Aluminum Doped MCM-41 Nanoparticles as Platforms for the Dual Encapsulation of a CO-Releasing Molecule and Cisplatin

Article abstract of DOI:10.1021/acs.inorgchem.7b01475

Mesoporous silica Al-MCM-41 nanoparticles have been used, for the first time, as vehicles for the single and dual encapsulation of the cationic CO-releasing molecule (CORM) [Mn(1,4,7-triazacyclononane)(CO)₃]⁺ (ALF472⁺) and the well-known antineoplastic drug, cis-[PtCl₂(NH₃)₂] (cisplatin). Thus, two new hybrid materials, namely, ALF472@Al-MCM-41 and ALF472-cisplatin@Al-MCM-41, have been isolated and fully characterized. The results reveal that the presence of CORM molecules enhances cisplatin loading 3-fold, yielding a cargo of 0.45 mmol g^-1 of ALF472⁺ and 0.12 mmol g^-1 of the platinum complex for ALF472-cisplatin@Al-MCM-41. It is worth noting that ALF472@Al-MCM-41 shows a good dispersion in phosphate buffered saline solution, while the dual hybrid material slightly aggregates in this simulated physiological medium (hydrodynamic size: 112 ± 23 and 336 ± 50 nm, respectively). In addition, both hybrid materials (ALF472@Al-MCM-41 and ALF472-cisplatin@Al-MCM-41) behave as photoactive CO-releasing materials, delivering 0.25 and 0.11 equiv of CO, respectively, after 24 h and exhibiting a more controlled CO delivery than that of the free CORM. Finally, metal leaching studies have confirmed the good retention capacity of Al-MCM-41 toward the potentially toxic manganese fragments (86% of retention after 72 h) as well as the low release of cisplatin (ca. 7% after 72 h).

Full text of DOI:10.1021/acs.inorgchem.7b01475

Article
pubs.acs.org/IC
Aluminum Doped MCM-41 Nanoparticles as Platforms for the Dual
Encapsulation of a CO-Releasing Molecule and Cisplatin
†
†
,†
†
‡,§
Francisco J. Carmona, Ignacio Jimen
́
ez-Amezcua, Sara Rojas, Carlos C. Romao,
̃
Jorge A. R. Navarro,† Carmen R. Maldonado,* and Elisa Barea*
,†
†
Department of Inorganic Chemistry, University of Granada, Av. Fuentenueva S/N, 18071 Granada, Spain
‡
ITQB NOVA, Instituto de Tecnologia Química e Biolog
́
ica Anton
Alfama Ltd., Instituto de Biologia Experimental e Tecnologica, IBET, Av. da Repub
S Supporting Information
́
́
io Xavier, Av. da Republica, 2780-157 Oeiras, Portugal
§
́
́
lica, 2780-157 Oeiras, Portugal
*
ABSTRACT: Mesoporous silica Al-MCM-41 nanoparticles have been
used, for the first time, as vehicles for the single and dual encapsulation
of the cationic CO-releasing molecule (CORM) [Mn(1,4,7-
+
+
triazacyclononane)(CO) ] (ALF472 ) and the well-known antineo-
3
plastic drug, cis-[PtCl (NH ) ] (cisplatin). Thus, two new hybrid
2
3 2
materials, namely, ALF472@Al-MCM-41 and ALF472-cisplatin@Al-
MCM-41, have been isolated and fully characterized. The results reveal
that the presence of CORM molecules enhances cisplatin loading 3-
−1
+
−1
fold, yielding a cargo of 0.45 mmol g of ALF472 and 0.12 mmol g
of the platinum complex for ALF472-cisplatin@Al-MCM-41. It is
worth noting that ALF472@Al-MCM-41 shows a good dispersion in
phosphate buffered saline solution, while the dual hybrid material
slightly aggregates in this simulated physiological medium (hydro-
dynamic size: 112 ± 23 and 336 ± 50 nm, respectively). In addition, both hybrid materials (ALF472@Al-MCM-41 and ALF472-
cisplatin@Al-MCM-41) behave as photoactive CO-releasing materials, delivering 0.25 and 0.11 equiv of CO, respectively, after
24 h and exhibiting a more controlled CO delivery than that of the free CORM. Finally, metal leaching studies have confirmed
the good retention capacity of Al-MCM-41 toward the potentially toxic manganese fragments (86% of retention after 72 h) as
well as the low release of cisplatin (ca. 7% after 72 h).
INTRODUCTION
inorganic platforms are the protection of the carbonyl metal
complexes toward their premature degradation in physiological
media, as well as the trapping of the corresponding toxic
decarbonylation fragments within their matrixes. In this
context, Mascharak et al. published the first example of a
photoCORMA based on a mesoporous silica. In particular, they
■
In spite of the well-known toxicity of CO at medium-high
concentrations, recent investigations have shown that this gas
can be exploited as a therapeutic principle, mainly in
inflammatory processes and cardiovascular diseases as well as
1
in transplantation and preservation of organs. In this context,
used the doped aluminum MCM-41 (Al-MCM-41) as a
the main drawbacks for its practical medical use are derived
from its lack of tissue specificity and the need for in-hospital
controlled administration of the gas. In order to overcome
these issues, different molecules capable of releasing CO
+
nanocarrier of the photoactive CORM, [Mn(pqa)(CO )]
3
(
pqa = (2-pyridylmethyl)(2-quinolylmethyl)amine), demon-
strating the capacity of this hybrid system to relax rat aorta
20
muscle rings in tissue bath experiments. In a later study, they
designed a trackable light triggered CO-delivery material, with
cytotoxic activity against human breast-cancer cells, by
(
CORMs) in a triggered manner have emerged in the past two
2
−6
decades.
In particular, special attention has been drawn to
those showing a turn on−off mechanism of CO delivery in the
4
,7,8
combining the same silica matrix (Al-MCM-41) and the
presence of light.
However, these photoactive CO-releasing
+
rhenium photoCORM, [Re(CO) (pbt)(PPh )] (pbt = 2-(2-
molecules have limited stability in biological media, and their
resulting photoproducts may exert undesirable toxic effects. To
tackle this problem, a wide range of vehicles have been explored
as platforms for these photoCORMs, leading to advanced solids
with CO-releasing properties upon irradiation (photoCORMA-
3
3
21
pyridyl)-benzothiazole). To the best of our knowledge, only
one other paper has been published to date regarding the use of
mesoporous silicas as platforms for the preparation of
CORMAs. Specifically, our group loaded the cationic photo-
CORM [Mn(tacn)(CO) ] (tacn = 1,4,7-triazacyclononane) in
18
+
9
−17
s).
Among these carriers, inorganic porous scaffolds, such as
3
mesoporous silicas and metal−organic frameworks (MOFs),
have recently emerged as promising candidates for this
Received: June 12, 2017
1
8−22
application.
The main advantages of the use of these
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.7b01475
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
the functionalized MCM-41-SO H and SBA-15-SO H silicas.
Scheme 1. Dual Encapsulation Strategy of the Cationic
3
3
+
+
However, the resulting materials showed a low CORM
retention in PBS (phosphate buffered saline), probably due
to cation exchange processes with this physiological buffer.
Therefore, with all these results taken into account, Al-MCM-
CORM ALF472 ([Mn(tacn)(CO) ] , tacn = 1,4,7-
3
triazacyclononane) and the Anticancer Drug Cisplatin (cis-
[PtCl (NH ) ]) in the Aluminum Doped Mesoporous Silica
2
3 2
Al-MCM-41
4
1 can be considered a promising platform for the preparation
of CORMAs, as it is a highly biocompatible mesoporous
material, which exhibits a remarkable stability in physiological
medium, and can be prepared as nanoparticles in a reproducible
manner. These features, together with the anionic nature of
its skeleton, make it a good candidate to encapsulate and
strongly retain the high payload of cationic species, such as
cationic CORMs, as has been previously demonstrated by the
2
3
20,21
Mascharak research group.
In another context, inorganic chemistry has made its most
important contribution toward fighting cancer with the
development of platinum complexes, which currently provide
highly effective chemotherapy for testicular, ovarian, neck,
24
cervical, and esophageal cancers. One of these platinum
complexes, cis-diamminedichloroplatinum(II) (cisplatin) is the
world’s most widely used chemotherapeutic drug and has had a
profound effect on the treatment of testicular cancer, making it
a model for a curable tumor. However, platinum complexes
experience loss of activity and side effects due to poor
circulation and delivery to the tumor, and high reactivity with
other molecules prior to reaching their primary biological target
cetyltrimethylammonium bromide (CTAB) was dissolved at 80 °C
in 480 mL of water and 3.5 mL of 2 M NaOH solution. Afterward, 98
mg of NaAlO and 4.52 mg of tetraethyl-orthosilicate (TEOS) were
2
added, and the reaction was kept at 80 °C for 2 h. The white powder
was filtered, washed with water, and dried at room temperature. The
surfactant was removed by calcination heating at 550 °C for 5 h with a
(DNA). In this context, the use of different biocompatible
−
1
ramp rate of 1 °C min . Anal. Calcd for SiO (AlO Na)0.106-
2
2
vehicles for platinum drugs may allow the active ingredient to
(
C H BrN)
(H O) (Al-MCM-41): C 1.15; H 1.44; N 0.19.
25
19 42
0.0065 2 0.45
reach intact the target cells in need of treatment.
Found: C 1.47; H 1.49; N 0.14. Residue after thermal treatment of Al-
MCM-41: SiO (Al O Na O) calcd 86.8%; found 88.1%. Al/Si
It has also been proven that carbon monoxide can exert a
2
2
3
2
0.053
1
cytoprotective action toward healthy cells, which may be useful
ratio determined by EDX: 0.106.
for the mitigation of undesirable side effects (namely,
cardiotoxicity, nefrotoxicity, etc.) associated with the admin-
istration of chemotherapeutic treatments. Thus, Piantadosi et
al. demonstrated that CO-inhalation ameliorated doxorubicin
Synthesis of cis-[Diamminedichloroplatinum(II)], cis-
[
PtCl (NH ) ] (Cisplatin). The compound was synthesized as
2
3 2
3
0
previously reported. All the manipulations were carried out in the
absence of light. In a typical reaction, 500 mg (1.2 mmol) of K PtCl
2
4
26
was dissolved in 5 mL of water. Then, 828 mg (5 mmol) of KI was
added, and the mixture was stirred for 10 min. Afterward, 0.4 mL of a
cardiomyophaty in mice, while Kim et al. reported the
protective effect of CO in vitro, as an inhibitor of the
mixture of H O/NH3 (1:1) was incorporated, and the resulting
2
2
7
doxorubicin-induced apoptosis in H9c2 cardiomyocytes.
solution was kept under stirring for 30 min. As a result, a precipitate of
28
Additionally, Motterlini et al. confirmed that the coadminis-
tration of cisplatin and CORM-3 decreased the in vitro
cytotoxicity of this antineoplastic agent against LLC-PK1
renal epithelial cells. Likewise, they demonstrated the benefit
of using CORM-3 in vivo, as a therapeutic adjuvant in the
treatment of cisplatin-induced nephrotoxicity.
[PtI (NH )] was obtained, which was filtered, washed with water (3 ×
2
3
5 mL), and collected. Then, a solution of Ag(NO ) (373 mg (2.2
3
mmol) in 10 mL of water) was mixed with 530 mg (1.1 mmol) of
[
1
°
PtI (NH )] and kept at 70 °C for 1.5 h. The solid was filtered, and
2 3
96 mg (2.6 mmol) of KCl was added to the solution and kept at 70
C for 1 h. The yellow precipitate (cisplatin) was filtered, washed with
water (3 × 10 mL), dried with diethyl ether (3 × 10 mL), and
With this background taken into account, in this study we
have taken advantage of the good retention properties of Al-
MCM-41 toward cationic species to prepare, for the first time, a
new CORMA encapsulating two bioactive molecules with
complementary therapeutic properties. With this aim, we have
performed the dual loading in Al-MCM-41 of the water-soluble
collected. Anal. Calcd for cis-[PtCl (NH ) ] (cisplatin): H 2.06; N
2
3 2
9
.34. Found: H 2.14; N 9.13. Residue after thermal treatment of
cisplatin: Pt calcd 65.02%; found 65.07%.
S y n t h e s i s o f [ 1 , 4 , 7 - ( T r i a z a c y c l o n o n a n e ) -
tricarbonylmanganese(I)bromide], [Mn(NC H ) (CO) ]Br
2 4 3 3
31
(
ALF472). The metal complex first described by Pomp et al. was
used as received from Alfama Ltd. Spectroscopic data for the
analytically pure compound not given in the original publication:
and air-stable cationic CO-releasing molecule, [Mn(tacn)-
+
(
CO)3] (tacn = 1,4,7-triazacyclononane), and the well-
−1 1
FTIR (KBr pellets) νCO = 2017 and 1895 cm ; H NMR (400 MHz,
δ ppm) (D O) 2.75 (m, CH ), 3.07 ppm (m, CH ), 6.07 ppm (NH).
known anticancer drug cis-[PtCl (NH ) ], cisplatin (Scheme
2
3 2
2
2
2
1
). In addition, both metal leaching and CO-releasing kinetics
−1
Solubility in water: 5 mg mL . Anal. Calcd for [Mn-
(HNC H ) (CO) ]Br (ALF472): C 31.06; H 4.34; N 12.07. Found:
studies of the resulting hybrid material have been evaluated in
simulated physiological conditions.
2
4
3
3
C 31.41; H 4.04; N 12.65%.
Evacuation/Activation of Al-MCM-41. Prior to the loading of
ALF472, the mesoporous silica material was outgassed (10− bar, 150
°C) for 24 h. Under these conditions, the complete removal of the
solvent guest molecules was achieved, and empty pores ready for drug
adsorption were obtained.
Single Encapsulation of ALF472 into Al-MCM-41. The
incorporation of ALF472 into the mesoporous silica material was
carried out by a solid−liquid impregnation method. In a typical
4
EXPERIMENTAL SECTION
■
All chemicals were commercially available and used without further
purification.
Synthesis of Al-MCM-41. The mesoporous silica Al-MCM-41 was
20
synthesized as previously reported, based on the procedure first
2
9
described by Cai et al.
In a typical synthesis, 1 g of
B
DOI: 10.1021/acs.inorgchem.7b01475
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
experiment, 100 mg of activated Al-MCM-41 (1.45 mmol, equivalent
to 0.15 mmol of exchangeable cationic sites) was suspended in a 10
mL aqueous solution of ALF472 (103.3 mg, 0.30 mmol). The mixture
was stirred and kept at room temperature and in darkness for 1 day in
order to ensure that equilibrium was reached. Afterward, the sample
was centrifuged (3500 rpm/5 min), and the resulting solid was washed
with water (3 × 10 mL) and dried at room temperature. Anal. Calcd
240, 300, 360, 480, 600, 720, and 1440 min) in order to determine the
released amount of CO and the kinetics of the process. At the end of
each experiment, the solutions were bubbled with CO and the
concentration of Mb-CO was calculated as previously reported. All
the experiments were performed in triplicate.
32
Manganese Lixiviation of ALF472@Al-MCM-41 in PBS. A 2.4
mg portion of ALF472@Al-MCM-41 was suspended in 1 mL of PBS
at 37 °C. After incubation in darkness for 72 h, the supernatant was
collected by centrifugation using a Vivaspin device (3500 rpm/10 min,
30 000 MWCO PES membrane). The concentration of Mn in the
supernatant was determined by ICP-MS. Moreover, a similar study
was performed under visible light in order to determine the lixiviation
of the encapsulated metal fragments (CORMs and inactivated
CORMs (iCORMs)) upon irradiation with light. Thus, suspensions
of ALF472@Al-MCM-41 (concentration based on 10 μM of ALF472)
were kept in PBS under visible light (using a fluorescent lamp,
luminous flux, 600 lm; color temperature, 4000 K, distance between
lamp and cuvette, 5 cm) at 37 °C for 72 h. At each time, the
supernatant was collected by centrifugation using a Vivaspin device
(3500 rpm/10 min, 30 000 MWCO PES membrane). The
concentration of Mn in the supernatant was determined by ICP-MS.
Platinum Lixiviation of ALF472-cisplatin@Al-MCM-41 in
PBS. Suspensions of ALF472-cisplatin@Al-MCM-41 (concentration
based on 10 μM of ALF472) were kept in PBS under visible light
(using a fluorescent lamp, luminous flux, 600 lm; color temperature,
4000 K, distance between lamp and cuvette, 5 cm) at 37 °C for 6, 24,
and 72 h. At each time, the supernatant was collected by centrifugation
using a Vivaspin device (3500 rpm/10 min, 30000 MWCO PES
membrane). The concentration of Pt in the supernatant was
determined by ICP-MS.
for SiO (AlO Na)
(AlO [Mn(HNC H ) (CO) ])
(H O)
2
2
0.059
2
2
4
3
3
0.037 2 0.65
(
ALF472@Al-MCM-41): C 4.51; H 2.11; N 1.75. Found: C 4.14;
H 1.82; N 2.00. Residue after thermal treatment of ALF472@Al-
MCM-41: (SiO (Al O ) )(Na O)0.030(MnO)0.037) calcd 78.1%;
2
2
3
0.048
2
found 73.9%. Ratio (EDX) Si/Mn: 0.030.
Dual Encapsulation of ALF472 and Cisplatin into Al-MCM-
1. The incorporation of both drugs was carried out in only one step,
4
by dissolving 15 mg (0.05 mmol) of cisplatin and 53.5 mg (0.15
mmol) of ALF472 in 15 mL of water. Then, 50 mg of activated Al-
MCM-41 (0.72 mmol, equivalent to 0.08 mmol of exchangeable
cationic sites) was added to the solution. The mixture was stirred and
kept at room temperature and in darkness for 1 day in order to ensure
that equilibrium was reached. Afterward, the sample was centrifuged
(
1
3500 rpm/5 min), and the resulting solid was washed with water (3 ×
5 mL) and dried at room temperature. Anal. Calcd for
SiO (AlO Na) (AlO [Mn(HNC H ) (CO) ])0.036[PtCl2-
2
2
0.059
2
2
4
3
3
(
NH ) ] (H O)
(ALF472-cisplatin@Al-MCM-41): C 3.44; H
3
2
0.01
2
1.85
3
.83; N 1.58. Found: C 3.60; H 3.34; N 1.53. Residue after thermal
treatment of ALF472-cisplatin@Al-MCM-41: (SiO (Al O )0.048)-
2
2
3
(
Na O)0.030(MnO)0.036Pt0.01), calcd 63.1%; found 58.0%. Ratio
2
(
EDX) Mn/Pt: 3.00.
Single Encapsulation of Cisplatin into Al-MCM-41. The
incorporation of cisplatin into the mesoporous silica material was
also carried out by a solid−liquid impregnation method. In a typical
experiment, 50 mg of activated Al-MCM-41 (0.72 mmol, equivalent to
Characterization. N adsorption isotherms were measured at 77 K
2
on a Micromeritics Tristar 3000 volumetric instrument. Prior to
measurement the powdered silica sample (Al-MCM-41) was activated
0
.08 mmol of exchangeable cationic sites) was suspended in a 15 mL
−
4
portion of an aqueous solution of cisplatin (15.0 mg, 0.05 mmol). The
by heating at 150 °C for 24 h and outgassing to 10 bar. The infrared
spectroscopy data were collected with a Fourier transform infrared
spectrophotometer Bruker Tensor 25. UV−vis spectra were collected
on a Shimadzu UV spectrophotometer. Elemental (C, H, N, S)
analyses were obtained in a Flash EA1112 CHNS-O (Centre of
mixture was stirred and kept at room temperature and in darkness for
1
day in order to ensure that equilibrium was reached. Afterward, the
sample was centrifuged (3500 rpm/5 min), and the resulting solid was
washed with water (3 × 15 mL) and dried at room temperature. Anal.
Calcd for SiO (AlO Na)
[PtCl (NH ) ]
(C H NBr)0.003-
19 42
Scientific Instrumentation of the University of Jaen
́
). Thermogravi-
2
2
0.106
2
3
2
0.003
(
H O) (cisplatin@Al-MCM-41): C 0.76; H 2.61; N 0.14. Found:
2 1.1
metric analysis were performed using a Mettler Toledo TGA/DSC
−1
C 0.75; H 2.60; N 0.20. Residue after thermal treatment of cisplatin@
Al-MCM-41: (SiO (Al O Na O)0.053Pt0.003), calcd 76.6%; found
STAR system under oxygen flow (20 mL min ) running from room
temperature (RT) to 900 °C with a heating rate of 2 °C min−
(Centre of Scientific Instrumentation of the University of Granada,
Figure S1). Inductively coupled plasma mass spectrometry (ICP-MS)
1
2
2
3
2
7
7.8%.
Hydrodynamic Size and Zeta-Potential Measurements. A 5
mg portion of Al-MCM-41, ALF472@Al-MCM-41, cisplatin@Al-
MCM-41, or ALF472-cisplatin@Al-MCM-41 was suspended in 10 mL
of PBS (10 mM, pH 7.4). The suspensions were homogenized before
DLS and Z-potential measurements by sonication (15 min, pulse on/
off 15 s, amplitude 30%, 20 W).
́
was carried out in an AGILENT 7500a (CICT, University of Jaen).
Energy-dispersive X-ray spectroscopy (EDX) analyses, mapping, and
TEM images were performed using a STEM PHILIPS CM20 HR
microscope equipped with an EDX spectrometer operating at an
accelerating voltage of 200 keV and a HAADF FEI TITAN G2
microscope also equipped with an EDX spectrometer (Centre of
Scientific Instrumentation of the University of Granada). Samples were
prepared by dispersing a small amount of the material (3 mg) in
absolute ethanol (1 mL) followed by sonication for 10 min and
deposition on a copper grid. DLS and Z-potential measurements were
carried out in a Zetasizer NanoZS (Malvern) instrument of the
Department of Applied Physics of the University of Granada.
CO-Delivery Studies. The amount of released CO was monitored
8
,32
as previously reported by others. Specifically, the CO delivery from
free ALF472 and the loaded materials ALF472@Al-MCM-41 and
ALF472-cisplatin@Al-MCM-41 were studied spectrophotometrically
by measuring the conversion of deoxy-myoglobin (deoxy-Mb) to
carbonmonoxy-myoglobin (Mb-CO). The amount of formed Mb-CO
was quantified by measuring the absorbance at 423 nm at different
times. Stock solutions of myoglobin from equine heart (100 μM,
−1
adjusted by UV−vis spectroscopy), sodium dithionite (40 mg mL ),
ALF472 (1 mM), and stock suspensions of the CO-releasing materials,
with a final concentration of 1 mM based on ALF472 (ALF472@Al-
RESULTS AND DISCUSSION
■
Synthesis and Characterization of the Drugs and Al-
MCM-41 Matrix. The cationic CORM used for our studies,
−1
MCM-41, 2.4 mg mL ; ALF472-cisplatin@Al-MCM-41, 3.1 mg
−1
mL ), were freshly prepared in degassed phosphate buffered saline
ALF472 ([Mn(tacn)(CO) ]Br, tacn = 1,4,7-triazacyclono-
3
(
PBS, 10 mM, pH 7.4). In a typical experiment, 100 μL of myoglobin
nane), is an air-stable and water-soluble manganese complex.
We have recently demonstrated that this CORM is photo-
activable upon irradiation with visible light, releasing 0.80 mmol
of CO per mmol of complex after 24 h, while the solutions are
stock solution, 100 μL of sodium dithionite stock solution, 790 μL of
degassed PBS, and 10 μL of ALF472 stock solution or CO-releasing
material stock suspensions were added in a sealed quartz cuvette (1400
μL) under inert atmosphere and kept at 37 °C under visible light
18
stable in darkness during the same time frame. In addition,
the well-known anticancer drug cisplatin, cis-[PtCl (NH ],
was prepared following a previously published protocol.
(
luminous flux, 600 lm; color temperature, 4000 K, distance between
lamp and cuvette, 5 cm) for 24 h. The solutions were analyzed by
means of UV−vis at different times (0, 15, 30, 60, 90, 120, 150, 180,
2
)
3 2
30
C
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Inorganic Chemistry
Article
The porous material Al-MCM-41 was synthesized according
to literature methods by doping the pristine MCM-41 matrix
can be explained taking into account the presence of a higher
amount of anionic exchangeable sites in Al-MCM-41 in
2
0
−1
with NaAlO . As a result, a negatively charged porous matrix
comparison to MCM-41-SO H (1.2 and 0.22 mmol g ,
2
3
4+
3+
(
with a Si /Al molar ratio of 1:0.106), ready to incorporate
respectively).
+
the cationic ALF472 , was obtained. The material was
characterized by elemental analysis (EA), infrared spectroscopy
Moreover, while the previously reported ALF472@MCM-
41-SO3 hybrid material was obtained as particles in the
micrometer range, ALF472@Al-MCM-41 was isolated as
nanoparticles. Indeed, TEM images revealed that the loaded
material showed a similar size (49 ± 11 nm, Figure S4) and
morphology to the empty matrix (64 ± 13 nm). In fact, the
nanometric nature of ALF472@Al-MCM-41 may add further
advantages to this new hybrid material in terms of its
administration through different routes (i.e., intravenous).
Regarding hydrodynamic size and Z-potential values, both
parameters confirmed the good dispersion of empty Al-MCM-
41 (93 ± 28 nm and −28 ± 1 mV) and loaded ALF472@Al-
MCM-41 (112 ± 33 nm and −23 ± 1 mV) in simulated
biological conditions. Finally, EDX elemental mapping of
ALF472@Al-MCM-41 nanoparticles corroborated the presence
of homogeneously distributed manganese, silicon, and alumi-
num as well as the absence of bromide (Figure S5). These
(
IR), thermogravimetric analysis (TG), N adsorption at 77 K,
2
transmission electron microscopy (TEM), and energy-
dispersive X-ray spectroscopy (EDX). Thus, while the nitrogen
adsorption/desorption isotherms confirmed the porosity of the
2
−1
silica material (SBET, 995 m g ; d (BJH), 2.97 nm, Figure
pore
S2), TEM images demonstrated the nanometric homogeneous
size of the particles (average size 64 ± 13 nm, Figure S3).
Single Encapsulation of ALF472 into Al-MCM-41. With
the aim of preparing new CO-releasing materials containing
multiple drugs with complementary effects, the suitability of the
anionic matrix Al-MCM-41 to incorporate the photoactive
+
cationic CORM ALF472 was first evaluated. Specifically, the
encapsulation process was performed by suspending, for 24 h
and in darkness, the anionic porous matrix in an aqueous
solution containing the double stoichiometric amount of
+
+
ALF472 , with regard to the available exchangeable extraframe-
results suggest that ALF472 is encapsulated into the pores of
work cations present in the matrix. Afterward, the suspension
was filtered, washed several times with water to remove the
nonencapsulated CORM, and dried at room temperature, thus
producing the loaded matrix ALF472@Al-MCM-41.
Infrared spectroscopy initially confirmed the successful
incorporation of the CO-prodrug within the porous nanoma-
terial (Figure 1). Indeed, the characteristic CO stretching bands
the matrix as a consequence of an ion exchange process and not
merely coprecipitated.
Dual Encapsulation of ALF472 and Cisplatin into Al-
MCM-41. Taking into account the successful incorporation of
ALF472 into the Al-MCM-41 nanoparticles, we decided to
further explore the ability of this matrix to encapsulate multiple
drugs, which could exert a synergistic or complementary
26−28
therapeutic effect.
As a proof of concept, we carried out,
for the first time, the dual encapsulation of our photoCORM,
ALF472, and the well-known antitumoral agent cisplatin. The
loading was performed by a one pot solid−liquid impregnation
process in water and in the absence of light in order to avoid
the photoactivation of the CORM. Once again, the successful
+
incorporation of ALF472 in the matrix was first confirmed by
infrared spectroscopy, showing the characteristic displacement
of CO stretching bands due to the confinement of the CORM
into the cavities of Al-MCM-41 (υ = 2031 and 1925 cm− for
ALF472-cisplatin@Al-MCM-41) (Figure 1). The cargo of both
drugs was quantified by elemental and thermogravimetric
1
analysis as well as EDX, yielding 0.45 mmol g of ALF472+
−1
and 0.12 mmol g− of the platinum complex. Regarding the
1
+
loading of ALF472 , the amount of encapsulated CORM was
−
1
similar to that of ALF472@Al-MCM-41 (0.48 mmol g ),
confirming that cisplatin does not hamper the cation exchange
encapsulation process in the pores of Al-MCM-41. In contrast,
the single loading of cisplatin into the matrix substantially
Figure 1. Infrared spectra of free ALF472 (turquoise line),
mesoporous silica Al-MCM-41 (gray line), and hybrid materials
ALF472@Al-MCM-41 (pink line) and ALF472-cisplatin@Al-MCM-
−1
decreased its cargo 3-fold (0.038 mmol g ). This result may
4
1 (yellow line). The displacement of the CO stretching bands in the
+
hybrid materials in comparison to the free CORM is highlighted.
suggest that the presence of ALF472 favors the loading of the
anticancer drug probably due to the formation of hydrogen
bonds between both guest molecules. Indeed, a similar trend
has been previously reported by Delgado et al. when using
of ALF472 appear slightly shifted in the hybrid material
−1
compared to pristine CORM (from υ = 2017 to 2029 cm and
−1
from υ = 1895 to 1927 cm ), as has been previously observed
for other related materials and attributed to the confinement of
MCM-41 and MCM-41-NH as hosts for the encapsulation of
2
33
cisplatin. In fact, the amino derivatized silica, ready to
establish hydrogen bonds, incorporated 0.204 mmol g of
1
8,20,21
−1
CORM molecules within the pores.
In addition, the amount of encapsulated ALF472+ was
quantified by a combination of techniques (namely, EDX and
elemental and thermogravimetric analysis). The results revealed
that the aluminum doped MCM-41 shows a better performance
in terms of loading (0.48 mmol [ALF472] g dried basis)
than the previously reported sulfonic-functionalized MCM-41
cisplatin while the nonfunctionalized matrix only loaded 0.029
mmol of this metallodrug per gram.
Moreover, TEM images confirmed that the dual encapsula-
tion process did not alter either the morphology or size of the
loaded nanoparticles (48 ± 14 nm, Figure 2), although a small
aggregation takes place when particles are suspended in a
physiological medium (hydrodynamic size 336 ± 50 nm, Z-
+
−1
+
−1
18
(
0.22 mmol [ALF472] g dried basis). This improvement
D
DOI: 10.1021/acs.inorgchem.7b01475
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Figure 2. TEM image of ALF472-cisplatin@Al-MCM-41 nano-
particles and size distribution profile (average size 48 ± 14 nm), n >
1
00.
potential −21 ± 1 mV). A similar trend was also observed for
cisplatin@Al-MCM-41 (hydrodynamic size 207 ± 23 nm, Z-
potential −29 ± 1 mV). In spite of this aggregation effect,
particle size is still appropriate for a potential biological
application, as it has been demonstrated that silica nanoparticles
ranging from 50 to 300 nm can be easily internalized by
3
4
endocytosis without any significant toxicity effects. Finally,
elemental mapping obtained by EDX further confirmed a
homogeneous distribution of both drugs in Al-MCM-41
nanoparticles (Figure 3) as well as the absence of bromide
counterions (Figure S6).
+
Figure 4. (a) Photoactivation process of [Mn(tacn)(CO) ]
(
3
+
ALF472 ) under simulated physiological conditions. (b) CO-
releasing kinetics upon irradiation with visible light of the free
complex ALF472 (turquoise ●) and the hybrid materials ALF472@
Al-MCM-41 (pink ) and ALF472-cisplatin@Al-MCM-41 (yellow
●). Black circles (●) demonstrate that there is no CO release from
ALF472 in darkness. The inset shows the evolution of the electronic
spectra of myoglobin upon CO coordination. Experimental conditions:
Ar atmosphere, 10 mM PBS, 37 °C, 10 μM of ALF472, and 10 μM of
myoglobin.
+
●
CO-Delivery Studies of the Loaded Materials. In order
to envisage a potential biological application as CORMAs of
ALF472@Al-MCM-41 and the dual hybrid system ALF472-
cisplatin@Al-MCM-41, the photoinduced CO-release proper-
ties of these materials were investigated in simulated biological
conditions (10 mM PBS, 37 °C) by UV−vis spectroscopy using
the myoglobin assay. For this purpose, a degassed PBS solution
of equine heart myoglobin was freshly reduced with an excess
of sodium dithionite under an inert atmosphere, and then, the
loaded matrix under study was added. As shown in Figure 4, the
CO-release kinetic profile of ALF472@Al-MCM-41 is quite
different from that of the free CORM. Indeed, a less steep slope
is observed for the encapsulated CO-prodrug
(kALF472@Al‑MCM‑41: 0.017 min ), which may allow a more
controlled CO supply than that of free CORM (kALF472: 0.031
min ). Moreover, the total amount of released CO after 24 h
accounts for 0.25 mmol of CO per mmol of encapsulated
ALF472 , while free CORM delivers 0.80 mmol of CO per
−1
−1
+
Figure 3. Elemental mapping obtained by EDX of the dual hybrid material ALF472-cisplatin@Al-MCM-41. The presence of silicon, aluminum,
manganese, and platinum elements into the nanoparticles is confirmed.
E
DOI: 10.1021/acs.inorgchem.7b01475
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
mmol of complex in the same time frame. Regarding the dual
material, ALF472-cisplatin@Al-MCM-41 shows a similar CO-
r e l e a s i n g r a t e t h a n A L F 4 7 2 @ A l - M C M - 4 1
morphology of the nanoparticles. It is worth noting that the
presence of CORM molecules increases significantly the cargo
of the platinum anticancer drug suggesting a synergistic loading
effect. In addition, both hybrid materials show a more
controlled CO release than the free CORM, efficiently trapping
most of the metal fragments, both loaded and generated. In
fact, it should be highlighted that the host−guest interactions
are strong enough to prevent the exchange of the CORM with
the ions present in the physiological medium as well as to avoid
the leaching of the potentially toxic decarbonylation fragments.
Although the dual hybrid material does exhibit a low release of
cisplatin, it may be considered as a proof of concept of the
feasibility of developing CORMAs with multiple therapeutic
effects.
−1
(
0
kALF472‑cisplatin@Al‑MCM‑41: 0.017 min ), although it delivers
.11 mmol of CO per mmol of CORM after 24 h, which
accounts approximately for the 50% of the total amount of CO
released from the single material ALF472@Al-MCM-41.
Hence, the lower capacity of ALF472-cisplatin@Al-MCM-41
to deliver CO upon activation with visible light may be related
to the stabilization of CO ligands due to the formation of H-
bonds involving both guest molecules.
Metal Leaching Studies of the Hybrid Materials. With
the aim of analyzing the ability of Al-MCM-41 to retain
+
ALF472 inside its channels, we then evaluated the manganese
leaching of a suspension of the hybrid material in saline
phosphate buffer (PBS) in darkness. In fact, the presence of
high concentrations of salts in the buffer could promote
cationic-exchange processes with the subsequent desorption of
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.7b01475.
■
*
S
+
ALF472 to the media. Thus, the results revealed that the
porous matrix Al-MCM-41 exhibited a better performance
+
toward the retention of ALF472 compared to the previously
Thermal analysis, nitrogen adsorption isotherms, EDX
analysis, TEM images, and size distribution profiles
reported MCM-41-SO H. Indeed, the aluminum doped silica
3
matrix is able to keep ca. 86% of CORM trapped after 72 h of
incubation in darkness, while the corresponding alkylsulfonic
(PDF)
+
18
matrix only retained the 14% of ALF472 after 1 h. These
+
AUTHOR INFORMATION
Corresponding Authors
E-mail: crmaldonado@ugr.es.
E-mail: ebaream@ugr.es.
ORCID
results suggest that the interaction between ALF472 and the
■
aluminum doped host is stronger than that established with the
alkylsulfonic-functionalized one, probably due to the different
location and distribution of the anionic sites within the matrix.
*
*
Indeed, in the case of MCM-41-SO H, the sulfonic groups
3
might be mainly located in the surface of the material while in
Jorge A. R. Navarro: 0000-0002-8359-0397
Elisa Barea: 0000-0001-9895-1047
Notes
4
+
3+
the case of Al-MCM-41 the substitution of Si by Al is likely
to occur along the framework with a more homogeneous
distribution. Regarding the metal leaching upon irradiation with
visible light, a similar manganese leaching from ALF472@Al-
MCM-41 was observed (20% after 72 h), which again confirms
the high ability of the doped matrix to keep trapped not only
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The Spanish Ministry of Economy and Competitivity and UE
Feder Program (Projects CTQ2014-53486R and CRM Juan de
la Cierva Contract), University of Granada (CRM Reincorpor-
+
ALF472 but also the corresponding decarbonylation fragments
produced after CO release.
After proving the good retention capacity of Al-MCM-41
toward the potentially toxic manganese fragments, we
quantified the delivery of cisplatin from the dual material in
the same experimental conditions used for monitoring CO
delivery (PBS, light). The results demonstrated that, approx-
imately, 5% of Pt is leached to the physiological medium after 6
h, and no significant increase was observed during the next 66 h
́
́
Plan Propio), Junta de Andalucia (Project P09-FQM-
acion
981 and S.R. postdoctoral contract), and COST action
4
CM1105 are gratefully acknowledged for generous funding.
The authors thank Alfama Inc. for providing ALF472. We thank
Helia Jeremias and Helena Laronha for the synthesis and
purification of ALF472 batches. The NMR data were acquired
at CERMAX (Centro de Ressonancia Magnetica Antonio
̂ ́ ́
Xavier), which is a member of the National NMR network.
(
total amount of leached Pt at 72 h: 7%). Hence, the high
retention of this anticancer drug within the cavities of the
anionic silica framework may be related to several issues, such
as the formation of hydrogen bonds between cisplatin and
CORM molecules as well as the generation of platinum cationic
species due to the exchange of chlorine ligands by water
molecules.
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