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Hybrid mesoporous silica nanoparticles
Elizabete Coutinho
e-mail: [email protected]
CQFM – Centro de Química-Física Molecular, Instituto Superior Técnico, 1049-001 Lisboa, Portugal.
ABSTRACT: A decrease in the side effects of a drug can be obtained if it is delivered in
an effective and timely manner to the needed location. Therefore, systems are needed to
improve the transport of the drug and the location of the target cells, resulting in an increase in
drug efficiency and decreased toxicity to healthy tissues. Nanoparticles have gained much
importance in medicine due to the fact that they have high area to volume ratio and, after
appropriate functionalization, the ability to recognize target sites.
In this work, hybrid spherical mesoporous silica nanoparticles (MSNs) were synthesized
with a core-shell design. The mesoporous silica core has a well-defined morphology with pores
that can incorporate molecules. The MSNs are characterized by ordered pores with diameters
arround 2-2.5 nm and volumes above 1 mL / g. RAFT polymerization (Reversible Addition-
Fragmentation chain Transfer), associated with the grafting-from method, were used to grow a
biocompatible polymer shell with temperature conformation dependence. This conformational
change from extended to collapsed can be used to control the release of molecules
incorporated in the pores, providing a convenient platform for controlled release of drugs. The
MSNs have diameters between 140 and 220 nm. We prove the successful release of molecules
from the MSNs pores through thermal stimulation of the polymeric shell. These nanoparticles
open new pathways in the development of controlled release systems.
KEYWORDS: mesoporous silica nanoparticles, core-shell nanoparticles, controlled
release, functionalization, RAFT.
1. INTRODUCTION
The hydrophobic nature of most
chemotherapeutic agents makes them poorly
soluble in water and therefore limits the
administration in higher doses. Thus, systems
that improve the transport of the drug and its
delivery to the target cells are required,
resulting in enhanced drug efficacy and
decreased toxicity to healthy tissues.These
ideas continue to be a priority in cancer
therapy. Mesoporous silica nanoparticles
(MSNs) have a mesoporous structure with
well-defined pore diameters between 2 and 4
nm, and volume exceeding 1 mL/g pore.1 In
this structure, the mesopores are aligned and
appear in form of honeycombs making it very
interesting and important for many
applications. The pores act as individual
reservoirs without links between them. These
internal structures can be controlled by the
initial reagents or the surfactant used. The
mesoporous silica nanoparticles have
diameters of 40 to hundreds of nanometers
and are a biocompatible three-dimensional
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network with silanol groups (≡Si-OH) on the
surface.2
The main objective of this work is to
develop hybrid MSNs with a thermoresponsive
polymer shell to obtain a smart release control
mechanism, able to accommodate large drug
loads and deliver their cargo on demand to a
desired location.3,4,5
The hybrid MSNs were
prepared by incorporating a perylenediimide
(PDI) derivative in the silica network (MSN-PDI)
for bioimaging. The surface of MSNs were
funcionalized with (3-aminopropyl)
triethoxysilane (APTES). The RAFT
polymerization method associated with the
grafting from method was used to growth the
polymeric shell.6,7
A fluorescent probe,
Sulforhodamine B (SRB), was incorporated into
the hybrid MSNs in order to study the controlled
release using fluorescence techniques.
2. MATERIALS AND METHODS
2.1. Materials
Absolute ethanol (EtOH, 99,9% Scharlau),
hydrochloric acid (HCl, 37% Panreac),
ammonium hydroxide solution (NH4OH, 25%
Fluka), tetraethyl orthosilicate (TEOS, 98%
Aldrich), N-cetyltrimethylammonium bromide
(CTAB, 99% Sigma), (3-aminopropyl)
triethoxysilane (APTES, 98% Sigma-Aldrich), N-
(3-dimethylaminopropyl) carbodiimide (EDC,
98% Sigma-Aldrich) were used without further
purification. The RAFT agent, 3-
(benzylsulfanylthiocarbonylsulfanyl) propionic
acid was used for RAFT polimerization, 2-(2-
methoxyethoxy)ethyl methacrylate (MEO2MA,
95% Sigma-Aldrich, Mn=188,22 g mol-1
) and
poly(ethylene glycol) methyl ether methacrylate
(OEGMA, 98% Sigma-Aldrich, Mn=475 g mol-1
),
2,2-azobis (2-methylpropionitrile) (AIBN, 99%
Sigma-Aldrich) were used without further
purification. Commercial toluene and
dichloromethane were distilled over calcium
hydride prior to use. The Perylenediimide
derivative was synthetized according to the
literature.8
2.2. Synthesis of the MSNs
The perylenediimide-functionalized mesoporous
silica nanoparticles (MSN-PDI) were prepared
by a sol-gel modified process.9 In a
polypropylene flask CTAB (0.113 g) was
dissolved in 58.7 mL of 0.5 M NH4OH at 50ºC.
2.5 mL of a TEOS solution (0.2 M in ethanol),
0.5 mL of a PDI solution (6 mg PDI and 15 mL
EtOH) and 9.2 mL of absolute ethanol were
added to the surfactant solution dropwise while
stirring. The stirring was continued for 5 h. TEOS
solution (2.5 mL, 0.2 M in ethanol) and PDI
solution (0.5 mL) were added in the mixture and
kept stirring for 1 h. The mixture was then aged
at 50º C for 24 h. The nanoparticles were
recovered by centrifugation at 19118 g for 20
min each cycle. The MSNs were washed 2 times
with ethanol/water and 3 times with ethanol. The
MSNs were dried at 50ºC. The amino
modification of the silica surface was performed
by dispersing the obtained nanoparticles in a
solution of APTES in dry toluene. The ratio of
the reaction mixture used was 0.2 g MSNs: 10
mL toluene: 0.468 mL APTES. The reaction
occurs at 125ºC under reflux and argon
atmosphere for 24 h. Finally, the nanoparticles
were centrifuged for three cycles (19118 g, 20
min), redispersed in absolute ethanol and finally
dried overnight at 50º C. To remove the
surfactant, the nanoparticles were placed in a
polypropylene flask with an acidic ethanol
solution (0.5 M HCl, 20 mL of this solution for
each 500 mg of particles) and stirred for 2 h at
40º C. The nanoparticles were centrifuged three
-
times and redispersed in ethanol (19118 g, 10
min each cycle) and subsequently dried
overnight at 50° C.
2.3. Synthesis of RAFT Agent Functionalized
MSNs (MSN-RAFT)
To immobilize the RAFT agent on the
nanoparticles surface, the EDC and dry
dichloromethane were added to the MSN-PDI-
APTES and RAFT agent. The mixture was
stirred under an argon atmosphere at room
temperature for 24 hours. The nanoparticles
functionalized with RAFT agent (MSN-PDI-
RAFT) were recovered by centrifugation in 3
cycles (19118 g, 20 min each cycle), and
redispersed in absolute ethanol.
2.4. Synthesis of MSN-Poly
The polymer shell was synthesized by
introducing MSN-PDI-RAFT, OEGMA, MEO2MA
and ethanol in the schlenk tube. The mixture
was placed in an ultrasonic bath for 30 minutes
and stirred for 45 minutes at room temperature
under argon atmosphere. A solution of AIBN (~8
mg, 10 mL ethanol) was stirred for 45 min at
room temperature under argon atmosphere.
After, the schlenk tube was placed in an oil bath
at 70° C and 30 µl of the AIBN solution was
added to the mixture. The reaction mixture was
maintained under argon atmosphere for 24
hours. At the end of the reaction, was
centrifuged in 3 cycles (8497 g, 10 min each
cycle) and redispersed in absolute ethanol.
2.5. Loading and Release of Sulforhodamine
B
For loading hybrid nanoparticles with
sulforhodamine B (MSN-POLY-SRB) a solution
of SRB (pH 7, 4.47 × 10-3
M) was prepared. First,
3 mg of MSN-POLY were added to 3 mL of the
SRB solution and the mixture was stirred
overnight at 20° C. The temperature was
changed to 50° C and mantained during 3 hours.
1 mL of this dispersion was centrifuged in 3
cycles (8497 g, 10 min each cycle, 40° C), and
redispersed in phosphate buffer to remove the
SRB which was not loaded in the MSNs.
Supernatants were stored to measure the SRB
which wasn´t incorporated in MSN-POLY. The
amount of SRB loaded into MSN-POLY was
calculated from the difference between the
concentration of SRB solution used in the loading
and the concentration the separated medium
after centrifugations. For the release experiment,
200 µL of the dispersion with MSN-POLY and
SRB were placed in a dialysis tube and 3.5 mL of
phosphate buffer were introduced in the cell with
a magnetic stirrer. A kinetic study was performed
by changing the temperature between 20° C and
50° C, each 20 minutes, during for 4 hours.
3. Methods
TEM images were obtained in a Hitachi
transmission electron microscope (model H-
8100 with a LaB6 filament) with an accelerator
voltage of 200 kV. One drop of particles
dispersion in ethanol was placed on a carbon
grid and dried in air before observation. Images
were processed with the Fiji software. Zeta
potential (ξ) and hydrodynamic diameter (Dh)
measurements of the particles were obtained
using a Zetasizer Nano ZS, model ZEN3600.
The absorption spectra were recorded on a
Jasco V-660 spectrophotometer with a Peltier
temperature control. Fluorescence emission and
excitation spectra were recorded on a Horiba-
Jobin Yvon Fluorolog 3 spectrofluorimeter and
Right Angle geometry was used. All spectra
were recorded using 1 cm x 1 cm quartz cells.
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4. RESULTS AND DISCUSSION
The fluorescent MSNs were obtained through a
sol–gel modified procedure, using CTAB as
template and ethanol as solvent. The excitation
and emission spectra of MSN-PDI dispersed in
ethanol are similar to the spectra of free PDI in
ethanol (Figure 1) meaning that the
incorporation of the PDI in the MSNs does not
affect the fluorescence properties of the dye.
Figure 1 - Normalized excitation (dashed lines, λemi =
560 nm) and emission (solid lines, λexc = 500 nm) for
PDI (green) and MSN-PDI 7a (blue).
The surface of the MSNs was modified with
APTES to incorporate primary amino groups on
the external surface.10
Then, the template was
removed using an acidic ethanol solution
allowing the pores to be completely available.
The synthesis of the polymer shell starts with the
anchoring of the RAFT agent on the external
surface of the MSN-PDI particles. The amino
groups on the external surface reacted with the
RAFT agent to obtain MSN-PDI-RAFT. These
nanoparticles were used on the RAFT synthesis
of the PEG-acrylate monomers, to obtain core-
shell silica-polymer nanoparticles (MSN-POLY)
(Figure 2).
Figure 2 - Different steps involved in the preparation of
hybrid MSNs coated with a thermoresponsive
biocompatible PEG-acrylate copolymer, incorporating a
PDI dye in the silica network.
The diameters of the mesoporous silica
nanoparticles were measured using TEM (DTEM)
and DLS (Dh). Table 1 summarizes the average
diameters obtained for the different synthesis
with both techniques. As seen in Table 1, the
hydrodynamic diameter is higher than the
diameter obtained by TEM, which shows the
tendency of nanoparticles to aggregate in
dispersion. However, the Dh is usually higher
than DTEM due to the hydration layer around the
nanoparticle in the particles dispersion.
0
0,2
0,4
0,6
0,8
1
1,2
400 500 600 700
Inte
nsi
ty (
a.u
)
Wavelength(nm)
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Table 1 - Average diameters and standard deviations
obtained by DLS and TEM for the core of MSNs at 25º
C.
Sample Dh (nm) DTEM (nm)
MSN-PDI 2a 210±7 150±32
MSN-PDI 3a 220±5 160±35
MSN-PDI 3b 180±14 170±27
MSN-PDI 5b 170±2 160±34
MSN-PDI 7a 160±2 140±38
MSN-PDI 7b 170±10 160±44
From the TEM images of MSN-PDI (Figure 3), it
is possible to observe the spheroid shape and
the mesopores network along the nanoparticles
(Figure 3 C). The RAFT polymerization was
conducted to synthesize a copolymer formed
from a mixture of two PEG-acrylates. The
thermossensitive behavior of the polymer shell
was tested by analyzing the Dh of the hybrid
nanoparticles at 25 and 40º C, for different
samples (Table 2).
Table 2- Average hydrodynamic diameters of different
samples of hybrid nanoparticles dispersed in water at
25 and 40° C.
The average Dh for sample MSN-POLY 3b was
290 ± 5 nm at 25ºC, decreasing to 180 ± 10 nm
at 40ºC, providing the temperature-switchable
collapsed/expanded conformation behavior. The
average hydrodynamic diameter for sample
MSN-PDI 3b at 25ºC (Table 1) was 180 ± 14
nm. This value is equal to that obtained for the
MSN-POLY at 40ºC, which indicates that at this
temperature the chains of polymer shell are
collapsed onto the silica surface.
Figure 3 - Transmission electron microscope images of
MSN-PDI, A and B showing the particle morphology
and C showing the structure mesoporous.
The Dh of MSN-POLY 7a and MSN-POLY 7a
at 40ºC are lower than Dh of MSN-PDI,
because the silica nanoparticles (MSN-PDI)
are more likely to aggregate than the silica
nanoparticles with polymer shell (MSN-POLY)
which are more stable and so are better
Sample Dh (nm) 25ºC Dh (nm) 40ºC
MSN-POLY 2a 270±21 180±26
MSN-POLY 3a 210±11 150±7
MSN-POLY 3b 290±5 180±10
MSN-POLY 5b 190±2 140±1
MSN-POLY 7a 310±16 180±12
MSN-POLY 7b 190±5 150±3
A
B
C
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dispersed. The TEM images for MSN-POLY 7b
(Figure 3 A) clearly show the polymer shell
around the silica nanoparticles. We also
measured the Zeta potencial in the different
steps of the synthesis of the nanoparticles
(Figure 4). The zeta potential of the silica
nanoparticles with polymer (MSN-POLY)
increased compared with the RAFT agent
nanoparticles (MSN-PDI-RAFT), suggesting
the surface modification of the nanoparticles
with the polymer.11
Figure 4- Average of Zeta potential of the different steps
of the synthesis of MSNs. The nanoparticles were
dispersed in water and measured at 25° C.
Figure 5 – Dinamic light scattering (DLS) of MSN-POLI
2a (A) and and MSN-POLI 7a (B) with LCST at 36,3ºC
and 35,6ºC, respectively.
The LCST of the polymer shell in MSN-POLY
was characterized by DLS, by measuring de Dh
from 20ºC to 50ºC (Figure 5). The polymer shell
present a LCST arround 36°C.12
To determine
the hysteresis we perform rapid cooling and
heating cycles (Figure 6). At temperatures below
the transition temperature (20°C), the diameter
was 218 nm, and above this temperature (50°C)
was 116 nm. When MSN-POLY was cooling, the
diameter was 114 nm at 50° C and 208 nm at
20°C, which indicates that the polymer chains
expanded at low temperature, but collapse onto
the silica surface at high temperature.13
Figure 6 - Transmission electron microscope image of
MSN-POLY 7b (A) showing a core-shell nanoparticle
and representation of the hydrodynamic diameters at
different temperature (B). The figure present a heating
cycle (20 - 50ºC, black) and followed by a cooling cycle
(50 - 20ºC, gray).
100
150
200
250
20 30 40 50
Dia
me
ter
(n
m)
Temperature ( °C)
A
B
A
B
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3.2. Release of sulforhodamine B
The MSNs coated with a polymeric shell were
tested as controlled release containers for
sulforhodamine B (SRB) molecules. The
emission (λexc = 520 nm) and excitation (λemi =
620 nm) spectra were recorded at 20° C and 50°
C (Figure 6), because this temperature will be
used in the release experiment. In figure 6, we
can observe that the fluorescence intensity at 50
° C is lower than at 20° C. This happens due to
the increase in the non-radiactive components
with temperature leading to a decrease in the
quantum yield. A ratio of fluorescence
intensities, RF = IF (50ºC) / IF (20ºC), was used to
correct the fluorescence intensities in the
release experiments. The SRB was loaded into
MSN-POLY and the study of release were
performed by switching the temperature
between 50 and 20° C, every 20 minutes. The
emission of free SRB and SRB loaded MSN-
POLY was followed for 4 hours. The kinetics
started at 50°C because at this temperature the
polymer shell was collapsed and the MSNs were
loaded. The fluorescence intensity obtained at
50ºC for the SRB and MSN-SRB were
normalized with the ratio RF (Figure 7).
Figure 7 - Excitation (dashed lines, λemi = 620 nm) and
emission (solid lines, λexc = 520 nm) spectra of SRB at
20° C (red) and 50° C (blue).
Figure 8 - Fluorescence intensity obtained for SRB
(blue) and MSN-SRB (green), measured continuously
during 4 hours at λemi = 585 nm and λexc = 565 nm. The
fluorescence intensities measured at 50° C for SRB
(red) and MSN-SRB (purple) were normalized with the
RF ratio.
The release profiles of MSN-POLY can be
observed by applying heating - cooling cycles
to the nanoparticles. The values at each
plateau (20ºC and 50ºC) were fitted with a 1st
order equation (Figure 9). The slope of the
different temperature for SRB and MSN-SRB
are compiled in Figure 10. The slopes of MSN-
SRB at 20°C are identical over time because
at this temperature the polymer chains are
hydrated and expanded allowing SRB
molecules to flow from the core to the shell but
forcing them to remain there due to the
electrostatic interations with the polymer
chains. The slopes for MSN-SRB at 50ºC are
higher than for free SRB at 50ºC, because by
increasing the temperature to 50ºC, the
extended polymer chains of MSN-SRB shrink
rapidly, releasing the SRB molecules that were
in the polymer shell and thus increasing the
fluorescent intensity in the cell bottom
compartment. The slopes decrease over time,
because the amount of SRB released
decreases as the particle load is depleted.
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Figure 9- Fluorescence intensities obtained at 20 ° C for
SRB (red) and MSN-SRB (blue) and normalized
fluorescence intensities obtained for the SRB at 50º C
(green) SRB and MSN-SRB at 50° C (purple). The
fluorescence intensities were measured to λemi= 585 nm
and λexc = 565 nm.
Figure 10 - Representation of the slopes obtained
from the adjustments made to the variations of the
fluorescence intensity of SRB (gray) and MSN-SRB
(blue) at 20° C, and SRB (yellow) and MSN-SRB
(orange) at 50° C.
The representation of the slopes versus time
(Figure 10) shows the free SRB slopes at 20°C
are similar over time (due to the slow diffusion of
free SRB molecules in solution by the
membrane). The free SRB slopes at 50°C not
significantly changed (although, the slopes were
higher than slopes at 20°C, due to the increased
porosity of the membrane with the temperature).
Figure 11 - Schematic representation of the behavior
of MSN-POLY loaded with sulforhodamine B. The
polymer at 50º C (a) is collapsed closing the pores.
Decreasing the temperature to 20ºC (b), the SRB
molecules flow from the pores to the hydrated
expanded polymer shell, remaining there duo to
electrostatic interations. Upon increasing the
temperature to 50ºC (c), the polymer shell collapsed
squeezing the SRB molecules.
The proposed mechanism of release is
presented in Figure 11. Initially, at 50ºC, the
MSNs were fully loaded and the polymer chains
are collapsed closing the pores entrance (Figure
11 a). By decreasing the temperature to 20°C,
SRB molecules flow to the polymer shell and
remain there (Figure 11 b). Increased the
temperature to 50°C, the polymer chains
collapsed and SRB is released (Figure 11 c). By
decreasing the temperature to 20°C again, a
new cycle starts (Figure 11 d), with the system
behaving as a nano-pump for the drug model.
CONCLUSIONS
A novel drug delivery carrier based on a
mesoporous silica core and a stimui-responsive
polymer shell has been successfully prepared.
The MSNs have incorporated in their structure a
0
500
1000
1500
2000
2500
3000
0-2
0
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47
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68
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90
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0
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0-1
20
13
0-1
42
15
0-1
60
17
0-1
80
19
0-2
00
Slo
pe
Time (min)
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fluorescent PDI derivative for bioimaging, and
after template removal the pores are completely
available to accommodate molecules. The
characterization by TEM shows that the
nanoparticles are mesoporous, monodispersed
and they have a spheroid shape. DLS
measurements showed the termoresponsive
behavior of the polymer shell. The release of a
model molecule (SRB) from the MSN-POLY
could be controlled by adjusting the
temperature. This hybrid nanocontainer have
potential applications as a nano-pump drug
delivery system controlled by temperature.
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