complement proteins bind to nanoparticle protein corona ... · i o ed. supplemental data for:...
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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)
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References:
1. Frens,G.ControlledNucleationfortheRegulationoftheParticleSizein
MonodisperseGoldSuspensions.Nature241,20-22(1973).
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