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Supporting Information Electronic Supplementary Material (ESI) for Nanoscale. This journal is © The Royal Society of Chemistry 2017 Supporting Information Visualization of the Protein Corona: towards a biomolecular understanding of nanoparticle-cell- interactions Authors: Maria Kokkinopoulou†1, Johanna Simon†1, Katharina Landfester1, Volker Mailänder*2, Ingo Lieberwirth*1 1Max Planck-Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany 2Clinic of Dermatology, University Medicine Mainz, Langenbeckstraße 1, 55131 Mainz, Germany † both authors contributed equally * Corresponding author Corresponding Authors: Volker Mailänder: [email protected] Ingo Lieberwirth: [email protected] 1 Contents Physicochemical properties of nanoparticles ............................................................................. 3 Visualizing protein nanoparticle interactions by electron microscopy ...................................... 3 Monitoring the protein corona with dynamic light scattering .................................................... 5 Quantification of adsorbed proteins and remaining content of proteins in the supernatant ....... 8 Classification of corona proteins ................................................................................................ 9 Cellular interactions of protein corona coated nanoparticles ................................................... 12 Methods ..............................................................................Fehler! Textmarke nicht definiert. Nanoparticle Synthesis .......................................................Fehler! Textmarke nicht definiert. LC MS Analysis ....................................................................................................................... 15 Literature .................................................................................................................................. 29 2 Physicochemical properties of nanoparticles Table 1: Physicochemical properties as hydrodynamic radius and ζ-potential of polystyrene nanoparticles determined by multi angle dynamic light scattering in aqueous solution and PBS. The fluorescence intensity of the BODIPY labeled nanoparticles (525/535 nm) was determined by Tecan Microplate Reader and normalized to the value for un-functionalized polystyrene nanoparticles. Rh (H2O) Rh (PBS) ζ-Potential Fluorescence Intensity Factor PS 73 ± 7.3 nm 75 ± 7.5 nm - 3.70 mV 1 PS- COOH 70 ± 7.0 nm 72 ± 7.0 nm - 7.21 mV 1.16 PS- NH2 74 ± 4.3 nm 79 ± 7.9 nm + 7.58 mV 1.05 Visualizing protein nanoparticle interactions by electron microscopy Figure 1: Cryo-TEM micrographs of un-functionalized PS nanoparticles incubated with gold-labelled bovine serum albumin visualize the structure of the protein corona. Nanoparticles (0.05 m2) were incubated with 100 µg of protein for 1h at 37°C before measurements were performed. 3 Figure 2: TEM micrographs of un-functionalized PS nanoparticles incubated with human serum and centrifuged once (A). Nanoparticles are surrounded by a huge protein cloud. Figure 3: TEM micrographs of un-functionalized PS nanoparticles incubated with human serum centrifuged and washed three times (D). The hard protein corona surrounding nanoparticles is visualized. 4 Figure 4: TEM micrographs of un-functionalized PS incubated with identified hard corona proteins. Nanoparticles (0.05 m2) were incubated with 100 µg of protein for 1h at 37°C before measurements were performed. Monitoring the protein corona with dynamic light scattering PS Figure 5: Autocorrelation function of un-functionalized PS nanoparticles incubated with human serum exemplary shown for a scattering angle of 30°. Upper graph: The blue line (–) represents the forced fit composed of the sum of the individual components whereas the red line (–) represents the fit with an additional aggregation function. Lower graph: Corresponding residuals resulting from the difference between the data and the two fits. The overlay of the red and blue fit indicates that there is no significant aggregation of un-functionalized PS nanoparticles incubated with human serum. Strong aggregation formation of nanoparticles in human serum would result in a great shift between the addition aggregation function (red) and the forced fit (blue). 5 PS- NH2 Figure 6: Autocorrelation function of PS -NH2 nanoparticles incubated with human serum exemplary shown for a scattering angle of 30°. Upper graph: The blue line (–) represents the forced fit composed of the sum of the individual components whereas the red line (–) represents the fit with an additional aggregation function. Lower graph: Corresponding residuals resulting from the difference between the data and the two fits. The overlay of the red and blue fit indicates that there is no significant aggregation of PS- NH2 nanoparticles incubated with human serum. PS- COOH Figure 7: Autocorrelation function of PS- COOH nanoparticles incubated with human serum exemplary shown for a scattering angle of 30°. Upper graph: The blue line (–) represents the forced fit composed of the sum of the individual components whereas the red line (–) represents the fit with an additional aggregation function. Lower graph: Corresponding residuals resulting from the difference between the data and the two fits. The overlay of the red and blue fit indicates that there is no significant aggregation of PS- COOH nanoparticles incubated with human serum. 6 Table 2: Soft corona hydrodynamic radii of polystyrene nanoparticles incubated within human serum measured by multi angle dynamic light scattering. The size of the nanoparticles was measured directly within human serum. Nanoparticles are surrounded by a huge protein causing a size increase. Values are exemplary given for a scattering angle at 30° as the system has the greatest sensitivity towards detection of aggregation formation. 1 Hydrodynamic radius (NP with soft corona) Intensity (Scattering angle 30°) (I%) PS 140 ± 14 nm 22 PS- COOH 178 ± 18 nm 21 PS- NH2 188 ± 19 nm 18 0,020 Rh = 73 nm (PS, in water) Rh = 92 nm (PS, after first centrifugation A) 0,016 0,012 1/Rh nm 0,008 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 q2/10E10cm-2 Figure 8: Incubation of un-functionalized PS with human serum and further centrifugation to isolate NPs with hard protein corona (PS; A) in comparison to PS in water. There is a size increase of 19 ± 2 nm monitored by multi angle dynamic light scattering attributed to the hard protein corona around nanoparticles. The angular dependency of the inverse hydrodynamic radii of un-functionalized PS is demonstrated. Table 3: Hard corona hydrodynamic radii of polystyrene nanoparticles incubated within human serum and centrifuged was measured by multi angle dynamic light scattering. Hydrodynamic radius (NP with hard corona) Rh (H2O) PS 92 ± 9.2 nm PS- COOH 80 ± 8.0 nm PS- NH2 88 ± 8.8 nm 7 0 -5 -3,7 -10 -15 Zetapotential / mV -20 -21 -22,1 -23,05 -22,6 -25 PS A B C D Figure 9: Zeta-potential after incubation, centrifugation and washing of un-functionalized PS nanoparticles incubated with human serum. Due to protein adsorption the zeta-potential is strongly decreased. Quantification of adsorbed proteins and remaining content of proteins in the supernatant Table 4: Amount of adsorbed protein in µg per 0.05 m2 NP determined after each purification step (A = First centrifugation, B = 1. Wash, C = 2. Wash, D = Third wash) Preparation PS Amount of adsorbed protein per 0.05 m2 NP A 781.9 ± 38.6 µg B 87.22 ± 2.42 µg C 50.51 ± 2.28 µg D 49.18 ± 3.00 µg Preparation PS- COOH Amount of adsorbed protein per 0.05 m2 NP A 1208.22 ± 101.38 µg D 137.35 ± 1.38 µg Preparation PS- NH2 Amount of adsorbed protein per 0.05 m2 NP A 1055.97 ± 18.78 µg D 66.70 ± 4.93 µg Table 5: Protein concentration in the remaining supernatant after centrifugation and washing. After the third wash the protein concentration is below the detectable range of the Pierce assay. Supernatant Concentration (mg/mL) 8 A 47.97 ± 1.71 mg/mL B 1.43 ± 0.03 mg/mL C 0.07 ± 0.02 mg/mL D * (below 0.05 mg/mL) Table 6: Amount of recovered nanoparticle after the first centrifugation and last washing steps for all nanoparticles measured via fluorescence intensity of nanoparticles at the initial concentration. Preparation PS PS- COOH PS- NH2 A 71.7 ± 4.8 % 83.6 ± 3.2 % 86.8 ± 5.9 % D 68.3 ± 3.4 % 72.7 ± 3.4 % 66.8 ± 1.2 % Classification of corona proteins After initial centrifugation 100 90 Tissue Leakage 80 Other plasma components Lipoproteine 70 Immunoglobulins 60 Complement system 50 Coagulation 40 Acute Phase 30 20 % based on all identified proteins identified all on % based 10 0 Human Serum PS PS-COOH PS-NH2 Figure 10: LC-MS Protein classification of proteins associated with nanoparticles after the first centrifugation (A). There is no significant difference in the protein pattern for all nanoparticles. After 3. wash 9 100 90 80 70 Tissue Leakage Other plasma components 60 Lipoproteine 50 Immunoglobulins 40 Complement system 30 Coagulation Acute Phase % based on all identified proteins identified all on % based 20 10 0 Human PS PS-COOH PS-NH2 Figure 11: LC-MS Protein classification of proteins associated with nanoparticles after the third wash (D). The distinct hard protein corona profile depends on surface functionalization of nanoparticles and highly differs from the protein composition in human serum. Figure 12: Evolution of the hard corona profil after each purification step for un-functionalized PS nanoparticles visualized by SDS-PAGE. (A = First centrifugation, B = 1. Wash, C = 2. Wash, D = Third wash) 10 100
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