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 fro
Trang 1Erik Hellstrand1, Iseult Lynch2, Astra Andersson3, Torbjo¨rn Drakenberg1, Bjo¨rn Dahlba¨ck3,
Kenneth A Dawson2, Sara Linse1,2and Tommy Cedervall1,2
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.
Trang 2with 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 lipo-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] Apolipolipo-protein 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 nanopartinanoparti-cles 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
Fraction number
120 nm
200 nm
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.
Trang 3tion difficult However, 1H 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 phosphatidylphosphatidyl-choline 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
0 1 2 3 4 5 6 7
8
0
2
4
6
8 ×10
7
4.1 4.3
4.5
0
8
16
×105
0.5 0.7
0.9 0 8 14
×106
Extract from plasma
p.p.m.
H
OCOR1
H
H
H
H
OCOR2
OCOR3
ROCO
CH3
p.p.m.
0 1 2 3 4 5 6 7
8
0
1
2
×10 9
4.1 4.3
4.5
5
15
25
×106
0.5 0.7
0.9 5 9 13
×107
Extract from 200 nm 50 : 50
incubated in plasma
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 pentagohexago-nal carbon rings in cholesterol and cholesterol ester.
Trang 4Copolymer 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 nanonano-particle pellet
One milligram of nanoparticles corresponds to
approx-imately 1.1· 1012 (1.8· 10)12mol) 120 nm particles
or 2.4· 1011 (4.0· 10)13mol) 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 adsorpsatura-tion 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% chocho-lesterol 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
0 10 20
Plasma conc / % NIPAM : BAM
Fig 3 Surface hydrophobicity is important, and the binding is
sur-face limited (A) Particle sursur-face 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)3nmol of 120 nm particles or 4.0 · 10)4nmol 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
Trang 5were 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 (apolipoproapolipopro-tein 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 apolipopropro-tein 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 apoliapoli-poprotein 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).
Nanoparticle corona Mass ratio of protein, cholesterol,
and triglyceride
Mass percentage of apolipoprotein
contributions
Trang 6VHDL 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 apolipolipo-protein 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
Elution volume/(mL)
A280
Apo A-I
Apo A-I
LDL-enriched fraction
HDL-enriched fraction
B-100
B-100
C
A
8 6 4 2 0
HDL Cholesterol/(a.u)
1
0
B-100 HSA
Apo A-I
130
72
36
28
17
D
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.
Trang 7chylomicron–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 apolipoproapolipopro-tein
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 corona in 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
Trang 8such 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
and 200 nm and with several different ratios of the
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
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
Polymerization was induced by adding 0.095 g ammonium
persulfate initiator in 10 mL of MilliQ water and heating at
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
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
experiment, plasma aliquots were centrifuged at 14 000 g to remove possible aggregates
Enzymatic determination of triglycerides and cholesterol
EDTA) and mixed with various amounts of plasma After
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
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
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
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
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
of human plasma, and incubated on ice After 1 h, the
nanoparticles, allowing pelleting by centrifugation The
Trang 90.8 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
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,
with 0.5 mL of lipoprotein particles, and incubated and
washed as described above The mixture was loaded onto a
frac-tions from each nanoparticle and lipoprotein fraction were
Extraction of lipids and detection by NMR
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
extracted against 1.2 mL of 0.5 m KH2PO4, 6 mL of
evaporated, and the remaining material was dissolved in
Ino-va spectrometer The chemical shift was referenced to the
residual chloroform signal (d 7.26)
Enzymatic determination of phosphatidylcholine
temperature The nanoparticles were then harvested by
centri-fugation at 14 000 g for two minutes, and washed three times
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
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
VLDL, LDL, HDL, and VHDL, respectively All fractions
the triglyceride concentrations were determined to be 1.5 and 6.8 mm in the chylomicron + VLDL fraction, 15 and 2.6 mm 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
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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
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