Chemicals

All phospholipids, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG-2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG-2000 NH₂), 1,2-distearoylglycero-3-phosphocholine (DSPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (gadolinium salt) (DMPE-Gd) were procured from Avanti Polar Lipids (Alabaster, AL). All other solvents were procured from Sigma-Aldrich, (St. Louis, MO) and were used without any further purification.

Tumor model system

We have recently described the procedures for establishment of primary patient tumor-derived HNSCC xenografts in SCID mice [6]. Using similar procedures, tumors were established in athymic nude mice. Female athymic nude mice (nu/nu, body weight 22-30 g) were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and housed in microisolator cages on-site at the Roswell Park Cancer Institute's Laboratory Animal Resource. The mice were provided standard chow/water, and maintained on 12-hour light/dark cycles in a HEPA-filtered environment. All procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at Roswell Park Cancer Institute.

Synthesis of Pt(TPNP)/Gd nanomicelles

We have recently described the procedure for synthesis of nanomicelles encapsulating the phosphorescent dye, Pt(II)-tetraphenyltetranaphthoporphyrin [Pt(TPNP)] in the hydrophobic core of the DSPE-PEG/DSPC nanomicelles [7]. We have modified the reported procedure to incorporate DMPE-Gd into nanomicelles. A schematic describing the encapsulation of the Pt(TPNP) into the DSPE-PEG/DSPC/DMPE-Gd phospholipid micelles is shown in Figure 2. Briefly, 100 μL of phosphorescent dye stock solution in toluene (0.12 mg/ml) was evaporated and dried under vacuum. The obtained solid mass was then resuspended in 1 mL chloroform with 6.33 × 10^-6 moles of phospholipids containing 17% of DSPE-mPEG-2000, 17% of DSPE-PEG-2000 NH₂, 13% of DMPE-Gd and 53% of DSPC. The solution was sonicated for 1 minute and the chloroform allowed to evaporate under vacuum, following which the residue was gently heated at 80°C and 2 mL of water was added to obtain an optically clear suspension containing Pt(TPNP)/DSPE-PEG/DSPC/DMPE-Gd (from here on referred as Pt(TPNP)-Gd nanomicelles). Dye/micelle formulation was purified by ultracentrifugation at 500,000 g for 2 hours. The supernatant was discarded and the pellet containing phosphorescent dye-micelles was resuspended in water. Samples of Pt(TPNP)-Gd nanomicelle formulation were stable in deionized water or phosphate buffered saline (PBS), with no observable aggregation, dissociation or bleaching for at least 1 month of storage. The samples were stored at 4°C for further use.

Characterization of Pt(TPNP)-Gd nanomicelles

The mean diameter of Pt(TPNP)-Gd polymeric micelles was evaluated by dynamic light scattering (DLS) measurements using a model 90 plus Zeta Sizer (Brookhaven Inc., Brookhaven, NY). Transmission electron microscopy was also performed using a JEOL model JEM-100CX electron microscope operating at an acceleration voltage of 80 kV. To measure the morphology and size distribution of polymeric micelles, a drop of sample solution (1 mg/ml) was placed on a 300-mesh copper grid coated with carbon. Approximately 2 minutes after deposition, the grid was tapped with filter paper to remove surface water and air-dried. Negative staining was performed using a droplet of 2% wt aqueous phospotungistic aciduranyl acetate. The optical absorption measurements were done using a Shimadzu model 3600 UV-Vis-NIR spectrophotometer. A SPEX 270M spectrometer (Jobin Yvon), equipped with an InGaAs TE-cooled photodiode (Electro-Optical Systems, Inc.) was used to obtain the photoluminescence (PL) spectra. A laser diode emitting at 630 nm was used as the excitation source. The sample in a quartz cuvette was placed directly in front of the entrance slit of the spectrometer, and the emission signal was collected at 90° relative to the excitation light.

Magnetic Resonance Imaging

Magnetic Resonance Imaging (MRI) studies were performed using a 4.7 T/33-cm horizontal bore magnet (GE NMR Instruments, Fremont, CA) incorporating AVANCE digital electronics (Bruker Biospec with ParaVision 3.0.2; Bruker Medical Inc., Billerica, MA) and a removable gradient coil insert (G060, Bruker Medical Inc., Billerica, MA) generating maximum field strength of 950 mT/m and a custom-designed 35-mm RF transmit-receive coil. In vitro MR relaxometry was performed in phantoms containing serial dilutions of the nanomicelle formulation over a Gd concentration range of 0-100 μM with varying phospholipid:Gd ratios. T1 relaxation rates (R1) were measured using a Fast Imaging with Steady-State Precession (True-FISP) imaging sequence with the following scan parameters: field of view (FOV) = 3.20 × 3.20 cm, matrix size = 128 × 128, TR/TE_eff. = 3.0/1.5 ms, NEX = 1, slice thickness = 1.50 mm, TI = 40.0 ms, flip angle = 60° and number of echoes = 60. R1 values were plotted as a function of concentration and linear regression analysis was performed to estimate T1-relaxivities. Estimates were compared to the clinically-approved gadolinium-based contrast agent, gadopentetate dimeglumine (Gd-DTPA; Magnevist^®).

In vivo MRI studies were performed using nude mice bearing subcutaneous primary patient tumor-derived HNSCC xenografts to examine the tumor imaging potential of the nanoparticles. Images were acquired at baseline (before nanoparticle injection), immediately after injection, 4 hours and 24 hours post-administration of (PtTPNP)-Gd nanoparticles (35 μmol Gd/kg). For these studies, animals were anesthetized using the inhalational anesthetic, Isoflurane (Abbott Laboratories, IL), induced at 4% in oxygen, and sustained 2-3% during imaging. The mice were secured in a form-fitted, MR-compatible sled (Dazai Research Instruments, Toronto, Canada) equipped with temperature and respiratory monitoring sensors. The sled, along with a phantom containing 0.15 mM Magnevist, was then positioned inside the scanner using a carrier tube composed of cellulose acetate butyrate plastic (Curbell Plastic, Orchard Park, NY). The animal body temperature was maintained at 37°C throughout the scanning process using an air heater system (SA Instruments Inc., Stony Brook, NY), and temperature feedback was automatically initiated due to thermocouples inherent to the sled, in conjunction with computer software supplied with the heater. Data acquisition consisted of localizer images followed by high resolution axial T2-weighted (T2W) images for anatomic imaging of tumor and normal tissues [FOV = 3.20 × 3.20 cm, matrix size = 256 × 192, TR/TE_eff. = 2500/41.0, NEX = 4, slice thickness = 1.00 mm, interslice distance = 1.25 mm, and number of echoes = 8]. T1-weighted (T1W) spin echo images were acquired for examining the tumor enhancement pattern at different times post nanoparticle injection [FOV = 3.20 × 3.20 cm, matrix size = 256 × 195, TR/TE_eff. = 404/7.8 ms, NEX = 4, slice thickness = 1.00 mm, interslice distance = 1.25 mm]. The change in T1-relaxation rate (R1 = 1/T1) of tumor and kidneys was measured using a T1-weighted saturation recovery fast spin echo (T1-FSE) sequence [FOV = 3.20 × 3.20 cm, matrix size = 128 × 96, NEX = 1, slice thickness = 1.00 mm, interslice distance = 1.25 mm, TE_eff = 25 ms and TR = 6000, 3000, 1500, 750, 500, 360.34 ms]. Finally, MR angiography was performed using a T1/flow-weighted 3D spoiled gradient echo (T1-SPGR) sequence with the following parameters: FOV = 4.80 × 3.20 × 3.20 cm, matrix = 192 × 96 × 96, TR/TE_eff. = 15.0/3.0, NEX = 1, flip angle = 40° and slice thickness = 32.00 mm. Image processing and analysis were carried out using the medical imaging software ANALYZE (Version 7.0; Biomedical Imaging Resource, Mayo Foundation, Rochester, MN). Regions of interest (ROIs) were manually drawn around tumor, muscle and kidney and object maps were created for calculation of T1 relaxation rates using MATLAB (Mathworks Inc., Version 7.1, Natick, MA).

In vivo Optical Imaging

Near-infrared optical imaging of tumor-bearing mice was performed at the 24 hour time point following completion of MRI studies. In vivo spectral imaging was carried out using the spectral imaging system, Maestro GNIR FLEX comprising of an optical head, an optical coupler and a cooled, scientific-grade monochrome CCD camera, along with image acquisition and analysis software. The polymeric micelles were excited at 650-700 nm using "deep red" excitation filter (CRi), transmitting light from the source (Xe lamp) in the range of 650-700 nm. An emission filter (800 LP) was used to cut off the leaking excitation light. A tunable liquid crystal filter in front of the imaging CCD camera was automatically stepped in 10-nm increments from 800 to 950 nm while the camera captured images at each wavelength with constant exposure. Overall acquisition time was about 10 seconds. The 16 resulting TIFF images were loaded into a single data structure in memory, forming a spectral stack with a spectrum at every pixel. Autofluorescence and the Pt(TPNP)-Gd nanomicelles phosphorescence spectra were obtained from the spectral image using the computer mouse to select appropriate regions. Spectral unmixing algorithms were applied to create the unmixed images of 'pure' autofluorescence and 'pure' phosphorescence signals.

Statistics

All measured values have been reported as mean ± standard error of the mean. P values less than 0.05 were considered statistically significant. All statistical calculations and analyses were performed using Graph Pad Prism (Version 5.00; Graph Pad, San Diego, CA). A two-tailed paired student's t test was used to compare tumor T1 relaxation rates at various time points in comparison to baseline pretreatment values.

Synthesis and characterization of (PtTPNP)-Gd nanoparticles

A schematic of the workflow involved in the design, synthesis and characterization (in vitro and in vivo) of dual modality (PtTPNP)-Gd nanoparticles is shown in Figure 1. Phospholipid nanomicelles encapsulating Pt(TPNP) were synthesized with gadolinium-functionalized surface as shown in Figure 2A. As one can see from the absorption and emission spectra of the micelles presented in Figure 2B, incorporation of DMPE-Gd to the nanomicellar formulation does not significantly affect the basic photophysical properties of Pt(TPNP) encapsulated in the hydrophobic core of the nanomicelles (7), and the Pt(TPNP)-Gd nanomicelles are phosphorescent. TEM analysis revealed a nearly spherical morphology of the nanoparticles with a narrow size distribution (Figure 2C). Zeta potential studies carried out with the Pt(TPNP)-Gd nanomicelles have shown an overall negative zeta potential owing to the presence of the PEG molecules on the surface of the nanomicelles. The net surface charge of the nanomicelles was found to be -38.24 mV. Consistent with the TEM observations, the DLS analysis of the nanoparticles revealed a unimodal distribution with the maximum percentage intensity to be of 103.6 ± 1.8 nm in size (Figure 2D).

In vivo MR-NIR optical imaging of HNSCC xenografts

Prior to in vivo investigation, studies were carried out in vitro to investigate the MR properties of the gadolinium containing nanomicelles. The T1 relaxivity of Pt(TPNP)-Gd nanomicellar formulation at 4.7T was markedly higher (10.4 mmol^-1*s^-1) than the clinically approved contrast agent, Gd-DTPA (3.6 mmol^-1*s^-1). Subsequently, in vivo imaging experiments were carried out using nude mice bearing primary patient tumor-derived human HNSCC xenografts. T1-enhancement characteristics and relaxation rate measurements of tumor and blood (kidneys) were performed before (precontrast) as well as 1, 4 and 24 hours after nanoparticle administration (35 μmol Gd/kg). Figure 3 shows colorized T1-relaxation maps (R1 maps) calculated from T1-weighted images of a mouse before (pre), immediately after (post), 4 hours and 24 hours after injection of the Pt(TPNP)-Gd nanomicelles. An axial slice showing the kidneys (upper panel) and the tumor (middle panel) is shown. An enlarged ROI of the tumor is also shown in the bottom panel. Signal enhancement was visible immediately after nanoparticle injection and persisted up to 24 hours post administration. Measurement of T1 relaxation rates (R1 = 1/T1) showed a significant increase (P < 0.05 at immediately post and 4 h post injection; P < 0.01 at 24 h) in tumor R1 at all 3 time points post nanoparticle administration, compared to preinjection values with peak enhancement in the tumor at 4 hours post injection (Figure 4A). A significant increase in the R1 of the kidneys was also observed immediately after injection and at 4 h post injection (Figure 4B, P < 0.001). Maximum intensity projection (MIP) images generated from 3D T1-weighted images acquired post contrast showed visible enhancement in contrast within the tumor and blood vessels in mice (Figure 4C).

NIR optical imaging, performed following the completion of MRI at the 24 hour time point, revealed high contrast phosphorescence signal and confirmed tumor localization of the nanoparticles (Figure 5A). The large spectral separation between the Pt(TPNP) absorption (~700 nm) and phosphorescence emission (~900 nm) allowed for a dramatic decrease in the level of background autofluorescence and scattered excitation light. Consistent with previous observations (7), ex-vivo NIR optical imaging revealed accumulation of nanoparticles in liver tissues, in addition to tumor localization (Figure 5A). No significant phosphorescence signal was seen from other dissected organs (heart, spleen, kidneys). Histologic evaluation of liver tissues did not show any evidence of toxicity following nanoparticle administration. Figure 5B shows haematoxylin and eosin stained liver section obtained from an animal 48 h following injection of Pt(TPNP)Gd nanomicelles. No visible change in the histologic architecture of the liver or evidence of necrosis, fibrosis, fatty changes or clear cell changes were seen in treated animals compared to controls. Corresponding liver section of an untreated control animal is also shown in Figure 5B.

Toxicologic evaluation of Pt(TPNP) nanoparticles was also performed by measuring the body weight of animals over a 2 week period. Animals injected with Pt(TPNP) formulation underwent physical and neurological evaluations. No changes in eating, drinking, grooming, exploratory behavior, activity, physical features (e.g., weight and skin color), and neurological status were observed in animals injected with the nanoformulation compared to control animals. No significant differences in average body weight were observed between control and treatment groups.