Perfluorooctylbromide (PFOB), 1H,1H,2H,2H-perfluorode-canethiol (PFDT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and cholesterol were supplied by Sigma-Aldrich (St. Louis, MO). Perfluoro-15-crown ether (PFCE) was purchased from SynQuest Laboratories (Alachua, FL). 3-(N-succinimidyloxyglutaryl) aminopropyl polyethyleneglycol-carbamyldistearoyl phosphatidyl-ethanolamine (DSPE-PEG3400-NHS) was supplied by NOF Co. (Tokyo, Japan) and CdSe/ZnS QDs was obtained from Evident Technologies (Troy, NY). L-α-phosphatidylcholine (lecithin, 95%, chicken egg) was purchased from Avanti Polar Lipids (Alabaster, AL). Antibodies were from following sources: anti-human EGF1R (AbD serotec, Oxford, UK), anti-human ErbB2 and anti-human IGF1R (R&D Systems, Inc., Minneapolis, MN). All compounds for the cell culture were supplied by Invitrogen (Carlsbad, CA).
2.2. Preparation of PFC/QD nanoemulsions
To fabricate PFC/QD nanoemulsions, two PFC materials [PFCE and PFOB] and two different colored QDs (emitting at 525, 606 nm) were used. For conventional hydrophobic ligand-capped CdSe/ZnS QDs to be compatible with PFC liquids, the hydrophobic ligands of QDs should be exchanged with PFDT. A 7 mg CdSe/ZnS QDs dispersed in toluene was mixed with 34 g PFC liquids containing 1 ml PFOT and methanol. After vigorous mixing of the two-phase solutions for 24 h, the CdSe/ZnS QDs were partitioned into the PFC phase. The QDs in PFC were then washed three times with methanol to remove the excess ligands from the solution. The PFC liquids containing CdSe/ZnS QDs were emulsified in an aqueous solution using surfactant mixtures. A surfactant mixture composed of 78.5 mol% lecithin, 20 mol% cholesterol, and 1.5 mol% DSPE-PEG3400-NHS were dissolved in chloroform, and the organic solvent was evaporated using a rotary evaporator and a freezing dryer for 24 h. After dispersing the surfactant mixture into sterilized distilled water, the solution was sonicated. PFC/QD solution (40% v/v), the surfactant mixture (2% w/v) and phosphate buffered saline were mixed for 4 min using a homogenizer. The mixture was followed by microfluidization. A Microfluidizer M-110S (Microfluidics, Inc., Newton, MA) operating at a liquid pressure of approximately 20000 psi was used for all reported nanoemulsion preparations. The fabricated PFC/QDs nanoemulsions were separated using the size-exclusion column and were stored at 4°C.
2.3. Preparation of antibody conjugated PFC/QD nanoemulsions
For targeting the breast cancer cells, three different antibodies [anti-human EGF1R, anti-human ErbB2 and anti-human IGF1R] were conjugated to the PFC/QD nanoemulsions. Antibody-conjugated PFC/QD nanoemulsions were developed by linking the carboxyl groups on the surface of PFC/QD nanoemulsions with the amine groups in antibodies. To the solution containing PFC/QD nanoemulsions and antibodies, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide (EDC) was added and permitted to react with the NHS ligand of PFC/QD nanoemulsions for 2 h at room temperature. To quench the reaction, add 2-mercaptoethanol to a final concentration of 10 mM. The antibody-coated PFC/QDs nanoemulsions were purified by size-exclusion with sepharose 4B column and concentrated using ultrafiltration for 30 min.
2.4. Characterization of PFC/QD nanoemulsions
The emission spectra of PFC/QD nanoemulsions were measured using a fluorescence spectrometer (LS 55, PerkinElmer Instruments, Wellesley, MA). The mean particle diameters and zeta (ζ)-potentials of the PFC/QD nanoemulsions were determined using a particle size analyzer (ELS-Z, Otsuka Electronics, Japan). Measurements were taken after diluting the nanoemulsion in water and equilibrating at room temperature for at least 30 minutes prior to each measurement. All measurements were taken at room temperature.
2.5. Cell viability assay
The cell viability was assessed by a modified MTT assay. Cell viability was measured for the following three cell lines: SKBR3, MCF-7, and MDA-MB 468 cells. These cell lines were obtained from the American Type Culture Collection (Rockville, MD). Human breast cancer cell lines SKBR3, MCF-7, and MDA-MB 468 were grown and maintained in each medium McCoy’s, MEM-α, RPMI1640 supplemented with 10% heat inactivated FBS, 50 IU ml−1 penicillin, and 50 μg ml−1 streptomycin, respectively. Each cell was plated into a 96-well plate (Corning Costar, Cambridge, MA) at 1 × 104 cells/well. After incubation for 24 h, the medium was removed and the prepared PFC/QDs nanoemulsions diluted to several different concentrations (0, 2.5, 7.4, 22.2, 66.7, 200 μl ml−1) were poured into the wells. After another 24 h incubation period, the residual nanoemulsions were removed and an MTT solution at a concentration of 2.5 mg ml−1 in magnesium- and calcium-free phosphate-buffered saline was added to each well. The wells were then incubated in a humidified CO2 incubator at 37°C for 1.5 h. 100 μl of acidified isopropanol/10% Triton X-100 solution was then added and the plates were shaken to dissolve the formazan products. The absorbance at 570 nm was measured with a microplate reader (Bio-rad, Hercules, CA) The cell survival rate in the control wells without the PFC/QD solutions was considered as 100% cell survival.
2.6. In vitro fluorescence and 19 F-MR imaging of breast cancer cells
For fluorescence imaging, SKBR3, MCF-7, or MDA-MB 468 cells were incubated with 50 μl ml−1 antibody-conjugated PFC/QD nanoemulsions (ErbB2-PFCE/QD606, EGF1R-PFOB/QD525, IFG1R-PFOB/QD606) for 24 h at 37°C. After being washed with PBS, the labeled cells were fixed with Cytofix/Cytoperm solution and stained with DAPI in PBS. Fluorescence images were obtained on a Deltavision RT deconvolution microscope (Applied Precision Technologies, Issaquah, WA) using emission filters (525WB20, 600WB20, Omega Optical, Brattleboro, VT). All 19 F-MR imaging experiments of PFC/QD nanoemulsions were performed with a 4.7 T Bruker scanner (Biospec, Rheinstetten, Germany) using a double-tuned 1H/19F quadrature birdcage RF resonator. A 19 F-MR image was captured with a FLASH sequence (128 × 128 matrix; 30 × 30 mm2 FOV; 50 ms TR; 2.6 ms TE; 12 mm slice thickness).
2.7. Statistical analysis
The statistical evaluations of the experiments were performed by ANOVA analysis followed by a Newman-Keuls multiple comparison test.