A summary of Methods is provided below and a detailed description of Methods is included in the Supplementary Information .
The toxin nanosponges were prepared by fusing RBC membrane vesicles on preformed poly(lactic-co-glycolic acid) (PLGA) nanoparticles through an established extrusion process17 (link). The size of the nanosponges was obtained from three dynamic light scattering (DLS) measurements using a Malvern ZEN 3600 Zetasizer. The morphology of the nanosponges after absorbing toxins was measured by transmission electron microscopy (TEM). For preparation of human RBC nanosponges, the RBCs were collected from whole human blood (Bioreclamation) and the characterization results were shown inFig. S9 . For lyophilization, nanosponges were prepared in 5% sucrose solution. Reconstitution of the lyophilized samples was performed by solubilizing the samples in water and the characterization results were included in Fig. S10 .
The in vitro toxin neutralization ability of the nanosponges was examined by mixing 3 μg of α-toxin with 200 μL of 1 mg/mL nanosponges for 30 min, followed by adding into 1.8 mL of 5% purified mouse RBCs. The released hemoglobin was then quantified to determine the degree of RBC lysis. The retention of α-toxin by the nanosponges was measured using SDS-PAGE. The in vitro toxin absorption capacity of the nanosponges was determined through titrating α-toxin to a fixed amount of nanosponges. The interaction of the nanosponges with cells was examined by a scanning fluorescence microscopy by incubating fluorescent nanosponges and RBC membrane vesicles with human umbilical vein endothelial cells (HUVEC). The in vitro cellular cytotoxicity of nanosponge-sequestered toxins was examined by incubating nanosponges of different concentrations with varied amounts of α-toxin, streptolysin-O, and melittin for 30 min, followed by adding to HUVECs for 24 hr. Then the cell viability was assayed using an MTT assay.
The in vivo toxin neutralization ability of the nanosponges was tested through subcutaneous injection of the nanosponge/toxin mixture to the flank region of nude mice, followed by histological analyses. On-site neutralization of α-toxin by the nanosponges was conducted by subcutaneously injecting 50 μL of 36 μg/mL of α-toxin solution, immediately followed by a 100 μL injection of 2 mg/mL nanosponges. The mice were imaged 3 days later for visualization of skin lesion formation (Fig S11 ). The in vivo detoxification efficacy was tested through intravenous injection of nanosponges before or after administration of a lethal dose of α-toxin to ICR mice, followed by monitoring the survival rate of the mice. For the in vivo hepatotoxicity study, one group of mice was sacrificed on day 3 following the injection of the toxin-bound nanosponges and another group was sacrificed on day 7. The livers were collected, sectioned, and stained with H&E for histological analyses.
The toxin nanosponges were prepared by fusing RBC membrane vesicles on preformed poly(lactic-co-glycolic acid) (PLGA) nanoparticles through an established extrusion process17 (link). The size of the nanosponges was obtained from three dynamic light scattering (DLS) measurements using a Malvern ZEN 3600 Zetasizer. The morphology of the nanosponges after absorbing toxins was measured by transmission electron microscopy (TEM). For preparation of human RBC nanosponges, the RBCs were collected from whole human blood (Bioreclamation) and the characterization results were shown in
The in vitro toxin neutralization ability of the nanosponges was examined by mixing 3 μg of α-toxin with 200 μL of 1 mg/mL nanosponges for 30 min, followed by adding into 1.8 mL of 5% purified mouse RBCs. The released hemoglobin was then quantified to determine the degree of RBC lysis. The retention of α-toxin by the nanosponges was measured using SDS-PAGE. The in vitro toxin absorption capacity of the nanosponges was determined through titrating α-toxin to a fixed amount of nanosponges. The interaction of the nanosponges with cells was examined by a scanning fluorescence microscopy by incubating fluorescent nanosponges and RBC membrane vesicles with human umbilical vein endothelial cells (HUVEC). The in vitro cellular cytotoxicity of nanosponge-sequestered toxins was examined by incubating nanosponges of different concentrations with varied amounts of α-toxin, streptolysin-O, and melittin for 30 min, followed by adding to HUVECs for 24 hr. Then the cell viability was assayed using an MTT assay.
The in vivo toxin neutralization ability of the nanosponges was tested through subcutaneous injection of the nanosponge/toxin mixture to the flank region of nude mice, followed by histological analyses. On-site neutralization of α-toxin by the nanosponges was conducted by subcutaneously injecting 50 μL of 36 μg/mL of α-toxin solution, immediately followed by a 100 μL injection of 2 mg/mL nanosponges. The mice were imaged 3 days later for visualization of skin lesion formation (
