Research Article

Cite This: ACS Appl. Mater. Interfaces 2019, 11, 27615−27623 www.acsami.org

Biomaterial Surface Hydrophobicity-Mediated Serum Protein

Adsorption and Immune Responses
Rahul M. Visalakshan,†,‡ Melanie N. MacGregor,‡ Salini Sasidharan,§ Artur Ghazaryan,∥
Agnieszka M. Mierczynska-Vasilev,⊥ Svenja Morsbach,∥ Volker Mailänder,∥,# Katharina Landfester,∥
John D. Hayball,¶,∇,○ and Krasimir Vasilev*,†,‡
†
School of Engineering and ‡Future Industries Institute, University of South Australia, Mawson Lakes, South Australia 5095,
Australia
§
Department of Environmental Sciences, University of California Riverside, Riverside, California 92521, United States
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

∥
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
⊥
The Australian Wine Research Institute, Waite Precinct, Adelaide, South Australia 5064, Australia
#
Department of Dermatology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstr. 1, 55131
Downloaded via UNIV OF TORONTO on October 22, 2020 at 22:42:56 (UTC).

Mainz, Germany
¶
School of Pharmacy & Medical Sciences, University of South Australia, Adelaide, South Australia 5001, Australia
∇
Experimental Therapeutics Laboratory, University of South Australia Cancer Research Institute, Adelaide, South Australia 5000,
Australia
○
Robinson Research Institute and Adelaide Medical School, University of Adelaide, Adelaide, South Australia 5005, Australia
*
S Supporting Information

ABSTRACT: The nature of the protein corona forming on biomaterial surfaces can
affect the performance of implanted devices. This study investigated the role of
surface chemistry and wettability on human serum-derived protein corona formation
on biomaterial surfaces and the subsequent effects on the cellular innate immune
response. Plasma polymerization, a substrate-independent technique, was employed
to create nanothin coatings with four specific chemical functionalities and a spectrum
of surface charges and wettability. The amount and type of protein adsorbed was
strongly influenced by surface chemistry and wettability but did not show any
dependence on surface charge. An enhanced adsorption of the dysopsonin albumin
was observed on hydrophilic carboxyl surfaces while high opsonin IgG2 adsorption was seen on hydrophobic hydrocarbon
surfaces. This in turn led to a distinct immune response from macrophages; hydrophilic surfaces drove greater expression of
anti-inflammatory cytokines by macrophages, whilst surface hydrophobicity caused increased production of proinflammatory
signaling molecules. These findings map out a unique relationship between surface chemistry, hydrophobicity, protein corona
formation, and subsequent cellular innate immune responses; the potential outcomes of these studies may be employed to tailor
biomaterial surface modifications, to modulate serum protein adsorption and to achieve the desirable innate immune response
to implanted biomaterials and devices.
KEYWORDS: protein adsorption, biomaterial, wettability, human serum, immune responses, plasma polymerization

1. INTRODUCTION nanoparticles, consists of a hard and soft layer.7 The “hard

The success of a biomedical device implantation and its
pathophysiology is dictated by the biological interactions at its
surface.1−4 This is because, immediately after implantation, a
biomaterial comes in contact with blood, and serum proteins
adsorb to surfaces, giving it a new biological identity almost
instantly.5,6 It is this protein layer and its composition, that
innate immune cells, such as macrophages, neutrophils, and
monocytes, detect rather than the pristine biomaterial
surfaces.6 Therefore, the protein layer formed on a biomaterial
surface has paramount importance in determining the fate of
an implanted biomaterial. This layer of adsorbed protein, also
referred to as protein corona, when they adsorb around
corona” results from the irreversible adsorption of proteins
while the dynamic exchange of proteins between biomaterial
surface and the medium is termed “soft corona”.6,8,9
Previously, we have reported on the role of hydrophilicity in
the reducing immune reaction with polyphosphoester-coated
nanocarriers, by encouraging selective adsorption of dysop-
sonin proteins in the corona.10 Whilst extensive research is
focused on understanding the protein corona formed around
Received: June 6, 2019
Accepted: July 16, 2019
Published: July 16, 2019

© 2019 American Chemical Society 27615 DOI: 10.1021/acsami.9b09900

ACS Appl. Mater. Interfaces 2019, 11, 27615−27623
ACS Applied Materials & Interfaces
various nanoparticles, the nature of the protein corona and the
surface parameters influencing its formation on the macro-
scopic surfaces of implantable biomedical device remain an
open question. Thus, in order to be able to design
immunomodulatory biomaterial surfaces capable of controlling
protein adsorption to modulate immune responses, it is
essential to understand the molecular mechanism driving
protein adsorption from human serum and how it dictates
behavior of innate immune cells, including macrophages as key
contributors.
In this study, we addressed the role of surface chemistry and
wettability in regulating the type and amount of adsorbed
protein, with the aim of interrogating how these biophysical
processes affect cellular innate immune responses, specifically
macrophage responses. To create model surfaces with a
desirable range of chemical and wetting properties, we
employed plasma polymerization.11 This technique was chosen
for surface modification because it provides a range of
attractive features; it facilitates the deposition of polymer-like
coatings that have a broad range of “tunable” chemical and
physical properties in a single step and is a solvent-free
process.2 The coating thickness is readily controllable, and can
be as little as just few nanometers thin.12 Most importantly,
plasma-derived coatings can be deposited on practically any
type of substrate material. The latter is attractive because any
particular surface property can be directly transferred to actual
functional products, including medical devices.13 The capacity
of plasma polymers to adhere well to most substrates can be
explained by the nature of the technique. Coatings are
deposited from a precursor which is electrically excited to
plasma phase, in our case, by radio frequency electromagnetic
field.14,15 Plasma consists of highly energetic molecules,
molecular fragments, ions, radicals, metastables, and free
electrons. These high energy species bombard the substrate
to be coated, creating reactive sites for chemical and physical
binding to the surface which results in strong adhesion of the
plasma-deposited film.16 In contrast, other methods for surface
preparation such as self-assembled monolayers and layer-by-
layer, are limited to specific types of substrate materials and
cannot be readily transferred to current medical devi-
ces.12,13,17,18 This creates a significant disconnection between
the results obtained using model substrates in the laboratory
and these from in vivo studies where surface properties need to
be transferred on only selected types of materials.12,19,20
Another reason for the discrepancy often observed between in
vitro and in vivo studies is that, in the laboratory, most protein
adsorption studies are conducted either using a single protein
or simple mixtures of selected proteins such as albumin,
fibrinogen, and immunoglobulin.21−24 These studies typically
fail to account for the dynamic processes and competing
protein adsorption events occurring in vivo.19,25 As a result, the
outcomes of studies involving clinically relevant bodily fluids
often appear contradictory to what is seen in single protein
adsorption studies. For instance, a study conducted on
polymer brushes surfaces demonstrated negligible protein
adsorption from a single fibrinogen buffer solution. However,
when incubated with undiluted plasma, the surface shows
enrichment in protein adsorption.26 Vitronectin adsorption
studies also showed altered adsorption behavior when
adsorbed from plasma, with considerable enrichment on to
various polymers surface as compared with its single protein
solution.27,28 The disconnection discussed above, points to the
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need for employing protein adsorption studies in biological
solutions that have greater relevance.
In this work, we provide an in-depth investigation of the
protein corona formed from full human serum on model
biomaterial surfaces with tailored chemical composition and
wettability. Human serum is a multiprotein solution that
exhibits complex and dynamic adsorption patterns compared
to model simple protein solutions.29 Thus, unlike adsorption
studies from reconstituted single or mixed protein suspensions,
this approach provides a practically relevant insight into the
nature of the protein layer that could form on the surface of an
implanted material. Furthermore, we attempted to derive a link
between the adsorbed protein corona and inflammatory
responses by measuring the production of pro and anti-
inflammatory cytokine signaling molecules from macrophages.
Macrophages are the sentinels of the body and can direct the
fate of an implanted material in different directionsfrom
successful integration to foreign body giant cells and
failure.30,31 The response of these cells is known to be
modulated by the material surface chemistry and the amount
of adsorbed proteins, their conformation, and orienta-
tion.3,4,32−34
In fact, this immune cell response arises from the interaction
between cells and surface bound proteins. Differences in the
type and amount of adsorbed protein, their unfolding and
orientation together determine the immune response.33,35 In
order to shed light on the host immune response as a whole to
different implant surface chemistry,6 we here investigated the
protein adsorption pattern from whole human serum and
correlated the findings with in vitro studies of macrophages
immune response.
2. MATERIALS AND METHODS
2.1. Materials for Plasma Polymerization. Acrylic acid (AC),
2-methyl-2-oxazoline (MEOX), allyalamine (AA), and 1,7-octadiene
(OD) and (Sigma-Aldrich, Australia) were used as received for
surface preparation.
2.2. Materials for Protein Corona Studies. Pooled normal
human serum was purchased from Innovative Research, Inc.
(Michigan, USA) and used for protein corona studies.
2.3. Materials for Immune Studies. Phorbol-12-myristate-13-
acetate (PMA) for THP-1 macrophage differentiation were purchased
from Sigma-Aldrich (Australia). Lipopolysaccharides (LPS) from
Escherichia coli O111:B4 were purchased Sigma-Aldrich (Austral-
ia).Cytokine production was quantified using human ELISA kits and
LEGENDplex from BioLegend (USA).
2.4. Surface Preparation. 2.4.1. Plasma Polymerization.
Surfaces with different chemical functionalities were coated on tissue
culture plates and silicon wafers using plasma polymer deposition in a
custom build reactor with a 13.56 MHz plasma generator.14 The
plasma chamber reactor was brought to vacuum, once the chamber
reaches a base pressure of 0.02 mbar, a 5 min air cleaning was carried
out at 0.11 mbar pressure with 50 W. Plasma deposition of allylamine
and AC was carried out at 0.13 mbar pressure for 2 min at 40 and 10
W, respectively, OD was deposited for 10 min at 20 W power. Methyl
oxazoline (MEOX) deposition was carried out at 0.08 mbar pressure
for 2 min at 50 W power. Using these plasma conditions, we obtained
stable polymer film coating with thickness in the range of 20−30 nm
as per ellipsometry measurements.
2.5. Surface Characterization. 2.5.1. X-ray Photoelectron
Spectroscopy. Surface chemical composition of plasma polymer-
deposited layer was analyzed using SPECS silicon and germanium
(SAGE) XPS spectrometer. X-ray photoelectron spectroscopy (XPS)
spectra were recorded with a monochromatic Mg radiation source
operated at 10 kV and 20 mA. The chemical composition of plasma-
deposited surfaces was identified from survey spectra over a 0−1000
DOI: 10.1021/acsami.9b09900
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Figure 1. (A) Photograph of the custom-built plasma polymerization chamber used to generate model nanothin coatings with controlled chemistry
from selected precursor and their respective predominant surface functional groups (B). (C) XPS survey spectra of the plasma polymer-coated
surfaces. (D) Water contact angle of the modified surfaces, data are shown as means ± standard deviation and the * significant difference compared
to AC (P < 0.05) using one-way ANOVA followed by the Dunnett’s multiple comparisons test. (E) Surface charge of modified surfaces.
eV range with a pass energy of 100 eV at a resolution of 0.5 eV.
CasaXPS software was used for data processing and curve fitting in
reference to the binding energy of neutral carbon C 1s peak at 285.0
eV.
2.5.2. Water Contact Angle. The water contact angle was
measured using a sessile drop contact angle goniometer in a class
1000 cleanroom. Plasma polymer-deposited silicon wafers were used
as substrates for the wettability measurements. Using a 50 μL
Hamilton microsyringe, a 5 μL Milli-Q water droplet was brought in
contact with the surface and imaged for the advancing contact angle.
The captured image of the droplet was processed with SCA20 Data
Physics software.
2.5.3. Zeta Potential Measurement. Plasma-coated silica particles
were used as substrates to measure the surface charge. Zeta potential
measurement was carried out using Zetasizer Nano ZS (Malvern, UK)
and transformed into zeta potential using the Smoluchowski equation
(Müller, 1991) for recognition. Measurements were carried out with
10−3 M KCl.
2.5.4. Ellipsometry. Silicon wafers were used as substrates for
plasma polymer film thickness measurements. An A J.A. Woollam Co.
variable angle spectroscopic ellipsometer imaging ellipsometer with
WVASE32 software was used to calculate thickness.
2.6. Serum Protein Adsorption Studies. 2.6.1. Protein Corona
Formation and Quantification. Human-pooled serum was purchased
from Innovative Research to study the protein corona formation. The
serum samples were centrifuged for an hour at 20 000 g to remove the
aggregated proteins. For serum protein adsorption studies, Serum
(500 μL) was added to the plasma polymer-deposited wells and
incubated for 1 h at 37 °C with constant shaking. After 1 h, the serum
was removed and the wells were washed three times using 1 mL of
phosphate-buffered saline (PBS) per well with 5 min shaking to
remove the loosely bound soft corona. To isolate the hard corona or
strongly bound serum proteins, 150 μL of sodium dodecyl sulphate
(SDS)−Tris HCl buffer (40 mg SDS + 125 μL Tris-HCl + Milli-Q
water up to a total of 2 mL) was added to the wells and incubated by
shaking at 95 °C for 15 min.36,37 The total protein concentration was
determined using a Pierce 660 nm protein assay according to the
manufacturer’s instructions.38−41
2.6.2. SDS Polyacrylamide Gel Electrophoresis. Gel electro-
phoresis was used for the visual representation of protein corona
contents based on molecular weights. The protein sample (16.25 μL),
NuPAGE LDS sample buffer (6.25 μL), and NuPAGE sample
reducing agent (2.5 μL) were mixed together and applied onto a
NuPAGE 10% bis−Tris protein gel (all Novex, Thermo Fisher
Scientific). The electrophoresis was carried out in NuPAGE MES
SDS running buffer at 150 V for 1.5 h and SeeBlue Plus2 prestained
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Standard (Invitrogen) was used as a molecular marker. The gel was
stained using Pierce silver stain kit (Novex, Thermo Fisher Scientific).
2.6.3. Liquid Chromatography−Mass Spectrometry Analysis. A
nanoACQUITY ultra performance liquid chromatography system
coupled with a Synapt G2-Si mass spectrometer (Waters Corpo-
ration) was used for performing quantitative analysis of protein
samples. Tryptic peptides were separated on the nanoACQUITY
system equipped with a C18 analytical reversed-phase column (1.7
μm, 75 μm × 150 mm, Waters Corporation) and C18 nano-
ACQUITY trap column (5 μm, 180 μm × 20 mm, Waters
Corporation).37
2.6.4. Immune Response Studies. Human monocytes, cell line
THP-1, were used in this study. RPMI 1640 (Sigma-Aldrich) was
used as the growth medium for THP-1 cells along with 10% fetal
bovine serum (FBS, Thermo Scientific) and 1% (v/v) penicillin/
streptomycin (Life Technologies). Cells were maintained in a
humidified atmosphere containing 5% CO2 at 37 °C. THP-1 cells
were differentiated into macrophages dTHP-1 using PMA according
to the protocol previously reported;42 briefly, 100 ng/mL PMA was
added to the media and incubated for 48 h and another 24 h with
PMA-free media. Differentiated dTHP-1 macrophages were seeded
on a plasma polymer-deposited 24-well plate at a density of 1 × 105
cells/mL. After overnight growth, the medium was removed and cells
were washed with PBS. The fresh medium was added to the wells
along with LPS (1 μg/mL) to activate macrophages. After 6 h of
incubation conditioned media were collected and centrifuged to
remove the cell debris. Supernatants were collected and analyzed for
pro and anti-inflammatory cytokines using LEGENDplex ELISA kits
(BioLegend, San Diego, CA, USA) following the manufacturer’s
instructions.
2.6.5. Statistical Analysis. All statistical computations were
performed using GraphPad Prism software, and the statistical
significance was analyzed using one-way analysis of variance
(ANOVA) followed by the Dunnett’s multiple comparisons test. All
of the data are shown as means ± standard deviation, and the level of
significance was set α (P < 0.05).
3. RESULTS AND DISCUSSION
To achieve the goal of this study, we used plasma
polymerization (Figure 1A) to generate four model surfaces
having tailored chemical functionality and wettability on tissue
culture plate and silicon wafer substrates. Schematically, these
surfaces are depicted in Figure 1B and were based on the
following precursors: AA (amineNH3, hydrophilic), AC
(carboxyl acidCOOH, hydrophilic), MEOX (C4H7NO,
hydrophilic), and OD (hydrocarbonCH3, hydrophobic).
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The surface chemical composition of the coatings was Wettability measurements shows that the static water
characterized by XPS and is shown in Figure 1C. The survey contact angle on the plasma-deposited coatings ranged
spectra of coatings deposited from AC and OD contain 72.1 between 49° and 92° (Figure 1D). AC coating had the most
and 92.5 at. % carbon (C 1s) and 27.8 and 7.4 at. % oxygen (O hydrophilic surface with a contact angle of 49°. MEOX and
1s), respectively (Table 1). However, the spectra for AA and AA-modified surfaces displayed similar hydrophilicity with a
water contact angle of 58° and 67°, respectively. The OD
Table 1. Atomic Concentration of Chemical Elements on surface was the most hydrophobic surface with a water contact
Plasma Polymer-Modified Surfacesa angle of 92°. These wettability behaviors are consistent with
surface C 1s [at. %] O 1s [at. %] N 1s [at. %] C/O N/C previously published reports for comparable plasma polymer
b b coatings.43,46 Plasma polymer surface modification also
AC 72.1 27.8 2.6
provided a range of surface charges at physiological pH =
MEOX 71.1 13.8 15 5.1 0.21
AA 77 8 15 9.6 0.19
7.4 (Figure 1E). AA surfaces were positively charged (+2.5
OD 92.5 7.4 b
12.5 b mV), MEOX and OD coatings had slightly negative charge
a b −18 and −19 mV, respectively, whereas the AC coatings had
All XPS data contain a standard error of 5%. Not present.
the highest negative charge of −28 mV.47,48 Thus, for the
following protein corona formation and immune response
studies, we had model surfaces covering a range of chemistry,
MEOX plasma polymer feature an additional nitrogen peak (N wettability, and surface charge, parameters which are all known
1s) of 15 at. % (Table 1), which is consistent with the to affect biological responses.
precursor’s chemical composition and previously published The model surfaces were incubated in human serum for 1 h
studies.43−45 and the unbound proteins or/and soft corona were removed

Figure 2. (A) Schematic illustration of the fate of a biomaterial post implantation. Blood−material interaction leads to the adsorption of serum
protein to the pristine biomaterial surfaces, which then attracts the subsequent immunological response. (B) Quantification of protein adsorption to
the modified surfaces along with classification of identified proteins according to their function. (C) Heat map of the most abundant proteins in the
protein corona of the modified surfaces determined by LC−MS. Only those proteins that constitute more than 1% of the protein corona on each of
the surfaces are shown in the heat map.

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by several rinsing steps. The remaining hard corona,
constituted of proteins strongly bound to the surface, was
then further interrogated. First, the serum proteins in the hard
corona were removed by treating the surface with SDS−Tris
HCl buffer at 95 °C and quantified with a colorimetric protein
assay as previously described.36 It is important to note that
both hard and soft corona take part in the interactions with the
pristine biomaterial surface. However, only a select set of
proteins, those with a high affinity for the surfaces will adhere
strongly enough to remain tightly bound for the extended
period of time. The soft corona, in contrast, is the dynamic
environment where rapid exchange of the biomolecule to and
from the surface occurs. The strongly bound proteins
constituting the hard corona stay in the vicinity of the surface
for a long time, and therefore, the immune cells are more likely
to “feel” the proteins that compose the hard corona than those
of the loose soft corona. In addition, the long residence times
of proteins in the hard corona make them easier to isolate and
identify than those from a soft protein corona.38,41
More than 100 proteins were identified in the hard protein
corona formed (Figure 2A) on the four different surface
chemistries using liquid chromatography−mass spectrometry
(LC−MS) (Table S1). The proteins present in the protein
corona were grouped according to their functions and shown
in Figure 2B. The total amount of the serum proteins adsorbed
on the AA and MEOX surface was lower compared to the AC
and OD surfaces. OD surfaces had the highest amount of
serum protein adsorbed (7.05 ± 0.9 μg cm−2), 2.07 μg cm−2 of
which was immunoglobulins. The AC surface showed the
second highest amount of protein (6.74 ± 0.8 μg cm−2), with
the main component being albumin at 3.06 μg cm−2. AA (5.07
± 0.2 μg cm−2) and MEOX (5.14 ± 0.8 μg cm−2)-modified
surfaces had the least serum protein adsorption, which
consisted of high amounts of lipoproteins of 3.06 and 2.73
μg cm−2, respectively. The type of protein bound to the
different surface chemistries differed significantly with no
straightforward link between the protein isoelectric point and
the surface charge.
Figure 2C shows the heat map of the most abundant
proteins in the protein corona formed on the modified
surfaces. Hydrophilic AC surface demonstrated higher affinity
to albumin, which constituted 45% of the total protein corona.
MEOX showed high affinity to clusterin and apolipoprotein B-
100, which contributed to 24 and 16% of the total serum
protein corona, respectively. AA surfaces had Ig gamma-2
chain C region (IgG2) (17%), clusterin (16%), and
apolipoprotein B-100 (16%) as the most prominent proteins.
Interestingly these proteins have also been found on the
poly(ethylene glycol)-coated surfaces of nanoparticles.37
However, hydrophobic OD surfaces had high affinity to
immunoglobulins IgG2 (26%). Interestingly, surface charge
did not offer any clear correlation to the type of protein
adsorbed. For instance, albumin, which is negatively charged at
physiological pH of 7.4 and has an isoelectric point (pI) of
5.86,49 was bound in large amount to the AC surface which
also have a net negative charge in these conditions. If protein
adsorption was purely driven by electrostatic interaction, one
would expect the negatively charged proteins to preferentially
bind to positively charged surface and vice versa. However, the
data show that this is not the case as noted previously for
nanoparticles.50 Neutral IgG2 (pI = 7.4) and negatively
charged apolipoprotein B-100 (pI = 6.58) and clusterin (pI =
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5.84) appear to adsorb strongly to the different surfaces with
no particular concern for their net charge.49
In contrast, surfaces with similar functionalities and
comparable wettability, namely, MEOX and AA, both having
nitrogen-containing chemical groups, attracted more apolipo-
protein B-100 and clusterin and in comparable amounts. On
the other hand, OD and AC surfaces, having different
functional groups, CH3 and COOH, respectively, had very
different patterns of serum protein adsorption. The OD and
AC also have wettability at the extreme of the spectrum
investigated in this work, that is, 92° and 49°, respectively. For
example, adsorption of IgG increased from 5 to 26% as the
surface becomes more hydrophobic. However, the adsorption
of albumin increased from 7 to 45% as the surface become
more hydrophilic (from OD to AC). Together, these results
indicate the specific serum protein adsorption patterns on
surfaces of different chemistry (nitrogen, carboxyl, and
hydrocarbon) and wettability (i.e., hydrophobic or hydro-
philic).
Previous reports involving model protein solutions have
investigated the role of surface hydrophobicity on the type and
amount of protein adsorbed. In single protein adsorption
studies, it has been previously reported that the adsorption of
human serum albumin (HSA) or bovine serum albumin was
enhanced on both hydrophilic25,51 or hydrophobic surfaces52
and that no significant difference existed between hydrophobic
and hydrophilic surfaces.53−55 While this may seem contra-
dictory, in their native conformation, the external and internal
surfaces of proteins are comprised of hydrophobic and
hydrophilic regions as well as charged and uncharged domains,
which can make adsorption energetically favorable on both
hydrophobic and hydrophilic substrates.55 Similarly, the
orientation of the adsorbing albumin molecule determines
the functional group (nonpolar CH3 group or polar −COOH)
exposed to the solid surface and thus its affinity with
hydrophobic or hydrophilic surfaces.51 These aspects become
particularly intricate in real body fluids because protein
arrangements and orientations are different in single and in
mixed protein solutions.25 Similarly, IgG also showed varying
adsorption behavior with respect to surface wettability when
experiments were conducted as a single protein solution or in
combination of multiple proteins.25,56 Our results uniquely
reveal preferential protein adsorption pattern on hydrophilic
and hydrophobic surfaces from human serum. Testing protein
corona in human serum is a scenario practically relevant for
subsequent correlation with both in vitro and in vivo tests, as it
accounts for competing adsorption−desorption event that
occur in the cell culture medium or blood. In the following,
immune cells were grown in FBS, which has a composition
comparable to that of human serum in an attempt to evaluate
the inflammatory responses arising as a consequence of the
protein corona formed on surfaces of different chemical and
wetting properties.
Human THP-1 cells were differentiated with PMA to
become macrophages and were cultured on the plasma
polymer-coated surfaces overnight with serum-containing
media.57,58 Macrophages are model cells used to study immune
responses because they can undergo measurable functional
changes in response to matrix clues which can be correlated
with in vivo results.30 Depending on the stimuli in the residing
environment, these cells can be polarized from the resting M0
phenotype to the proinflammatory M1 or anti-inflammatory
M2 subsets. M1 macrophages typically produce proinflamma-
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Figure 3. Cytokine expression of macrophage cultures on plasma polymer-coated surfaces having different surface chemistries. Production of
proinflammatory cytokines TNF-α (A) IL-6 (B) IL-1β (C) IP-10 (D), anti-inflammatory cytokine arginase (E), and IL-10 (F) as quantified by
multiplex ELISA. All the data are shown as means ± standard deviation and the * significant difference compared to OD (P < 0.05) using one-way
ANOVA followed by the Dunnett’s multiple comparisons test.

tory cytokines [e.g., tumor necrosis factor (TNF), IL-1β, IL-6, Scheme 1. Schematic Illustration of the Influence of Surface
and IP-10] which promote strong immune responses.59,60 Wettability on Serum Protein Adsorption to Modified
Conversely, M2 macrophages antagonize the inflammation by Surfaces and the Immune Responses Determined in Culture
enhancing the production of anti-inflammatory cytokines and of Macrophages; the Bottom Panel Shows Plasma Polymer-
other substances involved in repairing function, such as Coated Model Biomaterial Surfaces with Varying
arginase, TGF-β, IL-10, and mannose receptors. M2 are Wettability; the Middle Panel Illustrates How
present in the advanced stages of the healing process and Hydrophobicity Determines the Amount and Relative
participate in resolution of inflammation, tissue repair, and Abundance of Serum Proteins Adsorbed to the Modified
angiogenesis.59 Surfaces after Exposure to Serum; as the Surface Become
Figure 3 shows the effect of plasma polymer-derived model Hydrophilic, Dysopsonin Albumin Adsorption Increases
surface chemistry on cytokine production by LPS-activated While the Opsonin IgG2 Chain C Region Adsorption
macrophages obtained from ELISA. LPS is a polysaccharide Increases on Hydrophobic Surfaces; the Top Panel
present in the outer membrane of Gram-negative bacteria that Illustrates the Macrophage Immune Response toward M2
is known to elicit strong immune responses and is commonly Anti−Inflammatory and M1 Proinflammatory on
used for the activation of macrophage to assess the Hydrophilic and Hydrophobic Surfaces, Respectively
immunomodulatory properties of biomaterials. LPS addition
represents a strong inflammatory environment which mimics
bacterial infection or surgical trauma associated with
implantation of biomaterial.61 Hydrophobic OD surfaces
caused an increase in proinflammatory cytokine production
while hydrophilic AC surfaces resulted in the expression of
least proinflammatory cytokines, specifically, significantly lower
amounts of TNF-α and IP-10. Compared to OD surfaces, the
macrophages also produced lower amounts of proinflammatory
cytokines on the AA and MEOX surfaces. In perfect contrast,
anti-inflammatory cytokines arginase and IL-10 production was
higher on the hydrophilic AC surfaces and the least on the
hydrophobic OD surfaces. These results collectively highlight a
remarkable trend between immune responses and surface
wettability and chemical functionalities. The surface hydro-
phobicity appears to first dictate the type and amount of
protein adsorbing to the surface which in turn modulates the
following immune response, as summarized in Scheme 1. In
order to gain further insights into the connection between decelerate/inhibit phagocytosis, respectively.62 HSA is a
protein adsorption and immune response, in the following, we dysopsonin that can substantially inhibit phagocytosis even
consider the specific function of each protein adsorbing to the when combined with a strong opsonin such as α2GP or IgG.21
different modified surfaces. In contrast, opsonins, like IgG, signal for innate immunity and
In the field of immune response proteomics, some proteins inflammation by recognition through Fc receptors to antigen-
and biomolecules have been identified as opsonins or presenting cells (e.g., macrophages) and activate the immune
dysopsonins based on their tendency to promote or response or phagocytosis.21,63
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The protein corona study presented here showed that
hydrophilic AC surfaces having −COOH functional groups
displayed the highest affinity to albumin. However, hydro-
phobic OD surfaces rich in −CH3 functional groups displayed
highest affinity to IgG2. We then demonstrated that the AC-
modified surface, which attracted more serum dysopsonin
albumin, initiated the M2 pathway by upregulating the
production of anti-inflammatory cytokine and down regulating
the proinflammatory cytokines (Scheme 1, left). In contrast,
the OD-modified surface having high amounts of opsonin
IgG2 adsorbed appears to have initiated the M1 macrophage as
evidenced by an increase in the production of proinflammatory
cytokines and a reduction in the production of anti-
inflammatory cytokines (Scheme 1, right).
These results are in good agreement with published reports
which indicate that nanoparticles bound with immunoglobu-
lins (IgGs) played a critical role in activating immune
responses, opsonization, and phagocytosis.64,65 A recent
study from Simberg et al. (2019) reported that IgGs can
bind to foreign and self-antigens and can modulate comple-
ment activation through all three complement (classical,
alternative, and lectin) pathways.66 Therefore, the adsorption
of IgG on hydrophobic (OD, AA, and MEOX) surfaces is
likely to be responsible for the increase in proinflammatory
cytokines expression observed here. On the other hand,
surfaces that bind albumin reduced immune response or
phagocytosis.21,62,67 Because albumin adsorbed on surface does
not trigger immune responses, albumin is used as a
preadsorption strategy to reduce host response to foreign
biomaterials.68,69 Therefore, adsorption of albumin on hydro-
philic (AC) surfaces may be the reason for the reduction in
proinflammatory cytokines determined in our work. Similarly,
clusterin, an apolipoprotein that has a high affinity to nitrogen-
containing MEOX and AA surfaces, is also known for its
stealth properties and documented to help avoid unwanted
immune responses has also led to lower proinflammatory
cytokines expression on these surfaces.37 In summary, this
study demonstrates the influence of surface chemistry and
wettability on the selective attachment of proteins from serum
and how this further affects the inflammatory response of the
macrophage. Although this study is only focused on the
amount and type of adsorbed proteins from serum as function
of surface properties, it is worth noting that protein
conformation upon adsorption to surface also plays an
important role in defining the inflammatory consequences to
a biomaterial. We have previously reported on the role of
biomaterial surface nanotopography in fibrinogen unfolding
and the subsequent immune responses.33 We found that
surface nanotopography modulates the level of fibrinogen
unfolding which leads to the exposure of normally hidden
peptide sequences which activate the Mac-1 receptor of
inflammatory cells. Although protein unfolding is beyond the
scope of this work, the results presented here suggest that
future studies should be carried out to reveal the conforma-
tional changes not only of fibrinogen but also of other
abundant serum proteins such as albumin and immunoglobu-
lins and establish the links to the resultant inflammatory
cascades.
4. CONCLUSIONS
In summary, we demonstrated the role of surface chemistry
and wettability in total human serum adsorption to form the
hard corona and the following immune responses. A substrate-
27621
Research Article
independent plasma polymerization technique was utilized to
create nanothin model biomaterial coatings with different
chemical functionalities (AC, MEOX, AA, and OD) that
possess a range of wettability and surface charge properties. We
identified the effect of hydrophobicity in the selectivity of
serum protein adsorption. Enhanced dysopsonin albumin and
opsonin IgG2 adsorption was observed on hydrophilic and
hydrophobic surfaces, respectively. This in turn led to an
increase in anti-inflammatory cytokine on the hydrophilic
surface while proinflammatory cytokine production increased
on hydrophobic surfaces. These results lead to fundamental
insights that have to be considered for future biomaterial
design to control the protein adsorption process to modulate
immune response and govern the fate of implantable
biomedical devices.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.9b09900.
Ellipsometry thickness measurement; total serum
protein adsorption on the modified surfaces; SDS
PAGE gel electrophoresis; heat map for major protein
components of protein corona formed on the model
surfaces sorted against increasing isoelectric point and
sorted by increasing molecular weight; major protein
components of protein corona adsorbed on the modified
surfaces are grouped based on the lowest to highest
adsorbed concentration ranking between all four
surfaces; macrophage cell count; and list of all (111)
corona proteins identified by LC−MS (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: krasimir.vasilev@unisa.edu.au. Phone: +618-30-
25697 (K.V.).
ORCID
Melanie N. MacGregor: 0000-0002-8671-181X
Agnieszka M. Mierczynska-Vasilev: 0000-0002-4758-968X
Svenja Morsbach: 0000-0001-9662-8190
Krasimir Vasilev: 0000-0003-3534-4754
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
K.V. thanks ARC for DP15104212 and DP180101254,
NHMRC for Fellowship APP1122825 and Project grant
APP1032738, and the Alexander von Humboldt Foundation
for Fellowship for Experienced Researchers.
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