Intracellular Delivery: A Multifunctional and Modular Approach - Intracellular Delivery: Fundamentals and Applications - page 218

7

p = 0.004 p = 0.02

6

5

4

3

FL1

2

1

0

C 12 FDG

Fig. 12 Flow cytometry of lysosomal targeting by liposomes loaded with C 12 FDG. HeLa cells
were incubated with plain liposomes (20 and 200 mg/ml), Lip-RhB (200 mg/ml). The liposomes
were loaded with C 12 FDG (1.5% mol/mol), a fluorescent substrate for the intralysosomal
b-galactosidase. After 4 h incubation with liposomes, the cells were washed and additionally
incubated for 20 h with liposome-free DMEM. The fluorescence intensity of FITC (channel FL1)
was determined by flow cytometry. Each value is the mean ± SD of 2 experiments

C 12 FDG into the fluorescent C 12 FITC, which will be retained inside lysosomes
because of its lipophilic moiety. Thus, with a standard flow cytometry procedure, the
lysosomal targeting can be quantified by following the fluorescence intensity of live,
intact cells. We prepared plain and RhB-modified liposomes loaded with C 12 FDG.
The loading of C 12 FDG into RhB-modified liposomes was approximately ten times
less than into the plain liposomes. This decrease in the C 12 FDG loading can be
attributed to a stoichiometric competition between C 12 FDG and RhB in the lipo-
somal membrane leading to the partial loss of C 12 FDG due to its shorter lipophilic
moiety. Two different concentrations of plain liposomes (20 mg/ml and 200 mg/ml)
were used for cell treatment to achieve the same amount of C 12 FDG as with RhB-
modified liposomes (200 mg/ml). The treatment of cells with different concentra-
tions of C 12 FDG-loaded plain liposomes (20 and 200 mg/ml) led to a dose-dependent
increase in their FITC fluorescence relative to the control (untreated) cells (Fig. 12 ).
These data suggest that endocytosed liposomes actually deliver C 12 FDG into lyso-
somes. The cells treated with 200 mg/ml of RhB-modified liposomes demonstrated
significantly increased C 12 FITC fluorescence compared to the cells treated with both
20 and 200 mg/ml of the plain liposomes.
In any case, the development of organelle-specific drug delivery is only in its
early stages; however, it might have important clinical future.