Predicting the behaviour and selectivity of fluorescent probes for lysosomes and related structures by means of structure-activity models (original) (raw)
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Colloids and Surfaces B: Biointerfaces, 2007
In order to study mechanisms involved in liposome-cell interaction, this work attempted to assess the influence of vesicle composition on the delivery of liposomal content to Hela cells. In particular, to evaluate pH-sensitive properties and cell interaction of the prepared liposomes, the lipid formulations contained cholesterol (Chol) and they were varied by using phosphatidylcholines with different purity degree: soy lecithin (SL; 80% phosphatidylcholine), a commercial mixture of soy phosphatidylcholine (P90; 90% phosphatidylcholine) or dipalmitoylphosphatidylcholine (DPPC; 99% of purity). A second series of liposomes also contained stearylamine (SA). Dehydration-rehydration vesicles (DRV) were prepared and then sonicated to decrease vesicle size. Vesicle-cell interactions and liposomal uptake were examined by fluorescence microscopy using carboxyfluorescein (CF) and phosphatidylethanolamine-dioleoyl-sulforhodamine B (Rho-PE) as fluorescent markers. Fluorescence dequenching assay was used to study the influence of pH on CF release from the liposomal formulations. Liposome adhesion on the cell surface and internalization were strongly dependent on vesicle bilayer composition. SA vesicles were not endocytosed. DPPC/Chol liposomes were endocytosed but did not release their fluorescent content into the cytosol. SL/Chol and P90/Chol formulations displayed a diffuse cytoplasmic fluorescence of liposomal marker. (A.M. Fadda).
Endocytosis and intracellular fate of liposomes using pyranine as a probe
Biochemistry, 1990
Lipid vesicles (liposomes) containing pH-sensitive fluorophores were used as probes for the study of liposome entry and intracellular fate. Pyranine [8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS)] was entrapped in the liposome aqueous core during preparation to provide a means of detecting internalization into living cells. HPTS is highly water soluble and shows a strong pH-dependent shift in its fluorescence excitation spectrum. Fluorescence emission (FEM) is slightly pH dependent with excitation (AEx) at 350-415 nm but highly pH dependent with AEX at 450 nm. Liposomes bearing a net negative charge bound rapidly to CV-1 cells and underwent endocytosis. One hour after liposome addition, high FEM with XEx at 413 nm and low F E M with XEX at 450 nm suggest that most cell-associated liposomes had been internalized and resided at a mean pH of-6.6. Collapse of cellular H+ gradients with NH4Cl or monensin treatment rapidly and reversibly increased F E M with XEX at 450 nm. Direct examination by fluorescence microscopy corroborates the fluorometric data on internalization; over time, F E M remained high with AEX at 350-405 nm but decreased with XEx at 450-490 nm, showing that all lipid vesicles were internalized within 40 min at 37 OC. Acidification of intracellular liposomes increased over 3 h, reaching a minimum value of approximately pH 5.5. HPTS persisted within acidic cellular vesicles for 2-3 days, and cytoplasmic dye was observed infrequently, suggesting that liposome fusion with cellular membranes seldom occurs. Material delivered to the endocytic pathway via lipid vesicles labeled an assortment of intracellular organelles of varying motility and morphology, including dynamic tubular structures whose lumen is acidic.
Interaction of molecular probes with living cells and tissues. Part 2
Histochemistry, 1990
Cultured rat fibroblasts were exposed to 41 cationic fluorescent probes of very varied hydrophilicity/lipophilicity. Outcome of probe-cell interaction fell into one of the following categories: probe failed to enter the cells; probe accumulated on cell surfaces; probe accumulated in mitochondria, and/or in other intracellular regions. The observations were analysed using a Simplistic Chinese Box (SCB) approach, and the following conclusions were reached. It was the hydrophilic probes which failed to enter cells, whilst extremely lipophilic probes were retained on the cell surfaces. Only the slightly lipophilic cationic probes were permeant, and accumulated in mitochondria. Using the probes log P values to model hydrophilicity/lipophilicity, effective cationic mitochondrial stains can be specified numerically so: 0 < log/)probe < + 5. This SCB model was used to rationalise a variety of earlier observations on the action of mitochondrial probes. The applicability of the SCB approach to integrate image-based and biochemical investigations was demonstrated by using the action of chlorpromazine on mitochondrial action as a case example.
A Stability Test of Liposome Preparations Using Steady-State Fluorescent Measurements
Drug Delivery, 2001
The stability of liposome preparations under the action of the nonionic detergent Triton X-100 was measured using the fluorescent molecular probe octadecylrhodamine B (R18). The probe inserted in the lipid bilayer shows a self-quenched fluorescence and the degree of quenching depends both on the probe concentration and the phase state of the lipid membrane. The addition of detergent to the liposomes produces a steep decrease in self-quenching caused by dilution of the probe in the bilayer. The curves of steady-state fluorescence intensity show an abrupt change in slope that corresponds to the point at which liposomes break down into lipid-detergent mixed entities that are different from the earlier liposome-monodisperse population. The lytic process was followed in parallel by dynamic light scattering (DLS), and the analysis of the DLS results agree with the interpretation of the fluorescence measurements. The probe R18 therefore is a useful marker to test the stability of liposome ...
Journal of Biomedical Optics, 2005
Liposomes are known to be taken up by the liver cells after intravenous injection. Among the few techniques available to follow this process in vivo are perturbed angular correlation spectroscopy, nuclear magnetic resonance spectroscopy, and scintigraphy. The study of the intracellular pathways and liposomal localization in the different liver cells requires sacrifice of the animals, cells separation, and electronic microscopy. In the acidic intracellular compartments, the in situ rate of release of liposomes remains poorly understood. We present a new method to follow the in situ and in vivo uptake of liposomes using a fluorescent pH-sensitive probe 5,6carboxyfluorescein (5,6-CF). 5,6-CF is encapsulated in liposomes at high concentration (100 mM) to quench its fluorescence. After laparotomy, liposomes are injected into the penile vein of Wistar rats. Fluorescence images of the liver and the skin are recorded during 90 min and the fluorescence intensity ratio is calculated. Ratio kinetics show different profiles depending on the liposomal formulation. The calculated intracellular liver pH values are, respectively, 4.5 to 5.0 and 6.0 to 6.5 for DSPC/chol and DMPC liposomes. After sacrifice and flush with a cold saline solution, the pH of the intracellular site of the liver (ex vivo) is found to be 4.5 to 5.0. This value can be explained by an uptake of liposomes by the liver cells and subsequent localization into the acidic compartment. An intracellular event such as dye release of a drug carrier (liposomes loaded with a fluorescent dye) can be monitored by pH fluorescence imaging and spectroscopy in vivo and in situ.
Molecular Pharmaceutics, 2005
Many weakly basic drugs incubated with cells have been shown to specifically accumulate in lysosomes. The mechanistic basis and substrate specificity for this sequestration have not been rigorously evaluated; however, conditions are favorable for a pH-partitioning type accumulation. In some circumstances, this compartmentalization can be very extensive, which can impact the therapeutic efficacy of a drug. Despite the pharmaceutical importance, direct quantitative assessments of drug accumulation in lysosomes have not been previously described. We report here a novel magnetic capture technique that allows for quick and efficient isolation of lysosomes from cultured HL-60 cells that have been preincubated with model compounds. The amount of compound associated with the isolated fraction is determined by HPLC. Extensive biochemical and morphological characterizations of isolated lysosomes, together with HPLC data, allowed for estimates to be made regarding the concentration of model compounds in lysosomes. The corresponding theoretically determined concentration values, based on pHpartitioning theory, were also calculated for comparison purposes. Interestingly, experimentally determined values were approximately 3-15 times higher than theoretically predicted values. This finding suggests that mechanisms, in addition to pH-partitioning, may play a significant role in the accumulation of drugs in lysosomes.
Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities
Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane , defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleav-able functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker 1 Red DND-99 as well as with anti-LAMP1 Anti-body staining. When cell metabolism was inhibited with chloroquine, staining with an ester-ase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lyso-somal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and monitoring the effect of secondary therapeutic agents on lysosomal enzyme activity in drug development for the lysosomal storage disorders and allied diseases.
The response of fluorescent amines to pH gradients across liposome membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1972
Phospholipid liposomes were used to test atebrin and 9-aminoacridine as fluorescent probes for measuring pH gradients across membranes. Quenching of 9-aminoacridine fluorescence could be quantitatively related to the magnitude of pH gradients across liposome ...
Fluorescence Behavior of the pH-Sensitive Probe Carboxy SNARF-1 in Suspension of Liposomes¶
When exposed to the intracellular environment fluorescent probes sensitive to pH exhibit changes of photophysical characteristics as a result of an interaction of the dye molecule with cell constituents such as proteins, lipids or nucleic acids. This effect is reflected in calibration curves different from those found with the same dye in pure buffer solutions. To study an interaction of the probe 5(and 6)-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7,1(3H)-isobenzofuran]-3one (carboxy SNARF-1) with membrane lipids, we measured its fluorescence in model systems of large unilamellar vesicles (LUV) prepared by extrusion. When the dye was removed from the bulk solution by gel filtration the relative fluorescence intensity of the lipid-bound dye form was enhanced, showing a strong interaction of the dye molecule with LUV membrane lipids. Surprisingly, the dye molecules seem to be bound predominantly to the outer surface of the lipid bilayer. The same situation was found with small unilamellar vesicles prepared by sonication. This effect makes it difficult to use carboxy SNARF-1 for measurements of the internal pH in suspensions of liposomes.