WO2002086472A1 - Derives de rotors moleculaires et leurs procedes d'utilisation - Google Patents

Derives de rotors moleculaires et leurs procedes d'utilisation Download PDF

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WO2002086472A1
WO2002086472A1 PCT/US2002/000715 US0200715W WO02086472A1 WO 2002086472 A1 WO2002086472 A1 WO 2002086472A1 US 0200715 W US0200715 W US 0200715W WO 02086472 A1 WO02086472 A1 WO 02086472A1
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viscosity
plasma
membrane
intensity
dcvj
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Emanuel A. Theodorakis
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Regents Of The University Of California
Haidekkere, Mark, A.
Frangos, John, A.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D455/00—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
    • C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
    • C07D455/04—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing a quinolizine ring system condensed with only one six-membered carbocyclic ring, e.g. julolidine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
    • G01N2011/006—Determining flow properties indirectly by measuring other parameters of the system
    • G01N2011/008—Determining flow properties indirectly by measuring other parameters of the system optical properties

Definitions

  • the present invention generally concerns the development and use of fluorescent dyes known as molecular rotors which vary in fluorescence intensity based on the viscosity of the environment.
  • the invention further relates to a class of fluorescent dyes called molecular rotors that are modified with a hydrocarbon chain or hydrophilic group to allow for the measurement of membrane or liquid viscosity, respectively.
  • Molecular rotors are fluorescent molecules that exhibit viscosity-dependent fluorescence quantum yield, potentially allowing direct measurements of cell membrane viscosity in cultured cells.
  • Equation 4 Equation 4
  • a background intensity I 0 has been allowed for, which is the non- fluorescent amount of light caused by filter bleed-through, scatter, and ambient light.
  • the background intensity may be non-neglectable.
  • DCVJ For DCVJ, the constant x has been shown to be 0.6 [2]. Experiments that were conducted in media with different viscosities have confirmed the above equation [6]. The quantum yield increases approximately by a factor of 30 when dissolved in 1-propanol and glycerol, respectively [6]. Furthermore, DCVJ has been used to probe the microviscosity changes in phospholipid bilayers, showing a viscosity change between 70 and 120 cP at temperatures from 60 to 10°C [6].
  • Plasma Blood Viscometer For Resuscitation Therapy
  • This need continues to drive forward extensive efforts to develop blood substitutes and new plasma expanders.
  • recent experimentation shows that none of the commercially available plasma expanders or blood substitute products are able to reverse changes that occur in the microcirculation due to severe blood losses.
  • Blood is a viscous medium composed of cells and liquid plasma.
  • the hematocrit (Hct) of blood is the percent of the blood that is taken up by cells, predominantly red blood cells.
  • a hematocrit of 45% means that 45 percent of the blood volume is cells and the remainder is plasma.
  • Normal hematocrit ranges from approximately 38 for women to 42 for men.
  • Normal blood viscosity is a function of hematocrit squared and is about 4 cP (37 DC).
  • Normal plasma viscosity is in the range of 1.15 - 1.35 cP (37 DC; the viscosity of water at this temperature is 0.71 cP).
  • the viscosity of blood can be calculated if the hematocrit is known. Blood losses diminish the viscosity of both blood and plasma due to autotransfusion, a mechanism by which the organism causes relatively protein free tissue fluid to move into the vascular compartment.
  • transfusion trigger has been widely described and accepted as the maximum Aacceptable@ blood loss, below which whole blood transfusions are required.
  • the normal hemoglobin (Hb) content in adults is approximately 14 gm/dl and is important to consider since it is the Hb that carries the oxygen to be delivered to the tissues.
  • Hct 7 gm/dl
  • red blood cells must be distributed throughout the circulatory system, particularly in the capillary network where red blood cells transfer their oxygen to cells and carry away toxic waste products from cell metabolism
  • FCD functional capillary density
  • Blood and plasma viscosity plays a critical role in maintaining the microcirculation open and functional via two major mechanisms.
  • viscosity acts on the vasculature by stimulating the endothelial cells, which line the blood vessels, to produce crucial chemical signals, which maintain vessel patency and vasodilation.
  • the physical forces exerted by normal viscosity plasma result in open capillaries. Under conditions of reduced viscosity, the capillaries collapse on themselves, preventing further flow of plasma or red blood cells, impairing oxygen delivery and waste product exchange. In conditions under which the red blood cell mass is sufficient to deliver oxygen to tissues impaired capillary blood flow results in no removal of metabolic waste products.
  • volume replacement or volume expanding therapy does not addresses the reduction in plasma viscosity and the prevention of significant closure of capillary networks.
  • the new theory indicates that it is critical to maintain microvascular function as a primary endpoint of resuscitation therapy. If this is accomplished, oxygen delivery and clearance of metabolic waste products will be maintained even under conditions of extreme lowering of the red blood cell mass. This has wide raging implications for economizing blood transfusions, while improving the efficacy of current available plasma expanders, and blood substitutes under development.
  • fluid resuscitation indicates that information on plasma and blood viscosity will become an integral part of the decision making process on the form of resuscitation to be administered, and determine the selection of the physical properties of the fluid to be used.
  • Moderate losses of blood red blood cells
  • red blood cells can be safely corrected with simple fluids, and do not require special considerations in terms of blood viscosity, and in fact may be beneficial corrected with low viscosity plasma expanders.
  • red blood cells red blood cells
  • the cardiovasculature easily adapts to the lowered blood viscosity by increasing cardiac output with the net effect that the distribution of mechanical effects remains nearly normal up to this point.
  • a critical point is reached when blood losses exceed 50%.
  • conventional therapy demands the use of blood indicates to restore volume and oxygen carrying capacity, up to a reposition of hematocrit to about 75% of normal.
  • the same, or even improved resuscitation can be obtained by blood volume restitution with a high viscosity solution that restores the viscosity of the fluid in the circulation to a value intermediate between normal blood and the value prevalent in the hemorrhaged condition. This is significant because this procedure can be carried out in the field, does not require blood typing, and becomes life saving when blood is not available.
  • Blood plasma is an important factor in physiology and disease. Blood plasma viscosity anomalies are associated with diseases such as diabetes, hypertension, infections and infarctions [8-12], A direct cause and effect relationship has not been established between plasma viscosity and the development of these conditions, thus clinical medicine has not to date provided a strong stimulus for the development of practical techniques for characterizing either blood or plasma viscosity.
  • Those molecules commonly referred to as fluorescent molecular rotors, belong to the group of twisted intramolecular charge-transfer complexes (TICT).
  • Photoexcitation leads to an electron-transfer from the donor group to the acceptor group.
  • Relaxation can either occur through radiation (fluorescence) or intramolecular rotation (thermally induced non-fluorescent relaxation).
  • the preferred relaxation mechanism, intramolecular rotation is reduced in solvents with low free volume. Therefore, the quantum yield of a molecular rotor increases with decreasing free volume of the solvent [21]. Free volume and viscosity are related [22], which links quantum yield directly to the viscosity of the microenvironment.
  • the relationship between fluorescence quantum yield D and the viscosity D of the solvent has been derived analytically [21,2,6] and experimentally [40,42] and is known as the F ⁇ rster-Hofrmann-Equation [2 supra].
  • DCVJ When used for probing viscosity changes in cell membranes, however, DCVJ is associated with some problems.
  • DCVJ is water-insoluble.
  • a staining solution for the cells, though, is aqueous.
  • this difficulty can be overcome by preparing the phospholipid base readily stained before forming the liposomes.
  • DCVJ can be bound to serum proteins [3]. Depending on the serum, the binding capacity is limited.
  • protein-bound DCVJ migrates into the cytoplasm, thus adding a high level of constant background signal which reduces the sensitivity of the probe.
  • FRAP fluorescence anisotropy
  • the method of FRAP is based on directing a focused laser beam at the cell membrane, which destroys the fluorescent dye in a defined region. Diffusion within the membrane allows dye from the neighboring environment to migrate into the bleached spot.
  • the half-life of the fluorescence recovery is inversely proportional to the diffusion coefficient of the dye[46], which in turn is proportional to the fluidity.
  • FRAP is limited in its resolution, as reduction of the radius of the bleached spot increases the error associated with the measurement.
  • the bleaching pulse introduces power densities up to 1 MW/cm 2 to the membrane [46], leading both to the generation of free radicals by photolysis as well as local heating with potential damage to proteins [46].
  • the second effect may cause artefacts, because the resulting cross-linking of the proteins [46] leads to decreased membrane diffusivity.
  • Membrane viscosity measurements by fluorescence anisotropy utilize the fact that certain molecules can only be excited by light waves in one polarization plane.
  • the emitted light again, is polarized.
  • the molecule can rotate, thus shifting the plane of emitted light.
  • the ratio of emitted light parallel versus perpendicular to the plane of excitation is a nonlinear function of the viscosity [42]. While the spatial resolution is essentially determined by the limits of the microscope, rapid photobleaching of the probes both confines the maximum sensitivity and the exposure time of the probe to the excitation light.
  • the polarizing filters in the emission light path generally absorb a significant portion of the emitted light.
  • the assessment of membrane viscosity using molecular rotors overcomes the limits posed by FRAP or fluorescence anisotropy measurements.
  • the spatial resolution of the method is potentially as high as it is for fluorescence anisotropy, because it is primarily limited by the optics of the microscope.
  • the molecular rotor also responds to changes of viscosity almost instantaneously (within the nanosecond range) [5]. This allows a very high temporal resolution of the measurements, so that dynamic observations become possible.
  • FIG. 2 Excitation and emission spectra of CCVJ in pure plasma and a plasma:PS solution mixture. This figure shows representative excitation (dashed lines) and emission (solid lines) spectra for CCVJ, taken at 475 nm emission and 430 nm excitation wavelength, respectively. All other probes showed similar spectral behavior. It can clearly be seen that the fluorescence intensity is markedly higher in the high-viscosity sample (40% plasma and 60% PS solution; thick lines) as compared to pure plasma (thin lines).
  • Figure 4 Intensity values (average ⁇ SD) or 5 independent fluorescence-based viscosity measurements with smaller viscosity increments than those in Figure 2. Shown are the results of measurements taken with CCVJ (panel a) and CCVJ-ME (panel b). All average values are significantly different (p ⁇ 0.0001) from their neighbors. There is no overlap of the values between adjoining bars.
  • Figure 5 Apparent non-Newtonian behavior of blood plasma when tested mechanically. Shown are representative measurements for plasma (left graph) and a mixture of 60% pentastarch solution with 40% plasma (right graph). The measured viscosity decreases with increasing shear stress. At high shear stress values, the change is relatively small, the viscosities where data points below and above 450 s "1 normally deviate less than 5%. For this reason, viscosity values at 450 s "1 were used as viscosity reference values.
  • Figure 6 Calibration curves to obtain the constants and in Equation 2. Intensity is plotted over viscosity in double-logarithmic scale The slope of each line is lower in the high viscosity range, which may in part be attributed to the apparent saturation effect visible in the data point with the highest viscosity.
  • Figure 7 Chemical structure of 9-(dicyanovinyl)-julolidine (1) and the basic structure of the compounds synthesized (2).
  • One of the nitrile functionalities of DCVJ has been replaced by a carboxylic group, allowing the addition of hydrocarbon chains R.
  • Figure 7A Further synthesis as in Figure 7 resulting in a double side chain having one or two specificities.
  • Figure 8 Synthesis of the compounds 2a - 2g.
  • Julolidine 3 was formylated to afford julolidine aldehyde 4.
  • the beta-cyanosters 7 were obtained by esterification of cyanoacetic acid 5 with the corresponding alcohol 6. Condensation of the esters 7 with julolidine aldehyde 4 produced the desired probes 2.
  • Figure 9 Excitation spectra (dashed lines) and emission spectra (solid lines) for DCVJ and FCVJ in ethylene glycol. The spectra of the other compounds are similar. All probes exhibit a double excitation maximum in the blue range, and a single emission maximum in the green range.
  • Figure 10 Changes of fluorescence intensity as a function of the increase of shear stress for compounds 2b, 2c, 2d, and 2g (FCVJ), as well as for DCVJ. All compounds except 2c differ significantly (pO.OOOl) from DCVJ.
  • Figure 11 Measured signal intensity of cells stained with DCVJ and FCVJ (2g) as a response to different step stimuli of shear stress. Both curves were subjected to a noise filter and an exponential correction for photobleaching. FCVJ shows a dramatically higher response. This allows a significantly higher sensitivity, while the noise component is greatly reduced.
  • FIG. 12 Decay of fluorescence intensity of the probes in stained cells due to photobleaching.
  • FCVJ shows a significantly higher decay half-time than DCVJ (p ⁇ 0.0005).
  • Compound 2c is less stable than DCVJ.
  • Plasma viscosity was changed by adding the clinical-grade high-viscosity plasma expander pentastarch (10% by weight, average molecular weight 260 kD) in physiological saline solution (Pentaspan ® , DuPont Pharma, Wilmington DW). Fluorescence measurements were preformed on a Shimadzu RF-1501 fluorospectrometer (Shimadzu, Kyoto, Japan) using standard methacrylate cuvettes (Fisher Scientific, Pittsburg, PA). Viscosity was measured using a Brookf ⁇ eld DV-JJ+ cone-and-plate viscometer with CP-40 spindle (Brookfield, Middleboro, MA).
  • Viscosity values were obtained at discrete shear rate settings of 75, 90, 150, 225, 450, 750 s "1 (10, 12, 20, 30, 60, 100 rpm). Viscosities measured at 60 rpm (450 s "1 ) were used as a reference in order to avoid the apparent non-Newtonian behavior at low shear rates, since this was not present beyond this shear rate. This shear rate allowed to measure the viscosity of all the samples without exceeding the torque limitations of the instrument.
  • Table 2 gives an overview over the measured maximum intensities of the probes DCVJ, CCVJ, CCVJ-methylester, CCVJ-ethylester, and CCVJ-butylester, normalized by the intensity measured in pure blood plasma. Pure blood plasma and mixtures of 80% plasma with 20% PS solution, 60% plasma with 40% PS solution, 40% plasma with 60% PS solution and 20% plasma with 80% PS solution were used. The intensity increase of fluorescence intensity in a high-viscosity mixture containing 80% PS solution over pure plasma is about two-fold (with DCVJ and CCVJ) to 3.5-fold (with CCVJ-esters). Precision assessment of the measurement method
  • Equation 2 the measured intensity I was used, which is proportionally related to the quantum yield D, but also depends on geometry, concentration and incident light intensity.
  • Fluorescent molecular rotors used as viscosity probes, rely on a different mechanism than the application of shear forces. They provide two modes of relaxation, fluorescence and nonradiative intramolecular rotation. The latter is dependent on the free volume of the environment, which in turn is related to its viscosity. Through the free-volume theory of viscosity, photophysical measurements using molecular rotors become independent from mechanical forces. The fluid under observation is not sheared during the measurement, and protein-surface interactions that may lead to artifacts at low shear rates do not affect the result. Further advantages over mechanical measurements include small sample volumes needed to perform fluorescence measurements (microcuvettes typically have a volume of 200-250 Dl), and the high speed of the readout: Using fixed wavelength filters, intensity can be measured within fractions of a second.
  • Molecular rotors provide a measurement of viscosity for conditions at zero shear rate (zero flow), therefore the viscosity data is applicable solely if the fluid has
  • the viscosity measurements derived from molecular rotors obtained with plasma samples at different concentrations and mixtures with colloids to obtain different viscosities are linearly related to the viscosity measured in a conventional mechanical viscometer operated in the range of 450 s "1 .
  • Molecular rotors show the same difference in viscosity between different plasma samples as that shown by a mechanical viscometer used in the Newtonian range of plasma viscosity. Therefore, in principle for the samples used in this study the viscosity at zero shear stress is quantitatively and linearly related to the viscosity in the Newtonian regime for plasma. Consequently measurements with the rotors are representative of conventional measurements, when the rotors are calibrated against a sample that can be measured by both methods, mechanical and fluorescent.
  • the sample cuvette should be temperature-controlled, which is feasible due to the low amounts of volume used. Fluid turbidity, common in blood plasma samples, strongly affects fluorescence through absorption of excitation and emission light. Optimized cuvette geometries, such as a narrow rectangular cross- section, and offsetting the angle of excitation light, may solve this problem. Dye concentration linearly affects emission intensity, therefore the precision of the measurement depends on the precision of dye delivery. This was the main reason to use pre-stained plasma in this study as aliquots of 1 ml are easier to produce than aliquots of 10 Dl or less. A solution would be to measure absorption simultaneously to emission. Since absorption is not a function of viscosity, it should be dependent only on dye concentration and therefore provide a measurement standard. Another approach would be the simultaneous calibration with samples of known viscosity.
  • Membrane viscosity is a physical property of the cell, which describes the movement of molecules within the phospholipid bilayer. In general, membrane viscosity depends on the chemical composition of the bilayer and is shown to have optimum values for the proper function of various membrane-bound enzymes and receptors. For example, artificial phospholipid bilayers were found to have viscosity values between 70 and 120 cP, depending on the temperature [6], while in human epidermal cells, viscosity values ranging from 30 to 100 cP have been reported [26]. In liver cells, viscosities ranged from 108 to 217 cP [27].
  • membrane viscosity or membrane fluidity, which is its reciprocal
  • membrane viscosity phospholipids and membrane-bound proteins show different vertical and lateral displacements, as well as lateral and rotational diffusion behavior [28]. Consequently, changes in membrane viscosity have been linked with alterations in physiological properties, such as carrier-mediated transport, activities of membrane- bound enzymes and receptor binding [29].
  • variations in membrane viscosity are linked to a variety of diseases, such as atherosclerosis[30], cell malignancy [31], hypercholesterolemia [32] and diabetes [33,34]. Further examples include an increase in membrane viscosity in several blood cells and specific brain cells in patients with
  • Alzheimer's disease [35,36]. Increased membrane viscosity is also associated with aging [37]. Erythrocytes showed permanently increased membrane viscosity, thus altering hepatic microcirculation [38]. Furthermore, endothelial cells are able to sense fluid shear stress through the cell membrane [39] and effect changes that serve to maintain a specific level of flow in blood vessels [40] .
  • membrane viscosity in cellular biology and physiology led to the development of fluorescent-based methods for quantitative measurements.
  • FRAP fluorescence recovery after photobleaching
  • fluorescence anisotropy in which the out-of-plane rotation of a fluorophore is related to membrane viscosity [42]
  • use of environment-sensitive fluorescent probes in which fluorescence recovery after photobleaching (FRAP) is related to membrane viscosity [41]
  • FRAP fluorescence recovery after photobleaching
  • DCVJ Due to the above properties, DCVJ has been employed to address a wide variety of problems, including polymerization processes [44], tubulin remodeling [45], and membrane viscosity measurements in liposomes and micelles [6]. More recently, DCVJ was employed to probe changes of membrane viscosity induced by fluid shear stress in cultured cells [3]. Nonetheless, the use of DCVJ in membrane viscosity measurements is often limited by its poor water solubility, which renders the preparation of DCVJ-based staining solutions problematic. For experiments on liposomes [6], this difficulty can be overcome by preparing a DCVJ-stained phospholipid base before forming the liposomes. In cultured cells, DCVJ can be bound to serum proteins [3], but their binding capacity is limited.
  • compounds 2b-2g have very similar viscosity-dependent fluorescence properties with DCVJ, but improved membrane localization.
  • the farnesyl-containing probe 2g farnesyl-(2-carboxy-2-cyanovinyl)-julolidine, FCVJ
  • FCVJ farnesyl-(2-carboxy-2-cyanovinyl)-julolidine
  • Step-shaped flow profiles of a successively rising magnitude were applied to the cell layer. In all cases, a marked decrease of emission intensity could be observed when flow was turned on, followed by a recovery of the intensity when the flow was turned off.
  • the average intensity of each phase of shear stress was subtracted from the average intensity during low shear stress (0.07 Pa), resulting in data pairs of intensity change over shear stress increase Fig. 11. Linear regressions were computed from these data pairs, and tests for statistical significance (t-test) as well as the deviation from linearity (runs test) were performed.
  • FCVJ had the strongest response to shear stress.
  • the amplitude of the fluorescence change in stained cells exposed to shear stress was more than 20 times higher than it could be observed with DCVJ.
  • Fig. 6 shows the measured intensities of the fluorescence of DCVJ in comparison to FCVJ in stained cell membranes as a response to step changes of fluid shear stress of different magnitudes in an experimental set-up similar to the one described in [3].
  • the signal-to-noise ratio was significantly lower. While a statistically significant detection of shear stress with DCVJ was possible with values higher than 0.6 Pa [3], FCVJ allowed the detection of changes lower than 0.1 Pa (data not shown).
  • nucleole staining became visible for compounds with shorter chains, including DCVJ and compound 2c. Stained nucleoles were not visible for compounds 2d and FCVJ. This indicates lower intracellular migration rates of these probes.
  • FRAP fluorescence anisotropy
  • the method of FRAP is based on directing a focused laser beam at the cell membrane, which destroys the fluorescent dye in a defined region. Diffusion within the membrane allows dye from the neighboring environment to migrate into the bleached spot.
  • the half-life of the fluorescence recovery is inversely proportional to the diffusion coefficient of the dye [46], which in turn is proportional to the fluidity.
  • FRAP is limited in its resolution, as reduction of the radius of the bleached spot increases the error associated with the measurement.
  • the bleaching pulse introduces power densities up to 1 MW/cm 2 to the membrane [46], leading both to the generation of free radicals by photolysis as well as local heating with potential damage to proteins [46].
  • the second effect may cause artefacts, because the resulting cross-linking of the proteins [46] leads to decreased membrane diffusivity.
  • Membrane viscosity measurements by fluorescence anisotropy utilize the fact that certain molecules can only be excited by light waves in one polarization plane.
  • the emitted light again, is polarized.
  • the molecule can rotate, thus shifting the plane of emitted light.
  • the ratio of emitted light parallel versus perpendicular to the plane of excitation is a nonlinear function of the viscosity [42].
  • the spatial resolution is essentially determined by the limits of the microscope, rapid photobleaching of the probes both confines the maximum sensitivity and the exposure time of the probe to the excitation light.
  • the polarizing filters in the emission light path generally absorb a significant portion of the emitted light.
  • the assessment of membrane viscosity using molecular rotors overcomes the limits posed by FRAP or fluorescence anisotropy measurements.
  • the spatial resolution of the method is potentially as high as it is for fluorescence anisotropy, because it is primarily limited by the optics of the microscope.
  • the molecular rotor also responds to changes of viscosity almost instantaneously (within the nanosecond range) [5]. This allows a very high temporal resolution of the measurements, so that dynamic observations become possible.
  • Tests were performed to determine whether each compound is suitable for membrane viscosity measurements.
  • the fluorescent properties of each probe were determined. All compounds were similar in their physical properties.
  • solubility in aqueous media was determined, a prerequisite for the staining of attached cells.
  • Both BSA and FCS may be used as a carrier. BSA binding was possible for all compounds except 2f.
  • the probes with shorter chains, 2a through 2d, as well as FCVJ, were also soluble in media prepared with FCS. Due to its carboxylic acid, 2a is directly water-soluble. All compounds capable of binding to serum albumins were then tested for their cell staining capability. All compounds except 2a and 2f were able to stain cells.
  • FCVJ (2g) a rotor in which a farnesyl group was attached to the julolidine part via an ester bond, showed a superior response when probing cell membrane viscosity changes than all other probes, including DCVJ.
  • Farnesyl is a hydrocarbon chain that improves membrane localization [31,32].
  • Substituting R for farnesyl (Fig 7) resulted in a dye that shows an improved affinity for the cell membrane over DCVJ. This leads to a slower migration into the cytoplasm, a lower background signal and a higher signal-to- noise ratio in the measured intensity time-courses.
  • the confocal images show that FCVJ can be found in the membrane at a higher concentration.
  • DCVJ and the short-chained compound 2b showed a high affinity for the cytoskeleton, and both dyes can be found in the nucleoles.
  • compound 2d there is less tubulin staining and no nucleole staining. This suggests that longer chains allow a stronger binding of the dye to the membrane, and also reduces the migration into the inner compartments of the cell.
  • Molecular rotors are new and promising tools for cell membrane viscosity measurements.
  • One of the applications is the measurement of cell membrane viscosity in cultured cells or in vivo: membrane viscosity changes are crucial in the understanding of many signaling processes and diseases.
  • Commercially available rotors such as DCVJ, however, are associated with disadvantages such as binding to the cell cytoskeleton and migration into inner compartments of the cell. While DCVJ allows the measurement of cell membrane viscosity changes, a higher sensitivity can be expected from DCVJ- derived molecules that show a higher affinity to the membrane.
  • FCVJ This new molecular rotor, referred to as FCVJ, improved the signal-to-noise ratio, thereby allowing the measurement of far smaller viscosity changes than DCVJ.
  • FCVJ Yellow orange solid; Rp0.34 (silica, 30% ethyl ether in hexanes); IR (film) m a x 2931.4, 2210.6, 1708.0, 1613.4, 1571.6, 1522.3, 1442.9, 1316.5, 1227.1, 1163.1, 1095.8; 1H NMR (400 MHz, CDC1 3 ) 7.93 (s, IH), 7.50 (s, 2H), 5.42-5.43 (t, IH), 5.08-5.10 (m, 2H), 4.76-4.78 (d, 2H), 3.30-3.33 (m, 4H), 2.72- 2.76 (m, 4H), 1.90-2.20 (m, 8H), 1.74 (s, 3H), 1.67 (s, 3H), 1.60 (s, 6H); 13 C NMR (100 MHz, CDC1 3 ) 164.6, 154.1, 147.3, 142.1, 135.2, 131.4, 131.1, 124.2, 123.5, 120.6, 118.
  • human umbilical cord endothelial cells were harvested using fresh umbilical cords, where the veins were treated with a 0.02% solution of collagenase in phosphate-buffered saline solution for 30 min at 25°C [1].
  • the cells were suspended in culture medium (Medium M199 with 2 mM L-glutamine, 50 U/ml penicillin, 50 ⁇ g ml streptomycin and 20% FCS) and seeded on tissue culture dishes. These dishes were kept in the incubator at 37°C in CO 2 enriched air (5% CO 2 ) until confluency was reached.
  • the cells were lifted with cell dissociation solution and resuspended in Dulbecco's modified Eagle's medium with 20% FCS at a concentration of 2.5 ⁇ l0 6 cells/ml. Aliquots of 350 ⁇ l of the suspension were used to seed the cells on glass slides (dimensions 10x40 mm) that were previously treated for 2 h with 0.5 M NaOH. The cells attached and fo ⁇ ned a confluent layer within 24 h.
  • a staining solution was prepared from 50 ⁇ l stock solution of 20 mM of the examined probe in dimethyl sulfoxide (DMSO), which was dispersed in 2 ml FCS under vigorous stirring. 10 ml medium Ml 99 was added. The slides with the cells were covered with the staining solution. Incubation took place over 10 min in the dark at 37°C. After the incubation period, the cells were rinsed with HBSS, and the fluorescence checked under an epifluorescent microscope (Diaphot TMD, Nikon, Garden City, NY, USA) using the G2B filter set.
  • DMSO dimethyl sulfoxide
  • Flow chambers were assembled using a standard methacrylate spectroscopic cuvette (Fisher Scientific, Pittsburgh, PA, USA) and a parallel-plate black Delrin flow chamber with a channel width of 6 mm and a depth of 500 ⁇ m.
  • the flow channel was formed between the flow chamber wall and the glass slide with the confluent cell monolayer.
  • the parallel-plate flow chamber the following relationship between flow Q and wall shear stress holds: where ⁇ is the viscosity of the medium, h is the channel depth and w is the channel width.
  • excitation wavelength was set to 455 nm, and emission wavelength to 505 nm.
  • An additional 475 nm LP emission filter (Chroma, Brattleboro, VT, USA) blocked scattered blue light.
  • a microcontroller-driven syringe pump (Pump 22, Harvard
  • ester group also functions as electron acceptor group in a similar fashion as the CN group. Replacing both CN-groups with ester groups retains the viscosity dependent quantum yield, while at the same time it allows to attach two recognition elements (Ri and R 2 in Figure 2 (right)). We also confirmed the synthesis and the physical properties of a rotor moiety attached to a phospholipid.
  • outer leaflet is mainly composed of phosphatidyl choline (PC) and sphingomyelin (SP)
  • inner leaflet is mainly composed of phosphatidyl ethanolamine (PE) and phosphaidyl serine (PS).
  • PC phosphatidyl choline
  • SP sphingomyelin
  • PE phosphatidyl ethanolamine
  • PS phosphaidyl serine
  • PS phosphatidyl serine
  • the lipids are also organized in the lateral dimension due to preferential packing of sphingolipids and cholesterol into microdomains.
  • These microdomains which include detergent-resistant areas, lipid rafts, and caveolae, have been shown to play an essential role to a variety of biological and physiological functions of the cell.
  • caveolae are membrane micropatches enriched in cholesterol, glycosphingolipids, signaling molecules and caveolin (a 22-kDa protein, involved in the structure/formation of caveolae). It has been found that caveolae are a sort of transport vesicles involved in protein (and small molecules) trafficking from the Golgi to the cell membrane and back and are participating in transmembrane signaling.
  • Molecular rotors are unique fluorescent molecules that allow the real-time monitoring of the viscosity of their solvent. This measurement involves the determination of the quantum yield. Since the direct measurement of the quantum yield is a relatively complicated process, it would be desirable to use methods where the measurement of emission intensity is sufficient. Emission intensity depends on the molecule's quantum yield as well as the concentration of the fluorophore, the incident light intensity, and the absorption characteristics of the fluid. In order to relate viscosity to intensity instead of quantum yield, it is therefore necessary to eliminate the above influences. While the incident light can be kept constant by relatively simple means, dye concentration and fluid abso ⁇ tion require special techniques, which are the subject of this disclosure.
  • ⁇ is the viscosity of the solvent
  • ⁇ is the quantum yield
  • x is a dye dependent constant
  • C is a constant that reflects the rate of intramolecular rotation
  • the molecular rotor can be coupled to a fluorescent reference moiety, which is not viscosity-sensitive. Since the quantum yield of the reference moiety is independent of the environmental properties, and thus can be determined for any reference molecule, concentration can be directly determined from the ratio of incident to emitted light intensities.
  • the measurement process can further be simplified by computing the ratio of the emitted light at the wavelength of the molecular rotor to the emitted light at th wavelength of the reference moiety, requiring two successive measurements at two different wavelengths.
  • Candidates for the reference moiety are any fluorophores with non-overlapping spectra. Particularly, the emission wavelengths must be different, and each pair of excitation and emission wavelength mist be different to prevent fluorescent resonance energy transfer (FRET).
  • FRET fluorescent resonance energy transfer
  • an ideal fluorescent molecule is Dil-C 18 (1,1 - dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) with an excitation maximum at 540 nm and an emission maximum at 560 nm (DCVJ absorbs at 460 mn and emits at 500 nm).
  • Any fluorescent dye absorbs light at its abso ⁇ tion wavelength, part of which is emitted in fo ⁇ n of fluorescence. This abso ⁇ tion is concentration-dependent, but - in the case of molecular rotors - not viscosity dependent, as can be shown in an experiment.
  • a simultaneous abso ⁇ tion measurement at the excitation wavelength can be used to determine the dye concentration. Similar to the reference moiety, a ratio of the emission intensity to the abso ⁇ tion coefficient can be computed, which is independent of dye concentration.

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Abstract

L'invention concerne la mise au point et l'utilisation de matrices fluorescentes connues comme rotors moléculaires dont l'intensité de fluorescence varie d'après la viscosité de l'environnement. L'invention concerne en outre une classe de rotors moléculaires modifiés par une chaîne d'hydrocarbure ou un groupe hydrophile afin de permettre la mesure de la viscosité d'une membrane ou d'un liquide.
PCT/US2002/000715 2001-01-12 2002-01-12 Derives de rotors moleculaires et leurs procedes d'utilisation WO2002086472A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005073697A1 (fr) * 2004-01-20 2005-08-11 The Curators Of The University Of Missouri Capteur de contrainte locale d'ecoulement et de cisaillement fonde sur l'utilisation de rotors moleculaires
WO2005072216A2 (fr) * 2004-01-20 2005-08-11 The Curators Of The University Of Missouri Viscosimetres moleculaires de biofluides sir support a usage un vitro et un vivo
WO2010141263A1 (fr) * 2009-06-05 2010-12-09 Danisco Us Inc. Dosage viscométrique à haut débit de rotors moléculaires
RU2672806C1 (ru) * 2017-12-07 2018-11-19 Федеральное государственное автономное образовательное учреждение высшего образования "Национальный исследовательский Нижегородский государственный университет им. Н.И. Лобачевского" Способ фотодинамической терапии с контролем эффективности в режиме реального времени
WO2018229018A1 (fr) * 2017-06-12 2018-12-20 Biomillenia Sas Procédé de mesure de viscosité dans un système microfluidique
WO2020249823A1 (fr) * 2019-06-14 2020-12-17 Alcediag Biomarqueurs nano-rhéologiques pour suivi précoce et amélioré de pathologies associées à une modification de la déformabilité des globules rouges
CN114136940A (zh) * 2021-11-18 2022-03-04 江南大学 一种淀粉凝沉结晶程度的快速测定方法及其应用
CN118130194A (zh) * 2024-03-08 2024-06-04 四川轻化工大学 一种顶空法荧光可视化快速检测血氨的试剂盒制备及检测方法

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005073697A1 (fr) * 2004-01-20 2005-08-11 The Curators Of The University Of Missouri Capteur de contrainte locale d'ecoulement et de cisaillement fonde sur l'utilisation de rotors moleculaires
WO2005072216A2 (fr) * 2004-01-20 2005-08-11 The Curators Of The University Of Missouri Viscosimetres moleculaires de biofluides sir support a usage un vitro et un vivo
WO2005072216A3 (fr) * 2004-01-20 2005-12-01 Univ Missouri Viscosimetres moleculaires de biofluides sir support a usage un vitro et un vivo
WO2010141263A1 (fr) * 2009-06-05 2010-12-09 Danisco Us Inc. Dosage viscométrique à haut débit de rotors moléculaires
JP2020527726A (ja) * 2017-06-12 2020-09-10 ビオミレニア ソシエテ パ アクシオンス シンプリフィエ マイクロ流体システムにおける粘度を測定する方法
WO2018229018A1 (fr) * 2017-06-12 2018-12-20 Biomillenia Sas Procédé de mesure de viscosité dans un système microfluidique
US20210208045A9 (en) * 2017-06-12 2021-07-08 Biomillenia Sas Method Of Measuring Viscosity In A Microfluidic System
JP7003240B2 (ja) 2017-06-12 2022-02-10 ビオミレニア ソシエテ パ アクシオンス シンプリフィエ マイクロ流体システムにおける粘度を測定する方法
RU2672806C1 (ru) * 2017-12-07 2018-11-19 Федеральное государственное автономное образовательное учреждение высшего образования "Национальный исследовательский Нижегородский государственный университет им. Н.И. Лобачевского" Способ фотодинамической терапии с контролем эффективности в режиме реального времени
WO2020249823A1 (fr) * 2019-06-14 2020-12-17 Alcediag Biomarqueurs nano-rhéologiques pour suivi précoce et amélioré de pathologies associées à une modification de la déformabilité des globules rouges
CN114136940A (zh) * 2021-11-18 2022-03-04 江南大学 一种淀粉凝沉结晶程度的快速测定方法及其应用
CN114136940B (zh) * 2021-11-18 2023-01-31 江南大学 一种基于荧光光谱测定淀粉糊储藏凝沉过程中相对热焓值的方法及其应用
CN118130194A (zh) * 2024-03-08 2024-06-04 四川轻化工大学 一种顶空法荧光可视化快速检测血氨的试剂盒制备及检测方法

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