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. 2011 Oct 18;108(42):17450-5.
doi: 10.1073/pnas.1114518108. Epub 2011 Oct 3.

Targeted nanoparticle enhanced proapoptotic peptide as potential therapy for glioblastoma

Lilach Agemy  1 , Dinorah Friedmann-Morvinski, Venkata Ramana Kotamraju, Lise Roth, Kazuki N Sugahara, Olivier M Girard, Robert F Mattrey, Inder M Verma, Erkki Ruoslahti
Affiliations

Targeted nanoparticle enhanced proapoptotic peptide as potential therapy for glioblastoma

Lilach Agemy et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2014 Aug 12;111(32):11906

Abstract

Antiangiogenic therapy can produce transient tumor regression in glioblastoma (GBM), but no prolongation in patient survival has been achieved. We have constructed a nanosystem targeted to tumor vasculature that incorporates three elements: (i) a tumor-homing peptide that specifically delivers its payload to the mitochondria of tumor endothelial cells and tumor cells, (ii) conjugation of this homing peptide with a proapoptotic peptide that acts on mitochondria, and (iii) multivalent presentation on iron oxide nanoparticles, which enhances the proapoptotic activity. The iron oxide component of the nanoparticles enabled imaging of GBM tumors in mice. Systemic treatment of GBM-bearing mice with the nanoparticles eradicated most tumors in one GBM mouse model and significantly delayed tumor development in another. Coinjecting the nanoparticles with a tumor-penetrating peptide further enhanced the therapeutic effect. Both models used have proven completely resistant to other therapies, suggesting clinical potential of our nanosystem.

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Conflict of interest statement

Conflict of interest statement: E.R., V.R.K., and K.N.S. are shareholders in CendR Therapeutics Inc., which has rights to some of the technology described in this paper.

Figures

Fig. 1.
Fig. 1.
Homing of CGKRK peptide to GBM tumors and interaction of CGKRK peptide with mitochondria. (A) Mice bearing 005 glioma tumors in the right hippocampus were i.v. injected with 200 μg of CGKRK peptide labeled with Rd. After 3 h, the mice were perfused through the heart with PBS, and the tumor and normal brain tissue were collected. Inset, Right: Section of normal brain tissue. Red, CGKRK peptide; green, tumor cells; magenta, blood vessels; blue, nuclei. n = 3. (Scale bars, 200 μm.) (B) Proliferating human endothelial cells (resembling angiogenic endothelial cells) and U87 cells were incubated with FAM-CGKRK peptide (green) and MitoTracker (red) and examined by fluorescent microscopy. Yellow indicates colocalization. (C) FAM-CGKRK was incubated with purified mitochondria in the presence of increasing concentrations of either unlabeled CGKRK or an unrelated peptide (CREKA) as a control. Student t test (C). Error bars, mean ± SD; n.s., **P < 0.01; ***P < 0.001.
Fig. 2.
Fig. 2.
Homing of CGKRKD[KLAKLAK]2 NWs to GBM tumors. (A) General design of theranostic NWs. A chimeric peptide consisting of a tumor-homing peptide (CGKRK) and a proapoptotic peptide (D[KLAKLAK]2) is covalently coupled to iron oxide nanoparticles (NWs; length 80–100 nm, width 30 nm); FAM, carboxyfluorescein. (B) Iron oxide NWs coated with Rd-labeled CGKRKD[KLAKLAK]2 peptide through a 5KPEG linker were i.v. injected (5 mg iron per kg body weight) into mice bearing either 005 tumors or xenograft tumors generated with human GBM spheres or U87 cells. The tumor cells were injected into the right hippocampus. Five to six hours after the injection, the mice were perfused through the heart with PBS, and the organs were collected. Tumor sections were stained and examined by confocal microscopy. Red, CGKRKD[KLAKLAK]2-coated particles; green, tumor cells (both the human GBM spheres and U87 cells expressed green fluorescent protein); magenta, blood vessels stained with anti-CD31; blue, nuclei stained with DAPI. Arrows show representative blood vessels that accumulate CGKRKD[KLAKLAK]2-NW for each tumor model. (Scale bars, 100 μm.) (C) T2*-weighted MRI. Rd-labeled CGKRKD[KLAKLAK]2-NWs were i.v. injected into tumor-bearing mice. Grayscale images of axial planes through the tumors are shown. Gadolinium (Gd) and Feridex (Fe) were used as reference standards. n = 3–4.
Fig. 3.
Fig. 3.
D[KLAKLAK]2CGKRK-NW conjugates internalize into activated endothelial cells, colocalize with mitochondria, and induce cell death by apoptosis. (A) Confocal microscopic images of live HUVEC incubated for 2 h at 37 °C in the presence of fluorescein (FAM)-labeled NWs (green) and a marker for mitochondria (MitoTracker; red) for 15 min before the imaging. DNA was counterstained with Hoechst 33342 (blue). (Scale bars, 20 μm.) (B) FAM-CGKRKD[KLAKLAK]2 peptide was coupled onto the NWs via a reducible 5KPEG linker. The linker was cleaved from the NWs using DTT, and the amount of peptide present on the NWs was determined by fluorescence measurements in solution (to circumvent quenching on the NW surface) and used to calculate IC50. (C and D) HUVEC and T3 cells were left untreated (Control) or treated with a concentration of 10 μg/mL of NWs coated with either a control peptide (CREKA), D[KLAKLAK]2, or CGKRKD[KLAKLAK]2 for 24, 48, and 72 h (C), or the particles were washed away after 30 min and the incubation continued for 72 h (D). The cells were stained with annexin and analyzed by flow cytometry. The total percentage of annexin-positive cells (apoptotic and dead cells) is indicated. (E) Whole cell extracts (HUVEC) from the experiment in C were prepared and analyzed by immunoblotting using antibodies against cleaved caspase-3 and β-actin as loading control. (F) Confocal microscopy images of HUVEC treated with the indicated NWs and stained for cleaved caspase-3 (green), tubulin (red), and nuclei (blue). (Scale bars, 50 μm.)
Fig. 4.
Fig. 4.
CGKRKD[KLAKLAK]2-NW treatment of tumors induced by lentiviral injection. Mice bearing lentiviral (H-RasV12-shp53) induced brain tumors in the right hippocampus were i.v. injected with NW coated with peptides. The particles were administered every other day for 18 d, starting 3 wk after viral injection. (A) Survival curve of the nontreated and treated mice (n = 8–10 per group). (B) Mice were monitored for luciferase signal using the IVIS system (the lentiviral vector contains the luciferase reporter); one representative mouse from each of the indicated groups is shown. (C) H&E staining showing the lentiviral injection site in a representative control mouse and a mouse treated with CGKRK D[KLAKLAK]2-NWs at the end of treatment. (D) Confocal microscopy images of brain sections from a representative mouse at the end of the treatment with D[KLAKLAK]2-NWs (Di and Dii) and from a CGKRK D[KLAKLAK]2-NW–treated mouse that contained a small residual tumor (Diii and Div) (red); green, tumor cells. (Scale bars, 200 μm.)
Fig. 5.
Fig. 5.
Treatment of transplanted GBM tumors with CGKRKD[KLAKLAK]2-NWs. Tumors were developed by transplanting 3 × 105 005 cells into the right hippocampus of NOD-SCID mice. Ten days after tumor cell transplantation, the mice were i.v. injected with NWs. The NWs (5 mg iron/kg) were administered every other day for 3 wk or administered nonstop for the same period (n = 8 per group). (A) Survival curve of the treated mice. (B) Brain sections at the end of the treatment were stained: magenta, anti-CD31; red, CGKRKD[KLAKLAK]2-NWs or D[KLAKLAK]2-NWs; green, tumor cell; blue, DAPI. Arrows show representative blood vessels that accumulate CGKRKD[KLAKLAK]2-NW in the tumor. (Scale bars, 200 mm.) (C) Lectin perfusion of tumor mice at the end of the treatment. Vessels were stained by perfusion of biotinylated Lycopersicon esculentum lectin and visualized by confocal microscopy using anti-biotin. Green, tumor cells; red, perfused, lectin-labeled blood vessels; blue, nuclei (DAPI).
Fig. 6.
Fig. 6.
Enhanced antitumor effect of CGKRKD[KLAKLAK]2-NWs coinjected with iRGD. (A) Mice bearing orthotopic 005 tumors implanted 10 d earlier received every other day for 3 wk i.v. injections of either CGKRKD[KLAKLAK]2-NWs (5 mg iron/kg) or CGKRKD[KLAKLAK]2-NWs (5 mg/kg) mixed with 4 mmol/kg of iRGD or PBS. Survival curves are shown (n = 8–10 per group). (B) Mice bearing orthotopic 005 tumors were i.v. injected with CGKRKD[KLAKLAK]2-NW (5 mg iron/kg) in combination with 4 mmol/kg of either nonlabeled CRGDC (Upper) or iRGD (Lower) peptide. The tumors and tissues were collected 5–6 h later and analyzed by confocal microscopy. Red, CGKRKD[KLAKLAK]2-NWs; magenta, blood vessels; green, tumor cell; blue, DAPI. (Scale bars, 50 mm.)
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