Figure 1.
Development of nanoparticles incorporating a novel liposomal membrane destabilization peptide (LMDP) for efficient drug release into cancer cells.
This image depicts the concept of the carriers incorporating LMDP into the liposomal membrane (LMDP-lipo). The lipid bilayer membrane of LMDP-lipo is destabilized by cleaving LMDP as a trigger for membrane fusion. The destabilization promotes cargos release into the cell.
Figure 2.
(a) γ-secretase activity in cultured cells. The fluorescence peptide probe was incubated with membrane fractions from HUEhT-2, HeLa, MCF-7 and A549 cells. (b) Competitive inhibition of LMDP. The peptide probe and LMDP were incubated at the indicated concentrations in the membrane fraction of A549 cells at 37°C overnight. Values and bars represent the means and SD, respectively. (c) Representative chromatograms of LMDP with or without the membrane fraction as determined by HPLC analysis from three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001 versus HUEhT-2 (a), 0 µM LMDP (b), n = 3.
Table 1.
LMDP cleavage activity of γ-secretase assessed by HPLC analysis.
Figure 3.
Control-lipo consisted of EPC or EPC/DOPE/DOTAP (DOTAP), or EPC/DOPE/CHEMS (CHEMS). LMDP-lipo contained 5 mol% LMDP. They were incubated with the membrane fraction from A549 cells at 37°C for 1 h. White and black columns indicate Control-lipo and LMDP-lipo, respectively. Values represent the means of three individual experiments. Bars represent SD. *, P<0.05.
Table 2.
Lipid composition and physicochemical properties of LMDP-lipo.
Figure 4.
Characterization of LMDP-lipo cleavage by γ-secretase.
(a) Competitive inhibition of LMDP in liposomes. The fluorescence peptide probe was incubated with Control-lipo (EPC/DOPE/CHEMS) or LMDP-lipo (EPC/DOPE/CHEMS/3 mol% LMDP) at the indicated LMDP concentrations in the presence of the membrane fraction of A549 cells at 37°C overnight. White and black columns indicate Control-lipo or LMDP-lipo, respectively. (b) Representative chromatograms of LMDP-liposomes with or without the membrane fraction as determined by HPLC analysis from three independent experiments. (c) Size distribution of liposomes in the presence of the membrane fraction obtained from A549 cells with or without γ-secretase inhibitor was measured using the Zetasizer nano. Solid line and dotted line indicate Control-lipo and LMDP-lipo, respectively. (d) Polydispersity index (PDI) of the peak using Control-lipo or LMDP-lipo. White, gray and black columns indicate liposomes alone, liposomes in the presence of an A549 cell membrane fraction or liposomes in the presence of a membrane fraction with 10 µM DAPT, respectively. (e) Abundance distribution of the peak for Control-lipo (black circles) and LMDP-lipo (black triangles) under the same conditions as (d). (Values and bars represent the means and SD, respectively. *, P<0.05 and ***, P<0.001 versus 0 µM LMDP (a), Control-lipo (c, d), n = 3.
Table 3.
HPLC analysis of LMDP in liposomes after treatment with cell membranes containing γ-secretase.
Figure 5.
Selective calcein release from LMDP-lipo.
Control-lipo (EPC/DOPE/CHEMS) or LMDP-lipo (EPC/DOPE/CHEMS/5mol% LMDP) was incubated in the presence of the membrane fraction from HUEhT-2 cells (white columns), HeLa cells (dotted columns), MCF-7 cells (cross-hatched columns), A549 cells (black columns), or A549 cells with 10 µM DAPT (gray columns) at 37°C for 1 h. Data represent means ± S.D. (n = 3–7) *, P<0.05, **, P<0.01 and ***, P<0.001.
Figure 6.
Calcein release from LMDP-lipo into cells.
DiI-labeled and calcein encapsulated Control-lipo (EPC/DOPE/CHEMS) (a), LMDP-lipo (EPC/DOPE/CHEMS/5 mol% LMDP) (b), LMDP-lipo in the presence of 50 µM DAPT (c), LMDP-lipo in the presence of 0.4 M sucrose (d), LMDP-lipo in the presence of 2.5 mM amiloride (e) and LMDP-lipo in the presence of 5 µg/mL FilipinIII (f) were added to A549 cells and incubated at 37°C for 1 h. The intracellular location of CM-DiI (red) and calcein (green) was observed by CLSM. Red signal indicates liposomes. Scale bars, 10 µm.
Figure 7.
Time lapse imaging of LMDP-lipo by CLSM.
(a) DiI-labeled and calcein-encapsulated LMDP-lipo (EPC/DOPE/CHEMS/5 mol% LMDP) was added to A549 cells and incubated at 37°C. CM-DiI (red) and calcein (green) were observed at intervals of 30 sec by CLSM. Asterisks indicate the same LMDP-lipo. Scale bars, 2 µm. (b) Schematic image of fluorescence measurement. Fluorescence intensities of CM-DiI and calcein were measured at 0, 2 and 4 µm from the centers of dots indicating fluorescent-labeled liposomes taken up by the cells. Changes in fluorescence intensity in 3 dots/cell for 5 cells at the indicated times were analyzed for CM-DiI (c) and calcein (d) using NIS-Elements software.