Completehigh-densitylipoproteinsinnanoparticle corona
Erik Hellstrand
1
, Iseult Lynch
2
, Astra Andersson
3
, Torbjo
¨
rn Drakenberg
1
, Bjo
¨
rn Dahlba
¨
ck
3
,
Kenneth A. Dawson
2
, Sara Linse
1,2
and Tommy Cedervall
1,2
1 Biophysical Chemistry, Lund University, Sweden
2 School of Chemistry and Chemical Biology, University College Dublin, Ireland
3 Clinical Chemistry, University Hospital, Malmo
¨
, Lund University, Sweden
Nanoparticles entering any biological fluid will imme-
diately be covered by a corona of biomolecules. The
corona confers biological identity to the nanoparticles,
as it is this that interacts with the cellular machinery,
thereby determining the nanoparticle destiny in and
impacts on organisms. Previously, research has focused
on the protein composition of the corona, but many
other biomolecules, such as carbohydrates, nucleic
acids, and lipids, can also be contained in the corona
and play important biological roles that are different
from those of proteins. It is therefore essential to
extend the characterization of the corona to include
other biomolecules, as each plays a vital role in cellular
functionality and signaling. Complete characterization
of the nanoparticle biomolecule corona may be the
key to classifying nanoparticle risk and predicting
impacts.
The nanoparticles used in this study are copoly-
mers of N-isopropylacrylamide (NIPAM) and N-t-
butylacrylamide (BAM) at four ratios (85 : 15,
75 : 25, 65 : 35, and 50 : 50) and three sizes (70, 120
and 200 nm in diameter). These nanoparticles are
well characterized, and can be prepared monodisperse
with defined size and hydrophobicity (through
the NIPAM ⁄ BAM ratio), making them highly
suitable as model nanoparticles in biophysical and
biochemical studies. In addition, these polymer
particles have been suggested as drug delivery vessels
Keywords
apolipoprotein; HDL; lipids; nanotoxicology;
transport pathways
Correspondence
K. A. Dawson, School of Chemistry and
Chemical Biology, University College Dublin,
Belfield, Dublin 4, Ireland
Fax: +353 1 716 2415
Tel: +353 1 716 2447
E-mail: kenneth@fiachra.ucd.ie
T. Cedervall, Department of Biophysical
Chemistry, Lund University, Box 124,
SE-22100 Lund, Sweden
Fax: +46 46 222 4116
Tel: +46 46 222 8240
E-mail: tcedervall@yahoo.com
(Received 4 March 2009, revised 8 April
2009, accepted 15 April 2009)
doi:10.1111/j.1742-4658.2009.07062.x
In a biological environment, nanoparticles immediately become covered by
an evolving corona of biomolecules, which gives a biological identity to the
nanoparticle and determines its biological impact and fate. Previous efforts
at describing the corona have concerned only its protein content. Here, for
the first time, we show, using size exclusion chromatography, NMR, and
pull-down experiments, that copolymer nanoparticles bind cholesterol, tri-
glycerides and phospholipids from human plasma, and that the binding
reaches saturation. The lipid and protein binding patterns correspond clo-
sely with the composition of high-density lipoprotein (HDL). By using frac-
tionated lipoproteins, we show that HDL binds to copolymer nanoparticles
with much higher specificity than other lipoproteins, probably mediated by
apolipoprotein A-I. Together with the previously identified protein binding
patterns in the corona, our results imply that copolymer nanoparticles bind
complete HDL complexes, and may be recognized by living systems as
HDL complexes, opening up these transport pathways to nanoparticles.
Apolipoproteins have been identified as binding to many other nanoparti-
cles, suggesting that lipid and lipoprotein binding is a general feature of
nanoparticles under physiological conditions.
Abbreviations
BAM, N-t-butylacrylamide; HDL, high-density lipoprotein; HSA, human serum albumin; LDL, low-density lipoprotein; NIPAM,
N-isopropylacrylamide; SR-BI, scavenger receptor class B type 1; VHDL, very high-density lipoprotein; VLDL, very low-density lipoprotein.
3372 FEBS Journal 276 (2009) 3372–3381 ª 2009 The Authors Journal compilation ª 2009 FEBS
with potential for controlled release [1]. The identity
of proteins in the hard corona on these nanoparticles
is well established, and several biophysical parame-
ters, including stoichiometries, exchange rates, and
enthalpies of the interactions, have been determined
[2–4]. Surprisingly, most of the identified proteins are
apolipoproteins and other proteins associated with
lipoprotein particles. Serum albumin is also present,
but has a much higher dissociation rate and lower
affinity than the apolipoproteins, and will therefore
be replaced over time by apolipoproteins in plasma.
The selective binding of apolipoproteins raises many
interesting questions that this article begins to
address. For instance, it is not known whether the
nanoparticles bind only the apolipoproteins or the
complete lipoprotein particles, and, if the latter is
true, whether a specific lipoprotein particle is selec-
tively targeted. Additionally, we are interested in
knowing whether the binding is mediated by pro-
teins, lipids, or both, and whether the bound protein
or lipoprotein particle retains its receptor binding
and enzymatic activity.
Lipids in blood are transported via lipoprotein parti-
cles, of eight to several hundred nanometers in diame-
ter, containing lipids and proteins. Triglycerides and
cholesterol esters are found in the core of these lipo-
protein particles, surrounded by proteins and a mono-
layer of phospholipids. The proteins in the lipoprotein
particles are mainly apolipoproteins with a range of
structural and functional properties. The different clas-
ses of lipoprotein particles can be distinguished from
one another by the composition of apolipoproteins
and lipids. HDL is a heterogeneous population of lipo-
protein particles [5]. Apolipoprotein A-I is the main
protein component of HDL. In blood, apolipopro-
tein A-I recruits phospholipids and cholesterol to form
discoidal HDL particles that mature into larger, spher-
ical HDL particles. Apolipoprotein A-II and apolipo-
protein E are present in subfractions of HDL.
Additionally, proteins involved in lipid metabolism are
also associated with HDL (but not with other lipopro-
tein particles), including lecithin:cholesterol acyltrans-
ferase, cholesterol ester transfer protein, and
paraoxinase, which are involved in cholesterol meta-
bolism and transport.
Here we have investigated the binding to copolymer
nanoparticles of both lipids and proteins from whole
plasma and from isolated lipoprotein fractions, using
size exclusion chromatography, gel electrophoresis,
NMR, and enzymatic assays. We have investigated in
detail the lipid and protein binding pattern, and the
results imply that the copolymer nanoparticles bind
HDL lipoprotein particles.
Results
Lipid binding to nanoparticles determined by size
exclusion chromatography
Size exclusion chromatography was carried out to
establish whether lipids are associated with copolymer
nanoparticles, as shown in Fig. 1. This technique has
been previously used to determine interactions of pro-
teins with nanoparticles, as the elution behavior of the
proteins is shifted upon interaction with the nanoparti-
cles [3]. Copolymer nanoparticles with composition
50 : 50 NIPAM:BAM and diameters of 120 or 200 nm
were incubated with plasma, pelleted by centrifugation,
washed, and finally redispersed in buffer before being
loaded onto a Sephacryl S-1000 SF column. The pres-
ence of cholesterol in the eluted fractions was deter-
mined by an enzymatic assay. The results (Fig. 1)
show that cholesterol coelutes with nanoparticles of
different size at their respective elution volumes. This
clearly shows that plasma cholesterol associates with
the copolymer nanoparticles.
Lipids identified by NMR spectroscopy after
extraction
NMR spectroscopy was used to detect and identify the
lipids bound to the copolymer nanoparticles in plasma.
NIPAM ⁄ BAM 50 : 50 copolymer nanoparticles were
mixed with plasma, and unbound plasma lipids were
removed by repeated washing ⁄ centrifugation steps.
Lipids were then extracted from the nanoparticles
using chloroform ⁄ methanol extraction, and analyzed
by NMR spectroscopy (Fig. 2A). A substantial
amount of nanoparticles ended up in the chloroform
phase, disturbing the spectra and making quantifica-
0
0.2
0.4
0.6
0.8
1.0
010203040
Fraction number
120 nm
200 nm
Cholesterol/a.u.
Fig. 1. Cholesterol travels with nanoparticles in size exclusion chro-
matography. Copolymer particles, 50 : 50 NIPAM ⁄ BAM, with diam-
eters of 120 or 200 nm were mixed with human plasma. Unbound
plasma lipids were removed by centrifugation. The nanoparticle pel-
lets were washed and resuspended in buffer before being loaded
onto a Sephacryl S-1000 SF column. Each fraction was analyzed for
cholesterol by enzymatic assay. Arrows indicate the elution volume
of the nanoparticles.
E. Hellstrand et al. Completehigh-densitylipoproteinsinnanoparticle corona
FEBS Journal 276 (2009) 3372–3381 ª 2009 The Authors Journal compilation ª 2009 FEBS 3373
tion difficult. However,
1
H 1D-NMR spectra of lipids
extracted from nanoparticles incubated in plasma
consistently showed signals specific for cholesterol at
0.68 p.p.m. and for triglyceride at 4.1–4.3 p.p.m., as
shown in Fig. 2A. Lipids extracted directly from
plasma (Fig. 2B) gave the same peaks as in the spec-
trum of lipids extracted from nanoparticles. The peaks
were confirmed by reference spectra and through 2D
total correlation spectroscopy (TOCSY) NMR experi-
ments. In humans, about 80% of the phospholipid
content in lipoprotein complexes is phosphatidylcho-
line, which could be identified in the extract from
plasma from the nitrogen-attached methyl groups
visible at 3.4 p.p.m., but not in the extract from nano-
particles incubated in plasma. However, as we have
shown that lipids bind to the nanoparticles (both by
size exclusion chromatography and by enzymatic
assay), an enzymatic kit for detection of phosphatidyl-
choline was used to verify that phosphatidylcholine is
indeed bound to the nanoparticle pellet before extrac-
tion. The kit detected phosphatidylcholine in the pellet
of 50 : 50 NIPAM ⁄ BAM but not in the pellet of the
less hydrophobic 65 : 35 NIPAM ⁄ BAM. The lower
affinity for the less hydrophobic nanoparticles corre-
lates well with the results from protein, cholesterol and
triglyceride binding studies, where binding is seen only
with the more hydrophobic 50 : 50 particles, and
serves as a negative control (see below). This means
that phospholipids are adsorbed onto the nanoparticle
but are not detected in the NMR spectrum of the
extract, probably as a result of being bound to the
nanoparticles also in the chloroform phase and there-
fore not being visible, owing to slow tumbling.
Surface characteristics are important for lipid
binding
The surface hydrophobicity of the copolymer nano-
particles can be varied by changing the ratio of the
two comonomers. After incubation in plasma and
repeated washing ⁄ centrifugation, considerable amounts
of cholesterol were identified in the pellets of 50 : 50
NIPAM ⁄ BAM nanoparticles (Fig. 3A). In comparison,
the less hydrophobic 65 : 35 NIPAM ⁄ BAM nanoparti-
cles bound very little cholesterol (Fig. 3A). This shows
that the amount of lipids bound to the nanoparticles is
dependent on the hydrophobicity of the nanoparticle
surface. This behavior is similar to the hydrophobicity
dependence in protein binding reported previously [2].
The hydrophobicity dependence was also confirmed
in NMR experiments on extracts from 65 : 35
NIPAM ⁄ BAM nanoparticles, showing much lower or
no signals from cholesterol and triglyceride as com-
pared with extracts from 50 : 50 NIPAM ⁄ BAM nano-
particles. The same behavior was seen for phospholipid
binding as described in the previous paragraph.
0123456
7
8
0
2
4
6
8
×10
7
4.14.34.5
0
8
16
×10
5
0.50.70.9
0
8
14
×10
6
Extract from plasma
p.p.m.
Arbitrary units
H
OCOR
1
H
H
H
H
OCOR
2
OCOR
3
ROCO
CH
3
p.p.m.
012
34
5
6
78
0
1
2
×10
9
4.14.3
4.5
5
15
25
×10
6
0.50.70.9
5
9
13
×10
7
Extract from 200 nm 50 : 50
incubated in plasma
Arbitrary units
A
B
Fig. 2. Lipids identified by NMR spectroscopy after extraction. (A)
1
H-NMR spectrum of lipids extracted from 200 nm 50 : 50
NIPAM ⁄ BAM copolymer nanoparticles incubated in plasma. (B)
1
H-NMR spectrum of lipids extracted from plasma. Magnifications
to the left: Double quartet of peaks from the outer four hydrogen
protons in the glycerol part of triglycerides. Magnifications to the
right: Single peak from cholesterol and cholesterol esters at
0.68 p.p.m. from the methyl group positioned between the hexago-
nal and pentagonal carbon rings in cholesterol and cholesterol ester.
Complete high-densitylipoproteinsinnanoparticlecorona E. Hellstrand et al.
3374 FEBS Journal 276 (2009) 3372–3381 ª 2009 The Authors Journal compilation ª 2009 FEBS
Copolymer nanoparticles of even lower hydrophobicity
(75 : 25 and 85 : 15 NIPAM ⁄ BAM) were also tested,
and the amount of bound cholesterol was at back-
ground level (data not shown). However, these nano-
particles disperse more readily in aqueous buffers,
which makes any detailed comparison difficult. The
low amount of lipids bound to the less hydrophobic
nanoparticles serves as a good negative control for the
lipid binding detected on the 50 : 50 NIPAM ⁄ BAM co-
polymer nanoparticles.
Lipid binding is surface area dependent
Copolymer nanoparticles (50 : 50 NIPAM ⁄ BAM) of
two sizes, 120 and 200 nm in diameter, were incubated
in plasma, and the bound lipids were separated from
free lipids by repeated centrifugation and washing. The
two nanoparticles produce very similar pellets, but the
surface area is 1.7 times greater in the 120 nm nano-
particle pellet than in the 200 nm nanoparticle pellet.
One milligram of nanoparticles corresponds to approx-
imately 1.1 · 10
12
(1.8 · 10
)12
mol) 120 nm particles
or 2.4 · 10
11
(4.0 · 10
)13
mol) 200 nm particles. A
comparison of the amount of bound lipids in a nonsat-
urated system shows that 0.5 mg of 120 nm nanoparti-
cles binds about 1.4–1.9 times more cholesterol and
triglycerides than 0.5 mg of 200 nm nanoparticles, as
shown in Table 1. Consequently, the amount of lipids
bound depends on the total surface area rather than
on the pellet volume or the number of nanoparticles.
In control experiments using plasma without nanopar-
ticles, only minute amounts of lipids are detected, and
these are subtracted from the values reported in
Table 1. An additional factor that may influence the
cholesterol binding is the surface curvature ⁄ particle
size, as the naturally occurring lipoprotein complexes
range in size from 8–10 nm to 100 nm, and the
amount of cholesterol associated with each differs, as
shown in Table 1.
Lipid binding by copolymer nanoparticles reaches
saturation
If nanoparticles bind discrete lipoprotein particles, the
binding may reach saturation. This was tested in
experiments with increasing amounts of plasma added
to a constant amount of 200 nm 50 : 50 NI-
PAM ⁄ BAM nanoparticles. The cholesterol levels were
determined by enzymatic assay, as shown in Fig. 3B.
After a steep increase of the amount of bound choles-
terol, a plateau was reached at about 40% plasma,
indicating saturation. This is approximately the same
percentage of plasma as that at which protein satura-
tion was reached in protein adsorption experiments at
similar particle concentrations, suggesting a coupled
binding behavior [2]. At saturation, there is approxi-
mately 20 nmol cholesterol per mg 200 nm nanoparti-
cles, which corresponds to 50 000 cholesterol or
cholesterol ester molecules per nanoparticle, or 60 lg
HDL per mg nanoparticles (assuming 3.08 wt% cho-
lesterol and 17.6 wt% cholesterol ester in HDL). Using
a radius of 5 nm and a density of 1.14 gÆmL
)1
for
HDL, this can be estimated to be 400 HDL molecules
per 200 nm particle, or one-quarter of the theoretical
maximum coverage in one layer. The same experiments
0
0.2
0.4
0.6
50 : 50 65 : 35
nmol·mg
–1
nmol·mg
–1
0
10
20
020406080
Plasma conc. / %
NIPAM : BAM
AB
Fig. 3. Surface hydrophobicity is important, and the binding is sur-
face limited. (A) Particle surface hydrophobicity is important for
plasma lipid binding. Copolymer nanoparticles, 200 nm 50 : 50 or
65 : 35 NIPAM ⁄ BAM, were incubated with human plasma.
Unbound lipids were separated from the particles by centrifugation.
The particle pellets were washed three times, and the amounts of
cholesterol and triglyceride were determined by standard enzymatic
assays. (B) Plasma cholesterol binding by 200 nm 50 : 50
NIPAM ⁄ BAM nanoparticles reaches saturation at approximately
20 nmoL cholesterol per mg nanoparticles, which corresponds to
50 000 cholesterol or cholesterol ester molecules per nanoparticle.
Copolymer nanoparticles, 0.5 mg, were incubated with increasing
amounts of human plasma in a constant volume. Unbound lipids
were separated from the nanoparticles by centrifugation. The parti-
cle pellets were washed three times, and the amount of choles-
terol was determined by standard enzymatic assay.
Table 1. Amount and ratio ± standard deviation of lipids on 50 : 50
NIPAM ⁄ BAM copolymer particles with two different diameters and
at two plasma concentrations. One milligram of nanoparticles con-
tains approximately 1.8 · 10
)3
nmol of 120 nm particles or
4.0 · 10
)4
nmol of 200 nm particles, and the surface area is 1.7
times larger in 1 mg of 120 nm particles than in 200 nm particles.
Particles
Cholesterol
(nmolÆmg
)1
particles)
Triglyceride
(nmolÆmg
)1
particles)
Molar ratio of
cholesterol ⁄
triglyceride
120 nm 50 : 50,
33% plasma
11.1 ± 1.6 3.4 ± 0.1 3.2 ± 0.6
120 nm 50 : 50,
67% plasma
17.9 ± 0.9 6.1 ± 0.6 2.9 ± 0.4
200 nm 50 : 50,
33% plasma
5.9 ± 0.1 2.5 ± 0.3 2.4 ± 0.3
200 nm 50 : 50,
67% plasma
11.0 ± 0.4 4.0 ± 0.4 2.7 ± 0.4
E. Hellstrand et al. Completehigh-densitylipoproteinsinnanoparticle corona
FEBS Journal 276 (2009) 3372–3381 ª 2009 The Authors Journal compilation ª 2009 FEBS 3375
were performed with 70 and 120 nm nanoparticles, but
complete saturation could not be reached, owing to
the larger particle surface area. Fewer nanoparticles in
each sample will lead to small pellets that are difficult
to handle in a reproducible way.
The cholesterol
⁄
triglyceride ratio is increased in
the nanoparticle lipid corona
The molar ratio of cholesterol and triglyceride bound
to the nanoparticles was established for the 120 and
200 nm 50 : 50 NIPAM ⁄ BAM nanoparticles at two
plasma concentrations (Table 1). To ensure that there
were no differences in the experimental routine, the
pellets were split into two equal parts in the last wash
before the cholesterol and triglyceride levels were mea-
sured. The cholesterol ⁄ triglyceride molar ratios varied
between 2.4 and 3.2, but were within experimental
error for all conditions. The cholesterol ⁄ triglyceride
molar ratio measured in the same plasma was 1.5.
Thus, the cholesterol ⁄ triglyceride ratio was increased
by a factor of 2 following interaction with and binding
to the nanoparticles as compared with the ratio in
plasma, indicating that specific lipoprotein particles
were targeted. The specificity is further analyzed in
Table 2, where the ratios from Table 1, after conver-
sion to mass ratios, are compared to ratios for the
different lipoprotein classes. The approximate amount
of protein was determined by comparing bound apoli-
poprotein A-I with known amounts of apolipopro-
tein A-I by SDS ⁄ PAGE, as shown in Fig. S1. Table 2
also shows the apolipoprotein pattern estimated from
SDS ⁄ PAGE and compares it with the different lipo-
protein classes.
Bound protein and lipids from purified
lipoprotein particle fractions
Three fractions of lipoprotein particles ) chylomi-
crons + very low density lipoprotein (VLDL), low-den-
sity lipoprotein (LDL), and HDL – were obtained from
human plasma by ultracentrifugation in a salt gradient.
The HDL fraction was further fractionated into HDL
and very high-density lipoprotein (VHDL) fractions.
The proteins in the final four fractions were visualized
by SDS ⁄ PAGE (Fig. 4A, lanes 1–4), and the proteins
bound to 50 : 50 NIPAM ⁄ BAM 200 nm copolymer
nanoparticles from each lipoprotein particle fraction are
shown in Fig. 4A, lanes 5–8. The main proteins in the
chylomicron + VLDL fraction (Fig. 4A, lane 1) were
(in size order) apolipoprotein B-100 and ⁄ or apolipopro-
tein B-48 (apolipoprotein B-100 is not separated from
its truncated variant apolipoprotein B-48 in this sys-
tem), human serum albumin (HSA), apolipoprotein E,
and apolipoprotein A-I. The same proteins were present
on the nanoparticles from this fraction (Fig. 4A, lane 5),
but the relative apolipoprotein E and apolipoprotein
A-I as compared with apolipoprotein B-100 were much
greater on the nanoparticles, indicating preferential
binding of apolipoprotein A-1 and apolipoprotein E.
In the LDL fraction (Fig. 4A, lane 2), the main pro-
tein was apolipoprotein B-100, as expected, but visible
amounts of albumin, apolipoprotein E and apolipo-
protein A-I were also present. The relative amount of
apolipoprotein B-100 was much less on the copolymer
nanoparticles incubated in the LDL fraction (Fig. 4A,
lane 6), indicating that lipoprotein particles with apoli-
poprotein E or apolipoprotein A-I preferentially bind
to the copolymer nanoparticles. In the HDL and
Table 2. Protein and lipid composition of lipoprotein particles, and the biomolecule corona around the 200 nm 50 : 50 NIPAM ⁄ BAM nano-
particles following incubation in plasma. The lipoprotein compositions are from several references collected by LipidBank (http://lipidbank.jp).
Chylomicron VLDL LDL HDL
2
HDL
3
Nanoparticle
corona
Mass ratio of protein, cholesterol,
and triglyceride
Protein ⁄ cholesterol (gÆg
)1
) 0.3–0.7 0.6 0.4–0.6 1.3–2.1 2.5–6.1 1.3–2.4
Protein ⁄ triglyceride (gÆg
)1
) 0.01–0.02 0.2 1.7–4.2 5.8–13 7.5–18 2.2–3.6
Cholesterol ⁄ triglyceride (gÆg
)1
) 0.03 0.3 3.4–7.5 3.2–8.7 1.5–6 1.5–2.0
Mass percentage of apolipoprotein
contributions
Apolipoprotein A-I + 65 62 70
Apolipoprotein A-II 10 23 10
Apolipoprotein A-IV Trace 10
Apolipoprotein E 4 13 4 1 10
Apolipoprotein B-48 5–20 37
Apolipoprotein B-100 100
Apolipoprotein C 70–80 50 12 5
Complete high-densitylipoproteinsinnanoparticlecorona E. Hellstrand et al.
3376 FEBS Journal 276 (2009) 3372–3381 ª 2009 The Authors Journal compilation ª 2009 FEBS
VHDL fractions (Fig. 4A, lanes 3 and 4), the major
proteins were apolipoprotein A-I and HSA. On the
copolymer nanoparticles incubated in the HDL and
VHDL fractions (Fig. 4A, lanes 7 and 8), apolipo-
protein A-I dominated.
Previous studies of proteins bound to the 50 : 50
NIPAM ⁄ BAM copolymer nanoparticles in human
plasma did not identify apolipoprotein B in the hard
corona [2]. Here we observed, in small amounts, apo-
lipoprotein B on nanoparticles incubated in the lipo-
protein fractions in which apolipoprotein B is the
dominating protein (chylomicrons, VLDL, and LDL).
The ratio of apolipoprotein B to apolipoprotein A-I
was significantly lower on the nanoparticles relative to
the respective lipoprotein fraction, indicating that apoli-
poprotein B-100 or LDL bind to the copolymer nano-
particles with lower affinity than apolipoprotein A-I or
HDL. Size exclusion chromatography was used to
further study this competition. NIPAM ⁄ BAM 50 : 50
200 nm copolymer nanoparticles were mixed with lipo-
protein particle fractions and, after a washing step, par-
ticles and their bound proteins were loaded onto a
Sephacryl S-1000 column. The lipoprotein particles did
not affect the elution volume of the nanoparticles, and
free LDL and HDL clearly eluted separately from the
nanoparticles (Fig. 4B). Eluted nanoparticles were pel-
leted, and the associated proteins were separated by
SDS ⁄ PAGE (Fig. 4C). Only apolipoprotein A-I was
present on the eluted copolymer nanoparticles mixed
with the LDL fraction, indicating that apolipopro-
tein A-I–HDL has a much greater binding affinity than
apolipoprotein B-100–LDL for the copolymer nanopar-
ticles. As expected, only apolipoprotein A-I was recov-
ered from the nanoparticles after mixing with the HDL
or VHDL fractions. No proteins could be seen in
SDS ⁄ PAGE from the nanoparticles incubated with the
B
0
0.2
0.4
0.6
0 50 100 150 200
Elution volume/(mL)
A
280
Apo A-I
Apo A-I
LDL-enriched fraction
HDL-enriched fraction
B-100
B-100
C
A
LDL
HDL
8
6
4
2
0
Cholesterol/(µg)
Loaded Eluted
LDL
HDL
Cholesterol/(a.u)
1
0
B-100
HSA
Apo A-I
M
r
130
72
36
28
17
D
96 150141132123114105
96 150141132123114105
18765432
Elution volume/(mL)
LP-fractions
Adsorbed on NP
Fig. 4. Fractionated lipoproteins (LP) and their binding to copolymer particles. (A) SDS ⁄ PAGE (15% gel) of lipoprotein fractions and nanoparti-
cles (NP) incubated in lipoprotein fractions. Lanes 1–4: density fractions from human blood enriched in chylomicron + VLDL, LDL, HDL, and
VHDL, respectively. Lanes 5–8: proteins adsorbed to 200 nm 50 : 50 NIPAM ⁄ BAM copolymer nanoparticles incubated in the density frac-
tions loaded in lanes 1–4, respectively. Bound proteins were separated from unbound proteins by centrifugation, and desorbed by
SDS ⁄ PAGE loading buffer. (B) Size exclusion chromatography of lipoprotein fractions enriched in LDL or HDL and on 200 nm 50 : 50 copoly-
mer nanoparticles incubated in the same fractions, separated by centrifugation. Open circles: LDL fraction. Open triangles: HDL fraction.
Filled circles: nanoparticles incubated in LDL fraction. Filled triangles: nanoparticles incubated in HDL fraction. (C) SDS ⁄ PAGE on bound pro-
teins at the elution volumes from size exclusion chromatography corresponding to the elution volumes of copolymer particles. (D) Amounts
of cholesterol loaded in the size exclusion experiments in (A) with LDL and HDL fractions as compared with the relative amounts that
coelute with the nanoparticles.
E. Hellstrand et al. Completehigh-densitylipoproteinsinnanoparticle corona
FEBS Journal 276 (2009) 3372–3381 ª 2009 The Authors Journal compilation ª 2009 FEBS 3377
chylomicron–VLDL fractions, probably because the
amounts bound to the nanoparticles were too small. In
all cases of lipoprotein particles binding to the copoly-
mer nanoparticles, apolipoprotein A-I was identified,
suggesting that the binding is mediated by apolipopro-
tein A-1, although a similar role for apolipoprotein
A-IV and apolipoprotein E cannot be excluded.
The relative amounts of cholesterol on the nanopar-
ticles mixed with the HDL or LDL fractions were
determined after gel filtration. The volume of HDL or
LDL fraction mixed with the nanoparticles was the
same in each experiment, which means that there was
6.5 times more cholesterol available in the samples
incubated with the LDL fraction than in those incu-
bated with the HDL fraction. Nevertheless, there was
1.5 times more cholesterol on the eluted nanoparticles
mixed with HDL than on the nanoparticles mixed with
LDL, as shown in Fig. 4D. This shows that the nano-
particles bind both proteins and lipids with high speci-
ficity, supporting the conclusion that HDL rather than
LDL binds to the copolymer nanoparticles.
Discussion
This is, to our knowledge, the first time that lipids
have been detected in the biomolecular corona sur-
rounding nanoparticles in human plasma. Moreover,
we have found that intact HDL particles bind to nano-
particles. We show, with three different approaches,
that the plasma lipids, cholesterol and triglycerides, are
present on 50 : 50 NIPAM ⁄ BAM copolymer nanopar-
ticles incubated in plasma. First, cholesterol elutes
together with nanoparticles in size exclusion chroma-
tography, and the elution position of cholesterol
depends on the size of the nanoparticles. Second,
nanoparticles preincubated in plasma were extracted
with a mixture of chloroform and methanol. In the
extract, cholesterol and triglyceride were detected with
NMR spectroscopy. Third, cholesterol, phospholipids
and triglyceride were detected by enzymatic assay on
nanoparticles following incubation with human plasma
and separation of unbound lipids by centrifugation.
The amount of bound lipids depends on the surface
area presented by the nanoparticles and not on the
pellet size following centrifugation. Less hydrophobic
nanoparticles (65 : 35, 75 : 25 and 85 : 15 NI-
PAM ⁄ BAM) bind no or minute amount of lipids in
any of these methods, and therefore provide an excel-
lent negative control.
Lipoprotein particles can be distinguished from one
another by the identity and amount of proteins, and by
the amount and ratio of cholesterol and triglycerides.
We have previously characterized the protein profile
and shown that it includes apolipoproteins and enzymes
[2]. The identified apolipoproteins and enzymes found in
that study correspond to the proteins found mainly in
HDL and chylomicrons, which further strengthens the
present results. Furthermore, the protein ⁄ cholesterol
and protein ⁄ triglyceride ratios correspond well with the
ratios in HDL (Table 2), but not with the ratios in larger
lipoprotein particles. The protein ⁄ triglyceride and cho-
lesterol ⁄ triglyceride ratios are in the lower range, imply-
ing that a small number of triglyceride-rich lipoprotein
particles, like chylomicrons, also bind to the nanopar-
ticles. Chylomicrons, like HDL, contain apolipo-
protein A-I, which is identified as the main candidate
for mediating binding of lipoprotein complexes to the
nanoparticles. In experiments with plasma fractions
enriched in different lipoprotein classes, the 50 : 50
NIPAM ⁄ BAM nanoparticles show high specificity for
apolipoprotein A-I and bind lipids from the plasma
fraction enriched in HDL with higher affinity than
those from the plasma fraction enriched to LDL. In
conclusion, the results strongly suggest that the 50 : 50
NIPAM ⁄ BAM copolymer nanoparticles in plasma
are associated with intact lipid-loaded apolipoprotein
A-I-containing lipoprotein particles, preferentially
HDL. Apolipoproteins have also been detected on
other nanoparticles, e.g. polystyrene, solid lipid nano-
particles, and carbon nanotubes, raising the possibility
that binding of intact lipoprotein particles extends to
other classes of nanoparticles [6–15].
We may speculate a little on the role of the lipopro-
tein biomolecular coronain determining the destiny of
nanoparticles that enter the bloodstream. The size of
the nanoparticles used in this study is of the same
order as that of large lipoprotein particles, and there is
a possibility that the relative curvature between the
nanoparticles and lipoproteins favors the binding of
small HDL over larger lipoproteins, although no such
tendency can be seen in the results in Table 2. An
interesting aspect of the equal size of the nanoparticles
and the lipoproteins is, however, that it may open the
door to the lipoprotein transport system for the lipo-
protein-coated nanoparticles. Lipoprotein particles are
known to bind selectively to receptors expressed in
organs and cells. There are several receptors described
that mediate lipid transport and endocytosis of LDL.
In the field of nanomedicine, this has led to numerous
publications exploring the possibility of using LDL in
drug delivery systems [16,17]. The most common
receptor is scavenger receptor class B type 1 (SR-BI),
which mediates the bidirectional lipid transfer between
VLDL, LDL and HDL and cells [5]. SR-BI is
expressed mainly in the liver and steroidogenic glands,
but also in brain, intestine, and placenta, and in cells
Complete high-densitylipoproteinsinnanoparticlecorona E. Hellstrand et al.
3378 FEBS Journal 276 (2009) 3372–3381 ª 2009 The Authors Journal compilation ª 2009 FEBS
such as macrophages and endothelial cells [5]. Another
possible receptor is cubilin, which has been shown to
bind apolipoprotein A-I and HDL, and to mediate
endocytosis of HDL [18,19]. Cubilin is expressed in the
proximal tubule in kidney and in epithelial cells in yolk
sac and intestine [20,21]. As the copolymer nanoparti-
cles bind HDL, it is possible that there will be recep-
tor-mediated uptake and enrichment of the
nanoparticles in organs and cells rich in SR-BI and
cubillin. Thus, the discovery that nanoparticles can
bind intact lipoprotein complexes offers a new window
on nanomedicine, as nanoparticles may also hitch a lift
on existing cellular lipidic transport pathways. We are
currently investigating the biological fate of these
lipoprotein-binding nanoparticles in vitro.
We have, for the first time, detected lipids in the bio-
molecular corona surrounding nanoparticles and char-
acterized the lipid binding. The interaction with
lipoprotein particles is highly specific, and several
experimental findings suggest that the copolymer nano-
particles (50 : 50 NIPAM ⁄ BAM) bind complete and
intact lipoprotein particles with high specificity for
HDL. It is possible that lipoprotein particle binding is
a common feature of nanoparticles in a general sense,
which makes the mechanism of the binding and the
implications for nanoparticle fate and impacts in vivo
important topics for future study.
Experimental procedures
Copolymer nanoparticles
NIPAM ⁄ BAM copolymer nanoparticles of diameter 70, 120
and 200 nm and with several different ratios of the comono-
mers (85 : 15, 25 : 75, 65 : 35 and 50 : 50 NIPAM ⁄ BAM)
were synthesized in the presence of SDS as described previ-
ously, although higher SDS concentrations were used in the
present work, resulting in similarly sized nanoparticles [22].
The procedure for the synthesis was as follows: 2.8 g of
monomers (in the appropriate w ⁄ w ratio) and 0.28 g of cross-
linker (N,N-methylenebisacrylamide) was dissolved in
190 mL of MilliQ water with either 0.8 g of SDS (for the
70 nm nanoparticles) or 0.32 g of SDS (for the 200 nm nano-
particles), and degassed by bubbling with N
2
for 30 min.
Polymerization was induced by adding 0.095 g ammonium
persulfate initiator in 10 mL of MilliQ water and heating at
70 °C for 4 h [23]. The nanoparticles were extensively
dialyzed against MilliQ water for several weeks, with the
water being changed daily, until no traces of monomers,
crosslinker, initiator or SDS could be detected by proton
NMR (spectra were acquired in D
2
O using a 500 MHz
Varian Inova spectrometer). The nanoparticles were freeze-
dried and stored in the refrigerator until used.
Human plasma, and buffers
Blood was drawn from a healthy individual into tubes with
EDTA or heparin, and centrifuged at 14 000 g for 30 min.
The supernatants from several vials were combined, and
aliquots of 400 lL were stored at – 80 °C. Before each
experiment, plasma aliquots were centrifuged at 14 000 g to
remove possible aggregates.
Enzymatic determination of triglycerides and
cholesterol
Copolymer nanoparticles were dispersed on ice in NaCl ⁄
P
i
⁄ EDTA (10 mm phosphate, 150 mm NaCl, pH 7.5, 1 mm
EDTA) and mixed with various amounts of plasma. After
1 h of incubation on ice, the mixtures were heated to 23 °C
to promote aggregation of the nanoparticles. The samples
were centrifuged at 14 000 g for two minutes, and the nano-
particle pellets were saved and washed three times with
NaCl ⁄ P
i
⁄ EDTA. The amount of bound triglycerides was
measured by adding 150 lL of a 4 : 1 mixture of Free Glyc-
erol Reagent (Sigma, Stockholm, Sweden) and Triglyceride
Reagent (Sigma) to the pellets. The nanoparticle pellets were
dispersed, and incubated at 37 °C for about 30 min. After
incubation, the nanoparticles were pelleted by centrifugation
at 14 000 g for two minutes, and the absorbance of the super-
natant was measured at 495 nm. The reagents cleave the fatty
acids from the triglycerides, and the amount of free glycerol
is quantified and used as a reporter of the initial amount of
triglycerides, so a standard curve of glycerol was used in each
experiment. The amount of bound cholesterol was deter-
mined using an Amplex
Ò
Red Cholesterol kit (Invitrogen,
Stockholm, Sweden). The nanoparticle pellets were resus-
pended in 50 lL of cholesterol kit reaction buffer and 50 lL
of the cholesterol kit working solution. After 30 min of incu-
bation at 37 °C, the nanoparticles were pelleted by centrifu-
gation at 14 000 g for two minutes, and the fluorescence of
the supernatant was measured. The cholesterol kit deter-
mines the concentration of total cholesterol, including the
part that is present in the lipoprotein particle core as esters
with fatty acids.
Size exclusion chromatography of nanoparticles
and bound plasma lipids
Copolymer nanoparticles of 0.5 mL (10 mgÆmL
)1
) were
mixed with 0.4 mL of NaCl ⁄ P
i
⁄ EDTA or with 0.4 mL
of human plasma, and incubated on ice. After 1 h, the
samples were heated to 23 °C to promote aggregation of
nanoparticles, allowing pelleting by centrifugation. The
pellets were washed with 1 mL of NaCl ⁄ P
i
⁄ EDTA and
dispersed in 0.5 mL of NaCl ⁄ P
i
⁄ EDTA on ice. The mixture
was loaded onto a 30 · 1.5 cm Sephacryl S-1000 SF
column and eluted with NaCl ⁄ P
i
⁄ EDTA at a flow rate of
E. Hellstrand et al. Completehigh-densitylipoproteinsinnanoparticle corona
FEBS Journal 276 (2009) 3372–3381 ª 2009 The Authors Journal compilation ª 2009 FEBS 3379
0.8 mLÆmin
)1
, and 1.7 mL fractions were collected. The elu-
tion profile of the nanoparticles was obtained by recording
the scattering at 280 nm in a UV spectrometer after aggre-
gation of the nanoparticles at 37 °C. To obtain the elution
profiles of cholesterol or triglyceride, the nanoparticles in
each fraction were pelleted by centrifugation at 14 000 g
for two minutes, and the amount of lipid was determined
by enzymatic assays as described above.
In the experiments with fractionated lipoprotein particles,
1 mL of copolymer nanoparticles (10 mgÆmL
)1
) was mixed
with 0.5 mL of lipoprotein particles, and incubated and
washed as described above. The mixture was loaded onto a
95 · 1.5 cm Sephacryl S-1000 column and eluted with
NaCl ⁄ P
i
⁄ EDTA. To analyze the bound proteins, 2 mL frac-
tions from each nanoparticle and lipoprotein fraction were
spun down, and the proteins were desorbed by SDS ⁄ PAGE
loading buffer and separated by SDS ⁄ PAGE (15% gel).
Extraction of lipids and detection by NMR
Plasma (800 lL) was incubated with 800 lLof10mgÆ mL
)1
200 nm 50 : 50 copolymer nanoparticles in NaCl ⁄ P
i
⁄ EDTA
on ice for 1 h, and then for 30 min at room temperature.
The nanoparticles were then harvested by centrifugation at
14 000 g for two minutes, and washed three times with
800 lL of NaCl ⁄ P
i
⁄ EDTA. Plasma (800 lL) or nanoparti-
cle pellets, resuspended in 800 lL of NaCl ⁄ P
i
, were
extracted against 1.2 mL of 0.5 m KH
2
PO
4
, 6 mL of
CHCl
3
, and 2 mL of MeOH. The chloroform phase was
evaporated, and the remaining material was dissolved in
600 lL of deuterated (99.8%) chloroform.
1
H ⁄ 1D spectra
were recorded at 25 °C using a 600 MHz Varian Unity Ino-
va spectrometer. The chemical shift was referenced to the
residual chloroform signal (d 7.26).
Enzymatic determination of phosphatidylcholine
Plasma (200 lL) was incubated with 200 lLof10mgÆmL
)1
120 nm 50 : 50 or 65 : 35 copolymer nanoparticles in NaCl ⁄
P
i
⁄ EDTA on ice for 1 h, and then for 30 min at room
temperature. The nanoparticles were then harvested by centri-
fugation at 14 000 g for two minutes, and washed three times
with 400 lL of NaCl ⁄ P
i
⁄ EDTA. The pellet was analyzed
with the kit Phospholipids B no. 990-54009E (Wako, Neuss,
Germany) in a reaction volume of 1.5 mL.
Purification of lipoprotein particle fractions from
plasma
Lipoprotein particle fractions were purified as described by
Schumaker and Puppione [24]. Lipemic citrate plasma was
ultracentrifuged repeatedly at 147 000 g in an Optimal
L-70K Beckman centrifuge with a Ti 701 rotor, for 25 h at
12 °C. Before each centrifugation, the density was adjusted
with 5 m NaCl and saturated NaBr, both containing 0.04%
EDTA. After each centrifugation step, a lipoprotein fraction
was collected from the top of the centrifuge tubes. The cor-
responding densities from which the fractions were collected
are: 1.0068, 1.068, 1.21 and 1.25 gÆmL
)1
for chylomicron +
VLDL, LDL, HDL, and VHDL, respectively. All fractions
were dialyzed against NaCl ⁄ P
i
⁄ EDTA. The cholesterol and
the triglyceride concentrations were determined to be 1.5
and 6.8 mm in the chylomicron + VLDL fraction, 15 and
2.6 m m in the LDL fraction, and 2.4 and 0.73 mm in the
HDL fraction, respectively. In the VHDL fraction, the
cholesterol concentration was 0.073 mm, but the triglyceride
concentration was too low to be determined.
Acknowledgements
This work was funded in part by the EU FP6 projects
NanoInteract (NMP4-CT-2006-033231), BioNano-
Interact SRC and SIGHT (IST-2005-033700-SIGHT),
the Swedish Research Council (VR), and Science
Foundation Ireland.
References
1 Salvati A, Soderman O & Lynch I (2007) Plum-pudding
gels as a platform for drug delivery: understanding the
effects of the different components on the diffusion
behavior of solutes. J Phys Chem B 111, 7367–
7376.
2 Cedervall T, Lynch I, Foy M, Berggad T, Donnelly SC,
Cagney G, Linse S & Dawson KA (2007) Detailed iden-
tification of plasma proteins adsorbed on copolymer
nanoparticles. Angew Chem Int Ed 46, 5754–5756.
3 Cedervall T, Lynch I, Lindman S, Berggard T, Thulin
E, Nilsson H, Dawson KA & Linse S (2007) Under-
standing the nanoparticle-protein corona using methods
to quantify exchange rates and affinities of proteins for
nanoparticles. Proc Natl Acad Sci USA 104, 2050–2055.
4 Lindman S, Lynch I, Thulin E, Nilsson H, Dawson KA
& Linse S (2007) Systematic investigation of the ther-
modynamics of HSA adsorption to N-iso-propylacryla-
mide ⁄ N-tert-butylacrylamide copolymer nanoparticles.
Effects of particle size and hydrophobicity. Nano Lett 7,
914–920.
5 Zannis VI, Chroni A & Krieger M (2006) Role of
apoA-I, ABCA1, LCAT, and SR-BI in the biogenesis
of HDL. J Mol Med 84, 276–294.
6 Blunk T, Hochstrasser DF, Sanchez JC, Muller BW &
Muller RH (1993) Colloidal carriers for intravenous
drug targeting-plasma-protein adsorption patterns on
surface-modified latex-particles evaluated by 2-dimen-
sional polyacrylamide-gel electrophoresis. Electrophore-
sis 14 , 1382–1387.
Complete high-densitylipoproteinsinnanoparticlecorona E. Hellstrand et al.
3380 FEBS Journal 276 (2009) 3372–3381 ª 2009 The Authors Journal compilation ª 2009 FEBS
7 Gessner A, Lieske A, Paulke BR & Muller RH (2002)
Influence of surface charge density on protein
adsorption on polymeric nanoparticles: analysis by
two-dimensional electrophoresis. Eur J Pharm Biopharm
54, 165–170.
8 Gessner A, Waicz R, Lieske A, Paulke BR, Mader K &
Muller RH (2000) Nanoparticles with decreasing
surface hydrophobicities: influence on plasma protein
adsorption. Int J Pharm 196, 245–249.
9 Goppert TM & Muller RH (2003) Plasma protein
adsorption of Tween 80- and poloxamer 188-stabilized
solid lipid nanoparticles. J Drug Target 11, 225–231.
10 Goppert TM & Muller RH (2005) Polysorbate-stabi-
lized solid lipid nanoparticles as colloidal carriers for
intravenous targeting of drugs to the brain: comparison
of plasma protein adsorption patterns. J Drug Target
13, 179–187.
11 Goppert TM & Muller RH (2005) Protein adsorption
patterns on poloxamer- and poloxamine-stabilized solid
lipid nanoparticles (SLN). Eur J Pharm Biopharm 60,
361–372.
12 Gref R, Luck M, Quellec P, Marchand M, Dellacherie
E, Harnisch S, Blunk T & Muller RH (2000) ‘Stealth’
corona-core nanoparticles surface modified by polyeth-
ylene glycol (PEG): influences of the corona (PEG chain
length and surface density) and of the core composition
on phagocytic uptake and plasma protein adsorption.
Colloids Surf B Biointerfaces 18, 301–313.
13 Luck M, Paulke BR, Schroder W, Blunk T & Muller
RH (1998) Analysis of plasma protein adsorption on
polymeric nanoparticles with different surface character-
istics. J Biomed Mater Res 39, 478–485.
14 Muller RH, Ruhl D, Luck M & Paulke BR (1997)
Influence of fluorescent labelling of polystyrene particles
on phagocytic uptake, surface hydrophobicity, and
plasma protein adsorption. Pharm Res 14, 18–24.
15 Salvador-Morales C, Flahaut E, Sim E, Sloan J, Green
MLH & Sim RB (2006) Complement activation and
protein adsorption by carbon nanotubes. Mol Immunol
43, 193–201.
16 Chung NS & Wasan KM (2004) Potential role of the
low-density lipoprotein receptor family as mediators of
cellular drug uptake. Adv Drug Deliv Rev 56, 1315–1334.
17 Rensen PC, de Vrueh RL, Kuiper J, Bijsterbosch MK,
Biessen EA & van Berkel TJ (2001) Recombinant lipo-
proteins: lipoprotein-like lipid particles for drug target-
ing. Adv Drug Deliv Rev 47, 251–276.
18 Hammad SM, Stefansson S, Twal WO, Drake CJ,
Fleming P, Remaley A, Brewer HB & Argraves WS
(1999) Cubilin, the endocytic receptor for intrinsic
factor–vitamin B-12 complex, mediates high-density
lipoprotein holoparticle endocytosis. Proc Natl Acad Sci
USA 96, 10158–10163.
19 Kozyraki R, Fyfe J, Kristiansen M, Gerdes C, Jacobsen
C, Cui SY, Christensen EI, Aminoff M, de la Chapelle
A, Krahe R et al. (1999) The intrinsic factor–vitamin
B-12 receptor, cubilin, is a high-affinity apolipopro-
tein A-I receptor facilitating endocytosis of high-density
lipoprotein. Nat Med 5, 656–661.
20 Sahali D, Mulliez N, Chatelet F, Dupuis R, Ronco P &
Verroust P (1988) Characterization of a 280-kd protein
restricted to the coated pits of the renal brush-border
and the epithelial-cells of the yolk-sac – teratogenic
effect of the specific monoclonal-antibodies. J Exp Med
167, 213–218.
21 Seetharam B, Christensen EI, Moestrup SK, Hammond
TG & Verroust PJ (1997) Identification of rat yolk sac
target protein of teratogenic antibodies, gp280, as
intrinsic factor cobalamin receptor. J Clin Invest 99,
2317–2322.
22 Wu X, Pelton RH, Hamielec AE, Woods DR &
McPhee W (1994) The kinetics of poly(n-isopropyl-
acrylamide) microgel latex formation. Colloid Polym Sci
272, 467–477.
23 Lynch I, Dawson KA & Linse S (2006) Detecting cryp-
tic epitopes created by nanoparticles. Sci STKE 2006,
pe14.
24 Schumaker VN & Puppione DL (1986) Sequential flota-
tion ultracentrifugation. Methods Enzymol 128, 155–170.
Supporting information
The following supplementary material is available:
Fig. S1. Binding of apolipoprotein A-I to 200 nm
50 : 50 NIPAM ⁄ BAM nanoparticles monitored by
SDS ⁄ PAGE on a 15% polyacrylamide gel.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
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FEBS Journal 276 (2009) 3372–3381 ª 2009 The Authors Journal compilation ª 2009 FEBS 3381
. is
expressed mainly in the liver and steroidogenic glands,
but also in brain, intestine, and placenta, and in cells
Complete high-density lipoproteins in nanoparticle. by apolipoproteins in plasma.
The selective binding of apolipoproteins raises many
interesting questions that this article begins to
address. For instance,