Fluorescent liposomes

Further support to the notion that liposomal BPs exert their effects systemically was achieved through the use of liposomes loaded with the fluorescent marker, Rhodamine, with or without a BP (52,69). Marked reduction of the fluorescent signal was observed in blood monocytes (as well as reduced number) and in the liver and spleen of LBP-treated animals. Fluorescent liposomes (FL) were detected in injured but not in intact arteries. FL coadministered with LBPs significantly reduced the fluorescent signal in the injured arterial wall. The inactivation of monocytes after systemic administration of LBPs results in reduction of tissue macrophages in the injured artery. Thus, the outcome of systemic administration was manifested as local treatment for the injured artery. 

Choquette, S. J., Locascio-Brown, L., and Durst, R. A. (1992). Planar waveguide immunosensor with fluorescent liposome amplification. Anal. Chem. 64 55-60. 

The sites of targeting of IL on hypoxic cardiocytes was visualized by fluorescent microscopy, using rhoda-mine labeled antimyosin immunoliposomes. Fig. 17 shows that only cells treated with antimyosin-rhoda-mine labeled IL were still confluent in the culture and that almost all cells were labeled with fluoresent liposomes. Those cells treated with rhodamine labeled PL showed extremely sparse number of cells still attached to the culture plates at 24 h of incubation, with essentially no or minimal fluorescence. Confocal microscopic examination of the cultures treated with rhodamine labeled IL showed that the cells still retained their morphology and shape with scattered fluorescent liposomes attached to the cell membranes (Fig. 18A). Those cells treated with rhodamine labeled PL were shrunken and only a few random cells showed some non-specific attachment of fluorescent PL (Fig. 18B). In this study, untreated hypoxic cells were all dead and since there was no fluorescent compounds added in them, no micrographs were obtained. 

Fig. 1. Analysis of fluorescence colocalizafion Pearsons coefficient standard deviation (/ =6) for colocalizafion of rhodamine fluorescence (liposomal marker) with Mitofluor green fluorescence (mitochondrial marker) obfained with Imaged. Open bars indicate nonfargefed liposomes, shaded bars n(i ca e STPP liposomes. (Asferfs/rindicates a Pvalue of <0.005). (Reproduced wifh permission from ref. 8)...

S. Y Friesner, D. L. Mallik, S. Fluorescent liposomes for differential interactions with glycosaminoglycans. Anal. Chem. 2011, 83, 5989-5995. 

The experimental principle is illustrated in Fig. 3. The interaction of the polymer with the liposomal membranes causes the perturbation of the bilayer. This perturbation follows the leakage of calcein from the liposome. Calcein in high concentration in the liposome is self-quenched, but has strong fluorescence intensity by the leak from the liposome. Therefore, the extent of the membrane interaction can be estimated quantitatively from the fluorescence spectroscopy. 

An alternative approach is the use of pH-sensitive fluorophores (Lichtenberg and Barenholz, lOSS). These probes are located at the lipid-water interface and their fluorescence behavior reflects the local surface pH, which is a function of the surface potential at the interface. This indirect approach allows the use of vesicles independent of their particle size. Recently, techniques to measure the C potential of Liposome dispersions on the basis of dynamic light scattering became commercially available (Muller et al., 1986). 

Solubilization of an active H,K-ATPase is also a prerequisite for reconstitution of the enzyme into liposomes. With these H,K-ATPase proteoliposomes it is then possible to study the transport characteristics of pure H,K-ATPase, without the interference of residual protein contamination that is usually present in native vesicular H,K-ATPase preparations. Rabon et al. [118] first reported the reconstitution of choleate or n-octylglucoside solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. The enzyme was reconstituted asymmetrically into the proteoliposomes with 70% of the pump molecules having the cytoplasmic side extravesicular. In the presence of intravesicular K, the proteoliposomes exhibited an Mg-ATP-dependent H transport, as monitored by acridine orange fluorescence quenching. Moreover, as seen with native H,K-ATPase vesicles, reconstituted H,K-... 

Biochemical studies with purified preparations incorporated into liposomes have also been performed [32,33,96-98]. Reconstituted receptors from skeletal muscle bound DHPs, PAAs and diltiazem with high affinity and in a 1 1 1 stoichiometry [97], In general, the reconstituted proteins exhibit the characteristic pharmacological properties expected for these channels. In recent studies, our laboratory has reconstituted partially purified channels into liposomes containing the Ca -sensitive fluorescent dye, fluo-3 [33,96]. These channels exhibit Ca influx that is sensitive to activation by Ca channel activators and inhibitors with affinities similar to those observed in intact cells, and the Ca influx is dependent on the establishment of a gradient in the presence of valinomycin [132]. This assay provides a convenient and rapid approach to obtaining a macroscopic picture of the activity of the channels under different conditions, while the more complex studies in lipid bilayers provide a more complete analysis of the single channel behavior. 

A further partihon system based on the use of liposomes, and commercialized under the name Transil [110, 111], has shown its utiUty as a UpophiUcity measure in PBPK modeling [112]. Fluorescent-labeled liposomes, called fluorosomes, are another means of measuring the rate of penetration of small molecules into membrane bilayers [113, 120]. Similarly, a colorimetric assay amenable to HTS for evaluating membrane interactions and penetrahon has been presented [116]. The platform comprises vesicles of phospholipids and the chromahc Upid-mimehc polydiacetylene. The polymer undergoes visible concentrahon-dependent red-blue transformahons induced through interactions of the vesicles with the studied molecules. 

The permeability coefficient of 2.6x 10 locm/s at 296 K measured by Deamer is sufficient to supply the enzyme in the liposomes with ADP. How could it be shown that RNA formation actually does take place in the vesicles The increase in the RNA synthesis was detected by observing the fluorescence inside the vesicles. In the interior of the liposomes, the reaction rate is only about 20% of that found for the free enzyme, which shows that the liposome envelope does limit the efficiency of the process. The fluorescence measurements were carried out with the help of ethidium bromide, a fluorescence dye often used in nucleic acid chemistry. 

Pal, R., Barenholz, Y. and Wagner, R. R. (1988). Pyrene Phospholipid as a biological fluorescent-probe for studying fusion of virus membrane with liposomes. Biochemistry (Mosc). 27, 30-36. 

Razinkov, V. I., HernandezJimenez, E. I., Mikhalyov, 1.1., Cohen, F. S. and Molotkovsky, J. G. (1997). New fluorescent lysolipids Preparation and selective labeling of inner liposome leaflet. Biochim. Biophys. Acta-Biomembranes 1329, 149-158. 

Sipkins et al. [87] described the detection of tumor angiogenesis with an avp3-specific antibody that was conjugated to polymerized paramagnetic liposomes [87]. The red fluorescence represents the liposomes (Fig. 26c) and the green fluorescence represents blood vessels. In Fig. 26, we see that... 

Fig. 26 MR images of tumors of mice after they were injected with (a) paramagnetic av[33-specific RGD-liposomes and (b) nonspecific paramagnetic RAD-liposomes. (c, d) Fluorescence microscopy of 10 pm sections from dissected tumors revealed a distinct difference between tumors of mice that were injected with RGD-liposomes (c) or RAD-liposomes (d). Vessel staining was done with an endothelial cell-specific FITC-CD31 antibody. The red fluorescence represents the liposomes and the green fluorescence represents blood vessels. RGD-liposomes were exclusively found within the vessel lumen or associated with vessel endothelial cells (c), whereas RAD-liposomes (d) were also found outside blood vessels within the tumor (Adapted from [88])...

Covalent attachment of antibody molecules to liposomes can provide a targeting capacity to the vesicle that can modulate its binding to specific antigenic determinants on cells or to molecules in solution. Antibody-bearing liposomes may possess encapsulated components that can be used for detection or therapy (Figure 22.17). For instance, fluorescent molecules encapsulated within antibody-targeted vesicles can be used as imaging tools or in flow cytometry... 

Figure 22.17 Antibody-liposome conjugates may be used as targeting reagents for detection or therapeutic applications. The liposome may be constructed to contain fluorescent molecules for detection purposes or bioactive agents for therapy. The antibody component targets the complex for binding to specific antigenic determinants.

Figure 22.18 Biotinylated liposomes may be used in immunoassay systems to enhance the signal for detection or measurement of specific analytes. The liposome components may be constructed to include fluorescent molecules to facilitate detection of antigens within tissue sections.

Chen, R.F., and Knutson, J.R. (1988) Mechanism of fluorescent concentration quenching of carboxyfluo-rescein in liposomes Energy transfer to nonfluorescent dimers. Anal. Biochem. 172, 61. 

T. Ohyashiki, M. Nunomura, and T. Katoh, Detection of superoxide anion radical in phospholipid liposomal membrane by fluorescence quenching method using 1,3-diphenylisobenzofuran. Biochim. Biophys. Acta. 1421, 131-139 (1999). 

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