Cytoplasmic delivery of liposomes into MCF-7 breast cancer cells mediated by cell-specific phage fusion coat protein.

Materials and Reagents

Doxil® was purchased from Ben Venue Laboratories Inc. (Bedford, OH). L-α-phosphatidylcholine (egg; EPC); 1,2-dipalmitoyl- sn -glycero-3-[phospho- rac -(1-glycerol)] (sodium salt; DPPG); 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt; DOTAP); 1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)2000] (ammonium salt; PEG 2000 -PE ); cholesterol (98%); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD-DOPE); and 1,2-dimyristoyl- sn -glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt; Rho-PE) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). NH 4 Cl; Bafilomycin A1 and chloroquine diphosphate salt were from Sigma (St Louis, MO); trihydrochloride, trihydrate (Hoechst 33342); 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS; pyranine); p-xylene-bis-pyridinium bromide (DPX), and Alexa Fluor 488 transferrin were from Invitrogen Inc (Eugene, OR); Sodium cholate and BCA protein assay kits were from Pierce (Rockford, IL); Cell Titer Blue assay kit from Promega (Madison, WI); Fluor Mounting Medium was from Trevigen Inc (Gaithersburg, MD). MCF-7 human breast adenocarcinoma (HTB 22™) cells were obtained from the ATCC (Manassas, VA). All cells were grown as recommended by the ATCC at 37°C, 5% CO 2 .

Preparation of Liposomes

Phage-liposomes were prepared using a post-insert protocol 17 and liposomes with the compositions as shown in Table 1 . Briefly, plain liposomes were prepared by the hydration of the lipid film followed by a 30 min bath sonication and extrusion through 200 nm polycarbonate membrane. To prepare phage-liposomes, plain liposomes were incubated with phage fusion coat protein at the indicated phage protein-to-lipid weight ratios and with 15 mM final concentration of sodium cholate. After the overnight incubation at 37°C, the crude formulation was dialyzed at 4°C against PBS to remove sodium cholate. To prepare double-labeled liposomes, Rho-PE and NBD-DOPE were added to the liposome composition. The liposomes were dialyzed against PBS to remove non-incorporated markers. Phage-Doxil was prepared by incubating Doxil with the cholate-stabilized phage fusion coat protein at 0.5% protein-to-lipid weight ratio and subsequent dialysis as describe above. Liposomes encapsulating 35 mM HPTS and 50 mM DPX were prepared using a freezing-thawing protocol 18 .

Artificial Membrane Fusion

The fusion between the membranes of plain- or phage-liposomes and double-labeled liposomes with the FRET donor NBD-PE and acceptor Rho-PE was followed by monitoring the fluorescence resonance energy transfer between FRET pairs. Briefly, double-labeled liposomes with 0.1mol% NBD-PE and 0.5mol% Rho-PE were added to plain liposomes or phage-liposomes with a varying concentration of phage fusion coat protein in either citrate-PBS buffer (pH 5.3) or PBS buffer (pH 7.4) at a 1:4 molar ratio and a total lipid concentration of 100 µM. Samples were incubated at 37°C, and fluorescence intensities of the samples were measured. An emission spectrum between 500–650 nm was obtained at predetermined time points using the excitation wavelength of 470 nm. Changes in the fluorescence intensity ratio (R-value) of NBD-PE (530 nm) to Rho-PE (585 nm) served as an indicator of the occurrence of the fusion. The net membrane fusion activity mediated by phage protein was indicated by the normalized R value, which is the difference in R values between phage-liposomes and plain liposomes.

Intracellular Membrane Fusion

MCF-7 cells were grown in a 6-well microplate to 70–80% confluence, and then treated with 1µM of double-labeled phage-liposomes or plain liposomes at 4°C for 1h, then incubated at 37°C in the presence or absence of NH 4 Cl (20 mM) for 1h. Cells were scraped and suspended in 1 ml of PBS buffer, pH 7.4. Fluorescence intensities of the samples were monitored at excitation wavelength of 470 nm, emission wavelength of 530 nm and 585 nm. Changes in the fluorescence intensity ratio (R-value) of NBD-PE (530 nm) to Rho-PE (585 nm) served as an indicator of the fusion. The R-value was normalized to protein concentration of the sample detected by the BCA protein assay. Effect of Bafilomycin A1 and Chloroquine on Cytotoxicity of Doxil and phage-Doxil MCF-7 cells were seeded into 96 well microplates at a density of 4×10 4 cells/well and grown until cells reached 40–50% confluence. For Bafilomycin A1 (BFA) inhibition studies, MCF-7 cells were treated with 0.1 µM BFA for 30 min and then incubated with 51.7 µM of Doxil or phage-Doxil in a serum-free MEM containing 0.1 µM of BFA for 24 h. For chloroquine inhibition, MCF-7 cells were treated with 51.7 µM of Doxil or phage-Doxil for 24 h in a serum-free MEM containing 50 µM of chloroquine. As a control, MCF-7 cells were treated with 51.7 µM of Doxil or phage-Doxil, 0.1 µM of BFA or 50 µM chloroquine in a serum-free MEM for 24 h. After cells were washed 3 times with PBS, pH 7.4, cell viability was evaluated by the Cell Titer Blue assay as described in the manufacturer’s manual. Briefly, cells were incubated with the fresh complete MEM medium (100µl/well) along with the Cell Titer Blue assay reagent (20µl/well) at 37°C for 2h. The fluorescence intensity was measured using the multi-detection microplate reader (Bio-Tek, Winooski, VT) with 525/590 nm excitation/emission wavelengths. The percent of cell viability was calculated by dividing the treated sample value by the value for the untreated cell sample. The percent of cell viability upon the treatment with phage-Doxil with BFA or chloroquine inhibition was normalized to that with BFA or chloroquine treatment alone.

Fluorescence Microscopy

MCF-7 cells were seeded on sterile coverslips in 6-well plates at the density of 2.5 × 10 5 cells/well and grown to 70–80% confluence. To study the uptake mechanism of phage liposomes, cells were co-incubated with 1 µM of rhodamine-labeled phage-liposomes and 20 µg/ml of Alexa Fluor 488 transferrin in serum-free MEM medium for 30 min at 37°C. After triple washing with PBS at 4°C, the cells were fixed with 2.5% paraformaldehyde. To study the endosomal release of rhodamine-labeled phage- or plain liposomes, cells were incubated with 1 µM of rhodamine-labeled phage-liposomes or plain liposomes in a serum-free MEM for 30 min at 37°C, and washed once with PBS, pH 7.4. The cells were then incubated for an additional 2 h in a serum-free MEM at 37°C. Cell nuclei were counterstained with 5 µg/ml of Hoechst 33342 for 10 min. For determination of the cytosolic delivery of liposome-encapsulated HPTX-DPX, cells were incubated with 1 µM of HPTS-DPX encapsulating phage-liposomes or plain liposomes in a serum-free MEM for 1 h at 37°C, and washed with PBS, pH 7.4. Cells were incubated for additional 16 h in a serum-free MEM at 37°C. After triple washing with PBS, a coverslip was mounted on the glass slide over mounting medium and visualized with a fluorescence microscopy (Zeiss Co. Ltd. Germany) at 100 × magnification with FITC, TxR and DAPI filters. All images were taken with monochromatic CCD cameras, and the data was collected using Openlab software and exported as tagged image files (TIF).

Statistical Analysis

The statistical significance of the results was analyzed using the SPSS (version 16). Differences between experiment groups were compared using ANOVA followed by a Bonferroni post hoc test. The results were considered statistically significant if the p value was less than 0.05.

Materials and Reagents

Doxil® was purchased from Ben Venue Laboratories Inc. (Bedford, OH). L-α-phosphatidylcholine (egg; EPC); 1,2-dipalmitoyl- sn -glycero-3-[phospho- rac -(1-glycerol)] (sodium salt; DPPG); 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt; DOTAP); 1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)2000] (ammonium salt; PEG 2000 -PE ); cholesterol (98%); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD-DOPE); and 1,2-dimyristoyl- sn -glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt; Rho-PE) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). NH 4 Cl; Bafilomycin A1 and chloroquine diphosphate salt were from Sigma (St Louis, MO); trihydrochloride, trihydrate (Hoechst 33342); 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS; pyranine); p-xylene-bis-pyridinium bromide (DPX), and Alexa Fluor 488 transferrin were from Invitrogen Inc (Eugene, OR); Sodium cholate and BCA protein assay kits were from Pierce (Rockford, IL); Cell Titer Blue assay kit from Promega (Madison, WI); Fluor Mounting Medium was from Trevigen Inc (Gaithersburg, MD). MCF-7 human breast adenocarcinoma (HTB 22™) cells were obtained from the ATCC (Manassas, VA). All cells were grown as recommended by the ATCC at 37°C, 5% CO 2 .