Biomedicine & Pharmacotherapy 139 (2021) 111541

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Review

Research progress and application opportunities of nanoparticle–protein

corona complexes
Wei Huang a, b, Gao Xiao c, Yujuan Zhang b, *, Weiping Min b
a
Department of Pharmacy, The First People’s Hospital of Jiande, Jiande 311600, China
b
Department of immunology, School of Basic Medical Sciences and School of Pharmacy, Nanchang University, Nanchang 330006, China
c
College of Environment and Resources, Fuzhou University, Fuzhou 350108, China

A R T I C L E I N F O A B S T R A C T

Keywords: Nanoparticles (NPs) can be used to design for nanomedicines with different chemical surface properties owing to
Nanoparticles their size advantages and the capacity of specific delivery to targeted sites in organisms. The discovery of the
Protein corona presence of protein corona (PC) has changed our classical view of NPs, stimulating researchers to investigate the
Targeted drug delivery
in vivo fate of NPs as they enter biological systems. Both NPs and PC have their specificity but complement each
Immune
other, so they should be considered as a whole. The formation and characterization of NP-PC complexes provide
new insights into the design, functionalization, and application of nanocarriers. Based on progress of recent
researches, we reviewed the formation, characterization, and composition of the PC, and introduced those
critical factors influencing PC, simultaneously expound the effect of PC on the biological function of NPs.
Especially we put forward the opportunities and challenges when NP-PC as a novel nano-drug carrier for targeted
applications. Furthermore, we discussed the pros versus cons of the PC, as well as how to make better PC in the
future application of NPs.

1. Introduction
In recent decades, with the development of nanotechnology and the
invention of various nanomaterials, an increasing number of nano­
materials has been applied in the fields of biology and medicine. NPs
have been designed as nanomedicine delivery systems with different
surface chemistries for precise delivery to the diseased sites. Solid tu­
mors exhibit enhanced permeability and retention effects, while NPs are
small enough to leak through loose porous blood vessels and enter the
tumor tissue without penetrating into the normal tissue through the
dense normal blood vessels. This passively tumor-targeted strategy
prevents NPs from accumulating in the heart and other non-targeting
sites, thereby reducing the toxic side effects on normal tissues. Howev­
er, this strategy still cannot prevent the NPs from accumulating in
certain organs such as spleen and kidney. Another strategy is to modify
NPs with the targeting molecules, making NPs possessing an active
targeting function; however, this is difficult to be implemented. Warren
Chan and his colleagues have reviewed more than 100 nanomedical
papers published over the past 10 years and found that any
nanomaterial, whether active or not, has an average dose of only 0.7%
that reaches the tumors [1,2]. This reminds us that, even if the active
targeting strategy exists, the actual concentration of the material
reaching the target site may not be as good as expected. We need to take
it into consideration about the fate of nanomaterials as they enter living
systems.
Either passive targeting or active targeting strategy including the
design, synthesis and production of NPs can be achieved through in vitro
experimental manipulations. However, the fate and function of NPs
entering the biological system are unknown. According to the relevant
literatures, NPs have a large specific surface area and high surface free
energy. After entering the biological system, NPs are more likely to
adsorb various biological molecules, such as nucleic acids, cytokines,
amino acids, and proteins, to form a biomolecular layer on the surface,
which is mainly composed of proteins, so called a “protein corona”. PC
can affect the surface physicochemical properties of NPs, specific tar­
geting, toxicity, in vivo clearance, cellular uptake, signal transduction,
and immune response [3–6]. This review will begin with an explanation
of how the PC is produced and list the factors that influence its

Abbreviations: NP, nanoparticles; PC, protein corona; HSA, human serum albumin; AuNR, gold nanorods; MNPs, magnetic nanoparticles; BSA, bovine serum
albumin; FBS, fetal bovine serum; PEG, polyethylene glycol.
* Corresponding author.
E-mail address: yujuanzhang@ncu.edu.cn (Y. Zhang).

https://doi.org/10.1016/j.biopha.2021.111541
Received 27 November 2020; Received in revised form 22 February 2021; Accepted 23 March 2021
Available online 10 April 2021
0753-3322/© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
W. Huang et al.
formation. Then we will discuss whether a PC changes the properties of
NPs. Finally, we will summarize the interaction between NPs and cells,
to show how the NP-PC complex presents a challenge for the application
and development of new nano-drug delivery systems.
2. Formation, characterization, and composition of the PC
2.1. Formation and development
Owing to the high percentage of surface atoms and colloidal nature
of NPs, upon contacting with physiological media, the NPs will adopt
more stable thermodynamic states [7], which includes aggregation,
corrosion, dissolution and interaction with mediator proteins. Vorman
[8] found that at least 5 proteins were substituted with each other on one
surface in plasma within 1 min. This indicated that the surface contact of
inorganic NPs and proteins occurs promptly when NPs enter a physio­
logical environment, such as plasma, and the protein adsorbed on the
surface of the NP changes with the exposure time. At first, the proteins
transiently adsorbed are mostly those with a high-concentration and
low-affinity. At this stage, the adsorption is reversible and the proteins
can be desorbed. As time progresses, these proteins are gradually
replaced by high-affinity proteins. At the end, the protein adsorption
becomes irreversible. The continuous adsorption and desorption process
of this plasma protein on the surface involves factors such as incubation
time and surface properties, which is called the “Vorman effect” [8–10].
Like gas adsorption, proteins will randomly attach to the surface of NPs
at the beginning, then they will recombine on the surface of NPs through
a rearrangement reaction and crowding effect to form a domain [11],
which eventually exists in an irreversible minimum energy state.
The protein layer adsorbed on the surface of the NP is determined by
the surface properties of NPs, exposure time, protein concentration and
affinity of the protein. A protein layer, which has a high affinity, high
binding rate and irreversible adsorption on the surface of the NP, is
called a "hard corona". In contrast, a protein layer with low affinity and
reversible protein adsorption layer is called a "soft corona" [12,13]. If
the affinity of the protein is high enough and the exposure time is
Fig. 1. Hypothetical dynamic adsorption diagram of proteins on the surface of a NP. Proteins on the NP are always in the dynamic process of adsorption and
desorption. With the prolongation of exposure time, the low affinity and high concentration of proteins close to the NP would be replaced by proteins with high
affinity and low concentration, which rearrange at the surface of the particle and eventually form an irreversible stably hard corona composed of tightly bound
proteins, while the replaced proteins absorbed on the outside form a reversible unsteadily soft corona consisting of loosely bound proteins.
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Biomedicine & Pharmacotherapy 139 (2021) 111541
sufficient, the surface of the NPs will be completely occupied by hard
corona proteins, and the soft corona protein will adhere to the surface of
the hard corona protein. However, when the affinity of the protein is
low, it may cause the unstable NPs to aggregate or still adsorb some of
the low affinity, reversible soft corona proteins. The soft corona protein
changes with the biological environment, while the hard corona protein
adsorbed on the surface of the NPs maintains the initial binding status. A
schematic diagram of the dynamic adsorption of NPs and proteins is
shown in Fig. 1. Unlike most of the studies that separated NPs from
protein-containing fluids, Shang et al. used fluorescence correlation
spectroscopy (FCS) as an in situ technique to detect NP-protein in­
teractions while the NPs were suspended in biological fluids [14]. The
authors observed that the protein layer thickness always matched the
known molecular dimensions of the proteins binding in certain orien­
tations, such as human serum albumin (HSA). Notably, they pointed out
that while the corona formed from a single type of serum protein was
reversible, protein adsorption from a complex biological media, such as
blood serum, was entirely irreversible.
The rearrangement process that proteins undergo on the surface of
NPs is called the conformational change. Direct contact of hard corona
proteins with NPs typically causes changes in the secondary structure
(such as loss of α-helical and increase of β-sheets or β-turns), which can
result in a partial loss of biochemical activity [15–17]. The thickness of
the PC is dependent on many parameters such as protein concentration,
particle size, and surface properties of the particle [17].
2.2. Identification and analysis methods
Protein-bound NPs possess new roles, which need certain methods to
identify and analyze them. Some methods can qualitatively describe the
changes in the physicochemical properties of nanoparticles before and
after the coating of proteins. Generally, scanning electron microscopy
(SEM) and transmission electron microscopy (TEM) are the most com­
mon, direct, clear and persuasive methods to identify a new NP. TEM
can directly observe the morphology of nanomaterials, especially the
finer and smaller nanostructures [18,19]. Wang et al. [20] observed a
W. Huang et al.
thicker organic layer of gold nanorods (AuNRs) after protein adsorption
and measured the thickness of the PC through TEM images, and
concluded that the serum PC not only increased the stability of AuNRs,
but also prevented their directly destructive effects on membrane
structures by SEM and TEM images of cytoplasmic membrane structures.
Cryogenic transmission electron microscopy (Cryo-TEM) is more suit­
able for the observation of proteins, biological slices and other
temperature-sensitive samples, but has strict requirements on the tech­
nical operation of sample preparation [21]. Therefore, Cryo-TEM is
suitable to visualize the soft corona (weakly bound proteins) but little
studied for its poor stability. Kokkinopoulou et al. [22] studied the
evolution of soft plus hard coronas (in situ analysis) towards hard corona
(after three washing steps) on polystyrene nanoparticles by using
Cryo-TEM. Franqui et al. [23] observed a higher electronic density
distributed throughout the hard corona coated single-layer graphene
oxide (HC@SLGO) surface than bare SLGO by Cryo-TEM images, and
Cryo-TEM allowed the in-situ observation of GO sheets in DMEM me­
dium, once the sample was instantly frozen keeping its integrity in the
solution media (i.e., avoiding the drying effect and the structural
deformation of the sample by the microscope vacuum). In Franqui’s
study, atomic force microscopy (AFM) allowed a visualization of the
protein coating and its distribution on the GO surface, the measurement
of surface roughness and thickness of GO-protein complexes. It is an
important analysis method which can be used to evaluate the sizes of
particles and the thickness of PCs, as well as characterize the interaction
of proteins with NPs [24].
In addition to the methods mentioned above that can directly
observe or image the morphology of NPs, there are also some indirect
methods that can provide more information including charge, size,
structure, and so on. For charge analysis, zeta-potential analysis is the
most commonly used and indispensable method. As for size analysis, the
most conventional methods are dynamic light scattering (DLS) and
differential centrifugal sedimentation (DCS). DLS can measure the hy­
drodynamic size distribution of the NPs before and after protein
adsorption [25,26], while DCS is capable of separating the components
of a mixture based on their densities and sizes, which can be applied in
measuring the sizes of NP-PC complexes with a broad size range [27,28].
Moreover, small-angle X-ray scattering (SAXS) can be used to measure
the particles with the sizes in the range of 1–100 nm, especially ranging
from 5 to 25 nm [29]. Although SAXS applied to biological macromol­
ecules is a low-resolution structural technique, it is particularly accurate
in distinguishing between different structural models proposed from
higher resolution techniques. Spinozzi et al. [30] propose a novel
method for determining the structural and thermodynamic properties of
nanoparticle-protein complexes under physiological conditions by SAXS
and neutron-scattering measurements. Perez et al. [31] used
size-exclusion chromatography (SEC) and SAXS to model the detergent
corona around aquaporin-0, a membrane protein of known structure.
There are also some spectroscopic methods used to analyze the in­
teractions between NP and PC. UV–visible (UV–vis) spectroscopy is
usually used to record the absorption peak spectra generated by the
surface plasmon resonance of metal NPs, whose position and intensity
depend on NP size, shape, and dielectric constant of the medium in close
proximity to the surface of the NPs [32]. In UV-Vis absorption spectrum,
we can directly observe the red shift of the plasmonic resonance ab­
sorption peaks caused by the changes of size and surface of NPs after
proteins coated successfully [33], and the change of waveform caused
by the failure of coating [34]. In particular, the surface plasmon reso­
nance (SPR) mentioned in Cedervall’s study worked on the same prin­
ciple. By using gold surfaces with thiol-conjugated NPs, they studied the
kinetics of association and dissociation of plasma, HSA and fibrinogen
with NPs. Then they found the hydrophobicity of the particles depended
the association and dissociation rates [35]. In Maiorano’s study, based
on an enhanced plasmon resonance light-scattering peak at about
550 nm for the interparticle interactions of gold nanoparticles (AuNPs)
[36], so plasmon resonance light scattering (PRLS) as a very sensitive
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Biomedicine & Pharmacotherapy 139 (2021) 111541
tool can be used to monitor the physical status of protein-coated AuNPs
in the two media. Moreover, surface-enhanced Raman scattering (SERS)
was also often used in the study of NP-PC complexes. Winuprasith et al.
[37] observed protein-coated NPs after bile salt addition contained
bands from both protein and bile salts by SERS spectra, indicating that
the protein was not fully displaced by the bile salts. The SERS spectra of
bovine serum (BS) incubated particles showed peak broadening,
consistent with significant conformational changes to proteins in the PC
[38]. Fluorescence correlation spectroscopy (FCS) and fluorescence
cross-correlation spectroscopy (FCCS) are sensitive spectroscopy tech­
niques that measure fluorescence intensity fluctuations of single mole­
cules inside a femtoliter confocal volume, and they are suitable for
studying the formation of PC around NPs and for examining corona
stability in situ in biological matrixes [39]. Fourier transform infrared
spectroscopy (FTIR) is used to probe the absorption of infrared light on
NP surfaces and to provide information on protein adsorption, including
the evaluation of protein coatings on the surfaces of NPs and identifi­
cation of a protein’s secondary structure [40]. By comparing the FTIR of
human serum (HS), magnetic nanoparticles (MNPs) and HS@MNPs, the
characteristic peaks at 1651 cm-1 and 1548 cm-1 indicated the presence
of human serum protein on MNPs, and excessive weight loss of
HS@MNPs specifies human serum protein degradation in thermograms
[41].
The most commonly used methods for the identification and quan­
tification of PC components are sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and mass spectrometry (MS) or liquid
chromatography–tandem mass spectrometry (LC-MS/MS) [32,42].
In addition, techniques have been developed to determine the
conformation of the binding protein using circular dichroism spectros­
copy (CD), infrared (FTIR), SERS NMR, fluorescence quenching [43],
and computational simulations [17,44]. CD is sensitive to detect the NP
interaction-induced conformational changes of secondary structures and
the folding and binding properties of proteins according to chiral
properties [16,45,46]. SERS can provide information on the structure,
composition, and interaction of molecules in the proximity of gold NPs,
thereby enabling studies of adsorbed biomolecules in vivo [47]. It can
achieve in situ and real-time monitoring of ligand exchange reactions on
the gold surface [48]. By using SERS, Szekeres found bovine serum al­
bumin (BSA) and HSA interacted with the citrate ligands at the NP
surface via lysine residues [49]. Circu et al. [50] investigated the surface
interaction between the obtained nanosystems and BSA was by FTIR,
specially emphasized on the conformational changes of BSA induced by
vinylimidazole-rare earth complexes. The results obtained from FTIR
analysis showed the potential of these nanosystems to contribute to the
process of alpha/beta conversion of protein which highlights their ap­
plications in study of aspects regarding protein misfolding and aggre­
gation. Ban et al. [51] provides a method to accurately and
quantitatively predict the functional composition of the PC by inte­
grating a machine learning model and meta-analysis. Their study found
the functional protein compositions (e.g., immune proteins, complement
proteins, and apolipoproteins) in complex coronas were precisely pre­
dicted with good R-2 (most over 0.80). Moreover, the method success­
fully predicted the cellular recognition (e.g., cellular uptake by
macrophages and cytokine release) mediated by functional corona
proteins. Some methods can be used to study the formation of PC in
complex environments. Carril et al. [52] mentioned a methodology for
determining PC formation in complex media by using nuclear magnetic
resonance (NMR) spectroscopy: NPs are labeled with (19)F and their
diffusion coefficient measured using (19)F diffusion-ordered NMR. (19)
F diffusion NMR measurements of hydrodynamic radii allow for in situ
characterization of NPs in complex environments by quantification of
protein adsorption to the surface of NPs, as determined by increase in
hydrodynamic radius. Note that the methodology is not optics based,
and thus can be used in turbid environments, as in the presence of cells.
Moreover, the use of site-specific labeling with spin probes and fluo­
rophores combined with electron paramagnetic resonance (EPR)
W. Huang et al.
spectroscopy and fluorescence resonance energy transfer (FRET) can
reveal the structural and dynamic changes of NPs interacting with blood
proteins and in vivo [15].
Size exclusion chromatography (SEC), surface plasmon resonance
(SPR), and isothermal titration calorimetry (ITC) have also been re­
ported to estimate the affinity of binding proteins to the surface of
nanomaterials [13,14]. Cedervall et al. [35] determined the rates of
protein association and dissociation using SPR technology with nano­
particles that are thiol-linked to gold, and through SEC of
protein-nanoparticle mixtures. SEC can be used to isolate NP-associated
proteins as a systematic methodology. They also found ITC is suitable for
studying the affinity and stoichiometry of protein binding to NPs. By
using ITC, Danner et al. [53] found the protein affinity depends on the
number of phosphonate moieties on the particle surface, because initial
slope of the binding isotherm of ITC can present the binding affinity of
plasma proteins. It can also show the influence of the negative surface
charge of NPs toward the thermodynamic properties of the protein
adsorption. It is reported that FRET can also be used to study the affinity
and adsorption orientation of proteins in PC [46].
In general, one method is not enough to provide a comprehensive
and accurate description and characterization of the PC, and it is usually
combined with several different methods to analyze [54,55]. The above
methods for studying PC are summarized in Fig. 2.
2.3. Main protein composition
The composition and amount of proteins in the corona depends on
the physical and chemical characteristics of the NPs, the type of bio­
logical medium and the exposure time [56], so that each nanomaterial’s
PC are unique. In addition to the physical and chemical properties of the
NPs, the PCs determine part of the biological functions of the NP-PC
conjugates. NP-adsorbed proteins are identified to be important pro­
teins involved in key biological processes. Walkey and Chan [57] sum­
marized the composition and abundance of PCs formed by 63
nanomaterials in 26 studies. They found that a total of 125 unique
plasma proteins had been identified, of which 21 were high-abundance
Fig. 2. Graphic classification and summary of common methods for analyzing PC.
4
Biomedicine & Pharmacotherapy 139 (2021) 111541
proteins, and each adsorbent protein mass was observed to exceed 10%
of the total adsorbent protein mass on at least one of the nanomaterials.
The plasma PC appears to follow a general structure, with 2–6 proteins
adsorbed at high abundance, and many more adsorbed at low abun­
dance. The most abundant proteins accounted for average 29% of the
total adsorbed protein, and the top 3 most abundant proteins accounted
for average 56%. Some studies have also shown that among the
approximately 3700 proteins in human plasma, only a few tens, at the
most, are ever abundant in the hard corona. In addition, these proteins
rarely correspond to the most abundant proteins in plasma and are not
necessarily proteins with the highest individual affinity for the surface of
NPs [58,59]. When NPs such as iron oxide, silica, AuNPs, liposomes and
carbon nanotubes directly enter the blood environment, the following
proteins are mainly adsorbed: albumin, apolipoprotein, immunoglobu­
lins, fibrinogen, complement [57,60–63]. The physiological functions
corresponding to these adsorbed proteins are mainly lipid transport,
complement activation, coagulation, pathogen recognition and ion
transport. Although each nanomaterial can adsorb large amounts of
certain proteins, these abundant proteins are not always the same.
Additionally, even for a fixed material type, the size of the particle and
its surface modification are able to entirely change the nature of the
biologically active proteins in the corona, and thereby possibly also the
biological impacts [64].
The composition of the PC formed by a single protein is relatively
simple, and the hard protein of the PC is the determinate protein. To
better understand the process of PC formation, some scientists start by
modeling single protein, such as common albumin, immunoglobulin,
and uncommon soluble yeast protein extracts [65] and so on. By un­
derstanding the time and process of crowning of a single protein, we can
also infer the status of the protein in serum or plasma environment
where is more complex. Some single protein interacts with specific
materials, such as magnetic nanomaterials and transferrin. That iron
content governs the PC formation and induces a strong effect on the
thermal stability of the bound protein [66]. The study of a single protein
is beneficial for understanding its properties, but ultimately it will come
back to the study of NP and protein, protein and protein, protein and
W. Huang et al.
environment in the more complex environment of serum or plasma or
other biological fluid.
Serum stripped of fibrin contains 60–80 mg/mL protein, whose
57–71% is albumin, and followed by 8–26% are γ-globulins, especially
immunoglobulins G (IgG) [67]. Vitali et al. [63] confirmed that albumin
and serum can develop a PC evolving from unstable (soft) to stable
(hard) with AuNPs, while IgG incubation inevitably failed to form
corona, resulting in NP aggregation. Moreover, albumin proved to be the
most abundant protein in the hard PC formed by human serum and
AuNPs. This result has also been confirmed by other gold NPs in spite of
their size and shape [42]. Some studies have found that the most
abundant protein in certain silicon-based materials is apolipoprotein
[68]. Das et al. [69] found that both in the culture media Dulbecco’s
Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute
Medium (RPMI 1640) could find some cell matrix adhesion proteins on
AuNRs, but these proteins were missing on silica modified gold nanorods
(SiO2-AuNRs), which is a lateral reflection of the greater toxicity of the
naked AuNRs. In general, Cellular matrix adhesion proteins such as
immunoglobulins, complement proteins and other opsonins like plasma
fibrinogen are involved in the recognition and phagocytosis of NPs entry
into cells. Their preferential adsorption enhances phagocytosis and
trigger inflammation, determining NP toxic or non-toxic to cells, and
leading to NP removal from blood [70–72]. Conversely, serum albumin
and apolipoproteins are known to stabilize colloidal NPs and extend
their blood circulation [73–75]. A kinetic analysis of the PC of solid NPs
showed that the albumin was initially bound to the NPs and then
replaced by fibrinogen, after which fibrinogen was replaced by apoli­
poproteins [56,63]. An earlier study came to similar conclusions: when
proteins with different affinity competing for the available nanoparticle
surface area in a typical biological environment, HSA and fibrinogen
may dominate on the particle surface at short times, but will subse­
quently be displaced by lower abundance proteins with higher affinity
and slower kinetics, for example apolipoprotein A-I [35]. High plasma
concentrations of apolipoproteins have been shown to be able to replace
fibrinogen within seconds [76].
3. Critical factors influencing PC
3.1. Size of NPs
There are many other factors that influence the formation of PC. The
thickness and density of the PC formed on the surface of the NP are
directly related to the size of the NP. Piella et al. [33] studied the
interaction of citrate-stabilized AuNPs with proteins in the size at a
range of 3.5–150 nm, and found that the thickness and density of the PC
were strongly dependent on the size of the NPs. The particle size can be
determined by different modes of transition: (i) formation of an
incomplete corona, (ii) formation of a nearly single-density PC layer,
and (iii) formation of a multilayer corona. At first the authors chose an
appropriate exposure condition on the premise of stable NPs exist: for
the same citrate stabilized 10 nm AuNPs, when exposure experiments
performing in serum-free DMEM media, the immediate aggregation of
NPs can be clearly observed by UV–vis spectroscopy; while in the
complete cell culture medium (cCCM) composed of DMEM supple­
mented with 10% of fetal bovine serum (FBS), the main driver of
nanoparticle aggregation is particle concentration. The presence of
proteins is an important factor for the stabilization of NPs in high
strength electrolyte CCM. When AuNPs with different sizes were com­
bined with the CCM, the gold NP-PC, with a particle size ≤ 12 nm, was
smaller than the expected single-layer PC, while a single, full dense layer
was formed on the 12–80 nm AuNPs. The AuNPs with a particle
size > 80 nm formed a 2–3 layer multilayer PC, similar to that reported
in flat surfaces [77]. The protein adsorption kinetics showed that the
smaller the particle size, the faster the kinetic evolution and the thinner
(incomplete) the protein layer. Kihara et al. [78] structurally evaluated
the soft and hard components of the PC formed from polystyrene (PS)
5
Biomedicine & Pharmacotherapy 139 (2021) 111541
NPs and HSA. They found that the size of the NPs directly influenced the
nature of the PC, with larger particles favoring the formation of a soft
corona, owing to the decreased PS-HSA attraction.
3.2. Concentrations of NPs in medium
The concentration of NPs also greatly affects the formation of PC and
the properties of their surfaces. Zhang et al. [79] constructed four bio­
polymers (poly-(hydroxybutyrate-co-hydroxyhexanoate, PHBHHx) that
were composed of the same substance with similar particle sizes
(230 ± 20 nm) but different charges (− 30 to +40 mV), and studied the
protein adsorption of the four biopolymers at different concentrations
(125 μg/mL, 500 μg/mL, 1000 μg/mL) after incubation with 5% FBS.
The results indicated that the concentration of NPs led to a substantial
alteration in both the protein adsorption capacity and adsorbed protein
type. NPs with a lower concentration had a higher protein adsorption
capacity. When the concentration of the four biopolymers was less than
500 μg/mL, which was incubated in the serum protein at a concentra­
tion of 5% FBS, the zeta potential of all NPs-FBS was ~− 20 mV, which
indicated that the number of proteins in 5% FBS were sufficient to cover
almost all of the surface of the NPs. In contrast, when the concentration
of NPs was increased to 1000 μg/mL, the zeta potential of the four
polymers-FBS was altered, which resulted from the combination of the
associated proteins and uncovered part of the original NPs. This sug­
gested that the number of proteins in 5% FBS was insufficient to
completely cover the surface of the NPs.
3.3. Nanoparticle surface properties
3.3.1. Zeta potential and electrostatic force
It has been demonstrated that the zeta potential is a key factor
affecting NP-protein interactions [79]. The zeta potential not only plays
important roles in stabilizing NPs-based colloidal systems through the
electrostatic repulsive force, but is also a regulator for the particle size of
NPs made of the same material, where the larger the zeta potential, the
larger the electrostatic repulsion, and the smaller the particle size.
Moreover, in terms of protein adsorption, the composite charge of those
adsorbed serum proteins was negative, so that the larger the absolute
value of the zeta potential of the negatively charged NPs, the greater the
repulsive force and the less protein was absorbed [79]. The larger the
absolute value of the zeta potential of the positively charged NPs, the
greater the attraction and the more proteins were absorbed. However,
Shang et al. [14] proposed that local charge distributions of the binding
interfaces mediate electrostatic interactions rather than the net charge
of the proteins and NPs, and proteins adsorbed onto the NP surfaces in
certain orientations so as to allow efficient electrostatic interactions
between the charged patches on the proteins and the counter-charged
surfaces of the NPs.
Electrostatic force is one of the most important forces in the inter­
action between NPs and proteins. Kubiak and Mulheran [80] used
atomic molecular dynamics to study the adsorption of chicken protein
lysozyme (HEWL) on a negatively charged hydrophilic surface, and
concluded that the main force controlling the protein adsorption was the
electrostatic attraction of the protein fractions and surfaces. Ghosh et al.
[81] proposed the protein-nanoparticle-RCP (reverse charge
parity)-counterion model and demonstrated the electrostatic interaction
between negative surface NPs and positive surface HEWL. Additionally,
the irreversible denaturation of the protein conformation, owing to the
binding of counterions, was reported. They further used this model,
which involved reverse charge parity, to show the selective binding of
proteins on a charged surface iron oxide nanoparticles (IONPs) [82].
Dyawanapelly et al. [83] studied the interaction of lysozyme with
counterion-associated iron oxide nanoparticles (IONPs) using three
different types of NPs, namely, bare IONPs, low molecular weight chi­
tosan modified IONPs (LMWC-IONPs) and the counterion
(Na+)-associated sodium tripolyphosphate IONPs (STP-LMWC-IONPs).
 W. Huang et al.
Table 1
Overview of serum/plasma proteins adsorbed on the surface of different types of nanomaterials with varied surface chemistries.
Materials Type Size (TEM) Linker Medium Methods Main proteins brief description Refs

Polystyrene Ch-linPG21 170–280 nm Polyglycerol Plasma LC-MS/

[53]
MS
Ch-linP(G15-co- SDS-
GPE6) PAGE
Ch-linP(G12-co-
GPE9)
Polystyrene Galactose 33 nm,669 nm Galactopyranoside Normal human serum (incubated 1 h, MS Complement protein C3, Apolipoprotein A-I, Apolipoprotein E [101]
Glucose Glucopyranoside 37 ℃) Complement protein C3, Apolipoprotein A-I, Apolipoprotein E
SiO2 SiO2 22.4 ± 2.2 nm Fetal bovine serum LC-MS- Except for albumin, major proteins were apolipoproteins, [91]
MS
SiO2-NH2 –NH2 Alpha-2-HS-glycoprotein,complement C3 and hemoglobin.
SiO2-COO- –COO– PCs of three NPs changed dynamically during 1–48 h.
6

SiO2 UC-NPs 93.1 ± 14.1 nm Carboxylic acid Human plasma (incubated 1 h, 37 ℃) LC-MS/ Apolipoprotein E, Apolipoprotein E chain A, Alpha fibrinogen [94]
MS
HSA-NPs 98.0 ± 10.5 nm HSA pe-coated SDS- Apolipoprotein E, Apolipoprotein E4 chain A, Kininogen-1
PAGE
GG-NPs 101.9 ± 12.8 nm γ-Globulins pe-coated Apolipoprotein B100, Apolipoprotein E, Ig gmma-1 heavy chain
AuNPs BPEI-AuNP 40 nm, 80 nm Branched polyethyleneimine LFQ-LC-MS 40-BPEI-AuNP: albumin, Fibrinogen β chain, Fibrinogen α chain [102]
LA-AuNP Lipoic acid 55% human plasma (incubated SDS-PAGE 80-BPEI-AuNP: albumin, Fibrinogen α chain, Fibrinogen β chain
1 h)
PEG-AuNP Polyethylene 40-LA-AuNP: albumin, Talin-1, Myosin-9
glycol
80-LA-AuNP: albumin, Myosin-9, Talin-1
40/80-PEG-AuNP: albumin, Talin-1, Myosin-9
SPIONs CA-MF 10 nm Citric acid 10%/55% human plasma (incubated SDS- 10% plasma: CA-MF: Apolipoprotein B100, and Fibronectin [103]
1 h, 25 ℃) PAGE

Biomedicine & Pharmacotherapy 139 (2021) 111541

PAA-MF Poly(acrylic acid) MS OAOA-MF: Apolipoprotein A1
OAOA-MF Oleic acid 50% plasma: CA-MF: complement factor proteins and immunoglobulin
(CA and PAA SPIONs showed a very similar protein pattern) OAOA-MF: lipoproteins (Lipoprotein B100, ApoE, and ApoA)
W. Huang et al.
The experimental results showed that as a result of the binding of Na+
counter ions to proteins, the lysozyme fluorescence spectrum showed a
quenching effect after HEWL interacted with STP-LMWC-IONP (pH 9.0),
which indicated HEWL denaturation. The CD spectrum revealed a
conformational change in the HEWL secondary structure. In addition, a
counterion-induced lysozyme inactivation was confirmed by an enzyme
activity assay involving Micrococcus lysodeikticus. A pH of 9.0 was
observed to be a more favorable condition compared with pH 7.4
because of the strongest electrostatic interaction between lysozyme and
NP. Moreover, selective binding of LMWC-IONPs was observed with
protein at pH 9.0, which is in agreement with the earlier results using the
reverse charge parity model. Lysozyme, as an amyloid-forming model
protein, is also widely used as a model protein for studying folding and
aggregation mechanisms. This study assumed that counterions in the
surface coating of NPs can improve protein misfolding or unfolding, and
also prevent their aggregation, and thus can be considered as a powerful
and potential therapeutic strategy for the treatment of incurable
neurodegenerative diseases.
3.3.2. Hydrophobicity/hydrophilicity
The main forces that stabilize the structure of proteins are electro­
static interactions, which include hydrophobic, hydrogen bonding, co­
valent bonds, and weak van der Waals forces that play an important role
in stabilizing biopolymers. Shemetov et al. [84] reported that at the
atomic level, hydrophilicity is determined by the presence of polar
functional groups (such as Si2OH and Ti2OH) or under coordinating
metal ions (such as Fe2+ and Zr4+) in the outer surface layer. The
number of these sites determines the degree of hydrophilicity and hy­
drophobicity of the NPs. When a protein molecule approaches an NP, the
interaction of the protein with NP depends on the hydrophilicity and
hydrophobicity of the NP surface. In the case of a hydrophilic (polar)
surface, the interaction occurs directly through hydrogen bonds with
structures exposed on the surface of the protein. Whereas when the
surface is hydrophobic, the non-polar portion hidden in the dissolved
protein molecule may be exposed to the surface without making contact
with water, in which case, the interaction depends only on the degree of
hydrophobicity of the surface. Studies have shown that proteins adsor­
bed on hydrophobic surfaces have less natural structure than proteins
adsorbed on hydrophilic surfaces, which leads to severe protein dena­
turation [85].
Albumin and IgG tend to preferentially be absorbed by hydrophilic
nanomaterials, and the hydrophobic forces played a predominant role
[86]. While hydrophobic nanomaterials tend to preferentially adsorb
apolipoproteins, which may result from interactions between the lipid
binding domains of the apolipoproteins and the hydrophobic surface of
the nanomaterial. But not all apolipoproteins conform to this trend, for
example, ApoA-IV, ApoB, ApoC-III, and ApoE adsorb preferentially to
hydrophobic cholesterol or atheronal-B quantum dots, while ApoA-I,
ApoA-II, and ApoC-I adsorb preferentially to more hydrophilic
NH2-modified quantum dots [87].
3.3.3. Functional groups
The material and the functional groups on the surface of the NPs also
affect the formation of the PC. Park et al. [88] observed that FBS in cell
culture medium was preferentially adsorbed on aromatic thiol-modified
gold NPs relative to thiol-modified gold NPs with amino, carboxyl and
hydroxyl groups. Matczuk et al. [89] showed that the proteination of the
carboxyl-modified AuNP in a real serum environment occurred without
the participation of albumin, which abolished the PC under equilibrium
conditions. In contrast, AuNR with a surface amino group first formed an
albumin conjugate, but the albumin in this "soft" corona was gradually
replaced by other proteins that showed a higher affinity for the aminated
surface. The ease with which albumin can be lost depends on the surface
functional group-carboxylate or sulfonate, in particular aromatic sulfo­
nate. Delgado et al. [90] found that carboxylate permits easier loss of
albumin, which presumably allows the subsequent adsorption of
7
Biomedicine & Pharmacotherapy 139 (2021) 111541
proteins such as fibronectin, required for cell adhesion. Sulfonate holds
on to albumin more strongly, producing a persistent hard corona likely
to remain biocompatible. The mechanism is thought to be related to the
higher energy of interaction between sulfonate and amine than between
carboxylate and amine. Danner et al. [53] studied polystyrenes with
gradual increase of negative charges by modifying polyglycerol (PG),
and finally they concluded that high amount of phosphoric acid group is
not conducive to the stealth effect of nanocarriers. When the phosphate
content is low, clusterin is the dominant protein. As the phosphate
content increases, the proportion of clusterin drops to about 20%, which
seems to reach saturation. In addition, the fibrinogen and especially
immunoglobulin components were simultaneously increasing. The
introduction of phosphate not only resulted in a much higher absolute
adsorption capacity of the protein, but also a much lower specificity of
the protein. Fibrinogen and immunoglobulin were enriched with the
increase of phosphate groups; however, they are known to facilitate
uptake of phagocytic cells that remove the nanocarriers from the
bloodstream. These proteins identified may also be involved in the ag­
gregation process. Mortensen et al. [91] demonstrated that the PC
continues to change for over 48 hrs, being heavily influenced by the NP
surface chemistry. They found both native SiO2 NP and SiO2-COO- NP
had been identified with significantly higher levels of complement
pathway proteins in the PC. And serum albumin is believed to be a
dysopsonin, and both SiO2 and SiO2-NH2 NP had the highest count for
this protein in contrast to SiO2-COO- NP, which is consistent with Del­
gado’s conclusion. But it has been reported that apolipoproteins were
less frequently detected for PS-COO- NP than for the native PS NPs,
which is in contrast with their study [92].
Most functional groups designed for nanoparticles are intended to
enhance targeting and prolong blood circulation. However, in order to
better understand the interaction between the functional groups of
nanomaterials and proteins, cells and biological environment, we need
to enlarge the role of functional groups, and the proteins adsorbed and
the PC formed are also functional surfaces. Martens et al. [93] showed
that citrate and dextran are easily displaced by fibrinogen (Fb), which
upon binding to the surface stabilize the magnetic NPs from aggregation
over time. The PEG coating increased the stability of the magnetic NPs
by acting as a barrier for Fb adsorption on the surface of the NPs, and
make the Fb conjugates present a much higher binding to the integrin
containing membrane. It’s worth noting that their study provides an
evidence that the coating of NPs with Fb together with other substances
e.g., drugs might be applicable to enhance the targeted interaction with
membrane proteins. Moreover, Mirshafiee et al. [94] used γ-globulins
pre-coated SiO2 NPs to recruit opsonins and tested the ability of
phagocytes uptaking them. In the PCs, compared to the corona-UC-NPs
(carboxylic acid-functionalized silica NPs), pre-coating with HSA
increased the abundance of lipoproteins and reduced the abundance of
coagulation proteins, such as fibrinogen. Pre-coating with γ-globulins
had the opposite effect, reducing the wt% of lipoproteins and drastically
increasing the wt% of complement factors (e.g., complement C3, C4A,
C5, C1s, C8 subunits, C7, factor B, and C6) and immunoglobulins in the
corona as compared to UC-NPs. Their experiment proved that the
γ-globulins precoating promotes the recruitment of opsonins, namely
immunoglobulins and complement components, in the plasma to the PC,
but they did not enhance NP uptake by macrophages. It suggested that
other plasma proteins, such as apolipoproteins, covered the active sites
on the immunoglobulins in the PC and prevented them from binding to
receptors on immune cells.
The surface chemistry of NPs can easily be tuned using different
coating materials such as surfactants or polyelectrolytes. Polyelectrolyte
coatings can stabilize NP dispersions due to high charge densities and
can also be used for target, stealth and so on. In general, polyzwitterions
often show anti-fouling properties in terms of reduced unspecific protein
adsorption. Typical examples for polyzwitterions showing antifouling
behavior are poly(betaines) like poly(carboxybetaine acrylamide)
(pCBMA) [95]. There were exceptions, Gräfe et al. [96] detected
W. Huang et al.
Table 2
Overview of the degree of HSA conformational change induced by several gold
NPs modified with different functional group at different pH values.
Ligands PH
3.8 7.4 9.3
Change
Citrate Larger Larger Unaltered
PEG-OMe Smaller Unaltered Unaltered
PEG-NH2 Larger Larger Moderate
PEG-COOH Larger Unaltered Larger
Glycan Larger Unaltered Larger
increasing relative amounts of albumin (67 kDa) via specific Western
blot assays on polydehydroalanine-coated multicore iron oxide NPs. And
some block copolymer-stabilized soft NPs, even with fairly short hy­
drophilic chains, exhibit prominent protein repellent properties [97].
There have been many studies demonstrating that polyethylene glycol
(PEG) can reduce but not eliminate protein adsorption [16,57,98].
Specially, the better stealth property of the micelles with
alpha-glutamyl-terminated PEG shells was presumably attributed to the
zwitterionic property of the alpha-glutamyl groups [99]. The relevant
content is further explained in Section 5.1.
In conclusion, it can be found that the physical and chemical prop­
erties of nanomaterials and their surface modified functional groups or
coatings have a great influence on the formation of PCs (Table 1). Not
only that, the duration of incubation and serum or plasma concentration
also affect the composition of the PC. Most of the time, even with
different functional groups, the most abundant protein in the PC formed
for the same material during a short incubation period or at a low plasma
concentration is different in content but the same kind. The difference
mainly exists in some low abundance proteins [100]. With the prolon­
gation of incubation time, the composition of the PC changed obviously,
and the influence of specific functional groups on the PC was more
obvious [91].
3.4. pH
NPs may absorb proteins within a fairly narrow pH depending on
their isoelectric point. The closer the pH is to the isoelectric point, the
more protein is absorbed [104,105]. Most serum proteins have an iso­
electric point between pH 6 and 7, and therefore have a net negative
charge at pH 7.4. After an ion serum protein is adsorbed, it produces a
net negative charge to the NPs [106]. pH can affect the structure of
proteins. For example, the reduction of the α-helical structure of
AuNP-BSA bioconjugates is strongly dependent on pH [107]. Similarly,
Meesaragandla et al. [108] mentioned that HSA, the most abundant
protein in blood plasma, which is known to undergo pH-dependent
conformational changes [109]. The conformational changes of second­
ary structure of HSA in AuNP-HSA conjugates formed by different li­
gands coating AuNPs and HSA in different pH solutions were studied by
circular dichromatography. Compared with the original conformation of
HSA in different pH solutions, the general changes were shown in the
table below (Table 2). The ellipticity of HSA varied with the surface
ligand type of NPs and the pH value of the medium. Whether in physi­
ological pH 7.4, or in acidic or alkaline pH, there were always some
AuNP-HSA conjugates that showed a decrease in the percentage of
α-helix and an increase in the percentage of β-sheet content. The results
showed that both electrostatic and hydrogen bonding interactions had
an influence on the protein secondary structure which was further
influenced by the pH of the medium. Moreover, pH influences PC sta­
bility (via salt bridges and hydrogen bonding) and PC protein folding
[110]. Del Cano et al. [111] focused on the behavior of the bio­
conjugates made of the protein hemoglobin (Hb) and AuNP capped with
three different molecular layers (citrate anions (c), 6-mercaptopurine
(MP) and ω-mercaptoundecanoic acid (MUA)) in aqueous buffered
8
Biomedicine & Pharmacotherapy 139 (2021) 111541
solutions in a wide pH range. It had been found that these bioconjugates
were stable in neutral and alkaline solutions and, at pH lower than the
protein isoelectric point, aggregation took place.
3.5. Biological environment
The PC is formed in a biological fluid medium, so the biological fluid
environment also affects the formation and composition of the PC. The
formation of the PC has been observed in pure protein solution, cell
culture medium, full serum and also human body fluids [58,112–115].
The cell culture media DMEM and RPMI 1640, which mimic the in vivo
biological environment, were found to differ in the formation of protein
caps on the surface of NPs, and these differences also affected the
cellular responses. DMEM is more likely to form a large number of
time-dependent, stable and abundant PC, while RPMI shows different
protein adsorption kinetics and a reduced protein coating [32]. The
protein concentration in the biological fluid also affects the formation of
the PC, which includes the stability of the NPs [116,117]. Lundqvist
et al. [114] showed the evolution of the PC as a result of the transfer of
NPs from one biological fluid (plasma) into another (cytosolic fluid), a
simple illustrative model for the uptake of NPs into cells. Their results
supported that the “hard” PC formed around NPs evolved and changed
its identity by integrating new proteins when the protein-particle com­
plex was introduced into the second biological solution, which sug­
gested that the final corona contained a “fingerprint” of its history.
Therefore the corona composition could potentially depend not only on
the current environment of the nanoparticle, but also on all environ­
ments it has moved through [59].
In summary, the formation of PC has the following characteristics:
formation time, NP concentration, nanoparticle size, surface charge of
NPs, hydrophilic/hydrophobic properties, and biological fluid
environment.
4. Effect of PC on the biological function of NPs
It can be seen from the above that the formed PC is significantly
influenced by the physical and chemical properties of the NP in the
biological fluid such as particle size, morphology, surface charge, sta­
bility and biocompatibility. Simultaneously, the corona affects the bio­
logical effects of the NP and determines the result of the interaction
between NPs and cells. The adsorbed proteins, rather than the NP itself,
determine the cellular receptors used for binding, the internalization
mechanism, the intracellular transport pathway and the subsequent
immune response [16].
4.1. Effect on cells uptake and toxicity
It is well known that a series of reactive oxygen species (ROS) are
involved in aerobic cells during metabolism. Medium and high con­
centrations of ROS induce apoptosis and even necrosis through cellular
oxidative stress. Choi et al. [118] showed that the interaction of a
positively charged branched polyethylenimine-capped gold nano­
particle (BPEI-AuNP) with hepatocytes adversely affected cell uptake
and other cellular responses, but the presence of a PC played a protective
role. All naked BPEI-AuNP (40 nm and 80 nm) caused an increase in
oxidative stress in hepatocytes over time by sustained ROS/reactive
nitrogen species (RNS) production, but human plasma (HP) protein
significantly reduced ROS, RNS and SO levels in a size-independent
manner. Additionally, the naked BPEI-AuNP showed the highest
cellular uptake in human cells, but HSA and HP corona reduced uptake.
At cytotoxic levels, naked BPEI-AuNP induces antioxidant and
pro-apoptotic genes, but HP reduces other antioxidant genes. The results
showed that both HSA and HP can reduce the toxicity of BPEI-AuNP,
although the toxicological characteristics caused by HP are more
complicated. Li and Monteiro-Riviere [119] studied the uptake of
different sizes (40 nm and 80 nm) of gold nanorods by human epidermal
W. Huang et al.
keratinocytes with lipoic acid, PEG and BPEI surface modifications, and
found that PC reduced uptake. Moreover, in addition to 40-nm lipoic
acid-AuNP, AuNP uptake is energy dependent. Wang et al. [120]
demonstrated that when gold nanorod particles entered a
serum-containing medium or in vivo environment, a stable serum PC
could effectively prevent direct contact between the gold nanorod and
the cell membrane, which reduced the damage to the cell membrane,
thereby effectively reducing the toxicity of the gold nanorods. When
CTAB/AuNRs (cetyltrimethylammonium bromide-capped gold nano­
rods) penetrate into the cell membrane, the plasma membrane structure
becomes thinner then subsequently is destroyed, followed by an increase
in the number of bubbles and loss of microvilli. For FBS/AuNRs, the
membrane structure remains intact, and some gold nanorods can be
found around the plasma membrane, while the gold nanorods are
mainly located in the bubble-like lysosome.
4.2. Effect on drug release of drug-loaded NPs
The drug release profiles of nanocarriers depend on their interaction
with the PC, not only related to the type and amount of the adsorbed
proteins, but also to the size and type of the nanocarriers. It has been
reported that the PC forms a protein buffer layer on the surface of the
NPs in vivo, which shields and blocks drug release [121]. The PC
significantly affects the nanocarrier drug release profile, which is also
associated with the surrounding medium. Al-Ahmady et al. [122]
demonstrated that temperature-sensitive liposomes coated with a
plasma PC release doxorubicin slowly but not completely; but liposomes
recovered rapidly release doxorubicin without plasma protein media­
tors. This suggests that the plasma protein medium around the NP-PC
can adsorb some released drugs.
Fig. 3. Effects of PC on immune system and immunotoxicity. When NPs enter the biological system, they are quickly surrounded by various proteins, some of which
help them to be recognized by phagocytes, which belong to innate immunity. They are then cleared primarily by phagocytic cells in the lungs, liver, and spleen,
producing immunotoxicity.
9
Biomedicine & Pharmacotherapy 139 (2021) 111541
4.3. Effect on nanoparticle targeting
The surface of the NPs can modify various active moieties and
functional groups to selectively deliver the drug to a targeted site in the
specific cells and tissues. Several targeted functional biomolecules have
been reported, which include transferrin, insulin, folic acid [123],
mannose [124], apo A-1 and apo E. PC on the surface of NPs can reduce
the targeting of NPs. For example, the targeting ability of transfer pro­
teins was impaired when exposed to a biological environment (FBS),
owing to the shielding of plasma proteins in transferrin-conjugated silica
NPs (SiO2-PEG8-Tf), which reduced the specificity for transferrin re­
ceptors [4]. However, even if the surface of the NPs is coated with a
protein layer, it cannot completely cover the surface-modified func­
tional groups or targeting molecules [17]. Su et al. constructed twenty
types of cRGD-functionalized PEGylation Au-NPs by a combinatorial
strategy [125]. The surface chemistry of these NPs was diverse, which
included four different PEG lengths and five different targeting ligand
densities. When exposed to a serum-rich medium, a PC was formed on
these NPs. By incubation with targeted cells in a complete or serum-free
medium, the targeting abilities of cRGD were maintained even with a
density of 25%. However, the active targeting efficiency of
protein-bound NPs was remarkably reduced compared with the
non-protein bound NPs. When macrophages were incubated in complete
or serum-free medium, the PC provided a stealth effect on macrophage
cell recognition, which is beneficial for the passive targeting caused by
the EPR effect. Moreover, NPs conjugated with short PEG molecules and
moderate targeting ligand density might take on a lower macrophage
cellular uptake and a higher targeted cell internalization, thus exhibiting
a higher targeting efficiency. These surface chemistry results provide
guidelines for designing targeted NPs with improved targeting
efficiency.
W. Huang et al.
4.4. Effect on immune system and immunotoxicity
NPs as foreign substances entering a living body can induce complex
immune responses, including immunosuppression and immune activa­
tion [117]. These responses primarily begin in the innate immune sys­
tem. PC plays a critical role in making the particles easily recognized by
the innate immune system, especially by phagocytes, causing their quick
clearance by phagocytic cells located in organs such as the lungs, liver,
and spleen [126] (Fig. 3). This defines the immunogenicity and immu­
notoxicity of NPs. By using quantitative imaging based on laser ablation
inductively-coupled plasma mass spectrometry (LA-ICP-MS), Elci et al.
[127] found that neutral AuNPs are more likely to interact with the
immune system, as evidenced by their greater relative accumulation in
the marginal zone and white pulp regions of the spleen and with the
Kupffer cells of the liver. Positively-charged NPs accumulated exten­
sively in the glomeruli, liver hepatocytes, and red pulp of the spleen. The
negatively charged NPs accumulated extensively in the red pulp of the
spleen but had a broader distribution in the liver.
The complement system is a critical fluid component of innate im­
munity and is the first line of defense against foreign invaders including
nanomaterials, thus the complement-induced the immune response is
the most common reaction during defense immunity. Saha et al. showed
that macrophages uptake NPs by recognizing specific complement pro­
teins in the PC, and the higher the content of complement component
C4BPA, the greater the uptake of macrophages [128]. The complement
pathway activation is translated into an exacerbate inflammation
response [129]. It has been demonstrated that different types of gold
nanomaterials (AuNMs), such as spherical gold NPs (AuNP), gold
nanostars (AuNS) and gold nanorods (AuNR), can simultaneously acti­
vate the complement system through all three complement pathways
[130]. In the initial phase, non-specific adsorption of complement pro­
teins on the surface of NPs can determine the subsequent immune
cascade and can stimulate or attenuate the immune response, which
activates the three complement pathways downstream by recognition
and binding of the complement protein. Dai et al. [131] observed the
differences in proinflammatory cytokine secretion and immune cell
apoptosis when NPs interacted with immune cells in various biological
environments, where the PC formed determined whether the NPs
Fig. 4. NPs versus PC. NPs attempt to break through the barrier of the PC in different forms and have some potentially beneficial results.
10
Biomedicine & Pharmacotherapy 139 (2021) 111541
increased or decreased the secretion of specific cytokines.
The amount and identity of proteins adsorbed on the surface of the
different materials were strongly influenced by surface chemistry and
wettability, which led to a distinct immune response from macrophages
[132]. It was investigated how the surface chemistry-induced specific PC
affects the phagocytosis and immune responses of macrophages exposed
to the corona-nanoparticle complexes [133]. The PC alters the inter­
nalization pathways of gold nanorods by macrophages via the in­
teractions of the predominant coronal proteins with specific receptors
on the cell membrane. Serum proteins perturb the recognition of AuNPs
by scavenger receptors while promote the recognition by clathrin re­
ceptors in phagocytes. Therefore, clathrin-mediated endocytosis path­
ways dominated the internalization by phagocytic cells cultured in
serum-containing medium [134]. The cytokine secretion profile of
macrophages is also highly dependent on the adsorption pattern of the
PC. Therefore, proper design of NPs and control of PC components may
help to design drug carriers with prolonged circulation, enhanced
biocompatibility and reduced toxicity [135].
5. Opportunities and challenges: NP-PC as a novel nano-drug
carrier for targeted applications
In previous studies, some scientists have been aware of the existence
of the PC, and some targeted designs have been made for the hindrance
and influence of the PC (Fig. 4). These designs could reduce the surface
adsorption of proteins by NP particles, either to expose targeted mole­
cules to targeted cells and tissues, or to reduce some unnecessary bio­
logical reactions, such as complement reactions, or to reduce the
recognition and phagocytosis by phagocytes. These designs are effective
and play a significant role in providing enlightenment and guidance for
the subsequent application of NPs.
Even so, we should know that the adsorption of the PC is inevitable.
In recent years, researchers have identified that a PC can redefine and
improve the targeting of NPs known as "targeted drug corona effects",
which allows a nanoparticle-protein complex to target specific cell types
in a controlled manner [136,137]. It is more noteworthy that PC has
significant advantages in drug delivery due to their huge surface area
and excellent biocompatibility.
W. Huang et al.
5.1. Challenge to design protein-free corona
PC is often the prime reason for loss of NP stability, quick clearance,
and potentially harmful immunologic reactions [138], which has been
easily considered a “fluid biological barrier” blocking the delivery of NPs
to the tumor’s target site [126]. To design protein-free NPs has long been
a challenge for researchers. Although protein adsorption is inevitable,
the reduction of protein adsorption will maximize the surface chemical
biological effects of NPs. PEG modification is a classic method for
nanoparticle anti-biomolecule adsorption [139,140]. The PC of PEGy­
lated nanocarriers is enriched with certain so-called dysopsonins that
are responsible for the reduced uptake in immune cells [53,141,142]. In
some cases, PEG coating has a shielding effect, which possibly eliminates
the toxicity and immunogenicity of NPs in the body [93]. It has been
reported in the literature that gold NPs can resist the adsorption of
proteins by reducing the affinity for serum proteins through glutathione
coating [143] or functionalizing with lysine and cysteine [144]. Deco­
rating a branched or cyclic end group on the NP surface typically can
also adsorb less protein [128]. Moyano et al. [145] designed a series of
zwitterionic NPs with variable hydrophobicity. The zwitterionic func­
tional group can replace the PEG effect, which produces strong elec­
trostatic binding with water, and makes the zwitterionic system stable
and has the effect of a corona-less adsorption. These NPs do not adsorb
proteins on moderate levels of serum proteins and do not form protein
hard corona at physiological serum concentrations. Klepac et al. [146]
used EPR, ITC, and DLS methods to demonstrate that a hard corona is
not formed around the NP of HPMA amphiphilic polymer conjugates.
HPMA copolymers provide perfect "invisible" properties to NPs, which
prevent them from interacting with human blood plasma proteins and,
thus, keeps their functionality unchanged. The authors suggested that all
previous publications reporting a "hard corona-soft corona" should now
re-evaluate the types of NPs that had been used to study protein
adsorption. Knittel et al. [147] showed that ultrasmall AuNPs encom­
passing a zwitterionic glutathione monoethyl ester surface coating
(AuGSH(zwt)) were highly resistant to aggregation and serum protein
interactions. And the strep-tagged AuGSH(zwt) particles were not only
highly resistant to nonspecific protein interactions but also retained
their targeting capability in biological fluid, displaying efficient binding
to streptactin receptors in nearly undiluted serum. Moreover, it was
found that both zwitterion attachment and PEGylation could reduce PC
formation on AuNPs, and zwitterion attachment significantly prevented
PC formation, even more effectively than PEGylation [148]. In fact,
zwitterions or some specially coated nanomaterials have proposed new
strategies for the modification and application of NPs in biological
fluids, which deserves more attention. Reducing complement activation
by nanomaterials can also reduce protein adsorption, and recently some
scientists use membrane coatings to camouflage NPs to avoid immune
clearance, thus reducing complement activation by NPs [149,150].
On the one hand, the reduction of protein adsorption is to avoid the
function designed by NPs being concealed; on the other hand, it is also
conducive to the reduction of phagocytic cell recognition and clearance.
To design protein-free NPs is an important strategy for NPs to overcome
the clearance of the reticuloendothelial system (RES) or mononuclear
phagocytic system (MPS) [151,152]. Some scientists have proposed a
method of temporary blocking MPS to reduce consumption of phago­
cytes to NPs, such as modification of NPs with PEG [153]; grafting NPs
with polyglycerol [154]; injecting specifically modified liposomes to
enclose the cell membranes of phagocytes and thus reducing in­
teractions between phagocytes and subsequently injected NPs [155]; or
using antibody-mediated red blood cells to promote the absorption of
phagocytes and make the immune system saturated firstly and then
injecting NPs [156–158], etc. These measures could improve the
bioavailability of nano particles in the blood, extend biologic half-life of
the drug, and lower toxicity, is an effective strategy to improve the nano
drug delivery to target site.
11
Biomedicine & Pharmacotherapy 139 (2021) 111541
5.2. Protein-cell receptor-mediated targeting
Casals et al. [159] demonstrated the main presence of albumin (BSA)
on NPs using mass spectrometry. When NPs were mixed with a
BSA-specific antibody, the particle size increased, and this effect was not
obtained by the action of the control antibody IgG. This indicated that
the protein on the surface of the PC maintained a natural epitope. This
finding also suggested that proteins on the surface of the PC can serve as
specific design targets. The study by Barran-Berdon et al. [160]
demonstrated that the positively charged lipid nanocarrier
DC-Chol-DOPE (the cationic lipid 3β-[N-(N’,N’-dimethylaminoetha­
ne)-carbamoyl] combined with the zwitterionic lipid dio­
leoylphosphatidylethanolamine) was coated with proteins in HP for
1–60 min to form a PC, which was mainly enriched in apolipoprotein
(Apo AI, Apo C-II, Apo D and Apo E). It was demonstrated that
DC-Chol-DOPE/HP complex enters PC3 cells through receptor-mediated
endocytosis. With only weak endocytosis, even PC can inhibit endocy­
tosis mediated by acupoint-like invagination when NPs enter cell
membranes [119]. Fleischer et al. [16] used a set of polystyrene NPs to
adsorb protein of bovine serum albumin, which resulted in the binding
of two different cell surface receptors: the native albumin receptor and
the scavenger receptor. The BSA-NP complex formed by anionic NP
bound to the native albumin receptor, whereas the BSA-NP complex
formed by the cationic NPs bound to the scavenger receptor. It was also
demonstrated that the secondary structure of the adsorbed bovine serum
albumin protein controlled the cellular receptors used by the protein-NP
complex. Therefore, PC-cell receptor-mediated cell targeting should
monitor the structure of proteins, especially secondary structures.
5.3. NP-PC as a novel nano-drug delivery system
Serum proteins are multifaceted and can be used not only to stabilize
NPs, but also to make hydrophobic drugs or charged molecules soluble
[161–163]. Kah et al. [164] reported that the PC around the NPs has a
high load capacity like a sponge. It was also found that the ability of the
PC on the surface of cetyltrimethylammonium bromide-capped gold
nanorods (CTAB-GNRs) to load small molecules is 5–10 times greater
than covalent binding, either by loading a negatively charged DNA
oligonucleotide or a positively charged doxorubicin (DOX). The optical
properties of GNR resonance in near-infrared light can remotely control
and assist in the release of the PC payload. Ling Yeo et al. [58] used a
complex of mouse serum and gold nanorods to load and trigger the
release of the photosensitizer Chlorin e6 (Ce6), which was used to treat
NCr tumor mice by photodynamic therapy (PDT) and photothermal
therapy (PTT). The results showed that the temperature of the tumor
increased by 16.85 ◦ C under the irradiation of a local laser for 20 min,
and the tumor regression was completed within 19 days, which indi­
cated that the PC could successfully load and transport the drug for in
vivo treatment.
6. Conclusions and outlook
Nanoparticle-protein complexes are what biosystems ’see’ and
’respond to’ [112,165], which determines the fate of NPs in biosystems
rather than the bare NPs [59]. This raises new questions for the appli­
cation of NPs from ideal states to complex biological fluid environments
in vitro and in vivo: is a PC good or bad for NP applications? How can
one treat and fully use a NP-PC? The way in which NPs interact with
biological fluids greatly affects the properties and physiological func­
tions of the NPs. In the past, the successful application and failure
experience of nanomaterials in the biomedical field requires a
rethinking of the impact of the biological environment, and the perfor­
mance of the NPs needs to be re-evaluated. PC formed on the surface of
different nanomaterials are specific and cannot be generalized. There
are many kinds of proteins in the body of organisms, but they are limited
after all. The commonness and specificity of PC formed by different NPs
W. Huang et al.
remain to be studied. In the future, scientists should establish a
nanoparticle-PC database to better serve the research on the application
of NPs in biological systems.
There have been several reviews to summarize PC-NP complexes
from different aspects, including methods, composition, interactions, etc
[54,55,166,167]. This review redescribes the relevant content and
highlights the influence of PCs on the immune system and phagocytes. In
particular, the strategies for NPs to break through the barrier of PC and
the targeting application of PC-NP complexes as drug carriers are pro­
spected. The PC is a double-edged sword, seen as a biological barrier for
NPs, and could also play a key role in drug delivery. Weighing the pros
and cons and designing experiments properly is the key. When NPs are
designed as a drug delivery system, they should be viewed not only from
a chemical and engineering standpoint, but also from a biological
standpoint. The whole protein-covered nanoparticle system should be
viewed and studied as a biological delivery system, which requires us to
more accurately simulate the biological microenvironment. The com­
bination of data from biological experiments and computer analysis to
construct experimental frameworks will be one of the ways to build safe
and efficient nano-drug delivery systems in the future [168,169].
The development of new technologies and new instruments will
gradually unveil the mechanism of the interaction between PC and NPs.
Understanding the principles and influencing factors of the interaction
between PC and NPs facilitates the design of targeted nanomaterials,
which will make the PC controllable and functionalized, to construct
new drug delivery systems. These systems would be beneficial to
improve the biotoxicity and immunotoxicity, improve the targeting and
effectiveness, prolong the action time in vivo and improve the clearance
rate, so as to realize the accurate and safe use of nanomedicine.
Declaration of conflicting interests
The authors declare no conflict of interest.
Acknowledgments
The current study was supported by grants from the National Natural
Science Foundation of China (Grant Nos. 81803064, 81673009).
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