Distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoyl-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ammonium salt (PEG-DSPE) and distearoyl-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] were all purchased from Avanti Polar Lipids and used as received. The colloidal suspension of oleic acid coated magnetite (Fe3O4) particles of 3–15 nm in toluene was obtained from the “Polytehnica” of Timisoara (Romania). The Cy5.5 NHS ester was purchased from Amersham Biosciences (Piscataway, NJ). Cell culture supplies were purchased from Invitrogen (Carlsbad, CA).
2.2. Synthesis of the Cy5.5-PEG-DSPE lipid
73.5 mg NH2-PEG-DSPE was dissolved in a 500 μl 9:1 chloroform: methanol mixture and 10 mg Cy5.5 NHS ester in 500 μl DMSO. 25 ml of a 0.1 M sodium bicarbonate solution (pH~8.5) was prepared, heated to 70 °C and the NH2-PEG-DSPE solution added dropwise under vigorous stirring to form a micelle preparation. After cooling down to room temperature, the DMSO solution containing the Cy5.5 NHS ester was added to the micelle solution and stirred overnight in a beaker under N2. The preparation was washed extensively with millipore water by using a Vivaspin MW 10000 column concentrator. After 6 wash cycles the preparation was redispersed in 1 ml millipore water and freeze dried for 3 days. The residue was solubilized in a 2:1 chloroform: methanol mixture to a final concentration of 2 mg/ml Cy5.5 and stored at −80 °C.
2.3. Synthesis of the nanoemulsions
For the synthesis of the nanoemulsions, separate stock solutions of all the lipophilic components were prepared in chloroform. The concentrations were as follows: DSPC stock solution: 25 mg/ml, PEG-DSPE: 100 mg/ml, soybean oil: 0.5 g/ml, iron oxide particles: 100 mg/ml, Cy5.5 PEG-DSPE: 2 mg/ml. The composition of the formulation with the highest total amount of soybean oil was: 2.8 mg of DSPC, 10 mg of PEG-DSPE, 38 mg of soybean oil, 9 mg of iron oxide particles and 989 μg of Cy5.5 PEG-DSPE lipids. The formulation with the medium total amount of oil consisted of 2.8 mg DSPC, 10 mg PEG-DSPE, 20 mg soybean oil, 4.74 mg iron oxide particles and 760 μg Cy5.5 PEG-DSPE. The nanoemulsion with the lowest amount of oil contained 2.8 mg DSPC, 10 mg PEG, 10 mg soybean oil, 2.36 mg iron oxide particles and 502 μg Cy5.5 PEG-DSPE. The components were mixed together and added dropwise under stirring to 8 ml of boiling millipore water. Subsequently, the crude emulsion was homogenized by sonication using a thin sonicator tip (BioLogics, Inc., 3.9 mm). The preparation was sonicated for 20 min (level 20 %, pulse 70 %, device: Biologics, Inc., ultrasonic homogenizer model 150 V/T) while cooled with room temperature water. Finally, the preparation was concentrated to a final volume of 2.8 ml. The preparations were measured with a dynamic light scattering device before and after filtration through a 0.2 μl filter. The filtered nanoemulsions were stored in the dark at 4 °C.
2.4. Dynamic light scattering and zeta potential measurements
The hydrodynamic sizes of the particles were measured using a dynamic light scattering (DLS) device from Malvern Instruments (model HPPS). The sizes of the nanoemulsions were determined directly after they were synthesized. The formulations were diluted before the measurement as follows: 500 μl millipore water was mixed with 50 μl of the particles with the smallest amount of oil or 20 μl for the other two nanoemulsions. The mean sizes were determined with independent measurements from three drawn samples of the preparation with 5 runs per sample, 15 determinations in total. The Zeta potential was determined by using a device from Brookhaven Instruments Corporation (Zeta PALS, Zeta Potential Analyzer). Due to the interference of Cy5.5 fluorescence with the measurement, three nanoemulsions containing all the aforementioned components except the Cy5.5 lipids were used. In order to achieve measureable samples, 2000 μl of millipore water were mixed with 20 μl of the formulation with the highest content of oil, whereas 60 μl of the medium one and 150 μl of the smallest one were used. Three different samples of one nanoemulsion were measured.
2.5. Transmission electron microscopy
A Hitachi H7650 linked to a SIA (Scientific Instruments and Applications) digital camera run by Maxim CCD software was used. TEM was performed on the nanoemulsions diluted 1:10 and 1:20 in an ammonium acetate buffer using a 2 % sodium phosphotungstate (pH=7) negative stain as described by Forte and Nordhausen [34
In order to determine the longitudinal (r1) and transverse relaxivities (r2) of the particles, T1 and T2 measurements of solutions were performed on a 60 MHz Bruker Minispec (Bruker Medical BmbH, Ettingen) operating at 40 °C. Dilutions of 1:10, 1:20 and 1:40 of the nanoemulsions in millipore water were prepared and 200 μl were used for the measurement. R1 and r2 of the samples was then calculated from the slope of the graph of 1/T1 and 1/T2 plotted against the iron concentration.
2.7. Cell culture and tumor model
Human EW7 (Ewing’s sarcoma) cancer cells were maintained in RPMI 1640 medium supplemented with 10 % FCS. The cells were grown in a 5 % CO2, water saturated atmosphere at 37 °C and subculturing was performed twice a week by 1:10 dilution after trypsinization. Six-week-old male swiss nude mice were purchased from Taconic (Albany, NY) and all animal handling approved by the Mount Sinai School of Medicine Institutional Animal Care and Use Committee. To establish subcutaneous tumors in nude mice 2 million cells were injected. The tumors used in this study were in a volume range of 65–125 mm3.
2.8. Fluorescence imaging
Ex vivo and in vivo fluorescence imaging experiments were performed using an IVIS Imaging System 200 (Xenogen, Alameda, CA). To enable detection of the Cy5.5 fluorescence a 615–665 nm excitation filter and a 695–770 nm emission filter were used.
For the determination of the pharmacokinetics and the half-life of the three nanoemulsions, the compounds were intravenously administered and blood was drawn from the saphenous vein of the mouse leg after 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h and 24 h. The blood was collected in pre-weighed 1.5 ml cups containing 40 μl heparin (10 U/ml), centrifuged for 7 min at 3000 rpm with a table centrifuge and 20 μl of the supernatant pipetted into 0.2 ml cups. The photon count in the different samples was determined with the IVIS 200 using the following parameters: focus height of 0.3 cm, field of view 6.5 × 6.5 cm, and an excitation time of 0.5 s.
For the in vivo fluorescence imaging experiments, the highly fluorescent food of the nude mice was withdrawn at least 6 hours prior to the experiment. Moreover, remaining food powder traces in the fur were removed by wiping the mice with wet gauze. The mice were pre-scanned, injected with the nanoemulsions, and scanned at several time points post injection (5 min, 15 min, 30 min, 1 h, 2 h, 4 h and 24 h). A field of view of 12.2 × 12.2 cm was chosen and the focus height was set to 1.5 cm, while the excitation times varied from 1 s, up to 3 s and 5 s, depending upon the fluorescence intensity. After the imaging experiments the mice were sacrificed by saline perfusion under isoflurane anesthesia and the tumor, liver, lung, spleen and kidneys were excised and imaged together with the corresponding organs of a control mouse that was not injected. The parameters for the image acquisition were set as follows: focus height of 0.5 cm, field of view of 12.2 × 12.2 cm and excitation times of 0.5 s, 1 s and 3 s.
2.9. MR imaging
Nude mice under isoflurane anesthesia bearing subcutaneous EW7 tumors were scanned using a 9.4 T MRI system (Bruker Instruments). MR imaging was performed by using a spin-multiple echo sequence with a repetition time of 2500 ms and ten echos, i.e. 6.7 ms, 13.3 ms, 20 ms, 26.7 ms, 33.3 ms, 40 ms, 46.7 ms, 53.3 ms, 60 ms and 66.7 ms. The field of view was 2.6 × 2.6 cm, the matrix size 128 × 128, the no. of slices ten with a slice thickness of 1 mm and the no. of averages 4 which amounted to a total scan time of 16 min. After the pre-scans, mice were intravenously injected with one of the three nanoemulsions (equivalent of 16.7 mg/kg iron oxide) and scanned again 24 h afterwards according to the protocol above.