The poly(phenylene vinylene) derivative poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV; MW: 200,000 Da; polydispersity, 4.0) were purchased from ADS Dyes, Inc. (Quebec, Canada). Polystyrene (PS) and polystyrene graft ethylene oxide functionalized with carboxyl groups (PS-PEG-COOH; MW: 21,700 Da of the PS moiety; 1,200 Da of PEG-COOH; polydispersity, 1.25) were purchased from Polymer Source Inc. (Quebec, Canada). Silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775) was purchased from Sigma Aldrich, Inc. Cyclic RGD peptide was purchased from Peptides International, Inc. All the other chemicals were purchased from Sigma Aldrich, Inc. and used without purification. Luc8 was prepared according to our previously published procedures8
Synthesis of NIR nanoparticles
NIR775-doped NIR nanoparticles were prepared using a nanoscale precipitation technique30
. In a typical procedure, a solution of tetrahydrofuran (THF) containing 50 μg/mL of MEH-PPV, 50 μg/mL of PS-PEG-COOH, and 0.6 μg/mL of NIR775 dye was prepared. An aliquot of the mixture (5 mL) was then quickly dispersed into 10 mL of water under vigorous sonication. Extra THF was evaporated at an elevated temperature (below 90 °C) under the protection of nitrogen. The THF-free NPs solution was filtrated through a 0.2 μm cellulose membrane filter. Bioconjugation was carried out by the EDC-mediated coupling reaction between the carboxyl groups on the NPs and the amine-containing molecules (Luc8 and RGD peptide). In a typical conjugation reaction, 60 μL of concentrated HEPES buffer (1 M) were added to 3 mL of a solution of the carboxylate-presenting NP (50 μg/mL in water) and the amine-containing molecules (100 μL of Luc8 at 3 mg/mL and 30 μL of RGD peptide at 10 mg/mL), followed by vortex mixing. Then, 50 μL of freshly prepared aqueous EDC solution (10 mg) was added and the above mixture was magnetically stirred for 1 hour at room temperature. The uncoupled free Luc8 and excess EDC were removed by four washes using a 100 K Amicon Ultra filter (Millipore Corporation) under centrifugation at 3,000 rpm for 3 min at 4 °C. The final complex was kept in PBS buffer at 4 °C.
In vitro nanoparticles characterization
The size and morphology of the nanoparticles were investigated using Transmission Electron Microscope (TEM) (FEI Tecnai G2 F20 X-TWIN, 200 kV). TEM samples were prepared by dripping the NP solution onto a carbon-supported copper grid and drying it at room temperature before observation. The hydrodynamic size of the nanoparticles was also measured in aqueous solution by Dynamic Light Scattering (DLS) (Brookhaven 90 Plus Nanoparticle Size Analyzer). The absorption spectra were recorded on an Agilent 8453 UV-Vis spectrometer. Fluorescence and bioluminescence emission spectra were collected with a FluoroMax-3 (Jobin Yvon Inc.) and corrected for wavelength-dependent detector sensitivity as described by the company. In the case of bioluminescence, the excitation light was blocked.
Cell culture, cytotoxicity assay and cell imaging
U87MG (human glioblastoma, high αvβ3 expression) cells were grown in DMEM supplemented with 10% FBS. Cultures were maintained at 37 °C under a humidified atmosphere containing 5% CO2. Cytotoxicity in the U87MG cell line was measured using a methyl thiazolyl tetrazolium (MTT) assay. Cells growing in log phase were seeded into a 96-well cell-culture plate at 1×104 cells/well and then incubated for 24 h at 37 °C under 5% CO2. RET1IR NPs (100 μL/well) were added to the wells of the treatment group at varying concentrations, and 100 μL/well DMEM was added to the negative control group, followed by incubation of the cells for 24 h at 37 °C under 5% CO2. Subsequently, 10 μL of MTT (5 mg/mL) was added to each well of the 96 well assay plate and incubated for an additional 4 h at 37 °C under 5% CO2. After the addition of DMSO (200 μL/well), the assay plate was allowed to shake at room temperature for 20 min. A Tecan microplate reader was used to measure the OD570 (Absorbance value) of each well with the background subtraction at 690 nm. The following formula was used to calculate the viability of cell growth: cell viability (%) = (mean of Absorbance value of treatment group / mean of Absorbance value of control) × 100.
For cell imaging experiments, 5 × 105 cells per well were seeded on 18 mm glass coverslips and cultured for 24 h before imaging with a Zeiss Axiovert 200M Microscope (excitation: 480/30 nm; dichroic beamsplitter: Q570LP; emission: D755/40M; objective: 20x; acquisition time: 1 s).
Blood circulation half-life in mice
The RET1IR NPs (~20 μg) were injected intravenously into the tail veins of three six-week old nude mice. At various time points postinjection, ~20 μL of blood was collected from the tail into 50 μL of 0.9 % NaCl solution containing 1.5 mg/mL of EDTA. The NIR fluorescence intensity of blood samples was assayed on an IVIS spectrum imaging system (excitation: 465±15 nm; emission: 780±10 nm). Blood samples without the RET1IR NPs were measured to determine the blood autofluorescence level, which was subtracted from the fluorescence intensity of injected samples.
Mice were euthanized by cervical dislocation under deep isoflurane anesthesia. Urine samples were immediately collected, and lymph nodes, brain, spleen, pancreas, kidney, lung, heart, liver, bone (femur), muscle (hind leg), stomach (emptied), and dorsal skin were harvested; for tumor-bearing mice, tumors were also harvested. Tissues were subjected to fluorescence imaging using an IVIS spectrum imaging system immediately (excitation: 465±15 nm; emission: 780±10 nm).
Lymph node imaging
Mice were anesthetized with 2.5% isoflurane, and RET1IR NPs (~20 μg) were administered to nude mice by tail-vein catheterization using the Vevo MicroMarker TVA (Vascular Access) Cannulation Kit (VisualSonics). The tail vein was further flushed with 100 μL of PBS buffer. At 24 h after injection, mice were euthanized, dissected to locate the lymph nodes of interest, and imaged using an IVIS spectrum imaging system immediately (excitation: 465±15 nm; emission: 780±10 nm). Alternatively, ~ 10 μL of RET2IR NPs (~2 μg each) were administered to the forepaws via intradermal injections. Within 10 min of injection, mice received an intravenous injection of 10 μg of coelenterazine for in vivo bioluminescence imaging (acquisition time: 10 s; no emission filter). Following bioluminescence imaging, in vivo fluorescence imaging was carried out (excitation: 465±15 nm; emission: 780±10 nm).
Tumor implantation and in vivo imaging
Animal procedures were approved by the Institutional Animal Care Use Committee of Stanford University. Tumor cells were harvested by incubation with 0.05% trypsin-EDTA when they reached near confluence. Cells were pelleted by centrifugation and resuspended in sterile PBS. U87MG cells (2 × 106 cells/site) were implanted subcutaneously into the left shoulder of four- to five-week-old female nude mice (Charles River Breeding Laboratories). When the tumors reached the size of 2 to 8 mm in diameter (two to three weeks after implantation), the tumor-bearing mice were subjected to biodistribution and imaging studies. In vivo and ex vivo fluorescence imaging was performed with an IVIS spectrum imaging system (excitation: 465±15 nm filter; emission: collected from 520 nm to 840 nm with a bandwidth of 20 nm). For bioluminescence imaging, the mice were imaged after tail vein injection of coelenterazine (20 μg/mouse in 20 μL of methanol and 80 μL of phosphate buffer). Images were acquired without filters.
Tumor-bearing mice were sacrificed 48 h after injection with RET2IR@cRGD. Tumor tissues were collected, washed with PBS, frozen by dry ice and stored at –80 °C. Frozen samples were cryosectioned by microtome at –20 °C into slices of 5 μm thickness, and then fixed in cold acetone for 5 min (–20 °C). Nonspecific binding sites were blocked over 30 minutes with PBS containing 10% mouse serum. The sections were stained with 1 μg of Alexa Fluor 488 anti-mouse CD31 antibody (Biolegend Inc., San Diego, CA) in 100 μL PBS buffer for 1 h at 37 °C. The sections were washed with PBS and analyzed under a Zeiss Axiovert 200M Microscope.