complement proteins bind to nanoparticle protein corona ... · i o ed. supplemental data for:...

In the format provided by the authors and unedited.Supplemental data for:

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

© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2016.269

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 1

http://dx.doi.org/10.1038/nnano.2016.269

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 1013

Fe per particle, gram 2.51 x 10-17

Fe3O4 per particle, gram 3.46 x 10-17

Average number of Fe3O4 crystals per particle ~20

Fe atoms per 7 nm crystal ~11,000

Average crystal size, nm 7

Total particle mass, gram 5.14 x 10-17

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 10-21

DLS hydrodynamic volume (140 nm diameter), L 1.44 x 10-18

Shell volume, L 1.44 x 10-18

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-100

2 Fibrinogen beta chain Serum albumin

3 Fibrinogen alpha chain Complement C3





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-100

28 Prothrombin Serum albumin

29 Band 3 anion transport protein Complement C3

30 Spectrin alpha chain Apolipoprotein D





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.

3. Supplementary figures

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.

200 nm

c

30 50 70 90 110 130 150 170 190 2100

5

10

15

20

TEM contour length, nm

Num

ber

of p

artic

les

4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.00

5

10

15

TEM width, nm

Num

ber

of p

artic

les

a b

Protein per particle Serum Lepirudin-Plasma

Total protein, gram 2.2 x 10-17 3.6 x 10-17

C3 molecules/particle ~70 ~110

Fibrinogen molecules/particle ND ~80





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.

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.

50

25

12.5

6.25

3.125

1.56C3releasedfromSPIONWs

y = 0.793x - 0.2344 R² = 0.99653

0 5

10 15 20 25 30 35 40 45

0 20 40 60

Inte

grat

ed d

ensi

ty

C3 standard, ng per spot

C3 Integrateddensity 17.875Integrateddensity 17.846Integrateddensity 15.392C3perspot,ng 23.4909125C3perspot,ng 23.4512202C3perspot,ng 20.0924304C3moleculesperspot 7.87E+10C3moleculesperspot 7.86E+10C3moleculesperspot 6.73E+10parHclesperspot 6.10E+08parHclesperspot 6.10E+08parHclesperspot 6.10E+08C3/parHcle 129.18C3/parHcle 128.96C3/parHcle 110.49

a b c

a b





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.

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.

without s

erum

with se

rum

0

50

100

150

Dex

tran

imm

unor

eact

ivity

on

SPIO

NW

s

-SDS +SDS

Dextran

-SDS +SDS

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.

plasma

EDTA

iC3b

An0-P

75kDa

25kDa

100kDa

75kDa

25kDa

100kDa

a b c

plasma

EDTA

iC3b

An0-P

Non-reducing,C3

Reducing,C3





Supplementary Fig. 7: nanoworm-mediated generation of fluid-phase Bb in presence of

RAP (for Fig. 4).

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

fB#

C3#depletedserum+RAP#RAP##

Bb# 60kDa#

0 min

15 m

in

120 m

in0

2000

4000

6000

8000

10000P

rope

rdin

, in

tegr

ated

den

sity

Human properdin

Non-reducing

Seru

m C

3 Re

leas

ed C

3

b c NW S NW S NW S NW S 0 min 15 min 60 min 120 min

C3, reducing conditions

β-chain

a

250

150

75

50





coated SPIO nanoworms in PBS. Experiment was repeated 3 times with sera from

different healthy donors

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.

60 min incubation EDTA 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.

Lowexposure

marker

plasma

Plasma+ED

TA

C3b

iC3b

marker

marker

plasma

Plasma+ED

TA

C3b

iC3b

Highexposure

a)

marker

marker

0’

60’ Reincubated

b)





References:

1. Frens,G.ControlledNucleationfortheRegulationoftheParticleSizein

MonodisperseGoldSuspensions.Nature241,20-22(1973).





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