Research ArticleBIOPHYSICS

Neuropilin-1 and heparan sulfate proteoglycans cooperate in cellular uptake of nanoparticles functionalized by cationic cell-penetrating peptides

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Science Advances  06 Nov 2015:
Vol. 1, no. 10, e1500821
DOI: 10.1126/sciadv.1500821
  • Fig. 1 Utilization of NRP1- and HS-dependent pathways by TAT variants.

    (A) PPC1 cells were incubated with NRP1 blocking antibody or soluble HS (100 μg/ml) before the addition of AgNPs coated with the indicated peptides to measure NP internalization. The quantity of internalized AgNPs as fluorescence intensity per cell was normalized to that of control (no NRP1 blockade or soluble HS) and plotted on the y axis. (B and C) PPC1 cells were incubated at different concentrations of soluble HS (0, 10, 100, and 1000 μg/ml; B) or NRP1 blocking antibody (0, 20, 50, and 200 μg/ml; C) before the addition of AgNPs coated with the indicated peptides. The quantity of internalized AgNPs as fluorescence intensity per cell was normalized to that of control [no soluble HS (B) or NRP1 blocking antibody (C)] and plotted on the y axis. (D) PPC1 cells were treated with control or NRP1 siRNA before the addition of AgNPs coated with the indicated peptides for internalization. The quantity of internalized AgNPs as fluorescence intensity per cell was normalized to that of control (control siRNA) and plotted on the y axis. (E and F) The binding of AgNPs coated with the indicated peptides to immobilized NRP1 (E) or HS (F) was quantified as described in Materials and Methods. The fluorescence intensity of bound AgNPs was normalized to the average value of the control peptide [AgNP-RPARPAR (E) or AgNP-TAT-NH2 (F)] and plotted on the y axis. All experiments were independently carried out at least three times. The error bars indicate the SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test (compared to the control group of the indicated peptide).

  • Fig. 2 NRP1 dependence of NP internalization into HS-deficient cells.

    (A) CHO pgs745 cells transfected with NRP1 expression vector were incubated with AgNPs coated with the indicated peptides (red) in regular culture medium. Extracellular NPs were dissolved by etching, and live cells were stained for surface NRP1 (green) and nuclei (blue). Representative immunofluorescence images are shown. Scale bars, 10 μm. (B) The Ag intensity per cell in NRP1-positive cells was quantified using ImageJ for samples from (A). The values were normalized to that of Ag-RPARPAR (y axis). Error bars, SEM. **P < 0.01 and ***P < 0.001, Student’s t test. At least three independent experiments were carried out for each condition.

  • Fig. 3 Utilization of NRP1 and HS pathways by Sema C-terminal peptide variants.

    (A and B) The binding of AgNPs coated with the indicated peptides to immobilized NRP1 (A) or HS (B) was quantified as described in Materials and Methods. The fluorescence intensity of bound AgNPs was normalized to the average value of AgNP-RPARPAR (A) or AgNP-TAT-NH2 (B) and plotted on the y axis. (C) Internalization of AgNPs coated with the indicated peptides into PPC1 cells preincubated with NRP1 blocking antibody or soluble HS (100 μg/ml). After etching to dissolve extracellular NPs, the fluorescence intensity of the internalized NPs per cell was normalized to that of control (no NRP1 blockade, no soluble HS) and plotted on the y axis. (D) PPC1 cells were incubated with AgNPs coated with the indicated peptides in the absence or presence of soluble HS (100 μg/ml). After etching, AgNP internalization per cell was quantified, normalized to that of AgNP-Sema-OH, and plotted on the y axis. At least three independent experiments were carried out for each condition. Error bars, SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test [compared to the control group of the indicated peptide in (C)].

  • Fig. 4 Vascular permeability induced by peptide-neutravidin complexes.

    (A and B) Skin vascular permeability assay was carried out with VEGF or neutravidin complexed with the indicated peptides as described in Materials and Methods. The 600-nm absorbance was normalized to that of PBS control and plotted on the y axis. At least three independent experiments were carried out for each condition. Error bars, SEM. ns, not significant; *P < 0.05 and **P < 0.01, Student’s t test.

  • Fig. 5 Synchronization of cell entry and stepwise transport of peptide-coated AuNPs.

    (A) PPC1–green fluorescent protein (GFP) cells (green) were incubated with AgNP-RPARPAR (red) in hypo-K+ buffer for 30 min at 37°C. The same cells were imaged for surface-bound Ag (before etch) and their internalization (after etch). Representative images are shown. (B) PPC1 cells were incubated with AuNP-d-TAT (~17 nm in diameter) in hypo-K+ buffer for 30 min at 37°C. After washing to remove unbound particles, the cells were incubated in regular culture medium for 30, 60, and 240 min before subcellular transport was stopped by fixation. Representative TEM images at each of these time points (30 min, left; 60 min, middle; 240 min, right) are shown to indicate the major intracellular compartments containing NPs. MVBs, multivesicular bodies. Scale bars, 200 nm. (C) PPC1 cells were incubated with AuNP-d-TAT (~17 nm in diameter) and AuNP-RPARPAR (~50 nm in diameter) and processed as in (B). Scale bars, 200 nm.

  • Table 1 Abbreviations and sequences (or chemical composition) of the peptides used in this study.

    PeptideSequence
    TAT-OHYGRKKRRQRRR-COOH
    TAT-NH2YGRKKRRQRRR-CONH2
    d-TATYGRKKRRQRRR-CONH2 (d–amino acid)
    RPARPARRPARPAR-COOH
    Sema-OHGNKKGRNRR-COOH
    Sema-NH2GNKKGRNRR-CONH2
    AA-Sema-OHGNAAGRNRR-COOH

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/10/e1500821/DC1

    Fig. S1. Use of NRP1- and HS-dependent pathways by TAT variants.

    Fig. S2. AgNP internalization into HS-deficient cells.

    Fig. S3. Synchronization of NP cell entry using the hypo-K+ buffer.

    Fig. S4. Peptide-AgNP binding to cell surface receptors.

    Fig. S5. Subcellular localization of peptide-Au complexes.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Use of NRP1- and HS-dependent pathways by TAT variants.
    • Fig. S2. AgNP internalization into HS-deficient cells.
    • Fig. S3. Synchronization of NP cell entry using the hypo-K+ buffer.
    • Fig. S4. Peptide-AgNP binding to cell surface receptors.
    • Fig. S5. Subcellular localization of peptide-Au complexes.

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