1
Effect of Surface Chemistry and Associated Protein
Corona on the Long-Term Biodegradation of Iron
Oxide Nanoparticles In Vivo
Grazyna Stepien,
1,
‡ María Moros,
1,2,
‡
,*
Marta Pérez-Hernández,
1,3
Marta Monge,
4,5
Lucía
Gutiérrez,
1
Raluca M. Fratila,
6
Marcelo de las Heras,
7
Sebastián Menao Guillén,
8
Juan José
Puente Lanzarote,
8
Conxita Solans,
4
Julián Pardo,
1,3,9
and Jesús Martínez de la Fuente.
6,10,*
1-Institute of Nanoscience of Aragon (INA), University of Zaragoza, 50018 Zaragoza, Spain
2- Institute of Applied Sciences and Intelligent Systems-CNR, Via Campi Flegrei, 34, 80078,
Pozzuoli, Italy
3- Aragón Health Research Institute (IIS Aragón), Biomedical Research Centre of Aragón
(CIBA), 50009 Zaragoza, Spain
4- Institute of Advanced Chemistry of Catalonia (IQAC-CSIC) and CIBER in Bioengineering,
Biomaterials and Nanomedicine (CIBER-BBN), Jordi Girona 18-26, Barcelona 08034
5- Department of Pharmacy and Pharmaceutical Technology and Physical Chemistry, University
of Barcelona, Av/Joan XXIII s/n, 08028 Barcelona, Spain
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6- Aragon Materials Science Institute (ICMA), CSIC-University of Zaragoza and CIBER-BBN,
C/Pedro Cerbuna 12, 50009 Zaragoza, Spain
7-Department of Animal Pathology, Veterinary Faculty, University of Zaragoza, 50009
Zaragoza, Spain
8-Department of Clinical Biochemistry. H.C.U. Lozano Blesa, Zaragoza 50009, Spain.
9- ARAID foundation, 50018 Zaragoza, Spain
10-Institute of NanoBiomedicine and Engineering, Shanghai Jiao Tong University, Dongchuan
Road 800, 200240 Shanghai, PR China
ABSTRACT: Protein corona formed on the surface of nanoparticle in biological medium
determines its behavior in vivo. Here, iron oxide nanoparticles containing the same core and
shell, but bearing two different surface coatings, either glucose or poly(ethylene glycol), were
evaluated. The nanoparticles protein adsorption, in vitro degradation, and in vivo biodistribution
and biotransformation over four months, were investigated. Although both types of nanoparticles
bound similar amount of proteins in vitro, the differences in the protein corona composition
correlated to the nanoparticles biodistribution in vivo. Interestingly, in vitro degradation studies
demonstrated faster degradation for nanoparticles functionalized with glucose, whereas the in
vivo results were opposite with accelerated biodegradation and clearance of the nanoparticles
functionalized with poly(ethylene glycol). Therefore, the variation in the degradation rate
observed in vivo could be related not only to the molecules attached to the surface, but also with
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the associated protein corona, as the key role of the adsorbed proteins on the magnetic core
degradation has been demonstrated in vitro.
KEYWORDS: iron oxide nanoparticles, protein corona, biodistribution, nanoparticles
degradation, in vivo
INTRODUCTION
Up to day, iron oxide nanoparticles (IONPs) are one of the most studied nanomaterials for
biomedical applications. The considerable interest in IONPs results from the combination of
their unique magnetic properties, low toxicity and biodegradability.
1
Therefore, they present a
great potential for prominent nanomedicine applications such as external manipulation using
magnets, multimodal imaging, triggered drug delivery or hyperthermia induced tumor ablation.
2,3
However, satisfactory employment of nanoparticles (NPs) in any of those applications has not
been achieved yet mainly due to one hindrance, that is, controlling the behavior of NPs in vivo.
Once a NP is administered in vivo, it interacts with the components of the physiological
environment, specially with proteins, resulting in the formation of so-called protein corona
(PC).
4
PC can dramatically change the nanomaterial size, aggregation state and interfacial
properties, dominating in an uncontrolled way the biological behavior of NPs.
5
Although to date it is widely accepted that the presence of this PC would ultimately
determine the fate of the nanomaterial, its role in the biotransformation and degradation of NPs
in vivo has been scarcely investigated.
6
In vitro, it has been shown that the PC can enter the cell
attached to the NP, and later on, depending on the associated proteins, be destroyed with
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different kinetics in the lysosomes.
7
In vivo, it is known that once NPs are trapped in cells, their
biodegradation process would greatly depend on different factors such as the capping agent, the
clustering state or the recycling capacity of the organ.
8,9
Only recently, a comparative study of
two different coating shells for gold/iron heterostructures has demonstrated that poly(ethylene
glycol) (PEG)-coated NPs were more vulnerable to degradation in vitro and in vivo than those
NPs coated with oleic acid and a polymer, suggesting that this coating, and therefore the
associated PC would have a protective role in the degradation of the NPs.
8
However, the
composition of the shell was different between both types of NPs, and therefore, the role of the
diverse PCs cannot be clearly established.
Here, we provide evidence that the PC is the main responsible for the NPs fate and it is
implicated in the NPs degradation process. We report how the surface modification of identical
12 nm IONPs with either glucose or PEG affects the PC identity, the NPs biodistribution and
more importantly, the degradation over time.
RESULTS AND DISCUSSION
IONPs synthesis and functionalization
IONPs were synthesized following a previously reported seed-mediated growth method based on
the thermal decomposition of Fe(III) acetylacetonate,
10
as this method renders NPs with well-
controlled size and high crystallinity. In this case, 12 nm NPs were selected for the in vivo
studies (Fig. S1) as this diameter will likely avoid renal clearance,
11
allowing to track their
biodistribution in vivo. The synthesis method provides NPs dispersed in an organic solvent,
therefore a subsequent transfer to aqueous solution is required. For this purpose, modification of
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a previously reported protocol was applied, where NPs were coated with an amphiphilic polymer
shell (poly(maleic anhydride-alt-1-octadecene), PMAO).
12,13,14
Using this methodology, the
hydrophobic backbone of the polymer intercalates with the oleic acid chains that covers the NPs
core, leaving carboxylic groups exposed to the solvent. In this case, to be able to track the NPs ex
vivo, they were transferred into water using the PMAO polymer previously modified with a
fluorophore, 5-carboxytetramethylrhodamine (TAMRA) (NPs@PMAO, Scheme S1).
15
To
provide stability in biological media, NPs@PMAO were functionalized with either glucose
(NP@Glc) or PEG molecules (NPs@PEG), as those molecules have been previously
demonstrated to efficiently passivate NPs surface avoiding aggregation in cell culture medium in
vitro.
16,17,18
There is a general consensus that protein adsorption and consequently biodistribution
is highly dependent on the PEG molecular weight, therefore a long PEG of 5000 Da was chosen
to passivate the surface of the NP. In fact it has been described that maximal reduction in protein
adsorption is found for a PEG of 5000 Da.
19
Other examples have also linked this PEG MW with
the protein adsorption and consequently with the macrophage uptake. For instance, magnetic NP
uptake by macrophages is higher for NPs coated with a PEG of 600 Da when compared to a PEG
of 3000 Da.
20
It should be highlighted that NPs@Glc and NPs@PEG presented the same core size (Fig. S2a),
shell structure and stability (in water, phosphate buffered saline or cell culture medium
supplemented with 10% bovine serum), differing only in surface properties (Fig. S2b and S3).
Therefore, these NPs were perfect candidates for an in vivo behavior comparative study, as in
this case the adsorption of proteins and macrophage uptake would not be associated with the size
of the NPs as previously reported.
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