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How well can we characterize human serum

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Cite this: DOI: 10.1039/c9an01920k

transformations of magnetic nanoparticles?
Andrei R. Timerbaev

Received 26th September 2019, This mini-review summarizes analytical methods in use to uncover biochemical transformations that
Accepted 5th December 2019
magnetic nanoparticles (MNPs) are possibly undergoing while residing in human blood. Examples from
DOI: 10.1039/c9an01920k the recent literature are presented to illustrate what analytical challenges are to be addressed to shed light
[Link]/analyst on this important issue of biomedical application of MNPs.

Introduction lying biochemistry. It seems that such a situation has come

around because of the complexity of the biological matrix in
Among many classes of nanoparticles being researched for question and the associated challenges in the measurement of
biomedical applications, magnetic nanoparticles (MNPs) are MNPs in the presence of large amounts of biomolecules.9
perhaps one of the most advanced nanomaterials. This is due Traditional techniques commonly used for nanoparticle ana-
to their proven application as contrast agents for diagnostic lysis, such as dynamic light scattering and transmission elec-
purposes,1–3 the area that in combination with the therapeutic tron or confocal microscopy, have certain limitations in the
and drug-delivery potential of MNPs1,2,4,5 provides a wealth of assessment of blood serum-induced changes of MNPs.
medicinal opportunities. Therefore, there is a strong need for advanced ex vivo tools
The key developments of MNPs for use in nanomedicine capable of separating, detecting and possibly distinguishing
have been a focus of a large body of literature. Numerous the nanospecies, originating at the time of the bloodstream
recent reviews have described advances in the synthesis, transportation of MNPs. The main goal of this review is to pin-
surface modification, assembly, and different theranostic point such analytical approaches emerging to fill up the exist-
applications of MNPs, including imaging, drug delivery, target- ing gaps in the characterization and mode-of-action studies of
ing and cancer therapy (see ref. 3 and 6–8 to mention a few). MNPs designed for biomedical applications.
However, in author’s opinion, there is one important topic
related to nanoparticle processing in the body which received
less attention than it might deserve. It is commonly accepted Key milestones of studying nano–bio
that upon intravenous injection, the MNPs encounter the phys-
interactions in blood serum
iological environment rich in various biomolecules (mainly
proteins) that tend to adsorb onto the nanomaterial surface Prior to discussing the merits and current (or intrinsic) short-
and in this way induce a conversion from the original, syn- comings of individual or combined analytical techniques, it is
thetic (or assembled) identity to a new, biological one. The important to figure out what information is requested and
latter defines the biological response and hence dictates the supposed to be acquired from their application.
behavior and performance of MNPs in vivo. (a) Stability. Yet before becoming enveloped by proteins,
Despite a clear importance of comprehending the trans- MNPs face a serum matrix environment that can compromise
formations and properties of MNPs in the extracellular their colloidal stability and induce the formation of large
environment, there are only a handful of published reports aggregates10 or initiate a release of ionic metal forms.11 As one
dealing with real human blood (or serum) which will be high- of the key determinants for successful application of MNPs,
lighted in the present review. Most of the current research on stability is to be verified using phosphate-buffered saline (PBS)
the characterization of these materials is limited to probing or more reliably undiluted human serum.12 It should be noted
interactions with individual proteins (usually BSA) and thereby that in situ protein coating improves nanoparticle stability.10
cannot provide a fundamental understanding of the under- (b) Kinetics of transformation. Although in many contri-
butions it is stated that MNPs are immediately covered by
serum proteins, the formation of the protein corona is by far
Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, not a simple event. First, surface coating is a collective and
Moscow, 119991, Russian Federation. E-mail: [Link]@[Link] dynamic process whereby the proteins are adsorbed in a com-

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petitive manner. One of the possible binding scenarios is that worth trying. The knowledge of what conjugates serve as the
right upon intravenous injection the corona will be dominated form in which the MNPs reach the desired cells would help
by proteins (or other biomolecules; see below) whose attraction guide an improved design of next generation MNPs. Evidently,
on the nanoparticle surface is kinetically more favorable. the analytical task falls here into the realm of proteomic ana-
Then, over the time of blood exposure, high affinity but lysis presently dominated by mass spectrometry (MS)-based
(usually) lower abundance proteins are replaced by higher technologies.
abundance proteins (for MNPs, this is often albumin13,14). As a final remark of this section, each of the key para-
Second, some experimental evidence has been reported11 meters, characterizing the behavior of MNPs in human blood,
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which reveals that as a temporal situation after injection, other is overly dependent on their properties including the core/
serum biomolecules, e.g. sulfur-containing amino acids, may shell chemistry, shape and size, functionalization, type of mag-
participate in specific binding to MNPs thus initially compet- netism, etc. However, it is beyond the scope of this work to
ing with the proteins. analyze these manifold effects. Also being outside the focus of
It stands to reason that to gain knowledge on how fast and consideration are non-analytical methods, measuring magneti-
complete the conversion of investigational MNPs into a zation, magnetic relaxation, zeta potential, size deviation, etc.
protein-conjugated form takes place, a method of choice is to upon producing a protein corona around MNPs, and mole-
be able to resolve different nano–bio species, evolved upon the cular biology techniques like dot-blot analysis.
residence in human serum, and the intact particles, without
alterations in the separation (or isolation) system. It is worth-
while noting that there is no need to separate MNPs of
Gel electrophoresis
different sizes (see a recent summary of the state-of-the-art
techniques15) since only monodisperse particles are supposed Of the separation-based methods capable of probing protein-
to be administered. When monitoring the transformation of binding interactions, gel electrophoresis (GE) is perhaps the
MNPs, the time frame is of special consideration. It should be simplest approach that can provide initial information on the
complementary to (but not exceeding) the circulation time in composition of MNP–protein coronas.20 Typically, a mixture of
the blood, which varies – in terms of the blood half-life – from MNPs with a biofluid is subjected to heat treatment followed
several minutes (for example, the case of clinically approved by the separation of the denatured proteins by molecular
MNPs16,17) to a few hours, depending particularly on the weight using SDS-PAGE under reducing conditions. The advan-
surface modification,18 but rarely longer. tage of these types of nanoparticles can be taken to separate
(c) Composition of the protein corona. As a matter of fact, them from the excess of biomatrix proteins, prior to the GE
that is rather a loose concept as in a dynamic milieu of circu- analysis, by magnetic separation.21–25 Fig. 1 shows the setup
lating blood, the corona does not reach an equilibrium state.19 and the separation procedure, using a magnetic separator, for
After being continuously changed in composition due to con- removing first the excess (unbound) proteins and then the con-
vection and cellular metabolism, the blood surrounding nano- jugated MNPs. Alternatively, the protein conjugates may be iso-
particles constantly affects the protein corona composition. lated by a standard centrifugation method10,26 (see also a
Nevertheless, any attempt at identification of proteins prevail- study by Monopoli et al.27 on comparison of different isolation
ing in the corona after a certain time, prior to endocytosis, is methods tested with MNPs).

Fig. 1 Schematic representation of magnetic separation including the trapping, protein elution and protein–MNP conjugate elution. Reprinted with
permission from ref. 22. Copyright (2014) Springer Nature.

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In one of the few reports available on using GE, protein below).22,25,29 Similarly, this method was included as a separ-
binding dynamics of the iron oxide NPs of different sizes was ation tool in a western blot assay used to investigate the
investigated in human plasma,21 and the results are summar- binding of specific (complement) proteins to the serum-
ized in Fig. 2. It is evident that the protein adsorption protein corona around magnetic nanoworms and in vivo
increased upon increasing the incubation time, with a satur- dynamic protein exchange.24
ation state being reached after 1 h, and both the protein com- It is quite evident that the GE method, application of which
positions and abundances in the coronas are critically affected for MNPs still lacks two-dimensional mode and using MS-
by the particle size. Alterations in the corona composition and based peptide mass fingerprinting, bears only a supporting
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the amount of surface proteins as a function of exposure time character. Indeed, when applied alone GE could not achieve
were similarly recorded for poly(tert-butoxycarbonyl acrylic deep insight into the modulation of the MNP surface by
acid)-coated MNPs incubated in fetal bovine serum (FBS).23 human blood serum, let alone the exact identification of
However, as only common visualization by silver staining was nano–bio species evolved. The response may arrive from
employed, a global overview on the protein distribution advanced proteomic approaches, one of which based on
between three fractions (<30, 30–100, and >100 kDa) was LC-MS/MS has received some coverage in the area and is con-
merely feasible. For the same reason, no identification of sidered in the next section.
protein-corona composition was attained in other
contributions,10,28 with only different binding patterns being
revealed for particles with unlike coatings.10 Also to be men- Liquid chromatography-tandem mass
tioned are non-physiological conditions used by some authors spectrometry
to produce protein-conjugated particles, such as incubation at
temperatures other than 37 °C (ref. 23) or at varied serum con- This powerful coupling is a pillar technique for proteomics
centrations28 (both never occur in vivo). research and as such presents the most promising method-
As a sample preparation method, SDS-PAGE can be utilized ology for the identification of the protein corona of MNPs
to separate the MNP-binding proteins, isolated from MNPs, for formed in the blood environment. This can be done at the
the following in-gel digestion and LC–tandem MS analysis (see polypeptide level after trypsin digestion of MNP-binding pro-

Fig. 2 SDS-PAGE analysis showing the protein binding profiles of differently sized MNPs incubated in human plasma for different periods of time
(A–C) and the total optical intensities of gel lines (D). Reprinted with permission from ref. 21. Copyright (2014) Elsevier.

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teins, chromatographic separation (with a reversed-phase gra- Chen et al. performed the LC-quadrupole TOF-MS analysis
dient nanosystem) and the precise molecular mass determi- of serum and plasma proteins, bound to dextran-coated mag-
nation by MS, most commonly used in a tandem (MS/MS or netic nanoworms, after their isolation by the elution from par-
MS2) configuration and in combination with bioinformatics ticles with 2% SDS in PBS.24 Interestingly, the binding
computational tools to achieve higher degrees of identifi- outcome was quite different in different biofluids. While in
cation. Yet before such bottom-up profiling, the proteins, par- serum, albumin, immunoglobulins, apo-lipoproteins and C3
ticipating in the formation of the corona, should be isolated were among the most abundant proteins, fibrinogen was
from both MNPs and the biological milieu. highly enriched on the particle surface in plasma. Quantitative
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For this purpose, Bonvin et al. developed an original proteomic analysis showed about 1.6 times more protein
method for isolating human blood and lymph serum proteins bound per particle in plasma than in serum. In the same way,
interacting with uncoated iron oxide NPs, which comprises a Hu and co-workers determined the relative abundances of pro-
multi-step centrifugation procedure.29 This allowed a total of teins attached to the surfaces of iron oxide NPs varying in size,
over 200 proteins to be detected within the corona by LC-MS2 following incubation with human plasma for 1 h.21 The LC-
(Orbitrap technology). However, that study revealed several triple TOF-MS approach in use revealed the selective binding
complications in the application of the developed method- of proteins to the different sizes of MNPs. For instance, HSA
ology. There were certain deviations in the protein compo- was found as the second most abundant protein in the protein
sition between parallel measurements, notable even for the corona around 200 nm MNPs, while it was only the 12th and
20 most-abundant proteins, and these deviations became not among the 20 most abundant proteins for 400 and 30 nm
much more pronounced when an alternative magnetic iso- particles, respectively. As a case study where LC-MS2 and GE
lation method was applied for comparative reasons. Last but techniques were used in conjunction, this work deserves
not least, MNPs were incubated in human serum for 24 h special mentioning. Also, this is conceivably one of the few
which makes the identification results hardly relevant when reports where the authors tried to give an explanation how the
one is asked in what biological form MNPs enter the cell. corona composition is related to a cellular, metabolic,
Ashby and co-workers were able to differentiate the corona pro- immune system and other biological processes (shown in
teins with different binding kinetics toward MNPs, differing in Fig. 3), in which specifically bound proteins can participate.
surface ligand hydrophobicity and particle diameter, by apply- As expected, different surface coatings on MNPs affect the
ing flow field-flow fractionation.26 Due to nonequilibrium sep- composition of the corona of human plasma proteins. In line
aration conditions, only the hard-corona components were with this anticipation, the LC-MS2 assay employed by Vogt
coeluted with the particles from the column, then digested et al.30 provided evidence of very different protein corona sig-
and identified by 2-D LC-MS2. However, human serum natures for silica- versus dextran-coated particles with the same
depleted of HSA and immunoglobulin G used by this group as iron oxide core (and a similar overall size). Regardless of the
the biological medium could compromise identification. particle synthetic identity, the protein corona isolated by mul-

Fig. 3 Biological processes related to plasma proteins identified by LC-MS2 on (A) all three sizes of MNPs and (B) only one specific MNP size (30,
200 or 300 nm). Reprinted with permission from ref. 21. Copyright (2014) Elsevier.

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tiple centrifugation and washing steps was found to be

enriched in proteins involved in definite physiological func-
tions such as blood coagulation, lipid biosynthesis, immune
defense, etc. (cf. the above-mentioned study21). Similar examin-
ation of polyvinyl alcohol- and dextran-decorated MNPs in FBS
revealed that only high-abundance proteins, including serum
albumin, existed on the particles of all types and surface
charges.22 According to a recent kinetic study,25 HSA is also
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among proteins dominating the corona of MNPs with a meso-

porous silica shell at an initial stage of its formation, both in
human serum and FBS. However, desorption of the abundant
proteins took place during corona growth due to their lower
Fig. 4 Changes in the concentrations of low and high molecular-
affinities for such types of MNPs.
weight sulfur-containing compounds (black circles and open circles,
In summary, it is not surprising that LC-MS2 regarded as respectively) in human serum exposed to MNPs for different periods of
the state-of-the-art analytical platform in proteomic studies time. The sizes of the plotted dots vary, reflecting the error range of the
found an application niche for the characterization of MNPs ICP-MS data. Reprinted with permission from ref. 11. Copyright (2019)
in human blood. However, despite the well-established separ- The Royal Chemical Society.
ation and detection conditions, there is still no customary
approach to quantitatively isolate the tightly bound protein
corona and this circumstance may disqualify the protein composition is attained. However, at low incubation times, low
identification from being unequivocal (at least until such an molecular-weight compounds seem to play the role of major
approach is developed and put in practice). For instance, binding partners.
more than hundred diverse proteins, constituting the corona, Selected spectroscopic techniques can be used to under-
are typically documented but without a stoichiometric ana- stand some aspects of MNP–protein interactions with direct
lysis it is difficult to accept that this entire array is really analysis of the interaction mixtures. For instance, formation of
“hard” to accommodate (in different amounts!) around a the protein corona around differently decorated MNPs in FBS
single particle. was evidenced by Fourier transform IR spectroscopy.10 The
diffusion of MNPs in human blood (or serum) under the
action of an inhomogeneous magnetic field was examined by
Stand-alone (non-separation) recording electron spin resonance spectra and discussed by
taking into account the corona formation phenomenon.33
methods While apparently outdated, a photometric BCA protein assay
Alternative analytical techniques can be applied to characterize was shown feasible to measure the time-resolved amount of
the interactions of MNPs with serum components following proteins retained by the MNPs after their immersion in
off-line sample pretreatment or directly, using untreated human serum.25 However, overall, these methods are con-
samples. Both groups of non-separation methods are pre- sidered only complementary and not related to advanced
sented in this section. analytical methodologies.
It appears that relatively few kinetic studies have been per-
formed on the corona composition and some of these are
extended to several days, not matching thus the characteristic Concluding remarks
timescale of endocytosis. With the aim of elucidating how fast
MNPs confront biotransformations and become converted into In the past few years, profiling the protein corona around
the protein-conjugated form, Kuznetsova et al. have developed various types of nanomaterials has become an area of intense
a versatile approach, employing high-resolution ICP-MS to research, and plasma protein adsorption on a range of
monitor the changes in element concentrations in human different MNPs has been investigated by a number of groups.
serum incubated with poly(acrylic acid)-coated iron oxide While the ways can vary, the proteomic approach, which uses
NPs.11 In contrast to routine ICP-MS-based measurements of the cutting-edge LC-MS methodology often supported by pre-
metal-based NPs in biological matrices,31,32 the core metal was vious GE, governs such investigations. A representative
not quantified but biomolecular sulfur was after applying step- number of MNPs have been portrayed in terms of the number
wise ultrafiltration to separate serum biomolecules from the and identity of corona proteins (Table 1). As also witnessed by
nanoparticulate fraction (100 kDa) and then to differentiate this overview, the assembly of proteins covering MNPs is no
high ( proteinaceous) and low molecular-weight fractions ‘Madeira cake’ but has a multicomponent structure. It is likely
(30 kDa). From the data of Fig. 4, it is evident that the content for this reason that there is no general rule that could be
of free (unbound) proteins decreases as a function of time applied to every type of MNP to predict the biological outcome
(due to gradually enveloping the MNPs), but after 1 h the of its blood proteinization. Furthermore, a general lack of cor-
corona formation reaches the state in which a nearly stationary relation between the particle properties and the nature and

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Table 1 Selected examples of the MNP–protein interaction studies

MNPs (shape and core/size Sample preparation prior to

(nm)/functionalization)a Biofluid Method(s) the measurement Binding information Ref.

Spherical iron oxide/30–400/ Human plasma SDS-PAGE, Magnetic separation, digestion Adsorption kinetics, 21
stabilized by PEG LC-MS2 (by heating or trypsinization), identification and
and desalting by SPE (in the quantification of corona
case of LC-MS2) proteins
Spherical iron oxide/coated FBS SDS-PAGE, Magnetic separation and Amount and identity of 22
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with PVA or dextran with NH2, LC-MS2 digestion by heating adsorbed proteins
OH, or COOH groups
Spherical iron oxide/10 or 25/ Human serum depleted LC-MS2 Centrifugation or flow field- Binding kinetics, protein 26
coated with PAA, amphiphilic of HSA and flow fractionation, exchange rates, and the
block copolymer or azelaic immunoglobulin G trypsinization, and desalting composition of the soft and
acid hard corona
Spherical iron oxide/6 or 44/ Human plasma LC-MS2 Centrifugation and digestion Composition of the protein 30
coated with dextran or silica by trypsinization corona
Spherical iron oxide/10/coated FBS SDS-PAGE Digestion by heating Molecular weight distribution 23
with poly(tert-butoxycarbonyl of adsorbed proteins
acrylic acid)
Spherical iron oxide/182/ FBS SDS-PAGE Digestion by heating Amount and size distribution 28
coated with PEI of corona proteins
Worm-like iron-oxide strings/ Human serum and LC-MS Elution with 2% SDS in PBS, Identification and 24
103 in length and 7 in width/ plasma centrifugation, and quantification of corona
coated with dextran trypsinization proteins
Spherical iron oxide/103/ Human serum and FBS LC-MS2 Magnetic separation, Time-resolved number of 25
mesoporous silica shell SDS-PAGE, and trypsinization adsorbed proteins, corona
identification, and
adsorption/desorption
dynamics
Spherical iron oxide/17/ Human blood and LC-MS2 Magnetic separation, digestion Composition of the protein 29
uncoated lymph serum by heating, and SDS-PAGE corona
Spherical iron oxide/coated FBS SDS-PAGE Centrifugation and digestion Protein binding patterns 10
with PMAA or citric acid by heating
Spherical iron oxide/6/coated Human serum ICP-MS Ultrafiltration Binding kinetics 11
with PAA
a
PEG = polyethylene glycol; PVA = polyvinyl alcohol; PAA = poly(acrylic acid); PEI = polyethylenimine; PMAA = poly(methacrylic acid).

composition of the protein corona as of today makes in-depth Acknowledgements

characterization of each sort of MNP in question necessary.
This complicates the preclinical development of MNPs, This work is a contribution to research projects supported by
turning it to be laborious and expensive (given the number of the Russian Foundation of Basic Research (no. 19-03-00053)
MNPs currently tested). and the National Science Centre, Poland (no. 2018/29/B/ST4/
In our opinion, at the stage of extracellular assessment, i.e. 00178).
prior to internalization, more focus should be placed on a
high-throughput screening of investigational MNPs by measur-
ing their protein (or more generally biomolecular) kinetics and Notes and references
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