Supplementary information

Transcription

1 Supplementary information General Strategy for Direct Cytosolic Protein Delivery via Protein- Nanoparticle Co-Engineering Rubul Mout, Moumita Ray, Tristan Tay, Kanae Sasaki, Gulen Yesilbag Tonga, Vincent M. Rotello* Department of Chemistry, University of Massachusetts, 710 North Pleasant Street, Amherst, MA 01003, USA. *Corresponding author. S1

2 FIGURE LEGEND FOR SUPPLEMENTARY MOVIES Supplementary movie 1. Time-lapse confocal imaging of live nanoassembly-mediated GFP- E10 delivery. Supplementary movie 2. Membrane fusion of a single nanoassembly triggers direct cytosolic protein delivery. Supplementary movie 3. Nanoassemblies fused to cell membrane. SUPPORTING FIGURES Figure S1. Characterization of nanoparticles (ArgNPs), and GFP-E10:ArgNP nanoassemblies. TEM image of a) ArgNPs, and b) GFP-E10:ArgNPs assemblies. c) DLS size measurement of ArgNPs, indicating the hydrodynamic size (~10 nm in diameter). S2

3 Figure S2. E-tag engineering influences the net negative charge of GFP and hence the assembly formation and delivery efficiency. a) Addition of appropriate En to GFP, and their net negative charge. b) Flow cytometric assessment showing the comparison of GFP-En delivery efficiency as the length of E-tag was changed (also see Figure 2d for quantification). c) Flow cytometry data of individual GFP-En delivery, each in triplicate. S3

4 Figure S3. [ArgNP]/[GFP-E10] ratio determines delivery efficiency. Flow cytometry data showing the ratio dependent GFP-E10 delivery efficiency. (a) Mean fluorescence intensity (MFI) and (b) raw flow cytometry data showing the comparison of GFP-E10 delivery efficiency at various ArgNPs/GFP-E10 molar ratio. c) Individual raw flow cytometry data at different ratio, in duplicate. S4

5 Figure S4. Confocal microscopy Z-stacking images showing thorough cytosolar and nuclear delivery of GFP-E10. Images were taken at every 1 micron interval. 0 m indicates the bottom of the cell (at the dish surface). Scale bar= 20 m. Figure S5. Comparison of protein delivery using various delivery strategies. While supercharged GFP and cell penetrating peptide (CPP) based protein-delivery suffer from endosomal entrapment, our E-tag based nanoassembly method delivers proteins directly to the cytosol. Scale bar= 20 m. S5

6 Figure S6. 3D reconstruction of cells from z-stacking confocal images showing GFP-E10 delivery into the whole cell in different cell lines. Note that individual z-stacking images showed thorough cytosolic distribution of GFP-E10 (data not shown here). S6

7 Figure S7. Membrane fusion, but not membrane puncture is involved in nanoassemblymediated protein delivery. Assembled GFP-E10 was delivered into cells in the presence of free mcherry protein in the media. Free mcherry protein did not enter the cell, while GFP-E10 was delivered into the cell through nanoassembly fusion. Scale bar= 20 m. Figure S8. A membrane fusion like mechanism is involved in nanoassembly-mediated protein delivery. Confocal microscopy and flow cytometry show that endocytic inhibitors (wortmannin) did not block GFP-E10 delivery, whereas membrane fusion inhibitor (MBCD) completely blocked delivery. Scale bar= 20 m. S7

8 Figure S9. Delivered GzmA-E10 kills cells in a caspase 3/7 independent manner. Although GzmA-E10 delivery resulted in efficient cell death 24 h after delivery, the mechanism of cell death was caspase 3/7 independent; whereas, staurosporine caused caspase 3/7 dependent cell death in less than 3 h. Caspase 3/7 activation was measured using a standard fluorogenic peptide substrate. Scale bar= 20 m. Figure S10. Cell viability assay. Delivered GzmA-E10 kills HeLa cells efficiently. In contrast, no cell killing was observed with GFP-E10 delivery which was determined by Alamar blue test after 24 h of nanoassembly incubation. S8