Efficient dual sirna and drug delivery using engineered lipoproteoplexes

Transcription

1 Supporting Information Efficient dual sirna and drug delivery using engineered lipoproteoplexes Che Fu Liu, Raymond Chen, Joseph A. Frezzo, Priya Katyal, Lindsay K. Hill, Liming Yin, Nikita Srivastava, Haresh T. More, P. Douglas Renfrew, ^ Richard Bonneau ^ and Jin Kim Montclare a Department of Chemical and Biomolecular Engineering, NYU Tandon School of Engineering, Brooklyn, New York 11201, United States Department of Biomedical Engineering, State University of New York Downstate Medical Center, Brooklyn, New York 11203, United States Department of Radiology, New York University School of Medicine, New York, New York 10016, United States Department of Chemistry, New York University, New York, New York 10003, United States a Department of Biochemistry, SUNY Downstate Medical Center, Brooklyn, New York 11203, United States Center for Genomics and Systems Biology, New York University, New York Courant Institute of Mathematical Sciences, Computer Science Department, New York University, New York, NY 10009, United States ^Center for Computational Biology, Flatiron Institute, Simons Foundation, th Avenue New York, NY 10010, United States These authors contributed equally Doxorubicin docking in CSP. In the ligand conformer generating protocols, a total of 552 doxorubicin conformers passed the filtering criteria and were used in the docking simulations (Figure S1a). Low energy docked models tended to cluster near the N-terminus of the CSP helical bundle. The lowest energy docking conformation is shown in Figure S1. It has a binding energy of Rosetta Energy Units indicating that the energy of the system is lower when the ligand is present and binding is favorable. Doxorubicin can form hydrogen bonds with THR40. The CSP backbone requires little movement to accommodate the ligand and the CA RMSD between the model and the experimentally determined structure is 0.1 Angstroms. 1

2 Figure S1. a) Predicted docked conformation of doxorubicin in the central channel of CSP pentamer. b) Low energy surface charge of CSP pentamer. 2

3 1 2 3 Figure S2. Expression of CSP on 12% SDS-PAGE. Lanes 1, Ladder, lane 2 preexpression and lane 3 post expression. Figure S3. Purification profiles on 12% SDS-PAGE. Lane 1: Ladder, lane 2: Flow through, lane 3: wash, lane 4: 100mM Imidazole elution, lane 5-6: 200mM imidazole Elution, lane 7-9: 500mM Imidazole elution and lane 10: 1M imidazole elution fractions. 3

4 Figure S4. Confocal microscopy of MCF-7 cells treated with (a, b) Dox CSP and (c,d) Dox L2000 4

5 Figure S5. Histograms plots depict original flow cytometry analysis of Dox Cy5-siRNA CSP L2000. The percent of cells containing Cy5-siRNA was calculated from (a), and within sirna bearing cells, percent of cells bearing dox was calculated from (b). Similarly, percent of cells containing Dox was calculated from (c), and within dox bearing cells, percent of cells containing Cy5-siRNA was calculated from (d). Note: the cells were treated with Dox and Cy5-siRNA in the presence of CSP and L

6 Table S1. Values for Cell Viability Study CSP & Dox Treatment Equivalent Dox Conc. Actual Dox Conc. Actual CSP Conc. Neg Ctrl CSP CSP CSP CSP CSP CSP Dox Dox Dox Dox Dox Dox Dox CSP Dox CSP Dox CSP Dox CSP Dox CSP Dox CSP

7 Table S2. Values for Cell Viability Study -L2000 & sirna Treatment Equivalent Dox Conc. Actual Dox Conc. Actual CSP Conc. L2000:CSP:siRNA (wt/wt) Neg Ctrl (Opti) L2000 Dox :8:1 L2000 Dox :8:1 L2000 Dox CSP :8:1 L2000 Dox CSP :8:1 L2000 sirna :8:1 L2000 sirna :8:1 L2000 sirna CSP :8:1 L2000 sirna CSP :8:1 L2000 sirna Dox CSP 0.4 L2000 sirna Dox CSP :8: :8:1 7

8 Table S3. Values for Cell Viability Study -L2000 & sirna Treatment Equivalent CSP Mass (µg) Actual CSP Mass (µg) Actual L2000 Mass (µg) Actual sirna Mass (µg) Neg Ctrl (Opti) L2000 Dox L2000 Dox L2000 Dox CSP L2000 Dox CSP L2000 sirna L2000 sirna L2000 sirna CSP L2000 sirna CSP L2000 sirna Dox CSP L2000 sirna Dox CSP

9 Figure S6. Relative cell viability of MCF-7 cells after 48 hours treatment with L2000 transfection reagent controls based on MTT assay, normalized to the negative control which consists only of EMEM, Opti-MEM, and 50 mm Tris-HCl, 0.5 M NaCl ph 8 buffer. Data represented is one trial. Error bars represent standard deviation across four replicates. 9