Research ArticleMEDICAL ROBOTS

Soft erythrocyte-based bacterial microswimmers for cargo delivery

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Science Robotics  25 Apr 2018:
Vol. 3, Issue 17, eaar4423
DOI: 10.1126/scirobotics.aar4423
  • Fig. 1 RBC microswimmers for active cargo delivery.

    (A) RBC microswimmers are composed of an RBC, loaded with drug molecules and SPIONs, bound to a motile bacterium (bioengineered E. coli MG1655) via biotin-avidin-biotin binding complex. (B) SEM image (pseudocolored red, RBC; pseudocolored green, bacterium) of an example RBC microswimmer with an attached bacterium. (C) Example 2D propulsion trajectories of RBC microswimmers over time. The inset displays a bacterium attached to an RBC loaded with DOX molecules. Scale bar, 5 μm.

  • Fig. 2 RBC microswimmers loaded with DOX molecules preserve their membrane proteins after hypotonic treatment.

    (A) Loading of DOX molecules and SPIONs is achieved via hypotonic dilution technique. (B) RBCs (i) loaded with DOX via hypotonic dilution method (ii) preserve their membrane expression of TER-119 (iii and iv). DIC, differential interference contrast. (C) Flow cytometry density plots obtained from RBCs (i) after hypotonic-isotonic treatment, (ii) DOX encapsulation, (iii) anti–TER-119 staining, and (iv) both anti–TER-119 staining and DOX encapsulation show successful encapsulation of DOX and TER-119 expression. a.u., arbitrary units. (D) Cumulative released percentage of DOX molecules over time (5 days) from RBC cargoes at pH = 3.1, 5.0, 7.2, and 9.2.

  • Fig. 3 2D motility characterization and external magnetic steering of RBC microswimmers.

    (A to D) Mean speed distribution and 2D swimming trajectories of (A and B) free bacteria and (C and D) bacteria-driven RBC microswimmers, respectively. Free bacteria displayed a mean speed of 19.5 ± 9.2 μm s−1, whereas the mean speed of RBC microswimmers was 10.2 ± 3.5 μm s−1. (E) Magnetic steering of an RBC microswimmer and change in swimming direction upon change in magnetic field direction (i to iii). Red arrows indicate the direction of the magnetic field (B). The inset shows the five-coil setup used for magnetic steering of the RBC microswimmers. Scale bars, 10 μm.

  • Fig. 4 Deformability and stability of RBC microswimmers squeezing through confined spaces smaller than their size.

    (A) RBC microswimmers, fluorescently labeled, were able to squeeze with ease through microchannels with a width of 3 μm, which is smaller than the size of RBCs (4 to 6 μm). RBCs moving through a single channel over a time period of 0.56 s (i to iii) were highlighted with red circles. (B and C) RBC microswimmers preserved their integrity when (B) deformed inside the microchannels and (C) collected at the outlet. White arrows indicate intact RBC microswimmers, where bacteria preserved their attachment to RBCs. (D) Change in percentage of RBC microswimmers after squeezing through microfluidic channels was not statistically significant (Mann-Whitney test, P > 0.05). Error bars represent the SD. n.s., not significant.

  • Fig. 5 Active deformation of an RBC cargo propelled and pushed by a single bacterium through a 2-μm gap.

    RBC microswimmer approaches to a gap formed by two adjacent micropillars (i), bacterium pushes the RBC to deform within the microgap (ii and iii), and the RBC microswimmer moves out of the microgap while preserving its stability and motility (iv). Scale bars, 5 μm.

  • Fig. 6 On-demand, NIR light–triggered hyperthermia termination switch for RBC microswimmers.

    (A) Schematic RBC microswimmers, loaded with a photothermal agent (ICG) coupled with BSA, generating heat upon irradiation with NIR and resulting in termination of bacteria. (B) IR thermal images of RBC microswimmers loaded inside a capillary tube before and during NIR irradiation. (C) Live/dead staining of bacteria in RBC microswimmer samples before (i) and after (ii) NIR irradiation, respectively. Scale bars, 5 μm. (D and E) Quantitative measurements of (D) number of intact RBCs and (E) percent viable bacteria before and after NIR-activated hyperthermia termination, respectively. Error bars represent the SD.

Supplementary Materials

  • robotics.sciencemag.org/cgi/content/full/3/17/eaar4423/DC1

    Fig. S1. Bioengineered E. coli MG1655 for biotin attachment peptide expression of the cell membrane.

    Fig. S2. Characterization of TER-119 antigen presence on mouse RBCs and binding of biotin-conjugated TER-119 antibodies for streptavidin modification.

    Fig. S3. Bioengineered E. coli MG1655 attachment to DOX-loaded RBCs.

    Fig. S4. Fluorescence microscopy characterization of DOX loading into RBCs.

    Fig. S5. Flow cytometry density plots for RBCs without hypotonic isotonic treatment.

    Fig. S6. Flow cytometry population selection for RBCs.

    Fig. S7. SPION-loaded RBCs are attracted to a permanent magnet.

    Fig. S8. Characterization of the loading of citric acid–coated SPIONs into RBCs using EDS.

    Fig. S9. Photo of the custom five electromagnetic coils mounted to an inverted optical microscope and used for the magnetic guidance of RBC microswimmers.

    Fig. S10. FITC-labeled RBC microswimmers are shown at the entrance of a microchannel.

    Fig. S11. Deformation of FITC-labeled RBC microswimmers inside a microchannel with the attached bacterium.

    Movie S1. Bacteria-driven RBC microswimmers.

    Movie S2. Magnetic steering of bacteria-driven RBC microswimmers.

    Movie S3. Passive deformation of RBC microswimmers in microchannels.

    Movie S4. Active deformation of an RBC cargo propelled by single bacterium.

  • Supplementary Materials

    Supplementary Material for:

    Soft erythrocyte-based bacterial microswimmers for cargo delivery

    Yunus Alapan, Oncay Yasa, Oliver Schauer, Joshua Giltinan, Ahmet F. Tabak, Victor Sourjik, Metin Sitti*

    *Corresponding author. Email: sitti{at}is.mpg.de

    Published 25 April 2018, Sci. Robot. 3, eaar4423 (2018)
    DOI: 10.1126/scirobotics.aar4423

    This PDF file includes:

    • Fig. S1. Bioengineered E. coli MG1655 for biotin attachment peptide expression of the cell membrane.
    • Fig. S2. Characterization of TER-119 antigen presence on mouse RBCs and binding of biotin-conjugated TER-119 antibodies for streptavidin modification.
    • Fig. S3. Bioengineered E. coli MG1655 attachment to DOX-loaded RBCs.
    • Fig. S4. Fluorescence microscopy characterization of DOX loading into RBCs.
    • Fig. S5. Flow cytometry density plots for RBCs without hypotonic isotonic treatment.
    • Fig. S6. Flow cytometry population selection for RBCs.
    • Fig. S7. SPION-loaded RBCs are attracted to a permanent magnet.
    • Fig. S8. Characterization of the loading of citric acid–coated SPIONs into RBCs using EDS.
    • Fig. S9. Photo of the custom five electromagnetic coils mounted to an inverted optical microscope and used for the magnetic guidance of RBC microswimmers.
    • Fig. S10. FITC-labeled RBC microswimmers are shown at the entrance of a microchannel.
    • Fig. S11. Deformation of FITC-labeled RBC microswimmers inside a microchannel with the attached bacterium.
    • Legends for movies S1 to S4

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Bacteria-driven RBC microswimmers.
    • Movie S2 (.mp4 format). Magnetic steering of bacteria-driven RBC microswimmers.
    • Movie S3 (.mp4 format). Passive deformation of RBC microswimmers in microchannels.
    • Movie S4 (.mp4 format). Active deformation of an RBC cargo propelled by single bacterium.

    Files in this Data Supplement:

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