SPIO@SiO2-Re@PEG nanoparticles as magneto-optical dual probes and sensitizers for photodynamic therapy

Article abstract of DOI:10.1039/c6ra04332a

A dual magneto-optical nanoprobe, endowed with properties useful for photodynamic therapy, has been prepared. It is constituted by a superparamagnetic iron oxide (SPIO) core (diameter size distribution centered at ca. 10 nm), obtained by thermal decomposition of iron oleate, coated by a compact silica shell, grown in a reverse microemulsion environment. Luminescent [Re(phen)(CO)₃(py)]CF₃SO₃ complexes were covalently anchored to the silica shell, by functionalizing the pyridine ligand with a triethoxysilane moiety. Finally, the surface of the nanoparticles (NPs) was coated with a layer of polyethylene glycol (PEG), functionalized with triethoxysilane, to improve stability and stealthiness in physiological conditions. Transmission electron microscopy of these SPIO@SiO₂-Re@PEG nanocomposites showed a single population, with size distribution centered at ca. 40 nm. NPs showed nuclear relaxivity values that guarantee an appreciable contrast in magnetic resonance imaging (r₂ > 30 s^-1 mM^-1 at frequencies higher than 5 MHz). The presence of the Re complexes imparted photoluminescence to the NPs, with blue shifted emission and higher photoluminescence quantum yields with respect to the free [Re(phen)(CO)₃(py-upts)]⁺ complex (λ_em 553 vs. 580 nm, Φ 0.060 vs. 0.038, in aerated water solution). The complexes embedded into the NPs maintained a satisfactory efficiency toward ¹O₂ generation (quantum yields 0.21 vs. 0.26 for the free complex, as assessed using 1,5-dihydroxynaphthalene as indirect marker of the ¹O₂ presence). Preliminary cell penetration tests were performed on human lung adenocarcinoma A549 cells. Two photon excitation confocal microscopy showed that the NPs were easily internalized and accumulated in the perinuclear region of the cells already after 4 h of incubation. Increased cytotoxicity upon irradiation with respect to the dark was also observed, showing the potential of the nanocomposite for photodynamic therapy applications.

Full text of DOI:10.1039/c6ra04332a

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SPIO@SiO ꢀRe@PEG nanoparticles as magnetoꢀoptical
2
†
dual probes and sensitizers for photodynamic therapy
#
◊
#
‡
&
# &¥
Marco Galli, Elisa Moschini, Maria Vittoria Dozzi, Paolo Arosio, Monica Panigati,
Laura
§
◊
‡
&
#&
D’Alfonso, Paride Mantecca, Alessandro Lascialfari, Giuseppe D’Alfonso, and Daniela
#
&
Maggioni*
#
◊
Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy
Dipartimento di Scienze dell'Ambiente e del Territorio e di Scienze della Terra, Università degli Studi di Milanoꢀ
Bicocca, Piazza della Scienza 3, 20126 Milano, Italy
‡
Dipartimento di Fisica, Università degli Studi di Milano, Via Celoria 16, 20133 Milano, Italy
§
Dipartimento di Fisica, , Università degli Studi di MilanoꢀBicocca, Piazza della Scienza 3, 20126 Milano, Italy
Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali, Via Golgi 19, 20133 Milano, Italy
Istituto per lo Studio delle Macromolecole, Consiglio Nazionale delle Ricerche (ISMACꢀCNR), Via E. Bassini, 15,
&
¥
20133 Milano, Italy
†
Electronic supplementary information (ESI) available: Experimental details, DLS and TGA analyses, NMR, IR and
UVꢀVis spectra, confocal microscope images and MTT test graphs are presented.
1

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Abstract
A dual magnetoꢀoptical nanoprobe, endowed with properties useful for photodynamic therapy, has
been prepared. It is constituted by a superparamagnetic iron oxide (SPIO) core (diameter size
distribution centered at ca. 10 nm), obtained by thermal decomposition of iron oleate, coated by a
compact silica shell, grown in a reverse microemulsion environment. Luminescent
[
3 3 3
Re(phen)(CO) (py)] CF SO complexes were covalently anchored to the silica shell, by
functionalizing the pyridine ligand with a triethoxysilane moiety. Finally, the surface of the
nanoparticles (NPs) was coated with a layer of polyethylene glycol (PEG), functionalized with
triethoxysilane, to improve stability and stealthiness in physiological conditions. Transmission
2
electron microscopy of these SPIO@SiO ꢀRe@PEG nanocomposites showed a single population,
with size distribution centered at ca 40 nm. NPs showed nuclear relaxivity values that guarantee
ꢀ1
ꢀ1
an appreciable contrast in magnetic resonance imaging (r
2
> 30 s mM at frequencies higher than
5
MHz). The presence of the Re complexes imparted photoluminescence to the NPs, with blue
shifted emission and higher photoluminescence quantum yields with respect to the free
+
[
Re(phen)(CO) (pyꢀupts)] complex (λ 553 vs. 580 nm, Φ 0.060 vs. 0.038, in aerated water
3
em
1
solution). The complexes embedded into the NPs maintained a satisfactory efficiency toward O
2
generation (quantum yields 0.21 vs 0.26 for the free complex, as assessed using 1,5ꢀ
1
dihydroxynaphthalene as indirect marker of the O
2
presence). Preliminary cell penetration tests
were performed on human lung adenocarcinoma A549 cells. Two photon excitation confocal
microscopy showed that the NPs were easily internalized and accumulated in the perinuclear region
of the cells already after 4 h of incubation. Increased cytotoxicity upon irradiation with respect to
the dark was also observed, showing the potential of the nanocomposite for photodynamic therapy
applications.
2

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1
.
Introduction
The use of nanoparticles (NPs) in biomedicine has attracted much attention in the past decades,
1
for their remarkable potential in the resolution of many longꢀstanding problems. For this purpose,
the NPs diameters must be in the range 10ꢀ100 nm, since smaller particles would escape from the
circulatory system through renal clearance, whereas larger ones will be recognized by reticuloꢀ
endothelial system (RES), sequestered and mainly delivered to the liver. Preferential accumulation
in solid tumors can be observed for NPs with size smaller than 100 nm, for the so called EPR
(enhanced permeability and retention) effect, which can be further enhanced if the surface is
2
3
functionalized with molecules or polymers able to make the NPs stealth to the RES. NPs, thanks
to their large surface area, can be loaded with huge amounts of small molecules, and therefore can
act as efficient carriers of drugs or imaging agents toward their target, preventing their rapid
clearance. In this way different diagnostic and therapeutic functionalities can be integrated in a
single nanosized system. Since each imaging technique has pros and cons, nanoprobes provided
with different contrast agents exploitable in different imaging techniques could provide many
advantages over single monomodal probes.
4
ꢀ8
Many dual magnetoꢀoptical probes have already been reported, in which iron oxides magnetic
9
10
NPs bear light emitters, like quantum dots (QDs) or organic fluorophores. However the high
toxicity of QDs limits their application in diagnosis, while organic fluorescent molecules often
present drawbacks, such as high photobleaching, short lifetimes, and emission energy close to cell
autofluorescence. The use of organometallic complexes emitting from triplet states can overcome
these disadvantages, due to their generally high photostability, large Stock’s shifts, which prevent
selfꢀquenching and overlapping with cell autofluorescence, and long lifetimes, which allow to
improve the sensitivity by the use of timeꢀresolved techniques. For these reasons phosphorescent
1
1
metal complexes have been often proposed for imaging techniques.
+
Among these phosphorescent emitters, diimine complexes of facꢀRe(CO)
3
have been attracting
1
2
much interest since long time, for their remarkable photophysical properties, which have been
3

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1
3
14
exploited in a variety of fields, either for biomedical applications, or material science. Like other
6
d transitionꢀmetal complexes, these species exhibit an intense longꢀlived emission, originating
3
from triplet metalꢀtoꢀligand charge transfer ( MLCT) excited states. The strongest emissions occur
1
5,16
either from mononuclear cationic complexes
or from dinuclear species in which two metal
1
7
centers are bonded to the same chromophore. The direct conjugation of a luminescent
+
18
mononuclear rhenium complex “Re(L)(CO)
3
” to magnetite NPs has been recently reported, and
this nanosystem is proposed as multimodal probe (MRI T2 contrast agent, optical probe, and
186/188
potential β and γ emitter through the use of
tomography (γ) or radiotherapy (β).
Re hot isotopes, for singleꢀphoton emission
It has been recently proven that some of these rhenium complexes are endowed with another
important property for biomedical uses, i.e. they are efficient sensitizers for the generation of singlet
1
9 ꢀ 23
oxygen,
by tripletꢀtriplet energy transfer. Singlet oxygen production is at the base of
2
4 ꢀ 27
photodynamic therapy (PDT),
which has become more and more attractive as a valid
coadjuvant (or even alternative) to chemotherapy in cancer cure and as a new method for killing
pathogens in localized infections.
PDT requires a photosensitizer, i.e. a molecule that can be photoꢀexcited in a triplet state and is
able to transfer energy to molecular oxygen, converting it from triplet to singlet state (eq 1).
3
3
1
1
S * + O → S + O
(1)
1
2
0
2
2
8
Among the various species used as photosensitizers we can list organic dyes as methylene blue
2
9
24
26,30
24
31,32
or rose Bengal, as well as porphyrins, chlorines,
phthalocyanines, porphycenes,
and
Many different nanomaterials for PDT
containing transition metal complexes as photosensitizers have also recently appeared in the
2
4
33 ꢀ 35
36 ꢀ 37
19ꢀ23
metal complexes of Ru, Ir,
Pt
and Re.
38ꢀ42
literature,
One only of the above nanosized systems, constituted by silica NPs, involved the use
42
of rhenium complexes.
In this work we have prepared a dual probe nanocomposite, constituted by an iron oxide
magnetic core and a compact silica shell functionalized with a luminescent complex, viz.
4

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[
3 3 3
Re(phen)(CO) (pyꢀupts)]CF SO (pyꢀupts = 1ꢀ(pyridinꢀ4ꢀyl)ꢀ3ꢀ(3ꢀ(triethoxysilyl)propyl)urea). The
silica shell has been coated by a polyethyleneglycol (PEG) layer. Actually, pegylation, though
known to partly affect the efficiency of nanoparticles uptake, is a very effective method to reduce
4
3
their toxicity. Moreover, polyethylene glycols in vitro enhance the nanoparticles stability and in
vivo allow the nanoparticles to avoid macrophage recognition, uptake and clearance from systemic
4
4
circulation.
2
The photophysical and relaxivity properties of the SPIO@SiO ꢀRe@PEG
nanocomposites have been measured, to check the possible mutual interference between the
magnetic core and the luminescent shell. Preliminary cell penetration tests have been conducted on
2
human lung adenocarcinoma A549 cells to investigate the internalization of SPIO@SiO ꢀRe@PEG
NPs as well as the molecular probe, by means of two photon excitation (TPE) confocal microscopy.
The ability of the nanocomposites to act as PDT agents has also been investigated, both in cuvette
and in vitro.
2
.
Experimental
Materials, Instruments and Methods are detailed in Electronic Supporting Information,† as well as
the synthesis of the SPIO@OA NPs, of the pyꢀupts ligand and of PEG500ꢀSilane.
4
5
2
7
1
.1.
Synthesis of the [Re(phen)(CO)
3
(pyꢀupts)]OTf complex. Re(CO)
5
OTf
(37.2 mg,
ꢀ2
.83×10 mmol) was dissolved in dry toluene (8 mL) under nitrogen, and added with 15.5 mg of
ꢀ2
,10ꢀphenanthroline monohydrate (7.8×10 mmol). The mixture was heated at 100 °C for 5 h.
Finally, the temperature was lowered to 0 °C, giving an orange powder, from which the
intermediate product [Re(phen)(CO) (OTf)] was isolated by washing with toluene (3 mL, 2 times)
and drying under vacuum. Yield 96 %. The progress of the reaction was monitored by IR
spectroscopy and the final IR spectrum showed ν(CO) bands (CH Cl ) at 2035 (vs), 1935 (s) and
915 (s) cm . The pyridine ligand pyꢀupts (20.9 mg, 0.0644 mmol) was dissolved in 1 mL of
3
2
2
ꢀ1
1
anhydrous ethanol under nitrogen. Then, a sample of [Re(phen)(CO)
3
(OTf)] (38.6 mg, 0.0643
5

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mmol), dissolved in 2 ml of EtOH, was added. The initial suspension was heated at 50 °C and left
to react under magnetic stirring and nitrogen for 48 h, obtaining a clear and yellow solution. In
order to avoid the hydrolysis of the ethoxy groups, the resulting complex [Re(phen)(CO)
3
(pyꢀ
upts)]OTf was not isolated and was stored at 4 °C in ethanol solution under nitrogen. The
1
1
completeness of the coordination of pyꢀupts was assessed by HꢀNMR. H NMR (400 MHz,
MeOD) δ 9.72 (dt, J = 5.1, 1.2 Hz, 2H), 8.95 (dt, J = 8.3, 1.2 Hz, 2H), 8.25(s, 2H), 8.13 (dd, J = 8.3,
5
.1 Hz, 2H), 8.05 (d, J = 6.0 Hz, 2H), 7.19 (d, J = 6.0 Hz, 2H), 3.79 (quart, J = 7.2 Hz, 6H), 3.11
(
m, 2H), 1.53 (m, 2H), 1.18 (t, J = 7.2 Hz, 9H), 0.57 (m, 2H). FTIR (CO): 2034 (vs), 1934 (br, s)
ν
ꢀ1
ꢀ1
cm (ethanol); 2033 (vs), 1924 (br, s) cm (water).
2
.2. Coating of the SPIO NPs with a SiO shell. The silica coating was obtained by a synthesis
2
conducted in a reverse microemulsion. IGEPAL COꢀ520 (2.7385 g, 6.21 mmol) was dissolved by
sonication (20 min) in 22 mL of cyclohexane, in a Schlenk round bottom flask. Then SPIO@OA
3
NPs (0.82 mg, see ESI†) were added, together with aqueous NH (28%, 200 µL) and TEOS (150
µL, 0.677 mmol) to give a brown clear solution. The microemulsion was stirred for 16 h at room
temperature, then treated with ethanol (20 mL) and centrifuged (7550 g, 20 min). The precipitate
was recovered and easily reꢀsuspedend in 20 mL of ethanol by sonication. The purification
procedure was repeated twice.
2
.3.
PEG500ꢀSilane. The ethanol suspension of SPIO@SiO
the complex [Re(phen)(CO)
Functionalization of the SPIO@SiO NPs with [Re(phen)(CO) (pyꢀupts)]OTf and
2
3
2
NPs was treated with an ethanol solution of
3
(pyꢀupts))]OTf (310 µL, containing ca. 1 mg of complex, see ESI†)
and a few drops of aqueous NH (28%), as catalyst. The suspension was refluxed for 5 h, by
3
magnetic stirring under nitrogen, then the mixture was cooled to room temperature, and treated with
2 3
H O (4 mL), aqueous NH (28%, 600 µL) and PEG500ꢀSilane (60 µL, ca 0.126 mmol, see ESI†).
The reaction was allowed to proceed under magnetic stirring for 48 h. Then, the NPs were isolated
by centrifugation (15 min at 7550 g) and finally suspended in 20 mL of milliQ water and stored at 4
6

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ꢀ1
°
C for further uses. FTIR (water) ν(CO): 2033 (vs), 1924 (br, s) cm . The content of Fe and Re in
the mother solution was determined by ICPꢀAES on a sample digested as follows: 1.00 mL of the
NP suspension was dried on a sand bath, added with 1 mL of a 70:30 mixture of concentrated HCl
and HF (36% and 48%, respectively), and allowed to react for 30 min under sonication in a Teflon
vial at room temperature. The excess of HF was quenched by addition of H
3 3
BO (2 mmol). Then, a
mixture of concentrated HNO and H O (1:2 ratio, 1.5 mL) was added and left to react overnight at
3
2
2
ꢀ4
room temperature. [Fe] from ICPꢀAES = 1.2 × 10 M (6.7 ꢀg/mL Fe); [Re] from ICPꢀAES = 3.8 ×
ꢀ
5
1
0 M.
2
.4.
Photochemical stability test on [Re(phen)(CO)
3
(pyꢀupts)]OTf.
A
sample of
[
Re(phen)(CO)
3
2
(pyꢀupts)]OTf was dissolved in water saturated with O . The solution was exposed
2
to visible light (by using a 150 W / NDL lamp, 390 nm cutoff filter, 142 mW/cm see Fig S1†), and
UV−vis absorption spectra were recorded every 15 min for 1 h (Fig. S2†).
2
.5.
Singlet oxygen production. In a quartz cuvette 0.555 mL of the above described mother
ꢀRe@PEG NPs were added to 1.70 mL of a H O/MeOH 80:20 mixture,
affording a [Re] = 1×10 M. Subsequently 1,5ꢀdihydroxynaphthalene (DHN) (0.25 mL of a MeOH
solution of SPIO@SiO
2
2
ꢀ
5
ꢀ3
ꢀ4
solution 1×10 M) was added to give a [DHN]= 1×10 M. Oxygen was bubbled in the cuvette for
0 min. The solution was then irradiated with visible light (λ > 390 nm) and the reaction was
followed by acquiring UVꢀVis absorption spectra at different times. The same procedure was
followed for the photoreactions involving other photosensitizers, i.e. [Re(phen)(CO) (py)]OTf
1×10 M), and methylene blue (MB, 8×10 M, the much lower concentration being necessary to
balance the much higher molar absorptivity of MB).
.6. Cell uptake and cytotoxicity in dark and light conditions. SPIO@SiO
1
3
ꢀ5
ꢀ7
(
2
2
ꢀRe@PEGꢀNPs
were sonicated 30 minutes at 40 kHz (Sonica – Soltec) just before the use in order to obtain a
homogenous dispersion and then different volumes of the stock suspension were added directly in
the culture medium to obtain the working concentrations. The alveolar epithelial cells A549 from
7

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human lung carcinoma, purchased from ECACC (European Collection of Cell Cultures), were
routinely maintained in OptiMEM 10% FBS, at 37°C, 5% CO and seeded in 6 multiꢀwell plates for
2
the cell uptake assays. Images of treatedꢀ cells were recorded after exposure for 4h or 24h to: a) 50
ꢁL of a 760 ꢀg/mL suspension of NPs affording a NP concentration of 19 ꢀg/mL, corresponding to
ꢀ4
a [Re] = 0.95 ꢀM in the well; b) 50 ꢁL of a water solution (3.2 × 10 M) of the precursor molecule
[
Re(phen)(CO) (pyꢀupts)]OTf, affording a final [Re] = 8.0 ꢀM in the well. Two photon excitation
3
at 840 nm was exploited (with an excitation power on the sample of about 15 mW).
The A549 cells were also seeded in 12 multiꢀwell plates for cell viability evaluation. Cells were
exposed to increasing concentrations of NPs (1.9; 9.5; 19; 38; 76 ꢀg/mL) and then incubated for 4h
at 37 °C, 5% CO to allow NP internalization. At the end of this preꢀincubation period the A549
2
ꢀ2
cells were irradiated with the 150 W/NDL lamp light (200 mW cm ) for 10 min in order to
photoactivate the internalized nanostructured compound and then they were incubated again for 3 h
or 16 h. Not irradiated cells exposed at the same concentrations of NPs were used as reference (dark
conditions). At the end of the exposure, cells were rinsed with PBS and the working MTT solution
prepared in culture medium at a concentration of 0.3 mg/mL was added. After 2h the MTT solution
was removed and the purple MTT reduction product (formazan salts) was solubilized with DMSO.
The optical density of each sample, proportional to cell viability, was measured at 570 nm using
6
90 nm as reference wavelength with a multiplate reader. The experiments were replicated three
times and results were expressed as mean±SE. Statistical differences were verified using One way
ANOVA followed by Fischer LSD test (p < 0.05).
8

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3
.
Results and discussion
3
.1.
Synthesis of the complex [Re(phen)(CO)
3
(pyꢀupts)]OTf. As discussed in the
+
Introduction, a [Re(phen)(CO)
3
(py)] complex has been chosen as luminescent probe and singlet
oxygen photosensitizer. In order to avoid its slow release from the inorganic matrix, the pyridine
ligand was functionalized with a triethoxysilane moiety, to covalently bind the complex to the silica
4
6
shell. The ligand 1ꢀ(pyridinꢀ4ꢀyl)ꢀ3ꢀ(3ꢀ(triethoxysilyl)propyl)urea, hereafter pyꢀupts, was obtained
4
7
as depicted in Scheme 1, using a slightly modified literature procedure.
Scheme 1. Reaction scheme for the synthesis of the pyꢀupts ligand.
Then the [Re(phen)(CO)
3
(pyꢀupts)]OTf complex was prepared, by replacing with pyꢀupts the
4
8
labile triflate ligand in the precursor [Re(phen)(CO)
3
(OTf)] complex (Scheme 2). The reaction
was carried out in anhydrous ethanol, to avoid the early hydrolysis of the three ethoxy groups of the
1
silane moiety, and its progress was monitored by H NMR (Fig. S3†). The entry of pyridine in the
ligand sphere was accompanied by the progressive shift of the photoluminescence, from a faded
orange to a brilliant yellow color.
3
Scheme 2. Synthetic route to the complex [Re(phen)(CO) (pyꢀupts)]OTf.
9

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3
.2.
2
Synthesis of the magnetoꢀoptical SPIO@SiO ꢀRe@PEG nanocomposites. The synthesis
5
0
of iron oxide NPs stabilized by an oleic acid shell was performed by a literature procedure,
involving thermal decomposition of iron oleate in a high boiling solvent (here 1ꢀoctadecene). NPs
with a diameter size distribution centered at 10.4 ± 1.3 nm (from DLS analysis, Fig. S4†) were
obtained. The transmission electron microscopy (TEM) images (Fig. 1) were in good agreement
with the DLS data, showing a size distribution of the magnetic core centred at 9.4
±
1.3 nm.
5
1,52
The silica shell was grown in a reverse microemulsion environment,
and the reaction was
stopped after 6 hours, when DLS indicated the prevalence of NPs with a hydrodynamic diameter of
about 45 nm (Fig. S5†).
3
The luminescent [Re(phen)(CO) (pyꢀupts)]OTf complexes were then anchored to the silica shell,
by treating the SPIO@SiO NPs, dispersed in ethanol, with an ethanol solution of the complex, as
2
depicted in the second step of Scheme 3. The mixture was refluxed for 5 h, and the precipitate was
recovered by centrifugation (see Experimental part). The solid was strongly luminescent and was
also attracted by an external permanent magnet (Fig. S6†), thus immediately indicating the
formation of a dual magnetoꢀluminescent nanoꢀsystem (as subsequently demonstrated by the
detailed characterization described in the following sections).
O
Re(phen)(CO)3(py-upts)+
Ethanol / NH3(aq)
PEG500-Silane
O
O
TEOS / NH3(aq)
Si
NC
H
O
O
OH
44
O
Ethanol / NH3(aq)
Cyclohexane / Igepal-CO520
SPIO@SiO2
SPIO@SiO -Re
2
SPIO@SiO -Re@PEG
2
SPIO@OA
Oleic acid
=
Rhenium complex
=
2
Scheme 3. Schematic steps diagram of the synthesis of PEGꢀcapped magnetoꢀluminescent SPIO@SiO ꢀRe@PEG NPs.
Attempts to insert the luminescent probe directly in the waterꢀinꢀoil microemulsion, to condense
its silane terminals to the SPIO@SiO nanoparticles inside the nanoreactor, failed, since significant
2
aggregation was observed, likely due to destabilization of the microemulsion after the treatment
with the ethanol solution of the Re complex.
Upon water addition the SPIO@SiO
2
ꢀRe NPs were easily suspended, but the stability of the
colloidal dispersion was poor and in few hours the NPs completely settled. For this reason, and also
1
0

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to make them stealth to the reticuloꢀendothelial system for any in vivo future use, the NPs were
stabilized by capping their surface with short polyethylene glycol molecules (last step in Scheme 3),
previously functionalized with a triethoxysilane group through a carbamate link (Scheme 4 and Fig.
5
3
S7†). After the coating with the PEG ꢀsilane, the
ζ
potential of the NPs changed from ꢀ20 mV to
5
00
0
mV and the hydrodynamic diameter increased from 45 nm to about 80 nm (Fig. S8†). Moreover,
the thermogravimetric analysis of the nanocomposite (Fig. S9†) revealed an overall loss weight of
about 60%, ascribable to PEG and to the ligands on the rhenium complex.
The presence of the polymer shell strongly improved the stability of the colloid and reduced the
formation of irreversibly aggregated NPs.
Scheme 4. The preparation of the PEG silanized by formation of a carbamate link.
2
The morphology of the magnetoꢀluminescent SPIO@SiO ꢀRe@PEG nanocomposite was
analyzed by transmission electron microscopy (Fig. 1), which revealed a spherical form and a single
magnetic core per core/shell nanocomposite. The size distribution of the nanocomposite showed a
single population centered at 40 ± 3.5 nm.
The amount of Re on the NPs was estimated by ICPꢀAES analysis, corresponding to a loading of
ca. 6000 Re atoms per NPs. This loading well compares with the one obtained for related
luminescent complexes covalently embedded into silica NPs with diameters of ca. 60 nm (4600 Ru
54
atoms per NP).
1
1

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1
1
20
00
8
6
4
2
0
0
0
0
0
0
5
10
15
20
diameter (nm)
50 nm
18
16
14
12
10
8
6
4
2
0
0
20
40
60
80
diameter (nm)
100 nm
Fig. 1. TEM micrograph of the SPIO@OA nanoparticles (up) and of the core/shell SPIO@SiO
down), with the corresponding size distribution histograms.
2
ꢀRe@PEG nanoparticles
(
3
.3.
Re@PEG NPs. The photophysical characterization of the free complex [Re(phen)(CO)
upts)]OTf and of the corresponding SPIO@SiO ꢀRe@PEG nanocomposite was performed in dilute,
Photoluminescence properties of the precursor Re complex and of the SPIO@SiO
2
ꢀ
3
(pyꢀ
2
−
5
air equilibrated, water solution (about 1.0 × 10 M), as well as in the solid state. In order to
evaluate the influence of the urea link in the para position of the pyridine ligand, the photophysical
properties of the precursor complex were compared with those of the already known
16
[
3
Re(phen)(CO) (py)]OTf complex containing the unsubstituted pyridine ligand. The most relevant
photophysical data are listed in Table 1 and the corresponding absorption and emission spectra are
displayed in Fig. 2.
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2

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The UVꢀVis absorption spectra of the free complexes and the NPs showed a strong band at
about 275 nm, ascribed to ligandꢀcentered (LC) π−π* transitions, and a weaker broad band,
centered at about 370 nm. The latter band is ascribable to a spin allowed dπ(Re) → π*(phen) metalꢀ
1
toꢀligand charge transfer transition ( MLCT), by comparison with analogous cationic Re(I)
15,16
tricarbonyl complexes.
2.50
2.00
1.50
1.00
0.50
0.00
1.20
a)
b)
[
Re(CO)3(Phen)( Pp Uy -Pu Tp St )s] )O] OT Tf f
1
0
0
0
0
.00
.80
.60
.40
.20
Fe S3 POI O4 @SiO2-Re-PEG
0.00
1
90
390
590
790
430
530
630
λ (nm)
730
λ (nm)
Fig. 2. a) UV−vis absorption and b) normalized photoluminescence spectra (λex= 375 nm) of [Re(phen)(CO)
(pyꢀ
3
upts)]OTf (black dashed traces) and SPIO@SiO
temperature.
2
ꢀRe@PEG (gray traces) in air equilibrated water solution at room
Upon optical excitation the free complexes displayed a broad and featureless emission band in
the greenꢀyellow region of the visible spectrum, arising from an excited state that can be described
12
as a triplet MLCT state. This was confirmed by the quenching of the emission on going from deꢀ
aerated to air equilibrated solution (yields dropping from 0.117 to 0.070 for
[
Re(phen)(CO)
3 3
(py)]OTf, and from 0.053 to 0.038 for [Re(phen)(CO) (pyꢀupts)]OTf) and by the
solventꢀdependence of the emission maximum, which results redꢀshifted by about 20 nm on going
1
6
from CH
Re(phen)(CO)
The MLCT nature of the excited state was further supported by the influence of the pyridine
2
Cl
2
(
λ
= 544 nm)
to water (λ = 567 nm, Table 1) for the complex
[
3
(py)]OTf.
substituents on the absorption and emission spectra. Actually, the introduction of the electronꢀ
donating (by resonance effect) urea link in the para position of the pyridine ligand, afforded a redꢀ
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shift of about 13 nm of the emission maximum of [Re(phen)(CO)
3
(pyꢀupts)]OTf with respect to
[
Re(phen)(CO) (py)]OTf. This is in line with the presence of the electronꢀricher pyꢀupts ancillary
3
ligand, 55 which raises the HOMO level, then lowering the energy of the MLCT transition. The red
shift of the emission energy, observed for the complex [Re(phen)(CO) (pyꢀupts)]OTf, was
) and of the lifetime
3
accompanied by a slight decrease of the photoluminescence quantum yield (
Φ
of the excited state (Table 1), in agreement with the Energy Gap Law.56
The photophysical properties of the complexes were investigated also in the solid state, showing
a blue shift of the emission maxima with respect to the solution, in agreement with the
57
rigidochromic effect usually observed for mononuclear rhenium complexes. An increase of the
Φ
values was also observed, indicating that here the quenching processes often observed in the solid
state do not occur, and the dominant effect is the decrease, in the rigid environment, of the rotoꢀ
vibrational motions, responsible for the nonꢀradiative deactivation pathways of the excited
17a,c
states.
Some interesting differences in the photophysical behavior were observed on going from the
molecular complexes to the NPs. The emission maximum of the NPs in water solution was
+
considerably blueꢀshifted with respect to the corresponding [Re(phen)(CO)
3
(pyꢀupts)] complex in
the same conditions, to a value very similar to the one measured for this complex in the solid state.
Moreover, for the NPs the excited state lifetimes were longer and the emission quantum yields
higher than the values measured for the corresponding pyꢀupts complex in solution. This can be
attributed both to the confinement of the complex in the rigid silica environment and to its poor
exposition to the solution, hampering the deactivating action of water. However, the complex in the
(porous) silica shell remained sensitive to oxygen presence, as indicated by its ability to generate
singlet oxygen (see paragraph 3.6).
The two excited state lifetimes observed for the NPs, as well as for the complexes in the solid
state, result from the presence of complexes experiencing different environments, with different
available nonꢀradiative deactivation pathways.
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Table 1. Photoluminescence data for the molecular Re complexes and for the SPIO@SiO ꢀRe@PEG NPs,
2
in aerated water solution and in solid state (room temperature, λex = 375 nm).
Compound
Water solution, air
solid
τ / ns
λ
em/ nm
τ / ns
Φ
λem/ nm
Φ
7
40 (46%)
[
Re(phen)(CO)
3
(py)]OTf
567
580
553
454
0.070
0.038
0.060
530
553
0.158
0.119
2570 (54%)
9
93 (30%)
[
Re(phen)(CO)
3
(pyꢀupts)]OTf
257
2032 (70%)
4
1
09 (22%)
738 (78%)
2
SPIO@SiO ꢀRe@PEG NPs
1
3
.4.
transverse relaxation times of the SPIO@SiO
temperature in the frequency range to cover most of the typical fields for MRI tomographs, used
Relaxometry properties of SPIO@SiO
2
ꢀRe@PEG NPs. The H NMR longitudinal and
2
ꢀRe@PEG nanoparticles were measured at room
5
8
both in clinics (H = 0.2, 0.5, and 1.5 T) and in research laboratories, as specified in the
experimental part. The efficiency of the contrast agents was evaluated in the usual way: the nuclear
1 2
relaxivities, both r and r , were calculated as the inverse of the relaxation times normalized for
contrast agent concentration, i.e. the concentration of the magnetic part, once previously corrected
for the host diamagnetic contribution, according to equation 2.
r
i
= [(1/T
i
)
meas ꢀ (1/T
i
)dia]/c , i = 1, 2
(2)
where (1/T
i
)meas is the measured value on the sample with iron concentration c = 0.119 mmol/L,
and (1/Ti)dia refers to the nuclear relaxation rate of the milliQ water used as host solution.
Figure 3 shows the r
1
and r
2
nuclear relaxivities of SPIO@SiO
2
ꢀRe@PEG nanoparticles together
values for υ> 5MHz

with the values for the commercial contrast agent Endorem . The displayed r
2
ꢀ1
ꢀ1
(r
2
> 30 s mM ) guarantee an appreciable contrast in the MR images once the nanoparticles are
used, as some of us already demonstrated on other T and/or T relaxing nanoparticles of
1
2
5
9
completely different types.
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5

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Fig. 3. Longitudinal (top) and transverse (bottom) relaxivities of SPIO@SiO ꢀRe@PEG nanoparticles (red circles)
2
dispersed in water. Data are compared with the commercial compound Endorem® values (black squares).
3
.5.
2
Singlet oxygen generation by SPIO@SiO ꢀRe@PEG nanoparticles. The photochemical
stability of the photosensitizer was preliminarily tested, by irradiating for 1 h a sample of the free
complex [Re(phen)(CO) (pyꢀupts)]OTf, dissolved in water saturated with O (Fig. S2†). The
3
2
superposition of the absorption spectra recorded at different times indicated the high photostability
of the photosensitizer in these conditions.
The efficiency of the SPIO@SiO
2
ꢀRe@PEG nanoparticles toward singlet oxygen generation was
1
assessed by using 1,5ꢀdihydroxynaphthalene (DHN) as indirect marker of the O
2
presence.
1
Actually, DHN reacts quantitatively with O to give the oxidized species juglone (5ꢀhydroxyꢀ1,4ꢀ
2
3
5,6061
naphtalenedione, Scheme 5), according to equation 3.
1
3
DHN + O → Juglone + ½ O
(3)
2
2
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6

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Scheme 5. Photochemical oxidation of DHN to Juglone in the presence of a photosensitizer (PS).
1
Thus, the O
2
production was indirectly monitored by following the decrease of the UVꢀVis
absorption band of DHN at 297 nm (accompanied by the increase of the juglone band at 427 nm).
Fig. 4A shows the spectra recorded for the first 60 min of irradiation of a sample of NPs suspended
in a MeOH/H O (80:20) mixture, in the presence of DHN.
2
The reaction in the initial stages follows a pseudoꢀfirst order kinetics, ν = kobs[DHN], provided
61
that the oxygen concentration within this interval of time can be considered constant. By plotting
as a function of the irradiation time the values of ln(A/A ) for the DHN absorption at 297 nm (Fig.
B), the kinetic constant kobs for reaction 3 can be estimated.
The reaction was also carried out employing as photosensitizers the precursor complex
Re(phen)(CO) (pyꢀupts)]OTf, or the analogue [Re(phen)(CO) (py)]OTf complex (for comparison),
0
4
[
3
3
and methylene blue (MB) as standard.
The quantum yields for singlet oxygen generation (Φ
ꢁ
) were determined using equation 4:
std
ꢁ
std
std
i
Φ
ꢁ
= Φ
×
(ν
i
I / ν
I) (4)
where ν
i
is the initial rate of reaction (3), I indicates the photons absorbed by the sensitizer, and the
= 0.50 in
apex std labels the corresponding values for the standard (methylene blue in our case, Φ
ꢁ
ꢀ4
62
MeOH). The values of ν were provided by the product k [DHN] ([DHN] being 1×10 M),
i
obs
i
i
ꢀA( )
λ
while the values of I were estimated by numerical integration of Isource(λ)(1ꢀ10
), where Isource(λ)
is the intensity of the incident light at different wavelengths and A(λ) is the corresponding
absorbance of the considered sensitizer.
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1
.4
.2
1
A
1
0.8
0.6
0.4
0.2
0
2
50
450
650
λ / nm
0
0.02
0.04
0.06
0.08
B
-
-
-
-
-0.1
-0.12
-0.14
-0.16
-0.18
-0.2
0
5
10
15
20
25
t (min)
Fig. 4. A) UV−Vis absorption spectra recorded at different irradiation times (from 0 to 360 min, λ > 390 nm) on a
ꢀ
4
solution containing SPIO@SiO
bubbled with O for 10 min. B) Photoꢀoxidation of DHN in the presence of different sensitizers: methylene blue (∆),
Re(phen)(CO) (py)]OTf (O), [Re(phen)(CO) (pyꢀupts)]OTf (ꢀ), and SPIO@SiO ꢀRe@PEG NPs (◊). A and A
0
2
2
ꢀRe@PEG NPs (150 ꢀg/mL) and DHN (10 M) in 2.5 mL of MeOH/H O (80/20)
2
[
3
3
2
t
represent the absorbance measured at 297 nm (the maximum of the DHN absorption band) at time t and time 0,
respectively.
It must be pointed out that the Φ values estimated for dyes entrapped into nanoꢀsupports are
ꢁ
affected by significant uncertainties, because of the extinction arising from light scattering (which
6
3
varies with wavelength and can be only partly corrected) and also because of many other possible
matrix effects (including here the absorption of the iron oxide core), which are difficult to
6
4
accurately evaluate. All these contributions to the absorption spectra do not contribute to the
excitation of the sensitizer, and therefore their inclusion in the computation of the I value can lead
to severe underestimation of the quantum yields. For these reasons we preferred to obtain an
approximate value of the photons absorbed by the rhenium dye in the NPs by using the absorption
spectrum of the free complex, corrected for the different concentrations in the two solutions (that
were known from the ICP analysis of the rhenium content).
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The Φ
ꢁ
values estimated in this way for the SPIO@SiO
2
ꢀRe@PEG NPs resulted very similar to
the ones evaluated for the free Re complexes (0.26 – 0.29, see Table 2), which well compare with
2
0,65
those reported for analogous tricarbonyl Re complexes (0.20 – 0.26) in water solution.
Due to
the approximations involved in the evaluation of the light absorbed by the Re complexes on the
NPs, we cannot rule out that the actual quantum yields were somewhat lower than reported in Table
3
2
. In fact some decrease of the Φ
ꢁ
value might result from the fact that O
2
has to diffuse across the
NP coating in order to quench the triplet excited state of the anchored dyes and from the possibility
1
of entrapment of generated O
2
in the NP matrix. However, the significant point is that the NPs do
= 0.05) was computed
maintain a significant sensitizing activity. A lowest limit to this activity (Φ
ꢁ
using the unprocessed absorption spectra of the SPIO@SiO ꢀRe@PEG NPs. i.e. the spectrum
2
including all the ineffective contributions.
This is remarkably different from what observed for a very effective sensitizer as MB, for which
1
a strong decrease in the O
2
release (about two order of magnitude) when entrapped in a silica layer
6
7
was measured in a previous work. This might be due to some tripletꢀtriplet annihilation process,
arising from the close spatial confinement of a large number of emitters with very long excited state
lifetimes, as it is typical of the triplet states of organic dyes. On the contrary, for the rhenium
complex, which has a relatively short lifetime of the triplet state, the efficiency in singlet oxygen
generation was maintained.
1
Table 2. Pseudo firstꢀorder kinetics parameters and O
2
generation quantum yields for the photooxidation of
ꢀRe@PEG nanoparticles, in
DHN using the standard MB, the molecular complexes and the SPIO@SiO
2
ꢀ4
oxygenated water solution. Initial DHN concentration = 1×10 M. The values for the NPs are in parentheses
because evaluated by the approximation described in the text.
k
obs
I
3
ꢀ1
ꢀ1
Φ
ꢁ
×
10
(
min )
(mW cm )
4.63
Methylene Blue
17.8
5.30
2.91
0.50
0.26
0.29
[
[
Re(phen)(CO) (pyꢀupts)]OTf
2.64
1.31
3
Re(phen)(CO)
3
(py)]OTf
SPIO@SiO
2
ꢀRe@PEG NPs
0.925
(0.57)
(0.21)
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3
.6.
Cell uptake and PDT cytotoxicity of the SPIO@SiO
2
ꢀRe@PEG nanoparticles. The cell
uptake of the nanocomposites was preliminarily investigated, to find the time scale useful for the
PDT assays. The uptake by A549 cells was monitored by confocal microscopy, exploiting the
photoluminescence of the Re complexes, and in particular the possibility to promote their emission
6
8
by twoꢀphoton excitation (TPE). TPE microscopy (as described first by Denk et al.) uses nearꢀ
infrared laser light to excite chromophores with a (nonꢀlinear) quadratic dependence of the
excitation probability from the laser power, inducing optical sectioning without the need of a
pinhole (spatial filter). It presents many advantages for in-vivo applications: the nearꢀinfrared light
displays a deeper penetration in highly scattering tissues, and it induces a much lower photodamage
of the sample with respect to UV radiation. The intrinsic optical sectioning, moreover, limits the
possible phototoxic effect to the focal plane only, and increases the detection efficiency of the
whole system, since it does not require descanning optics nor pinholes in the revelation path,
allowing the use of large active area detectors.
Cells were incubated either with the molecular probe [Re(phen)(CO)
SPIO@SiO ꢀRe@PEG NPs, at different concentrations (see Experimental), and observed after both
h and 24 h through TPE confocal microscopy. Nuclei were stained with the viable nuclear dye
3
(pyꢀupts)]OTf or with the
2
4
Hoechst (blue), just before the microscopy evaluation.
The internalization of the free Re complex was hampered by its low solubility in aqueous media,
which led to the formation of microsized aggregates, well visible in the images acquired after 4 h of
incubation (Fig S10A†). However, a moderate cell uptake was observed at longer times (24 h, Fig
S10B†), and the complexes accumulated in the cytoplasm, forming small endosomalꢀlike clusters.
On the contrary, the nanoparticles were more easily internalized and accumulated in the
perinuclear region of the cells already after 4 h of incubation (see Fig. 5B and the movie of a zꢀscan
images sequence, downloadable from ESI†), although their persistence in the cytoplasm at longer
times was not confirmed. The feeble luminescence observed after 24 h (Fig. 5C) was mainly
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localized outside the cell membranes, suggesting that most of the probes had been excreted by the
cells. On the bases of these results an incubation time of 4 h was set for the cytotoxicity assays.
A
B
C
Fig. 5. TPE microscopy images, result of the projection and superposition on the xy plane of 10 single planes acquired
along the z axis at 0.5 ꢁm steps, of A549 cells incubated with SPIO@SiO ꢀRe@PEG NPs (19 ꢀg/mL, corresponding to
Re] = 0.95 ꢀM) for 4 h (Panel B) and 24 h (Panel C). Panel A shows untreated A549 cells used as control for the same
experiment. The blue color is due to the staining of nuclei with HOECHST.
2
[
The cytotoxic effect of the SPIO@SiO ꢀRe@PEG NPs was assessed both in dark and light
2
conditions, to investigate the ability of the NPs to induce an increase in cell death when photoꢀ
activated. Cells were incubated with different concentrations of NPs for 4 h, then were either
irradiated for 10 min with light from a 150 W NDL lamp or kept in the dark for the same time, and
finally incubated again for 3 h or 16 h. At the end, the cell viability was evaluated by the MTT test
(see Experimental part).
The results are summarized in Fig. 6. After 3 h and 16 h post irradiation (p.i.), a dose dependent
effect induced by the NPs was observed, with a statistically significant increase in cell mortality
after irradiation of the cells, compared to the dark exposure, testifying for an effective
photodynamic response.
The slight cytotoxic effect observed for cells exposed to the highest NP concentrations in the
dark should be attributed to the whole nanostructure, rather than to the sensitizer (i.e. the Re
3
complex). Indeed the [Re(phen)(CO) (pyꢀupts)]OTf complex alone did not induce any cytotoxicity,
as shown by viability tests performed by exposing A549 cells for different times (4h or 24h) to
increasing concentrations of the complex (see Fig. S11†).
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3
h (p.i) DARK
3h (p.i.) LIGHT
1
1
20
00
* #
*
*
#
*
8
6
4
2
0
0
0
0
0
*
*
0
1.9
9.5
19
38
76
SPIO@SiO -Re@PEG NPs (ꢀg/mL)
2
1
6h (p.i.) DARK
#
16h (p.i.) LIGHT
1
1
20
00
*
#
*
*
80
60
40
20
0
*
*
#
*
#
*
0
1.9
9.5
19
38
76
SPIO@SiO -Re@PEG NPs (ꢀg/mL)
2
Fig. 6. Cell viability (MTT assay) of A549 cells exposed to SPIO@SiO
2
ꢀRe@PEG NPs for 3h and 16h post irradiation
(p.i.). Black bars=dark conditions; Grey bars=light conditions: cells were exposed to NPs for 4h, irradiated for 10 min
#
and incubated for 3h or 16h again. *Significantly different from control; Significantly different light vs dark conditions
ANOVA + Fischer LSD test,p < 0.05).
(
4
.
Conclusions
A multifunctional nanocomposite, designed for being a bimodal imaging probe that can be
exploited also for PDT, has been prepared and characterized. As shown by the results, each
component of the nanocomposite preserved the ability to perform its function. The
superparamagnetic iron oxide cores, coated by the thick layer of silica and by PEG, to improve
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stability and stealthiness in physiological conditions, remained able to act as T2 agents for MRI,
providing a good contrast ability, as demonstrated by the relaxivity profiles.
At the same time, the luminescence properties, as lifetimes and photoluminescence quantum
yields, were even improved by the entrapment of the emitting rhenium complex in the silica shell,
due to increased rigidity of the environment and to reduced interaction with the water. Finally, the
1
Re complexes anchored to the NPs were still able to act as effective O
2
photosensitizer.
Preliminary investigation has revealed easy cellular uptake and increased cytotoxicity upon
irradiation with respect to the dark, even if with a low efficiency. This might be related to the NP
localization at subcellular level, which is known to have strong influence on cell response to
2
1,24,69
photoactivation.
Further studies on the internalization mechanism and on the subcellular
localization will be necessary to assess the true potential of these nanocomposites. In a parallel way
the performances of the diagnostic and therapeutic functionalities integrated in the SPIO@SiO2ꢀ
Re@PEG NPs can be optimized: the T2 contrast ability would benefit from a larger size of the
superparamagnetic core and a smaller thickness of the silica shell, while the luminescence
1
properties as well as the capability of O
2
generation would be improved by an increased Re
loading.
5
.
Acknowledgements
The use of instrumentation purchased through the Regione Lombardia – Fondazione Cariplo joint
SmartMatLab Project (2013ꢀ1766) is gratefully acknowledged. The authors also thank INSTMꢀ
Regione Lombardia (project MAGNano) for funding.
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1
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pp. 341–391.
1
2 For some pertinent reviews, see: (a) A. Kumar, S.ꢀS. Sun, A. J. Lees, Top. Organomet. Chem.,
010, 29, 1−35. (b) D. R. Striplin, G. A. Crosby, Coord. Chem. Rev., 2001, 211, 163−175. (c) D. J.
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Jr. Stukfens, A. Vlček, Coord. Chem. Rev., 1998, 177, 127−179. (d) A. J. Lees, Chem. Rev., 1987,
8
1
2
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5b
+
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8 Rillema et al.
bidentate ligand, and L’ is a py like ligand) by a route that involves the tetracarbonyl intermediate
Re(LꢀL)(CO) ]OTf, formed by stirring the precursor Re(CO) OTf at room temperature in a nonꢀ
3
obtained [Re(LꢀL)(L’)(CO) ] complexes (where LꢀL is a bpy or a phen
[
4
5
+
coordinating solvent, in the presence of the bidentate ligand LꢀL. The [Re(LꢀL)(L’)(CO)
3
] complex
was subsequently obtained by the addition of one equiv of the L’ ligand and refluxing for several
49
hours. Another synthetic strategy proposed by Wrighton et al. consisted in the substitution of the
ꢀ
Cl ligand with a pyridine in the precursor ReCl(CO)
3
(LꢀL), by refluxing in the presence of AgOTf.
We obtained the desired product by firstly preparing the precursor complex [Re(phen)(CO) (OTf)]
3
ꢀ
and then by easily substituting the OTf anion with the pyꢀupts ligand (Scheme 2).
2
8

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