Research ArticleBIOMEDICAL ENGINEERING

Chemotactic synthetic vesicles: Design and applications in blood-brain barrier crossing

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Science Advances  02 Aug 2017:
Vol. 3, no. 8, e1700362
DOI: 10.1126/sciadv.1700362
  • Fig. 1 Asymmetric polymersomes.

    (A) Schematic representation of a chemotactic polymersome using a combination of membrane topology formed by PEO-PBO copolymers mixed with either PMPC-PDPA or POEGMA-PDPA copolymers. The polymersomes encapsulate glucose oxidase and/or catalase enzymes. (B) 9:1 PMPC-PDPA/PEO-PBO polymersome imaged in positive staining exploiting the high affinity of PDPA with the staining agent phosphotungstic acid (PTA). (C) 9:1 POEGMA-PDPA/PEO-PBO polymersome imaged in the same staining agent for PDPA. (D) 9:1 PMPC-PDPA/PEO-PBO polymersome imaged in negative staining to highlight the differences in membrane thickness between the PDPA and the PBO membrane.

  • Fig. 2 Active diffusion studies.

    Normalized 1s-trajectories and corresponding MSDs for (A) symmetric PMPC-PDPA polymersomes loaded with glucose oxidase (Gox) and catalase (Cat) and responding to a glucose gradient (B) asymmetric PMPC-PDPA/PEO-PBO polymersomes loaded with catalase and responding to a hydrogen peroxide gradient, (C) loaded with glucose oxidase and responding to a glucose gradient, (D to E) loaded with glucose oxidase and catalase responding to a glucose gradient coming (D) from the right-hand side and (E) from the left-hand side and for (F) asymmetric POEGMA-PDPA/PEO-PBO polymersomes loaded with glucose oxidase and catalase responding to a glucose gradient coming from the right-hand side. Blue arrows indicate the direction of the substrate gradient. Scale bars, 20 μm. (G) The average drift velocity is plotted as a function of time after the substrate addition for the previous experiments. The error bars represents the SE calculated over n = 3 measurements. (H) Degree of polarization of the corresponding trajectories towards the chemical gradient plotted as percentage of particles versus the gradient angle. Perfect alignment with the gradient corresponds to θ = 0°. The dashed lines represent the SEs.

  • Fig. 3 Mechanism of chemotaxis.

    (A) Schematics of an asymmetric polymersome and its reference axis. We assumed the polymersome to be a sphere (R = 50 nm) with a smaller patch (r = 15 nm and sector angle α); the angle β represents the orientation of the unit vector n with respect to the chemical gradient ∇C here aligned to the x axis. We simulated the distribution of the products around the polymersome, and their normalized concentration is plotted (red line) alongside a fitting function ΔCp = A(cos(β/2))nint(2π/α) (blue line). n.u., normalized units. (B) Average MSDs for both experimental (circles) and simulated data (solid line) for asymmetric polymersomes loaded with Gox and Cat responding to a glucose gradient (purple line and data) or in PBS (orange line and data), loaded with Gox and responding to a glucose gradient (blue line and data), and loaded with Cat responding to a hydrogen peroxide gradient (red line and data). (C) Corresponding propulsion velocities calculated by the numerical fittings for the three different combinations of enzymes and substrates. The lines represent the average values, whereas the bars represent the range of minimum and maximum calculated velocity in the sample. (D) A total of 20 simulated trajectories of Gox + Cat–loaded polymersomes using the same temporal steps as in the experiments (30 fps). These are shown as a 3D axonometric projection view and in the corresponding xy plane to show the comparison with the experimental data. (E) A single simulated 3D trajectory shown with temporal steps of 33 ms (blue line) and 33 μs (orange line). The detail of a single trajectory is zoomed to show the succession of reorientation and running steps of the polymersome diffusion. (F) Schematics of the proposed mechanisms of asymmetric polymersome chemotaxis, which consists of an alternation of running and reorientation events.

  • Fig. 4 Collective chemotaxis.

    (A) Schematic of a petri dish where a cylindrical agarose gel soaked in glucose is placed. At time t = 0, a 1 mg ml−1 concentration of polymersomes is added in the dish center, and their concentration is sampled at different locations as indicated by the sampling map in (B). The dot labeled with “S” indicates the position of the source of glucose. (C to E) The resulting maps show the 2D distribution of asymmetric polymersomes (C) at time t = 0, and the distribution of polymersomes at time t = 10 min for (D) symmetrical PMPC-PDPA and (E) asymmetrical PMPC-PDPA/PEO-PBO polymersomes loaded with catalase and glucose oxidase. The isocratic white lines show the glucose gradient calculated by computational fluid dynamics. (F) A similar experiment is performed by adding glucose in the center of a petri dish containing fluorescently labeled polymersomes after they have thermalized in it. The imaging is performed with a fluorescence camera. (G) The corresponding fluorescence images are shown for both symmetric PMPC-PDPA and asymmetric PMPC-PDPA/PEO-PBO polymersomes loaded with catalase and glucose oxidase at different times: before the addition of glucose, at times t = 0, 10, and 15 min. The black bar indicates the needle for the injection of glucose over the imaging camera.

  • Fig. 5 Chemotaxis under flow and in vivo.

    (A) Normalized polymersome 1-s trajectories measured in the presence of steady-state flow (0, 0.5, and 3.5 μm s−1) and collected before and 1, 5, and 20 min after the glucose gradient addition for both PMPC-PDPA/PEO-PBO asymmetric and PMPC-PDPA symmetric polymersomes loaded with glucose oxidase and catalase. Red arrows denote the direction of the flow within the observation area, whereas the blue arrows denote the average direction of the glucose gradient within it. Scale bars, 20 μm. Pe, Péclet number. (B) Streamlines of flow observed in a capillary with a radius of 4 μm and a length of 800 μm calculated by computational fluid dynamics. The red cylinders represent erythrocytes (hematocrit H% = 10.7%), and the color map shows the normal velocity of the flow, that is, the component perpendicular to the vessel walls. (C) Simulated percentage of the total number of particles bound to the vessel surface as a function of their drift velocity in a gradient for 50-, 100-, and 250-nm asymmetric nanoparticles calculated with an agent-based model of chemotactic particles within a capillary such as in (B). Note that the error bars show the SE. (D) Frequency distribution of the crossing time from apical to basolateral of LA–POEGMA-PDPA polymersomes measured over 35 different measurements using the in vitro BBB model as shown in fig. S18 (note that one example measurement is shown in fig. S19). (E) Percentage of the injected dose found in the rat brain parenchyma and the capillary fraction 10 min after carotid artery in situ perfusion of LA–POEGMA-PDPA/PBO asymmetric polymersomes loaded with Gox + Cat and empty and LA–POEGMA-PDPA symmetric polymersomes loaded with Gox + Cat and empty, as well as pristine asymmetric POEGMA-PDPA/PEO-PBO polymersomes loaded with Gox and Cat (n = 6; ***P < 0.001 and ****P < 0.0001). The error bars show the SE. n.s., not significant. (F) Immunofluorescence histologies of rat hippocampus sections of animals treated with LA–POEGMA-PDPA/PBO asymmetric polymersomes loaded with Gox + Cat and pristine asymmetric POEGMA-PDPA/PEO-PBO polymersomes loaded with Gox and Cat.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/8/e1700362/DC1

    fig. S1. Number of encapsulated enzyme and polymersome size distribution.

    fig. S2. Schematic representation of the observation NanoSight chamber.

    fig. S3. Asymmetric polymersomes loaded with catalase in the presence of homogeneously dissolved hydrogen peroxide.

    fig. S4. Asymmetric polymersomes loaded with glucose oxidase in the presence of homogeneously dissolved glucose.

    fig. S5. Asymmetric polymersomes loaded with catalase and glucose oxidase in the presence of homogeneously dissolved glucose.

    fig. S6. Symmetric polymersomes loaded with catalase and glucose oxidase after the injection of PBS (that is, no gradient).

    fig. S7. Asymmetric empty polymersomes after the injection of PBS (that is, no gradient).

    fig. S8. Asymmetric polymersomes loaded with catalase and glucose oxidase after the injection of PBS (that is, no gradient).

    fig. S9. Symmetric PMPC-PDPA polymersomes loaded with catalase and glucose oxidase in the presence of a glucose gradient.

    fig. S10. Symmetric PEO-PBO polymersomes loaded with catalase and glucose oxidase in the presence of a glucose gradient.

    fig. S11. Asymmetric empty polymersomes in the presence of a glucose gradient.

    fig. S12. Asymmetric empty polymersomes in the presence of a hydrogen peroxide gradient.

    fig. S13. Asymmetric polymersomes loaded with catalase in the presence of a hydrogen peroxide gradient.

    fig. S14. Asymmetric polymersomes loaded with glucose oxidase in the presence of a glucose gradient.

    fig. S15. Asymmetric polymersomes loaded with catalase and glucose oxidase in the presence of a glucose gradient.

    fig. S16. Asymmetric PEO-PBO polymersomes loaded with catalase and glucose oxidase in the presence of a glucose gradient.

    fig. S17. Polymersome concentration in long-range chemotaxis.

    fig. S18. BBB in vitro model for polymersome qualification.

    fig. S19. Live cell imaging of LA polymersome real-time transcytosis.

    table S1. Diffusion simulation parameters.

    note S1. An agent-based model of nanoparticle propulsion in a capillary.

    note S2. Long-range chemotaxis.

    note S3. Simulation model and propulsion velocity fitting.

    note S4. Diffusion simulations.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Number of encapsulated enzyme and polymersome size distribution.
    • fig. S2. Schematic representation of the observation NanoSight chamber.
    • fig. S3. Asymmetric polymersomes loaded with catalase in the presence of
      homogeneously dissolved hydrogen peroxide.
    • fig. S4. Asymmetric polymersomes loaded with glucose oxidase in the presence
      of homogeneously dissolved glucose.
    • fig. S5. Asymmetric polymersomes loaded with catalase and glucose oxidase in
      the presence of homogeneously dissolved glucose.
    • fig. S6. Symmetric polymersomes loaded with catalase and glucose oxidase after
      the injection of PBS (that is, no gradient).
    • fig. S7. Asymmetric empty polymersomes after the injection of PBS (that is, no
      gradient).
    • fig. S8. Asymmetric polymersomes loaded with catalase and glucose oxidase after
      the injection of PBS (that is, no gradient).
    • fig. S9. Symmetric PMPC-PDPA polymersomes loaded with catalase and glucose
      oxidase in the presence of a glucose gradient.
    • fig. S10. Symmetric PEO-PBO polymersomes loaded with catalase and glucose
      oxidase in the presence of a glucose gradient.
    • fig. S11. Asymmetric empty polymersomes in the presence of a glucose gradient.
    • fig. S12. Asymmetric empty polymersomes in the presence of a hydrogen
      peroxide gradient.
    • fig. S13. Asymmetric polymersomes loaded with catalase in the presence of a
      hydrogen peroxide gradient.
    • fig. S14. Asymmetric polymersomes loaded with glucose oxidase in the presence
      of a glucose gradient.
    • fig. S15. Asymmetric polymersomes loaded with catalase and glucose oxidase in
      the presence of a glucose gradient.
    • fig. S16. Asymmetric PEO-PBO polymersomes loaded with catalase and glucose
      oxidase in the presence of a glucose gradient.
    • fig. S17. Polymersome concentration in long-range chemotaxis.
    • fig. S18. BBB in vitro model for polymersome qualification.
    • fig. S19. Live cell imaging of LA polymersome real-time transcytosis.
    • table S1. Diffusion simulation parameters.
    • note S1. An agent-based model of nanoparticle propulsion in a capillary.
    • note S2. Long-range chemotaxis.
    • note S3. Simulation model and propulsion velocity fitting.
    • note S4. Diffusion simulations.

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