Articles

https://doi.org/10.1038/s41565-018-0344-3

Immunoglobulin deposition on biomolecule

corona determines complement opsonization
efficiency of preclinical and clinical nanoparticles
Vivian P. Vu1, Geoffrey B. Gifford1, Fangfang Chen2, Halli Benasutti1, Guankui Wang1,3,
Ernest V. Groman1,3, Robert Scheinman3, Laura Saba4,5, Seyed Moein Moghimi 3,6,7
and Dmitri Simberg1,3*

Deposition of complement factors (opsonization) on nanoparticles may promote clearance from the blood by macrophages and
trigger proinflammatory responses, but the mechanisms regulating the efficiency of complement activation are poorly under-
stood. We previously demonstrated that opsonization of superparamagnetic iron oxide (SPIO) nanoworms with the third com-
plement protein (C3) was dependent on the biomolecule corona of the nanoparticles. Here we show that natural antibodies play
a critical role in C3 opsonization of SPIO nanoworms and a range of clinically approved nanopharmaceuticals. The dependency
of C3 opsonization on immunoglobulin binding is almost universal and is observed regardless of the complement activation
pathway. Only a few surface-bound immunoglobulin molecules are needed to trigger complement activation and opsonization.
Although the total amount of plasma proteins adsorbed on nanoparticles does not determine C3 deposition efficiency, the bio-
molecule corona per se enhances immunoglobulin binding to all nanoparticle types. We therefore show that natural antibodies
represent a link between biomolecule corona and C3 opsonization, and may determine individual complement responses to
nanomedicines.

A
dvances in the development of engineered nanomateri- of C5 convertase, which in turn cleaves the fifth complement pro-
als have resulted in several nanopharmaceuticals for clini- tein (C5) into C5a and C5b, thereby triggering activation of the ter-
cal use and many more in the clinical development stage1. minal pathway of the complement system. Both C3a and C5a are
Nevertheless, there are still important issues that need to be resolved, potent anaphylatoxins, which activate mast cells and other immune
including non-specific clearance of functional nanoparticles by dif- cells, and therefore induce proinflammatory reactions. Although
ferent immune cell types, and life-threatening infusion reactions the role of complement in infusion reactions to nanomedicine is
that occur in some patients receiving nanopharmaceuticals2. Better being debated11, there is a concern that uncontrolled complement
understanding of the immune mechanisms that lead to the clear- activation may affect the disease outcome and nanomedicine per-
ance and toxicities of nanomaterials in general, and of clinically formance in general12–14.
approved nanopharmaceuticals in particular, should be one of the As opsonization with C3 is the central event in complement cas-
primary tasks in nanomedicine research and development. Plasma cade and immune recognition, mechanisms of C3 deposition on
protein adsorption has long been debated to be an important fac- nanomedicines are of special interest for biological performance2.
tor affecting the functionality of intravenously injected nanomedi- Earlier, we found that C3b preferentially binds to the biomolecule
cines3–5. Recent studies have demonstrated the role of adsorbed corona on SPIO nanoworms in both plasma and serum. Moreover,
proteins in nanoparticle targeting6, immune responses7 and intra- C3 deposition was enhanced in the presence of the protein corona,
cellular toxicity8,9. Among the most interesting immunological con- underlying the dependence of the complement activation on other,
stituents of the protein corona formed around a nanoparticle are less characterized corona components15. SPIO is an important mag-
components of the complement system. Upon activation, all three netic resonance imaging contrast agent and a component of multi-
pathways (alternative, classical and lectin) of the complement sys- functional theranostic nanomedicines for imaging and treatment16,17.
tem converge at the point at which the third complement protein, Furthermore, we demonstrated a significant between-subject vari-
C3, is cleaved through assembly of C3 convertases, which gener- ability in levels of C3 deposition on SPIO nanoworms, as well as on
ate a common set of effector molecules including C3a and C3b2,10. clinically approved nanomedicines including the intravenous iron
The latter fragment covalently binds to activating surfaces, thereby supplement Feraheme, PEGylated liposomal doxorubicin LipoDox,
aiding their recognition by leukocytes and macrophages, a process and non-PEGylated liposomal irinotecan Onivyde18. We hypoth-
termed opsonization. C3b is further involved in surface assembly esize that complement activation efficiency by these nanomaterials

Translational Bio-Nanosciences Laboratory, The Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences,
1

University of Colorado Anschutz Medical Campus, Aurora, CO, USA. 2Department of Gastrointestinal Surgery, China–Japan Union Hospital, Jilin
University, Changchun, Jilin, China. 3Colorado Center for Nanomedicine and Nanosafety, University of Colorado Anschutz Medical Campus, Aurora, CO,
USA. 4Systems Genetics and Bioinformatics Laboratory, The Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical
Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA. 5Center for Translational Pharmacokinetics and Pharmacogenomics,
University of Colorado Anschutz Medical Campus, Aurora, CO, USA. 6School of Pharmacy, The Faculty of Medical Sciences, Newcastle University,
Newcastle upon Tyne, UK. 7Division of Stratified Medicine, Biomarkers and Therapeutics, Institute of Cellular Medicine, Newcastle University,
Newcastle upon Tyne, UK. *e-mail: [email protected]

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Articles NATure NAnOTeCHnOlOgy

is determined by differences in composition of the biomolecule of all complement pathways) inhibited C3 deposition by over 80%
corona. Among many plasma proteins, natural antibodies such (Fig. 1g; P <​ 0.0001). Previously, we demonstrated the involvement of
as immunoglobulin G (IgG) and immunoglobulin M (IgM) are the lectin pathway in complement activation by SPIO nanoworms28.
known to bind to foreign and self-antigens, and modulate comple- Nanoparticles can trigger complement activation through differ-
ment activation through the three complement pathways19–22. Here ent pathways in different subjects29, but notably immunoglobulins
we demonstrate that binding of only a few immunoglobulin mol- play a universal role in all tested samples regardless of the activation
ecules, and specifically IgG, determines the efficiency of C3 depo- pathway. Factor H (fH) is a negative serum regulator of comple-
sition on the above-mentioned nanomaterials in plasma and sera ment convertase that binds to C3b, promotes cleavage of C3b and
of healthy donors and in plasma of cancer patients, regardless of disassembly of the alternative pathway convertase. Genetic muta-
the complement activation pathway. Moreover, we demonstrate that tions in fH often lead to deficient binding and inhibition, and hence
the presence of the biomolecule corona enhances IgG binding to all differences in complement reactivity in general population (termed
tested nanomaterials. These results establish natural antibodies as a complotype)30. We measured binding of fH and C3 to SPIO nano-
critical factor that determines the efficiency of complement activa- worms in 12 healthy sera (7 females and 5 males). The amount of
tion in different subjects and by different nanomaterials, and could deposited C3 directly correlated with the amount of deposited fH
promote development of clinically approved nanoparticles with an (Fig. 1h; P =​ 0.0003), consistent with the ability of fH to bind the
improved safety profile. AP convertase. Addition of 100 µ​g ml−1 of purified fH to three sera
(Fig. 1i, dot blot) and three plasma samples (Supplementary Fig. 6,
Immunoglobulins promote C3 deposition Western blot) resulted in a minimal decrease in C3 opsonization of
We prepared SPIO nanoworms, ~110 nm hydrodynamic diameter, SPIO nanoworms, thereby ruling out the role of fH in the observed
coated with 20 kDa dextran (Fig. 1a and Supplementary Table 1), variability in C3 opsonization.
as described previously15,23. To understand the role of immuno- Feraheme is a clinically approved monocrystalline ultrasmall
globulins in C3 deposition on nanoparticles, we pretreated lepiru- SPIO coated with a reduced, negatively charged 10 kDa carboxy-
din plasma of three healthy donors with protein A sepharose beads. methyl dextran (Fig. 2a and Supplementary Table 1). Previously, we
Protein A strongly binds human IgG but weakly binds human IgM. demonstrated significant between-subject variability of C3 bind-
Following this pretreatment, there was minimal detectable level of ing to Feraheme18. C3 opsonization of Feraheme was significantly
immunoglobulin in plasma, but the quantities of C1q and C3 were decreased in three protein-A-depleted plasma samples (Fig. 2b and
close to physiological levels (Supplementary Fig. 1). According to Supplementary Fig. 2) and three sera (Fig. 2c; P =​  0.0029), and
Western blot analysis (Fig. 1b and Supplementary Fig. 2), the depo- reconstitution with polyclonal human IgG increased C3 opsoniza-
sition of C3 (predominantly iC3b) on nanoworms was decreased in tion in all plasma (Fig. 2b) and sera (Fig. 2c; P =​ 0.0315). In a healthy
all depleted plasma, and addition of polyclonal human IgG restored cohort of 12 sera (6 females and 6 males), the correlation between
C3 deposition. Compared with Western blot, dot blot is a more immunoglobulin and C3 binding to Feraheme was significant
quantitative assay to measure changes in the deposition of proteins (Fig. 2d; P =​ 0.002). C3 opsonization was not inhibited in three
on nanoparticle surfaces (Supplementary Fig. 3)15,18,23,24. Again, dot plasma samples treated with C1INH (Supplementary Fig. 7) and
blot analysis in sera of seven healthy donors (Fig. 1c) confirmed was not significantly inhibited in sera treated with EGTA/Mg2+
not only a significant decrease in C3 opsonization in depleted (Fig. 2e; P =​ 0.99). These data indicate that immunoglobulins trig-
sera (P <​ 0.0001) but also a significant increase in C3 opsonization ger C3 opsonization of Feraheme predominantly through the
(P <​ 0.0001) on addition of polyclonal human IgG. Because protein alternative pathway. Because Feraheme is administered as an iron
A can potentially deplete some IgM, we tested the ability of purified supplement to cancer patients31, we tested the effect of IgG on
polyclonal IgM to restore complement C3 deposition. Addition of C3 deposition in plasma from metastatic breast cancer patients.
IgM to three protein-A-depleted sera only marginally increased the Thus, we obtained matched lepirudin-anticoagulated and EDTA-
level of C3 deposited on SPIO nanoworms (Supplementary Fig. 4). anticoagulated plasma from a cohort of eight patients with breast
Also, addition of a monoclonal human IgG (trastuzumab) to the cancer (description of patients is in Supplementary Table 2).
depleted sera had no significant effect on C3 deposition (Fig. 1d). Depletion of immunoglobulins from lepirudin plasma significantly
Lastly, measurement of bound immunoglobulin and C3 in sera of decreased C3 opsonization of Feraheme (Fig. 2f; P =​  0.0016), and
12 healthy subjects (7 females and 5 males) showed a significant reconstitution with purified polyclonal IgG restored or increased
association between levels of immunoglobulin and C3 (Fig. 1e, complement activation in all samples (Fig. 2f; P <​  0.0001).
P =​ 0.0002). These data suggest that immunoglobulins, in particular There was little or no C3 detected on particles incubated in EDTA
IgG class, play an important role in the efficiency of complement C3 plasma (Fig. 2f).
deposition on SPIO nanoworms. Next, we tested the role of immunoglobulins in C3 opsonization
Surface-adsorbed antibodies can promote C3 opsonization of clinically approved liposomal nanomedicines. PEGylated liposo-
through all three complement pathways (Fig. 1f). The classical pathway mal doxorubicin (LipoDox, Doxil) is a ~100 nm liposome encap-
involves C1q binding to the Fc portion of closely adjacent antibodies25, sulating approximately 30,000 doxorubicin molecules (Fig. 3a and
whereas the lectin pathway may be triggered by binding of mannose- Supplementary Table 1) and has been used over 20 years for cancer
binding lectin, collectins and ficolins to sugar residues on IgG26,27. therapy32. C3 opsonization of LipoDox was significantly reduced in
In addition to these, IgG can enhance C3 deposition directly by act- three protein-A-depleted plasma (Fig. 3b and Supplementary Fig. 2)
ing as a scaffold for initial C3b binding and subsequently amplify and four sera (Fig. 3c; for dot blot P <​ 0.0001), and reconstitu-
C3b deposition through the alternative pathway turnover20,21. C3 tion with polyclonal human IgG restored C3 opsonization in both
deposition in the plasma of three donors (same donors as in Fig. 1b) plasma (Fig. 3b) and sera (Fig. 3c; P <​ 0.0001). In a cohort of 12
was not inhibited by the classical pathway inhibitor C1INH healthy sera (8 females and 4 males), there was a non-significant but
(Supplementary Fig. 5), suggesting minimal involvement of the suggestive association between levels of immunoglobulin and C3
classical pathway in these samples. Measurements of C3 deposi- (Fig. 3d; P =​ 0.062). C3 opsonization was not inhibited by C1INH
tion in a larger cohort of serum samples (n =​ 10 healthy donors) in three tested plasma samples (Supplementary Fig. 7), whereas
showed a significant decrease after treatment with EGTA/Mg2+ C3 opsonization was inhibited by 30–60% by EGTA/Mg2+ in five
(Fig. 1g; P <​ 0.0001) in 5 out of 10 sera, suggesting a role for cal- sera (Fig. 3e; P <​ 0.0001), suggesting that LipoDox triggers com-
cium-sensitive pathways in some of the samples, and predominantly plement activation in sera via the alternative pathway with some
the alternative pathway in other samples. EDTA (a global inhibitor involvement of calcium-dependent pathways. Involvement of the

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NATure NAnOTeCHnOlOgy Articles
a b c

Depl. + IgG

Depl. + IgG

Depl. + IgG
Non-depl.
Non-depl.

Non-depl.
Non-depl.

Non-depl.
Non-depl.
Depleted

Depleted

Depleted
SPIO NWs

+ EDTA

+ EDTA

+ EDTA
P < 0.0001 P < 0.0001

600

(Western blot)
75 kDa

Bound C3 (a.u.)
Bound C3
20 kDa dextran
400

200
25 kDa

!!!!!!!!100 nm 0
M61 F28 M56 C3b

ed
ed

G
Ig
t
et

le

+
pl

ep
e

ed
D
-d

t
le
on

ep
N

D
d e f
Lectin pathway
P = 0.0762
NP
P < 0.0001 Alternative
2,000 12.4 Ca2+ Sugars pathway
log10 molecules per dot

P = 0.0002 Classical MBL / ficolin / collectin

Bound C3 (a.u.)

1,500 12.2 pathway

MASP-1/2/3 C3 fB

Mg2+ fD
C3

1,000 12.0 NP
P
Ca2+ C4 Bb
500 11.8
C3b
C4b C2a
C1q fH,I
0 11.6 C2
NP
9.8 10.0 10.2 10.4 10.6 10.8
d

ab +
te

C1INH
Ig

um d
le

uz te

C3b C3b
+

Immunoglobulin
ep

st ple
d
D

te

tra e

log10 molecules per dot

le

D
ep
D

g h i
P < 0.0001
P = 0.0003 P = 0.006
800 P < 0.0001 P < 0.0001 12.2 800
log10 molecules per dot
Bound C3 (a.u.)

Bound C3 (a.u.)

600 12.0 600

C3

400 400
11.8

200 200
11.6
0 0
2+

TA
l

10.62 10.68 10.74 10.80

d
tro

tro

de
g

ED
on

/M

on

ad

fH
C

TA

log10 molecules per dot

EG

or
ct
Fa

Fig. 1 | Role of immunoglobulins in efficiency of C3 deposition on SPIO nanoworms. a, Schematic representation of SPIO nanoworms. Yellow indicates
dextran chains and brown represents iron oxide nanocrystals. b, Immunoglobulin depletion from plasma samples with protein A beads decreases
complement C3 deposition, and addition of polyclonal human IgG restores C3 deposition. The absence of α​′​chain (110 kDa) on SPIO nanoworms is due to
cleavage by factor I into iC3b/C3dg/C3c fragments. These experiments were repeated independently three times in three different plasma samples with
similar results. c, Depletion experiments as in b were performed in sera of healthy donors (n =​7 subjects), and C3 was analysed in a dot blot assay. Each
dot represents the mean of three technical replicates per sample. d, Addition of trastuzumab (monoclonal human IgG) to depleted sera (n =​ 4 subjects)
did not restore C3 deposition. Each dot represents the mean of three technical replicates per sample. e, Association between levels of immunoglobulin
and C3 on SPIO nanoworms in a cohort of healthy donors (n =​12 subjects) measured with dot blot assay. Red points indicate male samples; blue points,
female samples. Each point represents the mean of three technical replicates. f, Schematic diagram showing how nanoparticle-bound immunoglobulins
may trigger complement activation. These include the classical pathway by C1q binding to the Fc portion of surface-bound antibodies, the lectin pathway
via MBL/MASP-2 binding to glycosylated regions of antibodies and the alternative pathway, by deposition of antibody-bound C3. Natural inhibitors of
complement are shown as black objects with red arrows. g, Nanoparticles were incubated in sera supplemented with EGTA/Mg2+ (inhibitor of the classical
and lectin pathways) and EDTA (inhibitor of all pathways), and C3 deposition was measured with dot blot. In c and g, same colour refers to the same
individual (n =​10 subjects). Each dot represents the mean of three technical replicates per sample. h, Samples (as in e) were probed for fH binding.
fH is a serum inhibitor of convertase that binds to C3b. There was a positive association between levels of bound inhibitor and C3 in sera of healthy
donors (n =​12 subjects). Red points, male samples; blue points, female samples. Each dot represents the mean of three technical replicates per sample.
i, Addition of purified fH (100 µ​g ml−1) to sera with high and intermediate complement activation (n =​3 subjects) only minimally decreased C3 deposition
as measured with dot blot, suggesting that fH deficiency is not responsible for high level of C3 deposition. Each dot represents the mean of three technical
replicates per sample. In all experiments, lines connecting points indicate measures from the same subject. Colours designate different individuals.
The statistical analysis is explained in the Methods. a.u., arbitrary units; NP, nanoparticle; NW, nanoworm.

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Articles NATure NAnOTeCHnOlOgy

a b c

Depl. + IgG

Depl. + IgG

Depl. + IgG
Feraheme

Non-depl.
Non-depl.

Non-depl.
Non-depl.

Non-depl.
Non-depl.
Depleted

Depleted

Depleted
+ EDTA

+ EDTA

+ EDTA
O
O

H O P = 0.0029 P = 0.0315
H O
HO
HO
H OH
H 300
H
HO

(Western blot)

Bound C3 (a.u.)
O H O 75 kDa

Bound C3
O
O
HO
H OH
H 200
H OH

10 kDa CM dextran
100
25 kDa
0
100 nm F28 M61 M56 C3b

G
te

te

Ig
le

le

+
ep

ep

d
-d

te
le
on

ep
N

D
d e P < 0.0001 f
P= P<
P = 0.99 P < 0.0001 0.0016 0.0001 51
29
11.50 P = 0.0005 200 500
log10 molecules per dot

49
43
Bound C3 (a.u.)

Bound C3 (a.u.)
150 400
40
11.25 300 39
C3

100 37
200 33
50
11.00 100
0 2+ 0
9.2 9.4 9.6 9.8 10.0 10.2
l

TA
tro

TA
g

te

te

Ig
ED
/M
on

ED
le

le
Immunoglobulin

+
TA

ep

ep
C

d
-d

te
log10 molecules per dot
EG

le
on

ep
N

D
Lepirudin plasma

Fig. 2 | Role of immunoglobulins in efficiency of C3 deposition on clinically approved SPIO Feraheme. a, Schematic representation of Feraheme structure.
CM, carboxymethyl. b, Effect of immunoglobulin depletion and reconstitution in plasma of healthy donors. These experiments were repeated independently
three times in three different plasma samples with similar results. c, Effect of immunoglobulin depletion in sera of healthy donors (n =​3 subjects) measured
with dot blot. Each dot represents the mean of three technical replicates per sample. d, Correlation between levels of immunoglobulins and C3 bound to
Feraheme in healthy sera (n =​12 subjects). Red points, male samples; blue points, female. Each point is the mean of three experimental replicates and three
technical replicates per sample. e, Feraheme was incubated in sera with EGTA/Mg2+ or EDTA (n =​5 subjects). In c and e, same colour refers to the same
individual. Each dot represents the mean of three technical replicates per sample. f, Immunoglobulin depletion and reconstitution in lepirudin-anticoagulated
plasma from breast cancer patients (n =​8 subjects). Each dot represents the mean of three technical replicates per sample. EDTA-anticoagulated
plasma was from the same patient. In all graphs, lines connecting points indicate measures from the same subject. Colours designate different individuals.
Numbers in the legend are patient identifiers (Supplementary Table 2). The statistical analysis is explained in the Methods.

alternative and the lectin pathways in complement activation by suggesting that immunoglobulins trigger complement predomi-
PEG has been reported before33. In the cohort of lepirudin plasma nantly by the alternative pathway. In the cohort of lepirudin plasma
from eight breast cancer patients, depletion of immunoglobulins from eight breast cancer patients, depletion of immunoglobulins
significantly decreased C3 opsonization of LipoDox in all samples significantly decreased C3 opsonization of Onivyde in all samples
(Fig. 3f; P <​ 0.0001), and C3 deposition was increased or com- (Fig. 3l; p <​ 0.0001), and C3 deposition was increased upon IgG
pletely restored upon IgG reconstitution in five out of eight samples reconstitution in seven out of eight samples (Fig. 3l; p <​  0.0001).
(Fig. 3f; P =​ 0.0199). Notably, complete restoration of complement We previously found that C3 covalently binds to adsorbed serum
activation in samples 29 and 51 was achieved with a different batch proteins on the nanoparticle surface15. To determine whether C3
of polyclonal IgG, suggesting that this batch contained IgG clones attacks surface-adsorbed immunoglobulins, protein coronas formed
that could efficiently activate complement. in six healthy sera were eluted from SPIO nanoworms with 2%
Onivyde is a 120 nm non-PEGylated liposome internally loaded with SDS buffer, and analysed on a non-reducing gel (protein–C3 com-
~70,000 molecules of irinotecan (Fig. 3g and Supplementary Table 1), plexes should run as high-molecular-weight bands)35. In most
and was recently approved for gastrointestinal cancers34. Onivyde of the sera, the eluted immunoglobulin was localized in a high-
showed significant between-subject variability of C3 opsonization18. molecular-weight fraction (300–500 kDa) that co-localized with C3
C3 opsonization of Onivyde was decreased in two out of three pro- (Fig. 4a, upper arrow), consistent with C3–IgG complexes as
tein-A-depleted plasma samples (Fig. 3h and Supplementary Fig. 2) described previously36. In some samples, free IgG was also present
and in three depleted sera (Fig. 3i; P =​ 0.0018), and reconstitution (Fig. 4a, lower arrow). A similar result was obtained using lepirudin
with polyclonal human IgG restored or increased C3 opsonization plasma samples from five healthy donors (Supplementary Fig. 8).
in tested plasma samples (Fig. 3h) and sera (Fig. 3i; P =​  0.0253). In The fact that the majority of eluted C3 did not colocalize with IgG
a cohort of 12 healthy sera (5 females and 7 males), there was a sig- suggests that IgG serves as a trigger for C3 opsonization, rather than
nificant association between levels of bound immunoglobulin and a sole scaffold for C3 binding. According to quantitative stoichio-
C3 (Fig. 3j; P =​ 0.0003). C1INH did not decrease C3 opsonization in metric analysis of IgG and C3 binding in 12 sera (Fig. 4b), there
all the plasma samples (Supplementary Fig. 7), whereas EGTA/Mg2+ are significantly more C3 molecules than IgG molecules per SPIO
partially decreased it in two out of five sera (Fig. 3k; P =​  0.0264), nanoworm (C3/IgG ratio 51 ±​  17; P <​  0.0001, two-sided t-test),

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NATure NAnOTeCHnOlOgy Articles
a b c

Depl. + IgG

Depl. + IgG

Depl. + IgG
Non-depl.
Non-depl.

Non-depl.
Non-depl.

Non-depl.
Non-depl.
Depleted

Depleted

Depleted
+ EDTA

+ EDTA

+ EDTA
LipoDox
P < 0.0001 P < 0.0001
300

(Western blot)

Bound C3 (a.u.)
75 kDa

Bound C3
200

25 kDa 100

100 nm
0
M56 M61 F28 C3b

G
d
ed

te

Ig
et

le

+
pl

ep

ed
de

et
-
on

l
ep
N

D
P < 0.0001
d e f
P < 0.0001 P < 0.0001 P < 0.0001 P = 0.0199
P = 0.062 51
29
log10 molecules per dot

11.50 600 1,000

49

Bound C3 (a.u.)

Bound C3 (a.u.)
11.45 800 43
11.40 400 40
600 39
C3

11.35 37
400
11.30 200 33
200
11.25
0 0

2+

TA
11.1 11.2 11.3

TA
ro

te

te
g

Ig
ED
t

ED
/M
on

le

le

+
Immunoglobulin

ep

ep
TA
C

d
-d

te
log10 molecules per dot
EG

le
on

ep
N

D
Lepirudin plasma

g h i
Depl. + IgG

Depl. + IgG

Depl. + IgG
Non-depl.
Non-depl.

Non-depl.
Non-depl.

Non-depl.
Non-depl.
Depleted

Depleted

Depleted
+ EDTA

+ EDTA

+EDTA
Onivyde P = 0.0018 P = 0.0253
400

Bound C3 (a.u.)
300
(Western blot)
75 kDa
Bound C3

Irinotecan
200

25 kDa 100
100 nm
0

G
d

d
te

Ig
te
F28 M56 M61 C3b
le

le

+
ep

ep

d
te
-d

le
on

ep
N

j k P < 0.0001 l
P = 0.0264 P < 0.0001
P = 0.0003 P < 0.0001 P < 0.0001 51
29
log10 molecules per dot

12.0 250 500

49
Bound C3 (a.u.)
Bound C3 (a.u.)

200 400 43
40
11.8 150 300 39
C3

37
100 200
33
11.6
50 100
0 0
10.6 10.8 11.0 11.2 11.4 11.6
2+
l

TA
tro

TA
te

te

Ig
g
on

ED

ED
M

le

Immunoglobulin
+
pl
C

ep
TA

de

ed

log10 molecules per dot

D
EG

et
on

l
ep
N

Lepirudin plasma

Fig. 3 | Role of immunoglobulins in efficiency of C3 deposition on clinically approved liposomes. a, Schematic representation of LipoDox (a generic
version of Doxil, a regulatory approved liposomal doxorubicin). b, Effect of immunoglobulin depletion and reconstitution in plasma samples of healthy
donors. These experiments were repeated independently three times in three different plasma samples with similar results. c, Effect of immunoglobulin
depletion and reconstitution in healthy sera (n =​4 subjects), measured with dot blot. Each dot represents the mean of three technical replicates per
sample. d, Correlation between levels of immunoglobulin and C3 bound to LipoDox in healthy sera (n =​12 subjects). Red points, male samples; blue points,
female. Each point is the mean of three experimental and three technical replicates per sample. e, Nanoparticles were incubated in serum with EGTA/
Mg2+ or EDTA (n =​5 subjects). In c and e, same colour refers to the same individual. Each dot represents the mean of three technical replicates per sample.
f, Immunoglobulin depletion and reconstitution in lepirudin-anticoagulated plasma from breast cancer patients (n =​8 subjects). Each dot represents
the mean of three technical replicates per sample. EDTA-anticoagulated plasma was from the same patient. Numbers in the legend in f and l are patient
identifiers (Supplementary Table 2). g, Schematic representation of Onivyde, a clinically approved liposomal irinotecan. h, Effect of immunoglobulin
depletion and reconstitution in plasma of healthy donors. These experiments were repeated independently three times in three different plasma samples
with similar results. i, Effect of immunoglobulin depletion and reconstitution in healthy sera (n =​3 subjects). Each dot represents the mean of three
technical replicates per sample. j, Association between levels of immunoglobulin and C3 bound to Onivyde in healthy sera (n =​12 subjects). Red points,
male samples; blue points, female. Each point is the mean of three experimental and three technical replicates per sample. k, Effect of EGTA/Mg2+ or
EDTA on C3 deposition in healthy sera (n =​5 subjects). In i and k, the same colour refers to the same individual. Each dot represents the mean of three
technical replicates per sample. l, IgG depletion and reconstitution in lepirudin-anticoagulated plasma from breast cancer patients (n =​8 subjects). Each
dot represents the mean of three technical replicates per sample. EDTA-anticoagulated plasma was from the same patient. In all graphs, lines connecting
points indicate measures from the same subject. Colours designate different individuals. The statistical analysis is explained in the Methods.

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Articles NATure NAnOTeCHnOlOgy

a b
1,000

100
IgG C3b

C3/IgG
C3b 10
C3b

1
250
0.1
150

IO

de

ox
m
SP

D
vy
75

he

po
ni
ra

Li
Fe
50

IgG/NP C3/NP C3/IgG

Non-reducing SDS-PAGE SPIO NW 0.6 24.8 51.3
Feraheme 0.004 0.15 51.7
LipoDox 1.7 3.3 1.9
Onivyde 6.5 22.4 4.2

Fig. 4 | Association between immunoglobulin and C3 in the protein corona. a, Protein corona formed on SPIO nanoworms (six healthy donor sera) was
eluted with 2% SDS and analysed by non-reducing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The membrane was probed
with anti-C3 (IRDye 800, green) and anti-IgG (IRDye 680, red). IgG–C3 complexes in high-molecular-weight fraction are shown by the upper arrow.
Free IgG is shown by the lower arrow. IgG and C3b standards were run in parallel (left two lanes). Note the presence of several high-molecular-weight bands
for C3, suggesting binding to other corona proteins. The bulk of C3 is not bound to IgG. In the rightmost lane, there are more IgG–C3 complexes and also more
total C3, whereas the leftmost serum lane shows the opposite. The experiment was repeated three times in serum and once in plasma (Supplementary Fig. 7).
b, Stoichiometry calculations show more C3 than IgG molecules bound per nanoparticle. Feraheme shows the highest ratio, whereas LipoDox shows the
lowest ratio. Note that for Feraheme, not every nanoparticle contains C3 and IgG molecules. For other nanoparticle types, there are few IgG molecules per
nanoparticle. The results represent means ±​ s.d. of n =​12 sera for every nanoparticle type. Each dot represents the mean of the technical replicates.

per Feraheme (C3/IgG ratio 51.7 ±​  25.6; P <​ 0.0001, two-sided t-test), proteins and lipoproteins did not enhance or decrease the C3
per LipoDox (C3/IgG ratio 1.9 ±​  0.35; P <​  0.0001, two-sided t-test) deposition (Fig. 5d).
or per Onivyde (C3/IgG ratio 4.2 ±​  2.1; P <​  0.0001, two-sided t-test). The hallmark of natural antibodies is their ability to recognize
Collectively, the results suggest that just a few surface-bound self-epitopes on proteins, especially the denatured species41. We ques-
immunoglobulin molecules are needed to trigger complement tioned whether protein corona aids the binding of IgG to nanopar-
C3 opsonization. ticle surfaces. Purified human IgG was added to PBS, 50 mg ml−1
human serum albumin or protein-A-depleted sera (three healthy
Biomolecule corona promotes IgG binding to nanoparticles donors), and SPIO nanoworms, Feraheme, LipoDox or Onivyde
We questioned whether other corona proteins besides immuno- were added next. As shown in Fig. 5e,f and Supplementary Fig. 11,
globulin play a role in C3 opsonization. We incubated SPIO nano- in PBS there was minimal binding of IgG to all nanoparti-
worms with sera of six healthy donors and measured the total cle types, except for LipoDox. Importantly, IgG did not bind
amount of bound protein. The amount of bound C3 did not cor- to any nanoparticle type in presence of serum albumin,
relate with the amount of non-C3 protein in the coronas (Fig. 5a; whereas IgG showed efficient binding in the presence of protein
Pearson r2 =​  0.163; P =​ 0.4). Coronas with higher C3/particle density corona (Fig. 5e,f).
also contained a higher percentage of C3 (Supplementary Fig. 9).
These results clearly demonstrate that total bound protein does not Conclusions
determine the efficiency of C3 opsonization. At the same time, in In summary, we have demonstrated that in multiple sera and plasma
the same six sera, the amount of C3 showed correlation with pro- samples, natural antibodies play the critical role in complement
perdin, which is a positive regulator of the alternative pathway con- opsonization of diagnostic nanoparticles and clinically approved
vertase37 (Supplementary Fig. 10). Shotgun proteomics of protein nanomedicines, regardless of the activation pathway. More spe-
corona of SPIO nanoworms in six sera (Fig. 5b) and five lepirudin cifically, antibody binding to nanoparticles was dependent on the
plasma samples (Fig. 5c) showed enrichment with apolipoproteins, biomolecule corona and the binding of a few antibody molecules
albumin, complement factors, clotting cascade factors, fibronectin, was sufficient to trigger complement activation. Experiments with
immunoglobulins, carrier proteins and other regulatory proteins. depleted plasma samples and sera showed that in some samples the
These proteins have been reported to bind to nanoparticles38–40. C1q levels of C3 could not be restored to the original levels on addition
was not detected on any corona, thereby excluding possible involve- of polyclonal IgG (although sufficient quantities of C3 were pres-
ment of the classical pathway in complement activation. However, ent in such media). Indeed, non-specific deposition of antibodies
the corona obtained from sera incubations showed presence of may not necessarily trigger complement activation through any of
MBL-C and/or MASP-1 (where MBL is mannose-binding lectin and the three complement pathways. In the case of the alternative path-
MASP is MBL-associated serine protease). To test the role of key way, C3b attack and the convertase assembly may strictly depend
corona proteins in complement activation, SPIO nanoworms were on specific antibody–epitope binding, where a few antibodies could
briefly preincubated with fibrinogen, fibronectin, albumin, IgG, trigger the process. Thus, the aforementioned results are likely to
apolipoprotein B-100 (Apo-B), high-molecular-weight kininogens be due to different titre and/or absence of some specific immuno-
(HMWK), high-density lipoproteins (HDL), low-density lipo- globulin molecules in the polyclonal preparation and specific gly-
proteins (LDL), factor H and properdin, and then added (without cosylation patterns capable of triggering complement in the context
washing step) to sera of healthy donors (Fig. 5d). IgG promoted a of the adsorbed biomolecule corona. In terms of complement acti-
three-fold increase in complement C3 deposition compared with vation, this introduces considerable variability due to nanoparticle
non-preincubated particles (P <​ 0.0001). Fibronectin induced a surface heterogeneity, surface defects and solvation patterns within
minor increase in C3 deposition (P <​ 0.0001), whereas other tested a particular batch42, which in turn may control not only the extent of

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NATure NAnOTeCHnOlOgy Articles
a b Serum c Plasma
Apolipoprotein B-100 Complement C3*
Complement C3* Serum albumin*
Serum albumin* Immunoglobulin heavy constant mu*
Apolipoprotein E* Filamin-A
Immunoglobulin heavy constant mu* Talin-1
Mannose-binding protein C Plasma kallikrein*
Keratin, type I cytoskeletal 10 Kininogen-1*
Clusterin* Apolipoprotein E*
Apolipoprotein(a)* Properdin*
Alpha-2-macroglobulin Fibrinogen alpha chain

40 P = 0.426 0 400 800 Coagulation factor X

Kininogen-1*
Integrin alpha-IIb
Clusterin*
Confidence Keratin, type I cytoskeletal 9* Actin, cytoplasmic 2*
C3 molecules per NP

score Serotransferrin Immunoglobulin lambda constant 3*

Alpha-2-HS-glycoprotein Immunoglobulin heavy constant gamma 3*

30 Immunoglobulin heavy constant gamma 2*

Immunoglobulin kappa light chain*
Immunoglobulin heavy constant alpha 1*
Vinculin
Immunoglobulin heavy constant gamma 3* Complement C5
Mannan-binding lectin serine protease 1 Fibrinogen gamma chain
Immunoglobulin heavy constant gamma 1 Immunoglobulin heavy constant gamma 2*
20 Immunoglobulin lambda constant 3*
Immunoglobulin heavy constant alpha 1*
Integrin beta-3*
Erythrocyte band 7 integral membrane protein*
Actin, cytoplasmic 1 Myosin-9
Keratin, type II cytoskeletal 2 epidermal Immunoglobulin kappa light chain*
Vitronectin 14-3-3 protein zeta/delta
10 Haemoglobin subunit beta*
Prothrombin
Complement C4-A
Thrombospondin-1
Fibronectin* Complement component C7
Apolipoprotein C-III* Keratin, type I cytoskeletal 9*
Complement component C9* Platelet glycoprotein 4
0 Keratin, type II cytoskeletal 1
CD5 antigen-like
Immunoglobulin alpha-2 heavy chain
Haemoglobin subunit beta*
Haemoglobin subunit alpha* Fibrinogen beta chain*
0

7
8

Fibrinogen beta chain* Complement factor B*

–1

–1

–1
–1
0.

Apolipoprotein A-I Tubulin beta-1 chain

Integrin beta-3* Apolipoprotein A-IV*
10

10

10
10

Properdin* Complement component C9*

×

×
×

Haptoglobin Immunoglobulin J chain

Alpha-1-antitrypsin
0

0
0

Fibronectin*
1.

1.

2.
5.

Plasma kallikrein* L-lactate dehydrogenase A chain

Inter-alpha-trypsin inhibitor heavy chain H4 Complement component C8 alpha chain
Complement factor B* Haemoglobin subunit alpha*

Non-C3 protein per NP (g) Erythrocyte band 7 integral membrane protein*

Apolipoprotein A-II
Caveolae-associated protein 2
Immunoglobulin kappa variable 3-20
Alpha-2-antiplasmin Sarcoplasmic/endoplasmic reticulum calcium ATPase 3
Actin, cytoplasmic 2* Beta-2-glycoprotein 1
Protein AMBP Apolipoprotein C-III*
Antithrombin-III Apolipoprotein(a)*
Apolipoprotein A-IV* Coagulation factor XI
Apolipoprotein C-II Integrin beta-1

#1 #2 #3 #4 #5 #6

#7

#8

#9

1
#1

#1
e e e e e e
pl pl pl pl pl pl

e
pl

e
pl

pl

e
m m m m m m

pl

pl
m

m
Sa Sa Sa Sa Sa Sa

m
Sa

Sa

Sa

Sa

Sa
d e f

Wash,
measure C3
G

Add to
m d

m d

G
Ig

G
ru ete

ru ete

Preincubate
Ig
Ig

serum without
+
se epl

se epl

+
+

with protein
SA
S

washing
D

PB

P < 0.0001
H

400 P < 0.0001 SPIO NWs 400 SPIO NW LipoDox

Feraheme Onivyde
C3 (% of control)

300
Bound IgG (a.u.)

Feraheme 300

200 200
LipoDox
100 100
Onivyde
0 0
Nanoparticle-bound IgG M37 M40 M43 PBS+ HSA+
C IgG
H L
H L
Fi A SA
Ki ne B

l
er H
n
in n

Pr cto n
Fi ino tin

tro

Depleted sera + IgG IgG IgG

LD
D

di
br ge
Fa ge
o o

op r
n c
br p

on
o

Fig. 5 | Role of biomolecule corona in C3 and IgG deposition. a, Lack of correlation between the amount of total adsorbed (non-C3) protein and C3 on
SPIO nanoworms in sera (n =​6 subjects, repeated three times, Pearson correlation, two-sided t-test). NP, nanoparticle. b, Heat map of top 50 proteins
identified in coronas formed on SPIO nanoworms in sera (n =​ 6 subjects). c, Heat map of top 50 proteins identified in coronas formed on SPIO nanoworms
in plasma (n =​5 subjects). Proteins are ranked by the confidence score determined by the number of peptides (coverage) and the quality of the MS spectra.
The asterisk indicates that the protein was identified within the top 50 of both serum and plasma samples. d, Some of the proteins and lipoproteins
identified with proteomics were preincubated with nanoparticles, and the latter were added to sera without washing. Percentage change relative to buffer-
preincubated particles is shown. C3 deposition was significantly increased (P <​0.0001) after preincubation with IgG and fibronectin. (n =​3 sera, each dot
is the mean value and SD of three technical replicates). e, A representative dot blot showing nanoparticle binding of purified polyclonal IgG added either to
protein A-depleted serum or PBS or 50 mg ml−1 human serum albumin (HSA). Final IgG concentration was 5.5 mg ml−1 in all samples. The experiment was
reproduced twice. f, Densitometry quantification (integrated density) of binding of IgG to SPIO nanoworms, Feraheme and Onivyde shows that protein
corona enhances IgG binding compared with PBS or HSA. LipoDox showed significant binding in PBS and depleted sera, but no binding in presence of
HSA. Residual IgG binding in depleted sera was subtracted from IgG-reconstituted sera to obtain the ‘depleted serum +​IgG’ value. Labels designate serum
samples. M, male; number indicates age in years. Bars show mean values and s.d. of three technical replicates. The experiment was repeated twice.
The statistical analysis is explained in the Methods.

biomolecule deposition and retention, but also their projected con- of complement activation. Protein A is known to deplete serum/
formation and hence epitope exposure, which becomes the rate-lim- plasma from IgG1, IgG2, IgG3 and to some extent IgM43. Indeed,
iting factor in triggering alternative pathway activation. Therefore, binding of naturally occurring IgM antibodies against a com-
between-subject variability might be due to subtle conformational ponent of the pristine surface (for example, anti-PEG and anti-
differences in repertoire of nanoparticle-bound biomolecules and dextran IgM)44 could trigger complement activation through
concomitant presence of a specific immunoglobulin titre against the the classical pathway. Among the tested cohort of donors and
exposed antigenic epitope. nanoparticles, however, we excluded a major role of the classical
Although we demonstrated the role of IgG in complement acti- pathway in complement activation.
vation by nanomedicines, we did not identify the responsible IgG We excluded a direct and predominant IgG binding to the pristine
subclasses and cannot exclude the role of other classes of immu- surfaces, but the epitopes responsible for IgG docking are not known.
noglobulins, which could play an important role in the efficiency However, in the case of liposomes, earlier studies have suggested

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Articles NATure NAnOTeCHnOlOgy

C3b C3 corona can open new opportunities to improve haemocompatibility

and safety of nanomedicines.

Online content
C3b C3b Bb
Any methods, additional references, Nature Research reporting
C3b C3b P summaries, source data, statements of data availability and asso-
C3b
ciated accession codes are available at https://doi.org/10.1038/
s41565-018-0344-3

C3b Received: 7 April 2018; Accepted: 5 December 2018;

Published: xx xx xxxx
C3b
C3b References
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Postdoctoral Council. S.M.M. acknowledges support by International Science and
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Sci. Rep. 4, 5020 (2014). s41565-018-0344-3.
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nanoparticle pathophysiology. Nat. Nanotech. 8, 772–781 (2013). Correspondence and requests for materials should be addressed to D.S.
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Articles NATure NAnOTeCHnOlOgy

Methods 1:3 volume ratio with serum or plasma for 30 min in a 37 °C water bath. Following
Materials. Linear dextran (15–25 kDa molecular weight) and iron salts were incubation, samples were washed 5 times with PBS at 4 °C using ultracentrifuge
purchased from Sigma-Aldrich. Purified C3 (A113), fH (A137), C3b (A114), as described above for proteomic studies. To determine the amount of specific
iC3b (A115), C1 inhibitor (A140) and properdin (A139) were purchased from proteins bound to nanoparticles, a quantitative immuno dot blot assay was used
Complement Technology. All proteins were aliquoted at 1 mg ml−1 and stored at −​ as described15,18,24,28. Briefly, the pellets were resuspended in PBS, and 2 µ​l of each
80 °C. Every protein was exposed to no more than two freeze–thaw cycles. Purified sample was pipetted in triplicates onto a nitrocellulose membrane (0.45 µ​m pore,
human IgG (009–000–003, lots 137386, 134667, 138240), IgM (009–000–012, Bio-Rad). Standard 2-fold dilutions of purified protein standards were applied
lot 132713) and AffiniPure goat anti-human IgM (Fc5µ​specific; 109–005–129, to the same membrane (2 µ​l dots in triplicate). Calibrated 2 µ​l volume Eppendorf
lot 132466) were purchased from Jackson Immuno Research Laboratories. Goat pipette was used for dot application. The membranes were blocked with a blocking
anti-human C3 (55117, lot 07314) was from MP Biomedicals. Murine monoclonal buffer of 5% w/w nonfat dry milk in PBS-T (1x PBS and 0.1% v/v Tween-20)
anti-human C1 (A201, lot 062984) was from Quidel. Anti-human fH (A237, lot 4) for 1 h at room temperature, probed with the primary antibody for 1 h at room
and anti-factor P (A239, lot 4a) were from Complement Technology. C1q depleted temperature, followed by washes (three times) with PBS-T. Lastly, the membranes
human serum (A509, lot 100698) was from Quidel. Secondary donkey anti-goat were probed with corresponding secondary antibody labelled with IRDye 680 or
IRDye 800CW (926–32214, lot C80207–07) and goat anti-human IRDye 680CW IRDye 800 (1:20,000 in blocking buffer) for 1 h at room temperature and washed
(9266807, lot C7072015) were purchased from Li-COR Biosciences. Purified three times with PBS-T. The membranes were scanned using the Odyssey infrared
low-density and high-density lipoproteins were purchased from Athens Research. imager (Li-COR Biosciences) at either 700 nm or 800 nm depending on the label
Human serum albumin was obtained from Gemini Bioproducts. Fibronectin was of secondary antibodies. For dot blot quantification, the background of 16-bit
purchased from Upstate Cell Signaling. Feraheme (lot 10021802 and 10051302) was greyscale images was subtracted and integrated densities of the dots were measured
provided by N. Serkova, Department of Radiology, University of Colorado. Onivyde with ImageJ software. The number of protein molecules per dot was determined
(lot 1501279 A), LipoDox (lot 500592 and 500546) and Herceptin (lot 569330) from a standard curve of a protein dotted on the same membrane. Number of
were provided as sterile leftovers after administration to patients (free of charge) protein molecules per mg nanoparticle was calculated by dividing the number of
by the UC Denver Cancer Center Pharmacy. All liposomes were stored in original molecules/dot by the amount of nanoparticles (mg Fe or mg drug) applied per dot.
sterile vials at 4 °C before use. Herceptin (trastuzumab) was dialysed against PBS The previously calculated molar concentrations of nanoparticles and liposomes per
and stored in frozen aliquots at −​20 °C. Fibrinogen was purchased from Sigma- mg Fe or drug18 were used.
Aldrich. High-molecular-weight kininogen was provided by K. McCrae (Lerner For non-reducing Western blot, proteins were eluted from washed nanoparticle
Research Institute, Cleveland, OH). Protein A Sepharose CL-4B (dry beads) was pellet with 2% SDS in PBS for 1 h at room temperature, the particles were pelleted
from Sigma-Aldrich. All human studies were conducted in accordance with ethical with ultracentrifuge, the supernatant was mixed with Bio-Rad sample buffer
guidelines. Anonymous human sera and plasma from consented healthy donors without beta-mercaptoethanol and separated on a 4–20% Tris-Glycine SDS-PAGE.
(males and females, average age 47 ±​ 16 years) were obtained from the University For reducing Western blot, nanoparticles after washing were resuspended in
of Colorado Blood Donation Center using ‘no anticoagulant’ Z vacutainer tubes reducing sample buffer, boiled for 2 min at 95 °C and separated on a 4–20% Tris-
(BD) for sera, or Multiplate Hirudin (lepirudin) blood tubes (DiaPharma). Plasma Glycine SDS-PAGE. The proteins were transferred to nitrocellulose membrane, and
from consented breast cancer patients were obtained at the University of Colorado processed as described for dot blot assay above. For detection of C3 and IgG on
Cancer Center using K2EDTA vacutainer tubes (BD) and lepirudin tubes. The the same membrane, first C3 was detected with IRDye-800CW-labelled antibody,
plasma was collected under Colorado Multiple Institutional Review Board- then the membrane was washed and IgG was detected with IRDye-680-labelled
approved protocol 16–0610. Sera were processed as described by us previously18 antibody. Both dyes were detected by scanning membrane at 700 and 800 nm with
and stored in frozen aliquots at −​80 °C. Plasma was obtained by centrifugation of Li-COR Odyssey.
K2EDTA or lepirudin tubes at 5,000g for 20 min at room temperature immediately
after the collection and stored in frozen aliquots at −​80 °C. Preincubation of proteins with SPIO nanoworms. Purified protein solutions
in PBS or in the vendors’ buffers (kininogen 1 mg ml−1, fibronectin 1 mg ml−1,
SPIO nanoworm synthesis. Large SPIO nanoworms were synthesized from fibrinogen 1 mg ml−1, factor H 1 mg ml−1, properdin 1 mg ml−1, human serum
15–25 kDa dextran, Fe(iii) chloride and Fe(ii) chloride in a modified Molday albumin 50 mg ml−1, Apo-B100 1.9 mg ml−1, IgG 11.6 mg ml−1, HDL 1 mg ml−1 and
precipitation method in ammonia50 as described by us previously28. The ratio LDL 1 mg ml−1), or control PBS or the corresponding proteins’ buffer in the same
between dextran and iron salts determined the final size of the nanoparticles28. volume were preincubated with SPIO nanoworms (1 mg ml−1 in PBS) at a 1:1 v/v
Particles were resuspended in sterile water, filtered through 0.45 µ​m filter and ratio for 15 min at room temperature. Following preincubation, serum was added
stored at 4 °C. Size (intensity weighted diameter) was determined using Zetasizer to the nanoparticles at a 3:2 volume ratio, and the nanoparticles were incubated,
Nano ZS (Malvern Instruments). At least two batches were used in the study washed and assayed for bound C3 as described for dot blot assay. The experiments
(Supplementary Table 1). were performed in three different sera for each tested protein.

Label-free mass spectrometry. SPIO nanoworms (200 µ​l, 1 mg Fe ml−1) were Immunoglobulin depletion and reconstitution. To deplete serum or plasma
incubated with six sera types or 5 lepirudin plasma samples (600 μ​l each) for of immunoglobulin (predominantly IgG), 0.05 g of dry protein A sepharose was
15 min at 37 °C in Beckman 1.5 ml polyallomer tubes (Beckman-Coulter). added to a 1.7 ml Eppendorf tube. Next, 500 µ​l of PBS was added to each tube,
Nanoparticles were washed 5 times with 1 ml PBS using Beckman Optima and beads were allowed to swell for 30 min at room temperature. At the end of
ultracentrifuge (TLA-100.3 rotor, 450,000g, 8 min, 4 °C). The proteins were eluted the swelling period, tubes were centrifuged at 3,000g for 1 min and supernatant
from nanoparticles by incubation in 2% SDS in PBS for 1 h at room temperature was removed from the slurry with a capillary gel-loading tip until the beads
and additional ultracentrifugation. Nanoparticles were pelleted as described above, became almost dry (minimal carryover volume) and placed on ice. Freshly
and the protein concentration in the supernatant was measured using BCA Protein thawed cooled serum or lepirudin plasma (200 µ​l) was added and gently agitated
Assay (Thermo Fisher). Sample was processed at the Proteomics and Metabolomics on a thermal mixer for 30 min at 4 °C to limit complement activation. Following
core facility at the Skaggs School of Pharmacy, University of Colorado, using a agitation, samples were centrifuged for 1 min at 3,000g at 4 °C. Supernatant
previously described filter-aided sample purification method51. The sample was was carefully removed with a capillary gel-loading tip (without aspirating the
separated with a C-18 reverse-phase nano liquid chromatography column (100 µ​m slurry) and stored in aliquots at −​80 °C. For reconstitution, human IgG, IgM or
×​150 mm, 3.0 µ​m, 200 A; ProntoSil C18AQ by Nano LCMS Solutions). The sample trastuzumab (stock concentrations between 11.6 mg ml−1 and 14 mg ml−1)
was loaded onto a trap column of the same packing material for 5 min at 10 µ​ or PBS (control) were added to depleted sera or plasma in a 1:3 ratio and
l min−1 before the elution gradient. The flow rate was 800 nl min. One microliter of incubated at room temperature for 15 min, before addition of nanoparticles.
sample was injected. The mass spectrometry (MS) data were acquired in triplicate Adult serum IgG levels are 767–1,590 mg dl−1 or 7.67–15.9 mg ml−1. IgM
using a Bruker Impact HD Q-TOF mass spectrometer in nano-booster mode with serum levels are 37–286 mg dl−1, or 0.37–2.86 mg ml−1. Given the limitation
the following criteria: mass range 150–2,200 m/z, scan rate 2.0 Hz, precursor cycle of stock concentration of IgG and IgM (between 11.6 mg ml−1 and 14 mg ml−1),
time 3.0 s, absolute threshold 500 counts, 0% relative threshold, exclude after one the final immunoglobulin levels were 2.9–3.5 mg ml−1, which is lower than
spectrum and release after 2 min, and reconsider precursor if the ration of current native IgG but similar to native IgM levels. Three different batches of purified
intensity to previous intensity is 3.0. The MS/MS spectra were run against the IgG and one batch of purified IgM and one batch of trastuzumab were used for
Swiss-Prot human database with ProteinScape 4.0 (Bruker) using Mascot software. reconstitution experiments. The particles were incubated, washed and analysed
Trypsin cleavage specificity was set with a maximum of two missed cleavages with dot blot or Western blot as described above.
allowed, and variable cysteinyl carbamidomethylation, deamidation (NQ) and
oxidized methionine modifications were allowed. Peptide tolerance was set to Complement inhibition studies. To distinguish between pathways of complement
10.0 p.p.m. and a MS/MS tolerance to 0.5 Da. Peptides must be at least five amino activation, the inhibitor of Ca2+-sensitive pathways EGTA/Mg2+ (10 mM EDTA
acids long, and proteins were accepted with a Mascot score of >​40, and peptides and 10 mM MgCl final concentration), the inhibitor of both the alternative
were accepted with a Mascot score >​20. pathway and calcium-sensitive pathways EDTA (10 mM final concentration),
the inhibitor of classical pathway C1INH (2.2 µ​M final concentration) or 1× PBS
Assay of proteins bound to nanoparticles. A solution of PBS with 1 mg ml−1 (Fe (control) was added to sera or plasma samples at room temperature 30 min prior
or drug) of SPIO nanoworms, Feraheme, LipoDox or Onivyde was incubated at a to the experiment. Purified factor H was added to sera at 1:10 ratio for a final

Nature Nanotechnology | www.nature.com/naturenanotechnology

NATure NAnOTeCHnOlOgy Articles
concentration of 100 µ​g ml−1. Nanoparticles (1 mg ml−1 Fe or drug) were Reporting Summary. Further information on research design is available in the
then added to sera or plasma (1:3 nanoparticle:serum volume ratio) Nature Research Reporting Summary linked to this article.
and incubated at 37 °C for 30 min. Samples were processed in a dot
blot protocol listed above. Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange
Statistical analyses. Statistical analyses were done using R statistical software
Consortium via the PRIDE partner repository with the dataset identifier PXD011781.
(version 3.4.3) and RStudio (version 1.1.383), or Prism 7.0 (version 7.0a, GraphPad,
The raw data that support the graphs in Figs. 1c,d,e,g,h,i; 2c,d,e,f; 3c,d,e,f,i,j,k,l; and 5d
Dan Diego, CA). Graphics were created with R (heat map) or with Prism 7.0. Three
are available in extended Supplementary Information. Any additional data and data
technical replicates per sample were measured for each outcome. A linear mixed
analysis scripts for R are available from L.S. upon request.
model that accounted for repeated measures within a subject was used for IgG
versus C3 and IgG versus Factor H analyses and executed using the lme4 package
(version 1.1–15) in R. Statistical significance for pairwise post hoc testings of References
the marginal means from a linear mixed model was calculated using two-sided 50. Molday, R. S. & MacKenzie, D. Immunospecific ferromagnetic iron-dextran
t-test and a Tukey multiple testing correction in the emmeans (version 1.1) reagents for the labeling and magnetic separation of cells.
package in R. For Fig. 5d, statistical significance for pairwise post hoc testing J. Immunol. Methods 52, 353–367 (1982).
of the marginal means from a linear mixed model was calculated using two-sided 51. Wisniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample
t-test and a Dunnett multiple testing correction. preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).

Nature Nanotechnology | www.nature.com/naturenanotechnology

 nature research | reporting summary
Corresponding author(s): Dmitri Simberg

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Data analysis R software (version 3.4.3) and RStudio (version 1.1.383), Prism 7.0(v7a, GraphPad), ImageJ 1.49V, ProteinScape 4.0.3.315 and Bruker
Compass DataAnalysis 4.3(x64) (Bruker Daltonics), Mascot Search Engine (Matrix Science), lme4 package (version 1.1-15) in R, emmeans
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All studies must disclose on these points even when the disclosure is negative.
Sample size No sample size calculations were performed. The sample size was determined by material available. The sample size was sufficient. All
analyses indicated either clear dramatic differences (highly significant) or clear lack of difference (no significance and no observable trends in
the data).

Data exclusions No data were excluded from the analysis.

Replication For all analyses, three technical replicates were gathered for each sample and for most analyses assays were repeated across two or three
batches. Statistical analyses used all replicates in a mixed model to appropriately account for all sources of variance. All replications were
successful.

Randomization This was not a randomized study. For comparisons between IgG and C3, we used an observational design. For all other experiments, we
compared treatment conditions to untreated measurements within the same subject.

Blinding Blinding was not done for this experiment or analysis. The blinding was not possible due to nature of sample collection and processing in this
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Obtaining unique materials Plasma of breast cancer patients cannot be shared due to the COMIRB protocol limitations. Plasma and sera of healthy donors
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Antibodies

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Antibodies used All antibodies with the respective catalog numbers and lot numbers are listed in Methods section

Validation The antibodies were validated by the respective manufacturers (www.quidel.com, www.complementtech.com, licor.com,
www.jacksonimmuno.com). All the information including list of references is available on the respective websites. Anti-C3 and
anti-C1q antibodies were validated in this paper, either via complement inhibition by EDTA, or use of C1 depleted sera.

Human research participants

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Population characteristics For normal serum/plasma, both females and males were recruited. Age 18+. The patients were metastatic breast cancer
positive, females, age 18+. All ethnicities/races were included in the sample enrollment.

Recruitment Plasma and sera were collected from anonymous consented healthy donors at the University of Colorado blood bank. Both
genders were enrolled under standard Institutional Review Board protocol using the consent forms available at the University of
Colorado blood bank. Plasma from cancer patients were collected from anonymous deidentified patients undergoing exploratory
sample collection trial at the University of Colorado (trial 16-0610). All ethnicities/races were included in the sample enrollment.

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