Research ArticleHEALTH AND MEDICINE

Surface-anchored framework for generating RhD-epitope stealth red blood cells

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Science Advances  20 Mar 2020:
Vol. 6, no. 12, eaaw9679
DOI: 10.1126/sciadv.aaw9679
  • Fig. 1 Schematic illustration of the procedure for RBC surface engineering and the derived functions of individual cell-material hybrids in universal blood transfusions.

  • Fig. 2 Rational surface crosslinking framework for the construction of universal RBCs.

    (A) Schematic illustration of the procedure for RBC surface engineering. (B) Synthetic scheme for the anchor molecule conjugate (BAM-HRP). (C) Mechanism of the formation of PSA-tyramine conjugate and enzymatically crosslinked framework. SEM images of (D) human native RBCs and (G) surface-engineered human RBCs in the presence of BAM-HRP solution, PSA-tyramine (10 mg/liter), and H2O2 solution. TEM images of (E) human native RBCs and (H) surface-engineered human RBCs. Confocal images of (F) native RBCs and (I) engineered RBCs. (J) Optical images of human RhD-positive RBCs in the corresponding anti-typing sera. (K) BAM-tyramine-PSA engineered RhD-positive RBCs were mixed with equal concentrations of their anti-typing sera. (L) Flow cytometry analysis of native RBCs (left) and surface-engineered human RBCs (right) with fluorescein isothiocyanate–labeled anti-RhD antibodies.

  • Fig. 3 Physiochemical properties and biofunctional analysis of the native and engineered RBCs.

    (A) Osmotic fragility at 1 hour after the grafting of native RBCs (black) and RBCs derivatized with tyramine-PSA (10 mg/ml) (red). (B) Hydrophobicity tests based on the contact angle of the cell monolayer. (C) Cell electrophoresis assay characterized by zeta potential. (D) Deformability of the RBCs tested by ektacytometry. (E to H) Structural and functional analysis of the native RBCs (black) and engineered RBCs (red), including the ODC and the contents of Hill plot, 2,3-DPG, and ATP. (I to K) AI, AMP, and t1/2 of native RBCs, engineered RBCs, and mixtures of native and engineered RBCs in human plasma. (L) Comparison of the adhesions of the native RBCs, engineered RBCs, and mixtures of native and engineered RBCs to an endothelial cell–coated surface. a.u arbitary unit

  • Fig. 4 Usable and functional in vivo assay of the distribution of engineered RBCs after blood transfusion in organs in a mouse model.

    (A to D) Photoacoustic images of a mouse’s heart, liver, spleen, and kidney after injecting native and engineered RBCs labeled with DIR dye. Red, oxyhemoglobin; blue, deoxyhemoglobin; green, DIR dye. (E) Quantitative intensities of native and engineered RBCs labeled with DIR dye in the liver, spleen, and kidney after injection. (F) Quantitative intensity of oxyhemoglobin in the liver, spleen, and kidney after injection. (G) Survival profiles of PKH-26–labeled RBCs after blood transfusion in vivo.

  • Fig. 5 Complement and cytokine assay and biochemical analysis of organ functions following multiple transfusions.

    (A) Timeline of the three succeeding blood transfusions. (B) Life span of PKH-26–labeled native and engineered RBCs after blood transfusion in vivo. (C) Serum levels of complement 3 and complement 4 after multiple transfusions. (D) Serum levels of TNF-α and IL-6 after multiple transfusions. (E) Biochemical analysis of liver function after multiple transfusions. ALT (U/l), AST (U/l), ALP (U/l), TBIL (μM), DBIL (μM). (F) Biochemical analysis of kidney function after multiple transfusions. CREA (μM), BUN (mM), UA (μM).

  • Fig. 6 Immune responses and biochemical analysis of organ functions following immunostimulation in a rabbit model.

    (A) Timeline of prime, boost, and analysis steps of the immunostimulation. (B) Antibody titers in rabbits after receiving immunostimulation with native human RhD-positive RBCs, engineered human RhD-positive RBCs, and human RhD-negative RBCs at different time points. (C) Antibody titers in rabbits during the 5th week of immunostimulation after injection in the four groups. (D) Biochemical analysis of liver function during the 5th week of immunostimulation after injection in the four groups. ALT (U/l), AST (U/l), ALP (U/l), TBIL (μM), DBILALT (μM). (E) Biochemical analysis of kidney function during the 5th week in the four groups after immunostimulation. CREA (μM), BUN (mM), UA (μM).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/12/eaaw9679/DC1

    Supplementary Materials and Methods

    Fig. S1. Chemical characterizations of the enzymatically crosslinked framework for the encapsulation of the universal RBCs.

    Fig. S2. Stability of the engineered universal RBCs in human plasma.

    Fig. S3. Effects of the PSA-tyramine concentration on the optimized engineering of universal RBCs to prevent antibody-mediated aggregation in multiple blood types.

    Fig. S4. Anti-RhD antibody–mediated human RhD-positive RBC aggregation via different methods of cell surface engineering.

    Fig. S5. Cytomechanical and biochemical analysis, Bohr coefficient of the Hb forms in different circumstances, and blood clotting function of the native and engineered RBCs.

    Fig. S6. Organ distribution and in vivo functional profiles of the native and engineered RBCs illustrated by the imaging function of the photoacoustic system.

    Fig. S7. Biological safety evaluation of blood transfusion with native and engineered RBCs in a mouse model.

    Fig. S8. Rescue experiment for the hemorrhagic test via the transfusion of native and engineered RBCs in a mouse model.

    Table S1. Routine blood assays of the mice transfused with the native and engineered RBCs.

    Table S2. Routine blood assays for the hemorrhagic test after the transfusion with the native and engineered RBCs.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Chemical characterizations of the enzymatically crosslinked framework for the encapsulation of the universal RBCs.
    • Fig. S2. Stability of the engineered universal RBCs in human plasma.
    • Fig. S3. Effects of the PSA-tyramine concentration on the optimized engineering of universal RBCs to prevent antibody-mediated aggregation in multiple blood types.
    • Fig. S4. Anti-RhD antibody–mediated human RhD-positive RBC aggregation via different methods of cell surface engineering.
    • Fig. S5. Cytomechanical and biochemical analysis, Bohr coefficient of the Hb forms in different circumstances, and blood clotting function of the native and engineered RBCs.
    • Fig. S6. Organ distribution and in vivo functional profiles of the native and engineered RBCs illustrated by the imaging function of the photoacoustic system.
    • Fig. S7. Biological safety evaluation of blood transfusion with native and engineered RBCs in a mouse model.
    • Fig. S8. Rescue experiment for the hemorrhagic test via the transfusion of native and engineered RBCs in a mouse model.
    • Table S1. Routine blood assays of the mice transfused with the native and engineered RBCs.
    • Table S2. Routine blood assays for the hemorrhagic test after the transfusion with the native and engineered RBCs.

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