Preparation and study of the antibacterial applications and oxidative stress induction of copper maleamate-functionalized mesoporous silica nanoparticles

Article abstract of DOI:10.3390/pharmaceutics11010030

Mesoporous silica nanoparticles (MSNs) are an interesting class of nanomaterials with potential applications in different therapeutic areas and that have been extensively used as drug carriers in different fields of medicine. The present work is focused o

Full text of DOI:10.3390/pharmaceutics11010030

pharmaceutics
Article
Preparation and Study of the Antibacterial
Applications and Oxidative Stress Induction of
Copper Maleamate-Functionalized Mesoporous
Silica Nanoparticles
1
2
1
, Antonio Rodríguez-Diéguez 3
,
Diana Díaz-García , Perla R. Ardiles , Sanjiv Prashar
2
,
1,
Paulina L. Páez * and Santiago Gómez-Ruiz *
1
Departamento de Biología y Geología, Física y Química Inorgánica, ESCET, Universidad Rey Juan Carlos,
Calle Tulipán s/n, E-28933 Móstoles (Madrid), Spain; diana.diaz@urjc.es (D.D.-G.);
sanjiv.prashar@urjc.es (S.P.)
Departamento de Ciencias Farmacéuticas. Facultad de Ciencias Químicas,
Universidad Nacional de Córdoba. Ciudad Universitaria, Haya de la Torre y Medina Allende,
X5000HUA Córdoba, Argentina; pardiles@fcq.unc.edu.ar
Departamento de Química Inorgánica, Universidad de Granada, Facultad de Ciencias,
Campus de Fuentenueva, Avda. Fuentenueva s/n, E-18071 Granada, Spain; antonio5@ugr.es
Correspondence: plpaez@fcq.unc.edu.ar (P.L.P.); santiago.gomez@urjc.es (S.G.-R.);
Tel.: +54-351-5353850 (P.L.P.); +34-914888507 (S.G.-R.)
2
3
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 9 November 2018; Accepted: 10 January 2019; Published: 14 January 2019
Abstract: Mesoporous silica nanoparticles (MSNs) are an interesting class of nanomaterials with
potential applications in different therapeutic areas and that have been extensively used as drug
carriers in different fields of medicine. The present work is focused on the synthesis of MSNs
containing a maleamato ligand (MSN-maleamic) and the subsequent coordination of copper(II) ions
(MSN-maleamic-Cu) for the exploration of their potential application as antibacterial agents. The
Cu-containing nanomaterials have been characterized by different techniques and the preliminary
antibacterial effect of the supported maleamato-copper(II) complexes has been tested against two
types of bacteria (Gram positive and Gram negative) in different assays to determine the minimum
inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The biological
results showed a moderate antibacterial activity against Escherichia coli which motivated a more
detailed study of the antibacterial mechanism of action of the synthesized maleamate-containing
nanosystems and whose findings showed oxidative stress generation in bacterial cells. All the
prepared nanomaterials were also tested as catalysts in the “solvent free” selective oxidation of
benzyl alcohol, to observe if there is a potential correlation between the catalytic oxidation capacity
of the materials and the observed oxidative stress in bacteria. This may help in the future, for
a more accurate rational design of antibacterial nanosystems, based on their observed catalytic
oxidation activity.
Keywords: mesoporous silica nanoparticles; maleamates; copper; antibacterial; ROS; oxidation reactions
1
. Introduction
In recent years, mesoporous silica nanoparticles (MSNs) have been extensively used as supports
of a wide variety of therapeutic compounds, representing one of the most important support materials
in drug-delivery [ ]. In most cases, MSNs were loaded with cargo molecules, such as organic-based
anticancer drugs, to study their release profile and their potential therapeutic interest [5,6].
1–4
Pharmaceutics 2019, 11, 30; doi:10.3390/pharmaceutics11010030
www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2019, 11, 30
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However, mesoporous silica-based materials have also been functionalized with metallodrugs of
interest in cancer treatment, such as platinum compounds [ 14], titanium compounds [10 15 23],
tin compounds [22 24 25] and even ruthenium photosensitizers [26], observing promising anticancer
7–
, –
,
,
activity and a potential future application in clinical trials. The cellular action of these
metallodrug-functionalized nanostructured silica-based materials showed that, in most cases, the
nanosystems do not release the metallodrug to the biological medium (or the metallodrug release rates
are very low), and they usually act as “non-classical” drug-delivery systems, whose cytotoxic activity
is due to the action of the entire nanoparticle. Despite the lack of release of the metallodrug to the
medium and the fact that the cytotoxic action of these systems is due to the particle action, it seems
that the anticancer properties of these materials rely on the triggering of a cell death mechanism which
depends highly on the supported metallodrugs (even though they are not released to the medium),
as previously reported by our group [21].
We decided then to extend our studies of silica-based nanostructured materials functionalized
with metallodrugs to different therapeutic applications and distinct metal complexes. The use of new
antimicrobials and the confirmation of the importance of the eradication of microorganisms in the
first moments of bacterial colonization justify the need to find new therapeutic alternatives associated
with the eradication and control of infections. Hypermutability and high bacterial inoculum facilitate
the presence of selected multiresistant mutants during antimicrobial treatment. Thus, the general
objective of antimicrobial treatment is to minimize the bacterial cell mass, which implies the need to
use bactericidal drugs, which do not allow the selection of resistance mechanisms. The lack of effective
antimicrobials emphasizes the need for new approaches through the development of novel therapeutic
strategies that are capable of eliminating the microorganism by the use of novel nanomaterials.
Mesoporous silica nanoparticles (MSNs) and other mesoporous silica-based materials have usually
been studied in antibacterial studies loaded with different antibiotics [27] or other biocides [28].
In addition, mesoporous silicas have also been used as supports for the incorporation of silver or
copper nanoparticles [29] and have demonstrated interesting antibacterial properties. However, their
use as support of metal complexes with antibacterial properties is still very limited and only a very
recent study on the support of nickel and copper Schiff-base complexes with modest antibacterial
properties has been reported [30]. Thus, we have used MSNs of particle size below 100 nm to study the
incorporation of a maleamic acid fragment, as maleamates are able to perturb the biological action of
the enzyme maleamate amidohydrolase [31]. One should take into account that one of the steps in the
oxidative degradation of nicotinates in bacteria is the hydrolytic deamination of maleamate to maleate,
catalyzed by maleamate amidohydrolase. This might have a negative effect on bacterial viability [32],
which might be increased by increasing the concentration of maleamates in bacteria. Thus, we have
studied the coordination of copper to the maleamato ligand to obtain supported Cu complexes to
determine if there is a synergistic effect from both the copper center and the maleamato ligand that
may result in significant antibacterial properties, as it is well known that copper compounds present
interesting antimicrobial properties which have been extensively studied in recent years [33–38].
In particular, we became interested in copper(II) maleamates, because of the attractive properties
that these kinds of compounds have shown in previous antibacterial studies [39–43]. Although
these copper(II) compounds did not show a good degree of stability in physiologic medium, (even
though they incorporated auxiliary bipyridine and/or phenanthroline ligands in their structure), they
presented a potent antimicrobial activity which needs to be examined in detail. Thus, we decided
to prepare similar systems, but supported on MSNs, to study their antibacterial effect against two
types of bacteria (Gram positive and Gram negative) Escherichia coli and Staphylococcus aureus. In
addition, the induction of oxidative stress in the studied bacteria was also tested for the synthesized
nanosystems with interesting results that show that the maleamate-supported ligands, as well as the
copper complexes, are triggering a mechanism of oxidative stress in both E. coli and S. aureus.
Finally, bearing in mind that some of the current studies in biomedicine are focused on the
potential use of the catalytic properties of some made-to-measure catalytic metallodrugs, which are

Pharmaceutics 2019, 11, 30
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able to improve the therapeutic effects in different diseases through their catalytic reactivity [44
and that some copper species have already been supported onto MSNs showing interesting biological
and/or catalytic properties [49 52], we have evaluated the potential influence of the catalytic selective
–48];
–
oxidation properties of the studied materials in the induction of oxidative stress in bacteria. We have
tested the synthesized nanomaterials as oxidation catalysts in “solvent free” oxidation reactions, to try
to determine if there is a potential correlation between the catalytic oxidation capacity of the materials
in regular organic reactions and the observed oxidative stress in bacteria. The results indicate that,
with a thorough future study in this field, a rational design of antibacterial agents may be possible by
simply analyzing their catalytic oxidation capacity, prior to the evaluation of their antibacterial activity.
2
. Materials and Methods
2
.1. General Remarks on the Synthesis of Materials
All reactions were performed using standard Schlenk tube techniques in an atmosphere of dry
nitrogen. Solvents were distilled from the appropriate drying agents and degassed before use. The
reagents used in the preparation of the starting material (mesoporous silica nanoparticles (MSN)),
such as tetraethyl orthosilicate (TEOS) and hexadecyltrimethylammonium bromide (CTAB), were
purchased from Sigma Aldrich (Tres Cantos, Spain) and Acros Organics (Geel, Belgium), respectively.
Triethoxysilylpropylmaleamic acid (maleamic) used in the synthesis of MSN-maleamic was purchased
from Fluorochem (Derbyshire, UK). Copper(II) nitrate trihydrate and 5,5’-dimethyl-2,2’-bipyridine
used in the preparation of the final material (MSN-maleamic-Cu), were purchased from Scharlab
(Barcelona, Spain) and Fluorochem (Derbyshire, UK), respectively. All reagents were used directly
without further purification.
2
.2. General Remarks on the Characterization of Materials
1
3
C-CP MAS spectra, were recorded on a Varian-Infinity Plus Spectrometer (Varian Inc., Palo Alto,
◦
CA, USA) at 400 MHz operating at 100.52 MHz proton frequency (4
µs 90 pulse, 4000 transients,
spinning speed of 6 MHz, contact time 3 ms, pulse delay 1.5 s). X-ray diffraction (XRD) pattern of
the systems were obtained on a Philips Diffractometer model PW3040/00 X’Pert MPD/MRD (Philips,
Eindhoven, The Netherlands) at 45 kV and 40 mA, using a wavelength Cu Kα (λ = 1.5418 Å). Cu wt %
determinations by X-ray fluorescence were carried out with an X-ray fluorescence spectrophotometer
Philips MagiX (Philips, Eindhoven, The Netherlands) with an X-ray source of 1 kW and a Rh anode
using a helium atmosphere. Thermogravimetry analysis were obtained on a Shimadzu mod. DSC-50Q
◦
◦
(
Shimadzu, Kioto, Japan) operating between 30 and 950 C (ramp 20 C/min) at an intensity of 50 A.
N gas adsorption–desorption isotherms were performed using a Micromeritics ASAP 2020 analyzer
2
(Micromeritics, Norcross, GA, USA). Conventional transmission electron microscopy (TEM) was
carried out on a JEOL JEM 1010 (Jeol Ltd., Akishima, Japan), operating at 100 kV using copper
grids. For the preparation of the samples, suspensions in ethanol were sonicated for 10 min,
subsequently, a single drop of the suspensions was added to the grid, and the solvent was evaporated
at room temperature.
2
.3. Synthesis of Mesoporous Silica Nanoparticles (MSN Material)
The synthesis of MSN material was carried out from a modification of the experimental procedure
reported by Zhao et al. [44]. An aqueous solution of CTAB (1 g, 2.74 mmol) was prepared in 480 mL of
nanopure water. Sodium hydroxide (2.0 M, 3.5 mL) was added to the solution and the temperature
◦
adjusted to 80 C. The silicon source TEOS (5 mL, 22.4 mmol) was added dropwise under vigorous
stirring, and the solution was allowed to react for 2 h. The white precipitate was isolated by filtration
◦
and washed with abundant nanopure water and methanol and subsequently dried for 24 h at 80 C in
a stove. Finally, a calcination process was carried out at 500 C for 24 h.
◦

Pharmaceutics 2019, 11, 30
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2
.4. Preparation of MSN-Maleamic
First, MSN material (1.0 g) was dehydrated by treatment under vacuum at 80 C for 24 h.
◦
Subsequently, the dehydrated MSN material was suspended in toluene (50 mL) and, a solution of the
ligand triethoxysilylpropylmaleamic acid (2.0 g in 30 mL of dry toluene) was added to the mixture and
◦
stirred at 110 C for 48 h. The solution was then filtered, and the solid fraction washed with toluene
◦
and diethyl ether. The resulting white powder was dried for 24 h at 80 C.
2
.5. Preparation of MSN-Maleamic-Cu
A suspension of MSN-maleamic (0.85 g) in 20 mL of acetonitrile was stirred at room temperature
and subsequently, 357 mg (1.48 mmol) of copper(II) nitrate trihydrate and 272.3 mg (1.48 mmol) of
5
,’-dimethyl-2,2’-bipyridine was added (theoretical level of 10% Cu/SiO ). Subsequently, a solution of
2
◦
sodium hydroxide (5 mL, 2% NaOH w/w) was added and the reaction was stirred at 80 C for 24 h.
The solid product was isolated by centrifugation (6000 rpm, 10 min.) and washed several times with
acetonitrile and Milli-Q water for elimination the excess of reagents. The resulting light blue powder
◦
was dried for 24 h at 80 C.
2
.6. Antibacterial Studies
Minimum inhibitory concentration (MIC) and bactericidal concentration (MBC) were determined
for MSN-maleamic and MSN-maleamic-Cu by using the standard microdilution method on Mueller
Hinton (MH) broth in accordance with standards established by the Clinical and Laboratory Standards
Institute [53]. An overnight culture of Staphylococcus aureus ATCC 29213 or Escherichia coli ATCC 25922
5
7
was diluted to achieve a cell density in the range from 10 to 10 colonies forming units per milliliter
◦
(
CFU/mL) and incubated for 10 min at 37 C. Bacterial growth was observed at 18 h of incubation. The
lowest concentration of the complex that prevented bacterial growth was considered to be the MIC.
Viable bacterial counts were obtained for samples without visible bacterial growth by plating on MH
◦
agar plates, followed by aerobic incubation at 37 C for 18 h. The antimicrobial agent concentration
that produces the death of 99.9% of initial inoculum was considered the MBC.
2
.7. Oxidative Stress Studies
A bacterial suspension (100
µL) of Staphylococcus aureus ATCC 29213 and Escherichia coli
ATCC 25922 was incubated with 100
MSN-maleamic-Cu at MIC concentration for 1 h at 37 C. Subsequently, 20
µ
L of the corresponding materials MSN-maleamic and
◦
µL of H -DCFDA
2
20
µ
M aqueous solution were added. The fluorescence intensity was measured 30 min later with
a spectrofluorometer Biotek Synergy HT with the excitation and emission wavelengths at 480 and
20 nm, respectively. Non-treated bacterial suspensions were used as the control. The experiments
were carried out in triplicate.
5
2
.8. Catalytic Oxidation of Benzyl alcohol
MSN-maleamic and MSN-maleamic-Cu were tested as heterogeneous catalysts for benzyl alcohol
oxidation under regular catalytic conditions (without light activation) to determine their catalytic
activity and selectivity towards the formation of benzaldehyde. In a typical catalytic experiment, 5 mL
(
48.1 mmol) of benzyl alcohol, 0.5 mL (16.32 mmol) of hydrogen peroxide solution 30% (w/w) in H O,
2
and 25 mg of material were added under nitrogen atmosphere to a Schlenk tube. The mixture was
◦
stirred at 110 C during 6 or 24 h. Subsequently, the reaction mixture was filtered through a nylon filter
(
0.45
µ
m) and the filtrate was analyzed by GC-FID (Perkin-Elmer GC Clarus 580, Waltham, MA, USA)
®
with a Velocity column (dimethylpolysiloxane, 30 m, 0.25 mm, 1.00
benzaldehyde using the temperature program of Figure 1, at an injection temperature of 240 C and
with detector temperature of 300 C.
µ
m) to quantify the conversion to
◦
◦

Pharmaceutics 2019, 11, 30
5 of 18
Figure 1. Temperature ramp for gas chromatography (GC) quantification of catalytic products in the
oxidation of benzyl alcohol.
3
. Results and Discussion
3
.1. Synthesis and Characterization of Maleamic-Functionalized Mesoporous Silica Nanoparticles
MSNs were synthesized following the synthetic method described by Zhao and coworkers [54
]
with slight modifications on the purification steps as recently reported by our group [26].
◦
Dehydrated MSN material (treated overnight at 80 C under vacuum) were functionalized with
the maleamato ligand triethoxysilylpropylmaleamic acid (maleamic) to give MSN-maleamic (Scheme 1)
◦
by a simple grafting reaction in toluene at mild temperature (80 C).
Scheme 1. Grafting reaction of MSN material with triethoxysilylpropylmaleamic acid to give
mesoporous silica nanoparticles (MSN)-maleamic.
In a subsequent step, the material MSN-maleamic was reacted in acetonitrile with sodium
hydroxide and copper(II) nitrate in the presence of 5,5’-dimethyl-2,2’-bipyridine, to coordinate copper
centers to the maleamic-supported ligand, giving the copper-functionalized mesoporous silica material
MSN-maleamic-Cu (Scheme 2). The theoretical amount of Cu was adjusted to a theoretical level of 10%
Cu/SiO . All materials, MSN, MSN-maleamic, and MSN-maleamic-Cu, were characterized by several
2
techniques, such as X-ray diffraction (XRD), X-ray fluorescence (XRF), Fourier Transformed-infrarred
spectroscopy (FT-IR), solid-state NMR spectroscopy, diffuse reflectance ultraviolet spectroscopy
(DR-UV), and transmission electronic microscopy (TEM).

Pharmaceutics 2019, 11, 30
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Scheme 2. Coordination reaction of mesoporous silica nanoparticles-maleamato ligand MSN-maleamic
with copper(II) nitrate in the presence of sodium hydroxide and 5,5’-dimethyl-2,2’-bipyridine to
give MSN-maleamic-Cu.
3
.1.1. Quantification of the Functionalization Rate by Thermogravimetry and X-ray Fluorescence
The maleamic-functionalized material MSN-maleamic was characterized by thermogravimetry
(
TG) to determine the quantity of ligand that had been incorporated in the MSNs. The results
◦
◦
showed that, between 120 C and 650 C, the material presented a mass loss of ca. 46% (Table 1
and Figure S1 of supplementary material) which corresponds with a functionalization rate of the
maleamic ligand of ca. 1.45 mmol of maleamic ligand/gram of MSNs (Table 1). In addition, the
TG of the copper-functionalized material showed a similar behavior with a mass loss close to 42%
between 120 C and 650 C (Figure S2 of supplementary material). Thus, the copper-functionalized
material MSN-maleamic-Cu was also characterized by XRF to determine the quantity of supported
Cu wt % in MSN-maleamic-Cu. The results showed that the quantity of Cu was of 3.24% (Table 1)
which is a slightly higher incorporation rate than that found for other copper complexes supported on
mesoporous silica [30] and comparable with previous results obtained by our group when studying
◦
◦
the incorporation of metallodrugs of titanium [15,17,18,21,23] or ruthenium [26] in mesoporous silicas.
These showed functionalization rates between ca. 1% (for some substituted titanocene derivatives) to
ca. 7% (in the case of a grafting of a ruthenium polypyridyl complex).
Table 1. Determination of the quantity of or copper in mesoporous silica nanoparticles-maleamic
ligand (MSN-maleamic) or MSN-maleamic-Cu quantified by thermogravimetry or XRF, respectively.
mmol of Maleamic
or Cu/g Material
Material
Theoretical Cu wt % Experimental Cu wt % 2
Mass Loss % 1
1
MSN-maleamic
MSN-maleamic-Cu
-
10
-
46.3
42.1
1.45 (maleamic)
2
3.24
0.51 (Cu)
1
◦
◦
2
Determined by thermogravimetry between 120 C and 650 C. Determined by XRF.
The relatively low incorporation of Cu (0.51% wt in the material which is only ca. 32% wt of the
starting copper salt) could be due to the difficulty in the deprotonation of the supported maleamic
ligand to give the maleamate derivative. This deprotonation reaction was carried out using a diluted
solution of NaOH because higher concentrations of NaOH may affect the morphology of the MSNs.
Another plausible explanation to the relatively low incorporation of Cu to the material is that a partial

Pharmaceutics 2019, 11, 30
7 of 18
saturation of the pores of MSNs with the maleamic ligand limit the coordination of copper centers,
thus, decreasing the degree of Cu-functionalization.
3
.1.2. Characterization by Powder X-ray Diffraction Studies
The starting material MSN and the maleamate-functionalized systems MSN-maleamate and
MSN-maleamate-Cu have been characterized by XRD. The diffractogram of the unmodified material
◦
◦
◦
MSN (Figure 2) shows three peaks at 2.15 , 3.71 , and 4.28 that correspond to the Miller planes
100), (110), and (200), respectively. The intensity of the peak of the Miller plane (100) is very high
(
while the other two peaks are of low intensity, which is typical behavior for hexagonally ordered
mesoporous materials.
Figure 2. XRD diffractograms of MSN, MSN-maleamic, and MSN-maleamic-Cu.
It is important to note that, after the first step of functionalization with the maleamic ligand,
the intensity of the (100) peak in MSN-maleamic decreases, compared with that observed for the
unmodified MSNs. In addition, the position of the peak slightly shifts towards higher 2
θ angles
(Figure 2). The decrease in the intensity is usually associated with the functionalization inside the
pores of the mesoporous scaffold, which causes a partial blocking of the dispersion points (pores) of
the MSNs. In addition, the change of the position of the diffraction peaks to higher angles is indicative
of a pore size decrease. This corresponds to the functionalization with the maleamate ligand mainly
inside the pores of the nanoparticles.
Similar changes were observed for MSN-maleamic-Cu, as the diffractogram of this material, after
the functionalization with copper, show a decrease in the intensity of the (100) peak in comparison
with that of MSN-maleamic. In this case, the position (2
θ) of the (100) peak in MSN-maleamic-Cu
is practically the same as that of MSN-maleamic (Table 2). It is important to note that, after the
functionalization steps, the a parameter decreases (Table 2) as expected for a slight decrease of the
0
pore size.
Table 2. Powder XRD data of the synthesized compounds.
◦
Material
hkl
00
110
00
100
100
2θ ( )
dhkl (nm)
a0 (nm)
1
2.15
3.71
4.28
2.18
2.18
4.099
-
-
4.053
4.023
4.733
-
-
4.680
4.645
MSN-maleamic
2
MSN-maleamic
MSN-maleamic-Cu

Pharmaceutics 2019, 11, 30
8 of 18
3
.1.3. Nitrogen Adsorption–Desorption Isotherms
The starting MSN and the copper-modified nanostructured material (MSN-maleamic-Cu) have
been characterized by nitrogen adsorption/desorption isotherms. In all cases, one can observe that the
isotherms are between type IV and type VI (Figure 3) according to IUPAC classification [55] which
are typical of mesoporous silica nanoparticles. The isotherms show a hysteresis loop between P/P0
1
.0 and ca. 0.80 which is usually obtained because of the capillary condensation of nitrogen into the
straight pore of the system. Between P/P 0.8 and 0.4, the adsorption and desorption behave similarly,
0
however, an additional hysteresis loop (of minimal differences) was observed between P/P 0.4 and
0
0
.15, again, probably, due to capillary condensation.
Figure 3. N adsorption/desorption isotherms of MSN and MSN-maleamic-Cu.
2
The physical parameters were obtained after a careful analysis of the Brunauer–Emmett–Teller
2
−1
(
BET) isotherms (Table 3). The unmodified MSN material has a BET surface of 853 m
·
g
, a pore
−1
and a very narrow distribution of the pore diameter of 3.42 nm. After the
volume of ca. 0.7 cm3
·
g
functionalization reaction with maleamic ligand and the coordination of copper, the BET surface of
and the pore diameter to 3.11 nm, indicating that the
functionalization reaction mainly takes place inside the pores of the mesoporous silica nanoparticles.
MSN-maleamic-Cu is reduced to 414.8 m2
·
g
−1
Table 3. Physical parameters of MSN and MSN-maleamic-Cu measured by N2 adsorption–
desorption isotherms.
2
3
dp (BJH) 1 (nm)
Material
SBET (m /g)
Total Pore Volume (cm /g)
MSN
MSN-maleamic-Cu
853.03
414.80
1
0.73
0.32
3.42
3.11
Barrett, Joyner and Halenda.
3
.1.4. Solid-State NMR Spectroscopy
The material MSN-maleamic-Cu has been characterized by 13C CP MAS spectroscopy (Figure 4).
The spectrum shows the signals corresponding to the carbon atoms of the silylpropylmaleamato
moiety, the ethoxide groups, and the bipyridine ligands. Thus, a set of signals of high intensity at ca.
7
, 19, and 57 ppm corresponding to the carbon atoms of the Si–CH –CH –CH –N fragment of the
2 2 2
ligand is observed. In addition, two signals corresponding to the maleamate moiety are observed,
namely, a very broad signal of low intensity between 125 and 140 ppm due to the alkenylic carbon

Pharmaceutics 2019, 11, 30
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atoms (CH=CH), and a broad signal of very low intensity between ca. 190 and 200 ppm assigned to the
−
carbonyl carbon atoms of the carboxylate (COO ) or carboxamide (CONH) groups of the maleamate
fragment. Furthermore, the spectrum shows the resonances of the carbon atoms of the bipyridine
ligand as a very broad signal at ca. 172 ppm corresponding to the aromatic carbon atoms of the ring
and a signal at ca. 37 ppm assigned to the carbon atoms of the methyl groups bound to the aromatic
ring). Finally, the spectrum shows the signal of the pendant ethoxide groups at ca. 61 ppm (OCH2)
and 41 ppm (CH3).
Figure 4. 13C CP MAS NMR spectra of MSN-maleamic and MSN-maleamic-Cu.
3
.1.5. DR-UV and FT-IR Studies
The functionalized materials MSN-maleamic and MSN-maleamic-Cu have been characterized by
both DR-UV-vis spectroscopy and FT-IR. The DR-UV spectrum of MSN-maleamic (Figure 5) shows an
intense absorption peak at 210 nm and a small shoulder of adsorption recorded at ca. 250 nm, which
are associated with the maleamic ligand. After incorporation of copper nitrate and the coordination of
bipyridine, the spectrum of MSN-maleamic-Cu shows three clear absorption bands at ca. 210, 254, and
3
09 nm. The first two bands (210 and 254 nm) are due to the maleamato ligand, and the band at 309
is associated with the metal to ligand charge transfer (MLCT) which is usually observed in copper
complexes [56].
Figure 5. DR-UV spectra of MSN-maleamic and MSN-maleamic-Cu.

Pharmaceutics 2019, 11, 30
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FT-IR spectra of the materials MSN-maleamic and MSN-maleamic-Cu show the typical signals of
the mesoporous silica nanoparticles. The spectra present the O–H stretching of the silanol groups and
−
1
the adsorbed water molecules (between 3500 and 3200 cm ), the siloxane (Si–O–Si) groups (as a broad
−
1
−1
band at ca. 1100 cm ), the stretching band of the Si–O bonds (at ca. 900 cm ), and the deformation
−
1
vibrations of adsorbed water molecules at ca. 1625 cm
.
In addition to the signals of the silica, the appearance of some other bands of very low intensity
associated with both the maleamato ligand and/or the copper nitrate bipyridine fragment) are also
(
observed (See supplementary material Figure S3).
3
.1.6. TEM Studies
Finally, the starting material MSN and the copper-functionalized system MSN-maleamic-Cu,
were characterized by TEM. The TEM images (Figure 6a) of the unmodified MSN material show that
the nanoparticles are porous spheres with hexagonal ordered porous parallel channels with a narrow
®
particle size distribution (calculated by using ImageJ program) of ca. 89 ± 3 nm.
Figure 6. TEM images of MSN and MSN-maleamic-Cu.
After functionalization steps, the TEM images of the nanomaterial MSN-maleamic-Cu show that
the morphology of the nanoparticles has not significantly changed observing a similar distribution of
the pores and very similar particle size (92 ± 2 nm) than the unmodified MSNs.
3
.2. Biological Studies of the Synthesized Materials
3
.2.1. Antibacterial Tests
The coordination of copper to the maleamato ligand has been studied to obtain supported Cu
complexes to determine if there is a synergistic effect from both the copper center and the maleamato
ligand that may result in significant antibacterial properties. The MIC and MBC for MSN-maleamic and
MSN-maleamic-Cu by using the standard microdilution method on MH broth have been determined.
The results (Table 4) showed a low activity of MSN-maleamic and MSN-maleamic-Cu against the Gram

Pharmaceutics 2019, 11, 30
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positive bacteria S. aureus, while a good activity against the Gram negative bacteria E. coli was observed.
In general, the material MSN-maleamic (with a MIC of 62.5 g/mL and an MBC of 125 g/mL) is
more active than the material MSN-maleamic-Cu (MIC of 125 g/mL and an MBC of 125 g/mL)
µ
µ
µ
µ
in E. coli in terms of quantity of nanoparticles. However, when one analyzes the MIC referred to
the quantity of maleamic fragment in MSN-maleamic and copper centers in MSN-maleamic-Cu, the
results show a slightly lower MIC value for MSN-maleamic-Cu (Table 4, data in brackets). The MIC
and MBC of copper is similar, if not somewhat lower, than that found for similar systems based on
copper nanoparticles, confirming that the bactericidal activity synthesized material MSN-maleamic-Cu
in terms of copper MICs (4.1 to >8.1 µg/mL) is comparable, if not slightly higher, than those of other
Cu-based nanoparticulated systems [57–60] which showed MICs of ca. 6 to 20 µg/mL Cu.
Table 4. Minimum inhibitory concentration (MIC) and bactericidal concentration (MBC) for
MSN-maleamic and MSN-maleamic-Cu against Staphylococcus aureus and Escherichia coli. Data are
given in
µg/mL of material and data in brackets refer to
µg/mL of maleamato ligand or copper
centers, respectively.
Staphylococcus Aureus
Escherichia Coli
ATCC 25922
ATCC 29213
Material
MIC (µg/mL)
MBC (µg/mL)
MIC (µg/mL)
MBC (µg/mL)
MSN-maleamic
MSN-maleamic-Cu
250 [41.7]
>250 [>8.1]
>250 [>41.7]
>250 [>8.1]
62.5 [10.4]
125 [4.1]
125 [20.9]
125 [4.1]
When comparing the activity of the studied materials with some clinically used antibiotics,
such as ciprofloxacin, erythromycin, gentamicin, tetracycline, doxycycline, chloramphenicol or some
others against S. aureus populations, the synthesized materials MSN-maleamic and MSN-maleamic-Cu
showed a slightly higher activity than commercial antibiotics (with values from 0.5 to 128 µg/mL
depending on the antibiotic) [61], while the activity is similar or slightly higher against different E. coli
populations (with values from 0.5 to >256 µg/mL) [62].
3
.2.2. Oxidative Stress Studies
Oxidative stress is usually involved in the toxicity of different antibiotics used in the treatment
of bacterial infections [63] which leads to the oxidation of essential macromolecules and induces cell
death [64]. Determination of oxidative stress is usually interesting to observe if this is the main reason
of the antibacterial activity of the different agents. Thus, we studied the production of reactive oxygen
species (ROS) in bacterial suspensions incubated with 100
at 37 C.
µL of materials at MIC concentration for 1 h
◦
As can be seen in Figure 7, the synthesized materials MSN-maleamic and MSN-maleamic-Cu
generate in S. aureus ca. 50 and 30% more ROS, respectively, than untreated bacterial suspensions.
In E. coli, both materials generate an increment of ROS of 40% respect to control. Interestingly,
the material MSN-maleamic seems to generate more ROS than its analogue MSN-maleamic-Cu,
which is in agreement with a lower MIC and MBC found in the preliminary antibacterial tests and
which may indicate that the mechanism of bacterial death promoted by these kinds of functionalized
mesoporous silica-based materials is the oxidative stress generation mediated by an increment of ROS.
It is important to note that the major part of the external surface area of MSNs is in the pores of the
nanoparticles (higher than 95% of the surface as it corresponds to a mesoporous system), therefore,
the major part of the surface in contact with the bacteria is the external part of the pores which are
usually the areas with higher functionalization rates. The formation of ROS promoted by unmodified
MSN was also studied for both E. coli and S. aureus observing that the starting material produces much
lower quantity of ROS than the functionalized materials, the results observed for unmodified MSN are
in agreement with those observed previously in a recent report [65].

Pharmaceutics 2019, 11, 30
12 of 18
1
80
60
40
20
00
1
*
*
*
1
*
1
1
80
60
40
20
0
S
S
.
.
a
au
u
r
r
e
e
u
u
s
s
E. co
E. colili
Figure 7. Percentage reactive oxygen species (ROS) increase induced in bacterial suspensions incubated
with 100 L of MSN-maleamic and MSN-maleamic-Cu at minimum inhibitory concentration (MIC)
concentrations (the assays were performed at least in triplicate in three independent assays. Data
are expressed as means SD and analyzed by the Student’s t-test. p < 0.01 was used as the level of
µ
±
statistical significance compared with control; * = p < 0.01).
It is important to note that, in the synthesized nanostructured systems, the pores are functionalized
with either the maleamic ligand or the maleamato-Cu complex and they show influence on the
antibacterial behaviour of the materials, even though the bacteria may only be in contact with the
external part of the pores. However, in the culture medium, we expect that the nanoparticles may
internalize the bacteria and the reactions and formation of ROS may also be possible inside the pores
justifying the obtained MICs and ROS production results after functionalization of the materials with
the maleamic ligand or the maleamato-Cu complex.
3
.3. Study of the Catalytic Oxidation Activity of the Synthesized Materials
A topic of current interest in biomedicine is the potential use of the catalytic properties of some
made-to-measure catalytic metallodrugs in therapeutic applications. In view of the results obtained for
the generation of ROS in bacteria promoted by MSN-maleamic and MSN-maleamic-Cu nanomaterials,
the catalytic activity in a “solvent free” oxidation reaction of benzyl alcohol (Scheme 3) was studied to
determine if there is a potential correlation between the catalytic activity of the materials and their
production of ROS in bacteria.
Thus, the catalytic activity of MSN-maleamic and MSN-maleamic-Cu in the selective oxidation
of benzyl alcohol was tested using H O as an oxidant agent. Usually, hydrogen peroxide is a
2
2
more interesting oxidant than organic agents (such as tert-butyl hydroperoxide (TBHP)) in inorganic
materials because of the higher mobility of hydrogen peroxide on the surface of the materials, its
lower price and its lower environmental impact compared with most of the most widely used organic
◦
oxidants. The reactions were carried out at 110 C during 6 or 24 h, and the results are given in Table 5.

Pharmaceutics 2019, 11, 30
13 of 18
Scheme 3. Catalytic oxidation reaction of benzyl alcohol.
Table 5. Turnover number (TON), turnover frequency (TOF), selectivity to benzaldehyde or
benzyl benzoate and total TON in the oxidation of benzyl alcohol catalyzed by MSN-maleamic
or MSN-maleamic-Cu.
Benzaldehyde
Benzyl Benzoate
Material
Time (h)
Total TON
Selectivity
%)
Selectivity
(%)
TOF (h 1)
−
TON
TOF (h
−1
)
TON
(
MSN
0.34 ± 0.28
0.014
±
0.011 83.7 ± 16.1
0.041
0.014 54.2 ± 15.0
0.11 ± 0.04
0.005
±
0.002 16.3 ± 16.1
0.001
0.008 45.8 ± 15.0
24
0.45 ± 0.32
0
.69 ± 0.24
0.116
0.062
±
92.1 ± 1.9
0.06 ± 0.02
0.002
0.037
±
7.9 ± 1.9
6
24
0.75 ± 0.26
MSN-maleamic
1
.49 ± 0.34
±
0.89 ± 0.21
±
2.38 ± 0.55
MSN-maleamic
Cu
0.85 ± 0.06
0.56 ± 0.14
0.141
0.023
±
±
0.010 75.8 ± 10.6
0.23 ± 0.12
0.31 ± 0.19
0.012
0.009
±
±
0.001 24.2 ± 10.6
6
24
1.08 ± 0.18
0.87 ± 0.33
0.006 70.1 ± 10.9
0.005 29.9 ± 10.9
The catalytic study shows that both materials MSN-maleamic and MSN-maleamic-Cu present
similar oxidation activity in reactions carried out at 6 h with turnover number (TON) values between
ca. 0.6 and 0.9, while the oxidation capacity of MSN-maleamic is much higher at longer times
(
24 h). In addition, it is important to note that the unmodified MSN was slightly active at 24 h
as expected when compared with the activity reported for other mesoporous silica nanostructured
materials [66 67]. In addition, the selectivity of the oxidation reaction towards benzaldehyde is very
,
high at 6 h reaction for MSN-maleamic (higher than 90%) while it is much lower in the case of
MSN-maleamic-Cu (ca. 75%). The selectivity towards benzaldehyde clearly decreases after 24 h of
reaction for both catalysts MSN-maleamic and MSN-maleamic-Cu with a much higher decrease down
to ca. 55% in MSN-maleamic. When one compares the total TON (the sum of TON of benzaldehyde
and benzyl benzoate) observed in the reactions after 24 h, the results point towards a more efficient
oxidation capacity promoted by the maleamic-based material (MSN-maleamic with total TON of
ca. 2.4) than its copper analogue (MSN-maleamic-Cu with total TON of ca. 0.9), which is in agreement
with the higher ROS production observed and discussed in the previous Section 3.2. In this context,
the unmodified MSN has a low total TON of ca. 0.75 after 24 h which is also in agreement with the
−
1
observation of relatively low ROS in the antibacterial tests. Turnover frequency (TOF) values in h
are all following the same behavior than TON values as shown in Table 5.
It is noteworthy that the non-expected oxidation behavior of MSN-maleamic-Cu with time, as the
total oxidation (total TON) is higher at 6 h (1.08 0.18) than at 24 h (0.87 0.33). We then studied the
±
±
possible influence of the quantity of hydrogen peroxide in the oxidation capacity of the copper-based
material, observing that, in all cases, at higher quantities of hydrogen peroxide in the mixture (more
than 0.5 mL hydrogen peroxide in the reaction mixture) the oxidation capacity (TON) is higher at
6
h than at 24 h (See Table S1 of supplementary material). This behavior may be explained by the
possibility of Fenton-like degradation processes which may be present at high quantities of hydrogen

Pharmaceutics 2019, 11, 30
14 of 18
peroxide as observed previously in several other studies [68
,
69]. The Fenton-like degradation processes
may lead to decomposition of benzaldehyde and/or benzyl benzoate, reducing the apparent total
TON of the oxidation reactions. In this context, the degradation processes seem to be more abundant
with higher quantities of hydrogen peroxide and after 24 h of reaction.
4
. Conclusions
In conclusion, in this preliminary study we have carried out the synthesis and characterization of
MSNs functionalized with a maleamato ligand and the subsequent coordination of copper to give an
MSN-supported copper maleamate complex. These materials exhibit good activity against E. coli and
have been shown to generate a high quantity of ROS in the studied bacteria. In addition, our studies on
the oxidation of benzyl alcohol catalyzed by the synthesized materials indicates that there seems to be a
correlation of the catalytic activity and selectivity in oxidation reactions with the antimicrobial activity
and ROS generation, which motivates a more thorough study of a hypothetical structure-dependence
in oxidation and antibacterial activities promoted by functionalized mesoporous silica nanoparticles.
In this context, our future studies will focus, first, on a thorough study of the mechanism
of antibacterial action of the materials reported here, determining if they act as “classical” or
“
non-classical” drug-delivery systems. In addition, our work will also focus on the design and synthesis
of new maleamate-functionalized nanomaterials and the incorporation of other metal complexes
and ligands to try to clearly determine, in an extensive number of different nanomaterials, if the
catalytic activity can be correlated with the potential antibacterial activity. Moreover, the antibacterial
studies will extend to other informative bacterial assays, to determine which reactive oxygen species
are responsible for the oxidative damage to macromolecules, in addition, we will try to observe if
the synthesized materials promote resistance of the bacterial populations. Finally, bacterial DNA
fragmentation and membrane potential will also be examined. The results will be compared with
different selective catalytic oxidation reactions to support the hypothesis of a relationship between the
catalytic activity in oxidation reactions and the antibacterial properties.
Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/11/1/30/s1,
Figure S1: TG of MSN-maleamic; Figure S2: TG of MSN-maleamic-Cu, Figure S3: FT-IR spectra of MSN-maleamic
and MSN-maleamic-Cu; Table S1: TON, TOF, selectivity to benzaldehyde or benzyl benzoate in the oxidation of
a
benzyl alcohol catalyzed by MSN-maleamic-Cu with different H O quantities.
2
2
Author Contributions: Conceptualization, S.G.-R. and P.L.P.; methodology, D.D.-G., P.R.A. and A.R.-D.;
investigation, D.D.-G. and P.R.A.; resources, S.G.-R. and P.L.P.; writing—original draft preparation, S.G.-R.,
P.L.P., D.D.-G., P.R.A. and S.P.; writing—review and editing, S.G.-R., P.L.P., D.D.-G., P.R.A. and S.P.
Funding: This research was funded by Ministerio de Ciencia, Innovación y Universidades Spain-FEDER, grants
number CTQ2015-66164-R and CTQ2017-90802-REDT and by Agencia Nacional de Promoción Científica y
◦
Tecnológica (ANPCyT) (PICT 2015 N 1558).
Acknowledgments: We would like to thank to Consejo Nacional de Investigaciones Científicas y Técnicas de
Argentina (CONICET).
Conflicts of Interest: The authors declare no conflict of interest.
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