine Cy3 linked covalently to DNA. , 2007, The journal of physical chemistry. B.

[33]  D. Burgess,et al.  DNA-based therapeutics and DNA delivery systems: A comprehensive review , 2005, The AAPS Journal.

[34]  D. Lilley,et al.  The structure of cyanine 5 terminally attached to double-stranded DNA: implications for FRET studies. , 2008, Biochemistry.

[35]  M. Levitus,et al.  Nucleobase-Specific Enhancement of Cy3 Fluorescence , 2009, Journal of Fluorescence.

[36]  Pierre-Yves Bolinger,et al.  Encapsulation efficiency measured on single small unilamellar vesicles. , 2008, Journal of the American Chemical Society.

[37]  Huan‐Xiang Zhou,et al.  Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. , 2008, Annual review of biophysics.

[38]  T. Bonhoeffer,et al.  Live-cell imaging of dendritic spines by STED microscopy , 2008, Proceedings of the National Academy of Sciences.

[39]  Sune M. Christensen,et al.  A fluorescence-based technique to construct size distributions from single-object measurements: application to the extrusion of lipid vesicles. , 2008, Biophysical journal.

[40]  W. Meier,et al.  Observing Proteins as Single Molecules Encapsulated in Surface‐Tethered Polymeric Nanocontainers , 2009, Chembiochem : a European journal of chemical biology.

[41]  A. Bader,et al.  Homo-FRET imaging enables quantification of protein cluster sizes with subcellular resolution. , 2009, Biophysical journal.

[42]  J. Rädler,et al.  Diffusion and molecular binding in crowded vesicle solutions measured by fluorescence correlation spectroscopy , 2009 .

[43]  K. Kho,et al.  Confocal fluorescence polarization microscopy for linear unmixing of spectrally similar labels. , 2009, Micron.

[44]  D. Stamou,et al.  Quantification of nano-scale intermembrane contact areas by using fluorescence resonance energy transfer , 2009, Proceedings of the National Academy of Sciences.

[45]  T. Andresen,et al.  Drug delivery by an enzyme-mediated cyclization of a lipid prodrug with unique bilayer-formation properties. , 2009, Angewandte Chemie.

[46]  F. Caruso,et al.  Noncovalent liposome linkage and miniaturization of capsosomes for drug delivery. , 2010, Biomacromolecules.

[47]  J. Vörös,et al.  Liposome and lipid bilayer arrays towards biosensing applications. , 2010, Small.

[48]  M. Madsen,et al.  Liposomal formulation of retinoids designed for enzyme triggered release. , 2010, Journal of medicinal chemistry.

[49]  Sune M. Christensen,et al.  Sensing-Applications of Surface-Based Single Vesicle Arrays , 2010, Sensors.

[50]  P. Luisi,et al.  Spontaneous Protein Crowding in Liposomes: A New Vista for the Origin of Cellular Metabolism , 2010, Chembiochem : a European journal of chemical biology.

[51]  M. Levitus,et al.  Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments , 2010, Quarterly Reviews of Biophysics.

[52]  Y. Barenholz,et al.  Using liposomes to target infection and inflammation induced by foreign body injuries or medical implants , 2010, Expert opinion on drug delivery.

[53]  Joel M. Harris,et al.  Quantitative fluorescence microscopy to determine molecular occupancy of phospholipid vesicles. , 2011, Analytical chemistry.

[54]  N. Hatzakis,et al.  Observation of inhomogeneity in the lipid composition of individual nanoscale liposomes. , 2011, Journal of the American Chemical Society.