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Citation: Figari, G.; Gonçalves,
J.L.M.; Diogo, H.P.; Dionísio, M.;
Farinha, J.P.; Viciosa, M.T.
Understanding Fenofibrate Release
from Bare and Modified Mesoporous
Silica Nanoparticles. Pharmaceutics
2023,15, 1624. https://doi.org/
10.3390/pharmaceutics15061624
Academic Editor: Margherita
Morpurgo
Received: 20 April 2023
Revised: 19 May 2023
Accepted: 25 May 2023
Published: 30 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pharmaceutics
Article
Understanding Fenofibrate Release from Bare and Modified
Mesoporous Silica Nanoparticles
Giorgia Figari 1, JoséL. M. Gonçalves 1, Hermínio P. Diogo 1, Madalena Dionísio 2, JoséPaulo Farinha 1,*
and María Teresa Viciosa 1, *
1
Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, University of Lisbon, Avenida Rovisco
Pais, 1049-001 Lisbon, Portugal
2
LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade Nova
de Lisboa, 2829-516 Caparica, Portugal
*Correspondence: farinha@tecnico.ulisboa.pt (J.P.F.); teresaviciosa@tecnico.ulisboa.pt (M.T.V.)
Abstract:
To investigate the impact of the surface functionalization of mesoporous silica nanoparticle
(MSN) carriers in the physical state, molecular mobility and the release of Fenofibrate (FNB) MSNs
with ordered cylindrical pores were prepared. The surface of the MSNs was modified with either
(3-aminopropyl) triethoxysilane (APTES) or trimethoxy(phenyl)silane (TMPS), and the density of the
grafted functional groups was quantified via
1
H-NMR. The incorporation in the ~3 nm pores of the
MSNs promoted FNB amorphization, as evidenced via FTIR, DSC and dielectric analysis, showing no
tendency to undergo recrystallization in opposition to the neat drug. Moreover, the onset of the glass
transition was slightly shifted to lower temperatures when the drug was loaded in unmodified MSNs,
and MSNs modified with APTES composite, while it increased in the case of TMPS-modified MSNs.
Dielectric studies have confirmed these changes and allowed researchers to disclose the broad glass
transition in multiple relaxations associated with different FNB populations. Moreover, DRS showed
relaxation processes in dehydrated composites associated with surface-anchored FNB molecules
whose mobility showed a correlation with the observed drug release profiles.
Keywords:
mesoporous silica nanoparticles; confined water; fenofibrate; amorphous state; drug release
1. Introduction
Aiming to improve the therapeutic activity of already-established drugs, yet limited
in their activity, the pharmaceutical industry has redirected a good part of its research to
the development of new formulations, saving the cost associated with the discovery of
new ingredients.
Examples of this strategy are formulations that explore the possibility of using the drug
in metastable states or in the solid amorphous form, whose excess free energy in relation to
the more stable crystalline counterpart may result in improved solubility and/or dissolution
rate [
1
]. However, because the solid amorphous form is out of thermodynamic equilibrium,
it can evolve to the more stable crystalline state or, in the case of crystalline metastable
states, to another polymorph. Therefore, to benefit from amorphization, efforts have
been made to reduce the driving force to recrystallization, whether it takes place during
manipulation and storage or in the dissolution steps. This can be achieved via incorporation
in nanoporous matrices [
2
–
4
]. The amorphization of the guest occurs via a spatial limitation,
i.e., the pore size is small enough in relation to the critical nucleation size, preventing
the rearrangement of the disordered molecules into long-range ordered structures, or
inducing the formation of a different crystalline network (polymorphism) [
5
]. The reduced
cooperative mobility of the loaded drug may influence the tendency to organize in a definite
manner, impairing the formation of a crystal lattice structure [
6
]. In fact, low molecular
mobility, which is a measure of the rate of cooperative rearrangements in the amorphous
state, and high configurational entropy, the entropy difference between the disordered and
Pharmaceutics 2023,15, 1624. https://doi.org/10.3390/pharmaceutics15061624 https://www.mdpi.com/journal/pharmaceutics
Pharmaceutics 2023,15, 1624 2 of 27
crystalline states, were identified by Zhou and co-workers [
7
] as key factors governing
the avoidance of crystallization. This study was carried out on several drugs including
fenofibrate (FNB), the target compound tested in the present work, a pro-drug which
undergoes hydrolysis to active fenofibric acid, which has been prescribed since the 1980s to
treat hypercholesterolemia [8,9].
In accordance with the Biopharmaceutical Classification System, FNB (Scheme 1) be-
longs to class II due to its high permeability through lipid membranes (>90%) and its poor
aqueous solubility (0.8
µ
g mL
−1
) [
10
], a drawback that could reduce the drug’s bioavail-
ability. Therefore, many recent formulations have been developed [
9
,
11
,
12
]. Among these,
there have been several attempts reported in the literature to explore FNB in the amorphous
state by incorporating it in silica matrices [
13
,
14
]. These drug carriers present significant
low cytotoxicity [
15
–
17
] and a high surface area, allowing for a high dispersion of the
loaded compound. However, the crystallization tendency has not been totally suppressed
in silica-based FNB formulations [
18
,
19
]. Despite this, silica-based FNB formulations
have shown improved solubility and increased bioavailability, as well as high potential in
clinical studies [20,21].
Pharmaceutics 2023, 15, x FOR PEER REVIEW 2 of 28
the tendency to organize in a denite manner, impairing the formation of a crystal laice
structure [6]. In fact, low molecular mobility, which is a measure of the rate of cooperative
rearrangements in the amorphous state, and high congurational entropy, the entropy
dierence between the disordered and crystalline states, were identied by Zhou and co-
workers [7] as key factors governing the avoidance of crystallization. This study was car-
ried out on several drugs including fenobrate (FNB), the target compound tested in the
present work, a pro-drug which undergoes hydrolysis to active fenobric acid, which has
been prescribed since the 1980s to treat hypercholesterolemia [8,9].
In accordance with the Biopharmaceutical Classication System, FNB (Scheme 1) be-
longs to class II due to its high permeability through lipid membranes (>90%) and its poor
aqueous solubility (0.8 μg mL−1) [10], a drawback that could reduce the drug’s bioavaila-
bility. Therefore, many recent formulations have been developed [9,11,12]. Among these,
there have been several aempts reported in the literature to explore FNB in the amor-
phous state by incorporating it in silica matrices [13,14]. These drug carriers present sig-
nicant low cytotoxicity [15–17] and a high surface area, allowing for a high dispersion of
the loaded compound. However, the crystallization tendency has not been totally sup-
pressed in silica-based FNB formulations [18,19]. Despite this, silica-based FNB formula-
tions have shown improved solubility and increased bioavailability, as well as high po-
tential in clinical studies [20,21].
Scheme 1. Fenobrate (propan-2-yl 2-(4-[(4-chlorophenyl)carbonyl]phenoxy)-2-methylpropano-
ate) chemical structure with oxygen atoms numbered.
Motivated by such promising results using mesoporous silica as a carrier for FNB, it
is intended to deeply understand how the matrix characteristics in terms of pore geometry
and surface treatment inuence the guest’s physical state, molecular mobility and, ulti-
mately, its release under physiological conditions, trying to provide a rational basis for
the future design of drug delivery systems. Neat FNB can be taken as a model compound
due to its glass-forming ability, allowing it to test its molecular mobility in amorphous
form [22,23], but with a tendency for crystallization when kept close to room temperature.
Therefore, spherical mesoporous silica nanoparticles (MSNs) with cylindrical pore
diameters ~3 nm were prepared. The pore wall was functionalized to evaluate the impact
of the matrix surface on the drug release prole compared with the bare nanoparticles.
Aiming for this, the bare MSNs were chemically modied replacing some silanol groups
with, respectively, hydrophilic (3-aminopropyl) triethoxysilane (APTES) or hydrophobic
trimethoxy(phenyl)silane (TMPS); the functionalized nanoparticles will be hereafter des-
ignated as MSN_APTES and MSN_TMPS. As silanol density impacts water diusivity
and surface weability [24], the water dynamical behavior was probed via dielectric re-
laxation spectroscopy (DRS) as a mean to evaluate the inuence of APTES or TMPS graft-
ing.
lactone
ester
Scheme 1.
Fenofibrate (propan-2-yl 2-(4-[(4-chlorophenyl)carbonyl]phenoxy)-2-methylpropanoate)
chemical structure with oxygen atoms numbered.
Motivated by such promising results using mesoporous silica as a carrier for FNB, it is
intended to deeply understand how the matrix characteristics in terms of pore geometry and
surface treatment influence the guest’s physical state, molecular mobility and, ultimately,
its release under physiological conditions, trying to provide a rational basis for the future
design of drug delivery systems. Neat FNB can be taken as a model compound due to its
glass-forming ability, allowing it to test its molecular mobility in amorphous form [
22
,
23
],
but with a tendency for crystallization when kept close to room temperature.
Therefore, spherical mesoporous silica nanoparticles (MSNs) with cylindrical pore
diameters ~3 nm were prepared. The pore wall was functionalized to evaluate the impact
of the matrix surface on the drug release profile compared with the bare nanoparticles.
Aiming for this, the bare MSNs were chemically modified replacing some silanol groups
with, respectively, hydrophilic (3-aminopropyl) triethoxysilane (APTES) or hydrophobic
trimethoxy(phenyl)silane (TMPS); the functionalized nanoparticles will be hereafter desig-
nated as MSN_APTES and MSN_TMPS. As silanol density impacts water diffusivity and
surface wettability [
24
], the water dynamical behavior was probed via dielectric relaxation
spectroscopy (DRS) as a mean to evaluate the influence of APTES or TMPS grafting.
The bare and functionalized MSNs were loaded with FNB and the obtained composites
were characterized by a set of experimental techniques. FTIR and DSC were used to
access the physical state of the loaded FNB, and its mobility was probed via DRS, in both
hydrated and dehydrated states, allowing for the correlation of the guest dynamics with
the observed release profiles. Since the pore size is of the order of a few nanometers, the
Pharmaceutics 2023,15, 1624 3 of 27
work provides fundamental information regarding finite size effects from which a length
scale for crystallization and glass transition can be inferred.
2. Materials and Methods
2.1. Materials
Fenofibrate, IUPAC name propan-2-yl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoate
(CAS number 49562-28-9), was purchased from Sigma-Aldrich, St. Louis, MO, USA
(purity
≥
99%) and was used without further purification. The empirical formula is
C
20
H
21
ClO
4
, and the molecular weight is 360.83 g mol
−1
. Absolute ethanol (EtOH, >99.9%
Scharlau), tetraethoxysilane (TEOS,
≥
99%, Sigma-Aldrich GHEMIE GmbH, Steinheim,
Germany), cetyltrimethylammonium bromide (CTAB,
≥
99%, Sigma, St. Louis, MO, USA)
and sodium hydroxide solution (NaOH 1.4 M) were used without any purification in
the synthesis of the mesoporous silica nanoparticles. For the removal of the surfactant, a
solution of 0.5 M hydrochloric acid (HCl, 37% Panreac AppliChem, Darmstad, Germany)
in absolute ethanol was used. The deionized water was produced from a Millipore system
Milli-Q
≥
18 M
Ω
cm (with a Millipak membrane filter 0.22
µ
m). Regarding nanoparticle
functionalization, they were surface modified with trimethoxy(phenyl)silane (97% TMPS,
Sigma-Aldrich) and (3-Aminopropyl) triethoxysilane (98% APTES, Sigma-Aldrich) without
any treatment in dried toluene, which was distilled over calcium hydride before use. For
the NMR analysis samples, we used 1,3,5-trioxane (
≥
99.0%, Fluka, Seelze, Germany),
deuterium oxide (D
2
O, 99.9% atom, CIL) and dimethyl sulfoxide D6 (DMSO, 99.9%, CIL).
Disodium hydrogen phosphate (Na
2
HPO
4
, 99%, Riedel-de-Haën, Seelze, Germany) and
sodium dihydrogen phosphate monohydrate (NaH
2
PO
4
.H
2
O, 98%, Panreac, Castellar del
Vallès, Spain) were used to prepare the phosphate-buffered solution (PBS, pH 8) for the
release studies.
2.2. Equipment
Centrifugation. An Avanti J-301 (Beckman Coulter, Brea, CA, USA) was used for clean-
ing the MSNs after template removal. For the centrifugations, 50 mL centrifuge propylene
tubes were used. A Sigma 2k15 (B. Braun, Laguna Hills, CA, USA) centrifuge, rotor 12,141,
was used for washing the functionalized MSNs at 1100 min
−1
. Disposable 10 mL polypropy-
lene tubes were used for the centrifugations. A Hitachi Himac CT 15RE centrifuge was
utilized with 2 mL Eppendorf for washing bare and functionalized nanoparticles after drug
loading at 15,000 rpm.
pH measurements. The pH of the phosphate-buffered solution for the release studies
was measured using a pHenomenal®, 1000 L bench pH (mV◦C)−1meter pH.
Transmission electron microscopy (TEM). TEM images were obtained using a Hitachi
transmission electron microscope (Hitachi High-Technologies, Tokyo, Japan), model H-8100,
with a LaB6 filament (Hitachi) and an accelerator voltage of 200 kV. A camera KeenView
(Soft Imaging System, Münster, Germany) is incorporated into this equipment, which
via iTEM software allows one to acquire TEM images. MSNs dispersed in ethanol were
prepared and dried on a copper grid coated with carbon. The size/dimension, polydis-
persity and morphology of the particles were estimated using ImageJ 1.50i Fiji software
(Madison, WI, USA).
Nuclear magnetic resonance (
1
H NMR). The spectra were recorded using an AMX-400
instrument (Bruker, MA, USA) at 400 MHz. For this purpose, two solutions of NaOH
and 1,3,5-trioxane (internal standard, IS) in deuterated water (D
2
O) were prepared. In an
NMR tube, 5 mg of the sample, 400
µ
L of a solution of NaOH and 100
µ
L of a solution of
1,3,5-trioxane in D2O were mixed and sonicated.
Fourier transform infrared spectroscopy (FTIR). The FTIR spectra of pure MSNs, native
crystalline FNB and FNB loaded in nanoparticles, after solvent evaporation, were collected
using a Bruker (model: Alpha) over the range 4000–400 cm
−1
at room temperature in the
form of KBr pellets.
Pharmaceutics 2023,15, 1624 4 of 27
Differential scanning calorimetry (DSC). The thermal features were examined using
a differential scanning calorimeter DSC Q2000 from TA Instruments Inc. Guyancourt,
France (Tzero DSC technology), operating in the Heat Flow T4P option. Enthalpy (cell
constant) and temperature calibration were based on the melting peak of indium standard
(T
m
= 156.60
◦
C). Approximately 5 mg of each sample was introduced in an aluminum
hermetic pan with a Tzero hermetic lid and a pinhole to facilitate the exit of water. Thermo-
grams of all of the samples were obtained over a range of
−
90
◦
C to 120
◦
C at a heating
rate of 10
◦
C min
−1
under a nitrogen flow of 50 mL min
−1
and they were analyzed during
heating. Then, the analysis of data was carried out using the software Universal Analysis
2000 from Thermal Analysis. The melting point (T
m
) was determined as the minimum of
the endothermic peak, whereas the glass transition temperature (T
g
) was determined at the
onset of the glass transition.
Dielectric relaxation spectroscopy (DRS). Dielectric measurements were carried out
using the ALPHA-N impedance analyzer from Novocontrol Technologies GmbH, covering
a frequency range from 0.1 Hz to 1 MHz. The sample powder was placed between two
gold-plated electrodes of a parallel plate capacitor, BDS 1200, with two 50
µ
m thick silica
spacers, and the sample cell was mounted on a BDS 1100 cryostat and exposed to a heated
gas stream being evaporated from a liquid nitrogen dewar. The temperature was controlled
using the Quatro Cryosystem and performed at 0.5 ◦C.
To analyze the isothermal dielectric data, the Havriliak–Negami (HN) model func-
tion [
25
,
26
] was fitted to both imaginary and real components of the electrical modulus,
M∗
HN (ω). To account for the multimodal profile, a sum of HN functions was considered:
M∗
HN (ω)=M∞+∑
j
∆M
(1+ (−i(ωτH N)−1)αHN )βHN (1)
where jis the index over which the individual processes are summed,
∆
M=M
0−
M
∞
,
τHN
is the characteristic HN relaxation time and
αHN
and
βHN
are shape parameters
(0 <
αHN
<1and0<
αHN βHN ≤
1) describing, respectively, the symmetric and asymmetric
broadening of the complex dielectric function [27].
From the estimated values of
τHN
and the shape parameters, a model-independent
relaxation time was determined from [25]:
τmax =τHN sinαHN π
2+2βHN −1
αHN sinαH N βHN π
2+2βHN −1
αHN (2)
The temperature dependence of the relaxation times could be described using the
Arrenhius equation in the case of thermally activated processes (linear T-dependence) or the
well-known Vogel/Fulcher/Tammann/Hesse equation [
28
–
30
] when curved, respectively;
see Equations (3) and (4).
τ=τ0expEa
RT (3)
where
τ0
is the relaxation time at infinite T,Ris the ideal gas constant and E
a
is the activation
energy, and
τ(T)=τ0expB
T−T0(4)
where
τ0
and Bare parameters and T
0
is the so-called Vogel temperature (found around 50
degrees below Tg).
2.3. Methods
Synthesis and characterization of the MSNs. Mesoporous silica nanoparticles (MSNs)
were synthesized following a method reported in [
31
,
32
]. Briefly, 1.75 mL NaOH 1.4 M
and 240 mL H
2
O milli-Q were mixed in a polypropylene flask with a flat bottom, which
was transferred to an oil bath and heated to 32
◦
C. The reaction was performed with
Pharmaceutics 2023,15, 1624 5 of 27
homogeneous magnetic stirring to obtain a stable mixture and avoid a larger distribution
of diameters. Afterward, 500 mg of cetyltrimethylammonium bromide (CTAB) was added
to the solution and maintained under stirring for 30 min. Then, 2.5 mL of tetraethyl
orthosilicate (TEOS) was added, drop by drop, and the solution was kept under continuous
stirring for 3 h at 35 ◦C. The particles were recovered via centrifugation and the solid was
washed with a mixture of ethanol and water (50% v/v). The solid (MSNs-CTAB) was
dried at 50
◦
C overnight and the latter was placed under vacuum. Then, 0.5 M HCl in
ethanol (25 mL per 500 mg of MSNs) was added to solubilize the CTAB and the solution
was sonicated for 15 min and left stirring overnight at 50
◦
C. After 24 h, the particles were
washed 3 times. First, with a solution of ethanol and water (50% v/v), and then twice
with absolute ethanol. Finally, the nanoparticles were dried overnight in a ventilated oven
at 50 ◦C.
Modification of the MSNs pores. The modification of the MSNs’ surface with
(3-aminopropyl)triethoxysilane (APTES) was performed by suspending 0.166 g of the
MSNs in anhydrous toluene (10 mL) under an argon atmosphere into a 25 mL round
bottom flask. This flask was sonicated under argon atmosphere for 15 min. Subsequently,
APTES (0.10 mL) was added dropwise, and the resulting mixture was maintained at 130
◦
C
under reflux in an argon atmosphere for 24 h. A solid product (MSN_APTES) was obtained
via centrifugation (1100 min
−1
for 10 min) and washed three times with absolute ethanol,
discarding the supernatant each time. The particles were dried in a ventilated oven for 24 h
at 50 ◦C.
The surface of the MSNs was also covalently modified with trimethoxy(phenyl)silane
(TMPS). For this purpose, 0.169 g of nanoparticles was dispersed in 15 mL of dry toluene.
Then, 50
µ
L of TMPS was added to the mixture and it was maintained at 125
◦
C under
reflux in an argon atmosphere for 24 h. The remaining steps were like those carried out
with MSN_APTES. Solid MSN_TMPS was recovered.
Loading MSNs with FNB. To remove any impurities, or residual solvents including
water, the mesoporous nanoparticles were placed in a glass cell under vacuum (10
−4
bar)
and heated up to 150
◦
C via the immersion of the cell in an oil bath, for 8 h. After this
period, the cell was allowed to cool down to room temperature. FNB dissolved in 2 mL
of acetone was injected in the cell container hosting the outgassed matrix and then stirred
for 3 h to increase the uptake of the drug. Finally, the solvent was allowed to evaporate
for one day under gentle stirring at room temperature, and after which a dry powder was
obtained (FNB@MSNs). Table 1presents the drug loading conditions used for all of the
silica nanocarriers.
Table 1. Drug loading conditions.
Sample FNB Loaded
(% w/w) *
Available Vp
(cm3)VFNB (cm3) ** Pore Volume Occupied
by FNB (% v/v)
MSN 22.8 0.095 0.035 37
MSN_APTES 22.8 0.096 0.036 37
MSN_TMPS 33.3 0.081 0.050 62
* %FNB mass fraction in the sample; ** volume estimated for crystalline form I of FNB.
The theoretical maximum of the loading was estimated as in [33]:
maximum loading =Vp∗ρFN B
1g+Vp∗ρFNB ∗100% (5)
where
ρFNB
is the density of the fenofibrate (1.177 g cm
−3
taken from reference [
34
] for form I
of crystalline FNB) and V
p
is the pore volume of the silica. The latter (V
p
= 0.68 cm
3
g
−1
)
was estimated from BET data for similar mesoporous silica nanoparticles, as described by
some of us in reference [
32
]. The maximum theoretical load calculated from Equation (5)
was approximately 44 wt%. However, it must be noted that the maximum loading may
Pharmaceutics 2023,15, 1624 6 of 27
have been overestimated, because the density of the crystalline FNB, used to determine it,
was higher than that of the amorphous form [34].
In the functionalized MSNs, the pore diameter can slightly decrease due to the anchor-
ing of the modifying agent. This effect has been reported for mesoporous silica modified
with methyltrimethoxysilane, which reduced the pore diameter by 0.2 nm [
35
]. Having
this in mind, FNB was loaded significantly below the theoretical maximum predicted by
Equation (5) to minimize the deposition outside the pores (both FNB mass and % occupied
volume are included in Table 1).
Monitoring drug release. Measurements were performed at room temperature using
a UV-660 UV-Vis Spectrophotometer (JASCO International, Tokyo, Japan) with a double
monochromator and a photomultiplier detector. The assays were carried out at room
temperature in a quartz cuvette with 1 cm
×
1 cm dimension using a polypropylene dialysis
device with a cellulose membrane (Slide-A-Lyzer Mini Dialysis Devices, 10 K MWCO,
0.5 mL) inserted in its top (a schematic representation of this setup is illustrated in the SI
of ref. [
36
]). The solution medium for drug release was composed of phosphate buffer
at pH 7.4 and ethanol in the w/w proportion of 90:10; a calibration curve at
λ
= 289 nm
was constructed by dissolving known FNB amounts in the same solution medium with
the following linear regression parameters: A= (15.2
±
0.5)
×
10
3×
C+ (0.005
±
0.003),
where the slope represents
ε
(molar absorptivity coefficient) and C is the concentration.
Ethanol was chosen due to the higher solubility of FNB in this medium [
37
]. Briefly,
200
µ
L of PBS/EtOH was added to around 1.1 mg of loaded nanoparticles and placed
in the top chamber. The bottom chamber was filled with 2.8 mL of the buffered solution
media and kept under continuous stirring at 400 rpm. Drug release was monitored at
room temperature (~25
◦
C) by collecting successive UV-Vis spectra (200 to 700 nm) at an
acquisition rate of 400 nm min
−1
. During the first 4 h, spectra were taken every 60 s and
then every 600 s until completion at 8 h.
3. Results
3.1. Structural Characterization of MSNs
Mesoporous silica nanoparticles were observed via TEM (representative image in
Figure 1) which confirmed the spherical shape of the nanoparticles and allowed for the
determination of their size. The analysis of diameter using ImageJ software provided a
value of 49.0 nm (inset of Figure 1) from a fitting with a Gaussian function. The average
size estimated was that expected, in accordance with the synthesis procedure described
before [31].
Pharmaceutics 2023, 15, x FOR PEER REVIEW 7 of 28
Figure 1. Transmission electron microscope image illustrating the regular shape and size of the mes-
oporous silica nanoparticles. Inset: probability distribution of nanoparticles’ radius obtained from a
sample of 30 nanoparticles; the red line corresponds to the Gaussian ing function of μ = 49 and
σ2 = 32.6, corresponding to a particle radius R = 49 ± 6 nm.
3.2. Quantication of Grafted Functional Groups
The amount of functional groups in the MSNs was determined using NMR following
the procedure previously described [38]. The 1H-NMR spectra obtained for both of the
modied silicas are represented in Figure 2. All of the peaks have been assigned and as-
sociated with specic atom positions in the corresponding structures. At 1.2 ppm and 3.6
ppm, the resonance peaks observed correspond to residual ethanol from washing the
MSNs after synthesis.
Figure 2. Solution 1H-NMR (silica structure destroyed at pH = 13) of functionalized MSN with (a)
APTES and (b) TMPS groups. Peaks are identied with the corresponding functional groups in ac-
cordance with chemical structures in the insets. Residual ethanol protons are denoted by (*).
30 35 40 45 50 55 60 65
0.0
0.1
0.2
0.3
0.4
Probability
RTEM (nm)
a)
b)
c)
Figure 1.
Transmission electron microscope image illustrating the regular shape and size of the
mesoporous silica nanoparticles. Inset: probability distribution of nanoparticles’ radius obtained
from a sample of 30 nanoparticles; the red line corresponds to the Gaussian fitting function of
µ
= 49
and σ2= 32.6, corresponding to a particle radius R = 49 ±6 nm.
Pharmaceutics 2023,15, 1624 7 of 27
3.2. Quantification of Grafted Functional Groups
The amount of functional groups in the MSNs was determined using NMR following
the procedure previously described [
38
]. The
1
H-NMR spectra obtained for both of the
modified silicas are represented in Figure 2. All of the peaks have been assigned and
associated with specific atom positions in the corresponding structures. At 1.2 ppm and
3.6 ppm, the resonance peaks observed correspond to residual ethanol from washing the
MSNs after synthesis.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 7 of 28
Figure 1. Transmission electron microscope image illustrating the regular shape and size of the mes-
oporous silica nanoparticles. Inset: probability distribution of nanoparticles’ radius obtained from a
sample of 30 nanoparticles; the red line corresponds to the Gaussian ing function of μ = 49 and
σ2 = 32.6, corresponding to a particle radius R = 49 ± 6 nm.
3.2. Quantication of Grafted Functional Groups
The amount of functional groups in the MSNs was determined using NMR following
the procedure previously described [38]. The 1H-NMR spectra obtained for both of the
modied silicas are represented in Figure 2. All of the peaks have been assigned and as-
sociated with specic atom positions in the corresponding structures. At 1.2 ppm and 3.6
ppm, the resonance peaks observed correspond to residual ethanol from washing the
MSNs after synthesis.
Figure 2. Solution 1H-NMR (silica structure destroyed at pH = 13) of functionalized MSN with (a)
APTES and (b) TMPS groups. Peaks are identied with the corresponding functional groups in ac-
cordance with chemical structures in the insets. Residual ethanol protons are denoted by (*).
30 35 40 45 50 55 60 65
0.0
0.1
0.2
0.3
0.4
Probability
RTEM (nm)
a)
b)
c)
Figure 2.
Solution
1
H-NMR (silica structure destroyed at pH = 13) of functionalized MSN with
(
a
) APTES and (
b
) TMPS groups. Peaks are identified with the corresponding functional groups in
accordance with chemical structures in the insets. Residual ethanol protons are denoted by (*).
The integral of the 1,3,5-trioxane (used for calibration) peak at 5.17 ppm was compared
to the integral of the protons from the
α
-carbon to the silicon atom at 0.38 ppm, to estimate
the amount of organic APTES molecules grafted onto the surface. Regarding TMPS, the
peaks between 8 and 7 ppm were assigned to the aromatic protons (see Figure 2b). The
integrated intensity of the peaks from the aromatic ring was normalized to account for
its protons.
In both of the modified silica samples, the density of the grafted groups (see Table 2) is
lower than the density of silanol in fully hydroxylated silica (~5–7 OH nm
−2
[
39
–
41
]). This
is the result of the reaction between each APTES or TMPS molecule through one, two or
three hydrolyzed ethoxy moieties with the same number of silanol groups of the MSNs’
surface [
42
]. Nevertheless, the functionalization with TMPS leads to a significantly lower
density relative to APTES, possibly due to the bulkiness of the phenyl group turning the
adjacent reaction sites inaccessible.
Table 2. APTES and TMPS concentration on the MSNs calculated from 1H NMR spectra.
Sample mmol g−1MSNs Modifying Molecules/nm−2
MSN_APTES 3.14 1.93
MSN_TMPS 0.98 0.60
Pharmaceutics 2023,15, 1624 8 of 27
3.3. FTIR Analysis of MSNs Loaded with FNB
The modification of the MSNs with APTES or TMPS introduced changes in the infrared
spectra of the pristine silica matrix (Figure S1). The band at 1086 cm
−1
was assigned to the
Si–O–C asymmetry stretching vibration and Si–OH stretching vibration [
43
]. Additionally,
a large band was detected between 3600 and 3000 cm
−1
, assigned to hydroxyl groups
of adsorbed water (O-H stretching vibrations), and the respective bending modes were
detected at 1636 cm−1[44,45].
The modified MSNs_APTES shows additional bands relative to the bare MSNs (see
green arrows in Figure S1): (i) a structured band around 2938 cm
−1
attributed to the C-H
stretching vibration [
46
], and (ii) at 1554 cm
−1
, related to the N-H bending vibrations of the
aminopropyl moiety [
47
]. These bands confirm that the amino-propyl groups of APTES
were successfully grafted onto the MSNs.
The presence of the TMPS modifying agent in the silica matrix leads to the appearance
of two small bands at 702 and 745 cm
−1
(see a scale up in the inset of Figure S1), which, in
accordance with [48], are assigned to rocking motions of C-H in the phenyl group.
To evaluate whether FNB was successfully loaded, the spectrum of the neat crystalline
drug was collected and compared with that of FNB loaded into the MSNs (Figure 3). The
presence of FNB in the loaded nanoparticles was detected via several small sharp bands,
characteristic of the neat drug superimposed to that of the unloaded matrices. The regions
due to the C-H and C=O vibrations, located, respectively, between 3800 and 2600 cm
−1
(Figure 3a) and 1800 and 1550 cm
−1
(Figure 3b), will now be analyzed in more detail and
compared with the spectra of the loaded materials.
Spectral region of 4000–2600 cm
−1
. In the wavenumber region between 3000 and
3100 cm
−1
, crystalline FNB presents multiples weak to moderate bands due to the C-H
stretch of the FNB aromatic rings [
49
,
50
]. Immediately below this region, three sharp bands
are detected, with the ones located at 2986 and 2938 cm
−1
being assigned to saturated
aliphatic C-H stretching vibrations [
49
,
51
]. These can be distinguished in the spectra of
the FNB-loaded nanoparticles; however, those found for the neat amorphous FNB were
slightly broadened and their relative weights had changed [
22
,
52
]. Moreover, a close
inspection of the 2936 and 2874 cm
−1
bands of the neat FNB (see Figure S2) reveals a shift
to lower wavenumbers upon loading. This could have arisen from amorphization and the
building up of interactions with the pore walls’ silanol groups. It has been reported that
weak CH
···
O intermolecular interactions are known to exist in FNB polymorphs linking
either aromatic (most stable polymorph I, IIa and IIb [53]) or isopropyl methyl hydrogens
(polymorph IIb and III [
10
]) and ketone oxygen. Upon incorporation into MSNs, this kind
of interaction could arise between the CH groups of FNB molecules adsorbed close to the
pores’ surface, and -OH from the silica matrices. In the case of the MSN-APTES matrix,
this region is also affected by the superposition of the CH from the aminopropyl moiety
absorption band (Figure S1).
Around 3500 cm
−1
, a broad band is detected in the loaded matrices’ spectra, also
observed in the unloaded matrices, which are absent in bulk FNB. In both the unloaded and
loaded matrices, the spectral response is perfectly simulated by two Gaussian functions (see
Figure S3) centered at 3455
±
4 and 3235
±
4 cm
−1
, which are in good accordance with the
wavenumber found by D. Ngo et al. [
54
] and assigned, respectively, to hydrogen bonding
between water molecules and Si-OH hydrogen bonded to water. The absence of bands cen-
tered at higher wavenumbers means that neither free water (3540 and 3625 cm
−1
[
55
]) nor
isolated Si-OH (3740 cm
−1
[
54
]) exist in the composites. The normalization of the spectral re-
sponse for the unloaded and loaded matrices (see Figure S4) shows that the high wavenum-
ber flank remains unaffected upon drug incorporation. Nevertheless, the low wavenumber
side is sensitive to the type of matrix, being slightly depleted in FNB@MSN_TMPS, while
it broadens in FNB@MSN and even more so in FNB@MSN_APTES, relative to the respec-
tive empty matrices’ spectra; this contribution was considered in the overall simulation
by a band included at ~3100 cm
−1
(see blue band in Figure S3). The widening to low
Pharmaceutics 2023,15, 1624 9 of 27
wavenumbers could reflect the existence of strong HBs involving FNB, water molecules
and silanol/amino groups of the host.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 9 of 28
Figure 3. FTIR spectra of FNB@MSNs (blue), FNB@MSNs_APTES (green) and FNB@MSNs_TMPS
(pink) in (a) 4000–2600 cm−1 hydroxyl and aliphatic CH regions and (b) 1800–1550 cm−1 carboxylic
region. See Figure S2 as well, in which the resulting spectra after subtracting the weighted spectrum
of the corresponding unloaded MSNs are represented. Spectrum of bulk FNB (black) is displayed
on right axis.
Spectral region of 4000–2600 cm−1. In the wavenumber region between 3000 and 3100
cm−1, crystalline FNB presents multiples weak to moderate bands due to the C-H stretch
of the FNB aromatic rings [49,50]. Immediately below this region, three sharp bands are
detected, with the ones located at 2986 and 2938 cm−1 being assigned to saturated aliphatic
C-H stretching vibrations [49,51]. These can be distinguished in the spectra of the FNB-
loaded nanoparticles; however, those found for the neat amorphous FNB were slightly
broadened and their relative weights had changed [22,52]. Moreover, a close inspection of
the 2936 and 2874 cm−1 bands of the neat FNB (see Figure S2) reveals a shift to lower wave-
numbers upon loading. This could have arisen from amorphization and the building up
of interactions with the pore walls’ silanol groups. It has been reported that weak CH…O
intermolecular interactions are known to exist in FNB polymorphs linking either aromatic
(most stable polymorph I, IIa and IIb [53]) or isopropyl methyl hydrogens (polymorph IIb
and III [10]) and ketone oxygen. Upon incorporation into MSNs, this kind of interaction
could arise between the CH groups of FNB molecules adsorbed close to the pores’ surface,
and -OH from the silica matrices. In the case of the MSN-APTES matrix, this region is also
Figure 3.
FTIR spectra of FNB@MSNs (blue), FNB@MSNs_APTES (green) and FNB@MSNs_TMPS
(pink) in (
a
) 4000–2600 cm
−1
hydroxyl and aliphatic CH regions and (
b
) 1800–1550 cm
−1
carboxylic
region. See Figure S2 as well, in which the resulting spectra after subtracting the weighted spectrum
of the corresponding unloaded MSNs are represented. Spectrum of bulk FNB (black) is displayed on
right axis.
Spectral region of 1800–1550 cm
−1
. The carbonyl stretching region displays two well-
defined bands in crystalline FNB, as is shown in Figure 3b, centered at 1728 cm
−1
and
1650 cm
−1
. These are assigned to the C=O stretching of, respectively, the ester (C=O2-O1)
and the ketone groups (C=O4) [
52
,
56
]; see Scheme 1for the identification of these functional
groups. A shift to higher wavenumbers, to (1731
±
1) and (1656
±
1) cm
−1
, respectively, is
observed for the loaded MSNs, together with band broadening relative to the spectrum of
crystalline FNB; such modifications are still detected even when the weighted contribution
of the unloaded matrices (which exhibit a broad band in this region) is removed from the
spectra (see Figure S4). These changes are similar to the ones reported in the literature
for amorphous FNB [
27
,
57
], acting as first evidence that incorporated FNB exists in the
amorphous form. However, in particular for the lactone carbonyl band, although a similar
shift is observed for three nanocomposites, it shows a broadening (see Figure 3b and S4)
Pharmaceutics 2023,15, 1624 10 of 27
mainly toward the low wavenumber side, being more pronounced in FNB@MSN_TMPS,
suggesting the existence of FNB interactions with the holding matrices.
Below the C=O absorption region, crystalline FNB shows a structured band with a
maximum at 1599 cm
−1
associated (Figure S5) with vibrational modes of the aromatic rings
(in-plane stretching and deformation of benzene unities) [
52
]. In the three loaded silicas, a
depletion of the low wavenumber shoulder is observed, while the sharp and well-defined
1599 cm−1absorption band prevails, also observed upon neat FNB amorphization [52].
3.4. Differential Scanning Calorimetry (DSC)
To evaluate the thermal transformations of the incorporated drug, differential scanning
calorimetry (DSC) was used, and the resulting behavior was compared with that of the
neat drug and the unloaded matrices. The nanoparticles only show the endotherm signal
due to water removal (see Figure S6 and Table S1), whose amount was estimated from the
weight loss after calorimetric analysis. Lower water content was found for MSN_APTES,
which is not unexpected due to the high density of APTES grafting (see Table 2) that
could replace up to three silanol groups, leaving less available sites for water adsorption.
However, it should be noted that the terminal amine moieties of APTES must interact more
strongly than silanol with water molecules, as inferred by the MSN_APTES calorimetric
endotherm that extends to higher temperatures relative to unmodified MSN and also the
TMPS-functionalized matrix. In the latter, much less of a density coverage was achieved
and, therefore, the water uptake was similar to that of the bare matrix.
Concerning neat FNB, the calorimetric analysis showed that it melts at 81.4
◦
C, which
corresponds to crystalline form I [
23
,
58
]. It easily vitrifies by cooling from the melt, even at
low cooling rates such as 1 ◦C min−1[23]; nevertheless, upon heating at 5 ◦C min−1, after
the glass to supercooled transition (T
g-on
=
−
17.7
◦
C), it tends to recrystallize with a cold
crystallization temperature onset slightly above room temperature ([23], Figure S7).
When FNB is incorporated into unmodified MSN, the thermogram obtained at 10
◦
C min
−1
shows the step of the glass transition below
−
20
◦
C, and at higher temperatures shows a
large exothermal peak related to water evaporation (Figure 4a). This behavior is similar in
both FNB@MSN_TMPS and FNB@MSN_APTES; however, in the latter, a small and sharp
endothermal peak at 81.2
◦
C overlaps with the water release signal, likely related to a small
FNB crystalline fraction (commented on in the next section). In subsequent cooling/heating
scans, no evidence of recrystallization/melting was detected; however, the glass transition
step was hardly distinguished.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 11 of 28
at low cooling rates such as 1 °C min−1 [23]; nevertheless, upon heating at 5 °C min−1, after
the glass to supercooled transition (Tg-on = −17.7 °C), it tends to recrystallize with a cold
crystallization temperature onset slightly above room temperature ([23], Figure S7).
When FNB is incorporated into unmodied MSN, the thermogram obtained at 10 °C
min−1 shows the step of the glass transition below −20 °C, and at higher temperatures
shows a large exothermal peak related to water evaporation (Figure 4a). This behavior is
similar in both FNB@MSN_TMPS and FNB@MSN_APTES; however, in the laer, a small
and sharp endothermal peak at 81.2 °C overlaps with the water release signal, likely re-
lated to a small FNB crystalline fraction (commented on in the next section). In subsequent
cooling/heating scans, no evidence of recrystallization/melting was detected; however, the
glass transition step was hardly distinguished.
Figure 4. Heating thermograms of FNB@MSNs obtained at 10 °C min−1 with the following: (a) Two
successive cycles with nal temperature of 100 °C (water content ~4.9%); inset shows a scale up of
the glass transition region for bulk amorphous FNB (black) and hydrated FNB@MSN (light blue).
(b) Evolution of the glass transition step by varying the nal temperature from Tend-1 = 50 °C to Tend-
2 = 70 °C, Tend-3 = 100 °C and Tend-4 = 100 °C; the arrows indicate the onset of each step associated to
the bimodal glass transition. The inset shows the schematic temperature vs. time procedure applied.
To understand how water inuences the thermal behavior of MSN-incorporated
FNB, a gradual drying procedure was applied to a fresh sample of each nanocomposite,
consisting of successive cooling/heating cycles to increase the nal temperatures (see inset
in Figure 4b). Representative thermograms collected upon heating for the FNB@MSNs are
shown in Figure 4b. The step associated with the glass transition, clearly observed in the
rst heating, is progressively depleted upon water removal with a concomitant emergence
of a second discontinuity at higher temperatures (the arrows in Figure 4b indicate the
respective onsets); this will be commented upon further when analyzing the dielectric re-
sults. The onset temperatures of the rst step (Tg-on) are summarized in Table 3.
From Figures 4 and S8, two main characteristics are observed for the three samples:
(i) a shift in the glass transition to higher temperatures under drying, and (ii) a signicant
broadening of the glass transition step relative to the neat drug. Regarding observation (i),
this means that the glass transition temperature depends on the hydration level, being
lower for higher water contents, caused by water plasticization, as observed for other low-
molecular-weight compounds [59,60]. This eect is more striking for the FNB@MSNs, for
which a decrease close to 20 degrees was observed, for a sample with a hydration level
close to 5% (see the inset of Figure 4a).
Figure 4.
Heating thermograms of FNB@MSNs obtained at 10
◦
C min
−1
with the following: (
a
) Two
successive cycles with final temperature of 100
◦
C (water content ~4.9%); inset shows a scale up of
the glass transition region for bulk amorphous FNB (black) and hydrated FNB@MSN (light blue).
(
b
) Evolution of the glass transition step by varying the final temperature from T
end-1
= 50
◦
C to
T
end-2
= 70
◦
C, T
end-3
= 100
◦
C and T
end-4
= 100
◦
C; the arrows indicate the onset of each step
associated to the bimodal glass transition. The inset shows the schematic temperature vs. time
procedure applied.
Pharmaceutics 2023,15, 1624 11 of 27
To understand how water influences the thermal behavior of MSN-incorporated
FNB, a gradual drying procedure was applied to a fresh sample of each nanocomposite,
consisting of successive cooling/heating cycles to increase the final temperatures (see inset
in Figure 4b). Representative thermograms collected upon heating for the FNB@MSNs are
shown in Figure 4b. The step associated with the glass transition, clearly observed in the
first heating, is progressively depleted upon water removal with a concomitant emergence
of a second discontinuity at higher temperatures (the arrows in Figure 4b indicate the
respective onsets); this will be commented upon further when analyzing the dielectric
results. The onset temperatures of the first step (Tg-on) are summarized in Table 3.
Table 3.
Onset of the glass transition temperature (T
g-on
) values of FNB in MSN, MSN_APTES and
MSN_TMPS under successive cooling/heating cycles with different final temperatures; water content
was estimated from the weight loss after DSC runs.
Sample
Tg-on (◦C)
Heating 1
Tend-1 = 50 ◦C
Tg-on (◦C)
Heating 2
Tend-2 = 70 ◦C
Tg-on (◦C)
Heating 3
Tend-3 = 100 ◦C
Water Content
(%)
FNB@MSNs −23.5 −21.5 −21.5 4.1
FNB@MSNs_APTES
−20.9 −18.6 −19.8 5.7
FNB@MSNs_TMPS
−14.7 −12.5 −9.4 2.0
FNB Tg-on (◦C) = −17.7
From Figure 4and S8, two main characteristics are observed for the three samples:
(i) a shift in the glass transition to higher temperatures under drying, and (ii) a significant
broadening of the glass transition step relative to the neat drug. Regarding observation
(i), this means that the glass transition temperature depends on the hydration level, being
lower for higher water contents, caused by water plasticization, as observed for other
low-molecular-weight compounds [
59
,
60
]. This effect is more striking for the FNB@MSNs,
for which a decrease close to 20 degrees was observed, for a sample with a hydration level
close to 5% (see the inset of Figure 4a).
Concerning the broadening of the heat flow step (ii), it suggests that, upon loading,
the guest drug is able to explore a variety of configurations, ranging from the pore core
to the silica surface. Given that all matrices have comparable pore size dimensions, the
differences persisting in the glass transition temperature of the nanocomposites in the dried
state, i.e., after eliminating the water effect, provide a picture of the drug distribution inside
the pores and/or of the drug–silica interactions. This will be discussed in more detail when
analyzing the dielectric data.
Additionally, it is clear that the magnitude of the glass transition step decreases when
going from the hydrated to the dried state, for each sample. Since the heat capacity is
a measure of the configurational degrees of freedom [
61
], their loss when crossing the
glass transition during cooling (as the supercooled liquid becomes frozen) determines the
magnitude of the calorimetric step at T
g
. In the supercooled liquid regime, it is expected
that the hydrated sample possesses a higher number of degrees of freedom than in the
corresponding dried state, as the water molecules decrease guest–guest interactions and
prevent, to some extent, the drug adsorption at the pore wall. Indeed, the interaction with
the host surface reduces the guest degrees of freedom, and, therefore, the glass transition
step during vitrification. Furthermore, the anchoring of a fraction of the drug molecules
to the pore walls, leading to the formation of an almost immobile layer, could cause the
decrease in the step associated with the glass transition after drying. Ultimately, if the
incorporated drug forms a thin monolayer directly interacting with the inner pore wall,
it is expected that it will not respond calorimetrically, as found for other drugs loaded on
similar silica matrices [62].
Pharmaceutics 2023,15, 1624 12 of 27
For a deeper understanding of the dynamic behavior of FNB loaded in MSNs, around
the glass transition, embracing both supercooled and glass states, dielectric relaxation
spectroscopy (DRS) was used.
3.5. Dielectric Relaxation Spectroscopy (DRS)
Due to the dipolar moment of FNB, DRS can be used for characterizing the molecular
motions of FNB in the mesoporous matrices over a large frequency range, from 10
−1
to 10
6
Hz. Spectra were collected upon heating from
−
150
◦
C up to 150
◦
C regarding
the unloaded nanoparticles, and up to 120
◦
C for samples with fenofibrate (to avoid the
degradation of the guest molecule). The temperature was increased in different steps: from
−150 ◦C to 50 ◦C in steps of 5 ◦C and in the remaining temperature range, every 2 ◦C.
3.5.1. Unloaded Hydrated Matrices
As shown via DSC, water influences the glass transition and hence the dynamical
behavior of FNB in the silica matrices. Thus, the water response in unloaded MSNs was
prior dielectrically characterized. Each unloaded matrix was submitted to two successive
series of isothermal spectra from
−
100 to 150
◦
C; the second run was collected after keeping
the sample at 150
◦
C for 20 min to ensure water evaporation. It should be noted that at
the end of the chemical modification of the MSNs’ surface, the matrices were submitted to
severe dehydration (see Section 2.3), and, thus, the water detected in the first run of the
dielectric measurements was water reabsorbed during storage.
Several relaxation processes are identified for each MSN in the hydrated state, located
at different temperatures, and they have different relative intensities (see a representative
ε00
(f) spectrum at
−
90
◦
C in Figure 5a and S9, in the M
00
(f) representation). Except for
MSN_APTES, the dielectric loss (
ε00
) becomes negligible in the second-series spectra, re-
inforcing the assignment of the relaxations detected in the first series to reorientational
processes involving water. In the APTES-modified matrix, the persistence of a dielectric
response means that there is still some water retained in the silica nanoparticles. The
fact that different spectra were obtained for each matrix means, on the one hand, that
there was an effective modification of the surface of the nanoparticles, either by TMPS or
APTES, and, on the other hand, that such a modification impacted the dynamics of water
in the nanoparticles.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 13 of 28
severe dehydration (see Section 2.3), and, thus, the water detected in the rst run of the
dielectric measurements was water reabsorbed during storage.
Several relaxation processes are identied for each MSN in the hydrated state, located
at dierent temperatures, and they have dierent relative intensities (see a representative
ε″(f) spectrum at −90 °C in Figures 5a and S9, in the M″(f) representation). Except for
MSN_APTES, the dielectric loss (ε″) becomes negligible in the second-series spectra, rein-
forcing the assignment of the relaxations detected in the rst series to reorientational pro-
cesses involving water. In the APTES-modied matrix, the persistence of a dielectric re-
sponse means that there is still some water retained in the silica nanoparticles. The fact
that dierent spectra were obtained for each matrix means, on the one hand, that there
was an eective modication of the surface of the nanoparticles, either by TMPS or
APTES, and, on the other hand, that such a modication impacted the dynamics of water
in the nanoparticles.
To calculate the relaxation times of the water reorientational motions, the imaginary
component of the complex electric modulus, M*(ω) = M′(ω) + iM″(ω), was analyzed using
the corresponding part of a sum of Havriliak–Negami ing functions (Equation (1)), and
a model-independent relaxation time was determined by Equation (2). The obtained re-
laxation times for all of the detected processes allowed for the construction of the relaxa-
tion map, presented in Figure 5b.
Figure 5. (a) Permiivity spectra (ε″) collected at −90 °C for hydrated unloaded MSNs, and (b) re-
laxation map of all of the processes detected in hydrated unloaded matrices (ing in M″(f)).
Straight lines represent the ing with an Arrhenius function for processes I, I* and II, respectively,
in light gray, dark gray and white shadowed regions. An Arrhenius function for process II was only
ed for the low-temperature branch.
The relaxation times (in a logarithmic plot) of the processes in the tested frequency
window above −100 °C for the three nanoparticles (hereafter designated as process I) fol-
low a linear temperature dependence described by the Arrhenius equation (3). Regarding
the hydrated unmodied silica, the estimated activation energy is 44 kJ mol−1, compatible
with the process assigned to reorientations of ice-like water cluster structures found in
bulk ice by Johari et al. [63]. Nevertheless, the chemical modication of the MSNs impacts
the activation energy, since when silanol groups of the bare matrix are capped by amine
moieties (APTES), the activation energy increases, while functionalization with the more
hydrophobic (but at a lower coverage) TMPS leads to a decrease in Ea (Table 4). This must
reect the extent and surface-directing water hydrogen-bonded (HB) arrangement. For
water in porous glass [64], and in non-porous silica nanoparticles [65], a proportionality
was found between the activation energy and the number of HBs connecting the clustering
water molecules, for an equivalent low-T relaxation process. Concomitantly to the Ea in-
crease, the Arrhenian pre-exponential factor, τ (see Table 4), decreases, becoming signif-
icantly lower than the time corresponding to local non-cooperative relaxation (Debye
(b)
Figure 5.
(
a
) Permittivity spectra (
ε00
) collected at
−
90
◦
C for hydrated unloaded MSNs, and (
b
) relax-
ation map of all of the processes detected in hydrated unloaded matrices (fitting in M
00
(f)). Straight
lines represent the fitting with an Arrhenius function for processes I, I* and II, respectively, in light
gray, dark gray and white shadowed regions. An Arrhenius function for process II was only fitted for
the low-temperature branch.
To calculate the relaxation times of the water reorientational motions, the imaginary
component of the complex electric modulus, M*(
ω
)= M
0
(
ω
)+ iM
00
(
ω
), was analyzed using
the corresponding part of a sum of Havriliak–Negami fitting functions (Equation (1)), and
a model-independent relaxation time was determined by Equation (2). The obtained relax-
Pharmaceutics 2023,15, 1624 13 of 27
ation times for all of the detected processes allowed for the construction of the relaxation
map, presented in Figure 5b.
The relaxation times (in a logarithmic plot) of the processes in the tested frequency
window above
−
100
◦
C for the three nanoparticles (hereafter designated as process I) follow
a linear temperature dependence described by the Arrhenius Equation (3). Regarding the
hydrated unmodified silica, the estimated activation energy is 44 kJ mol
−1
, compatible
with the process assigned to reorientations of ice-like water cluster structures found in
bulk ice by Johari et al. [
63
]. Nevertheless, the chemical modification of the MSNs impacts
the activation energy, since when silanol groups of the bare matrix are capped by amine
moieties (APTES), the activation energy increases, while functionalization with the more
hydrophobic (but at a lower coverage) TMPS leads to a decrease in E
a
(Table 4). This must
reflect the extent and surface-directing water hydrogen-bonded (HB) arrangement. For
water in porous glass [
64
], and in non-porous silica nanoparticles [
65
], a proportionality
was found between the activation energy and the number of HBs connecting the clustering
water molecules, for an equivalent low-T relaxation process. Concomitantly to the E
a
increase, the Arrhenian pre-exponential factor,
τ∞
(see Table 4), decreases, becoming
significantly lower than the time corresponding to local non-cooperative relaxation (Debye
time,
τ≈
10
−13
s) [
66
]. This indicates that there is some degree of cooperativity between
the relaxing dipoles that are involved in process I. The change in both E
a
and
τ∞
for the
three MSNs agrees with the rationalization proposed in [
65
] (herein labeled as process 2),
and allows for the ordering of the extent of cooperativity of HB water clusters relaxing in
the different environments as MSN_APTES > MSN > MSN_TMPS.
Table 4.
Activation parameters of all processes found in unloaded silicas. The relaxation times of
process I and I* were fitted with Equation (3).
Process I Process I* Process II
Ea
(kJ mol−1)τ∞(s) Ea
(kJ mol−1)τ∞(s) Ea
(kJ mol−1)τ∞(s) Tmax *
(◦C)
MSN 44 8.0 ×10−19 – – 44 2.9 ×10−12 9.0
MSN_APTES 55 4.7 ×10−21 57 2.1 ×10−19 60 6.3 ×10−18 21.8
MSN_TMPS 37 1.8 ×10−17 29 1.1 ×10−10 n.a. n.a. 4.2
* Temperature of the log(τ) maximum of the saddle-like profile taken directly from the data.
As process I shifts to higher frequencies with the temperature increase, a weak relax-
ation emerges in the low-frequency range of the spectra of functionalized MSNs, hereafter
named process I*. The corresponding relaxation times follow an Arrhenius tendency from
which the E
a
and
τ∞
values were extracted (Equation (3)). The estimated activation param-
eters are also influenced by the surface functionalization (see Table 4), and, therefore, the
underlying relaxation mechanism must involve joined reorientations of the surface chem-
ical units and the interacting water molecules. In the case of hydrophobic MSN_TMPS,
water molecules interact preferentially with bare silanol groups, whereas in the more
hydrophilic MSN_APTES, the water molecules also interact with the grafted groups. In
unmodified MSN, the process is not detected due to a conjunction of different factors: the
high intensity of process I, the high dispersion of water molecules in the homogeneous
inner pore wall (only silanol groups) and the closeness of the incoming low-frequency side
process, hereafter designated as process II; see Figure S10, whereby the individual HN
fitting functions for spectra at
−
90
◦
C are included, allowing one to visualize the relative
positions of process I and I*. Process I* in MSN_APTES is the one still detected after heating
up to 120
◦
C, which is a clear indication that the thermal treatment was insufficient for
achieving full dehydration, as previously observed via calorimetric analysis, due to strong
APTES–water interactions.
Upon the temperature increasing, process II emerges with a high intensity presenting a
singular behavior with temperature in the activation map, whereby the profile is referred to
Pharmaceutics 2023,15, 1624 14 of 27
in the literature as being saddle-like [
64
,
67
–
69
]. This particular contour results, on the one
hand, from the increase in the mobility of loosely bound water with increasing temperature,
evolving as a simply thermally activated process (low-T branch). On the other hand (high-T
branch), it is due to the contribution of two mechanisms: (i) the removal of nearly free
water by a joint effect of temperature increase and the nitrogen flow circulating through
the sample cell that drags water molecules out during each isothermal spectral acquisition,
and (ii) the slowing down of the reorientational motions of the remaining water molecules,
which are more strongly bound to the pore wall.
The 3D representation of M
00
(f,T) mirrors the concurrence of all of these effects (Figure S11).
The temperature dependence of the relaxation times of the low-T branch of process II obeys an
Arrhenius law, allowing one to estimate both the pre-exponential factor (
τ∞
) and activation
energy, which follow the same trend as in I and I* (respective parameters included in
Table 4). The kink point of the saddle-like profile is located at higher temperatures
for APTES (T
max
in Table 4), in agreement with its higher ability to retain water, as
mentioned above.
The results reported in this section show how the hydration level and surface modifi-
cation influence water mobility, even at a low functionalization density, such as the one
achieved via TMPS grafting.
3.5.2. Bulk FNB
The molecular mobility of bulk amorphous FNB was probed via dielectric spectroscopy
to recognize its signature in the loaded matrices. The drug amorphization was achieved by
cooling the corresponding crystalline form from the melt at c.a. 8
◦
C min
−1
. The dielectric
spectra in both of the
ε00
and M
00
representations were fitted via the superposition of
HN functions.
Two main relaxation processes were detected, a dominating process associated with the dy-
namic glass transition, a designated
α
-process (visible in the frequency window above ~
−
30
◦
C)
and a secondary
γ
relaxation (emerging at temperatures as low as
−
110
◦
C), attributed to the
rotation of the C
6
H
4
O
3
(CH
3
)C=O2-O1(CH
3
)
2
group (Scheme 1), relative to the dihedral
angle between two aromatic groups; [
22
,
70
] the intramolecular nature of this process was
demonstrated according to its low activation energy (close to 30 kJ mol
−1
) and pressure
insensitivity by Szklarz et al. [
71
]. The here-obtained parameters (Table S2) for both
α
relaxation and the
γ
-process are similar to the ones reported in the literature [
22
,
71
].
A third process is required in the fitting analysis, partially overlapping with the
α
peak,
whose features allow for the identification of it as a Johari–Goldstein (JG) process (
βJG
),
as previously suggested in the literature, [
70
] being pressure-sensitive as a characteristic
of a process with an intermolecular nature [
71
]. The detailed analysis for the bulk FNB is
shown in the Supplementary Materials section Dielectric analysis of amorphous bulk FNB.
3.5.3. FNB-Loaded Matrices
After FNB incorporation, the dielectric response of the loaded silica MSNs was mod-
ified, relative to the unloaded matrices, as exemplified in the isochronal representation
displayed at 10
4
Hz in Figure 6of the real,
ε0
(left side), and imaginary,
ε00
(right side),
parts of the complex permittivity for both the first (hydrated; open symbols) and second
(dehydrated; filled symbols) series of spectral collection; data for the neat FNB are included
at the top of Figure 6(black symbols). The comparison of the
ε0
(T) and
ε00
(T) traces of the
three composites with the ones corresponding to the neat drug, allows for the assigning
of the dielectric response observed between 0 and 40
◦
C to FNB. In the hydrated loaded
matrices, the already-characterized water processes are also visible mainly in the low-T
region (open symbols in Figure 6); after dehydration, these processes vanish (FNB@MSN
and FNB@MSN_TPMS) or become highly depleted (FNB@MSN_APTES), allowing one to
better disclose the FNB contribution.
Pharmaceutics 2023,15, 1624 15 of 27
Pharmaceutics 2023, 15, x FOR PEER REVIEW 16 of 28
Figure 6. Isochronal representation of ε′ (left panels) and ε″ (right panels) vs. T at f = 104 Hz for (a,b)
native FNB, illustrating the eect of cold crystallization and melting, (c,d) FNB@MSNs, (e,f)
FNB@MSNs_APTES and (g,h) FNB@MSNs_TMPS; open and lled symbols for hydrated and dried
data, respectively.
The previous calorimetric analysis showed that amorphous FNB undergoes cold
crystallization above 40 °C, and melting close to 80 °C (Figure S14). These phase transi-
tions are also observed in DRS via the sharp decrease (crystallization) and subsequent
increase (melting) in both ε′(T) and ε″(T) traces (indicated by vertical arrows in Figure
6a,b). Upon FNB incorporation, no clear signal of melting was found for the studied ma-
terials; however, for FNB@MSN_APTES, minor discontinuity was visible in ε′(T) and im-
perceptible in ε″(T), around 80 °C. This could have originated from the melting of a resid-
ual recrystallization of FNB, coherent with DSC analysis (see also Figure S14). It should
be noted that FNB recrystallization is observed when it is loaded in silica matrices with
larger pore sizes (≥18 nm), with a lower melting temperature for smaller pore sizes [19].
The absence of melting in FNB@MSN and FNB@MSN_TMPS means that FNB has been
incorporated within the pores and, furthermore, the pore size (~3 nm) lies below the crit-
ical diameter for FNB crystal growth. In the case of FNB@MSN_APTES, the minute endo-
therm signal at the same neat FNB melting temperature indicated that a rather small frac-
tion was deposited outside of the pores during preparation. After heating up to 120 °C,
liquid FNB should enter inside the pores, as no evidence of either recrystallization/melting
was detected.
In the following sections, the dielectric response of FNB incorporated into each MSN
sample will be analyzed. Despite a severe drying procedure prior to FNB loading, the
loaded matrices easily reabsorbed water (explaining why two series of spectra were col-
lected). In the rst series (hydrated), the ing analysis was only carried out for spectra
acquired between −110 °C and ~30 °C (ending T, depending on the material), as above this
temperature, the sample dielectric response continuously changes due to progressive wa-
ter release (the dielectric cell is not isolated, and it is uninterruptedly submied to nitro-
gen ux).
Figure 6.
Isochronal representation of
ε0
(left panels) and
ε00
(right panels) vs. T at f= 10
4
Hz
for (
a
,
b
) native FNB, illustrating the effect of cold crystallization and melting, (
c
,
d
) FNB@MSNs,
(
e
,
f
) FNB@MSNs_APTES and (
g
,
h
) FNB@MSNs_TMPS; open and filled symbols for hydrated and
dried data, respectively.
The previous calorimetric analysis showed that amorphous FNB undergoes cold
crystallization above 40
◦
C, and melting close to 80
◦
C (Figure S14). These phase transitions
are also observed in DRS via the sharp decrease (crystallization) and subsequent increase
(melting) in both
ε0
(T) and
ε00
(T) traces (indicated by vertical arrows in Figure 6a,b). Upon
FNB incorporation, no clear signal of melting was found for the studied materials; however,
for FNB@MSN_APTES, minor discontinuity was visible in
ε0
(T) and imperceptible in
ε00
(T),
around 80
◦
C. This could have originated from the melting of a residual recrystallization
of FNB, coherent with DSC analysis (see also Figure S14). It should be noted that FNB
recrystallization is observed when it is loaded in silica matrices with larger pore sizes
(
≥
18 nm), with a lower melting temperature for smaller pore sizes [
19
]. The absence of
melting in FNB@MSN and FNB@MSN_TMPS means that FNB has been incorporated
within the pores and, furthermore, the pore size (~3 nm) lies below the critical diameter for
FNB crystal growth. In the case of FNB@MSN_APTES, the minute endotherm signal at the
same neat FNB melting temperature indicated that a rather small fraction was deposited
outside of the pores during preparation. After heating up to 120
◦
C, liquid FNB should
enter inside the pores, as no evidence of either recrystallization/melting was detected.
In the following sections, the dielectric response of FNB incorporated into each MSN
sample will be analyzed. Despite a severe drying procedure prior to FNB loading, the
loaded matrices easily reabsorbed water (explaining why two series of spectra were col-
lected). In the first series (hydrated), the fitting analysis was only carried out for spectra
acquired between
−
110
◦
C and ~30
◦
C (ending T, depending on the material), as above
this temperature, the sample dielectric response continuously changes due to progressive
water release (the dielectric cell is not isolated, and it is uninterruptedly submitted to
nitrogen flux).
Non-functionalized MSN loaded with FNB. The isothermal spectra collected for
FNB@MSN, both hydrated (first run) and dehydrated (second run), were analyzed us-
ing a sum of HN functions (Equation (1)). Representative fitting curves and a summary
Pharmaceutics 2023,15, 1624 16 of 27
of obtained parameters are included in ESI (Figure S15 and Table S3). The temperature
dependence of the obtained relaxation times for all of the detected processes are repre-
sented in Figure 7a (hydrated—open symbols; dehydrated—filled symbols), with neat
FNB and unloaded MSN data included as black and orange symbols, respectively. For
the process detected at the lowest temperatures, log
τ
(T) evolves linearly, and so, it can be
fitted by the Arrhenius equation. An activation energy of 33 kJ mol
−1
was obtained with
a pre-exponential factor of 1.4
×
10
−14
s. This relaxation process could be attributed to
either a modified
γ
-process or to process I observed for hydrated unloaded MSN (orange
circles in Figure 7a). Since after dehydration this process is absent, it is most probably
assigned to the water relaxation. Nevertheless, relaxation involving reorientational motions
of the (C=O2-O1) ester group according to the attribution of the secondary
γ
in neat FNB
should not be discarded. Given that the drug is a proton acceptor species [
57
,
72
] and the
matrix has both a proton donor and acceptor silanols [
73
], it is reasonable to assume that
part of the FNB C=O groups interact via hydrogen bonding with the silanol groups of the
MSNs’ surface. In this sense, it is important to note that the FTIR analysis provided evidence
of strong interactions involving the Si-OH groups (Figure S3a). After dehydration, with
the water release, more adsorption sites become free to interact with FNB molecules, thus
strengthening the drug–host interaction, and, therefore, strongly depleting the amplitude
of the unbonded FNB
γ
dipolar motion, impairing its dielectric detection in the second run.
At higher temperatures, an intense and broad process emerges on the low-frequency
side, which is adequately simulated by a sum of two relaxations (see illustration of the
individual fitting functions in Figure S15a,b). The assignment of these processes becomes
clear via the analysis of their temperature dependencies in the relaxation map (Figure 7a).
The more intense process follows saddle-like temperature dependence, vanishing after
dehydration. It is assigned to process II, previously observed in the hydrated unloaded
matrix (included in Figure 7a as orange symbols). The T-dependence of the high-frequency
process is curved, bending off at ~10
◦
C, a temperature at which water starts to be removed
from the material (confirmed by the coincidence with the temperature corresponding to
the
τ
maximum of the FNB@MSN process II). This behavior can be rationalized as in the
lowest T-range: water is relaxing with FNB, enhancing the drug mobility relative to the
neat drug (black symbols), resulting in a plasticizing effect. From here,
τ
(1/T) follows the
temperature dependence of the bulk FNB
α
-relaxation (black symbols), and, since water
is removed as the temperature increases, a shift in the relaxation times toward slower
values occurs. For the highest temperatures, the
τ
(1/T) dependence becomes linear. This
last temperature dependence is similar for that of the dehydrated FNB@MSN (filled blue
symbols in Figure 7a), with both traces superimposing in the high T-range. The latter is
unequivocally resolved over a large T-range, due to the absence of water process II in the
second run (see isotherm at 0 ◦C in Figure S15c).
The continuous deviation in the relaxation time mirrors the decreasing FNB mobility,
concomitant with water release. This may have originated from a progressive adsorption of
FNB to the silica pore walls at sites previously occupied by water molecules, resulting in a
dynamical hindrance relative to the neat drug, as the relaxation times of this process lie on
the low-frequency/high-temperature side of the bulk
α
-process. Moreover, the temperature
dependence after water removal is Arrhenius-like (activation parameters in Table 5), in
line with the features exhibited by a surface (S) process found in low-molecular-weight
glass formers adsorbed at the silica inner pore walls [
74
–
76
]. A close inspection of the
T-dependence of the relaxation times reveals a bending at 12.7
◦
C (see inset in Figure 7a)
near/above the region where the
αFNB
-trace is crossed. Additionally, the temperature
dependence of the magnitude of the electrical modulus (
∆
M) reveals a crossover occurring
at a similar temperature (Figure S16), reinforcing the alteration in the overall dynamics,
probably caused by the freezing of the surface population. The latter could have originated
from the second high-T step detected in the thermogram (Figure 4b).
Pharmaceutics 2023,15, 1624 17 of 27
Pharmaceutics 2023, 15, x FOR PEER REVIEW 19 of 28
Figure 7. Relaxation maps of (a) FNB@MSN, (b) FNB@MSN_APTES and (c) FNB@MSN_TMPS.
Open symbols correspond to hydrated series data and lled ones correspond to dried series. In each
gure, the relaxation times of dierent processes detected in the corresponding unloaded matrix
(orange symbols) and those present in bulk FNB (black symbols) are included. Solid lines are the
ing functions with Arrhenius or VFTH estimated for each process. The inset in (a) is a scale up of
the second run (dried) of the bending at ~12.7 °C (1000/T = 3.5 K−1) from α-fast to the S-process
compared to αFNB-trace (see text).
The relaxation times of the two processes, observed in the second run of both modi-
ed MSNs, exhibit a non-Arrhenius temperature dependence that can be described by the
Figure 7.
Relaxation maps of (
a
) FNB@MSN, (
b
) FNB@MSN_APTES and (
c
) FNB@MSN_TMPS.
Open symbols correspond to hydrated series data and filled ones correspond to dried series. In each
figure, the relaxation times of different processes detected in the corresponding unloaded matrix
(orange symbols) and those present in bulk FNB (black symbols) are included. Solid lines are the
fitting functions with Arrhenius or VFTH estimated for each process. The inset in (
a
) is a scale up
of the second run (dried) of the bending at ~12.7
◦
C (1000/T = 3.5 K
−1
) from
α
-fast to the S-process
compared to αFNB-trace (see text).
Pharmaceutics 2023,15, 1624 18 of 27
Table 5.
Estimated parameters of the Arrhenius fit (Equation (3)) and the VFTH fit (Equation (4)) to
the relaxation times for all of the detected processes in the dehydrated samples. Data corresponding
to bulk FNB have been included for comparison. All relaxation times were extracted from the fitting
of the M00 spectra.
Sample Process Function τ0(s) Ea(kJ mol−1) B (K) T0(K)
FNB
γArrhenius 3.2 ×10−14 28.7
αVFTH 4.0 ×10−15 1598 205
σVFTH 1.3 ×10−6635 273
FNB@MSN
αfast Arrhenius 7.7 ×10−19 79.4
SOH Arrhenius 8.2 ×10−24 106.5
MWS VFTH 1.4 ×10−92395 204
σArrhenius 3.6 ×10−15 90.5
FNB@MSN_APTES
SOH/αVFTH 8.7 ×10−13 1508.8 198.6
SAPTES VFTH 2.1 ×10−11 801.8 276.4
MWS/σArrhenius 1.5 ×10−14 98.8
FNB@MSN_TMPS
SOH/αVFTH 1.5 ×10−20 4978.2 151.9
STMPS VFTH 1.7 ×10−23 8846.7 107.2
MWS Arrhenius 1.7 ×10−16 100.7
σArrhenius 5.1 ×10−14 88.5
Below this crossover region, the relaxation times become lower than those of the bulk
αFNB
-process. This acceleration of the relaxation rate may be interpreted as a manifestation
of finite size effects of a distinct population being localized in the pore core. Indeed, when
a material is confined to dimensions of the order of the cooperative rearranging regions
(in the framework of the Adam–Gibbs model) [
77
], their relaxation times can undergo an
acceleration effect with a decrease in the glass transition temperature (chapter 6 in [
27
,
76
]),
an effect compatible with the reduction in T
g-on
observed via DSC (Table 3). Consequently,
this low-T relaxation can be designated as
αfast
. Ultimately, if the pore diameter is lower
than the correlation length associated with the size of the cooperatively rearranging regions,
the T-dependence converts from non-Arrhenius (continuous growth of cooperativity) to
Arrhenius (constant or absent cooperativity) [
78
]. Apparently, the T-dependence of the
detected
αfast
process is linear, although to confirm the Arrhenian behavior, the scanned
range should be extended to even lower frequencies.
The enhancement in the relaxation rate at low temperatures and the lowering of the
onset of T
g
(onset of the first step in Figure 4b) found for FNB incorporated into MSN were
also reported for the same drug loaded in nanoporous AAO membranes with higher pore
diameters [
19
]. While crystallization was observed in the AAO membranes, it was avoided
in the tested MSN matrices with pore sizes ~3 nm. Above 60
◦
C, an additional process
emerges, which will be further discussed when analyzing the behavior of the three systems.
Functionalized MSNs loaded with FNB. It has already been shown that surface func-
tionalization affects the dynamics of water processes and, therefore, it is expected to also
influence the molecular mobility of the guest drug. This will be evaluated in the present
section by comparing the dielectric response of matrix-functionalized composites with
that of an unmodified matrix. At first glance, the main differences are as follows: (i) the
detection of a single process in the low-T region (whose ill definition only makes possible
the fitting in the second series of FNB@MSN_APTES); (ii) the lack of the water saddle-like
process at intermediate temperatures for both matrices (in spite of comparable water con-
tents), meaning that loosely bound water is absent in these samples and (iii) the detection
of a relaxation process clearly comparable to the FNB αbulk-process.
Pharmaceutics 2023,15, 1624 19 of 27
The assignment of the different processes detected in functionalized MSNs will be
made according to the temperature dependence of their relaxation times included in the
activation maps in Figure 7b,c (Figures S17 and S18 illustrate the fitting procedure of
isothermal spectra for a set of representative temperatures). With regard to the low-T
relaxation in FNB@MSN_APTES (Figure 7b) observed in the second run, the overlapping
of the respective log(
τ
(T)) trace with that observed for the MSN_APTES unloaded matrix
allows one to undoubtedly assign it to process I* of water. This confirms the strong tendency
of the APTES-modified MSNs to retain water, differently from the other composites, in
which water was merely reabsorbed after FNB loading. In the first run, FNB@MSN_APTES
shows a clear process above the glass transition of bulk FNB (the corresponding relaxation
times are included in Figure 7b as open circles), although affected by the progressive water
release during acquisition data upon heating. In the second series of measurements, an
additional relaxation is visible at higher temperatures. For FNB@MSN_TMPS (Figure 7c),
two processes are also needed to deconvolute the M
00
spectra in both hydrated and dry
states. Their T-dependence of relaxation times is very similar for both hydrated and dry
series of measurements coherent with a low water uptake.
The relaxation times of the two processes, observed in the second run of both modified
MSNs, exhibit a non-Arrhenius temperature dependence that can be described by the
VFTH fitting function (fitting parameters are summarized in Table 5). The emergence of
the processes at temperatures higher than the T-range at which
α
bulk FNB is observed
reflects a hindrance of the underlying molecular motions, and so they can be identified
as surface processes. To explain their origin, it should be considered that two types of
guest–host interactions can arise in the modified MSNs: (i) between silanol and FNB, and
(ii) between grafted APTES or TMPS groups with FNB. The former is expected to exist
in modified and unmodified composites, giving origin to a surface process common to
three systems (hereafter designated as S
OH
). The position of the relaxation times of this
S
OH
-process in the modified MSNs relative to the one observed in FNB@MSN reflects a
weakening of the silanol–FNB interactions due to the presence of APTES or TMPS. The
extrapolation of the VFTH fitting functions to
τ
= 100 s [
79
] provides an estimate of the glass
transition temperature,
−
28
◦
C and
−
23
◦
C, respectively, for APTES and TMPS; considering
the corresponding dielectric glass transition temperature for the bulk FNB (
−
25.5
◦
C; see
Table S2), the T
g-dielec
was thus estimated, keeping the same relative order found for the
calorimetric T
g-onset
. For the case of FNB incorporated into an APTES-functionalized matrix,
both DSC and DRS concur disclosing a dynamically accelerated population relative to
the neat drug at low temperatures (decreased T
g
), however to a lower extent, relative
to what was found for FNB@MSN. The fact that the shape parameters that simulate the
FNB@MSN_APTES dielectric spectra remain almost constant for all of the temperature
ranges suggests that the same kind of cooperative rearrangement is being probed, being
less (low T) or more (high T) influenced by the surface.
On the other hand, the second process (located at slower relaxation rates relative to
the S
OH
-process) may have originated from interactions involving not only the silanol
groups but also the grafted groups. Besides the Si-OH
···
O=C interactions, N-H
···
O=C
interactions in the APTES matrix or
π
-
π
stacking between the TMPS matrix and FNB
aromatic units could also arise. This anchoring of the same FNB molecule at two sites
of the pore wall will further constrain the guest dynamics. Two reasons can sustain this
hypothesis: (i) the lengthening of APTES and bulkiness of TMPS moieties relative to Si-OH
and (ii) the high flexibility of the FNB aliphatic chains allowing the guest molecule to adopt
different conformations, as already reported for this drug [
52
]. Both (i) and (ii) favor the
binding via a non-covalent interaction at multiple sites, which is already known to occur
in other guest–host systems [
80
]. The slower relaxation rate of the MSN_APTES surface
process and the near absence of a calorimetric response (blue DSC curve in Figure 4a)
indicate FNB-APTES as being the strongest guest–host interaction.
The present investigation clearly demonstrates that DRS analysis allows disclosing
several relaxation processes which are in the origin of the widening and multimodal
Pharmaceutics 2023,15, 1624 20 of 27
characteristics of the glass transition region observed via DSC (remember the DSC curves
in Figure 4b and S8).
3.5.4. Conductivity of the Loaded Samples
At temperatures above reorientational dipole motions, DRS mainly probes the migra-
tion of charge carriers. The conductivity behavior gives an insight on how the modifications
in the confining media reflect the long-range charge mobility.
The second run of the isothermal measurements of FNB@MSN revealed a highly
asymmetric peak in the modulus representation (Figure 8a) and multiple regimes in the
conductivity spectra,
σ0
(f) (Figure 8b). The latter shows three different regions: a dc plateau
at low frequencies (1), which bends off to a frequency dependent regime (2), distinct from
the ac high-frequency behavior (3). The dielectric response at intermediate frequencies
must have originated from a Maxwell–Wagner–Sillars (MWS) effect [
81
], as expected in
heterogenous systems in which charges accumulate at interfaces (with discontinuities in the
dielectric properties) [
82
]. The absence of this effect in bulk FNB (Figure S19a) supports the
attribution to MWS relaxation in the composite. The effect is clearly seen in Figure 8b, with a
strength that can be quantified by the difference between the experimentally measured con-
ductivity (symbols) and the isotherm predicted by the Jonscher law [
83
]. The latter just takes
into account diffusive (at low frequencies, at which the plateau emerges) and semi-diffusive
(ac frequency response at high frequencies) charge transport mechanisms, according to
(
σ0(ω)=σ01+(ω/ωcross)s
), in which
σ0
is the direct current conductivity (estimated in
the frequency-independent regime), sis a material and temperature-dependent parameter
(0.5
≤
s
≤
1) and
ωcross
is the angular frequency at which the dc plateau changes to an ac
regime. In the FNB@MSN M
00
(f) spectra, both dc conductivity and the MWS process emerge
as the corresponding peaks overlap in this temperature range, causing the abovementioned
asymmetric M
00
(f) peak. This peak can be deconvoluted in two HN functions: one at the
lowest frequencies (fitted with shape parameters
αHN
=
βHN
= 1 as expected for a pure dc
conductivity process (chapter 3 in [
27
])), and another function which takes into account the
interfacial MWS contribution (see the thin solid lines in Figure 8a and S19b as examples).
The same effect is observed in the TMPS functionalized silica (Figure 8a).
For MSN_APTES, the MWS contribution is mainly visible, as confirmed by the shape
parameters (
αHN
= 0.85 and
βHN
= 0.5), with the conductivity contribution lying in the
low flank of the frequency window. The absence of a well-defined dc plateau reflects
the hindrance to long-range charge migration imposed by the presence of the APTES
moiety. The observed behavior for the functionalized matrices loaded with FNB results
from the balance between the degree of grafting—higher for APTES, increasing MWS
contribution—and the amount of incorporated drug—higher for TMPS, increasing the
conductivity contribution.
The temperature dependence of the MWS and
σdc
-processes is well described by
either an Arrhenius or a VFTH plot, depending on the sample. The corresponding fitting
parameters are presented in Table 5.
Differently to what happens in bulk FNB, for which the Debye–Stokes–Einstein corre-
lation is obeyed (inset of Figure S12), pointing to a coupling between translational (
σdc
) and
reorientational dipolar motions (
τα
), for FNB loaded in the nanoparticles, it is not possible
to test the proposed relationship, due to the large temperature gap between the dipolar
processes and the emergence of dc conductivity.
3.6. Release Kinetics
Samples previously investigated via DRS were used in the FNB release tests. To
analyze the release profile, absorbance at
λ
= 289 nm, in which the FNB spectrum shows a
maximum, was represented as a function of time (Figure 9). The masses used in these tests
were very similar (0.00113
±
0.00003 g), facilitating the direct comparison of the results (see
the Experimental section for the specific details of the drug delivery assays).
Pharmaceutics 2023,15, 1624 21 of 27
Pharmaceutics 2023, 15, x FOR PEER REVIEW 22 of 28
Figure 8. Isothermal spectra at 120 °C of the FNB-loaded nanoparticles (a) in the M″(f) representa-
tion, with the spectra deconvoluted in two HN functions (MWS and σ) for FNB@MSN and
FNB@MSN_TMPS (thin lines), and only one HN function for FNB@MSN_APTES (overall t shown
as thick lines); (b) σ′(f) spectra for the three composites, with the isotherm σ′(f) predicted by the
Jonscher law for FNB@MSN (solid lines).
3.6. Release Kinetics
Samples previously investigated via DRS were used in the FNB release tests. To an-
alyze the release prole, absorbance at λ = 289 nm, in which the FNB spectrum shows a
maximum, was represented as a function of time (Figure 9). The masses used in these tests
were very similar (0.00113 ± 0.00003 g), facilitating the direct comparison of the results
(see the Experimental section for the specic details of the drug delivery assays).
At the initial stages (up to ~50 min), almost no FNB was released in any of the sam-
ples. Considering that most of the water was removed during the dielectric measure-
ments, this time lag is likely related to the slow diusion of FNB from within the pores of
the matrix, driven by the intrusion of the releasing medium into the nanoparticles, ex-
plaining the absence of a burst release. This supports the previous indication that FNB
molecules are mostly located near the pore walls, rather than being in the core or outside
of the pores.
After this initial step, the increase in the absorbance was very pronounced in the
MSN_APTES sample, while for MSN and MSN_TMPS the release rate of the loaded FNB
was signicantly slower. The FNB release prole from MSN_APTES can be described us-
ing the empirical Hill model (see ref. [84] and references therein), which adapted to abso-
lute values of absorbance reads:
𝐴(𝑡)= 𝐴𝑚𝑎𝑥𝑡𝛾
𝐴50% +𝑡𝛾
(6)
where Amax is the maximum absorbance aained (corresponding to a certain value of dis-
solved drug), A50% is the time required to reach 50% of Amax and γ is a sigma factor. The
Figure 8.
Isothermal spectra at 120
◦
C of the FNB-loaded nanoparticles (
a
) in the M
00
(f) repre-
sentation, with the spectra deconvoluted in two HN functions (MWS and
σ
) for FNB@MSN and
FNB@MSN_TMPS (thin lines), and only one HN function for FNB@MSN_APTES (overall fit shown
as thick lines); (
b
)
σ0
(f) spectra for the three composites, with the isotherm
σ0
(f) predicted by the
Jonscher law for FNB@MSN (solid lines).
Pharmaceutics 2023, 15, x FOR PEER REVIEW 23 of 28
Hill ing function well describes the very slow FNB release prole from MSN_APTES
(solid line in Figure 9—ing parameters presented in the caption).
Figure 9. Release prole measured from absorbance at λ = 289 nm of FNB from MSN (blue),
MSN_APTES (green) and MSN_TMPS (pink). The data corresponding to the FNB@MSN_APTES
sample were modeled using the Hill equation (Equation (6)) with Amax = 0.17, γ = 1.77 and A50% =
32,751 min1.77 as the ing parameters. Inset: relaxation map including only surface processes (col-
ored symbols) and α of bulk amorphous FNB (black markers).
Although only a small fraction of FNB was released at the end of the 8 h for all of the
composites (Table 6), the way in which the drug is released depends on the matrix, with
the larger dierence observed for MSN_APTES. The amount of FNB released from
MSN_APTES at the end of the 8 h is about ten times larger than that from the unmodied
MSNs. This behavior can be rationalized considering the relaxation times estimated from
the dielectric analysis of the dried nanocomposites (i.e., series 2). The FNB molecules in-
volved in the SOH-process are expected to be released rst (see a close-up of the activation
map of the surface processes depicted in the inset of Figure 9). In relation to this process,
the FNB relaxes more slowly in the unmodied matrix, causing the lower FNB release
rate. On the other hand, the dierences observed in the drug release proles of the two
functionalized silica matrices indicate that, in addition to guest mobility (where the relax-
ation times are comparable), another parameter must be involved. This is probably asso-
ciated with topological dierences on the inner surface of the pore. The type and density
of the grafted groups aect the architecture of the pore wall, altering the homogeneity of
the adsorbed FNB layer. Indeed, as previously mentioned (Table 2), the density of the
grafted groups is not enough to uniformly cover the pore wall surface. These inhomoge-
neities can weaken the adsorption of FNB to the silanol groups relative to the adsorption
occurring in high-density and homogeneously silanol-covered unmodied MSNs, ulti-
mately determining the release rate of the FNB population that leaves the pores rst.
Figure 9.
Release profile measured from absorbance at
λ
= 289 nm of FNB from MSN (blue),
MSN_APTES (green) and MSN_TMPS (pink). The data corresponding to the FNB@MSN_APTES
sample were modeled using the Hill equation (Equation (6)) with A
max
= 0.17,
γ
= 1.77 and
A
50%
= 32,751 min
1.77
as the fitting parameters. Inset: relaxation map including only surface processes
(colored symbols) and αof bulk amorphous FNB (black markers).
Pharmaceutics 2023,15, 1624 22 of 27
At the initial stages (up to ~50 min), almost no FNB was released in any of the samples.
Considering that most of the water was removed during the dielectric measurements, this
time lag is likely related to the slow diffusion of FNB from within the pores of the matrix,
driven by the intrusion of the releasing medium into the nanoparticles, explaining the
absence of a burst release. This supports the previous indication that FNB molecules are
mostly located near the pore walls, rather than being in the core or outside of the pores.
After this initial step, the increase in the absorbance was very pronounced in the
MSN_APTES sample, while for MSN and MSN_TMPS the release rate of the loaded FNB
was significantly slower. The FNB release profile from MSN_APTES can be described using
the empirical Hill model (see ref. [
84
] and references therein), which adapted to absolute
values of absorbance reads:
A(t) = Amax tγ
A50% +tγ(6)
where A
max
is the maximum absorbance attained (corresponding to a certain value of
dissolved drug), A
50%
is the time required to reach 50% of A
max
and
γ
is a sigma factor. The
Hill fitting function well describes the very slow FNB release profile from MSN_APTES
(solid line in Figure 9—fitting parameters presented in the caption).
Although only a small fraction of FNB was released at the end of the 8 h for all of
the composites (Table 6), the way in which the drug is released depends on the matrix,
with the larger difference observed for MSN_APTES. The amount of FNB released from
MSN_APTES at the end of the 8 h is about ten times larger than that from the unmodified
MSNs. This behavior can be rationalized considering the relaxation times estimated from
the dielectric analysis of the dried nanocomposites (i.e., series 2). The FNB molecules
involved in the S
OH
-process are expected to be released first (see a close-up of the activation
map of the surface processes depicted in the inset of Figure 9). In relation to this process,
the FNB relaxes more slowly in the unmodified matrix, causing the lower FNB release rate.
On the other hand, the differences observed in the drug release profiles of the two function-
alized silica matrices indicate that, in addition to guest mobility (where the relaxation times
are comparable), another parameter must be involved. This is probably associated with
topological differences on the inner surface of the pore. The type and density of the grafted
groups affect the architecture of the pore wall, altering the homogeneity of the adsorbed
FNB layer. Indeed, as previously mentioned (Table 2), the density of the grafted groups is
not enough to uniformly cover the pore wall surface. These inhomogeneities can weaken
the adsorption of FNB to the silanol groups relative to the adsorption occurring in high-
density and homogeneously silanol-covered unmodified MSNs, ultimately determining
the release rate of the FNB population that leaves the pores first.
Table 6.
Release data obtained via UV-Vis: mass of sample (m
sample
); weight fraction of FNB loaded
in MSNs (FNB
loaded
); mass of FNB in MSNs (m
FNB
); moles of FNB in MSNs (n
FNB
); mass of MSNs
(m
MSN
); moles of FNB in the MSNs per gram of nanoparticles (n
MSN
); moles of FNB released after 8 h
per gram of MSNs (nrel); mole fraction released after 8 h relative to loaded FNB (nreleased).
Sample msample (g) FNBloaded
(wt%) mFNB (g) nFNB (mol) mMSN (g) nMSN
(mol g−1)
nrel
(mmol g−1)nrel (mol) nreleased
(%)
FNB@MSN 1.11 ×10−322.8 2.53 ×10−47.01 ×10−78.57 ×10−48.18 ×10−42.36 ×10−32.02 ×10−90.29
FNB@MSN_APTES 1.13 ×10−322.8 2.58 ×10−47.14 ×10−78.72 ×10−48.18 ×10−42.17 ×10−21.89 ×10−82.65
FNB@MSN_TMPS 1.16 ×10−333.3 3.86 ×10−41.07 ×10−67.74 ×10−41.38 ×10−31.60 ×10−21.24 ×10−81.15
4. Conclusions
Fenofibrate (FNB) was incorporated into spherical mesoporous silica nanoparticles
(MSNs) with external diameters of ~50 nm and pore diameters of ~3 nm. The tested MSNs
were either unmodified or modified via the grafting of APTES or TMPS groups (with 1.93
and 0.60 molecules nm−2, respectively).
The presence of grafted groups in the MSNs has a noticeable effect on the dynamics of
adsorbed water, as observed via dielectric analysis. This allowed for the distinction of three
Pharmaceutics 2023,15, 1624 23 of 27
relaxation processes associated with the following: (i) reorientational motions in ice-like
clusters (process I), (ii) joined reorientations of the surface chemical units and the interacting
water molecules (process I*) and (iii) the relaxation of loosely bound water (process II).
After FNB loading, calorimetric, infrared spectroscopy and dielectric analysis showed
that the drug was stabilized in the amorphous state, proving that the pore size was below
the critical nucleus diameter for FNB crystallization.
Broad glass transition was observed via DSC (for the three composites), assigned
to the loaded FNB drug. Moreover, the glass transition onset (T
g,ons
) slightly shifted
to lower temperatures for FNB@MSN and FNB@MSN_APTES, while it increased for
FNB@MSN_TMPS. Calorimetric glass transition was sensitive to readsorbed water, mainly
for FNB@MSN. DRS confirmed this sensitivity to water content, as the FNB relaxation rate
slowed down upon water removal. After dehydration, the mobility at temperatures around
the calorimetric glass transition still revealed an accelerated dynamic. The evolution of
the respective relaxation times with temperature showed a kink to a more constrained
process, assigned to surface silanol groups interacting with FNB molecules (S
OH
-process).
In the functionalized matrices, a correspondent S
OH
-process was detected, although it
exhibited higher mobility due to a weakening of the silanol–FNB interaction caused by
the presence of the grafted groups. Additionally, a high-temperature surface process was
detected, associated with FNB interacting mainly with the APTES and TMPS capping
groups. Furthermore, the existence of guest–host interfaces was found in the beginning of
the MWS process, detected in all composites, affecting the conductivity response.
The drug delivery profiles showed no initial burst release. Instead, drug release was
only detected after a significant time interval (~50 min). FNB is released faster from the
APTES-functionalized matrix which is interpreted in terms of the higher molecular mobility
of the FNB population associated with the S
OH
-process, and heterogeneities in the surface
coverage imprinted by the grafted groups.
The work presented here allowed a number of key parameters to be correlated with
FNB release behavior, enabling a more rational design of mesoporous silica nanoparticles
for drug amorphization and delivery.
Supplementary Materials:
The following supporting information can be downloaded at https://
www.mdpi.com/article/10.3390/pharmaceutics15061624/s1. References [
22
,
23
,
70
,
71
,
79
,
85
–
89
] are
cited in the Supplementary Materials.
Author Contributions:
Conceptualization, J.P.F. and M.T.V.; formal analysis, G.F. and M.T.V.; inves-
tigation, G.F., J.L.M.G. and H.P.D.; writing—original draft preparation, G.F.; writing—review and
editing, M.T.V., M.D. and J.P.F.; funding acquisition, J.P.F. All authors have read and agreed to the
published version of the manuscript.
Funding:
This work was supported by Fundação para a Ciência e a Tecnologia (FCT-Portugal) and
COMPETE (FEDER), projects UIDB/00100/2020 and UIDP/00100/2020 (CQE).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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