Research ArticleMATERIALS SCIENCE

Rapid transport of germ-mimetic nanoparticles with dual conformational polyethylene glycol chains in biological tissues

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Science Advances  07 Feb 2020:
Vol. 6, no. 6, eaay9937
DOI: 10.1126/sciadv.aay9937
  • Fig. 1 Characterization of NPs and detection of the anisotropy of NR.

    (A) Scanning electron microscopy (SEM) images of NR with a size of about 78 nm by 220 nm, SNS with a size of about 80 nm, and LNS with a size of about 140 nm. (B) Hydrodynamic diameter of NPs. (C) Zeta potentials of NPs. (D) Porous structures of NPs in high-resolution SEM images. Yellow arrows indicate the tip-specific porous structure on the NR. (E) Schematic illustration of detecting hydroxyl densities via the friction between the ─OH groups and COOH-modified AFM probe. (F) Height and adhesion mapping via AFM. Top left, NR body; top right, NR tip; bottom left, SNS; bottom right, LNS. The regions on the rod body showed significantly lower adhesion than the tip regions and the surface of the spherical NPs. (G) Quantification of hydroxyl groups on MSN via thermogravimetric analysis (TGA). The yellow and blue areas indicate the weight loss due to the dehydroxylation and condensation of the hydroxyl groups on the silica surface, respectively. (H) Adhesion force and the inferred hydroxyl density of different regions on MSN. Representative images are presented. Data are means ± standard error of mean. *P < 0.05 and ***P < 0.001, one-way analysis of variance (ANOVA) and Bonferroni’s test.

  • Fig. 2 Detection of HPEGC via AFM.

    (A) Non-PEGylated NPs showed limited adhesion force and apparently lacked a corona. (B) Compared to the non-PEGylated NPs, the SNS and NR with 2% PEGylation showed increased adhesion force resulting from the interactions between the probe and PEG chains. The negligible HPEGC indicated the mushroom structure of PEG. (C) With 4% PEGylation, the NPs showed a clear HPEGC. The SNS showed an isotropic corona, while the NR exhibited anisotropy, in which a thick corona could be detected at the tips and a thin corona was present on the rod body (as indicated by the arrows), structurally mimicking germs. (D) NPs with 6% PEG content showed a uniform, thick HPEGC around them. In particular, PEG brushes were observed under this condition on the rod body (as indicated by the arrow). (E) With a much higher PEG content of 20%, the HPEGC became more rigid. However, the corona could be observed again as the peak force was increased from 1.5 to 5.0 nN. Representative images are presented. Scale bar, 100 nm.

  • Fig. 3 In vitro functional evaluation of GMNP.

    (A) Ensemble-averaged MSD of NPs diffusing in freshly obtained rat intestinal mucus. (B) Effective diffusivities of NPs. Arrows indicate a rapid growth in diffusivity. (C) The negative impact of PEGylation on uptake by E12 cells with preremoval of mucus (n = 4). (D) Uptake by mucus-containing E12 cells that simulated the multiple barriers of intestinal mucosa (n = 4). (E) Three-dimensional (3D) view of the transport of NPs across multiple E12 barriers at different times. Scale bar, 15 μm. (F) Different cell entry patterns of NPs. The anisotropic GMNPs adhered to the membrane via their body side, while the isotropic NRs were wrapped through the tip first. MEM, membrane. Scale bar, 500 nm. (G) Schematic of the superior efficiency of GMNPs over their isotropic counterparts in traversing across multiple barriers. (H) Transportation of GMNPs and their isotropic counterparts with optimized 4% PEGylation in tumor spheroids. Scale bar, 100 μm. (I) Quantification of fluorescent coverage of NPs at different depths of spheroids. Representative images are presented. Data are means ± standard error of mean. ns, no significant difference; **P < 0.01 and ***P < 0.001, one-way ANOVA and Bonferroni’s test.

  • Fig. 4 In vivo transportation of NPs in mucosal and tumor tissues.

    (A) NP transportation through and retention in rat small intestines. (B) Intestinal uptake examined by CLSM after intragastric (i.g.) administration and quantification of relative fluorescent intensity. Scale bar, 300 μm. (C) Tumor permeation and retention effect of NPs in tumor-bearing nude mice. (D) Quantification of total radiant efficiency. *P < 0.05 and **P < 0.01 for comparison of GMNP and SNS and ##P < 0.01 for comparison of GMNP and LNS, one-way ANOVA and Bonferroni’s test. (E) Tumor slices examined under CLSM at 8 hours after peritumoral injection. Representative images are presented (n = 1 for saline control group and n = 3 for other groups). Scale bar, 200 μm.

  • Fig. 5 Mechanistic insights into the superiority of the anisotropic HPEGC.

    (A) Diffusion patterns of GMNP, SNS, and LNS in mucus under STED microscopy. As indicated by the yellow arrows, the position of the GMNP could suddenly change after being trapped by mucin for some time, displaying a strong hopping pattern. (B to E) Interactions of the HPEGC on different NPs with the cell membrane detected via AFM. Particles were modified onto the AFM probe, as shown by the SEM images. The average adhesive forces indicated that the body of the GMNP with mushroom-like PEG chains had much stronger interactions with the cell membrane than did the rod tip, SNS, and LNS. Data are means ± standard deviation.

  • Fig. 6 The diffusivity and internalization processes of different types of NRs from molecular simulation.

    Typical 3D centroid trajectories, MSD values, and endocytosis pathway of the NRs with high PEG density (A, D, and E), anisotropic PEG density (B, E, and H), and low PEG density (C, F, and I). In the simulation of the diffusivity of the NPs, αA and αB represent the interactions between the chain nodes and two tip regions and between the chain nodes and body region of the NRs, respectively. For the simulations of the internalization process, the effect of the PEG density was represented by the interaction between the NPs and chain network/membrane bilayer, where εA and εB are the interactions between the receptors and two tip regions and between the receptors and body region of the NRs, respectively. The green and blue dotted lines in (D to F) are the slopes of the plots of the MSD versus time and the time scale of the hopping, respectively. The insets in (G to I) are the stable states of the endocytosis of different NRs.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/6/eaay9937/DC1

    Fig. S1. Characterization of NPs.

    Fig. S2. Schematic illustration and detection of HPEGC of LNS.

    Fig. S3. Distribution of logarithmic effective diffusivities for NPs with various PEG degrees at a time scale of 1 s.

    Fig. S4. Additional in vitro data.

    Fig. S5. Superiority of GMNP for oral delivery.

    Fig. S6. Superior tumor permeation of GMNP compared with the NR-2%PEG and the NR-6%PEG.

    Fig. S7. Mechanistic studies.

    Fig. S8. Protein corona-related and biodistribution studies.

    Fig. S9. The molecular models used in the CGMD simulation.

    Fig. S10. Diffusion and internalization simulations.

    Movie S1. Diffusion pattern of GMNP in the mucus detected by STED.

    Movie S2. Diffusion pattern of SNS in the mucus detected by STED.

    Movie S3. Diffusion pattern of LNS in the mucus detected by STED.

    Movie S4. Diffusion pattern of NR-2%PEG in the mucus detected by STED.

    Movie S5. Diffusion pattern of NR-6%PEG in the mucus detected by STED.

    Movie S6. Simulation of NR with anisotropic PEG density diffusing in biological hydrogels.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. Characterization of NPs.
    • Fig. S2. Schematic illustration and detection of HPEGC of LNS.
    • Fig. S3. Distribution of logarithmic effective diffusivities for NPs with various PEG degrees at a time scale of 1 s.
    • Fig. S4. Additional in vitro data.
    • Fig. S5. Superiority of GMNP for oral delivery.
    • Fig. S6. Superior tumor permeation of GMNP compared with the NR-2%PEG and the NR-6%PEG.
    • Fig. S7. Mechanistic studies.
    • Fig. S8. Protein corona-related and biodistribution studies.
    • Fig. S9. The molecular models used in the CGMD simulation.
    • Fig. S10. Diffusion and internalization simulations.

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Diffusion pattern of GMNP in the mucus detected by STED.
    • Movie S2 (.avi format). Diffusion pattern of SNS in the mucus detected by STED.
    • Movie S3 (.avi format). Diffusion pattern of LNS in the mucus detected by STED.
    • Movie S4 (.avi format). Diffusion pattern of NR-2%PEG in the mucus detected by STED.
    • Movie S5 (.avi format). Diffusion pattern of NR-6%PEG in the mucus detected by STED.
    • Movie S6 (.gif format). Simulation of NR with anisotropic PEG density diffusing in biological hydrogels.

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