Animal experimentation was performed according to procedures approved by the Animal Research Committees at the University of California, Santa Barbara, San Diego, and San Francisco, and the Burnham Institute for Medical Research. Xenografts were created by injecting nude mice with 106
human cancer cells orthotopically, subcutaneously, or into the tibia and the brain: prostate cancers PC-3 (Yang et al., 1999
), PPC1 (Zhang et al., 2006
), and 22Rv1 (Drake et al., 2005
), pancreatic cancer MIA PaCa-2 (Sugahara et al., 2008
), and breast cancer BT474 (Rusnak et al., 2001
). Disseminated prostate tumors were generated by injecting 106
GFP-PC-3 cells (Yang et al., 1999
) into the left ventricle of the heart of nude mice. Tumors were monitored with the X-ray system of the Image Station In Vivo FX (Eastman Kodak Company, Rochster, NY) or the Illumatool Bright Light System LT-9900 (Lightools Research, Encinitas, CA). Transgenic mice were maintained as described (Arbeit et al., 1994
; Hanahan, 1985
; Hezel et al., 2006
In vivo peptide and phage homing
Approximately 200 µg of FAM-labeled synthetic peptides (Karmali et al., 2009
) were intravenously injected into tumor-bearing mice and allowed to circulate for 15 min to 2 hrs. Tissues were collected and observed under UV light (Illumatool Bright Light System LT-9900), and processed for immunofluorescence (Karmali et al., 2009
) or immunohistochemistry (Sugahara et al., 2008
). To quantify the homing area of peptides within tumors, cryo-sections immunohistochemically stained with an anti-FITC antibody were scanned with the Scanscope CM-1 scanner and analyzed with the ImageScope software (Aperio Technologies, Vista, CA; Fogal et al., 2008
). To assess phage homing (Zhang et al., 2006
plaque-forming units (pfu) of T7 phage were intravenously injected into tumor-bearing mice, and allowed to circulate for 15 min. The mice were perfused through the heart with PBS containing 1% BSA and tissues were harvested for immunofluorescence. In some experiments, 50 µg of function-blocking anti-neuropilin-1 antibody (R&D Systems, Minneapolis, MN) or goat IgG (Abcam, Cambridge, MA) was intravenously injected into the tumor mice 15 min prior to the phage injections. The phage were allowed to circulate for 10 min prior to perfusion and collection of the tumors and other tissues.
Preparation of micelles
Lipids were purchased from Avanti Polar Lipids (Alabaster, AL).
DSPE-PEG2,000-iRGD(FAM) was prepared by coupling FAM-iRGD peptide bearing a cysteine on its N-terminus to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-maleimide(polyethylene glycol)2,000 (DSPE-PEG2,000-maleimide) at 1:1 molar ratio at room temperature for 4 hrs.
DSPE-PEG2,000-FAM was prepared by coupling 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol)2,000 (DSPE-PEG2,000-amine) with NHS-Fluorescein (Pierce Biotechnology, Rockford, IL) at a 1:1 molar ratio for 1 hr at room temperature. DSPE-PEG2,000-Cy7 was prepared similarly using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol)2,000 and Cy7-NHS ester (GE Healthcare, UK).
DSPE-PEG2,000-iRGD(FAM), DSPE-PEG2,000-amine, and DSPE-PEG2,000-Cy7 in 3:6.7:0.3 molar ratios were dissolved in chloroform/methanol (3:1, v/v). The solvent was evaporated, and the dried lipid film was kept under vacuum for 8 hrs and allowed to swell in PBS for 2 hrs at 60°C. The vial was vortexed and sonicated to produce micelles. The micelles were sequentially filtered through 0.2 µm and 0.1 µm filters, and washed with sterile PBS to remove unreacted peptides. Control Cy7 micelles were prepared using DSPE-PEG2,000-FAM in place of DSPE-PEG2,000-iRGD(FAM). The micelles were 15–25 nm in diameter as measured in deionized water by dynamic laser light scattering (refractive index, 1.59; viscosity, 0.89) on a Malvern Zetasizer Nano (Malvern, UK).
Optical in vivo imaging of micelle-peptide conjugates
PDAC mice were injected with 100 µl of 1 mM micelles in PBS. After 3 hrs, the mice were anesthetized, shaved, and subjected to whole body imaging with the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
Tissue sections were processed as described (Karmali et al., 2009
). Cells were grown on collagen I-coated coverslips (BD Biosciences, San Jose, CA) overnight, and incubated with 108
pfu/ml of T7 phage for 30 min. The cells were fixed in 4% paraformaldehyde, and stained with antibodies and DAPI (Molecular Probes, Eugene, OR). The primary antibodies were rat anti-mouse CD31 monoclonal antibody (BD Biosciences), and rabbit anti-human αv integrin (Chemicon, Temecula, CA), rabbit anti-human neuropilin-1 (Chemicon), mouse anti-human neuropilin-1 (Miltenyi Biotec, Auburn, CA), and rabbit anti-T7 phage (Teesalu et al., 2009
) polyclonal antibodies. The secondary antibodies, Alexa594 goat antibodies to mouse, rat, and rabbit IgG and Alexa488 donkey anti-rabbit antibody were from Molecular Probes. Cells and tissue sections were examined by a Fluoview 500 confocal microscope (Olympus America, Center Valley, PA).
In vitro phage binding and penetration assays
Suspended cells (106 cells in DMEM containing 1% BSA) were incubated with 108 pfu/ml of T7 phage for 1 hr at 4°C. The cells were washed 4 times with the binding buffer, lysed with lysogeny broth containing 1% NP-40, and titrated. Phage penetration assays used the same procedure, except that the cells were incubated with phage at 37°C, and that an acidic buffer (500 mM sodium chloride, 0.1 M glycine, 1% BSA, pH 2.5) was substituted for the binding buffer in the second wash to remove the phage that bound to the cell surface. Inhibitors of binding and penetration were added 20 min prior to incubation with phage. Non-infectious phage were prepared by treating phage with UV for 8 min in DMEM containing 1% BSA. The UV-inactivated phage particles expressing about 200 peptides per particle were used as multivalent inhibitors. Free synthetic peptides, mouse antibodies against human α1, α2, αvβ3, αvβ5, α5β1, α4, or αv integrins and integrin subunits (Chemicon), goat anti-rat neuropilin-1 (R&D Systems), with mouse and goat IgG isotype controls (Abcam) were also tested. In some cases, 109 pfu phage were treated with 50 μg/ml of crystalline trypsin for 5 min at 37°C before use. The proteolytic reaction was terminated with 5 mg/ml of soy-bean inhibitor.
The experiments were performed as described (Sugahara et al., 2003
) except that 1 mM of MgSO4
, and MnCl2
were added to the buffer containing the integrin antibodies. The antibodies were the same as in the cell binding assays, and were detected with an Alexa488 goat anti-mouse or goat anti-rabbit antibody (Molecular Probes). The cells were analyzed with an EasyCyte Plus System (Guava Technologies, Hayward, CA).
FAM-iRGD fragment isolation
PPC1 cells (107 cells in DMEM) were treated with 10 µM carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132; EMD Chemicals, Gibbstown, NJ) for 30 min at 37°C to inhibit proteasomes, and incubated with 20 µM of iRGD peptide labeled with FAM at the N-terminus or C-terminus. The cells were washed once with acidic buffer and lysed in MPER (Pierce Biotechnology) containing protease inhibitors (Complete Mini EDTA-free; Roche Applied Science, Indianapolis, IN) on ice for 30 min. The sample was centrifuged for 30 min at 12,000 rpm. The supernatant was applied onto an anti-FITC affinity column, and after washing, bound peptides were eluted with glycine-HCl, pH 2.8. The eluate was subjected to mass spectrometry.
Binding affinities of iRGD and CRGDK to αvβ3 and αvβ5 integrins (US Biological, Swampscott, MA) and to neuropilin-1 (R&D Systems) were quantified by an ELISA by measuring IC50
. First, saturation binding assays were performed. Microtiter wells coated with 5 µg/ml of the purified proteins were incubated for 1 hr at room temperature with various concentrations of biotinylated iRGD or CRGDK peptide in a HEPES-based buffer containing 1 mM of MgSO4
for integrin binding and PBS for neuropilin-1 binding. After washing with the same buffer added with 0.01% Tween 20, streptavidin-conjugated horseradish peroxidase (Vector laboratories, Burlingame, CA) was added to the wells and incubated for 30 min at room temperature. Peptide binding was quantified with 2,2-azino-bis(3-etylbenzthiazoline-6-sulfonic acid; Sigma-Aldrich, St. Louis, MO) as a substrate. In subsequent competition studies, microtiter wells coated with the proteins were incubated with various concentrations of non-labeled test peptides and a biotinylated reporter peptide at a concentration that gave half maximal binding in the saturation binding assay. After 1 hr incubation at room temperature, the binding of the biotinylated peptide was quantified as above. Affinities were determined from the inhibition data as described (Müller, 1980
Magnetic Resonance Imaging
Nude mice bearing 22Rv1 orthotopic human prostate tumors were injected intravenously with superparamagnetic iron oxide nanoworms (Park et al., 2009
) coated with iRGD or CRGDC peptides, or untargeted-nanoworms at a dose of 5 mg/kg of iron. Each animal received Ni-liposomes (0.2 µmol of Ni) intravenously 1 hr prior to the nanoworms to increase the half-life of the nanoworms (Simberg et al., 2007
). The mice were repeatedly imaged before and 3 and 7 hrs after injection of the nanoworms. For each scan, the mice were anesthetized with isoflurane and repositioned into a 30 mm diameter mouse coil. The axial plains were carefully matched to previous scans by measuring the height of the sections and comparing the vascular patterns in the images. Iron sensitive MRI scans consisting of T2-weighted fast spin-echo were acquired using a 3-Tesla MR imager (GE Healthcare, Milwaukee, WI). The conditions used were; repetition time/echo time = 6.4 s/70 ms, echo train length = 32, readout bandwidth = ± 15.6 kHz, in-plane spatial resolution = 220 µm, field of view = (3.5 cm)2
, slice thickness = 1 mm, number of excitation = 3. After imaging, tissues of interest were harvested without perfusion and processed for immunofluorescence.
Tumor treatment studies
Peptide-conjugated abraxanes were prepared and characterized as described (Karmali et al., 2009
). For in vitro cytotoxicity studies, 22Rv1 or BT474 cells were seeded in 96-well culture plates (5 × 104
cells per well) and incubated overnight. The cells were incubated with various concentrations of the conjugates for 30 min at room temperature, and washed with fresh culture media. MTT assays (Invitrogen) to assess cell viability were performed on the cells 48 hrs later. For in vivo tumor treatment studies, nude mice bearing 2 week-old 22Rv1 orthotopic xenografts (typically about 250 mm3
in tumor volume) were intravenously injected with the abraxane conjugates. The conjugates were given every other day for 14 days at a paclitaxel equivalent of 3 mg/kg/injection. The iRGD peptide control was administered in an equivalent amount of iRGD in each iRGD-abraxane dose. Mice bearing subcutaneous 22Rv1 tumors were treated similarly for 12 days, and orthotopic BT474 tumors for 20 days. The experiments were terminated according to the guidelines by the Animal Research Committee at the University of California, Santa Barbara. To study the homing pattern of the abraxane conjugates in the 22Rv1 orthotopic tumors, the conjugates were intravenously injected to tumor bearing mice at a dose of 3 mg/kg, and allowed to circulate for 3 hrs. The mice were perfused through the heart, and tissues of interest were harvested and processed for immunofluorescence.
Mice bearing 22Rv1 or BT474 tumors were intravenously injected with the abraxane conjugates at a paclitaxel equivalent of 9 mg/kg/injection. After 3 hours, the mice were perfused through the heart, and tissues of interest were harvested. The tissues were homogenized in cold RIPA buffer (Pierce Biotechnology) containing protease inhibitors (Complete Mini EDTA-free) and kept on ice for 30 min. The samples were then centrifuged for 30 min at 14,000 rpm. The abraxane concentration in the supernatant was quantified with an ELISA: abraxane was captured with a taxol antibody (Novus Biologicals, Litleton, CO) coated onto a 96-well plate and detected with a human albumin antibody labeled with biotin (US Biological).
Data were analyzed by two-tailed Student’s t
-test and one-way analysis of variance (ANOVA) followed by suitable post-hoc test. The details are given in Table S3