Fig 1.
Analyses of stable cell lines.
(A) Determination of relative PLA2G2A mRNA levels in L2P2 and control cells, normalized to GAPDH and ACTB levels. Values are referred to Colo 205 cells with high endogenous PLA2G2A mRNA levels [35]. (B) Relative luciferase activity of L2 and L2P2 cells normalized to cell numbers as determined by a cell viability assay as substitute readout. Error bars represent standard error of the mean (SEM).
Fig 2.
Schematic illustration of the principle of the developed firefly luciferase bioassay for following in situ sPLA2-assisted release of luciferin from liposomes.
(A) Engineering of a stable MCF-7 breast cancer cell line to produce firefly luciferase and sPLA2 enzymes. The sPLA2 enzyme will be secreted to the extracellular fluid. (B) Preparation of luciferin remote-loaded liposomal formulations. (C) Plating of the engineered MCF-7 cell line, and incubation of the seeded plate for 48–72 hours. (D) The sPLA2-assisted hydrolysis of liposomes and the release of the encapsulated luciferin. The liberated luciferin can freely translocate across the cell membrane, and will be oxidized quickly by the intracellular luciferase enzyme in a light-emitting reaction. (E) Typical readout of the bioluminescence signal from the release assay.
Fig 3.
Testing of the bioassay using free luciferin.
Dependence of the maximum of the luminescence profile generated upon the addition of luciferin to L2 cells on (A) number of seeded cells (0–50,000 L2 cells, 10 mM luciferin), (B) luciferin concentration (0–5 mM luciferin, 10,000 L2 cells), and (C) number of days after the seeding of 2,500 L2 cells (1–10 days, 1 mM luciferin). Lines are inserted only as a guide to the eye. Error bars represent the standard deviation (SD).
Fig 4.
Luminescence profiles generated upon the addition of 141 μM luciferin to L2 and L2P2 cells.
Fig 5.
Testing of lysed luciferin-loaded liposomes.
Luminescence profiles generated upon the addition of luciferin-loaded liposomes lysed with 0.5 wt.% Triton X-100 to L2 cells. Two formulations were tested (100 nm diameter liposomes), DSPC/DSPG 7:3 (PCPG, 117 μM luciferin and 880 μM lipid) and DSPC/DSPG/cholesterol 4:3:3 (PCPGch, 74 μM luciferin and 1220 μM lipid).
Fig 6.
Testing of luciferin-loaded liposomes.
Luminescence profiles generated upon the addition of luciferin-loaded liposomes to L2P2 and L2 cells. Two formulations were tested (100 nm diameter liposomes), DSPC/DSPG 7:3 (PCPG, 33 μM luciferin and 833 μM lipid) and DSPC/DSPG/cholesterol 4:3:3 (PCPGch, 33 μM luciferin and 265 μM lipid).
Fig 7.
Testing of the effect of PEGylation.
Luminescence profiles generated upon the addition of PEGylated luciferin-loaded liposomes to L2P2 and L2 cells. The lipopolymer DPPE-PEG 2000 (5 mol%) was added via post insertion to the loaded liposomes. Two formulations were tested (100 nm diameter liposomes), DSPC/DSPG 7:3 with (PCPG, 33 μM luciferin and 833 μM lipid) and DSPC/DSPG/cholesterol 4:3:3 (PCPGch, 107 μM luciferin and 833 μM lipid).