Materials
Perfluoro-15-crown ether (PFCE) was obtained from SynQuest Laboratories Inc. (Alachua, FL). L-a-phosphatidylcholine (Egg PC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (DSPE-mPEG2000) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Indocyanine green, and cholesterol were obtained from Sigma-Aldrich Co. (St. Louis, MO).
Preparation of PFC/ICG nanoemulsions
To synthesize the PFC/ICG nanoemulsions, PFCE liquids were emulsified in an aqueous solution using a lipid mixture. The lipid compositions of the PFC/ICG nanoemulsions were PC/cholesterol/DSPE-mPEG2000 in a molar ratio of 70:20:10, respectively. The lipid mixture was reacted for 1 h at room temperature by the addition of 2 mg ICG, evaporated with a rotary evaporator to ensure the production of a thin lipid film, and dried in a vacuum oven (25°C) for 24 h. The lipid film was rehydrated with phosphate-buffered saline (PBS), and the resulting solution was sonicated in a bath sonicator followed by five cycles of freezing and thawing. The rehydrated lipid mixture (2% w/v) and PFCE solution (20% v/v) were mixed for 4 min using a homogenizer, followed by microfluidisation [37]. A M-110S microfluidiser (Microfluidics Inc., Newton, MA) operating at a liquid pressure of approximately 20,000 psi was used for nanoemulsion preparations. The PFC/ICG nanoemulsions were stored at 4°C.
Characterization of PFC/ICG nanoemulsions
To evaluate the characteristics of the PFC/ICG nanoemulsions, a JEOL FE-TEM (transmission electron microscope) was utilized, and the TEM images were captured at 200 kV using a device from Tecnai. The PFC/ICG nanoemulsions were drop-cast onto carbon-coated TEM grids preliminarily stained with 2% uranyl acetate, and the solution was dried in a vacuum oven.
The emission and absorption spectra were obtained on a Perkin-Elmer LS-55 and a Beckman Coulter UV–VIS spectrophotometer (DU 800). The size of the PFC/ICG nanoemulsions was analyzed via dynamic light scattering using an electrophoretic light scattering photometer (ELS-Z, Otsuka Electronics, Osaka, Japan). The NIR fluorescence images of the PFC/ICG nanoemulsions were obtained using the IVIS Lumina imaging system (Caliper Life Science, MA) with an ICG filter set.
ICG and PFC loading efficiency
The ICG loading efficiency was analyzed using a previously reported method [29]. The quantity of ICG loaded into the PFC/ICG nanoemulsions was determined from the free ICG that was not incorporated into the PFC/ICG nanoemulsions. A 1-mL sample of the PFC/ICG nanoemulsion was centrifuged, and the supernatant was removed and stored in a centrifuge tube; the PFC/ICG nanoemulsion was dispersed in a PBS solution. The centrifugation was repeated, and the collected supernatants were combined. The ICG concentration was quantified via UV–Vis spectroscopy. The quantity of ICG inside the PFC/ICG nanoemulsions was also measured to verify the accuracy of the method. Selected PFC/ICG nanoemulsion samples were treated with an HNO3 solution to induce capsule disassembly and to release the ICG into the solution. For all tested samples, the quantity of ICG released and the unencapsulated ICG equaled the quantity of the ICG precursor, indicating that the mass balance was conserved. The loading efficiency was calculated as the mass of ICG incorporated by the PFC/ICG nanoemulsions divided by the total ICG mass added to the nanoemulsion aggregate suspension.
To evaluate the PFC loading efficiency in PFC/ICG nanoemulsions, 19 F-MR imaging was performed on serial dilutions (0 – 0.4 ml) of PFCE liquids using a 4.7 T Bruker scanner (Biospec, Rheinstetten, Germany). The 19 F MR signal intensity was determined from the PFC signals originating from the PFCE liquids within a region of interest (ROI). We generated a calibration curve from the serial dilutions of the PFCE liquids and calculated the PFC loading efficiency in PFC/ICG nanoemulssions. The loading efficiency of PFC onto the PFC/ICG nanoemulsions was approximately 75.5 ± 3.2%.
Physicochemical stability of PFC/ICG nanoemulsions
The ICG and PFC/ICG nanoemulsions were diluted with distilled water to a final concentration of 1 μg/ml and were loaded onto a 12-well plate. The samples were irradiated with 760 nm NIR light from an LED for a predetermined time of 10, 20, 30, 60, or 120 min at room temperature. The effect of the light exposure on the degradation of the PFC/ICG nanoemulsions was determined with visible light at room temperature. The fluorescence intensity was measured for up to 6 days. After incubation, the remaining fluorescence of each sample was measured using a spectrofluorometer with excitation and emission wavelengths of 760 nm and 820 nm, respectively. For the quantitative analysis, we normalized the fluorescence signal intensity. This processing normalized the signal data points to the range [0, 1].
(1)
Y denotes the y values of input curve, and Y’ is the normalized curve.
Cell culture
The HeLa (human cervical cancer cells) and Raw264.7 (Murine macrophage cells) cell lines were obtained from the American Type Culture Collection (Rockville, MD). These cell lines were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 IU/ml penicillin, and 50 μg/ml streptomycin. The cultures were maintained at 37°C/5% CO2 in tissue culture plates. The DC2.4 cells, previously characterized as an immature murine dendritic cell line, were obtained from Dr. Kenneth L. Rock (Dana-Farber Cancer Institute, Boston, MA) [38]. This cell line was grown and maintained in DMEM supplemented with 10% heat-inactivated FBS, 50 IU/ml penicillin, and 50 μg/ml streptomycin.
Cell fluorescence imaging
To determine the intracellular delivery capacity of PFC/ICG nanoemulsions, the HeLa, Raw264.7, and DC2.4 cells were incubated with 10 μl/ml PFC/ICG nanoemulsions in μ-slide 8-well microscopy chamber at a density of 1 × 104 cells per well for 6 h at 37°C. The culture medium was then carefully aspirated, and the cells were washed three times. The labeled cells were fixed with 4% paraformaldehyde and stained with DAPI. The NIR fluorescence images were obtained on a Deltavision RT deconvolution microscope (Applied Precision Technologies, Issaquah, WA) using a filter set (excitation: 775/50, emission: 845/55; Omega Optical, Brattleboro, VT).
In vitro 19 F-MR and NIR fluorescence imaging
HeLa, Raw, or DC2.4 cells (1 × 106) were seeded on each well of a 6-well plate and grown for 24 h. The cells were then incubated with a medium containing 10 μl/ml PFC/ICG nanoemulsions. After 6 h, the medium was removed, and the cells were washed three times with PBS. The cell pellets were suspended with a 2% solution of low-melting agarose. The cells were collected in 0.2-mL tubes, and the MR and NIR fluorescence signals were measured. All 19 F-MR imaging of the PFC/ICG nanoemulsions was performed with a 4.7 T Bruker scanner using a double-tuned 1H/19F quadrature birdcage RF resonator. The 19 F-MR image was captured with a FLASH sequence (128 × 128 matrix; 30 × 30 mm2 FOV; 50 ms TR; 2.6 ms TE; 10 mm slice thickness; 256 NEX). The NIR fluorescence images were obtained using the IVIS Lumina imaging system (Caliper Life Science, MA) with an ICG filter set.
Cell cytotoxicity assays
The cell cytotoxicity was assessed using a modified 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Raw, HeLa, or DC2.4 cells were seeded in a 96-well plate (Corning Costar, Cambridge, MA) at 1 × 104 cells/well. After incubation for 24 or 48 h, several different concentrations of the prepared PFC/ICG nanoemulsions (0.38 ug/ul of ICG, 0.3 ul/ul of PFC) were poured into the wells. After incubation for a predetermined time, the residual nanoemulsions were removed, and a 2.5 mg/ml MTT solution was added to each well. The wells were then incubated in a humidified CO2 incubator at 37°C for 2 h. An acidified isopropanol/10% Triton X-100 solution (100 μl) was then added, and the plates were shaken to dissolve the formazan products. The absorbance was measured using a microplate reader at 570 nm. The cell survival rate in the control wells without the PFC/ICG nanoemulsions was considered 100% cell survival. The cytotoxic concentration (CC80) was defined as the concentration of the compound that reduced the absorbance of the control samples by 80%.
In vivo tracking of PFC/ICG nanoemulsions using NIR fluorescence and 19 F-MR imaging
Female hairless mice, 5–6 weeks of age, were purchased from SLC, Inc. (Japan). The mice were maintained at the KRIBB animal facility under pathogen-free conditions. All animal care and experimental procedures were approved by the Animal Care Committees of the KRIBB.
For the in vivo NIR fluorescence and 19 F-MR imaging of the sentinel lymph nodes, hairless mice were injected with 20 μl (25 μM of ICG, 15 ul/ml of PFC) of the PFC/ICG nanoemulsions (n = 5) or free ICG solutions (n = 5) in the footpad of the foreleg. Prior to the fluorescence imaging experiments, the mice were anesthetized with 200 μl of a 2.5% avertin solution (2, 2, 2-tribromoethanol-tert amyl alcohol, Sigma) throughout the experiments. After a predetermined time, the fluorescence intensity was quantitatively analyzed using the IVIS Lumina imaging system. Thereafter, the 19 F-MR images of the mice were obtained with a 4.7 T Bruker scanner using a double-tuned 1H/19F Birdcage coil design (inner diameter: 35 mm; length: 78 mm). After acquiring the morphological 1H images, the resonator was tuned to 19 F. For the 19 F-MR image, the mouse was imaged with a gradient echo sequence (128 × 128 matrix; 3 cm FOV; 56.0 ms TR; 2.6 ms TE; 20 mm slice thickness; 60° flip angle; 256 NEX; 30 min total scan time).
Statistical analysis
The statistical evaluations of the experiments were performed by ANOVA analysis followed by a Newman-Keuls multiple comparison test.