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1 In the format provided by the authors and unedited. DOI: /NNANO Complement proteins bind to nanoparticle protein corona and undergo dynamic exchange in vivo Fangfang Chen #, Guankui Wang #, James I. Griffin, Barbara Brenneman, Nirmal K. Banda, V. Michael Holers, Donald S. Backos, LinPing Wu, Seyed Moein Moghimi and Dmitri Simberg * # Equal contribution * Corresponding author 1. Supplemental methods Atomic force microscopy was performed at the Nanomaterials Characterization Facility, University of Colorado Boulder. Highly diluted samples were dried on a cleaned borosilicate glass surface and imaged using a Nanosurf EasyScan 2 AFM (110-µm scan head) with an Aspire Conical AFM probe tip (CT170R) using intermittent contact (dynamic force mode) to avoid damage to the samples. Citrate capped gold nanoparticles (Au NPs, 30 nm of diameter) were freshly prepared using a published method 1, and were sterilized, filtered and finally stored in sodium citrate buffer (0.1 mm) before use. For PEGylation, 5kDa methoxy PEG-thiol (Nectar) was used. PEG was dissolved in distilled water at 10 mm concentration and 10 µl of PEG was incubated with 0.5 ml gold solution at approximate concentration of 0.3 mm under shaking for 1 h at RT. After conjugation, Au-PEG was washed three times and resuspended in sodium citrate buffer (0.1 mm). For C3 binding studies, Au-PEG NPs were mixed with lepirudin plasma as described in main Methods, washed by centrifugation and either analyzed with SDS PAGE and reducing western blot, or NATURE NANOTECHNOLOGY 1

2 incubated in 2% SDS to elute the proteins and C3, and analyzed with non reducing SDS- PAGE and western blot. 2. Supplementary tables Supplementary Table 1. Physicochemical parameters (calculated and measured) of SPIO nanoworms: For experimental details see Methods. Particle concentration experiments (Nanosight) and size measurements (DLS) were repeated at least 3 times. Dextran quantification (gravimetric analysis) was repeated twice. Particle concentration in 1 mg/ml Fe 3.99 x Fe per particle, gram 2.51 x Fe 3 O 4 per particle, gram 3.46 x Average number of Fe 3 O 4 crystals per particle ~20 Fe atoms per 7 nm crystal ~11,000 Average crystal size, nm 7 Total particle mass, gram 5.14 x Dextran % (dry weight, w/w) 33% Dextrans per particle 500 Core volume (100 nm x 7 nm x 7 nm, TEM), L 4.90 x DLS hydrodynamic volume (140 nm diameter), L 1.44 x Shell volume, L 1.44 x Supplementary Table 2. Proteomic identification of plasma and serum proteins (top 30 hits) adsorbed to SPIO nanoworms: Full list of the identified proteins (158 for serum and 227 for plasma) and raw mass spectrometry data are provided in the supplementary dataset. Rank Plasma Serum 1 Complement C3 Apolipoprotein B Fibrinogen beta chain Serum albumin 3 Fibrinogen alpha chain Complement C3 NATURE NANOTECHNOLOGY 2

3 4 Fibronectin Apolipoprotein A 5 Myosin-9 Serotransferrin 6 Filamin-A Alpha-2-macroglobulin 7 Talin-1 Fibronectin 8 Fibrinogen gamma chain Complement C4-B 9 Thrombospondin-1 Plasma kallikrein 10 Serum albumin Complement factor B 11 Apolipoprotein B-100 Fibrinogen alpha chain 12 Integrin beta-3 Apolipoprotein E 13 Complement factor H Apolipoprotein A-I 14 Vinculin Apolipoprotein A-IV 15 Complement C4-A Kininogen-1 16 Complement C4-B Ceruloplasmin 17 Complement C5 Fibrinogen beta chain 18 Alpha-2-macroglobulin Ig gamma-1 chain C region 19 Alpha-actinin-1 Vitamin D-binding protein 20 Actin, cytoplasmic 1 Ig mu chain C region 21 Complement component C9 Complement factor H 22 Apolipoprotein A-I Plasminogen 23 Ferritin family homolog 3 Mannose-binding protein C 24 Apolipoprotein E Transferrin receptor protein 1 25 Kininogen-1 Clusterin 26 Clusterin Apolipoprotein D 27 Complement component C7 Apolipoprotein B Prothrombin Serum albumin 29 Band 3 anion transport protein Complement C3 30 Spectrin alpha chain Apolipoprotein D NATURE NANOTECHNOLOGY 3

4 Supplementary Table 3. Quantification of protein absorbed per nanoparticle in human serum and plasma: Data from a representative experiment (out of 2 using 2 different sera and plasma samples from matched donors) are shown. ND = non-detectable with immune dot-blot assay. For calculation details see Methods. Protein per particle Serum Lepirudin-Plasma Total protein, gram 2.2 x x C3 molecules/particle ~70 ~110 Fibrinogen molecules/particle ND ~80 3. Supplementary figures a Number of particles Number of particles TEM contour length, nm TEM width, nm b c 200 nm Supplementary Fig. 1: Characterization of SPIO nanoworms (supplement for Fig. 1): a) Distribution of nanoworm core length (upper graph) and width (lower graph) based on counting of TEM images of ~100 particles; b) Dynamic light scattering measurement (screen shot) of particle size (intensity weighted) using Zetasizer Nano; c) AFM image of round-shaped SPIO nanoworms reflects the overall shape of core shell particles. Larger micron-sized particles are likely aggregates formed during sample drying; Inset shows an individual particle. NATURE NANOTECHNOLOGY 4

5 a b c C3 Integrated density y = 0.793x R² = C3 released from SPIO NWs Integrated density C3 standard, ng per spot Integrated density Integrated density C3 per spot, ng C3 per spot, ng C3 per spot, ng C3 molecules per spot 7.87E+10 C3 molecules per spot 7.86E+10 C3 molecules per spot 6.73E+10 parhcles per spot 6.10E+08 parhcles per spot 6.10E+08 parhcles per spot 6.10E+08 C3/parHcle C3/parHcle C3/parHcle Supplementary Fig. 2: Quantitative dot blot assay to determine C3 number per nanoworm. The results are representative of a typical experiment. SPIO nanoworms were incubated in serum/plasma, washed and incubated in 2% SDS to elute the proteins. a) Dot blot of C3 probed with a polyclonal antibody; b) Standard curve of C3 on the membrane (all in 2% SDS); c) spreadsheet showing quantification of C3 molecules/particle. a b Supplementary Fig. 3: Whole-field view of FIB-SEM (a) and TEM (b) of SPIO nanoworms in human serum corresponding to cropped images in Fig. 2. Bar = 100 nm for both images. NATURE NANOTECHNOLOGY 5

6 Dextran immunoreactivity on SPIO NWs without serum with serum Supplementary Fig. 4: Dextran immunoreactivity of SPIO nanoworms before and after incubation in human serum as measured with dot blot. Dextran immunoreactivity is not decreased in serum, suggesting that adsorbed proteins do not mask dextran chains. Data are means and s.d., n=3, repeated 3 times. Dextran C3 -SDS +SDS -SDS +SDS Supplementary Fig. 5: SPIO nanoworms were incubated in 2% SDS/PBS for 1h to elute the bound proteins. The particles were washed by ultracentrifugation and the amount of dextran and C3 on particles was compared using dot blot assay. While the treatment removed the majority of C3, it did not decrease the amount of immunoreactive dextran on particles. NATURE NANOTECHNOLOGY 6

7 DOI: /NNANO b ic3b An0-P EDTA plasma ic3b An0-P EDTA c plasma a 100kDa Non-reducing, C3 100kDa 75kDa 75kDa 25kDa 25kDa Reducing, C3 Supplementary Fig. 6: Complement activation and assembly of PEGylated gold nanoparticles in human plasma. a) Images of particles by non-contrast TEM. Main size bar: 200nm, inset size bar: 50nm; b) C3 binding to particles in plasma was analyzed after elution in 2% SDS and running non-reducing western blotting. Considerable fraction of of C3 appears to be bound to proteins. Anti-properdin antibody blocked over 80% of C3, suggesting involvement of the AP; c) reducing western blot shows that majority of C3 on gold particles is in the ic3b form, along with other cleaved fragments likely due to factor I activity. Experiment was repeated 2 times using different plasma. NATURE NANOTECHNOLOGY 7

8 fb# C3# depletedse RAP## rum+rap# Bb# 60kDa# Supplementary Fig. 7: nanoworm-mediated generation of fluid-phase Bb in presence of RAP (for Fig. 4). a 0 min 15 min 60 min 120 min NW S NW S NW S NW S b Serum C3 Released C3 c Human properdin C3, reducing conditions β-chain Non-reducing Properdin, integrated density min 15 min 120 min Supplementary Fig. 8: Spontaneous release of C3 and properdin from particles upon incubation. a) SPIO nanoworms were incubated in normal human serum, washed and further incubated at room temperature for different times; Gel shows levels of residual C3 on nanoworms and in the supernatant (S) as a function of time. C3 (mostly ic3b and intact C3) was gradually detached from nanoworms over time; b) some of the released C3 is bound to serum proteins as evidenced by the appearance of high molecular weight fraction of C3 in non-reducing conditions. Some C3 appeared to run similarly to the serum C3, likely due to self-cleavage of the C3b covalent bond over time; c) Properdin levels in the supernatant show gradual release over time following incubation of serum NATURE NANOTECHNOLOGY 8

9 coated SPIO nanoworms in PBS. Experiment was repeated 3 times with sera from different healthy donors 60 min incubation Reincubated Supplementary Fig. 9: Images of magnetically labeled leukocytes corresponding to Fig. 5C. Representative grey microscopic images (40x magnification) show magnetically isolated leukocytes with nuclei stained by Hoechst (white dots). There was a significantly enhanced uptake after reincubation of particles in fresh serum, demonstrating the dynamic nature of complement opsonization and critical role of the removable fraction in the uptake. NATURE NANOTECHNOLOGY 9

10 a) marker plasma Plasma+EDTA C3b ic3b marker marker plasma Plasma+EDTA C3b ic3b marker High exposure Low exposure b) marker 0 60 Reincubated Supplementary Fig. 10: Fill-length gel images corresponding to: a) Fig. 3b, plasma panel, and b) Fig. 5b, serum panel. Relevant lanes are inside the boxes. Unrelated lanes are marked with cross. Gel image in (a) is shown at 2 different exposures due to different amounts of C3 protein on particles and C3b/iC3b standards. The lower exposure was used for C3b/iC3b panel in Fig. 3b. NATURE NANOTECHNOLOGY 10

11 References: 1. Frens, G. Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions. Nature 241, (1973). NATURE NANOTECHNOLOGY 11