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Krasnov and L. M. Ogorodova, RSC Adv., 2016, DOI: 10.1039/C6RA13178F.

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pHLIP-Modified Magnetic Nanoparticles for Targeting Acidic

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Diseased Tissues
Received 00th January 20xx,

RSC Advances Accepted Manuscript

a‡ b,c‡ b b d
Accepted 00th January 20xx A. M. Demin, A. G. Pershina* K. V. Nevskaya, L. V. Efimova, N. N. Shchegoleva, M. A.
d e e a b
Uimin, D. K. Kuznetsov, V. Ya. Shur, V. P. Krasnov and L. M. Ogorodova
DOI: 10.1039/x0xx00000x

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A covalent immobilization of a pH-low insertion peptide
(pHLIP) to Fe3O4 magnetic nanoparticles was carried out
resulting in formation of MRI-visible materials able to
specific accumulation in the acidic damaged tissues. The
pH-dependent pHLIP-mediated binding of the obtained
nanoconjugates to the cell in acidic environment was
demonstrated on HTC cells in vitro and in a mouse LLC
tumour model in vivo.
1
pHLIPs (pH Low Insertion Peptides) are a class of peptides
possessing pH-dependent transmembrane activity; these peptides
are able to insert into the cell membrane at slightly acidic pH (<7.0).
Acidification of the intercellular space is a property of tumour,
inflammatory, ischemic and other damaged tissues. Therefore,
pHLIPs have been successfully used for targeting the therapeutic
2, 3 4, 5 6 12,13
molecules and tracers to acidic tissues.
The purpose of the work was to develop an approach for
production of a new nanoconjugate based on the Fe3O4 magnetic
nanoparticles (MNPs) modified by 3-aminopropylsilane (APS) and
conjugated to pHLIP, and to test it as magnetic resonance imaging
(MRI) visible contrast agent for acidic tissues (eg tumour) targeting.
One of the most effective methods for modification of MNPs for
biomedical applications is covalent binding of organic molecules
7 Fe3O4 (Fig. 1A) were prepared by co-precipitation from Fe /Fe
that are pre-modified by alkoxysilane reagents, for example, by 3-
aminopropyltrimethoxysilane (APTMS). To the best of our
knowledge, there is a limited number of publications on
immobilization of pHLIP on the surface of inorganic nanoparticles
8,9
for their selective transport in the acidic tissues. In particular,
there are no information about obtaining of the MNP–pHLIP
nanoconjugates. Taking into account high potential of magnetic
10
nanoparticles for diagnosis (e.g. MRI) as well as theraphy, for
instance by magnetic hyperthermia in high frequency magnetic field
or magneto-mechanical actuation in low frequency alternating
11
magnetic field, the design of such nanoconjugate seems to be
promising. In order to preserve the biological activity, it is accepted
to conjugate pHLIPs at the amino group of L-Ala (N-terminal amino
acid) or the thiol group of L-Cys.
In the work a method for immobilization of pHLIP to the APS-
modified MNPs (MNPs-APS) through covalent binding to the L-Cys
thiol group using 6-maleimidohexanoic acid N-hydroxysuccinimide
ester (EMCS) as a linker is offered (Scheme 1).
Derivatives containing the maleimide moiety have been used for
modification of the SH groups in biomolecules, and their
14,15
conjugation to nanoparticles. The nucleophilic addition reaction
of thiols to double bond of the maleimide residue results in the
stable 3-thio-succinimidyl ester. Thus, the proposed approach of a
covalent binding of pHLIP to the MNP surface provided a
nanoconjugate stability in the biological environments is carried
out.
MNPs with an average diameter of 10 nm and a spinel phase state
3+ 2+
salt solutions. At the first step, the surface of nanoparticles 1 was
16,17
modified using APTMS (2) in a similar to previously described
(Scheme 1). The number of APS residues immobilized on the
–1
surface was 0.75 mmol g according to the elemental analysis
based on the measurments of carbon content in the sample, as well
18
as the IR spectroscopy data (Fig. 2).
Then, EMCS (4) cross-linker was attached to MNPs-APS through the
coupling of the hydroxysuccinimide-activated carboxyl group to the
amino groups on the MNPs surface followed by conjugation with
pHLIP (Scheme 1).
Immobilization of pHLIP on MNPs was confirmed by the IR
spectroscopy (Fig. 1). In the FTIR spectra of nanoconjugate 7 the
–1
characteristic absorption bands of MNPs ( 546 cm , Fe-O) and
–1 -1
pHLIP ( 3272 cm , stretching vibrations of NH; 2922 cm ,

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–1 –1
stretching vibrations of CH; 1648 cm , amide I; 1533 cm , amide II; explained by the fact that the shell formed by a peptide had a
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–1 low contrast in the TEM images and was almost invisible.
DOI: 10.1039/C6RA13178F
1453 and 1390 cm amide III) were indicated. Small shifts in the
–1
absorption bands position were observed due to pHLIP binding to The specific magnetization of MNPs-pHLIP was 52 emu g and
–1
the MNP surface (S1). The number of pHLIP residues immobilized was lower than the magnetization of initial MNPs (81 emu g ),
on the surface of MNPs was calculated from the carbon content in which can be explained by the presence of a silane coating and
17 -6 –1 peptide on the surface of MNPs (S5).
the sample similar to and amounted to 6.7 10 mol g (27.6 mg
–1
g MNPs) (S2).
According to the thermogravimetric analysis (TGA) (Fig. 2) heating
of the obtained MNPs to 150 C resulted to a weight loss (1.2%),
which is associated with the thermodesorption of physically
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adsorbed water from the nanoparticle surface. Further heating to

530 C leaded to the decomposition and removal of organic

RSC Advances Accepted Manuscript

molecules bound to the surface of MNPs. The weight loss of those
samples was related with decomposition of 3-aminopropylsilane, 6-
maleimidohexanoyl and peptide fragments and with their partial
removal as simple volatiles (for example, H2O, CO2, NH3) in the
gaseous phase.
The comparison between values of the weight loss of MNPs-EMCS
and MNPs-APS, MNPs-pHLIP and MNPs-EMCS allows to calculate
the amount of EMCS and pHLIP (20.8 and 19.8 mg/g MNPs
correspondingly). The less amount of a peptide on MNPs surface
according to TGA data in comparison with the elemental analysis
Fig. 1 FTIR spectra of initial MNPs (1), MNPs-APS (2), MNPs-
data can be explained by incomplete decomposition and removal of
pHLIP (3) and pHLIP (4).
organic molecules fragments from MNPs surface.
The hydrodynamic characteristics of MNPs-pHLIP 7
suspensions in aqueous media were studied using of dynamic
light scattering method. It has been shown that the obtained
nanoconjugates form stable suspensions in water (mean
particle hydrodynamic diameter, Dh = 155 nm; polydispersity
index, PdI = 0.2; zeta potential = –22 mV), and in DMEM with
10% fetal bovine serum (Dh = 180 nm, PdI = 0.2, zeta potential
= –10 mV) (S3).
According to the transmission electron microscopy (TEM) data,
significant changes in the morphology of MNPs after surface
modification and conjugation with the peptide did not occur:
the average diameter was 10 nm, the nanoparticle phase state
remained unchanged (Fig. 3 B–D). According to scanning Fig. 2 TGA data of MNPs-APS (1), MNPs-EMCS (2) and MNPs-
electron microscopy (SEM), MNPs-pHLIP particles have a
pHLIP (3).
spherical shape with an average diameter of about 12 nm (Fig.
3 E, F); (S4). The slight difference between these data can be

Scheme 1 Immobilization of pHLIP on MNPs.

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The ability of nanoconjugate to accumulate in acidic tissue in

vivo was studied on the model of LLC tumour in mice. Forty
hours after nanoconjugate intravenous administration the
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intensive accumulation of nanoparticles in tumour however

RSC Advances Accepted Manuscript

not in liver was evidenced by histological analysis (Fig. 4 D). In
the meantime, in 40 hours after intravenous injection of
parent (non-targeted) MNPs-APS, no iron accumulation in
tumour tissue was observed (S6).
21
According to the MTT test, the obtained nanoconjugate at a
–1
concentration up to 100 mg L did not exhibit a cytotoxicity in the
HTC cells (Fig. 4 C).

Fig. 3 A: TEM image and the electron diffraction pattern of

initial MNPs; B: TEM image, С: electron diffraction pattern and
D: the size distribution of MNPs-pHLIP; E: SEM image and F:
the size distribution of MNPs-pHLIP.

Relaxivity r2 is a key feature of MNPs used for MRI. The higher

relaxivity is, the less amount of nanoconjugate can be detected in
the tissues by MRI. To characterize the MR-contrasting properties
of MNPs-pHLIP the T2 relaxivity for suspensions of nanoconjugate Fig. 4 A: T2-weighted MR images and T2 relaxivity plots of
with different concentrations were registered at 11.7 T (Fig. 4 A). aqueous suspensions of MNPs-PHLIP at concentrations of 2–40
–1 –1
The calculated value of r2 was equaled to 117.21 mmol s and M (11.7 T, Biospec 117/16 USR, Bruker); B: binding of MNPs-
exceeded that of some commercial contrast agents approved by pHLIP to the HTC cells in vitro; the cells were incubated with
–1
FDA (USA), for example, Feridex and Combidex (98 and 60 mmol MNPs-pHLIP at pH 6.0 (blank columns) and pH 7.4 (gray
–1 19
s , respectively).
columns); asterisks denote to the statistically significant
Efficiency of the MNPs-pHLIP nanoconjugate binding to the cells at differences based on the Student’s t-test (p  < 0.01); С: MTT
normal (7.4) and weakly acidic pH (6.0) was studied in vitro in the assay of cell viability; HTC cells were incubated with MNPs-
8
rat hepatoma (HTC) cell line (Fig. 4 B). The cells incubated with pHLIP for 2 (blank columns) and 24 (gray columns), pH 7.4; D:
MNPs-pHLIP at pH 6.0 showed a 1.7-2.7-fold higher cell uptake in Prussian blue stained liver and LLC-tumour sections; the mice
comparison with the cells incubated at pH 7.4 (according to the were intravenous administrated with MNPs-pHLIP in dose of 2
measurements of iron concentration in cell lysate by ferrozine- mg/kg, the tissues were extracted in 40 hours after
20
based assay ). These data evidenced the pH-dependent pHLIP- administration.
mediated uptake of nanoconjugate by the cells.

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Conclusions Nikiforidis, S. Xanthopoulos, D. Psimadas, M. Paravatou-

Immobilization of pHLIP on the Fe3O4-based MNPs was carried
out for the first time. pHLIP was fixed on the APS-modified 16
MNPs surface using a heterofunctional linker EMCS through
17
the conjugation to the peptide thiol group. It has been shown
that the resulting nanoconjugate exhibits good MRI
contrasting properties, binds efficiently to cells in a weakly 18
acidic environment both in vitro and in vivo and has no
cytotoxic effect on the cells. Thus, the obtained pHLIP- 19
20
modified MNPs can be used as MR-visible contrast agent for
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Petsotas, L. Palamaris, J. D. Hazle and G. C. 10.1039/C6RA13178F
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Interface Sci., 2014, 433, 163.
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A. M. Demin, A. Yu. Vigorov, I. A. Nizova, M. A. Uimin, N. N.
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Charushin, Mendeleev Commun., 2014, 24, 20.
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Y.-X. J. Wang, Quant. Imaging Med. Surg., 2011, 1, 35.
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tumours targeting. 21 T. Mosmann, J. Immunol. Methods, 1983, 65, 55.

RSC Advances Accepted Manuscript

Acknowledgements
This work was supported by the Russian Science Foundation
(project no. 14-15-00247).
The authors thank Prof. Sergey V. Vtorushin for conducting of
histological analysis, Dr. Igor A. Klimov for helping in animal
experiments, Artyom A. Minin for providing DLS analysis and
Oleg B. Shevelev for assisting in MRI scanning.

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