ARTICLES

PUBLISHED ONLINE: 15 FEBRUARY 2016 | DOI: 10.1038/NNANO.2015.330

Protein adsorption is required for stealth effect of

poly(ethylene glycol)- and poly(phosphoester)-
coated nanocarriers
Susanne Schöttler1,2, Greta Becker1, Svenja Winzen1, Tobias Steinbach1, Kristin Mohr1,
Katharina Landfester1, Volker Mailänder1,2*† and Frederik R. Wurm1*†

The current gold standard to reduce non-specific cellular uptake of drug delivery vehicles is by covalent attachment of
poly(ethylene glycol) (PEG). It is thought that PEG can reduce protein adsorption and thereby confer a stealth effect.
Here, we show that polystyrene nanocarriers that have been modified with PEG or poly(ethyl ethylene phosphate) (PEEP)
and exposed to plasma proteins exhibit a low cellular uptake, whereas those not exposed to plasma proteins show high
non-specific uptake. Mass spectrometric analysis revealed that exposed nanocarriers formed a protein corona that
contains an abundance of clusterin proteins (also known as apolipoprotein J). When the polymer-modified nanocarriers
were incubated with clusterin, non-specific cellular uptake could be reduced. Our results show that in addition to reducing
protein adsorption, PEG, and now PEEPs, can affect the composition of the protein corona that forms around nanocarriers,
and the presence of distinct proteins is necessary to prevent non-specific cellular uptake.

dsorption of proteins from physiological fluids to nano-

A
carriers leads to the formation of a protein shell1,2. This
rapidly forming protein corona has previously been shown
to be responsible for the biological fate of nanocarriers3–5. Poly-
(ethylene glycol) (PEG) is widely used to suppress any non-specific
protein adsorption, and PEGylated drugs and (nano)carriers show
longer blood half-lives and less non-specific cellular uptake com-
pared to unmodified drugs6,7. This is a prerequisite for specific
targeting8,9. For surfaces, PEGylation is also the typical approach
to reduce protein adsorption10,11. This feature, usually referred to
as the ‘stealth’ effect, is generally explained by the high level of
hydration of the hydrophilic polyether backbone, which also pre-
vents protein adsorption on typically hydrophobic polymer surfaces
by means of steric repulsion12,13. When the stealth properties of PEG
are discussed it is often neglected that even if the overall protein
adsorption is reduced, it cannot be fully suppressed; a certain
amount of serum proteins, for example, is always detected on the
drug carriers, thus altering the surface properties14,15.
PEG is a non-biodegradable polyether and its accumulation in the
body must be prevented. This is especially important when drug–PEG
conjugates are administered for chronic diseases over a period of
several years. In such cases, an accumulation of PEG in the body is
likely, and can cause unwanted side effects13. Indeed, the development
of PEG antibodies16,17 and severe hypersensitivity reactions have been
reported18. Antibody formation can lead to an accelerated blood clear-
ance following repeated systemic administration19. Furthermore, PEG
has been shown to trigger complement activation, which can lead to
anaphylactic reactions in sensitive individuals20. These factors have
given rise to a search for alternatives to PEG, and other biocompa-
tible polymers have been recognized to improve the in vivo proper-
ties of pharmaceuticals. For example, zwitterionic molecules such as
polybetaines or polysaccharides can also generate hydrophilic shells
when coupled to nanoparticles21. Degradable polymers that have
been proposed as PEG alternatives include hydroxyethyl starch
(HES), polysialic acid and dextrin13. However, another very interest-
ing polymer class—the poly(phosphoester)s (PPEs)—has never
been investigated with respect to stealth behaviour. In recent
years, we have been studying PPEs with respect to novel synthetic
protocols and biomedical applications22,23. The chemical structure
of PPEs is highly modular, they are degradable, and their degra-
dation products and time can be adjusted by means of precise
chemistry24,25. Several studies have dealt with the preparation of
PPE-based drug carriers, but none has investigated the protein
interactions of the hydrophilic PPEs with blood proteins26,27.
Three distinct findings are reported herein. First, the polymer-
modified nanocarriers (with PEG and PPE) exhibit decreased
protein adsorption after incubation in human plasma compared
to unmodified particles. Second, and more importantly, mass spec-
trometric analysis of the protein corona generated on the particles’
surface after plasma incubation revealed a similar pattern of proteins
on PEGylated and ‘PPEylated’ surfaces. Clusterin—also termed apo-
lipoprotein J (ApoJ)—was identified as a major component on both
surfaces. Third, interestingly, a high non-specific cellular uptake for
both polymer-modified nanocarriers was found without previous
plasma incubation, indicating the requirement for distinct proteins
to prevent non-specific cellular uptake. This non-specific cellular
uptake could eventually be reduced by non-covalent ‘pre-loading’
with clusterin onto the nanocarriers, demonstrating a strong effect
of reduced cell internalization. Unravelling the effects of protein
type and stealth polymer structure will produce new efficient drug
delivery devices.
PEEP as novel stealth polymers
The nanocarriers (Fig. 1) used in this study were monodisperse
polystyrene nanoparticles with diameters of ∼100 nm, which were
prepared by free radical terpolymerization of three monomers

1
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. 2 Johannes Gutenberg University Mainz, University Medical Center,
Department of Dermatology, Langenbeckstr. 1, 55131 Mainz, Germany. †These authors jointly supervised this work. * e-mail: mailaender@mpip-mainz.mpg.de;
wurm@mpip-mainz.mpg.de

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NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2015.330 ARTICLES
a b H2N NH2 c
PS-NH2
H2N NH2
x y z
O O
O O O O
NO R
O
NH3CI
R = PEG R = PEEP

N O H
H
O N
F
B N
O
O N OP O
O nO 100 nm
F nO
m m

Ø (nm) 106 117 119 117 122

n − 44 110 49 92

m − 3,500 2,900 3,200 2,400

ζ (mV ) 46 15 8 −9 −10

PS-NH2 PS-PEG44 PS-PEG110 PS-PEEP49 PS-PEEP92

Figure 1 | Characteristics of nanocarriers. Amine-functionalized polystyrene nanoparticles were modified with PEG or PEEP. a, The terpolymer of styrene,
2-aminoethyl methacrylate hydrochloride and BODIPY-methacrylate, used as the carrier material for the nanoparticles investigated here. b, PEG and PEEP
attachment to the amino groups of polystyrene nanoparticles. c, Scanning electron microscopy image of PS-PEEP49. d, Schematic representation of
nanocarriers (average diameter Ø determined by dynamic light scattering (DLS), number of repeating units n, number of polymer chains per nanoparticle m,
and zeta potential ζ).

via miniemulsion28. Styrene was copolymerized with 2 wt%
2-aminoethyl methacrylate hydrochloride to introduce reactive
groups for subsequent polymer attachment and in the presence of
a fluorescently active boron-dipyrromethene (BODIPY)-methacrylate
to label the nanocarriers for further biological studies (see
Supplementary Section ‘Synthesis’ for details). The nanoparticles
were characterized by standard procedures (Fig. 1: light scattering,
zeta potential, number of polymer chains per particle, Mn of each
chain = 190,000 g mol–1). N-hydroxysuccinimide-functionalized
PEG (Mn = 2,000 g mol–1, degree of polymerization (DPn) = 44
and Mn = 5,000 g mol–1, DPn = 110) and the water-soluble PPE-
derivative PEEP (poly(ethyl ethylene phosphate), Mn = 7,600 g mol–1,
DPn = 49 and Mn = 14,100 g mol–1, DPn = 92) were coupled to the
amino-functionalized nanocarriers. Attachment of the PEG and
PEEP chains to the nanocarriers was demonstrated by the reduction
in the zeta potential (PEGylated particles, ∼+10 mV; PPEylated
particles, ∼–10 mV). The number of attached PEG or PEEP chains
on each particle could be determined accurately via 1H NMR spectro-
scopy, and was found, typically, to be in the range 2,000–4,000,
corresponding to a degree of functionalization of the amine groups
of 11–16% (for the list of results see Supplementary Tables 7 and 8).
After 15% grafting of the surface amino groups, an area of ∼10 nm2
was available for each polymer chain. Kuga29 has reported the hydro-
dynamic radii of different PEGs to be 1.3 nm (PEG Mn = 2,000) and
2.7 nm (PEG Mn = 6,000), resulting in surface areas of up to 23 nm²
for these polymers. Assuming a PEG chain to have the same confor-
mation and size when on the surface of a particle as is found in sol-
ution, the calculations show that the maximum number of polymer
chains is almost reached for PEG110. Gel permeation chromato-
graphy (GPC) analysis of the dissolved modified nanocarriers,
that is, grafted polystyrene, proved that no unreacted polymer was
in the mixture (compare Supplementary Fig. 19).
To investigate the stealth effect of the polymer-modified nano-
carriers, the amount of proteins adsorbed after plasma incubation
was analysed (Fig. 2a). The nanocarriers were incubated with
human blood plasma for 1 h and separated from unbound proteins
by centrifugation and several washing steps. The adsorbed proteins
were removed from the surface by treatment with urea and quanti-
fied with a colorimetric protein assay. The adsorption of plasma
proteins was significantly reduced by surface functionalization
with PEG and PEEP. Decreases of 79% (PS-PEG44), 66%
(PS-PEG110), 73% (PS-PEEP49) and 70% (PS-PEEP92) were
observed compared to the adsorption of proteins onto PS-NH2
nanocarriers (Fig. 2a). The reduction in protein adsorption to a
value similar to that for PEG is the first indication of a stealth
effect provoked by PPEs. The adsorbed protein patterns were
further visualized using SDS–polyacrylamide gel electrophoresis
(PAGE; Supplementary Fig. 28). At first glance, a similar protein
pattern can be observed for all stealth nanocarriers, with enrichment
of a 38 kDa protein, but several differences between PEGylated and
PPEylated surfaces are also detected. The molecular weight differ-
ences of PEG and PEEP seem not to play a crucial role. The
amino-functionalized control nanocarrier is mainly covered by
albumin, the most abundant protein in human blood plasma (the
band at ∼62 kDa). Again, the overall stronger protein adsorption
to PS-NH2 is clearly illustrated. In addition to the covalently
PEG- and PPEylated nanocarriers, Lutensol AT50 (a PEG-based sur-
factant) was self-assembled on amino-functional and neutral PS
nanoparticles as controls. With different concentrations of surfactant,
similar protein patterns were identified by SDS–PAGE or mass

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ARTICLES NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2015.330

a c −Plasma +Plasma
5

4
PS-NH2

Proteins (mg m−2)

PS-PEEP49

PS-PEEP92
3

PS-PEG110
PS-PEG44
PS-NH2
2

1 PS-PEG44

b 1,250
Plasma −Plasma
Relative fluorescence intensity

1,000 PS-PEG110

750

500

250 PS-PEEP49

0
-P 44

-P 44
0

0
-P 49

PS 92

-P 49
92
-P 2

-P 2
PS H

PS H
-P 11

-P 11
PS EG

PS EG
P

P
PS EEP

PS EEP
PS EG

PS EG
-N

-N
EE

EE
PS

PS-PEEP92

Figure 2 | Cellular uptake of PEEP and PEG nanocarriers. Functionalization of nanoparticles with PEEP and PEG inhibits uptake into macrophages only in the
presence of proteins. a, Quantification of human plasma proteins adsorbed to the nanocarriers’ surface (mg protein m–²). Values are expressed as mean ± s.d.
of biological triplicates. b, Flow cytometry analysis of RAW264.7 cells incubated with different nanocarriers for 2 h. The nanocarriers were incubated in human
plasma or in water (–plasma) for 1 h at 37 °C and separated from residual proteins by centrifugation before being added to cells. Values are expressed as
mean ± s.d. of biological triplicates. The experiment was further replicated three times in the laboratory. c, Laser scanning microscopy images of RAW264.7
cells incubated with PS-NH2 , PS-PEEP49, PS-PEEP92, PS-PEG44 and PS-PEG110 for 1 h in 100% human plasma or DMEM without plasma. The cell membrane
is stained with CellMask Orange (red) and nanocarriers are shown in green. Scale bars, 10 μm.

spectrometry (Supplementary Figs 29 and 30). The lower protein proteins alone that is responsible for the inhibition of cellular inter-
binding of the stealth nanocarriers compared with the unmodified nalization by the stealth polymers. It is, instead, a secondary effect;
control was also confirmed by isothermal titration calorimetry that is, the specific protein patterns of PEG- or PEEP-functionalized
(Supplementary Fig. 31). nanocarriers consist of ‘don’t-eat-me’ proteins. Laser scanning
microscopy (Fig. 2c) also shows this effect clearly: strong inter-
Proteins are necessary for the stealth effect nalization of the unmodified nanocarriers (PS-NH2) and both
The advantage of stealth nanocarriers is their ability to remain in the PEEP-modified nanocarriers (PEEP-NP) into RAW264.7 cells was
blood circulation for extended periods of time by evading clearance observed (even after only 1 h) when the cells were cultured in
by the immune system30. Macrophages are important components culture medium without plasma proteins. In contrast, macrophages
of the immune defence system and play a major role in clearing cultured with plasma did not internalize PEGylated or PPEylated
foreign molecules from the blood. Only by eluding these phagocytic nanocarriers, but the unmodified nanoparticles were taken up.
cells can nanocarriers reach their destination within the body and Even after 24 h incubation, no significant uptake of the stealth nano-
fulfil their task. Therefore, the internalization of the described nano- carriers pre-coated with plasma proteins was observed, in contrast
carriers into a murine macrophage-like cell line, namely RAW264.7, to the control nanocarrier (Supplementary Fig. 32). Similar results
was studied. Flow cytometry analysis revealed that uptake of the were also obtained for the cervical cancer cell line HeLa cultured
‘naked’ nanocarriers (PS-NH2) is inhibited by the attachment of in medium with or without serum (Supplementary Fig. 33a,c). A
PEEP to the same degree as by PEG when the nanocarriers are high uptake of PEGylated and PPEylated nanocarriers without the
previously incubated with plasma (Fig. 2b). presence of proteins, and no uptake after incubation with 10%
Most intriguingly, a high uptake of all nanocarriers in protein- fetal bovine serum (FBS), was observed by flow cytometry analysis
free medium was observed. As these nanocarriers carry no adsorbed and live cell imaging. Furthermore, human immature dendritic
proteins, this strongly indicates that it is not the reduced amount of cells, also professional phagocytes in the human body, did not

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NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2015.330 ARTICLES
a PS-NH2 PS-PEG44 PS-PEG110 functions shows that the control nanocarrier is mainly covered by
albumin (as visualized by SDS–PAGE) and the proteins involved
in coagulation, and lipoproteins bind preferentially to the polymer-
modified nanocarriers. Interestingly, for PEEP-modified nanocarriers,
lipoproteins constitute up to 85% of the protein corona.
Figure 3b depicts the most abundant proteins identified in the
so-called ‘hard’ corona of the nanocarriers (Supplementary
Acute phase
PS-PEEP49 PS-PEEP92 Table 9). Albumin, fibrinogen and vitronectin are the most promi-
Coagulation
Complement system
nent proteins on the control nanocarrier (PS-NH2 , >80%), but these
Immunoglobulins proteins are strongly depleted on PEGylated and PPEylated nano-
Lipoproteins carriers. In these latter nanocarriers, clusterin—an apolipoprotein—
Other plasma components is the major protein in the hard corona, and can be assigned to the
Tissue leakage
38 kDa band in the SDS–PAGE results (Supplementary Fig. 28). It
makes up 77% of the protein corona on the PEEP-functionalized
PS-PEEP49

PS-PEEP92
nanocarriers, with all other proteins contributing less than 2%. This
PS-PEG110
PS-PEG44
PS-NH2

b significant enrichment of one single protein is remarkable. In previous

publications, apolipoproteins have been found to be enriched3,31,32,
Apolipoprotein A-I 80 some literature has described that hydrophobic materials adsorb
Apolipoprotein A-IV apolipoproteins even more33, and hydrophilic nanomaterials have
Apolipoprotein C-III been reported to adsorb mainly albumin, fibrinogen and IgG as
Apolipoprotein D more abundant proteins. As apolipoproteins have lipid-binding
Clusterin
domains, their attraction to hydrophobic surfaces is readily concei-
Percentage of total protein corona

Complement C1q subcomponent 60

subunit B
Complement C1q subcomponent
subunit C
Fibrinogen alpha chain
Fibrinogen beta chain
Fibrinogen gamma chain
Fibronectin
Ig gamma-1 chain C region
Ig gamma-2 chain C region
Ig kappa chain C region
Ig mu chain C region
Serum albumin
Serum amyloid P-component
Vitronectin
Figure 3 | Proteomic analysis of protein corona on the surface of
nanocarriers. The protein corona of PEG- and PEEP-functionalized
nanoparticles mainly consists of the protein clusterin. a, Classification of
protein corona components identified by quantitative LC-MS. The average
amount of each protein (in fmol) was calculated from three biological and
two technical replicates. Identified proteins were grouped according to their
function in biological processes, and the amounts of proteins were summed.
b, Heat map of the most abundant proteins in the protein corona of PS-NH2 ,
PS-PEG44 , PS-PEG110 , PS-PEEP49 and PS-PEEP92 determined by proteomic
mass spectrometry. Values were calculated from the molar masses of each
protein identified by LC-MS. Only those proteins that constitute at least 1%
of the protein corona on one of the nanocarriers are shown.
show any significant internalization of the four stealth nanocarriers
when incubated with serum (Supplementary Fig. 33b). All nanocar-
riers were investigated with respect to their toxicity. The PEGylated
and PPEylated nanocarriers did not promote the cell death of HeLa
cells, but the control nanocarrier (PS-NH2) showed minor toxicity
(Supplementary Fig. 34).
From these results, fundamental conclusions can be drawn. It is
not only the reduction in protein adsorption that explains the stealth
effect of both PEG- and PEEP-functionalized nanocarriers; more
importantly, the type of protein must be highly important. This
renders the stealth effect a secondary effect caused by selective
protein adsorption.
Analysis of protein corona reveals high clusterin content
With liquid chromatography–mass spectrometry (LC-MS) analysis,
a total of 171 proteins were identified in the corona of all five nano-
carriers (Fig. 3). Grouping the identified proteins according to their
vable. Interestingly, PEG is supposed to be a rather hydrophilic
material. However, different apolipoproteins have different
propensities to adsorb: Walkey and Chan found that ApoA4,
ApoB, ApoC3 and ApoE adsorb preferentially to hydrophobic
40
quantum dots, while ApoA1, ApoA2 and ApoC1 adsorb preferen-
tially to more hydrophilic NH2-modified quantum dots33.
Apolipoproteins therefore appear to be a class of proteins of interest
for different nanocarriers, and will need to be further explored for
20
their different functions.
Clusterin has been reported to adsorb to the surface of several
nanocarriers with varying affinities. Relatively high amounts of clus-
terin were detected on solid lipid nanoparticles, with an enhanced
0 quantity with increasing incubation time34. Furthermore, a signifi-
cantly enhanced affinity was detected for silica nanoparticles31.
On polystyrene nanoparticles with distinct surface characteristics,
clusterin has been identified as one of the most abundant proteins32,
and an increasing amount of clusterin has been reported for nano-
particles with increasing hydrophobicity35,36. To our knowledge,
such a high abundance of clusterin has not been described pre-
viously for other nanocarriers. On PEGylated nanocarriers, apolipo-
protein A1 (as well as clusterin) is also found (∼11%, corresponding
to the band at ∼28 kDa in the SDS–PAGE results).
These differences in the composition of the protein corona for
two different hydrophilic polymers is an additional handle for the
future design of novel drug carriers or diagnostic devices, if the mol-
ecular structure of the stealth polymer can be tailored in a way to
control not only the amount but also the type of plasma protein,
for example by copolymerization.
The significant enrichment of clusterin on PEG-functionalized
nanocarriers (and even more on PEEP-functionalized nanocarriers)
suggests its important function in stealth properties. In blood, the
native glycoprotein clusterin is assembled with other apolipopro-
teins, such as ApoA1, into high-density-lipoprotein (HDL) par-
ticles. The clusterin/ApoJ gene encodes for a different protein
isoform: a highly conserved disulphide-linked heterodimeric
secreted glycoprotein of ∼75–80 kDa (sCLU)37, as well as cyto-
plasmic (cCLU) and a nuclear (nCLU) isoform38. The secreted
form of clusterin in plasma has a concentration of 40–60 μg ml–1
and is thus not among the most abundant of proteins, but it is
still a protein with a relevant concentration. sCLU was first
described in testis fluid39 and, as well as in blood plasma, sCLU is
also found in other body fluids including milk, urine and cerebro-
spinal or lacrimal fluid37,40. Various physiological functions have
been proposed for clusterin: complement attack prevention,

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ARTICLES
Water
HSA
PS-PEEP92
800
700
Relative fluorescence intensity
600
Plasma
500 or
clusterin
400
300
200
100
0
rin
A
a
2O
HS
te
H
as
us
+
+
Pl
Cl
+
+
Figure 4 | Clusterin reduces non-specific cellular uptake. Clusterin
functions as a dysopsonin and assists the ‘stealth effect’. Cellular uptake of
PS-PEEP92 into RAW264.7 cells after incubation in water, human plasma,
clusterin (66.6 μg ml–1) or human serum albumin (HSA, 66.6 μg ml–1).
The nanocarriers were incubated in each medium for 1 h at 37 °C before
being added to the cells maintained in DMEM. After 1 h, the cells were
analysed by flow cytometry. Values are expressed as mean ± s.d. of
biological triplicates. The experiment was further replicated three times
in the laboratory.
promotion of cell aggregation, regulation of apoptosis and lipid
transport, but all are mainly based on binding partners41. The
secreted form of clusterin has been described to prevent protein
aggregation and precipitation by binding to hydrophobic regions
of misfolding-prone proteins42. It is therefore believed to be the
first secreted mammalian chaperone to be identified. One could
speculate that PEG and PEEP appear to clusterin to be such
proteins, although it remains unclear how they are structurally
related. Alternatively, other proteins that can be detected under
these conditions in our proteomic approach could have been mis-
folded during binding to the nanoparticles’ surfaces, and sub-
sequently attracted clusterin. However, binding to polymeric
surfaces of this protein has not been studied to date.
Clusterin reduces non-specific cellular uptake
To further analyse the effect of clusterin, PPEylated nanocarriers were
incubated with native clusterin at its physiological concentration43,
and uptake into RAW264.7 cells was studied. Figure 4 confirms
that incubation with clusterin reduces the uptake of the nanocarriers
into macrophages by 75.4% (in the absence of plasma or clusterin a
high cellular uptake is detected). To prove that this effect cannot be
provoked by any plasma protein, human serum albumin (HSA)
was used as control. Interestingly, HSA incubation increased the
internalization by 34%, although albumin is often described as a dys-
opsonin44. Although pure clusterin does not have the same effect as
whole plasma, which decreases the uptake by as much as 99.5%, it
shows that clusterin functions as a strong dysopsonin for macro-
phages and supports the idea of its predominant participation in
the stealth effect of PEG- and PPEylated nanocarriers. The lower effi-
cacy of clusterin alone in hindering nanoparticle uptake compared to
the complex protein corona formed in whole plasma indicates the
376
© 2016 Macmillan Publishers Limited. All rights reserved
NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2015.330
necessity for a mixture of dysopsonins on stealth nanocarriers. Due
to the washing of the nanocarriers before the proteomic analysis, pro-
teins in the soft corona are not identified but may also contribute to
the stealth effect. Although some first steps have been taken45,
methods to investigate the soft corona in its complexity are not yet
as elaborate as those carried out for the hard corona. These comp-
lementary methods are urgently needed to add to the knowledge
about the protein corona. While dysopsonization has also been
achieved by the coupling of self-peptides derived from cell surface
proteins46, this is the first report on a polymeric surface resulting in
a specific plasma adsorption.
Although, opsonization of nanocarriers with plasma proteins is
thought to be the major cause for clearance from the bloodstream,
we have shown that stealth nanocarriers functionalized with either
PEG or PEEP were internalized by macrophages when no proteins
were present, and so opsonization cannot have occurred. Walkey
and colleagues have suggested that PEG does not eliminate serum
protein adsorption in total, but selectively suppresses adsorption
of specific proteins, resulting in a minimized macrophage
uptake47. We want to go even further: we postulate that it is not
only the selective reduction of opsonizing proteins such as immuno-
globulins and complement factors, but in fact the selective enrich-
ment of a single type of protein, depending on the polymer
structure, that triggers the (secondary) stealth effect.
Apolipoproteins have been recognized previously as an impor-
tant class of proteins governing the in vitro and in vivo fate of nano-
carriers1,4,48. ApoE, in particular, has been identified as an
interesting molecule, because it enhances the passage of nanocarriers
through the blood–brain barrier49, while ApoA1 and ApoB-100
also promote transport into the central nervous system50. Other
apolipoproteins, such as ApoA4 and ApoC3, have been described
to function as dysopsonins32. Clearly, these proteins could also con-
tribute to the stealth effect and may even substitute for clusterin on
other stealth nanocarriers. Nevertheless, we found clusterin to
be such a prominent protein in the proteome of the nanoparticle
corona that it appears to be the most important one.
In summary, PEGylated and PPEylated (PEEP-modified) model
nanocarriers demonstrate a distinct reduction and modification of
protein adsorption after incubation with human plasma when com-
pared with non-modified nanocarriers. The uptake of the
PEGylated and PPEylated nanocarriers by different cell lines,
including a macrophage-like cell line, was completely inhibited.
We identified clusterin as the major protein in the corona of both
polymer-modified nanocarriers, with its highest enrichment on
PPEylated surfaces. Furthermore, pre-loading of the PPEylated
nanocarriers with clusterin reduced macrophage uptake (while
albumin pre-loading increased the uptake), providing evidence for
a dysopsonizing function of clusterin concerning internalization
into macrophages. When comparing these results with other
attempts to pursue a ‘stealth effect’, for example, by Discher and
colleagues46, who used peptides of hCD47 to prohibit clearance by
phagocytes, our work can be seen to report a facile way to enrich
the ‘stealth effect’ of nanoparticles, just by mixing with
human plasma.
These results lead to fundamental insights that have to be con-
sidered for future nanocarrier design: (1) the stealth effect is not
a polymer effect alone, but a secondary effect; (2) degradable
poly(phosphoester)s are potential PEG alternatives; (3) the chemical
structure of the stealth polymer can trigger the amount and type of
plasma protein adsorbed and thus the biological fate of the
nanocarrier; (4) clusterin is an interesting candidate for further
investigations into the stealth effect.
Methods
All experimental details, materials, and methods can be found in the
Supplementary Information.
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NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2015.330
Received 9 March 2015; accepted 10 December 2015;
published online 15 February 2016
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Acknowledgements
T.S. and F.R.W. acknowledge support from the Max Planck Graduate Center (MPGC). G.B.
and T.S. acknowledge support from the Graduate School of Excellence ‘MAINZ’ (Materials
Science in Mainz). This work was supported by the DFG/SFB1066 (‘Nanodimensionale
polymere Therapeutika für die Tumortherapie’, Q1 and Q2). F.R.W. thanks the DFG WU
750/6-1 for funding.
Author contributions
F.R.W., V.M. and K.L. supervised the project. F.R.W., V.M., S.S. and G.B. conceived and
designed the experiments. G.B. and T.S. synthesized the polymers and nanoparticles. S.S.
performed the cell experiments and proteomic analysis. S.W. and K.M. performed the
isothermal titration calorimetry measurements. F.R.W. and S.S. wrote the manuscript. All
authors discussed the results and commented on the manuscript.
Additional information
Supplementary information is available in the online version of the paper. Reprints and
permissions information is available online at www.nature.com/reprints. Correspondence and
requests for materials should be addressed to V.M. and F.R.W.
Competing financial interests
The authors declare no competing financial interests.
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