FITC-labeled liposomes

With the optimized lipid composition (opDC ePC DOPE eSph Choi DC-Chol = 5 5 5 12 3), the HVJ-cationic liposomes showed 100 to 800 times greater transfection efficiency in vitro compared with the conventional HVJ-PS liposomes. The presence of serum (10% FCS) in the transfection mixture did not decrease luciferase activity significantly. Even 70% FCS reduced the activity by less than 40%. LacZ gene expression showed that transfection efficiency of BHK-21 cells by optimized HVJ-cationic liposomes (opDC) and by conventional HVJ-cationic liposomes (DC) was 90-100% and 50-60%, respectively. With conventional HVJ-anionic liposomes (PS), LacZ expression was found in only 1-3% of the cells. The optimized HVJ-cationic liposomes were also much more effective for the transfer of FITC-labeled ODNs to cultured cells [16]. 

In order to observe the behavior of chitosan-coated liposomes more precisely, chitosan was labeled with fluorescein isothiocyanate (FITC) via chemical reaction at the isothiocyanate group of FITC and the primary amino group of chitosan the liposomes (Lips) were marked by incorporation of Dil into the liposomal formulation. FITC-labeled chitosan (FITC-CS), non-coated liposomes, and FITC-labeled chitosan-coated liposomes (FITC-CS-Lips) were intragastrically administered into male Wistar rats, and then the behavior of the molecules was visualized by CLSM (Thongborisute et al. 2006a). The results demonstrated that the chitosan molecules themselves, as well as the liposomes, could penetrate across the intestinal mucosa. Moreover, the CLSM images demonstrated a lack of separation of the chitosan molecules from the surface of the liposomes after the administration of chitosan-coated liposomes. 

An avidin-biotin system has been used to attach antibodies in the bilayer of DDSs. Xiao et al. developed a three-step strategy to improve the tumor-to-tissue ratio of anticancer agents [184], Two antibodies specific for the CA-125 antigen that is highly expressed on NIH OVCAR-3 cells were used. These cells were prelabeled with biotinylated anti-CA-125 antibody and fluoroscein isothiocyanate (FITC)-labeled streptavidin (SAv) prior to administration of biotinylated liposomes. Both antibodies were specifically bound to the cell surface of OVCAR-3 cells but not to SK-OV-3 cells, which do not express the specific antibody. Antibody biotinylation did not affect its immunoreactivity. 

Form liposomes by hydration of the film with either 1 mL of HBS pH 6.5 or 1 mL of HBS (pH 7.0) containing 10 mg/mL FITC dextran in case of rhodamine-PE labelled or DiD labelled liposomes, respectively. 

Liposomes (without peptide) were labelled with rhodamine-PE in the lipid bilayer and in the inner compartment using FITC-dextrane 9000 and incubated with DCs for one hour. Figure 3 shows clearly that only in the case of AVE 3 and AVE 43 could a significant uptake be observed. The fluorescence inside the DCs is in the case of AVE 3 homogenously distributed in the cytosol, whereas in the case of AVE 43 the liposomes seem to be caught in granular structures, presumably endosomes. The PS causes the liposomes... 

Fig. 8.3. FCS Proton exchange kinetics measurements at biological membranes, (a) Principal design of experiment. Liposomes were labeled with one FITC fluo-rophore undergoing fluorescence fluctuations due to protonation/deprotonation. (b) Collection of FCS curves of the vesicles at different pH. The FCS curves reflect singlet-triplet transitions in the microsecond time range, protonation kinetics in the 10-100 ps time range and translational diffusion in the milliseconds time range. Inset measured protonation relaxation rates vs. proton concentration, (c) Principle of the proton collecting antenna effect...

Although outside the scope of this paper, it is of relevance to state that next to the HRP-conjugates we have prepared similar conjugates to a number of other reporter labels and enzymes, including FITC and )3-galactosidase, as well as CRM197-coated liposomes, and similar results were obtained (unpublished observations). 

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