Stock solutions of all the components in chloroform were prepared. Typically a total of 20 µmoles of the amphiphillic lipids (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (PEG2000-DSPE), maleimide-PEG2000-DSPE, purchased from Avanti Polar Lipids) were mixed at molar ratios as indicated in . The amount of soybean oil was defined as mg per µmole of the amphiphillic lipid mixture. For fluorescence imaging, 0.1 mol% of NIR664-PEG2000-DSPE (SyMO-Chem) and/or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt (rhodamine-PE, Avanti Polar Lipids) was added, and for MRI 25 mol% gadolinium- diethylenetriaminepentacetate-bis( stearylamide) (Gd-DTPA-DSA, Avanti Polar Lipids) was added at the expense of DSPC. Subsequently the chloroform was evaporated with rotation evaporation (60 min at maximum vacuum and room temperature) and the resulting lipid film was hydrated with hepes buffered saline (HBS, 2.38 g/L Hepes and 8 g/L NaCl, pH 6.7). The obtained crude emulsion was sonicated for 20 min (Heat Systems-Ultrasonics, W-225R, duty cycle 35%, 30 Watt and 20 kHz) in a water bath keeping the emulsion at ambient temperature. Half of the final nanoemulsions were conjugated with c(RGDf(-S-acetylthioacetyl) K) (Ansynth) (RGD, 13.5 µg RGD per µmole lipid). Before adding the peptide to the maleimide functionalized nanoemulsion, the thiol group on the RGD peptide was deacetylated at pH 7 for 1 h. The activated peptide was added to the nanoemulsions and the resulting mixture was left to react overnight at 4 °C. The nanoemulsions were concentrated when necessary with vivaspin concentrators (100 kDa MWCO) and finally dialysed (Spectra/Por Float-A-Lyzer G2, 100 kDa MWCO, Spectrum Laboratories) against HBS of pH 7.4 to remove unconjugated RGD and to obtain physiological pH. After preparation, the nanoemulsions were stored at 4 °C for a minimum of 5 days before using them in experiments to assure hydrolysis of unreacted maleimide groups.
Figure 1 Nanoemulsion schematics and characteristics. A: Cartoons of the nanoemulsions with the different PEG2000-DSPE content and mushroom/brush configuration indicated. B: Lipid and soybean oil content of the different nanoemulsions. C: Hydrodynamic diameters (more ...)
Dynamic light scattering and zeta potential measurement
The hydrodynamic size and size distribution, and the zeta potential were measured using dynamic light scattering techniques (Malvern, Zetasizer Nano PS). For size measurements 8 µl of nanoemulsion suspension was dispersed in 800 µl HBS in an ordinary cuvette. Reported values were the average of approximately 50 measurements. The reported values are the means and PDIs after combining the 50 measurements with the Malvern zetasizer series software. For zeta potential measurements 20 µl of nanoemulsion suspension was dispersed in 1.5 ml dH2O in a capillary cell (Malvern, DTS1061). Reported values were the average of 300 measurement runs.
Cell culturing and incubations
HUVEC (Lonza Bioscience) were cultured in gelatin coated cell culture flasks using Dulbecco’s modified eagle medium (DMEM, Gibco/Invitrogen) supplemented with endothelial cell growth medium (EGM) BulletKit (1% FBS, Lonza Bioscience). The cells were used for experiments at passage 4 to 7. In all experiments, the cells were incubated in medium containing either no (blank), RGD or CTRL (non-targeted) nanoemulsions at a 1 mM lipid concentration for 3 h, followed by 3 times washing with PBS. For live cell imaging with CLSM, HUVEC were grown in gelatin coated 8-well µ-slides (Ibidi). After the incubation and washing, the cells were maintained in fresh medium. For flow cytometry experiments, HUVEC were grown in gelatin coated 12-well plates. After nanoemulsion incubation, the cells were detached from the wells with 0.25% trypsin-EDTA solution (Sigma-Aldrich), spun down at 1500 rpm for 5 min, resuspended in ice cold PBS and placed on ice. In the endocytosis inhibition assay, the cells were pre-incubated with either chlorpromazine (10 µg/ml) or genistein (70 mg/ml) for 30 min before incubating with medium containing both the endocytic inhibitor and the nanoemulsions.
Cellular uptake of nanoemulsions was measured by flow cytometry (Gallios, Beckman Coulter). The 633 nm laser line was used to excite the NIR664 fluorochrome and the fluorescence was detected using a 650–670 bandpass filter. The flow cytometry data was analyzed using the software Kaluza (Beckman Coulter). Cellular fragments and debris were excluded from the analysis by gating the fluorescence on side scatter vs forward angle light scatter signal. Within one experiment all the samples were analyzed using the same gate. The cellular uptake was measured both as the percentage of cells with higher fluorescence intensity than cellular autofluorescence as well as the amount of internalized nanoemulsion, which was estimated as the median fluorescence intensity divided by the median of the autofluorescence and normalized to the observed maximum cellular uptake. The targeting effect was defined as the ratio between the normalized fluorescence intensity of cells incubated with RGD or CTRL nanoparticles.
Live cell CLSM
Live HUVEC were imaged by CLSM (Zeiss LSM 510 META) using an apochromate 40×/1.2 water immersion objective and a frame size of 1024×1024 pixels. The 8-well µ-slide was placed in an incubation chamber (PECON, CTI-controller 3700 and temp control 37-2) mounted on the CLSM object stage, and the cells were imaged at 37° C and 5% CO2. NIR664-PEG-DSPE was excited at 633 nm and the fluorescence detected using a meta-detector at 676–719 nm, and rhodamine-PE was excitated at 543 nm and detected using a meta-detector 569–612 nm.
To assess particle integrity, the cells were incubated with P5, P10, and P20 CTRL and RGD nanoemulsions containing both NIR664-PEG-DSPE and rhodamine-PE and imaged up to 3 h post incubation.
Intracellular localization of the P5 RGD and CTRL nanoemulsions were compared by co-incubating the cells with CTRL nanoemulsions labeled with rhodamine-PE and RGD nanoemulsions labeled with NIR664-PEG2000-DSPE.
To assess lysosome colocalization of the nanoemulsion, the cells were incubated for 45 min with medium containing 50 nM lysotracker green (Invitrogen) and washed before the incubations with nanoemulsions. Lysotracker green was excited at 543 nm and detected at 565–615 nm
For intravital CLSM, tumors grown in dorsal window chambers in mice were used. The window chambers (made of polyoxymethylene, build in house) were implanted as previously described 45
in male athymic Balb/c Nu/nu mice of 20 to 25 gram. The mice were anesthetized with a subcutaneous injection of 12 mg/kg midazolam/fentanyl/ haloperidol /water (3/3/2/4). 24 h after implanting the chambers, 2–3·106
HeLa cells (from the cell line of human cervical carcinoma cells) were injected in the center of each chamber. The surgical procedures were performed under sterile conditions. The animals were kept under pathogen-free conditions at a temperature of 19 to 22 °C, 50 to 60% humidity, and 65 air changes per h, and animals were allowed food and water ad libitum
. The drinking water contained 2% sucrose and 67.5 mg/L Baytril (enrofloxacin). After 12–16 days when the tumors were 0.2 to 0.7 cm thick and filled 30 to 100% of the window area, the mice were used for experiments. The mice were anesthetized by subcutaneous injections of 12 mg/kg midazolam/fentanyl/Haldol/water (3/3/2/4) and either P5 or P50 nanoemulsions were intravenously (i.v.) injected at a dose of 80 µmole lipid/kg bodyweight. Six mice received each PEG formulation and from these 2 mice received RGD (NIR664-PEG-DSPE labeled), 2 mice CTRL (NIR664-PEG-DSPE labeled), and 2 mice both RGD (rhodamine-PE labeled) and CTRL (NIR664-PEG-DSPE labeled) nanoemulsions. This allowed us to study the distribution of each agent in 4 mice. For visualization of the vasculature, 100 µl of 20 mg/ml 2MDa FITC labeled Dextran (Sigma-Aldrich) was injected i.v. The anesthetized mice were placed on a heating pad at 37°C which was fixed to the microscope stage in a specially designed mouse holder. The tumors were imaged using a C-Apoplan 40×/0.8 water objective with long working distance. NIR664 and rhodamine were excited and detected as for imaging of cells. FITC-dextran was excited at 488 nm and detected with a bandpass filter at 500–550 nm. The tumors were imaged from the coverslip and approximately 100 µm into the tissue with a frame size of 512×512 pixels. 3D visualizations of intravitally acquired z-stacks were obtained using Amira (Visage Imaging)
All the MRI experiments were performed in a Bruker Biospec 7.05 Tesla horizontal bore magnet. The T1 relaxivity of the Gd in the nanoemulsions was obtained using a dilution series in HBS ( pH 7.4) of 6 different Gd concentrations ranging from 0.05 to 1 mM in 2 ml eppendorf tubes. The tubes were placed in an in house built sample holder which was imaged with a volume resonator. The T1 relaxation time was measured using a spin-echo protocol: echo time (TE) of 8.4 ms; repetition times (TR) of 15, 25, 50, 75, 100, 200, 300, 400, 600, 800, 1200, 1600, 3200, 6400, 12800, 18000 ms; field of view (FOV) of 60×40 mm; matrix (MTX) of 128×128; slice thickness of 2mm; 2 averages.
For P5 nanoemulsion detection in vitro in cell pellets, HUVEC were incubated with nanoemulsions for 3 h (n=3 in each group: blank, RGD nanoemulsion, and CTRL nanoemulsion), washed and detached with trypsin. The cell suspension was centrifuged and the pellet fixed with 150 µl 4% paraformaldehyde and transferred to a 200 µl eppendorf tube. In the eppendorf tube a cell pellet was formed by gravity. The cell pellets were imaged using a quadrature surface coil with a T1-weighted spin echo sequence: TE of 7.7; TR of 1000 ms; FOV of 20×16 mm, MTX of 100×80, slice thickness of 0.5 mm; 20 averages. Subsequently a T1-map was obtained with a spin echo protocol: TE of 8.4 ms; TR of 42, 80, 160, 320, 640, 1280, 2000, 3500, 5000, 8000, 12000, 16000 ms; FOV of 16×16 mm; MTX of 80×80; slice thickness of 0.6 mm; 4 averages.
For DCE-MRI, the xenografts of the ovarian cancer cell line TOV21G in 12 week old female athymic Balb/c Nu/nu mice were used. Tumors were imaged around 4 weeks after subcutaneous inoculation of 1.5·106 tumor cells in the flank. The animals were kept under pathogen-free conditions at a temperature of 19 to 22 °C, 50 to 60% humidity, and 65 air changes per h, and animals were allowed food and water ad libitum. The mice were anesthetized with isoflurane (2% in 67% N2 / 33% O2) and the tail vein was canulated. Respiration rate and body temperature were monitored using pressure-sensitive and rectal temperature probes (SA Instruments, New York, NY, USA) and hot air flow and isoflurane flow were adjusted accordingly. The tumors were imaged with a quadrature surface coil using a dynamic imaging sequence with a temporal resolution of 21.6 s and using 60 repetitions it lasted 21.6 min (RARE pulse sequence with TE of 7 ms; TR of 300 ms, RARE factor 2, zero filling acceleration of 1.34, FOV25.2×14.4 mm, MTX of 56 × 32, slice thickness of 1 mm, 1 slice, 6 averages). The nanoemulsions were injected i.v. at the start of the 11th repetition as a bolus lasting approximately 20 s. Either P5 RGD nanoemulsions (n=3) or P5 CTRL nanoemulsions (n=3) were injected at a dose of 80 µmole of lipid/kg bodyweight, resulting in 20 µmole Gd per kg bodyweight. In the frames with the highest relative signal enhancement in the DCE-MRI, pixels in the tumor with at least 6 % relative signal enhancement were color coded using Matlab (The MathWorks, Natick, MA, USA). Those color coded relative signal enhancement maps were merged with high resolution T1-weighted post-injection images to visualize the distribution of signal enhancement throughout the tumor. The high resolution T1-weighted images were obtained 25 minutes post nanoemulsion injection (FLASH pulse sequence, TE of 5.4 ms, TR of 350 ms, FOV 25.2×14.4 mm, MTX of 56 × 32, slice thickness of 1 mm, 1 slice, 4 averages). Those images were also used as a reference when defining ROIs in the dynamic sequence. The analysis of the T1-maps and the DCE-MRI curve plotting was performed using Matlab.
Where appropriate, statistical testing was performed using 2 sample, upper tailed student t-tests with Minitab software (Minitab Inc., State College, PA, USA). The significance criterion was p ≤ 0.05.