Foroozandeh and Aziz Nanoscale Research Letters (2015) 10:221

DOI 10.1186/s11671-015-0922-3

NANO REVIEW Open Access

Merging Worlds of Nanomaterials and Biological

Environment: Factors Governing Protein Corona
Formation on Nanoparticles and Its Biological
Consequences
Parisa Foroozandeh1* and Azlan Abdul Aziz1,2

Abstract
Protein corona has became a prevalent subject in the field of nanomedicine owing to its diverse role in determining the
efficiency, efficacy, and the ultimate biological fate of the nanomaterials used as a tool to treat and diagnose various
diseases. For instance, protein corona formation on the surface of nanoparticles can modify its physicochemical properties
and interfere with its intended functionalities in the biological microenvironments. As such, much emphasis should be
placed in understanding these complex phenomena that occur at the bio-nano interface. The main aim of this review
is to present different factors that are influencing protein-nanoparticle interaction such as physicochemical properties
of nanoparticle (i.e., size and size distribution, shape, composition, surface chemistry, and coatings) and the effect of
biological microenvironments. Apart from that, the effect of ignored factors at the bio-nano interface such as temperature,
plasma concentration, plasma gradient effect, administration route, and cell observer were also addressed.
Keywords: Protein corona; Nanoparticle; Bio-nano interface; Ignored factors

Review science, chiefly in the field of medicine and biomedical

Introduction science, which has given birth to the term nanomedi-
Nanoscience is recognized as a promising field of science cine. Application of NP in medical biology arises from
that can overcome several scientific shortcomings in their ability to encounter cellular machinery and poten-
diverse scientific fields, such as physics, biology, chemistry, tially access to unreachable targets like the brain due to
and materials science [1, 2]. The breakthroughs achieved their small size [6, 7]. Consequently, they have shown
in the field of nanoscience are mainly attributable to the promising application in various branches of biomedical
changes in the properties of materials as they are reduced science such as drug delivery [8], gene delivery [9–11],
to the size of nanometer from their bulk form. The tissue repair [12], cancer therapy, [13] disease diagnoses
materials assume novel mechanical, chemical, electrical, and therapy [14], hyperthermia [15], magnetic resonance
optical, magnetic, electro-optical, and magneto-optical spectroscopy [16], and as contrast agents for magnetic
properties as compared to bulkier counterparts [3–5]. resonance imaging (MRI) [17].
For instance, gold in bulk form is inert and conducts NPs owing to their large surface-to-volume ratio have
electricity, however gold shrunk into “nano” form becomes a very active surface chemistry in comparison to bulk
a very good catalyst and turns into a semiconductor materials. When they come into contact with biological
instead. One of the most exciting prospects that have milieu, they seek to lower their high surface energy by
emerged from the field of nanoscience is nanoparticle (NP) adsorbing biomolecules, resulting in the creation of
technology that are currently being incorporated and uti- complex layer of biomolecules that would cover the sur-
lized to solve many intricate technical problems in modern face of NP [18–20]. More specifically, when NPs are ex-
posed to a biological medium, physical and chemical
* Correspondence: [email protected]
1
interactions occur between the surfaces of NPs and dif-
School of Physics, Universiti Sains Malaysia, 11800 Penang, Malaysia
Full list of author information is available at the end of the article
ferent biological components within the medium such as

© 2015 Foroozandeh and Aziz ; licensee Springer. This is an Open Access article distributed under the terms of the Creative
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Foroozandeh and Aziz Nanoscale Research Letters (2015) 10:221 Page 2 of 12

proteins, peptides, and glycolipids. Due to these interac- administration of the NPs, blood plasma proteins adsorb
tions, a “bio-nano interface” develops at the point where to the surface of NP to form protein corona [23, 30–32].
the two entities come into contact [21–23]. The formed In the case of other administrative routes, NPs will react
interface covers the surface of NP, thereby modifying its with other biomolecules of the body fluids primarily
quality and endowing it an identity within a biological before reaching the blood plasma. Protein corona is a
framework, the so-called protein corona. Interestingly, the dynamic layer and its composition changes with time due
biological identity dictates the cellular/tissue responses to ongoing protein absorption and desorption [19, 29]. It
such as cellular uptake, kinetics, signalling, accumulation, is worth remarking that protein corona is the primary
transport, and toxicity [19, 22, 24, 25]. Also, the protein contact to the cell. Therefore, what the biological entity
corona gives information about the interface formed be- sees when it comes into contact with NPs is the protein
tween the NP and the biological milieu [25]. Walkey and corona formed at that specific time [19, 29, 33]. The com-
his fellow researchers employed protein corona fingerprint position of protein corona for each nanomaterial is unique
to establish a quantitative model which will predict the and is influenced by many parameters such as physico-
cell association of various composition of gold NPs [26]. chemical properties of NPs and characteristics of the
Their findings suggest that this model is 50 % more accur- environment [25].
ate compared to the one which applies NP parameters The formation of protein corona causes a reduction in
such as size, aggregation state, and surface charge indicat- the surface energy and toxicity of the NP as compared
ing that the protein corona gives more information about to the “bare” particle. In general, protein corona changes
the biological behavior of NP rather than its physical the size, surface chemistry, and surface charge of the NP,
properties. thereby affecting its uptake, biodistribution, and cellular
Protein corona can exist in two different forms on the fate [18, 24]. Protein corona is formed at the bio-nano
surface of NPs, determined by the type of layers formed. interface by the aid of several forces such as hydro-
Essentially, two different type of layers can be formed, dynamic, electrodynamic, and electrostatic or steric
namely “soft” and “hard” coronas, the former consisting of forces and solvent and polymer bridging. These forces
loosely bound proteins with short lifetime and the latter also determine the kind of structure that protein corona
consisting of tightly bound proteins with long lifetime may form at these interfaces. Structure of protein corona
[27]. Composition of protein corona is influenced by the can be evaluated in the physiological environment (i.e.,
physicochemical properties (i.e., composition, size, shape, in situ) or after isolation from the physiological environ-
and surface properties) of NPs and the characteristic of ment (i.e., ex situ). Techniques such as differential cen-
biological environment in which the NPs are dispersed. It trifugation (DC) or size exclusion chromatography (SEC)
is noteworthy to mention that the cell perceives protein are utilized for isolation of protein corona. Different pa-
corona as opposed to the bare surface of NPs as they rameters of protein corona such as thickness, protein
come into contact [21, 24, 28]. Thus, understanding identity, protein quantity density, protein-NP affinity,
protein corona which confers the biological identity to NP and protein conformation can be analyzed and quanti-
is important as it will have major repercussions in the field fied using various analytical tools [24, 25].
of nanomedicines whereby toxicological and physiological
responses to NPs are studied. Composition and Structure of Protein Corona
This review presents different factors that are influencing The protein corona evolves over time from what was
protein-NP interactions, including the effect of physico- formed at the initial stage of NPs’ interaction with bio-
chemical properties of NP (i.e., size and size distribution, logical medium. Initially, when the NPs come into contact
shape, composition, surface chemistry, and coatings), effect with biological medium, the most abundant proteins with
of environment, and the effect of ignored factors at the low affinity adsorb to the NP surface and form the layer
bio-nano interface such as temperature, plasma concentra- which is called soft corona. Over time, those proteins
tion, plasma gradient effect, administration route, and cell would be replaced by high affinity proteins that have a
observer. Moreover, the impact of these parameters on the lower abundance in the medium and form the hard
composition of protein corona and fate of NPs in biological corona layer [22, 30, 34, 35].
environment will be addressed. The coronas may also be classified based on their ex-
change rates. In particular, hard corona possess long life-
Creation of Protein Corona time which shows slow exchange rate with the medium
It is now well understood that upon coming into contact while soft corona has faster exchange rates. Hard corona
with NPs in the biological medium, different biomolecules consists of tightly bound proteins that do not easily de-
such as proteins, lipids, and glycans will compete to inter- sorb, in contrast to weakly bound proteins that consti-
act with the NP surface to form a layer called protein cor- tute the soft corona. It is well accepted that owing to the
ona [23, 24, 29]. For instance, in the case of intravenous longer half-life of hard corona in the biological medium,
Foroozandeh and Aziz Nanoscale Research Letters (2015) 10:221 Page 3 of 12

its interaction with cellular receptors will determine the adsorption of high abundance proteins such as albumin,
fate of NPs as depicted in Fig. 1 [20, 25, 36, 37]. IgG, and fibrinogen which is called “early” stage. The
It is now hypothesized that either the proteins in the “late” stage of the Vroman effect occurs when those
hard corona adsorb directly to NPs surface while pro- proteins will be replaced by high affinity proteins such
teins in the soft corona bind to the hard corona via weak as apolipoproteins and coagulation factors [41–44].
protein–protein interaction or the hard and soft corona Goppert and co-workers investigated “Vroman effect”
proteins may both bind directly to the NPs surface with on solid lipid nanoparticle (SLN) over a period of time
distinct binding energies [25]. Hard corona does not (i.e., 0.5 min to 4 h) [45]. They reported that at the early
exist for all nanoparticles. Predominantly for NPs that stages, albumin was replaced by fibrinogen. The longer
are coated with functional group such as PEGylated incubation time resulted in the replacement of fibrino-
nanoparticles, only the soft corona can be observed [38]. gen with IHRP (inter-α-trypsin inhibitor family heavy
Due to long residence time of proteins in the hard cor- chain-related protein) and apolipoproteins. Their study
ona, it is considered to be the main component in defin- have demonstrated that protein desorption did not occur
ing the biological identity for NPs. from SLN throughout the time period of their investiga-
Simberg and co-workers introduced a model for the tion on the protein adsorption kinetics. They also ob-
protein corona which includes “primary binders” that served an increase in the total amount of proteins that
interact with the NPs surface at first followed by “sec- were adsorbed onto the surface of SLN after more than
ondary binders” that binds to the primary binders by 4 h of incubation with plasma. The on oil-in-water
way of protein–protein interactions [39]. This multi- nanoemulsions (o/w nanoemulsions) showed a markedly
layered structure plays an important role in the physio- different adsorption behavior as compared to SLN
logical response as the interaction of the primary whereby the originally adsorbed proteins were replaced
binders can be changed by the secondary binders or by proteins having a higher affinity to the surface (“Vro-
being “masked” by them, thereby hindering their inter- man effect”). No Vroman effect could be observed on
action with the biological environment. oil-in-water nanoemulsions (o/w nanoemulsions). Fur-
Walky and Chan summarized the composition of the thermore, increasing plasma concentration leads to an
protein corona across 26 studies for 63 nanomaterials, increment in the amount of adsorbed apolipoproteins A-
and they have identified a subset of 125 unique plasma I, A-IV, C-II, and C-III [42]. The Vroman effect on ultra-
proteins that have adsorbed to at least one nanomaterial small superparamagnetic iron oxide (USPIO) nanoparti-
[25]. This subset of plasma proteins was identified and cles has been assessed by Jansch and co-workers [46].
categorized as “adsorbome.” One could observe that Their study showed that no Vroman effect on USPIO
each “adsorbome” have different physiological roles, but can be determined and no replacement of higher affinity
they commonly participate in lipid transport, comple- protein with high abundance protein on USPIO could be
ment activation, pathogen recognition, blood coagula- detected. Moreover, with prolonging incubation time,
tion, and ion transport. The authors have established the amount of fibrinogen and immunoglobulins in-
that at high abundance roughly 2–6 proteins are creased while the relative amount of major proteins,
adsorbed in a “typical” plasma protein corona and at low such as apolipoproteins, fibrinogen, and albumin,
abundance, more protein are absorbed. A review study remained constant over time.
summarized the types of protein binding to different
nanoparticles to determine the composition of protein Protein Corona Conformation
corona [40]. This study postulates that albumin, im- When proteins adsorb to NP, structural rearrangements
munoglobulin G (IgG), fibrinogen, and apolipoproteins may take place within the protein molecules. These
can be detected in the corona of all the nanoparticles “conformational changes” can render the protein to
due to their high abundance in plasma. become dysfunctional due to loss of native form or
Variation in composition of protein corona over time thermodynamically favorable if it allows either charged
is explained by the Vroman effect. The Vroman effect or hydrophobic regions of proteins to interact with
states that the composition of protein corona may vary either hydrophobic or charged NPs [25, 47, 48]. It is
over time whereas the total amount of protein remains remarkable to note that conformational changes of pro-
relatively constant. In particular, the Vroman effect elu- teins after desorption are usually irreversible. However,
cidates how low affinity but highly abundant proteins the structure of protein and the surface properties of
that are adsorb first to the surface of the NP will be re- NPs are key factors in determining the level of conform-
placed by higher affinity proteins. The Vroman effect is ational change. For instance, hydrophobic nanoparticles
the function of concentration of protein, incubation undergo the conformational change more than hydro-
time, and affinity of proteins. Upon intravenous adminis- philic NPs [49]. Mahmoudi and co-workers illustrated
tration of NPs, the Vroman effect in plasma involves the the conformational changes of iron-saturated human
Foroozandeh and Aziz Nanoscale Research Letters (2015) 10:221 Page 4 of 12

Fig. 1 (See legend on next page.)

Foroozandeh and Aziz Nanoscale Research Letters (2015) 10:221 Page 5 of 12

(See figure on previous page.)

Fig. 1 Schematic representation of exchange/interaction scenarios and of the structure of protein-nanoparticle. a Schematic representation of the possible
exchange/interaction scenarios at the bionanointerface at the cellular level. b Schematic drawing of the structure of protein-nanoparticle in blood plasma
confirming the existence of various protein binding (e.g., an outer weakly interacting layer of protein (full red arrows) and a hard slowly exchanging corona
of proteins (right) (adapted with permission from [35]))

transferrin protein due to interaction with superpara- protein corona considerably evolves in the second bio-
magnetic iron oxide NPs (SPIONs) [50]. Different sizes logical medium. Yet, the final corona preserves a “finger-
of bare SPIONs and PVA-coated SPIONs were incu- print” of prior history. This can be beneficial to determine
bated with iron-saturated human transferrin protein. the transport pathways of NPs.
Analyzing the resulting complex by fluorescence and The changes in the adsorption pattern of serum proteins
UV-vis spectroscopy, SDS-PAGE gel electrophoresis and over time from 5 to 360 min were quantitatively and
circular dichroism revealed irreversible changes in con- qualitatively investigated by Nagayama and co-workers
formation of iron-saturated human transferrin protein [53]. They used SDS-PAGE and Western blotting to asses
due to the release of iron. Particularly, conformation of 50-nm lecithin-coated polystyrene NP at different times.
human transferrin changes from a compact structure to The quantitative study revealed that the total amount of
an open structure. Moreover, it was found that conform- adsorbed proteins increased over time. The qualitative
ational changes of transferrin depend on the surface study indicated variation in the kinds of proteins adsorbed
properties and size of the SPIONs. For instance, more since the amount of some proteins increased over time
conformational changes were detected on the bare NPs whereas others decreased. Complement C3, IgG, apolipo-
than PVA-coated NPs. protein E (ApoE), and immunoglobulin A (IgA) showed
Shang and co-workers studied the conformational increment over time. On the contrary, concentration of
changes of bovine serum albumin (BSA) in the albumin albumin remained constant.
to gold nanoparticle bioconjugates at different pH values
(i.e., 2.7 (E form), 3.8 (F form), 7.0 (N form), and 9.0
Effect of Different Parameters on the Composition of
(B form) by employing different spectroscopic tech-
Protein Corona
niques such as UV-vis, fluorescence, circular dichroism,
Several factors affect the manner by which NPs interact
and Fourier transform infrared spectroscopies [51]. The
with biomolecules and composition of the resulting pro-
results show that the changes in the conformation of
tein corona. In view of the fact that protein adsorption
BSA occur at the secondary and the tertiary structure
takes place at the interfacial region between NPs and its
levels. In addition to that, studies on the effect of envir-
surroundings, the physicochemical properties of NPs and
onmental pH on the conformational changes indicated
the biological environment are vital parameters governing
that higher pH causes larger changes.
protein corona formation. Therefore, analyzing and un-
derstanding each of these parameters are essential for safe
Time Evolution of the Protein Corona
design of NPs.
The time evolution of the protein corona formed on
different-sized gold NPs ranging from 4 to 40 nm in the
cell culture media with 10 % fetal bovine serum (FBS) Effect of Nanoparticle Composition
was demonstrated by Casals and co-workers [52]. Pro- NP composition and its surface chemistry are crucial fac-
tein corona formed around gold NPs were analyzed by tors in determining the affinities and identities of proteins
zeta potential measurements, UV-vis spectroscopy, dy- that bind to NPs. Deng and co-workers have studied the
namic light scattering, and transmission electron mi- binding of human plasma proteins to commercially avail-
croscopy which revealed a reduction of surface charge able metal oxide NPs such as titanium dioxide, silicon
and an increment in the thickness of protein corona dioxide, and zinc oxide with the same surface charge
layer. Albumin was shown to be the most abundant pro- [54]. The authors revealed that similar proteins adsorb
tein on the surface of NPs by mass spectrometry analysis to titanium and silicon dioxide NPs, whereas signifi-
of the protein corona. Furthermore, they reported that cantly different proteins composed the hard corona of
loosely bound proteins will over time evolve to form an zinc oxide NPs. In particular, clusterin, apolipoprotein
irreversible bound protein. The evolution of protein cor- D, and alpha-2-acid glycoprotein were detected in the
ona after NPs were transferred from plasma into cyto- corona of titanium and silicon dioxide NPs while those
solic fluid was illustrated by Lundqvist and co-workers were not observed in the corona of zinc oxide. Interest-
[38]. Various NPs such as 9-nm silica, 50-nm polystyr- ingly, some other proteins like transferrin, Ig heavy
ene, and 50-nm carboxyl-modified polystyrene particles chain alpha, and haptoglobin (alpha) only were found
were employed in this study. It was observed that the in the corona of zinc oxide NPs alone.
Foroozandeh and Aziz Nanoscale Research Letters (2015) 10:221 Page 6 of 12

Effect of Nanoparticle Size adsorption of lysozyme on 100-nm NP causes more

The surface curvature of NP, which is associated to the protein unfolding than on 4-nm NP. In particular, the
size of the NP, plays a key role on the adsorption of size of the NPs was found to be a crucial factor in de-
proteins, conformational changes and composition of termining the structure and function of lysozyme upon
protein corona [32, 55]. Due to high surface curvature of adsorption onto silica NPs.
NP as compared to the bulk materials, their protein
binding affinities are distinct from bulk materials of the
same composition [56]. More specifically, protein– Effect of Nanoparticle Shape
protein interactions reduce at high curved surfaces of The manner by which proteins adsorb onto the surface
nanoparticle leading to different composition of protein of NP as well as the biological responses to NP is
corona. In addition, adsorbed proteins at highly curved strongly influenced by the shape of NP. For instance,
surface NPs undergo less conformational changes than the shape of gold NPs has a huge effect on their inter-
adsorbed proteins at flat surfaces of the same material actions with cell layers; in particular, spherical gold NPs
[19, 24]. Tenzer and co-workers investigated the protein has higher association in cell as compared to rod-
corona formation on silica NPs (SiNP) of various sizes shaped gold NPs [60]. The effect of shape of titanium
upon exposure to blood plasma [57]. They observed that dioxide NPs on protein binding was investigated by
the size of SiNP drastically affected the binding of 37 % Deng and co-workers [54]. The authors found that
of the identified proteins. Interestingly, even 10-nm vari- clusterin and apolipoprotein D were only observed on
ations in particle size notably influence protein corona spherical NPs and were not detected on nanorods or
composition. Likewise, lipoprotein clustsacaerin bound nanotubes.
to the small SiNP while prothrombin or the actin regula-
tory protein gelsolin were absorbed on the larger SiNP.
However, no correlation to NP size was observed for the Effect of Surface Functional Group and Coating
adsorption of several proteins such as immunoglobulin In order to prevent absorption of proteins and to control
(IgG) or actin. Dobrovolskaia and co-workers examined the protein corona composition, the surface of NP can
30- and 50-nm colloidal gold incubated in human be functionalized with different groups. This confers a
plasma, and they found more proteins were adsorbed on “stealth character” to the surface of NP, hence eluding
the 30 nm than on the 50 nm gold NPs [58]. from being observed by immune cells. Appropriate poly-
In a recent work, the effect of NP size on binding con- mers such as polyethylene glycol (PEG) can also be ap-
stant and hill constant (i.e., the degree of cooperatively plied to coat the surface of NPs to decrease protein
of protein-NP binding) was probed [31]. The authors binding and prevent them from being recognized by the
used gold NPs of various sizes ranging from 5 to 100 RES, the so-called PEGylation. Density of PEG on the
nm and incubating them with common human blood surface of NPs can be controlled in order to prolong
proteins: albumin, fibrinogen, γ-globulin, histone, and circulation time in blood. “Siliconate” has also been used
insulin. It was observed that the size of gold NPs signifi- to coat the surface of NPs and hinder protein adsorption
cantly correlate to binding constant, K, as well as the [61, 62]. Polystyrene NPs with different functional
degree of cooperatively of particle-protein binding (Hill groups (i.e., PEG, amidine, carboxyl, amine, lysine, me-
constant, n), In particular, the binding constant increases thyl, and cysteine) were used in cultured endothelium
with NP size, whereas the Hill constant seeks to de- cells [41]. It was concluded that the protein binding cap-
crease with NP size. Additionally, they have also realized acity to these functionalized surface of NPs demonstrate
that the thickness of protein corona progressively in- their tendency to interact with the cells. Additionally,
creases as the size of NP increases. Moreover, conform- NP-cell association is not influenced by the identity of
ational change upon association with the NPs showed bound proteins.
enhancement with the size of NP. Studies on poloxamine 908 coating polystyrene nano-
In an analysis of protein corona formed on 50- and spheres revealed a reduction of fibronectin adsorption
100-nm polystyrene NPs upon exposure to human compared to uncoated nanospheres [63]. Coating of
plasma, Lundqvist and co-workers showed that the size both single-walled carbon nanotubes and amorphous sil-
of NP affect the type of adsorbed proteins to the ica particles with Pluronic F127 resulted in the enhance-
surface of NP to form corona and subsequently com- ment of dispersion of the NPs but notably decreasing
position of the protein corona [32]. For instance, apoli- adsorption of serum proteins [64]. Aggarwal et al. has
poprotein B-100 did not adsorb to 50-nm NPs but summarized the effects of various coatings such as PEG,
binds to 100-nm NPs. The impact of the size of silica poloxamer, poloxamine, dextran, Pluronic F127, polysor-
NPs on the adsorption of lysozyme was studied by bate, and poly(oxyethylene) on the protein binding and
Vertegel and co-workers [59]. They concluded that biodistribution of NPs [40].
Foroozandeh and Aziz Nanoscale Research Letters (2015) 10:221 Page 7 of 12

Effect of Surface Charge [22]. The impact of media composition on the formation
Surface charge of the NP is a crucial factor in determining of protein corona was studied by Maiorano and co-
the protein corona composition and consequentially its workers [67]. They incubated various sized citrate-capped
eventual fate in the biological system. Positively charged gold NPs with cellular media such as Dulbecco Modified
NPs are easily recognized by opsonins resulting in the Eagle’s Medium (DMEM) and Roswell Park Memorial
elimination of these particles by the reticuloendothelial Institute medium (RPMI) that were supplemented with
system (RES) and its eventual concentration in the liver the fetal bovine serum (FBS). These are the commonly
and spleen [40, 35]. In order to prevent opsonization, NPs used cell culture media and they differ in glucose, salt
surface can be coated with negatively charged groups lead- composition, and amino acids. A number of techniques
ing to a negative zeta potential in the range of 30–50 mV (dynamic light scattering, UV-visible, and plasmon reson-
in physiological conditions. When the surface-coated NPs ance light scattering) were used to evaluate the corona
are exposed to biological medium, the adsorbed proteins formation on gold NPs mediated by DMEM and RPMI. It
on their surface cause a large decrease of their zeta poten- was concluded that formation of protein corona by utiliz-
tial to 5–10 mV negative [41]. Therefore, the colloidal sta- ing DMEM is significantly time dependent, while using
bility of those complexes is directly related to the nature RPMI leads to distinct dynamics and reduction of protein
of the protein corona. A study on gold NPs with positive, corona. Protein-NP complexes were also characterized by
negative, and neutral ligands show that in the case of sodium dodecyl sulfate polyacrylamide gel electrophoresis
charged ligands (both positive and negative), protein de- (SDS-PAGE) and mass spectroscopy, and it was found
naturation occurs while the neutral ligands retain the that protein corona composition does not relate to the
structure of proteins [55]. amount of serum proteins. Viability assays in both cul-
Gessner and co-workers studied the impact of surface tured media DMEM and RPMI were performed on two
charge density of negatively charged polymeric NPs and cell lines HeLa (human epithelial cervical cancer cell line)
found enhancement in plasma protein absorption with and U937 (human leukemic monocyte lymphoma cell
an increase in the surface charge density of NPs [65]. line) for 15-nm gold NPs. Interestingly, significant differ-
Studies on polystyrene NPs demonstrated that proteins ences in dynamics, cellular uptake, and biodistribution of
with isoelectric points (PI) of less than 5.5 like albumin protein-NP complexes were observed. More specifically,
adsorbed on positively charged particles whereas pro- internalization of protein-NP complexes in cells that were
teins with isoelectric points of higher than 5.5 like IgG formed in RPMI media was notably higher than those
bound to negatively charged particles. formed in DMEM, resulting in higher cytotoxic effects.
Moreover, the protein corona formed in DMEM was more
Effect of Hydrophilicity/Hydrophobicity abundant and stable compared to protein corona formed
More proteins can adsorb onto the surface of hydropho- in RPMI. These differences in the protein-NP complexes
bic NPs than their hydrophilic counterparts. Moreover, mediated by different environments would affect the cel-
due to high affinity of proteins to hydrophobic NPs ra- lular response. Therefore, apart from NP characterization,
ther than hydrophilic NPs, many more adsorbed pro- assessment of cell culture media must be made in order to
teins undergo protein denaturation on the surface of understand its subsequent interaction with NPs.
hydrophobic NPs and lose their native structure [66]. The conditioning of cell culture medium and its effect
Likewise, the binding of apolipoproteins were found to on the biological identity of NPs was investigated by
be a major part of the formation of protein corona on Albanese and co-workers [68]. Complete growth medium
hydrophobic NPs whereas hydrophilic NPs typically that are used for culturing cells in vitro are commonly
adsorb IgG, fibrinogen, and albumin [30, 47]. supplemented with serum that contains varying amount
Cedervall and co-workers employed ITC to study the of proteins or peptides that have the propensity to form
affinity and stoichiometry of protein binding [34]. The corona on the surface of NPs, thereby altering their physi-
authors revealed that as the hydrophobicity of particles cochemical properties and interaction with the cells. How-
increase, it promotes the stoichiometry of proteins. They ever, as the cells are cultured or incubated with the
found that albumin on hydrophobic particles has shorter complete growth medium, the composition of these pro-
residence time than hydrophilic ones. Furthermore, tein or peptides along with other components may change
surface of hydrophobic particles has higher coverage at over time due to cellular metabolic activity of the cells,
equilibrium point. hence affecting the corona formation on the NPs. Condi-
tioning of cell culture medium refers to exposure of the
Effect of Biological Environment NPs in an environment that the cells have been cultured
In addition to the characteristics of NP, composition of for a certain amount of time, thereby containing all the
the biological medium in which they interact is a critical by-products of cellular metabolic activity. This mimics the
factor in determining the composition of protein corona dynamics of the in vitro cellular environment that NPs are
Foroozandeh and Aziz Nanoscale Research Letters (2015) 10:221 Page 8 of 12

exposed to more accurately. From the study, it was shown traverse in their path to reach the intended target site,
that cell conditioning causes gold NP aggregation which the concentration and the type of proteins they encoun-
in turn varies the composition of protein corona that ter will also differ depending on the administrative route
relies on NP size, surface chemistry, and cell phenotype. (e.g., subcutaneous, intradermal, intramuscular, intraven-
Moreover, it was demonstrated that dynamic extracellular ous, intraosseous, intralumbar, and inhalation). Hence,
environment may alter the initial biological identity and the resulting corona formation on NPs would also be
consequently the cell uptake. different [21, 24]. For example, the difference in the pro-
tein corona composition can be observed in the case of
Effect of Ignored Factors inhalation and intravenous route. In the case of NPs ad-
Besides the effect of NP characteristics and biological ministered via the inhalation route, it will reach the lung
environment, there are several other influential hidden cell barrier which contains different biological fluids,
factors at the bio-nano interface which are significantly ef- therefore accumulating totally different plasma proteins
fective in determining the composition of protein corona than the blood for NPs administered via the intravenous
and their subsequent cellular responses. Recently, research route as depicted in Fig. 2.
has been focused to asses these parameters which are gen- Based on their work, Ghavami and co-workers hypothe-
erally called “ignored factors,” and it includes gradient sized that plasma protein gradient has great impact on the
plasma, plasma concentration, cell observer, temperature composition of protein corona and the biological fate of
and cell membrane composition. Detailed research must NPs in vivo [21]. They have employed two NPs, hydropho-
be carried out to understand these ignored factors as to bic carboxylated polystyrene (PSOSO3) and hydrophilic
enable the development of better and effective nano- silica (SiO2) particles, to probe the effect of the plasma
medicine while preventing unanticipated consequences concentration gradient. They used one-dimensional so-
due to poor formulation of nano-based drugs. dium dodecyl sulfate polyacrylamide gel electrophoresis
(1D PAGE), liquid chromatography mass spectrometry
Gradient Plasma Effect and Nanoparticle (LC-MS/MS), dynamic light scattering (DLS), zeta poten-
Administrative Route tial, differential centrifugal sedimentation (DCS), and
It was shown that assessment of protein corona compos- transmission electron microscopy (TEM) techniques to
ition in gradient plasma media is critical to understand characterize NP-protein complexes. They have concluded
what the cell “see” in vivo due to different pathways of that the composition and the quantity of proteins existing
NPs. When NPs enter the body, they will come into con- in the hard corona vary between the gradient plasma
tact with a multitude of biological components in the media and non-gradient plasma media. More specifically,
bodily fluid before contacting the target cell. As they the quantity of low molecular weight proteins (˂25 kDa)

Fig. 2 Schematic illustration of difference in the hard corona composition on nanoparticles depending on the route of administration; intravenous
and inhalation. The proteins that adsorbs on nanoparticle may vary depending on its exposure to the different types of biological fluids in the
human body
Foroozandeh and Aziz Nanoscale Research Letters (2015) 10:221 Page 9 of 12

SPIONs with different surface chemistries are interacted

with a number of cell lines such as Capan-2, Panc-1,
Hela, and Jurkat cells. It was reported that each cell line
interacted with the NPs in a different way. For instance,
while high level toxic effects could be observed on the
brain–derived neuronal and glial cells and lung cells, same
SPIONs caused moderate level toxic effects on the other
cell types. In particular, both the uptake and toxicity of
SPIONs are significantly dependent on the cell type.

Plasma Concentration Effect

The effect of plasma concentration on the composition
of protein corona was investigated by Monopoli and co-
workers [27]. They employed PSOSO3 NPs and hydro-
Fig. 3 Graphical representation of plasma concentration effect on the
philic silica (SiO2) NPs to study the protein adsorption
thickness of hard corona formation on nanoparticles. The thickness of
hard protein corona increases reciprocally as the concentration of and protein corona. They characterized NP-protein
plasma increases complex by DCS, DLS, and zeta potential, whereas com-
position of the hard corona was determined semi-
quantitatively by using 1D PAGE and electrospray liquid
in the corona decreased compared to the amount that was chromatography mass spectrometry (LC MS/MS). They
forming in non-gradient plasma media [21]. found that increasing the concentration of plasma in-
creases the thickness of the hard protein corona as
Cell Observer shown on Fig. 3. In addition, it was observed that the
Another remarkable factor in determining the fate of structure of NP-protein complexes in situ is roughly the
NPs in vivo is “cell observer” (i.e., cell types). The first same with the structure of those in vitro after separation
contact point of cells with the surface of NP is the cell from excess plasma. More specifically, the concept that
membrane which is distinct for each cell type due to the the hard corona may evolve remarkably as a function of
difference in surface proteins, sugars, and phospholipids. protein concentrations will have significant impact when
Large variation in cell membranes leads to different studies conducted on in vitro cell culture conditions
cellular uptake and toxicity mechanisms [29, 69]. The were used to extrapolate over to in vivo conditions.
concept of “cell observer” should be taken into account
in order to interpret the toxicity data as well as to deter- Temperature
mine the dose of NP per cell in therapeutic application It is notable that the human body temperatures differ ac-
of NPs. The impact of “cell observer” on the uptake and cording to the parts of body [70], gender, and physical
toxicity of superparamagnetic iron oxide NPs (SPIONs) activities. When peripheral parts of the body are exposed
were demonstrated by Laurent and co-workers [69]. to cold weather, body temperature decreases to 28 °C

Fig. 4 Scheme showing positively charged nanoparticles having a greater efficiency in cell membrane penetration and cellular internalization than
negatively charged nanoparticles
Foroozandeh and Aziz Nanoscale Research Letters (2015) 10:221 Page 10 of 12

[71]. It has been shown that even the temperature of positively charged NPs to have a greater efficiency in cell
intracellular living cells is not homogeneous [71, 72]. membrane penetration and cellular internalization than
Female’s body temperature is higher than the male body negatively charged NPs as depicted in Fig. 4. In other
temperature, and it also changes with female’s hormonal words, the negatively charged NPs have lower level of CM
cycle. Physical activities cause an increment of 2 °C and adsorption, which consequently decreases the probability
during sleep the temperature of body drops to a lower of cellular uptake. Nevertheless, the cellular uptake rate of
state. Moreover, fever causes the temperature of body to positively charged NPs can significantly disrupt the CM
increase to 41 °C [73]. Hence, body temperature varies and as a result increases its toxicity [76, 77].
in the range of 35 to 39 °C. Mahmoudi and co-workers
have studied the effect of temperature variation on the Conclusions
formation and composition of protein corona [74]. Fluo- In this review, we have highlighted the factors that affect
rescently labeled, negatively charged polymer-coated protein corona formation on NPs based on the current
FePt NPs were applied to incubate with human serum knowledge and understanding of this unique phenomenon.
albumin (HSA) and fluorescence correlation spectros- Moreover, the section on the ignored factors has schemed
copy (FCS) was used to quantify protein absorption at through some of the issues and precautions that must be
different concentrations and temperatures. Furthermore, considered prior to the application of NPs in humans for
Dextran-coated FeOx NPs with different surface charges medical treatment and diagnosis. Emphasis should be
were incubated with FBS at different temperatures. It placed on more research to realize other hidden factors
was concluded that the protein corona composition is governing protein corona formation. With more informa-
influenced by variation in incubation temperature and tion, the existing problems faced by researchers in this field
has great impact on the cellular uptake as well. could be rectified or solved and smarter solution are hoped
The effect of plasmonic heat induction on the protein to be found. Understanding protein corona formation and
corona composition of gold nanorods was investigated by its biological consequences will be pivotal as the field of
Mahmoudi and co-workers [75]. They have incubated nanomedicine is set to dominate in the near future.
cetyltrimethylammonium bromide (CTAB)-stabilized gold
nanorods with FBS at different concentrations. Thereafter, Competing Interests
The authors declare that they have no competing interests.
protein corona composition before and after plasmonic
heating induced by continuous laser irradiation were stud- Authors’ Contributions
ied. UV-vis absorbance spectroscopy, transmission electron PF and AAA wrote the manuscript. Both authors read and approved the final
microscopy, ζ potential, and LC-MS/MS analysis were used manuscript.

to characterize the NP-protein complexes. They have re- Acknowledgements

vealed that the composition of hard protein corona was The authors appreciate useful discussions with Dr. Shaharum Shamsuddin.
changed noticeably by applying both irradiation and ther- The authors would like to thank Universiti Sains Malaysia for funding this
research work through Fundamental Research Grant Scheme (203/PFIZIK/
mal heating while there was no significant effect on the 6711351).
surface charge of protein corona. In contrast, differences in
the composition of protein corona formed on gold nano- Author details
1
School of Physics, Universiti Sains Malaysia, 11800 Penang, Malaysia.
rods were observed when different forms of heating were 2
Nano-Biotechnology Research and Innovation (NanoBRI), Institute for
used as in the case of plasmonic heating (i.e., photoin- Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800
duced) and conventional thermal heating. They have con- Penang, Malaysia.
cluded that alteration in the protein corona composition as Received: 23 February 2015 Accepted: 1 May 2015
a result of photoinduced local heating might affect the bio-
logical fate of NPs. Hence, these changes that will affect
the final biological fate of plasmonic NPs should be taken References
1. Albanese A, Tang PS, Chan WC. The effect of nanoparticle size, shape, and
into account for biological safety design and application of surface chemistry on biological systems. Annu Rev Biomed Eng.
NPs for hyperthermia treatments. 2012;14:1–16.
2. Rauch J, Kolch W, Laurent S, Mahmoudi M. Big signals from small particles:
regulation of cell signaling pathways by nanoparticles. Chem Rev.
Cell Membrane Composition 2013;113(5):3391–406.
In biological environments, surfaces of NPs are signifi- 3. Steigerwald ML, Brus LE. Semiconductor crystallites: a class of large molecules.
cantly modified by the adsorption of proteins leading to Acc Chem Res. 1990;23(6):183–8.
4. Wang Y. Nonlinear optical properties of nanometer-sized semiconductor
the creation of an interface between NPs and the cell clusters. Acc Chem Res. 1991;24(5):133–9.
membrane (CM) [25]. Therefore, to better understand the 5. Weller H. Quantized semiconductor particles: a novel state of matter for
interactions that occur at the bio-nano interface, the effect materials science. Adv Mater. 1993;5(2):88–95.
6. Mahmoudi M, Sant S, Wang B, Laurent S, Sen T. Superparamagnetic iron oxide
of cell membrane should also be considered. For instance, nanoparticles (SPIONs): development, surface modification and applications in
it is well known that negatively charged CM causes chemotherapy. Adv Drug Deliv Rev. 2011;63(1–2):24–46.
Foroozandeh and Aziz Nanoscale Research Letters (2015) 10:221 Page 11 of 12

7. Ragnaill MN, Brown M, Ye D, Bramini M, Callanan S, Lynch I, et al. Internal 31. Lacerda SHDP, Park JJ, Meuse C, Pristinski D, Becker ML, Karim A, et al.
benchmarking of a human blood–brain barrier cell model for screening Interaction of gold nanoparticles with common human blood proteins. ACS
of nanoparticle uptake and transcytosis. Eur J Pharm Biopharm. Nano. 2009;4(1):365–79.
2011;77(3):360–7. 32. Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA. Nanoparticle
8. Cho K, Wang X, Nie S, Chen Z, Shin DM. Therapeutic nanoparticles for drug size and surface properties determine the protein corona with possible
delivery in cancer. Clin Cancer Res. 2008;14(5):1310–6. implications for biological impacts. Proc Natl Acad Sci.
9. Ditto AJ, Shah PN, Yun YH. Non-viral gene delivery using nanoparticles. 2008;105(38):14265–70.
Expert Opin Drug Deliv. 2009;6(11):1149–60. 33. Walczyk D, Bombelli FB, Monopoli MP, Lynch I, Dawson KA. What the cell
10. Rosi NL, Giljohann DA, Thaxton CS, Lytton-Jean AK, Han MS, Mirkin CA. “sees” in bionanoscience. J Am Chem Soc. 2010;132(16):5761–8.
Oligonucleotide-modified gold nanoparticles for intracellular gene regulation. 34. Cedervall T, Lynch I, Foy M, Berggård T, Donnelly SC, Cagney G, et al.
Science. 2006;312(5776):1027–30. Detailed identification of plasma proteins adsorbed on copolymer
11. Tanaka T, Mangala LS, Vivas-Mejia PE, Nieves-Alicea R, Mann AP, Mora E, nanoparticles. Angew Chem Int Ed. 2007;46(30):5754–6.
et al. Sustained small interfering RNA delivery by mesoporous silicon 35. Mahmoudi M, Sahraian MA, Shokrgozar MA, Laurent S. Superparamagnetic
particles. Cancer Res. 2010;70(9):3687–96. iron oxide nanoparticles: promises for diagnosis and treatment of multiple
12. Xu C, Mu L, Roes I, Miranda-Nieves D, Nahrendorf M, Ankrum JA, et al. sclerosis. ACS Chem Neurosci. 2011;2(3):118–40.
Nanoparticle-based monitoring of cell therapy. Nanotechnology. 36. Mahmoudi M, Simchi A, Imani M, Shokrgozar MA, Milani AS, Häfeli UO, et al.
2011;22(49):494001. A new approach for the in vitro identification of the cytotoxicity of
13. Liang XJ, Meng H, Wang YZ, He HY, Meng J, Lu J, et al. Metallofullerene superparamagnetic iron oxide nanoparticles. Colloids Surf B: Biointerfaces.
nanoparticles circumvent tumor resistance to cisplatin by reactivating 2010;75(1):300–9.
endocytosis. Proc Natl Acad Sci. 2010;107(16):7449–54. 37. Saptarshi SR, Duschl A, Lopata AL. Interaction of nanoparticles with proteins:
14. Yan L, Zhao F, Li S, Hu Z, Zhao Y. Low-toxic and safe nanomaterials by relation to bio-reactivity of the nanoparticle. J Nanobiotechnol. 2013;11:26.
surface-chemical design, carbon nanotubes, fullerenes, metallofullerenes, 38. Lundqvist M, Stigler J, Cedervall T, Berggård T, Flanagan MB, Lynch I, et al.
and graphenes. Nanoscale. 2011;3(2):362–82. The evolution of the protein corona around nanoparticles: a test study. ACS
15. Lee JH, Jang JT, Choi JS, Moon SH, Noh SH, Kim JW, et al. Exchange-coupled Nano. 2011;5(9):7503–9.
magnetic nanoparticles for efficient heat induction. Nat Nanotechnol. 39. Simberg D, Park JH, Karmali PP, Zhang WM, Merkulov S, McCrae K, et al.
2011;6(7):418–22. Differential proteomics analysis of the surface heterogeneity of dextran iron
16. Kircher MF, de la Zerda A, Jokerst JV, Zavaleta CL, Kempen PJ, Mittra E, et al. oxide nanoparticles and the implications for their in vivo clearance.
A brain tumor molecular imaging strategy using a new triple-modality Biomaterials. 2009;30(23):3926–33.
MRI-photoacoustic-Raman nanoparticle. Nat Med. 2012;18(5):829–34. 40. Aggarwal P, Hall JB, McLeland CB, Dobrovolskaia MA, McNeil SE.
17. Rogers WJ, Meyer CH, Kramer CM. Technology insight: in vivo cell tracking Nanoparticle interaction with plasma proteins as it relates to particle
by use of MRI. Nat Clin Pract Cardiovasc Med. 2006;3(10):554–62. biodistribution, biocompatibility and therapeutic efficacy. Adv Drug Deliv
18. Eigenheer R, Castellanos ER, Nakamoto MY, Gerner KT, Lampe AM, Wheeler Rev. 2009;61(6):428–37.
KE. Silver nanoparticle protein corona composition compared across 41. Ehrenberg MS, Friedman AE, Finkelstein JN, Oberdorster G, McGrath JL. The
engineered particle properties and environmentally relevant reaction influence of protein adsorption on nanoparticle association with cultured
conditions. Environ Sci: Nano. 2014;1(3):238. endothelial cells. Biomaterials. 2009;30(4):603–10.
19. Mahmoudi M, Lynch I, Ejtehadi MR, Monopoli MP, Bombelli FB, Laurent S. 42. Harnisch S, Müller RH. Adsorption kinetics of plasma proteins on oil-in-water
Protein—nanoparticle interactions: opportunities and challenges. Chem Rev. emulsions for parenteral nutrition. Eur J Pharm Biopharm. 2000;49(1):41–6.
2011;111(9):5610–37. 43. Göppert TM, Müller RH. Polysorbate-stabilized solid lipid nanoparticles as
20. Monopoli MP, Åberg C, Salvati A, Dawson KA. Biomolecular coronas colloidal carriers for intravenous targeting of drugs to the brain: comparison
provide the biological identity of nanosized materials. Nat Nanotechnol. of plasma protein adsorption patterns. J Drug Target. 2005;13(3):179–87.
2012;7(12):779–86. 44. Vroman L, Adams A, Fischer G, Munoz P. Interaction of high molecular
21. Ghavami M, Saffar S, Abd Emamy B, Peirovi A, Shokrgozar MA, Serpooshan weight kininogen, factor XII, and fibrinogen. Blood. 1980;55(1):156–9.
V, et al. Plasma concentration gradient influences the protein corona 45. Göppert TM, Müller RH. Adsorption kinetics of plasma proteins on solid lipid
decoration on nanoparticles. RSC Adv. 2013;3(4):1119–26. nanoparticles for drug targeting. Int J Pharm. 2005;302(1–2):172–86.
22. Nel AE, Mädler L, Velegol D, Xia T, Hoek EM, Somasundaran P, et al. 46. Jansch M, Stumpf P, Graf C, Rühl E, Müller RH. Adsorption kinetics of plasma
Understanding biophysicochemical interactions at the nano-bio interface. proteins on ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles.
Nat Mater. 2009;8(7):543–57. Int J Pharm. 2012;428(1–2):125–33.
23. Treuel L, Nienhaus GU. Toward a molecular understanding of 47. Gessner A, Waicz R, Lieske A, Paulke B, Mader K, Muller RH. Nanoparticles
nanoparticle-protein interactions. Biophys Rev. 2012;4(2):137–47. with decreasing surface hydrophobicities: influence on plasma protein
24. Rahman M, Laurent S, Tawil N, Yahia LH, Mahmoudi M. Nanoparticle and adsorption. Int J Pharm. 2000;196(2):245–9.
protein corona. In: Protein-nanoparticle interactions. New York: Springer; 48. Treuel L. How protein adsorption shapes the biological identity of NPs—where
2013. p. 21–44. do we stand? J Phys Chem Biophys. 2013;3:e113.
25. Walkey CD, Chan WCW. Understanding and controlling the interaction of 49. Buijs J, Vera CC, Ayala E, Steensma E, Håkansson P, Oscarsson S.
nanomaterials with proteins in a physiological environment. Chem Soc Rev. Conformational stability of adsorbed insulin studied with mass spectrometry
2012;41(7):2780. and hydrogen exchange. Anal Chem. 1999;71(15):3219–25.
26. Walkey CD, Olsen JB, Song F, Liu R, Guo H, Olsen DWH, et al. Protein corona 50. Mahmoudi M, Sardari S, Shokrgozar MA, Laurent S, Stroeve P. Irreversible
fingerprinting predicts the cellular interaction of gold and silver changes in protein conformation due to interaction with
nanoparticles. ACS Nano. 2014;8(3):2439–55. superparamagnetic iron oxide nanoparticles. Nanoscale. 2011;3(3):1127–38.
27. Monopoli MP, Walczyk D, Campbell A, Elia G, Lynch I, Bombelli FB, et al. 51. Shang L, Wang Y, Jiang J, Dong S. pH-dependent protein conformational
Physical-chemical aspects of protein corona: relevance to in vitro and in vivo changes in albumin: gold nanoparticle bioconjugates: a spectroscopic study.
biological impacts of nanoparticles. J Am Chem Soc. 2011;133(8):2525–34. Langmuir. 2007;23(5):2714–21.
28. Lesniak A, Fenaroli F, Monopoli MP, Åberg C, Dawson KA, Salvati A. Effects 52. Casals E, Pfaller T, Duschl A, Oostingh GJ, Puntes V. Time evolution of the
of the presence or absence of a protein corona on silica nanoparticle nanoparticle protein corona. ACS Nano. 2010;4(7):3623–32.
uptake and impact on cells. ACS Nano. 2012;6(7):5845–57. 53. Nagayama S, Ogawara K-i, Fukuoka Y, Higaki K, Kimura T. Time-dependent
29. Laurent S, Burtea C, Thirifays C, Rezaee F, Mahmoudi M. Significance of cell changes in opsonin amount associated on nanoparticles alter their hepatic
“observer” and protein source in nanobiosciences. J Colloid Interface Sci. uptake characteristics. Int J Pharm. 2007;342(1–2):215–21.
2013;392:431–45. 54. Deng ZJ, Mortimer G, Schiller T, Musumeci A, Martin D, Minchin RF.
30. Cedervall T, Lynch I, Lindman S, Berggard T, Thulin E, Nilsson H, et al. Differential plasma protein binding to metal oxide nanoparticles.
Understanding the nanoparticle-protein corona using methods to quantify Nanotechnology. 2009;20(45):455101.
exchange rates and affinities of proteins for nanoparticles. Proc Natl Acad 55. Lynch I, Dawson KA. Protein-nanoparticle interactions. Nano Today.
Sci. 2007;104(7):2050–5. 2008;3(1–2):40–7.
Foroozandeh and Aziz Nanoscale Research Letters (2015) 10:221 Page 12 of 12

56. Hill HD, Millstone JE, Banholzer MJ, Mirkin CA. The role radius of curvature
plays in thiolated oligonucleotide loading on gold nanoparticles. ACS Nano.
2009;3(2):418–24.
57. Tenzer S, Docter D, Rosfa S, Wlodarski A, Kuharev J, Rekik A, et al.
Nanoparticle size is a critical physicochemical determinant of the human
blood plasma corona: a comprehensive quantitative proteomic analysis.
ACS Nano. 2011;5:7155–67.
58. Dobrovolskaia MA, Patri AK, Zheng J, Clogston JD, Ayub N, Aggarwal P,
et al. Interaction of colloidal gold nanoparticles with human blood: effects
on particle size and analysis of plasma protein binding profiles. Nanomed
Nanotechnol Biol Med. 2009;5(2):106–17.
59. Vertegel AA, Siegel RW, Dordick JS. Silica nanoparticle size influences the
structure and enzymatic activity of adsorbed lysozyme. Langmuir.
2004;20(16):6800–7.
60. Cox MC, Barnham KJ, Frenkiel TA, Hoeschele JD, Mason AB, He Q-Y, et al.
Identification of platination sites on human serum transferrin using 13C and
15N NMR spectroscopy. JBIC J Biol Inorg Chem. 1999;4(5):621–31.
61. Gref R, Lück M, Quellec P, Marchand M, Dellacherie E, Harnisch S, et al.
‘Stealth’ corona-core nanoparticles surface modified by polyethylene 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. 2000;18(3–4):301–13.
62. Perry JL, Reuter KG, Kai MP, Herlihy KP, Jones SW, Luft JC, et al. PEGylated
PRINT nanoparticles: the impact of PEG density on protein binding,
macrophage association, biodistribution, and pharmacokinetics. Nano Lett.
2012;12(10):5304–10.
63. Moghimi SM, Muir I, Illum L, Davis SS, Kolb-Bachofen V. Coating particles
with a block co-polymer (poloxamine-908) suppresses opsonization but
permits the activity of dysopsonins in the serum. Biochimica et Biophysica
Acta (BBA)-Molecular. Cell Res. 1993;1179(2):157–65.
64. Dutta D, Sundaram SK, Teeguarden JG, Riley BJ, Fifield LS, Jacobs JM, et al.
Adsorbed proteins influence the biological activity and molecular targeting
of nanomaterials. Toxicol Sci. 2007;100(1):303–15.
65. Gessner A, Lieske A, Paulke BR, Müller RH. Influence of surface charge
density on protein adsorption on polymeric nanoparticles: analysis by
two-dimensional electrophoresis. Eur J Pharm Biopharm. 2002;54(2):165–70.
66. Roach P, Farrar D, Perry CC. Interpretation of protein adsorption: surface-
induced conformational changes. J Am Chem Soc. 2005;127(22):8168–73.
67. Maiorano G, Sabella S, Sorce B, Brunetti V, Malvindi MA, Cingolani R, et al.
Effects of cell culture media on the dynamic formation of protein −
nanoparticle complexes and influence on the cellular response. ACS Nano.
2010;4(12):7481–91.
68. Albanese A, Walkey CD, Olsen JB, Guo H, Emili A, Chan WCW. Secreted
biomolecules alter the biological identity and cellular interactions of
nanoparticles. ACS Nano. 2014;8(6):5515–26.
69. Laurent S, Burtea C, Thirifays C, Hafeli UO, Mahmoudi M. Crucial ignored
parameters on nanotoxicology: the importance of toxicity assay
modifications and “cell vision”. PLoS ONE. 2012;7(1):e29997.
70. Rhoades R, Pflanzer RG. Human physiology. 4th ed. Fort Worth: Saunders
College Publication; 1989.
71. Petersdorf R. Chap 12. Chills and fever. In: Wintrob MM, Thorn GW, Adams
RD, Braaunwald E, Isselbacher KJ, Petersdorf RG, editors. Harrison’s principles
of internal medicine. 7th ed. New York: McGraw-Hill; 1974. p. 58–60.
72. Yang J-M, Yang H, Lin L. Quantum dot nano thermometers reveal
heterogeneous local thermogenesis in living cells. ACS Nano.
2011;5(6):5067–71.
73. Hasday JD, Singh IS. Fever and the heat shock response: distinct, partially
overlapping processes. Cell Stress Chaperones. 2000;5(5):471–80.
74. Mahmoudi M, Abdelmonem AM, Behzadi S, Clement JH, Dutz S, Ejtehadi Submit your manuscript to a
MR, et al. Temperature: the “ignored” factor at the nanobio interface. ACS journal and benefit from:
Nano. 2013;7(8):6555–62.
75. Mahmoudi M, Lohse SE, Murphy CJ, Fathizadeh A, Montazeri A, Suslick KS. 7 Convenient online submission
Variation of protein corona composition of gold nanoparticles following
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plasmonic heating. Nano Lett. 2014;14(1):6–12.
76. Mahmoudi M, Meng J, Xue X, Liang XJ, Rahman M, Pfeiffer C, et al. 7 Immediate publication on acceptance
Interaction of stable colloidal nanoparticles with cellular membranes. 7 Open access: articles freely available online
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