Research ArticleHEALTH AND MEDICINE

Tumor-homing peptides as tools for targeted delivery of payloads to the placenta

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Science Advances  06 May 2016:
Vol. 2, no. 5, e1600349
DOI: 10.1126/sciadv.1600349
  • Fig. 1 Tumor-homing sequences CGKRK and iRGD target the mouse placenta.

    (A) Pregnant mice (n = 3 per group) were intravenously injected with phage bearing the surface peptides CGKRK or iRGD or the control sequence G7 (1.5 × 1010 colony-forming units per mouse). After 30 min, mice were subjected to cardiac perfusion; phage were recovered from individual organs and quantified; results are expressed as fold titers relative to those of the control sequence G7. (B to J) Synthetic peptides (200 μg) were injected into the tail vein of pregnant mice. After 3 hours, mice were subjected to cardiac perfusion to remove unbound peptide. Placentas were collected, fixed, and frozen; evidence of peptide binding was assessed by fluorescence microscopy. n = 3 placentas were examined from each of n = 4 pregnant mice. Representative images are shown. (B to D) FAM-CGKRK. (E to G) FAM-iRGD. (H to J) FAM-ARA (control). Green, FAM-labeled peptides; blue, 4′,6-diamidino-2-phenylindole (DAPI; nuclei). JZ, junctional zone; Lab, labyrinth; SA, spiral artery.

  • Fig. 2 Administration of tumor-homing peptides does not alter reproductive outcome.

    Pregnant mice were intravenously injected with PBS (100 μl), acetyl (Ac)–iRGD, or Ac-CGKRK (100 μg) at E11.5, E13.5, and E15.5. (A to F) Mice were sacrificed at E18.5, and the following variables were measured: number of fetuses per litter (A), number of resorptions per litter (B), fetal weight (C), placental weight (D), fetal/placental (F/P) weight ratio (E), and percent increase in maternal body weight from E10.5 to E18.5 (F). Data points represent mean value per litter; horizontal line represents median. **P < 0.01, Kruskal-Wallis test. PBS (n = 9), iRGD (n = 9), CGKRK (n = 10).

  • Fig. 3 Tumor-homing peptides accumulate in the syncytium of human placental explants.

    (A to F) First-trimester (A, C, and E) or term placental explants (B, D, and F) were incubated with peptide (20 μM) for 0 to 3 hours. For pulse-chase (P/C) experiments, explants were incubated with peptide for 3 hours, then transferred to fresh medium, and cultured for a further 21 hours. Binding and uptake were assessed by fluorescence microscopy (n = 3). (A and B) FAM-CGKRK. (C and D) FAM-iRGD. (E and F) FAM-ARA. Green, FAM-labeled peptides. VS, villous stroma. Scale bars, 50 μm. (G and H) First-trimester placental explants were serum-starved for 24 hours and then incubated with vehicle control (PBS), Ac-CGKRK, or Ac-iRGD (20 μM). CTB proliferation and apoptosis were quantified at 24 and 48 hours, respectively, by immunostaining for Ki67 and M30, respectively. Median (n = 4 placentas). *P < 0.05, Kruskal-Wallis test.

  • Fig. 4 CGKRK binds to membrane-associated calreticulin.

    Mouse placental homogenates were applied to a chromatography column containing immobilized CGKRK peptide. Bound proteins were eluted with excess CGKRK. (A) Immunoblot of sequential column eluent fractions. (B to G) Immunostaining of mouse placenta (B to D), human first-trimester placenta (E and G), or human term placenta (F) for calreticulin (B, C, E, and F) or control immunoglobulin G (IgG) (D and G). Dec, decidua. Scale bars, 50 μm (representative images, n = 4). (H) Affinity of CGKRK for recombinant calreticulin. Binding of increasing concentrations of FAM-CGKRK to immobilized calreticulin was measured and normalized to nonspecific peptide binding. Mean ± SEM (n = 4). (I) Human term placental explants cultured with nontargeting (NT) siRNA or calreticulin-specific siRNA sequences; following calreticulin knockdown, explants were incubated with FAM-CGKRK (20 μM; 30 min). Fluorescence of tissue supernatants was quantified. Median (n = 4). (J) Human first-trimester placenta incubated with FAM-CGKRK (20 μM; 30 min; green) and immunostained with an antibody to calreticulin (red). Blue, DAPI (nuclei). Scale bars, 10 μm; n = 4. (K) MVM vesicles labeled with calreticulin antibody (green) or control IgG (black); binding was quantified by flow cytometry (representative histogram, n = 4). (L) MVM vesicles incubated with FAM-CGKRK (20 μM) and bovine serum albumin (BSA) (control; 10 μg) or recombinant human calreticulin (10 μg) for 30 min. FAM-CGKRK binding was quantified by flow cytometry. Median (n = 4).

  • Fig. 5 Tumor-homing peptides facilitate delivery of iron oxide nanoworms to the mouse placenta.

    Peptide-coated iron oxide nanoworms (5 mg of iron/kg body weight) were injected into the tail vein of pregnant mice. After 3 hours, mice were subjected to cardiac perfusion to remove unbound nanoworms. Placentas were collected, fixed, and frozen; evidence of nanoworm binding was assessed by confocal microscopy. Representative images are shown. (A to C) FAM-CGKRK (n = 3 placentas from n = 4 mice). (D to F) FAM-iRGD (n = 3 placentas from n = 3 mice). (G to I) FAM-ARA (n = 3 placentas from n = 3 mice). Green, FAM-labeled nanoworms; blue, DAPI (nuclei). Scale bar, 50 μm.

  • Fig. 6 Liposomes decorated with tumor-homing peptides facilitate targeted delivery of CF to the placenta.

    (A) Size distribution (in nanometer) of peptide-decorated liposomes. (B to J) Peptide-decorated liposomes (red) containing CF (green) were injected (100 μl per mouse) into the tail vein of pregnant mice at E12.5. After 24 hours, mice were subjected to cardiac perfusion to remove unbound liposomes. Placentas were collected, fixed, and frozen; evidence of liposome binding was assessed by confocal microscopy. n = 3 placentas from n = 3 mice were examined. Representative confocal images are shown. (B to D) TAMRA-CGKRK. (E to G) Rhodamine-iRGD. (H to J) TAMRA-ARA. Red, peptide; green, CF cargo; blue, DAPI (nuclei). Scale bars, 50 μm. (K to M) Term placental explants were incubated with peptide-decorated liposomes (100 μl) for 24 hours. Binding and uptake were assessed by fluorescence microscopy (n = 3). (K) TAMRA-CGKRK (n = 3). (L) Rhodamine-iRGD (n = 3). (M) TAMRA-ARA (n = 3). Red, peptide; green, CF cargo; blue, DAPI (nuclei). Scale bars, 50 μm. AU, arbitrary units.

  • Fig. 7 Targeted delivery of IGF-2 increases placental weight in wild-type C57BL/6J mice.

    Pregnant mice (N = dams, n = fetuses) were intravenously injected with 100 μl of PBS (N = 8, n = 59), plain (undecorated) liposomes (N = 8, n = 49), empty ARA–decorated liposomes (N = 8, n = 53), iRGD-decorated liposomes (N = 8, n = 56), CGKRK-decorated liposomes (N = 9, n = 62), free IGF-2 (1 mg/kg maternal body weight; N = 8, n = 43), plain liposomes (N = 9, n = 68), ARA liposomes (N = 8, n = 45), or iRGD liposomes (N = 8, n = 43) containing IGF-2 (approximately 0.3 mg/kg maternal body weight) on E11.5, E13.5, E15.5, and E17.5. (A and B) Mice were sacrificed at E18.5; fetal (A) and placental (B) weights were measured. Data points represent individual conceptuses; horizontal line represents median. (A) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to animals treated with iRGD liposomes containing IGF-2; (B) **P < 0.01, ***P < 0.001 compared to animals treated with plain liposomes; Kruskal-Wallis test with Dunn’s multiple comparison post hoc test.

  • Fig. 8 Targeted delivery of IGF-2 improves fetal weight distribution in the P0 mouse model of FGR.

    Pregnant mice (N = dams, n = fetuses) were injected on E11.5, E13.5, E15.5, and E17.5 with 100 μl of free IGF-2 (1 mg/kg maternal body weight; N = 8, n = 53) or iRGD-decorated liposomes containing IGF-2 (approximately 0.3 mg/kg maternal body weight; N = 8, n = 60), or were untreated/PBS-injected controls (N = 8, n = 56). (A to C) Mice were sacrificed at E18.5; placental (A) and fetal (B) weights were measured, and a fetal weight population distribution curve was plotted (C). Data points in (A) and (B) represent individual conceptuses; horizontal lines represents mean (A) or median (B); horizontal dashed lines represent mean/median value of the wild-type (WT) and P0 controls. Vertical dashed line in (C) represents the fifth weight centile (in milligram) of control WT mice. P0 fetal weights were significantly increased in mice treated with iRGD-decorated IGF-2 liposomes compared to controls or mice treated with free IGF-2 (P < 0.05); weight variation was significantly reduced (P < 0.05, F test of equality of variances). Data obtained from PBS-injected mice (n = 3) were combined with those from untreated animals (n = 5) because mean fetal and placental weights were not significantly different.

  • Table 1 Peptides identified by MALDI-TOF mass spectrometry.
    ProteinGene IDTotal number of peptides identifiedNumber of unique peptides identified
    CalreticulinCalr214
    Aspartyl aminopeptidase ADNPEP32
    Proteasome subunit β type-6PSMB632
    Protein disulfide isomerase 6Pdia662
    Triosephosphate isomeraseTpi133

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/5/e1600349/DC1

    fig. S1. The tumor-homing peptides FAM-CGKRK and FAM-iRGD do not accumulate in maternal organs of pregnant mice.

    fig. S2. FAM-CGKRK and FAM-iRGD colocalize with endothelial cell and trophoblast markers in the mouse placenta.

    fig. S3. FAM-CGKRK and FAM-iRGD colocalize with markers of the mouse placental labyrinth.

    fig. S4. Administration of placental homing peptides does not alter cell turnover in the developing mouse placenta.

    fig. S5. FAM-CGKRK and FAM-iRGD colocalize with markers villous trophoblast.

    fig. S6. FAM-iRGD colocalizes with αV integrin in mouse decidual spiral arteries.

    fig. S7. Calreticulin knockdown in the STB layer of human term placental explants.

    fig. S8. Biodistribution of tumor-homing peptide–decorated nanoworms in pregnant mice.

    fig. S9. Biodistribution of tumor-homing peptide–decorated liposomes in pregnant mice after 24 hours.

    fig. S10. Biodistribution of tumor-homing peptide–decorated liposomes in pregnant mice after 72 hours.

    fig. S11. Biodistribution of liposomes decorated with a control peptide in pregnant mice.

    fig. S12. Biodistribution of plain liposomes (no targeting peptide) in pregnant mice.

    fig. S13. Targeted delivery of IGF-2 to wild-type C57BL/6J mice does not alter litter size, resorption number, or weight of maternal clearance organs.

    fig. S14. Targeted delivery of IGF-2 to P0 mice does not alter litter size, resorption number, or weight of maternal clearance organs.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. The tumor-homing peptides FAM-CGKRK and FAM-iRGD do not accumulate in maternal organs of pregnant mice.
    • fig. S2. FAM-CGKRK and FAM-iRGD colocalize with endothelial cell and trophoblast markers in the mouse placenta.
    • fig. S3. FAM-CGKRK and FAM-iRGD colocalize with markers of the mouse placental labyrinth.
    • fig. S4. Administration of placental homing peptides does not alter cell turnover in the developing mouse placenta.
    • fig. S5. FAM-CGKRK and FAM-iRGD colocalize with markers villous trophoblast.
    • fig. S6. FAM-iRGD colocalizes with αV integrin in mouse decidual spiral arteries.
    • fig. S7. Calreticulin knockdown in the STB layer of human term placental explants.
    • fig. S8. Biodistribution of tumor-homing peptide–decorated nanoworms in pregnant mice.
    • fig. S9. Biodistribution of tumor-homing peptide–decorated liposomes in pregnant mice after 24 hours.
    • fig. S10. Biodistribution of tumor-homing peptide–decorated liposomes in pregnant mice after 72 hours.
    • fig. S11. Biodistribution of liposomes decorated with a control peptide in pregnant mice.
    • fig. S12. Biodistribution of plain liposomes (no targeting peptide) in pregnant mice.
    • fig. S13. Targeted delivery of IGF-2 to wild-type C57BL/6J mice does not alter litter size, resorption number, or weight of maternal clearance organs.
    • fig. S14. Targeted delivery of IGF-2 to P0 mice does not alter litter size, resorption number, or weight of maternal clearance organs.

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