Functional magnetic mesoporous silica microparticles capped with an azo-derivative: A promising colon drug delivery device

Article abstract of DOI:10.3390/molecules23020375

Magnetic micro-sized mesoporous silica particles were used for the preparation of a gated material able to release an entrapped cargo in the presence of an azo-reducing agent and, to some extent, at acidic pH. The magnetic mesoporous microparticles were loaded with safranin O and the external surface was functionalized with an azo derivative 1 (bearing a carbamate linkage) yielding solid S1. Aqueous suspensions of S1 at pH 7.4 showed negligible safranin O release due to the presence of the bulky azo derivative attached onto the external surface of the inorganic scaffold. However, in the presence of sodium dithionite (azoreductive agent), a remarkable safranin O delivery was observed. At acidic pH, a certain safranin O release from S1 was also found. The pH-triggered safranin O delivery was ascribed to the acid-induced hydrolysis of the carbamate moiety that linked the bulky azo derivatives onto the mesoporous inorganic magnetic support. The controlled release behavior of S1 was also tested using a model that simulated the gastro intestinal tract.

Full text of DOI:10.3390/molecules23020375

molecules
Article
Functional Magnetic Mesoporous Silica
Microparticles Capped with an Azo-Derivative:
A Promising Colon Drug Delivery Device
Adrián H. Teruel 1,2 ID , Carmen Coll 1,2, Ana M. Costero 1,2,3 ID , Daniel Ferri 1,3
,
Margarita Parra 1,2,3, Pablo Gaviña 1,2,3, Marta González-Álvarez 4, Virginia Merino 1,5
,
M. Dolores Marcos 1,2,6,7, Ramón Martínez-Máñez 1,2,6,7,
*
and Félix Sancenón 1,2,6,7
ID
1
Interuniversity Research Institute for Molecular Recognition and Technological Development (IDM),
Polytechnic University of Valencia, University of Valencia, 46100 Valencia, Spain; adherte@upv.es (A.H.T.);
macolme1@upvnet.upv.es (C.C.); ana.costero@uv.es (A.M.C.); daniel.ferri@uv.es (D.F.);
margarita.parra@uv.es (M.P.); pablo.gavina@uv.es (P.G.); Virginia.merino@uv.es (V.M.);
mmarcos@qim.upv.es (M.D.M.); fsanceno@upvnet.upv.es (F.S.)
CIBER of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain
Department of Organic Chemistry, University of Valencia, 46100 Valencia, Spain
Engineering Department. Pharmacy and Pharmaceutical Technology Section, Miguel Hernandez University,
03550 Alicante, Spain; marta.gonzalez@umh.es
Pharmacy and Pharmaceutical Technology and Parasitology, University of Valencia, 46100 Valencia, Spain
Joint Research Unit in Nanomedicine and Sensors. Polytechnic University of Valencia, Health Research
Institute Hospital La Fe, 46100 Valencia, Spain.
Joint Unit CIPF-UPV of Mechanisms of Diseases and Nanomedicine, Valencia, Polytechnic University of
Valencia, Prince Felipe Research Center, 46100 Valencia, Spain
2
3
4
5
6
7
*
Correspondence: rmaez@qim.upv.es; Tel.: +34-963-877-343
Received: 15 January 2018; Accepted: 8 February 2018; Published: 10 February 2018
Abstract: Magnetic micro-sized mesoporous silica particles were used for the preparation of a gated
material able to release an entrapped cargo in the presence of an azo-reducing agent and, to some
extent, at acidic pH. The magnetic mesoporous microparticles were loaded with safranin O and the
external surface was functionalized with an azo derivative 1 (bearing a carbamate linkage) yielding
solid S1. Aqueous suspensions of S1 at pH 7.4 showed negligible safranin O release due to the
presence of the bulky azo derivative attached onto the external surface of the inorganic scaffold.
However, in the presence of sodium dithionite (azoreductive agent), a remarkable safranin O delivery
was observed. At acidic pH, a certain safranin O release from S1 was also found. The pH-triggered
safranin O delivery was ascribed to the acid-induced hydrolysis of the carbamate moiety that linked
the bulky azo derivatives onto the mesoporous inorganic magnetic support. The controlled release
behavior of S1 was also tested using a model that simulated the gastro intestinal tract.
Keywords: magnetic mesoporous silica; azo derivatives; pH triggered; azo reductor; colon release
1. Introduction
In recent years, the blending of coordination, molecular, supramolecular and biomolecular
chemistry concepts with porous materials has led to the development of smart functional gated
materials with multiple applications in different scientific and technological fields [1]. These hybrid
materials are mainly composed by two subunits: (i) an inorganic porous support for cargo loading;
and (ii) certain molecules acting as “molecular gates” for controlled cargo release upon the application
of external stimuli [2].
Molecules 2018, 23, 375; doi:10.3390/molecules23020375
www.mdpi.com/journal/molecules

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One of the most commonly used inorganic supports in gated materials is mesoporous silica due to
its outstanding features such as biocompatibility, inertness and chemical stability, high loading capacity
and ease of functionalization using the well-known alkoxysilane chemistries [3]. Besides, mesoporous
silica can be prepared as micro- or nanoparticles, the diameter of the pores can also be adjusted from
the typical 2–3 nm range to ca. 6–10 nm (to include inside the porous network biomolecules such
as small peptides, DNA fragments or enzymes) and can be prepared including other nanoparticles
(for example, magnetite) in their structure endowing the final support with novel features [4–6].
These “gated materials” have been extensively used in controlled release processes [7–10] as
well as in sensing/recognition protocols (when a dye or fluorophore is released in the presence of a
target molecule) [11] and, very recently, in abiotic communication networks involving several gated
nanodevices [12,13]. Dealing with controlled drug release, the preparation of carriers able to deliver
certain drugs at-will and on the site of action, minimizing secondary effects, is a benchmark in the
treatment of different diseases [14].
From another point of view, colon drug delivery systems (CDDS) have been developed for their
use in two main applications: (i) to deliver macromolecular drugs, such as protein or peptides that
found in the colon a better environment to be absorbed without losing activity because of the relatively
low proteolytic enzyme activity and good absorption (e.g., insulin, calcitonin, and vasopressin);
and (ii) to increase the efficacy in prevention and treatment of colon related diseases (e.g., irritable
bowel syndrome, inflammatory bowel diseases (IBD), and colorectal cancer) [15,16]. In fact, the
development of CDDS is an excellent strategy to deliver certain drugs in colon, decreasing adverse
effects and enhance drug efficacy. With this aim, smart formulations for targeting colon such as
pH-responsive biodegradable polymers, time-dependent formulations, pressure responsive systems,
osmotic controlled materials, bio-adhesive systems, enzyme triggered prodrugs or drugs coated with
enzyme-sensitive polymers have been recently developed [17–19].
Despite these examples, the use of hybrid organic–inorganic gated materials based on mesoporous
silica for the development of CDDS is still an almost unexplored field. For the preparation of these
CDDS silica-based nanodevices, enzymes produced by colon microbiota (azoreductase,
-xylosidase, nitroreductase, glycosidase deaminase, etc.) are ideal candidates to be used as external
triggers [20,21].
Bearing in mind our interest in the development of silica-based gated materials for controlled
release applications [22 29], we report herein the preparation of capped a micro-sized silica mesoporous
β-galactosidase,
β
–
solid containing magnetic nanoparticles (S1) as a suitable carrier to release an entrapped cargo in
colon. Although not specifically tested in this present work, the magnetic character of the prepared
nanodevice may be useful to enhance the retention time of the particles in the part of interest of the
intestinal tract (for instance in colon). Moreover, particles S1 are loaded with a dye and capped with
an azoderivative attached to the external surface of the inorganic matrix by means of a carbamate
group. We demonstrated that the presence of the bulky azoderivative onto the external surface of S1
inhibited cargo release, whereas in the presence of a reducing agent delivery was observed. The solid
was also able to deliver the cargo to some extent at acidic pH. A study on payload release from S1 in a
simulated digestive process in mouth, stomach, small intestine and colon is also reported.
2. Results and Discussion
2.1. Synthesis of Gated Magnetic Micro-Sized Mesoporous Silica Particles
Magnetic nanoparticles (MNPs) coated with oleic acid were prepared by a co-precipitation
procedure that used a mixture of FeCl3 and FeCl2 and ammonium hydroxide [30]. Moreover,
mesoporous silica was prepared following the “atrane route” with small modifications [31,32]. In this
synthetic protocol, n-cetyltrimethylammonium bromide was used as structural directing agent and
tetraethylortosilicate as silica source. MNPs were suspended in water and incorporated into the
synthesis crude before the addition of NaOH. The resulting brownish powder was washed and the

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surfactant was subsequently removed by calcination. This procedure yielded the final magnetic
micro-sized inorganic solid (S0). Once the magnetic mesoporous support was synthesized, the pores of
the siliceous phase were loaded with safranin O (as a model drug in our study) and the external surface
functionalized with the bulky azo-containing derivative
1. This synthetic protocol yielded micro-sized
magnetic solids S1 (see Scheme 1). It was expected that the bulky azo-containing derivative
1
would
inhibit dye leaching from the pores, whereas safranin O delivery should be observed in the presence of
sodium dithionite (able to hydrolyze azo linkages) or partially at acidic pH (due to the hydrolysis of
the carbamate moiety in 1).
Scheme 1. Magnetic micrometric silica mesoporous supports loaded with safranin O and capped with
azo derivative (1). Safranin O is released in the presence of an azoreductor.
The synthesis of azo-containing alkoxysilane derivative 1 (see Scheme 2) starts with the mesylation
of 2-methoxyethanol (1a) in dichloromethane that yielded 1b [33]. Then, in a second step, 1b was
reacted with the disodium salt of N-phenyldiethanolamine (1c) obtaining the oligoethylenglycol
derivative 1d
.
Afterwards, 1d was reacted with the diazonium salt of 4-aminobenzyl alcohol
was obtained
(1e) yielding azo derivative 1f. Finally, azo-containing trialkoxysilane derivative
1
upon reaction between 1f and (3-isocyanatopropyl) triethoxysilane (1g). The final azo-containing
trilakoxysilane derivative (1) was fully characterized using 1H- and 13C-NMR and HRMS.
Scheme 2. Synthetic route used for the preparation of azoderivative 1. 2-methoxyethanol (1a);
2-methoxyethyl methanesulfonate (1b); N-phenyldiethanolamine (1c); oligoethylenglycol derivative
(1d); 4-aminobenzyl alcohol (1e); azo derivative (1f); (3-isocyanatopropyl) triethoxysilane (1g).

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2.2. Characterization of the Prepared Materials
S0 and S1 were characterized using powder X-ray diffraction (PXRD), transmission electron
microscopy (TEM), scanning transmission electron microscopy (STEM), N2 adsorption–desorption
isotherms and thermogravimetric analysis. Figure 1 shows the PXRD patterns (at high and low angles)
of oleate-stabilized MNPs, the magnetic mesoporous microparticles as-made, S0 and S1. PXRD of
oleate-coated MNPs (see also Figure 1) showed the five typical high-angle reflections of magnetite
phase indexed as (220), (311), (400), (511) and (440) Bragg peaks. The same peaks were observed for
the as-made microparticles, S0 and S1 materials but with a marked decrease in intensity (Figure 1).
This intensity loss was ascribed to the inclusion of MNPs into the inorganic mesoporous matrix.
Low angle PXRD pattern of as-made microparticles (Figure 1) displayed the typical reflections of a
hexagonal-ordered mesoporous system that can be indexed as (100), (110), (200) and (210) Bragg peaks.
A shift of the (100) peak in calcined S0 solid was clearly observed. This displacement is consistent with
an approximate cell contraction of 3.64 Å and is attributed to the condensation of silanol groups during
the calcination step. The presence of the characteristic (100) reflection in the diffraction spectrum of
S1 clearly indicated that the mesoporous structure was preserved throughout the filling process with
safranin O and the anchoring of the azo derivative 1.
Figure 1. Powder X-rays patterns at: (
a) high angles; and (b) low angles of oleate stabilized magnetite
(Fe3O4) nanoparticles and magnetic micrometric mesoporous silica particles as-synthesized, S0 and S1
.
Figure 2 shows transmission electron microscopy (TEM) images of solid S0. As could be
seen, the typical porosity associated with the mesoporous inorganic support was present. Besides,
MNPs were observed as small dark dots. Scanning transmission electron microscopy (STEM)
images of solid S0 (iron mapping) indicated that MNPs are randomly distributed through the
mesoporous silica microparticles (Figure 3). Moreover, to assess the magnetic behavior of S0, field

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dependent magnetization curves measured at 298 K were obtained. The obtained curves (see Figure 4)
displayed no hysteresis loops and showed coercivity values near zero, in agreement with a typical
superparamagnetic behavior at room temperature. The saturation magnetization value for S0 was
1.5 emu/g. This magnetization value is not far from those reported in the literature for other magnetic
mesoporous silica particles, which are typically in the 1.7–10 emu/g range [30].
Figure 2. TEM image of micrometric mesoporous silica particles containing magnetite nanoparticles (S0).
Figure 3.
(a) STEM images of micrometric mesoporous silica particles containing magnetite
nanoparticles (S0); (b) silica mapping; and (c) iron mapping.
m (emu/g)
1.5
1.0
0.5
0.0
-60000
-40000
-20000
0
20000
40000
60000
H (A/m)
-0.5
-1.0
-1.5
Figure 4. Field-dependent magnetization curves at room temperature of S0 microparticles.
N2 adsorption–desorption studies of S0 and S1 were also carried out (Figure 5). As could be
seen, solid S0 showed a typical curve for mesoporous silica materials, with an adsorption step at
intermediate P/P0 values (0.2–0.4). This isotherm is classified as type IV in which the absorption step
deals with nitrogen condensation inside the mesopores. The pore diameter of S0 was calculated by
the Barret-Joyner-Halenda (BJH) method [34]. The narrow BJH pore distribution observed and the
absence of a hysteresis loop in the 0.2–0.4 P/P0 interval suggested the existence of uniform cylindrical
mesopores. Values of pore diameter of 2.83 nm and pore volume of 0.94 cm3 g−1, calculated on the
adsorption branch of the isotherm, were found. Pore diameter estimated from TEM images agree
with this value. The application of the Brunauer-Emmett-Teller (BET) model [35] gave a value of
1057 m2 g−1 for the total specific surface. On the other hand, the N2 adsorption–desorption isotherms

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of solid S1 are typical of mesoporous systems with partially filled mesopores, and a decrease in both
the adsorbed N2 volume and the specific surface area was clearly observed (Table 1). This reduction in
the BET surface, compared with that of S0, was ascribed to the loading of pores with safranin O and
the functionalization of the surface with the bulky azo derivative 1 in S1.
Figure 5. N2 adsorption–desorption isotherms for S0 and S1 materials.
Table 1. BET specific surface values, pore volumes and pore sizes for S0 and S1.
−1
−1
)
Solid
SBET (m2 g
)
pore volume 1 (cm3 g
pore size 1,2 (nm)
S0
S1
1057
834
0.94
0.68
2.83
-
1
2
Total pore volume according to the BJH model. Pore size estimated by using the BJH model applied on the
adsorption branch of the isotherm, for P/P0 < 0.6, which can be associated to the surfactant generated mesopores.
Total organic matter on solid S1 was determined by thermogravimetric analysis (see
1
Supplementary Materials) and H-NMR (Table 2). The amount of anchored azo derivative onto
S1 was determined by 1H-NMR upon dissolving the corresponding sample in HF/D2O in the presence
of tetraethyl ammonium bromide as internal standard (Table 2) [36]. Besides, energy dispersive
X-ray spectroscopy (EDX, 20 kV) was used to determine Si and Fe content in S1 microparticles. EDX
measurements showed a value of 5.9 for the Si/Fe ratio in the final S1 solid.
Table 2. Content of total organic matter (in g) per gram of SiO2, content of azo derivative (in g) per
gram of SiO2 and dye released (in µg) per mg of solid in S1.
Solid
S1
Organic Content (g/g SiO2)
Azo Derivative (g/g SiO2)
Dye Release (µg/mg solid)
0.0469
0.043
0.69
2.3. Cargo Release from S1
Payload release from solid S1 at acidic (2.0 and 4.5) and neutral (7.4) pH in the presence or absence
of the azoreductor agent sodium dithionite was studied [37 38]. These pH values were selected taking
,
into account the possible use of S1 as nanodevice for controlled release in colon: pH 2.0 is typical of
gastric juices, pH 4.5 is found in the transition from stomach to intestines (this pH can also be found in
the colon of IBD patients), and a neutral pH ranging 6.5–7.4 is typically found in the intestine. In a
typical experiment, 2 mg of S1 were suspended in water at selected pH values, the final suspensions
◦
stirred at 37.5 C and aliquots were taken at scheduled times. The aliquots were centrifuged to remove
the solid and the release of safranin O to the solution monitored by measuring fluorescence at 571 nm
upon excitation at 520 nm. Cargo release profile for solid S1 is shown in Figure 6. Aqueous suspensions

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of S1 showed negligible safranin O release at neutral pH. However, as a clear contrast, a marked
delivery of entrapped safranin O was seen in the presence of sodium dithionite (more than 80% of
maximum dye delivered after 4 h). The observed delivery is attributed to the presence of sodium
dithionite, which can reduce azo groups in the capping molecule
safranin O release [37 38]. At acidic pH (2.0 and 4.5), a sustained safranin O release was also observed.
Delivery of entrapped safranin O was ascribed to a hydrolysis of carbamate moieties, which linked the
bulky azo derivative onto the external surface of S1. Maximum release of safranin O for S1 was ca.
0.51 µg/mg solid at pH 7.4 in the presence of sodium dithionite.
1 resulting in pore opening and
,
1
Figure 6. Release kinetics of safranin O from S1 in water at pH
≈
2 (square), pH
≈
4.5 (circle), pH
≈
7.4
(triangle) and pH 7.4 in the presence of azoreductor agent sodium dithionite (SD, 57.4 mM) (cross).
≈
2.4. In Vitro Digestion Model Assay
Despite the partial payload delivery from S1 at acidic pH shown above, it has to be noted that
permanence of the particles in the stomach is usually less than 2 h and therefore only a partial cargo
delivery from S1 is expected to occur before targeting colonic tissues. In fact, to study in depth the
possible application of azo-gated magnetic microparticles as suitable particles for cargo delivery in
colon, payload delivery from S1 was further tested in a model of digestion using simulated solutions.
The model used was a modification of that introduced by Oomen and co-workers [39,40] that is a
three-step procedure simulating digestive process in mouth, stomach and small intestine. Moreover,
we introduced a fourth step to simulate the digestive process in colon. In a typical experiment, 2 mg
of solid (S1) were suspended in simulated saliva (5 min at 37.5 ◦C) and then simulated gastric juice
(stomach) was added and the mixture stirred 2 h. Afterwards, duodenal simulated juice, bile and
bicarbonate solution (small intestine) were added and the suspension stirred for two more hours.
Finally, sodium dithionite was added and it was left for 24 h to mimic the colon environment and the
presence of azoreductases. Safranin O release in the entire process was evaluated via monitorization of
the emission at 571 nm upon excitation at 520 nm. The obtained results are shown in Figure 7.
As shown in Figure 7, a nearly “zero release” was observed during the first 5 min when solids were
in contact with simulated saliva. In contact with gastric juice, a safranin O delivery of ca. 0.3 µg/mg
solid was found and ascribed to partial pore opening due to the acidic environment. In small intestine
conditions, no further delivery of safranin O was observed. However, a marked cargo release was
found in simulated colon conditions and related with the presence of sodium dithionite, which can
reduce azo bonds of the capping ensemble in S1.

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Figure 7. Release kinetics of Safranin O from S1 in simulated gastro-intestinal tract (GIT) fluids.
3. Materials and Methods
3.1. General Techniques
Transmission electron microscopy (TEM), scanning transmission electron microscope (STEM),
powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), N2 adsorption–desorption,
nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR) and fluorescence
spectroscopy techniques were employed to characterize the synthesized materials. TEM and STEM
images were acquired using a JEOL JEM-1010 and a JEOL JEM 2100F microscopes (Akishima, Japan),
respectively. PXRD measurements were performed on a Bruker D8 Advance diffractometer (Billerica,
MA, USA) using CuKα radiation. TGA were carried out on a TGA/SDTA 851e Mettler Toledo balance
(Greifensee, Switzerland), using an oxidant atmosphere (air, 80 mL/min) with a heating program
◦
consisting on a heating ramp of 10 C per minute from 393 to 1273 K and an isothermal heating step at
this temperature for 30 min. N2 adsorption–desorption isotherms were recorded with a Micromeritics
ASAP 2010 automated sorption analyzer (Norcross, GA, USA). The samples were degassed at 120 ◦C
under vacuum overnight. The specific surface areas were calculated from the adsorption data in
the low pressure range using the Brunauer-Emmett-Teller (BET) model. Pore size was determined
1
following the Barrett-Joyner-Halenda (BJH) method. H NMR spectra were recorded using a Bruker
AV400 spectrometer (Billerica, MA, USA). Infrared spectra were recorded using a Bruker Tensor
27 equipment (Billerica, MA, USA). Fluorescence measurements were carried out in a PerkinElmer
EnSpire Multimode Plate Reader (Waltham, MA, USA).
3.2. Chemicals
The chemicals tetraethylorthosilicate (TEOS), n-cetyltrimethylammonium bromide (CTABr),
sodium hydroxide, triethanolamine (TEAH3), (3-isocyanatopropyl) triethoxysilane, iron(III) chloride
hexahydrate, iron(II) chloride tetrahydrate, oleic acid, safranin O dye, 2-methoxyethanol, triethylamine,
methanesulfonyl chloride, hydrochloric acid, N-phenyldiethanolamine, anhydrous acetonitrile, sodium
hydride, 4-aminobenzyl alcohol, sodium nitrite, sodium dithionite and all chemicals for the digestive
fluids were provided by Sigma-Aldrich (Madrid, Spain). Magnesium sulfate, sodium carbonate, and
all the analytical-grade solvents were purchased from Scharlab (Barcelona, Spain). Aluminum oxide
was acquired from Merck (Madrid, Spain). All products were used as received.

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3.3. Synthesis of Oleic Acid-coated Magnetic Nanoparticles (MNPs)
MNPs were prepared using a modified co-precipitation method. In a typical procedure,
FeCl3
·
6H2O (24.0 g, 0.089 mol) and FeCl2
·
4H2O (9.8 g, 0.049 mol) were dissolved in deionized water
◦
(100 mL) under nitrogen atmosphere with vigorous stirring at 80 C. Then ammonium hydroxide
(50 mL) was added quickly into the solution. The colour of the solution immediately turned black
and then oleic acid (3.8 g, 0.013 mol) was added after 30 min. The reaction was kept at 80 ◦C for 1.5 h.
The final oleic acid-coated MNPs were washed with deionized water until neutral pH. Afterwards, the
MNPs were transferred into a chloroform solution (30 mg MNPs/mL CHCl3).
3.4. Synthesis of Mesoporous Silica Microparticles Containing MNPs (S0)
A chloroform suspension of MNPs (6 mL, 30 mg MNPs/mL CHCl3) was mixed with an aqueous
solution of CTABr (8 mL, 10 mg/mL) and the resulting suspension was treated with ultrasounds and
heated. Finally, ultrasounds were applied again and the suspension was passed through nylon filters
(0.45 µM) to ensure MNPs homogeneity. Magnetic micro-sized MCM-41 particles were synthesized
following the so-called “atrane route”. For this purpose, a solution of TEAH3 (25.79 g, 0.173 mol) and
NaOH (2 mL of a 6 M solution) was heated to 120 ◦C and then cooled down to 70 ◦C, at this moment
◦
TEOS (11 mL, 0.045 mol) was added and the crude reaction was heated up to 120 C. The crude
reaction was left cooling down again and CTABr (4.68 g, 0.013 mol) was added at 118 ◦C. Next, water
containing MNPs (74 mL of distilled water + 6 mL of the MNPs CTABr-water suspension) was slowly
◦
added with vigorous stirring at 70 C. Besides, NaOH (10 mL of a 0.1 M solution) was added to obtain
a pH
≈
10.9. After a few minutes, a brownish suspension was formed. This mixture was aged in an
autoclave at 100 ◦C for 24 h. The resulting powder was collected by filtration and washed with water.
Finally, the solid was dried at 70 ◦C. To prepare the final solid (S0), the as-synthesized microparticles
were calcined at 550 ◦C using oxidant atmosphere for 5 h to remove the template.
3.5. Synthesis of 1b
2-Methoxyethanol (1a, 16 mL, 0.2 mol) was dissolved in dichloromethane (270 mL) in a 2 L
round-bottomed flask. The solution was kept in an ice bath for 15 min, and then triethylamine
(55.2 mL, 0.4 mol) was added to the solution. Afterwards, a solution of methanesulfonyl chloride in
dichloromethane (0.32 mol in 48 mL) was added dropwise for 60 min to the initial reaction crude.
After this addition, reaction was stirred at room temperature for another 90 min period. Then, the
crude was poured onto a water/ice mixture containing concentrated hydrochloric acid (50 mL), and
the organic layer was separated, washed three times with brine and dried with anhydrous MgSO4.
Dichloromethane was eliminated in a rotary evaporator to give 1b as yellow oil (25.16 g, 0.18 mol, 91%
yield). Spectroscopic data were coincident with those reported in the literature.
3.6. Synthesis of 1d
N-phenyldiethanolamine (1c, 3.90 g, 0.021 mol) was dissolved in dry acetonitrile (70 mL) and then
the flask was purged several times with argon to remove oxygen and water. Then, sodium hydride
(1.2 g, 0.05 mol) was gradually added at room temperature, after which a white precipitate appeared.
On the other hand, 1b (6 g, 0.043 mol) was dissolved in anhydrous acetonitrile (30 mL) and then added
dropwise to the generated dianionic salt of 1c. After this addition, the crude reaction was heated at
reflux for 24 h. Then, the reaction mixture was filtered off and the organic solvent was eliminated using
a rotary evaporator, yielding a yellow oil. The product 1d (1.25 g, 4 mmol, 20% yield) was purified
using column chromatography with aluminum oxide and hexane/ethyl acetate (8:2 v/v) as the eluent.
Spectroscopic data were consistent with those reported in the literature.

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3.7. Synthesis of 1f
4-aminobenzyl alcohol (1e, 414 mg, 3.36 mmol) was dissolved in hydrochloric acid (150 mL,
4 mol L−1). Th◦is solution was slowly added to water (25 mL) containing sodium nitrite (232 mg,
3.36 mmol) at 5 C. The generated diazonium salt of 1e was immediately added to an aqueous solution
containing 1d (1 g, 3.36 mmol). The reaction was allowed to react for 30 min at 5 ◦C and for 30 min
at room temperature. The final dark red crude was neutralized with a saturated sodium carbonate
solution and the organic product was extracted with dichloromethane. Organic layers were dried
with MgSO4 filtered off and the solvent was eliminated in a rotary evaporator. Product 1f (250 mg,
0.58 mmol, 17% yield) was isolated as a dark red-orange solid through column chromatography with
1
aluminum oxide and hexane/ethyl acetate (3:2 v/v) as the eluent. H-NMR (400 MHz, CDCl3): δ = 7.84
(d, 4H), 7.46 (d, 2H), 6.79 (d, 2H), 4.75 (s, 2H), 3.74–3.49 (m, 16H), 3.38 ppm (s, 6H); 13C:
58.1, 67.3, 69.7, 70.9, 110.6, 121.2, 126.5, 127.4, 141.1, 154.9, 156.4 ppm.
δ = 50.2, 57.4,
3.8. Synthesis of 1
In a first step, 1f (520 mg, 1.2 mmol) was dissolved in chloroform (20 mL). Then, excess of
alkoxysilane derivative 1g (468 µL, 1.8 mmol) and triethylamine (468 µL, 0.003 mmol) were added
under argon atmosphere. The crude reaction was allowed to react for 72 h at 60 ◦C. Afterwards, the
solvent was eliminated in a rotary evaporator and the reaction crude was washed 3 times with cold
hexane to remove impurities. Compound
1 was isolated as orange oil (550 mg, 0.810 mmol, 67% yield).
1H-NMR (400 MHz, CDCl3):
δ
= 7.76 (d, 4H), 7.38 (d, 2H), 6.72 (d, 2H), 5.06 (s, 2H), 4.68 (s, 1H), 3.75
(q, 6H), 3.62–3.47 (m, 16H), 3.31 (s, 6H), 3.14 (t, 2H), 1.56 (m, 2H), 1.15 (t, 9H), 0.56 (t, 2H) ppm; 13C:
δ = 160.9, 142.3, 139.4, 129.9, 128.5, 127.6, 122.4, 119.1, 114.2, 111.8, 100.2, 72.1, 70.8, 68.5, 65.3, 59.2, 51.4,
32.1, 29.9, 29.5, 22.8, 14.3, 1.2 ppm. HRMS-EI m/z: calcd for C33H54N4O9Si 678.3660; found: 677.2403
(M − H+).
3.9. Synthesis of S1
Magnetic mesoporous silica microparticles (S0, 270 mg) were suspended in an acetonitrile solution
(10 mL) of safranin O (75 mg, 0.8 mmol/g S0) under argon atmosphere. The obtained suspension was
then stirred at room temperature overnight to achieve maximum loading of the pores. Afterward,
1
(550 mg, 3 mmol/g S0) was dissolved in anhydrous acetonitrile (8 mL) and was added to the
microparticle/dye suspension. The mixture was stirred at room temperature for 6 h in an argon
atmosphere. S1 was isolated by centrifugation, washed several times with acetonitrile and was finally
dried at 40 ◦C for 24 h.
3.10. Controlled Release Studies
Controlled release studies from S1 were tested at different pH values (i.e., 2.0, 4.5 and 7.4) and in
the presence of a reductive environment (sodium dithionite, which is known to reduce azo linkages).
In a typical experiment, 2 mg of the corresponding solid were suspended in water at the selected pH
(2 mL) and aliquots were extracted at given times (0, 5 min, 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, 8 h, and
24 h), centrifuged to remove the solid and supernatants were loaded into a 96-well plate to measure
safranin O fluorescence at 571 nm (excitation at 520 nm). Controlled release from S1 microparticles
were also tested in in vitro digestion conditions using simulated solutions for saliva, gastric juice,
duodenal juice and bile [39
37 C (temperature of the human body) and stirring at 12,000 rpm (to ensure the correct suspension of
,
40]. In vitro digestion experiments were carried out with S1 (2 mg) at
◦
the microparticles). Digestions started by adding simulated saliva fluid (320
5 min. Then, simulated gastric juice (630 L) was added, and the mixture was stirred for 2 h. Later,
simulated duodenal juice (630 L), bile (320 L) and a bicarbonate solution (1 M, 100 L) were added
µL) and incubating for
µ
µ
µ
µ
simultaneously and the mixture was stirred for another 2 h. Finally, colon fluid was simulated by
adding sodium dithionite (20 mg, 57.4 mM/2 mL). Aliquots were taken at given times (0, 5, 20, 35, 65,

Molecules 2018, 23, 375
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95, 125, 155, 185, 215, 245, 260, 275, 305, 365, 425, 485, 1320 and 1685 min), centrifuged and supernatants
were loaded into a 96 well plate to measure safranin O fluorescence. Calibration curves, in water,
simulated small intestine fluid and simulated colon fluid, were performed to assess the amounts of
safranin O released from solid S1 in the different environments.
4. Conclusions
As summary, here we have reported the synthesis and characterization of magnetic mesoporous
silica microparticles loaded with safranin O and capped with bulky azo derivative bearing
pH-hydrolysable moieties (carbamate group). At neutral pH, the capped material showed a negligible
safranin O release. However, at neutral pH, addition of a reducing agent such as sodium dithionite
(mimicking azoreductase enzyme) induced a marked safranin O release. The observed delivery was
ascribed to a reduction of the azo bonds that induced a fall in the steric crowding around pore outlets
with subsequent dye delivery. Moreover, a partial payload release was also observed at acidic pH.
Delivery studies in a digestion model indicated an important part of the payload was released in colon.
These particles are expected to find applications as smart controlled drug delivery systems in the colon
mucosa (e.g., for IBD patients) due to the presence of azoreductase enzymes produced by bacteria
in the colon microbiota. Besides, its ability to release the cargo at acidic pH, opens the possibility to
improve the performance of these carriers for drug delivery in ulcerative colitis and Crohn’s disease
in which a marked decrease in the pH of the colon happens [41
,42]. Moreover, these microparticles
(S1) were equipped with magnetic nanoparticles to potentially apply magnetic fields to enhance the
retention time of the solids in colon. Similar magnetic microparticles to those reported here and loaded
with a drug (e.g., 5-ASA) are promising materials for the oral treatment of colon related diseases.
Further in vivo studies in this line will be performed in due course.
Supplementary Materials: The Supplementary Materials are available online.
Acknowledgments: We thank the Spanish Government (projects MAT2015-64139-C4-1-R and AGL2015-70235-
C2-2-R (MINECO/FEDER)) and the Generalitat Valenciana (project PROMETEOII/2014/047) for support.
AHT thanks to the Spanish MEC for his FPU grant. The authors also thank the Electron Microscopy Service at the
Universitat Politècnica de València for support. SCSIE (Universitat de València) is also gratefully acknowledged
for all the equipment employed. NMR was registered at the U26 facility of ICTS “NANBIOSIS” at the Universitat
de València. The authors thanks L. A. Villaescusa for his helpful discussion about the 1H-NMR analysis of the
composition of loaded and functionalized supports.
Author Contributions: Adrian H. Teruel
, Ana María Costero, Daniel Ferri, Marta Gonzalez-Alvarez,
Virginia Merino and Ramón Martínez-Máñez conceived and designed the experiments; Adrian H. Teruel
,
Carmen Coll, Daniel Ferri, Margarita Parra and Pablo Gaviña performed the experiments; Adrian H. Teruel
,
Ana María Costero, Daniel Ferri, Margarita Parra, Pablo Gaviña, Marta Gonzalez-Alvarez, Virginia Merino,
M. Dolores Marcos, Ramón Martínez-Máñez and Félix Sancenón analyzed the data; and Adrian H. Teruel,
Ana María Costero, Ramón Martínez-Máñez and Félix Sancenón wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).