Supporting information Chiral Surface of Nanoparticles Determines the Orientation of Adsorbed Transferrin and its Interaction with Receptors Xinyi Wang,,,# Mingzhe Wang,,# Rong Lei, Shui fang Zhu, Yuliang Zhao, Chunying Chen,* CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, & CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China and University of Chinese Academy of Sciences, Beijing 100190, China College of Science, Shenyang Agricultural University, Shenyang 110866, China Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, 100029, China AUTHOR INFORMATION Corresponding Author *E-mail: chenchy@nanoctr.cn Author Contributions # Xinyi Wang and Mingzhe Wang contributed equally. S1
Figure S1. Representative TEM image of Cit-AuNPs. (a) The average diameter of AuNPs was measured to be 13.6±0.9 nm (n=100). (b) The average diameter of AuNPs was measured to be 23.3±1.8 nm (n=100). S2
Figure S2. FT-IR spectra of penicillamine (a) and penicillamine-modified AuNPs (b). The S-H stretching vibration peak of penicillamine at ~2600 cm -1 disappeared in the spectrum of the penicillamine-modified AuNPs, indicating Au-S bond formation. S3
Normalized absorbance 1.0 0.8 0.6 AuNPs AuNPs(L) AuNPs(D) AuNPs(D/L) AuNP-Tf AuNP(L)-Tf AuNP(D)-Tf AuNP(D/L)-Tf 0.4 0.2 400 440 480 520 560 600 Wavelength (nm) Figure S3. The plasmon absorption spectra of AuNPs with various surface modifications. The characteristic peak location of citrate-protected AuNPs was centered at 519 nm, and appeared red shifted resulting from the modification of various ligands. S4
Figure S4. CD spectra of AuNPs modified with chiral Penicillamines (Pen). (A) CD spectra of pure D-Pen, L-Pen and D/L-Pen. (b) CD spectra of pure Cit-AuNPs. (c) CD spectra of AuNPs modified with D-Pen, L-Pen and D/L-Pen after subtracting backgroud signal of Cit-AuNPs. (d) CD spectra of centrifugal supernatants of chiral Pen-modified AuNPs. S5
Figure S5. Modified Stern-Volmer plots for the fluorescence quenching of Tf by chiral AuNPs at different temperatures. The concentration of Tf was fixed at 1.5μM and the concentrations of various chiral AuNPs were from 0 to 15 nm. S6
Figure S6. PRLS spectra of AuNPs (10 nm) with different chiral surfaces upon interaction with Tf at different molar ratio. S7
Zeta Potential (mv) -40-35 -30 AuNPs(D) AuNPs(L) AuNPs(D/L) -25-20 -15 1:0 1:5 1:10 1:50 1:100 1:500 1:1000 Molar Ratio (AuNPs:Tf) Figure S7. Surface zeta potential measurement of chiral Pen-AuNPs and Tf system with different moalr ratio. The zeta potential of Tf is ~-10.1 mv. S8
Figure S8. Distribution of transferrin surface charges. (a) Space-filling models of Tf (PDB ID: 4X1B) with color-coded types of charge (red, negative charge; blue, positive charge; grey, no charge). (b) The distribution of amino acids with charges on the surface of Tf (protein sidechains are hidden for simplification), based on solvent-accessible surface area (ASA) calculation (calculated online at http://mobyle.rpbs.univ-paris-diderot.fr/). S9
Frequency (Hz) Energy dissipation ( 10-6 ) 0-5 8:2 POPC:POEPC F n=3 /3 D n=3 5 4-10 3-15 2-20 -25-30 0 2 4 6 8 10 Time (min) 1 0 Figure S9. QCM-D monitoring of SLB (8:2 DOPC:DOEPC) formation on the silicon oxide substrate through surface-mediated vesicle fusion. All data presented here were measured at the third overtone. S10
Figure S10. QCM-D measurement of the frequency variation (Δf) for interaction of Tf-adsorbed chiral AuNPs with POPC/POEPC SLBs on a silica coated quartz crystal sensor. (a)-(c) represent results of three parallel experiments. All nanoparticle concentrations were 180 µg/ml and the solutions were injected at a flow rate of 20µL/min. S11
Figure S11. Quantitative analysis of Tf corona formation on 23 nm Pen-AuNPs. a-c denote the hydrodynamic radius changes of 23 nm Pen-AuNPs after incubating with Tf; The curves (red solid lines) were fitted according to the Hill equation, and the best-fit parameters are listed on the right. The final concentrations of all Pen-AuNP solutions were 45 µg/ml, and the DLS was measured after mixing AuNPs with the desired concentrations of Tf for 2 h at room temperature. (d) The 3-D structure of Tf was described using a space-filling model of Tf (PDB ID: 4X1B) with color-coded types of charge (red, negative charge; blue, positive charge; grey, no charge). (e) Statistical distribution of charged amino acids on each side of the virtual cube of Tf. (f) Schematic of interaction between Tf adsorbed on Pen-AuNPs and SLBs (POPC:POEPC, 8:2) monitored by QCM-D. (g) Statistical results of the adsorption of Tf-adsorbed 23 nm Pen-AuNPs onto SLBs based on Figure S12. All data are the mean± SD of three replicates. * P< 0.05 and ** P< 0.01. S12
Figure S12. QCM-D measurement of the frequency variation (Δf) for interaction of Tf-adsorbed Pen-AuNPs with POPC/POEPC SLBs on a silica coated quartz crystal sensor. (a)-(c) represent results of three parallel experiments of 23 nm Pen-AuNP. All concentrations of NPs were 45 µg/ml and the solutions were injected at a flow rate of 20µL/min. S13
Volume (%) 18 15 12 9 6 3 0 0 500 1000 1500 2000 Size (d. nm) Figure S13. The hydrodynamic size of cell-derived liposomes. The liposomes were obtained by repeatedly extruding HEK293A cells more than 10 times through a filter membrane with 0.8µm pore diameter. S14
Figure S14. QCM-D monitoring of the frequency variation (Δf) for Steps 1-3 sequentially. The EDC/NHS-actived carboxyl coated sensor crystals were exposed in order to: chiral AuNPs (Step 1); transferrin (Step 2); HEK293A cell-derived liposomes (Step 3). The concentrations of all chiral AuNPs were 150µg /ml and the solutions were injected at a flow rate of 20µL/min. The HEPES buffer solution was flowed to remove previous solution in the interval between two steps. TfR on HEK293A cells were treated using RNAi-mediated gene silencing (a-c), wide type (d-f), and transient transfection with a TfR-overexpression plasmid (g-i), respectively. S15
Figure S15. AFM imaging of the microstructures of modified chiral AuNPs on the sensor surface. (a)-(d) denotes blank (no modification of chiral AuNPs), AuNPs(D), AuNPs(L) and AuNPs(D/L) respectively. The concentrations of all chiral AuNPs were 150µg /ml and the scan sizes were 1 µm. S16
Figure S16. Flow cytometry analysis of the expression of TfR in HEK293A cells. (a) Typical flow cytometric histograms for the detection of TfR with three expression levels involving knockdown (RNAi-mediated gene silencing), wide type, and over-expression (transient transfection of TfR cdna). (b) The quantification of TfR expression in a. For each sample, more than 1 10 4 events were measured. Data was the mean± SD of three replicates. * was for P< 0.05, and ** was for P< 0.01. S17
Figure S17. QCM-D measurement of the frequency variation (Δf) for the interaction between Tf absorbed onto chiral AuNPs and TfR expressed in cell-derived liposomes. TfR had three levels of expression via being treated by RNAi-mediated gene silencing (a1-a3), wide type (b1-b3), and transient transfection with a TfR-overexpression plasmid (c1-c3), respectively. The concentrations of all chiral AuNPs were 150µg /ml, and the solutions were injected at a flow rate of 20µL/min. S18
F (Hz) 0-40 -80-120 AuNPs(D) AuNPs(L) AuNPs(D/L) -160 0 2 4 6 8 Time (h) Figure S18. QCM-D measurement of the frequency variation (Δf) for the interaction between Tf-adsorbed chiral AuNPs and HEK293A cell-derived liposomes pre-blocked by Tf. TfR on HEK293A cell-derived liposomes (transient transfection of TfR-expression plasmid) were blocked by excess Tf (100µg /ml) for 2h before injected into sensor. The concentrations of all chiral AuNPs were 150µg /ml and the solutions were injected at a flow rate of 20µL/min. S19
Table S1 Assignment of amide I band frequencies to secondary structure of protein. Mean frequencies(cm -1 ) Secondary structures 1,624 ± 1.0 β-sheet 1,627 ± 2.0 β-sheet 1,633 ± 2.0 β-sheet 1,638 ± 2.0 β-sheet 1,642 ± 1.0 β-sheet 1,648 ± 2.0 Random 1,656 ± 2.0 α-helix 1,663 ± 3.0 3 10 -helix 1,667 ± 1.0 β-turn 1,675 ± 1.0 β-turn 1,680 ± 2.0 β-turn 1,685 ± 2.0 β-turn 1,691 ± 2.0 β-sheet 1,696 ± 2.0 β-sheet S20