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1 Supporting Information Agemy et al /pnas SI Methods Cell Lines and Tumors. HUVEC (Lonza) were cultured using EBM- 2 medium with endothelial cell growth supplement (Lonza). Human astrocytoma cell line (U87) was grown in DMEM plus 10% FBS and 1% glutamine pen-strep (Invitrogen). Mouse GBMinitiating 005 cell line was established as previously described (1). The 005 cells were maintained in N2 medium, which contains DMEM/F-12 (Omega Scientific), 1% N2 supplement (Invitrogen), 20 ng/ml human FGF-2 (Peprotech), 20 ng/ml human EGF (Promega), and 40 μg/ml heparin (Sigma-Aldrich). T3 cells were obtained by differentiation induction of 005 cells cultured in EGM-2 (Lonza). Human GBM spheres were obtained and cultured as described previously (2). Mouse GBM-initiating 005 cells were transplanted into brains of nonobese diabetic (NOD)-SCID mice. A total of cells were suspended in 1.5 μl of PBS and injected stereotaxically in the right hippocampus. Human GBM spheres xenografts were created by injecting cells orthotopically into NOD-SCID mice in 1.5 μl of PBS.Animal experimentation was performed according to procedures approved by the Animal Research Committee at the University of California, Santa Barbara, The Sanford-Burnham Medical Research Institute, and The Salk Institute for Biological Research, San Diego, CA. Preparation of NWs. NWs coated with peptides were prepared as previously described (3, 4). Aminated NWs were pegylated with maleimide-5kpeg-nhs (JenKem Technology). Peptides were conjugated to the nanoparticles through a thioether bond between the cysteine thiol from the peptide sequence and the maleimide on the functionalized particles. Regarding quantification peptide on NWs, the aminated NWs were pegylated with OPSS-5KPEG-NHS (JenKem Technology). Peptides were conjugated to the nanoparticles through a disulfide bond between the cysteine thiol from the peptide sequence and the pyridyl sulfenyl protected thiol on the functionalized particles. The CGKRK D [KLAKLAK] 2 -NWs linked covalently through the disulfide linkage were treated with DTT. The concentration of free peptide thus obtained in the solution was estimated using fluorescence spectroscopy with a peptide standard curve. Magnetic Resonance Imaging. Mice bearing orthotopic 005 tumors were i.v. injected with CGKRK D [KLAKLAK] 2 -NW (5 mg iron/ kg). Approximately 5 h after NW injection, the mice were anesthetized with isoflurane and subjected to T2*-weighted MRI using the following parameters: 3D spoiled gradient echo sequence, time to repetition 40 ms, time to echo 18.6 ms, flip angle 15, bandwidth 115 Hz/pixel, 0.16 mm in-plane resolution, and 0.3-mm slice thickness (three slices averaged offline for improved signal/noise ratio). Mice were scanned in a Sigma HDx 3T whole body scanner (GE Healthcare) using a custom-made 3- cm-diameter volume coil. After imaging, tissues of interest were harvested and processed for H&E staining. In Vivo Matrigel Angiogenesis. Two-month-old BALB/c nu/nu mice were s.c. injected bilaterally in the inguinal area with 500 μl of Matrigel (Becton Dickinson) with or without 500 ng of recombinant human bfgf (Becton Dickinson) as an angiogenesis stimulant. The mice were treated every other day with PBS or CGKRK D [KLAKLAK] 2 nanoworms (NWs) (5 mg/kg) for 2 wk. At the end of treatment the mice were killed under anesthesia by perfusion through the heart with far-red fluorescent Alexa Fluor 647 isolectin GS-IB4 conjugate (Molecular Probes), followed by further perfusion with 10 ml of 4% paraformaldehyde. The Matrigel plugs were imaged by the Odyssey Infrared Imaging System (LI-COR Biotechnology), and for histological analyses, 7- to 10-μm sections were cut and viewed under a Fluoview 500 confocal microscope (Olympus America). The treatment and PBS control groups consisted of two mice (four plugs) in each of three independent experiments. Tube Formation Assay. Wells in 24-well plates were coated with 250 μl Matrigel (Becton Dickinson). human umbilical vein endothelial cells (HUVEC) were detached with trypsin (Lonza), washed with PBS, and 10 5 cells in EBM2 media supplemented as mentioned above were subsequently plated on top of the Matrigel in the presence of 5 or 10 μg/ml of NWs. The cells were incubated at 37 C for 24 h and photographed under a brightfield microscope at 40 magnification (Leica Microsystems). NW Toxicity Studies. Serum was collected from mice before treatment, 1 d after treatment ended, and 2 wk after treatment was concluded. L-Alanine-2-oxoglutarate aminotransferase (ALT) levels in serum were determined using Infinity ALT (GPT) Reagent (ThermoScientific) according to the manufacturer s instructions. The same serum samples were used to determine the levels of IL-6 using the Mouse IL-6 ELISA Set (BD OptEIA; BD Biosciences). Isolation of Mitochondria and Peptide/Phage Binding. Mitochondria were isolated from livers of BALB/c mice using differential centrifugation with buffers from a Pierce mitochondrial isolation kit for tissue according to the manufacturer s instruction (Pierce Biotechnology). To test the binding of the CGKRK peptide to mitochondria, purified mitochondria were preincubated with various concentrations of nonlabeled CGKRK or control peptide (CREKA) for 30 min at 4 C. FAM-CGKRK was then added and incubated for an additional 1 h. The binding of the FAM peptide was quantified by fluorescence. In phage binding assays, purified mitochondria were suspended in 10 ml DMEM supplemented with 1% BSA and incubated with pfu of peptide-displaying phage overnight at 4 C. The mitochondria were washed three times with DMEM/BSA, and the phage were collected with lysogeny broth containing 1% Nonidet P-40 and quantified by plaque assay. Immunoblot Analysis of NW-Bound Proteins. HUVEC were incubated 24 and 48 h with 10 mg/ml CGKRK D [KLAKLAK] 2 - NWs or CREKA-NW and lysed with RIPA buffer (Pierce) according to the manufacturer s instructions. The lysates were separated by SDS/PAGE. After transfer of the proteins onto nitrocellulose membranes for 2 h at 200 ma, the membrane was treated for 1 h at room temperature with TBS-0.05% Tween containing 5% milk and incubated with 1 mg/ml anti-caspase 3 (Cell Signaling). Biophotonic Tumor Imaging. Mice bearing luciferase-labeled GBM tumors received injections of 3 mg per mouse of freshly prepared luciferin substrate (Promega) suspended in PBS. The mice were then anesthetized with isofluorane and imaged using the Xenogen IVIS 100 Imaging System (Xenogen, Caliper Life Sciences), 10 min after i.p. injection of luciferin at a 1-min acquisition time in a small binning mode. Cell Proliferation Assay and Imaging. The MTT [3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide] assay (Mo- 1of6

2 lecular Probes) was used to quantify cell proliferation. Cells were seeded in complete medium into 96-well plates ( cells per well) and allowed to attach overnight at 37 C in a humidified 5% CO 2 atmosphere. The culture medium was then removed, and various concentrations of peptides or NWs were added. After 48 and 72 h, 10 μl of the MTT reagent (5 mg/ml in PBS) was added to each well. The medium was removed from cells after 3 h, and 100 ml of DMSO:MEOH (1:1 vol/vol) were added to each well. The plates were read at a wavelength of 595 nm. For live cell imaging, cells were seeded on glass-bottom plates (Willco), and 24 h later the cells were washed and incubated with FAM-labeled peptides or NWs for 45 min at 37 C. The cells were then rinsed three times with PBS and incubated for an additional 15 min at 37 C with 500 nm MitoTracker Red (Molecular Probes) followed by nuclear staining with Hoechst DNA dyes (Molecular Probes) for 12 min. The cells were analyzed with Fluoview FV 500 confocal microscopy (Olympus America). 1. Marumoto T, et al. (2009) Development of a novel mouse glioma model using lentiviral vectors. Nat Med 15:110e Soda Y, et al. (2011) Transdifferentiation of glioblastoma cells into vascular endothelial cells. Proc Natl Acad Sci USA 108:4274e Agemy L, et al. (2010) Nanoparticle-induced vascular blockade in human prostate cancer. Blood 116:2847e Park JH, et al. (2009) Systematic surface engineering of magnetic nanoworms for in vivo tumor targeting. Small 5:694e700. Fig. S1. CGKRK peptide in normal organs. Mice bearing 005 tumors in the right hippocampus were i.v. injected with 200 μg of rhodamine-labeled CGKRK peptide. After 3 h the mice were perfused through the heart with PBS, and tissues were collected, sectioned, and analyzed for rhodamine fluorescence. Red, CGKRK peptide; blue, nuclei. n = 3. (Scale bars, 200 μm.) Fig. S2. CGKRK D [KLAKLAK] 2 -NWs in normal tissues of tumor-bearing mice. CGKRK D [KLAKLAK] 2 -NW (red) were injected into mice bearing 005 tumors, and tissues were collected and analyzed by confocal microscopy. Red, CGKRK D [KLAKLAK] 2 -NWs; blue, nuclei stained with DAPI. (Scale bars, 200 μm.) 2of6

3 Fig. S3. Homing of CGKRK-NWs to GBM tumors. (A) Iron oxide NWs coated with rhodamine-labeled CGKRK or D [KLAKLAK] 2 peptide were i.v. injected (5 mg iron per kg body weight) into mice bearing 005 tumors. Five to six hours after the injection the mice were perfused through the heart with PBS, and tissues were collected. Tumor sections were stained with anti-cd31 and examined by confocal microscopy. The NWs are red, tumor cells green, blood vessels magenta, and nuclei with DAPI blue. Arrows point at a representative tumor blood vessel that has accumulated CGKRK-NWs. (Scale bars, 200 μm.) (B) Quantification of NWs in 005 tumors. Percentage of particle-positive blood vessels was measured by confocal microscopy by analyzing five random areas in each tumor section (n =3). 3of6

4 Fig. S4. CGKRK D [KLAKLAK] 2 -NW conjugates induce cell death by apoptosis. HUVEC cells (A) and T3 cells (B) were left untreated (Control) or treated with 10 μg/ml of NWs coated with CGKRK D [KLAKLAK] 2 -NWs for 48 (A) or72h(b). (C) Cells were incubated with the indicated NWs, and after 30 min the cells were washed to remove excess NWs and then incubated for 72 h. Annexin V staining and analysis by flow cytometry were used to measure apoptosis in the cultures. Representative images indicating the percentage of annexin-positive cells (apoptotic and dead cells). Fig. S5. CGKRK D [KLAKLAK] 2 -NW conjugates inhibit in vitro and in vivo angiogenesis. (A) Tube formation assay using primary HUVEC plated on growth factorreduced Matrigel in 5% FCS medium alone (Control) or containing the indicated NW constructs. The formation of networks of capillary-like structures was viewed by phase-contrast microscopy at 40 magnification 24 h after plating. (B) Inhibition of in vivo Matrigel angiogenesis by CGKRK D [KLAKLAK] 2 -NWs. Matrigel plugs with or without bfgf were s.c. injected into BALB/c nu/nu mice. The mice were treated every other day with i.v. injections of either CGKRK D [KLAKLAK] 2 -NWs (5 mg/kg) or PBS. The mice were killed 14 d later and perfused with 647 Alexa isolectin. The Matrigel plugs were removed from the mice and viewed macroscopically under fluorescent light (Upper) or by confocal microscopy analysis of sections of the plugs (Lower). 4of6

5 Fig. S6. Toxicology analyses of mice treated with CGKRK D [KLAKLAK] 2 -NWs. (A) ALT levels measured before (Pre-treatment), after completion of a 3-wk treatment course (After treatment), and after a subsequent 2-wk recovery period (2 weeks after treatment). (B and C) Testing for possible active and innate immune responses against NW by measuring antibody (B) and IL-6 levels (C). The measure was done from serum of mice collected as in A. (D) H&E staining of the kidneys in PBS-treated control mice and mice treated with CGKRK D [KLAKLAK] 2 -NWs at the end of the treatment. Fig. S7. Apoptosis analysis of tumors from mice treated with D [KLAKLAK] 2 -NWs (control) and CGKRK D [KLAKLAK] 2 -NWs. Apoptosis was analyzed by TUNEL staining (magenta); GFP-expressing 005 tumor cells are green. (Scale bars, 50 μm.) 5of6

6 Fig. S8. Treatment of mice bearing intracranial U87 tumors with CGKRK D [KLAKLAK] 2 -NWs. Tumors were induced by injecting GFP-expressing U87 cells into the right hippocampus of mice. Treatment with i.v. injections of CGKRK D [KLAKLAK] 2 -NWs and control NWs was started 10 d after the tumor cell injection and continued every other day for 3 wk (n = 5 per group). Table S1. Quantification of NWs outside tumor vessels Percentage of penetrating NW Variable Excluding CD31 area Excluding twofold CD31 area CGKRK D [KLAKLAK] 2 -NWs + CRGDC 27% ± 10% 17% ± 6% CGKRK D [KLAKLAK] 2 -NWs + irgd 71% ± 4% 57% ± 15% CGKRK D [KLAKLAK] 2 -NWs were injected into mice bearing 005 tumors combined with a conventional RGD peptide (CRGDC; control) or irgd. NWs were detected in tumor sections by rhodamine fluorescence, and blood vessels were stained with anti-cd31. Percentage of penetrating NWs was calculated using the MetaMorph program. 6of6