Confocal liposomes

Beyer, U., Rothen-Rutishauser, B., Unger, C., Wunderli-Allenspach, H., Kratz, F., Differences in the intracellular distribution of acid-sensitive doxorubicin-protein conjugates in comparison to free and liposomal formulated doxorubicin as shown by confocal microscopy. Pharm Res 18, 29-38 (2001). 

Figure 3 Binding and internalization of different liposomal formulations by bone marrow-derived dendritic cells (BmDQ from mice. BmDC were incubated for one hour with liposomes at 37 C and liposome uptake was analyzed by confocal laser scanning microscopy.

Integrin receptor-binding peptides have been used to enhance liposome binding, uptake, and expression (25,47 9). The inclusion of an 0(5pi integrin-targeted peptide into a liposomal complex enhanced transfection efficiency four- to five-fold in Jurkat cells and 10- to 13-fold in TF-1 cells (48). Confocal and electron microscopy revealed that the mechanism of cell entry conferred by RGD peptides on liposomes is predominantly by clathrin-coated endocytosis rather than by phagocytosis (50). 

In common with the polylysine DNA-condensing peptide, the p peptide has also been shown to have nuclear localization properties. Confocal microscopy revealed that p peptide in a complex containing fluorescent lipid- and dye-labelled DNA associates with the nuclei and nucleoli of both dividing and nondividing cells within 15 minutes of exposure to the complex, thus suggesting an NLS function. However, this property was not detectable when the peptide became incorporated into a 3p-[N-(N, N -dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol)/DOPE cationic liposome (149). It may be the case that the lipids may mask the critical residues. 

A Lundqvist, G Ocklind, L Haneskog, P Lundahl. Freeze-thaw immobilization of liposomes in chromatographic gel beads evaluation by confocal microscopy and effects of freezing rate. J Mol Recogn 11 52-57, 1998. 

Yomo, Urabe and coworkers (Yu et al, 2001), for example, reported the expression of a mutant GFP (actually the pET-21-GFPmutl-His6 mutant) in lecithin liposomes. Large GFP-expressing vesicles, prepared by the film hydration method, were analyzed using flow cytometry as well as confocal laser microscopy. 

M. E. M. J. van Kuyk-Meuwissen, H. E. Junginger, and J. A. Bouwstra, Interactions between liposomes and human skin in vitro, a confocal laser scanning microscopy study, Biochim. Biophys. Acta 7377 13-39 (1998). 

Verma, D.D., Verma, S., Blume, G. et al. Liposomes increase skin penetration of entrapped and non-entrapped hydrophilic substances into human skin a skin penetration and confocal laser scanning microscopy study. Eur. J. Pharm. Biopharm. 2003 55 271-7. 

Fig. 10.5 Protocol for measurement of the mucoadhesive behavior of polymer-coated liposomes with confocal laser scanning microscopy (CLSM)...

Fig. 10.6 Confocal laser scanning microscopy photographs of various parts of the intestinal tract of the rat at 2 h after intragastric administration of ssCS-Lips (taken from Takeuchi et al. 2005a). The measured mean diameter is 281.2 nm. The formulation of liposome is DSPC DCP cholesterol = 8 2 1...

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. 18 Confocal micrographs of 24 h hypoxic H9C2 cardiocytes treated with rhodamine labeled IL (B) or rhodamine labeled PL (A). The micrographs are shown in pseudocolors. Cells treated with IL showed retention of membrane integrity and cell morphology (A). Liposomes represented as yellow colored regions are also discernable on the cells. Cells treated with PL showed presence of only dead cells with only a few cells having non-specifically attached PL (B). (From Ref...

Confocal fluorescence micrographsobtained upon incubation of cells with STPP liposomes (see Note 5) are subjected to fluorescence colocalization analysis using ImageJ. Pearsons coefficient values obtained for STPP liposomes are compared to non-targeted liposomes prepared by replacing 1.5% STPP with 1.5%DOTAP(Fig.l). 

TAT-peptide modified liposomes were prepared and cellular association and intracellular distribution of (double) fluorescently labelled particles were assessed by flow cytometry and confocal laser scanning microscopy. 

Directly visualize the samples using a confocal microscope equipped with a 568 nm laser, suitable for monitoring Rhodamine-PE labelled liposomes (see Fig. 3). 

Fig. 2. Intracellular localization of TAT-liposomes. OVCAR-3 cells are incubated with 150 nmol of double fluorescently labelled TAT-liposomes for 1 h and subsequently incubated for 1 h (a) or 23 h (b) in liposomes-free medium. Thirty minutes before visualization the endocytic pathway is labelled with Lysotracker Red. Live cell imaging is performed with confocal laser scanning microscopy. Double labelled liposomes are used to study the integrity of the liposome during the uptake process co-localization of both liposomal labels would indicate that the liposomes are intact. 1 h both liposomal labels are localized at the plasma membrane, which represent intact cell-bound TAT-liposomes. The electronically merged image clearly shows lack of co-localization with the endocytic pathway marker, Lysotracker Red. This opposite to the 24 h incubation, both liposomal labels can be seen intracellularly in a punctuate pattern. In the electronically merged image, co-localization with Lysotracker Red is clearly visible. This indicates that the TAT-peptide modified liposomes bind to the plasma membrane and after internalization are present in endocytic vesicles. Therefore, we conclude that the liposomes are internalized by endocytosis. (Reproduced from (12) with permission from Elsevier Science)...

In a subsequent study, van Hal et al. [40] reported that a decrease in cholesterol content in liquid state bilayers, which increases bilayer fluidity, resulted in an increase in estradiol transport across SC. With confocal laser scanning microscopy, Meuwissen et al. examined the diffusion depth of gel- vs. liquid-state liposomes labeled with fluorescein-dipalmitoylphosphatidylethanolamine (fluorescein-DPPE) with human skin in vitro [41] (Figure 3) and rat skin in vivo [42] and found that the lipophilic label when applied in liquid-state bilayers onto the skin penetrated deeper into the skin than when applied in gel-state liposomes. Recently, Fresta and Puglisi [43] reported that corticosteroid dermal delivery with skin-lipid liposomes was more effective than delivery with phospholipid vesicles, both with respect to higher drug concentrations in deeper skin layers and therapeutic effectiveness. This is a very surprising result, because skin lipid liposomes are rigid and form stacks of lamellae on the surface of the skin [44]. From the previously mentioned studies it seems clear that the thermodynamic state of the bilayer plays a crucial role in the effect of vesicles on dmg transport rate across skin in vitro. 

In skin research, drug and chemical targeting to various layers of skin is of great interest. For this piupose, various approaehes have been tried. Confocal laser scanning microscopy has been employed to study the influence of permeant lipophilicity and vehicle composition on local chemical acciunulation (Grams et al., 2003). In these studies, fluorescently labeled liposomes were applied on skin in vivo, and the penetration pathway and penetration depth of the lipophflie fluorescent label into the skin was visualized by confocal laser scanning microscopy (Van Kuijk-Meuwis-sen et al., 1998). 

Jelinek and co-workers demonstrated that the changes in fluorescence of PDA can be used to detect the interactions of drug molecules with the membranes of live cells. Diacetylene patches containing 10,12-tricosadiynoic acid were transferred from diacetylene vesicles to the membranes of leukemia cells and were polymerized with UV radiation. This fusion efficiency was found to depend on the lipid composition of the liposomes and the presence of cholesterol on the plasma membrane. The interactions of these cells with lidocaine (a local anesthetic), polymixin-B (an antibiotic), and oleic acid were studied by confocal fluorescence microscopy. The PDA patch on the live cells showed bright red fluorescence when the cell membranes were perturbed. The blue to red color change in the presence of oleic acid was apparent when the cells were sedimented by centrifugation. 

Uster, P. S., Working, P. K. and Vaage, J., Pegylated liposomal doxorubicin (DOXIL , CAELYX ) distribution in tumour models observed with confocal laser scanning microscopy, Int. J. Pharm., 162, 77-86 (1998). 

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