Orthogonal Clickable Iron Oxide Nanoparticle Platform for Targeting, Imaging, and On-Demand Release

Article abstract of DOI:10.1002/chem.201800389

A versatile iron oxide nanoparticle platform is reported that can be orthogonally functionalized to obtain highly derivatized nanomaterials required for a wide variety of applications, such as drug delivery, targeted therapy, or imaging. Facile functional

Full text of DOI:10.1002/chem.201800389

A Journal of
Accepted Article
Title: Orthogonal Clickable Iron Oxide Nanoparticle Platform for
Targeting, Imaging, and On-Demand Release
Authors: Noelia Guldris, Juan Gallo, Lorena García-Hevia, José Rivas,
Manuel Bañobre-López, and Laura M. Salonen
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To be cited as: Chem. Eur. J. 10.1002/chem.201800389
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Orthogonal Clickable Iron Oxide Nanoparticle Platform for
Targeting, Imaging, and On-Demand Release
Noelia Guldris,[a,b] Juan Gallo,[a] Lorena García-Hevia,[a] José Rivas,[a,b] Manuel Bañobre-López,*[a] and
Laura M. Salonen*[a]
Abstract: A versatile iron oxide nanoparticle platform is reported,
which can be orthogonally functionalized to obtain highly derivatized
nanomaterials as required for a wide variety of applications such as
drug delivery, targeted therapy, or imaging. The facile
functionalization of the nanoparticles using two ligands with
isocyanate moieties allows for high coverage of the surface with
on command through triggered heat production.[9–12] Therefore,
magnetic IONs can act as theranostic probes, serving both
therapy and diagnosis simultaneously.[11,13] ION properties can
be tailored by functionalizing their surface, for example, to
enhance water solubility with hydrophilic polymers, obtain
therapeutic activity by loading drug derivatives, and allow
maleimide and alkyne groups. As
a
proof-of-principle, the
detection
by
several
imaging
modalities
through
nanoparticles were subsequently functionalized with a fluorophore
as a drug model and with biotin as a targeting ligand towards tumor
cells using DielsAlder and azidealkyne cycloaddition reactions,
respectively. The thermoreversibility of the DielsAlder product was
exploited to induce the on-demand release of the loaded molecules
by magnetic hyperthermia. Additionally, the nanoparticles were
shown to target cancer cells by in vitro experiments, as detected by
magnetic resonance imaging.
functionalization with imaging probes.[5,14,15] Therefore, it is of
high interest to develop chemistry strategies for the
functionalization and stabilization of IONs that are simple,
reproducible, scalable, and orthogonal.[16]
Herein, we have employed isocyanate-bearing ligands to
synthesize a bifunctional ION platform with both maleimide and
alkyne moieties (Figure 1), which can undergo orthogonal
reactions with thiols (thiolene and thiolyne reactions),[17]
conjugated dienes (DielsAlder cycloaddition),[18] and azide
functional groups (1,3-dipolar azidealkyne cycloaddition).[19]
Due to the limitations in the characterization and purification of
IONs, click reactions constitute an ideal scenario for
functionalization towards biomedical applications.[20] We
demonstrate the applicability of this platform by loading a
fluorophore as a drug model using the DielsAlder reaction[21–
Introduction
Iron oxide nanoparticles (IONs) feature attractive properties for
application in biomedicine due to the low cost associated to their
production, high biocompatibility, and great magnetic
response.[1] Nano-sized materials have emerged as promising
vehicles for drug delivery[2] as they have the potential to reduce
side effects by increasing the selectivity towards the target
tissue. This can be especially beneficial in the case of drug
candidates that exhibit good therapeutic efficacy but fail in
systemic toxicity.
The magnetic response of IONs can be exploited for detection
by magnetic resonance imaging (MRI), as IONs can be
designed to create different contrasts.[3,4] Similarly, the magnetic
properties can be used to label stem cells for cell tracking
applications,[5] to guide the nanomaterials to the desired
location by the application of magnetic fields,[6] to increase
temperature by magnetic hyperthermia,[7,8] or to release a drug
25]
and reacting the alkyne moieties by azidealkyne
cycloaddition with a PEG-bearing biotin ligand for targeting. The
thermoreversible character of the DielsAlder reaction enabled
a temperature-triggered release of the fluorescent molecules
through the application of an external alternating magnetic field.
Additionally, the capability of the IONs to enhance the contrast
in MRI was key to validate their tumor cell-targeting ability,
which was found to be enhanced by biotin.
[a]
N. Guldris, J. Gallo, L. García-Hevia, J. Rivas, M. Bañobre-López,
L. M. Salonen
Figure 1. The developed bifunctional ION platform with maleimide and alkyne
groups can be functionalized by DielsAlder and azidealkyne cycloadditions,
respectively.
International Iberian Nanotechnology Laboratory (INL)
Av. Mestre José Veiga
Braga 4715-330, Portugal
E-mail: laura.salonen@inl.int, manuel.banobre@inl.int
N. Guldris, J. Rivas
[b]
Department of Applied Physics, Technological Research Institute,
Nanotechnology and Magnetism Lab,
Universidade de Santiago de Compostela, Spain
Supporting information for this article is given via a link at the end of
the document.
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Results and Discussion
basic pH, with -potential values of +20 and 90 mV found at
pH 2 and 12, respectively (Figure S3). To allow for the reaction
of the naked IONs with the isocyanate ligands, the pH of the
aqueous ION solution was set to pH 6.5 prior to transfer of the
IONs to toluene in order to ensure the absence of free hydroxide
ions and sufficient nucleophilicity of the hydroxyl groups.
The functionalization of naked Fe3O4 was first performed with
maleimide isocyanate 1 (Scheme 1). The IR spectrum of
Maleimide-Fe3O4 (Figure S4) featured vibrational bands at
1602, 1514, 1240, and 584 cm1 corresponding to the stretching
bands of C=O, NH, CO, and FeO, respectively. At the same
time, as compared to the IR spectrum of ligand 1, the band at
2306 cm1 corresponding to the stretching of the isocyanate
moiety disappeared after the reaction with the IONs, confirming
the successful reaction between the hydroxyl and isocyanate
functional groups, in agreement with the literature.[26] We have
also successfully carried out the functionalization using IONs
prepared by the method of Hayashi et al.,[32] suggesting that the
functionalization of iron oxides with isocyanates can be used
with nanoparticles synthesized through different protocols
(Figure S5).
The functionalization of naked Fe3O4 was also carried out with
novel alkyne isocyanate 2, and with an equimolar mixture of
maleimide isocyanate 1 and alkyne isocyanate 2 to give access
to Alkyne-Fe3O4 and Maleimide-Alkyne-Fe3O4, respectively.
The IR spectrum of novel nanoconjugate Alkyne-Fe3O4
(Figure 3) showed characteristic bands at 3250, 1520, 1239,
and 571 cm1, corresponding to the stretching of -C≡CH, –C=O,
CO, and FeO, respectively, along with the disappearance of
the NCO stretching at 2278 cm1 with respect to the IR spectrum
of 2, similarly to that observed for Maleimide-Fe3O4. The IR
spectrum of Maleimide-Alkyne-Fe3O4 featured mixed bands
from both maleimide and alkyne ligands that were difficult to
assign to one particular ligand (Figure S6), with the exception of
the vibration band at 3250 cm1 attributed to the stretching of
the alkyne moiety.
Synthesis and characterization
Isocyanate ligands, recently reported by Licandro and
coworkers as a new ION functionalization strategy,[26] were
chosen for the functionalization of the nanoparticle surface,
where they form carbamates upon reaction with the hydroxyl
groups on the nanoparticle.[27] The advantages of this method
include short reaction times, mild reaction conditions, and easy
access to ligands with a variety of functional groups. Maleimide
isocyanate 1 (Scheme 1) was synthesized via a literature-
known procedure[28] in five steps with an overall yield of 35%,
and all intermediates were obtained without the need of
chromatographic purification. Novel alkyne isocyanate 2 was
obtained in three steps from 4-hydroxybenzoic acid with an
overall yield of 55% (for details, see the Experimental Section
in the supporting information (SI)).
For functionalization with isocyanate alkyne and maleimide
ligands, naked IONs, which feature hydroxyl groups on the
surface,[29][30] were synthesized in water in the absence of
surfactants following the coprecipitation method of Stroeve and
coworkers (Naked Fe3O4).[31]
Transmission electron microscopy (TEM) images showed that
the IONs were spherical with a mean size of 7.7 ± 3 nm (Figure
S1 in the SI). Fourier transform infrared (FT-IR) spectroscopy
showed two strong vibrational bands at 3395 and 587 cm1
assigned to OH and FeO vibrations, respectively (Figure S2).
No other bands of significant intensity, other than a minor C=O
band, were found in the spectrum, indicating the absence of
organic coating. This was further confirmed by elemental
analysis (EA) experiments that showed a carbon content of 0.05
mol% (Table 1). In agreement, the total organic content was
found to be 3 wt% by thermogravimetric assay (TGA, Figure 2).
As expected,[29] the -potential results showed that the naked
IONs are positively charged at acidic and negatively charged at
Scheme 1. Synthesis of monosubstituted nanoconjugates and the bifunctional iron oxide platform Maleimide-Alkyne-Fe3O4 and their conversion using the
DielsAlder cycloaddition and copper catalyzed azidealkyne click reaction (CuAAC). Reaction conditions: i)iii) IONs with either isocyanate 1, 2, or an equimolar
mixture of 1 and 2, respectively, sonicated in dry toluene, 4 h, 60 ºC; iv) and v) pyrene furfuryl 3 and Maleimide-Fe3O4 or Maleimide-Alkyne-Fe3O4 in toluene,
3 days, 65 ºC; vi) pyrene azide 4 and Alkyne-Fe3O4 in THF/H2O mixture 1:0.95, 24 h, rt; vii)viii) N3-PEG-Biotin and Alkyne-Fe3O4 or Pyrene-Maleimide-Alkyne-
Fe3O4 in THF/H2O mixture, CuSO4, sodium ascorbate, 24 h, rt.
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unknown final ratio, it was not possible to derive the number of
moles of the ligands from the elemental analysis results.
Table 1. Elemental analysis of naked Fe3O4, Maleimide-Fe3O4,
Alkyne-Fe3O4, and Pyrene-Alkyne-Fe3O4.
Sample
%C
%H
%N
Naked Fe3O4
0.054
23.1
18.9
36.1
0.460
1.27
1.86
2.23
-
Maleimide-Fe3O4
Alkyne-Fe3O4
4.28
2.38
6.47
Maleimide-Alkyne-Fe3O4
Figure 2. TGA of Naked Fe3O4 (black), Maleimide-Fe3O4 (red), Alkyne-
Fe3O4 (blue), Maleimide-Alkyne-Fe3O4 (green).
Pyrene-Alkyne-Fe3O4
26.4
2.14
4.13
1H NMR spectrum of Maleimide-Fe3O4 (Figure 4, red) was in
agreement with that reported by Licandro and coworkers.[26] The
spectrum of Alkyne-Fe3O4 (Figure 4, blue) features a peak at
8.45 ppm corresponding to the NH proton, a pair of broad
signals at 7.37 and 6.91 ppm corresponding to the phenyl
protons, and a singlet at 4.74 corresponding to CH2 in α-position
to the alkyne. The proton corresponding to C≡CH is hidden
under the water signal from the solvent. Notably, in both
samples the presence of the NH proton indicates the formation
of the carbamate on the NP surface. The 1H NMR spectrum of
Maleimide-Alkyne-Fe3O4 (Figure 4, black) featured a set of
proton signals for both maleimide and alkyne ligands. The
similar intensities of the signals from both ligands suggest that
they are present on the ION surface in a nearly equimolar ratio.
Interestingly, in addition to the previously observed signals for
the NH protons, two new peaks appeared at 8.77 and 8.57
ppm. They could stem from the presence of hydrogen-bonded
dimers between the carbamate moieties on the surface of the
nanoparticles. Each carbamate could interact either with a
similar ligand (maleimidemaleimide or alkynealkyne), which
would result in two signals for NH protons, or with a different
ligand (maleimidealkyne), which would result in two additional
proton signals. The nearly equal intensity of the four NH proton
signals in the Maleimide-Alkyne-Fe3O4 spectrum would
account for a statistical distribution of the hydrogen-bonded
dimers.
Figure 3. FT-IR spectra of naked Fe3O4 (black), Alkyne-Fe3O4 (blue), and
alkyne isocyanate 2 (green).
TGA experiments showed a 51 and 63 wt% weight retention for
Maleimide-Fe3O4 and Alkyne-Fe3O4, respectively (Figure 2).
With 36 wt%, Maleimide-Alkyne-Fe3O4 featured the highest
organic content. The increase in the organic content, as
compared to the singly substituted nanoparticles, could be due
to changes in packing when both ligands are present, stemming
for example from hydrophobic interactions or hydrogen bonding.
Both Maleimide-Fe3O4 and Alkyne-Fe3O4 featured two strong
weight losses, at 200 °C and 600 °C for Alkyne-Fe3O4 and at
350 and 700 °C for Maleimide-Fe3O4 (Figure 2). Interestingly,
Maleimide-Alkyne-Fe3O4 clearly showed three strong weight
losses at 200, 350, and 700 °C, indicating the presence of both
maleimide and alkyne moieties on the ION surface.
Elemental analyses (EA) of the C, H, and N content of the
samples were found to be in agreement with the TGA results
(Table 1). Maleimide-Fe3O4 features a total molar organic
content of 38%, which corresponds to 1.66 mmol of maleimide
ligand per g of IONs, in good agreement with the results of
Licandro and coworkers.[26] For Alkyne-Fe3O4, the total molar
organic content was 31%, which corresponds to 1.64 mmol of
ligand per g of NP (details SI page 23). Since Maleimide-
Alkyne-Fe3O4 has two different ligands on the surface in an
Figure 4: 1H NMR spectra (500 MHz, DMSO-d6, 25 °C) of i) Maleimide-Fe3O4,
ii) Alkyne-Fe3O4, and iii) Maleimide-Alkyne-Fe3O4.
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DielsAlder and CuAAC cycloadditions
stable in water after the functionalization procedure, allowing
purification by dialysis. This is an additional verification of the
success of the procedure, since the starting material
Alkyne-Fe3O4 can only be dispersed in organic solvents due to
the apolar nature of the ligands on the nanoparticle surface.
TGA measurements of Biotin-Alkyne-Fe3O4 showed an
increase of 29 wt% in organic content after the CuAAC reaction
and two additional weight losses at 100 and 350 °C as
compared to Alkyne-Fe3O4 (Figure S11), confirming the
success of the reaction. A concentration of 90 µmol of biotin per
g of ION was found attached to the surface of Biotin-Alkyne-
Fe3O4 using a commercial kit based on the formation of a
complex between 4-hydroxyazobenzene-2-carboxylic acid
(HABA) and avidin (for details, see the SI).[41]
The reaction conditions employed with the singly substituted
Maleimide-Fe3O4 and Alkyne-Fe3O4 were then used
sequentially to functionalize Maleimide-Alkyne-Fe3O4 by both
DielsAlder and CuAAC reactions to give Pyrene-Biotin-Fe3O4.
First, the maleimide moieties were reacted with pyrene furfuryl
3 to give access to Pyrene-Maleimide-Alkyne-Fe3O4 (Scheme
1). The presence of pyrene after the reaction was confirmed by
UV-Vis spectroscopy that showed absorption bands from the
maleimide moiety at 261 and 210 nm and those from the pyrene
ligand at 343, 327, 313, and 234 nm (Figure 5, green). An
increment in the organic content of 22 wt% was calculated from
TGA measurements, with one additional weight loss featured at
400 °C in comparison with Maleimide-Alkyne-Fe3O4 (Figure
S12).
The maleimide moiety can react in the thermally reversible
DielsAlder cycloaddition.[21] This reaction has advantageous
characteristics for cargo release: it is self-contained, i.e. all the
atoms present in the diene and dienophile remain in the
resulting products, and the displacement of the equilibrium is
governed by temperature, solvent, or reactant concentration,
and not by external molecules such as water, metal catalyst, or
base/acids.[22] IONs are known to produce heat when placed
under an alternating magnetic field in a phenomenon known as
magnetic hyperthermia,[33] which can be used to induce the
release of cargo molecules attached on the nanoparticles
through thermosensitive bonds.[23,24,34–36] The hyperthermia
temperature profile is dependent on the distance from the ION
surface, with the highest temperature reached at the surface,
and decreasing exponentially with increasing distance.[37]
Maleimide-Alkyne-Fe3O4 is designed to place the cargo
molecules only a few ångstroms away from the surface of the
inorganic core and allow magnetic hyperthemia to sufficiently
increase the local temperature to trigger the release.
To study the DielsAlder reaction with the IONs, we prepared
furfuryl pyrene 3 (for test reactions and synthetic procedures,
see the SI), the release of which could be quantified by
fluorescence measurements. The cycloaddition between 3 and
Maleimide-Fe3O4 occurred in toluene at 65 °C in 3 days to yield
Pyrene-Maleimide-Fe3O4 (Scheme 1). This long reaction time
and low temperature were opted for to hinder cleavage of the
maleimide carbamate from the surface of nanoparticles (Figure
S7). A reaction time of one day did not yield the product Pyrene-
Maleimide-Fe3O4, as verified by the absence of increase in the
organic content by TGA. On the other hand, after 3 days an
increase in organic content of 9 wt% was observed in TGA
measurements as compared to Maleimide-Fe3O4 (Figure S8).
The UV-Vis spectrum of Pyrene-Maleimide-Fe3O4 in ethanol
showed the characteristic bands of the furfuryl pyrene moiety at
342, 326, 267, and 202 nm (Figure S9).
The azidealkyne cycloaddition conditions were tested on
Alkyne-Fe3O4, which was reacted with pyrene azide 4, allowing
characterization of the resulting Pyrene-Alkyne-Fe3O4 by
fluorescence
spectroscopy.
The
copper-catalyzed
azidealkyne click reaction (CuAAC) reaction was carried out in
a mixture of THF and water,[38] with copper sulfate and sodium
ascorbate as catalyst for 24 h. Pyrene-Alkyne-Fe3O4 dispersed
in THF showed UV-Vis absorption bands at 344, 328, 276, 265,
243, and 205 nm, characteristic of the pyrene moiety (Figure
S10, left), and fluorescence emission bands of pyrene at 416,
394, and 374 nm (Figure S10, right). An increase of 7 wt% in
organic content as compared to Alkyne-Fe3O4 was observed
by TGA (Figure S11). EA measurements of Pyrene-Alkyne-
Fe3O4 showed a 3.7 wt% increment in organic content when
compared to Alkyne-Fe3O4 (Table S1), in agreement with the
TGA results.
Using the same reaction conditions, Alkyne-Fe3O4 was
functionalized with a commercially available targeting ligand
N3-PEG-Biotin to give Biotin-Alkyne-Fe3O4 (Scheme 1). The
biotin moiety has been shown to target several tumor cells,[39]
and the polyethylene glycol was envisioned to enhance water
solubility of the nanoconjugate and impart stealth properties
towards biomolecules.[40] Indeed, the IONs were found to be
Figure 5: UV-Vis spectra of a solution of naked Fe3O4 (grey), pyrene furfuryl
3 (blue), Maleimide-Alkyne-Fe3O4 (red), and Pyrene-Maleimide-Alkyne-
Fe3O4 (green) in THF.
Subsequently, the alkyne moieties were functionalized with
N3-PEG-Biotin and resulting water-dispersible Pyrene-Biotin-
Fe3O4 was purified by dialysis. A concentration of 200 µmol of
biotin per g of ION was found in Pyrene-Biotin-Fe3O4 by the
HABA/avidin assay. Fluorescence spectroscopy of the doubly
functionalized nanoconjugate showed emission bands at 416
nm, 394, and 375 nm, confirming that the previously loaded
furfuryl pyrene remains attached to the nanoparticle surface
(Figure S13).
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Magnetic Properties
ångstroms from the surface to promote its release without a
macroscopic temperature increase, thus avoiding possible
systemic toxicity from protein denaturation.[44] Therefore, a
diluted aqueous solution of Pyrene-Biotin-Fe3O4 (0.84 mg mL1
in water) was subjected to an alternating magnetic field (AMF)
of 863 kHz and 15.9 kAm1 for 1 h (for details, see the
Experimental section). Only a slight macroscopic temperature
increase was observed, with a maximum recorded temperature
of 27 °C.
After the treatment, the sample was centrifuged and the
supernatant analyzed by fluorescence spectroscopy to quantify
the release of pyrene furfuryl 3 (Figure S15). The quantification
was performed using a calibration curve of pyrene furfuryl 3 in
water at λexc = 305 and λemis = 375 nm. A total concentration of
5.2 nM of the fluorophore was found to be released from
Pyrene-Biotin-Fe3O4. This value is within the range of half-
maximal inhibitory concentrations (IC50) for several anticancer
drugs, such as commercially available paclitaxel with IC50 of
2.57.5 nM,[45] and novel taxoid drugs bearing targeting biotin
ligands with IC50 of 3.22−9.80 nM.[46] Moreover, the
concentration released is in the range of those found in the
literature for magnetic-hyperthermia-induced release, with
3105 nM reported by Fontaine and co-workers[36] depending
on the experimental hyperthermia conditions, and 0.63 nM
reported by Kirschning and co-workers,[34] placing Pyrene-
Biotin-Fe3O4 among the thermosensitive platforms from which
loaded molecules can be released by AMF-mediated
hyperthermia.
In order to quantitatively evaluate the behavior of
Pyrene-Biotin-Fe3O4 as an MRI contrast agent and verify its
ability to reduce the transverse relaxation time of water protons,
a T2 map of the sample was acquired at Fe concentrations
between 0 and 550 µM (Fe) at the clinical field of 3 T at room
temperature. Figure 6A shows a significant concentration-
dependent reduction of the T2 relaxation time, which linearly
decreases as the Fe concentration increases. These T2
relaxation values were used to calculate the transverse
relaxivity parameter, r2, which reflects how the T2 relaxation
rates of a solution change as a function of concentration,
thereby describing the efficacy of the system as a T2 MRI
contrast agent. The r2 value for Pyrene-Biotin-Fe3O4 was found
to be 160 mM1s1 at room temperature and 3.0 T (Figure S14),
superior to the relaxivity of commercial T2 contrast agents such
as Endorem® (93 mM1s1) or Resovist® (143 mM1s1) at the
same field strength.[42]
Biotin-Mediated Targeting
Pyrene-Biotin-Fe3O4 was designed to bear biotin as tumor-
targeting moiety. In order to verify the targeting capability of the
sample, the internalization of the nanoparticles was evaluated
with human lung cancer cells A549, which are known to
overexpress biotin receptors.[43] The dark T2 contrast in MRI of
Pyrene-Biotin-Fe3O4 was taken advantage of to demonstrate
the cellular uptake (Figure 6B). MR phantom images of control
cells without nanoparticles showed a nearly double intensity of
signal to those incubated at 4 ºC with Pyrene-Biotin-Fe3O4,
suggesting that the nanoparticles were internalized by the cells.
As expected, the presence of iron oxide nanoparticles induces
a strong reduction of the T2-weighted MRI signal. Incubation
with the nanoparticles at low temperature minimizes cellular
energy-dependent trafficking processes, thus helping to reduce
the non-mediated uptake of nanoparticles by the cells. In order
to further evaluate the role of the biotin moieties in the cell
targeting of Pyrene-Biotin-Fe3O4, different concentrations of
free biotin (100, 200, and 300 µM) were added to the incubation
mixture to pre-saturate a part of the biotin receptors. Analysis of
the T2-weighted images showed a concentration-dependent
recovery of the signal with increasing free biotin concentration
as a consequence of the lower number of biotin receptors
available to mediate the uptake of Pyrene-Biotin-Fe3O4 (Figure
6B). This is in agreement with a biotin-mediated interaction
between the cells and Pyrene-Biotin-Fe3O4 nanoparticles.
Magnetic Hyperthermia
Finally, magnetic hyperthermia experiments were conducted to
study the on-demand release of pyrene from Pyrene-Biotin-
Fe3O4 nanoparticles. The temperature profile achieved in
magnetic hyperthermia[33] is known to be dependent on the
distance from the ION surface, being the highest at the surface
and decreasing exponentially with increasing distance.[37]
Therefore, in order to promote the release of a molecule by
magnetic hyperthermia, it is of fundamental importance to place
the delivery molecules close to the surface of the IONs. In
Pyrene-Biotin-Fe3O4, pyrene furfuryl 3 is located only a few
Figure 6. A: colored T2 maps of water solutions of Pyrene-Biotin-Fe3O4 at
different concentrations showing the good performance of the nanoparticles
as MRI contrast agents. B: T2 signal intensity obtained from T2-weighted MR
images of A549 cell phantoms incubated with Pyrene-Biotin-Fe3O4
(50 µg mL1) in the presence or absence of increasing concentrations of free
biotin.
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Conclusions
Pyrene-Biotin-Fe3O4 as an aq. solution (0.84 mg NP/mL, 5.5 mL).
Quantification of biotin was performed by HABA/avidin bioassay (For
more details, see the SI).
In summary, we have developed a versatile bifunctional ION
platform with two orthogonal reacting groups, maleimide and
alkyne. As a proof-of-principle, we have functionalized the IONs
with a targeting ligand towards cancer cells through 1,3-dipolar
azidealkyne cycloaddition and a model fluorophore through
DielsAlder reaction, and demonstrated the utility of the
platform in targeted therapy as verified by MRI and on-demand
delivery upon external command. In combination with its
demonstrated capability to enhance the contrast in T2 MR
images, this hybrid platform shows potential to be further
exploited as a theranostic agent for combined imaging and
therapy applications.
Cell targeting experiments
A549 cells (106) were incubated in 60 mm Petri dishes 24 h before the
tests. After 24 h, the cell cultures were incubated for 30 min at 4 C in
the presence of different concentrations of free biotin (0, 100, 200, and
300 µM). After the incubation, cells were washed with PBS, and
50 µg mL1 of Pyrene-Biotin-Fe3O4 nanoparticles were added and
incubated for 30 min at 4 C. Cells were washed again with PBS,
dissociated with trypsin, and then pelleted via centrifugation (600 g, 60
s). The supernatant was discarded and the cells resuspended in warm
agar solution (1%, 50 µL). 30 µL of this suspension were transferred to
the bottom of a small (300 µL) microtube and immediately placed on ice
to solidify the gel before cell precipitation. The gels were stored in the
fridge for at least 4 h prior to imaging.
Experimental Section
Magnetic Hyperthermia
Pyrene-Biotin-Fe3O4 in Milli-Q water at pH 7 (0.84 mg mL1 of NPs, 1
mL) was subjected to an AMF of 863 kHz and 15.9 kAm1 for 1 h. As
control, a sample at the same concentration was left at rt for 1 h. Then,
the two samples were placed in centrifugal filters (Amicon with a cut-off
of MW 10000 Da) and subjected to centrifugation (4000 rpm, 10 min).
The supernatants from the centrifugation were analyzed by fluorescence
spectroscopy at λexc = 305 and λemis = 375 nm. The concentration of the
released fluorophore was derived from the calibration curve of furfuryl
pyrene 3 by means of fluorescence intensity (Figure S15 in the SI).
Synthetic protocols are provided below for key samples. Additional
synthetic procedures for all other derivatives, equipment, and conditions
used for the measurement of physical data are found in the Supporting
Information.
Maleimide-Alkyne-Fe3O4
Naked iron oxide NPs (27 mg) were suspended in dry toluene (1.4 mL)
and subjected to sonication for 5 min. Maleimide isocyanate 1 (24 mg,
0.11 mmol) and alkyne isocyanate 2 (19 mg, 0.11 mmol) were added to
the suspension and the mixture was subjected to sonication at 60 °C for
4 h. Then, the IONs were isolated magnetically and washed with toluene
(4 times) to yield Maleimide-Alkyne-Fe3O4 (20 mg). 1H NMR (500 MHz,
[D6]DMSO, 25 ºC, TMS): δ = 8.90 (s), 8.77 (s), 8.56 (s), 8.43 (s), 7.55
(br. s, 2H), 7.37 (br. s, 2H), 7.24 (br. s, 2H), 7.17 (br. s, 2H), 6.92 (br. s,
2H), 4.74 (br. s, 2H), signal of C≡CH overlaps with the water signal from
the deuterated solvent; 13C NMR (125 MHz, [D6]DMSO, 25 ºC, TMS): δ
= 134.5, 127.5, 119.8, 118.2, 115.3, 55.6 (data from HSQC experiment);
IR (KBr) = 576, 1025, 1211, 1503, 1563, 1593, 1644 (C=O), 3282
(C≡CH) cm1; UV/Vis (EtOH): λmax = 264 nm; elemental analysis found
C 36.06, H 2.23, N 6.47; TGA: weight retention 36%.
Acknowledgements
We would like to thank Dr. M. F. Cerqueira (University of Minho,
Portugal) for the Raman analysis. This work was partially
supported by the NORTE 2020 (2014–2020 North Portugal
Regional Operational Programme) and the ERDF (European
Regional Development Fund) under the Grant NORTE-45-
2015-02, and by European Union's Horizon 2020 research and
innovation programme under grant agreement Nº686009. J.G.
and L.M.S. acknowledge the financial support from the Marie
Curie COFUND Programme (NanoTRAINforGrowth) under
Grant No. 600375.
Pyrene-Maleimide-Alkyne-Fe3O4
Maleimide-Alkyne-Fe3O4 (50 mg) was suspended in toluene (1.6 mL)
and subjected to sonication for 5 min. Then, furfuryl pyrene 3 (19 mg,
81 µmol) was added and the reaction mixture was stirred for 3 d at 65 °C.
The NPs were isolated magnetically and suspended in toluene
(repeated 4 times) to yield Pyrene-Maleimide-Fe3O4 (40 mg).
Quantification of the unreacted pyrene furfuryl 3 in the collected toluene
supernatant was carried out by UV-Vis spectroscopy. The amount of
pyrene furfuryl 3 attached to the NP was estimated by subtracting the
unreacted amount to the amount added to the reaction mixture. Results
Keywords: iron oxide • nanoparticle • click chemistry •
DielsAlder reaction • hyperthermia
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Title
Let’s click again! A versatile iron oxide platform was developed bearing orthogonal reacting groups, which were converted by two
consecutive cycloaddition reactions. Using magnetic resonance imaging, the utility of the functionalized platform was shown for targeted
therapy. Additionally, the on-demand release of the model fluorophore was effected using magnetic hyperthermia.
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