129
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica Chapter 5
SYNTHESIS OF IRON OXIDE (Fe2O3) INCORPORATED
MAGNETIC MESOPOROUS SBA–15 SILICA
5.1.
Introduction
Magnetic materials have a range of applications in the field of biomedicine that include
their use as contrast agents in magnetic resonance imaging (MRI), as drug delivery
systems that can be targeted to desired locations by an applied magnetic field, as a
controlled delivery system where the release can be triggered by an external magnetic
field, in magnetic separations, as a hyperthermic agent for annihilating cancer cells and in
biosensors [1–7].
The suitability of a magnetic material for a specific biomedical
application depends upon a number of factors. These include the dimensions of the
particle, its shape and magnetization values that will enable the magnetic field to guide
the material to the desired location. However, once the magnetic field is removed, the
particles should not exhibit a tendency to form agglomerates as it may lead to formation
of blocks in the blood vessels, which may turn fatal. A low remanence and a low
coercivity value for the material will prevent agglomeration after removal of the applied
magnetic field [8]. Additional requirements for these magnetic particles to be used
in vivo are biocompatibility, surface functionalities that can be easily modified to impart
specific properties such as target specificity, desired residence time, enhanced cell
permeability and optical visibility [9–12]. In addition, the material should be inert and
should neither involve in undesirable reactions with the biological components nor elicit
an adverse biological response against it [13].
130
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica Thus far, different types of magnetic micro– and nanoparticles have been developed and
efforts are on to improve existing materials for biological compliance. In particular,
applications of porous magnetic materials have attracted great attention due to their
potential to become multi–functional [14]. Not only can these materials be surface
modified with suitable ligands to confer specificity, non–immunogenicity, enhance cell
permeation and optical visibility, the pores can serve to accommodate suitable guest
molecules [15] that can be used for therapeutic purposes. The introduction of magnetic
property to these porous materials can confer magnetic visibility to these structures
thereby enabling manipulation of its location, kinetics, visibility and release properties by
the magnetic field. Such types of multi–functional materials belong to a class, popularly
known as ‘theranostic materials’, which is a hybrid material capable of both diagnosis
and therapy. Mesoporous silica possesses tunable pores, functionality and morphology
and are likely candidates that can fulfill the requirements of a theranostic agent and hence
has become a hot topic for research in the present day.
Incorporation of magnetic properties in porous materials has been through introduction of
magnetic nanoparticles into the materials. Research for the development of porous
magnetic materials has primarily been centered on two approaches:
(i)
Post–synthesis or grafting technique which involves the introduction of magnetic
nanoparticles in a pre–formed porous matrix
(ii)
Pre–synthesis or co–precipitation technique which involves synthesis of the porous
template around the magnetic nanoparticles
A few attempts have also been developed to include a magnetic precursor into the porous
template either by post–synthesis or pre–synthesis method followed by the conversion of
131
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica the incorporated magnetic precursor in to the magnetic nanoparticle through chemical
reactions. The most commonly employed magnetic nanoparticle for modification has
been iron and its oxides though cobalt doped mesoporous silica materials have also been
reported widely in literature [16, 17]. But most biomedical applications that are being
explored currently prefer the iron–based porous materials probably due to their ease of
synthesis and controllable properties and relatively lesser toxicity. Many groups have
investigated the magnetic functionalization of mesoporous materials such as SBA–15,
MCM–41, and MCM–48 by the grafting technique [18–21]. These ordered mesoporous
silica materials have gained considerable attention for the following reasons:
(i)
Mesoporous materials possess a network of channels and voids of well–defined
size in nanoscale range (2–50 nm). This highly regular pore architecture makes
them suitable candidates for hosting a variety of molecules whose dimensions can
be tuned by modifying the pore dimensions
(ii)
They can easily be functionalized because of their high specific surface area and
abundant –Si–OH groups on the pore walls
(iii) Silica is generally considered non–toxic and bioinert and hence may be explored
for applications in biomedical field [22–23]
Hierarchical structures of metal oxides in the mesoporous silica framework have been
developed using various methods. The co–precipitation techniques have been used to
develop composite frameworks consisting of metal–oxygen–silicon links [24]. Some
groups have attempted to transform iron metal into iron oxide through chemical reactions
after entrapping them into the mesopores [25]. Garcia et al. reported a method based on
the sol–gel technique for fabricating ordered mesoporous aluminosilicate materials with
superparamagnetic γ–Fe2O3 particles embedded in the walls [26]. In this method, a sol
132
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica prepared by hydrolysis of (3–glycidoxypropyl) trimethoxysilane and aluminum
secondary butoxide (9:1 molar ratio) was added in a solution containing iron(III)
ethoxide and a structure–directing agent of a block copolymer. After stirring for a few
hours followed by evaporation of solvent and calcination, mesoporous aluminosilicate
materials with superparamagnetic γ–Fe2O3 particles embedded in the walls were
obtained. This throws a new light on a facile synthesis route for the preparation of
nanocomposites with nanocrystals in the silica walls.
Wang et al. [27] have suggested that the –OH groups on the matrix surface generated by
hydrolysis of a silica source, e.g., TEOS, were favorable for nucleation of iron oxide
particles. Yiu et al. have reported the synthesis of iron oxide–silica nanoparticles (γ–
Fe2O3–maghaemite and α–Fe2O3–haematite) with the iron oxide inside the mesochannels
of silica. Internalization of these particles inside mesenchymal stem cells and human
osteoblasts has also been reported [28]. Further, iron metal–silica and magnetic silica
(Fe3O4–SBA–15) have been synthesized by temperature programmed reduction [29].
The iron–to–silica ratio is a critical factor in determining the magnetization property of
the nanoparticles. Alam et al. have used mesoporous silica as template to synthesize
Fe2O3 nanoparticles with uniform and tunable size by a ‘nanosieve’ approach [30].
Yiu et al. have demonstrated that the iron oxide SBA–15 nanoparticles coated with
cationic polyethylene imine could be used for magnetofection of DNA into cells [31].
Vinu et al. have synthesized iron oxide nanoparticles that were confined in the
mesoporous silica framework of KIT–6, a type of mesoporous silica with three–
dimensional pores and large pore volumes [32]. Wang et al. have reported the synthesis
of a core–shell iron oxide silica mesoporous structure by post–synthesis for protein
133
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica separation [33]. A novel approach to incorporate iron oxide inside the mesopores of a
silica framework was reported by Hess et al. Here, an iron containing protein ferritin
was incorporated in to a mesoporous silica framework followed by calcinations during
which the organic template and the protein were eliminated leaving behind the iron as
iron oxide in the mesopores [34].
An interesting facet that emerges from these studies is that the magnetic property as well
as the morphology and pore architectures are highly dependent on the reaction precursors
and reaction conditions which in turn will have a major influence not only on the
magnetic properties of the nanoparticles confined in the mesopores but also on the
biological relevance of these materials. An important factor that also influences the
magnetic properties of the mesoporous samples is the metal to silica ratio. Li et al. had
investigated the influence of different ratios of iron to silica ranging between 0.1 and 0.3
on the textural properties of the mesoporous silica membrane developed using a porous
alumina template [35].
However, effects of high loads of iron on mesoporous
architecture have not been explored thoroughly yet. Another major concern in designing
strategies for incorporating magnetic properties to the mesoporous silica is in preserving
the highly ordered framework. Though numerous reports are available on the synthetic
strategies for structural and textural control of the mesoporous silica framework and
incorporation, not much attention has been focused on the compatibility of these
materials in vivo. This work aims to systematically investigate the effects of pH on the
structural and textural properties of magnetic mesoporous silica synthesized using the
pre–synthesis route and employing different ratios of iron to silica. The pH of the
reaction was also varied to understand the conditions that would promote high levels of
iron oxide incorporation in to the mesopores without compromising on the oriented pore
134
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica architecture of the silica framework. A comparison with a modified post–synthesis
method has also been done. The tissue compatibility of these iron doped mesoporous
silica materials, which have not been reported before, has been evaluated to understand
the response of the biological system to these magnetic mesoporous silica.
5.2.
Materials & Methods
5.2.1. Materials
Tetraethylorthosilicate (TEOS), sulphorhodamine–B and pluronics P–123 were
purchased from Sigma Chemicals, USA. Hydrochloric acid, methylene blue, methyl
orange and iron(III) chloride were purchased from Merck, India. All chemicals were of
GR grade and were used as such without any further purification.
Iron oxide
nanoparticles of dimensions 6–8 nm used in the post–synthesis method were a kind gift
from Prof. M. S. Ramachandra Rao, Indian Institute of Technology (Madras), Chennai.
5.2.2. Synthesis of iron oxide incorporated SW through grafting technique
In this method, mesoporous SBA–15 silica and iron oxide synthesized separately were
used to obtain the final product.
Initially, the mesoporous SBA–15 silica (SW)
synthesized as discussed in earlier chapters was dispersed in deionized water followed by
addition of nanostructured iron oxide particles with constant stirring. The final product
was filtered, dried and subjected to analysis. The various stages used for the synthesis
are presented as a flow sheet in Figure 5.1.
135
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica Pluronic P-123 + Solvent
Acidified with HCl to
obtain pH < 1
Stirred well for three hours
Addition of TEOS
o
Refluxed at 80 C for 24 hours
Filtered, washed & dried for 8 hours at
o
100 C in vacuum
Addition of Iron oxide
Stirred or sonicated
Physico-chemical characterization
Figure 5.1: Post–synthesis of iron oxide incorporated mesoporous SBA–15 silica
5.2.3. Synthesis of iron oxide incorporated SW through co–precipitation technique
Five grams of P–123 was added to 60 g of water and stirred at room temperature until a
clear solution was obtained. The pH in different trials was maintained below 1, 3 and 5
by addition of HCl. Calculated amount of iron(III) chloride dissolved in 5 mL of
deionized water and 9 g of TEOS were added as precursors. The silica to iron ratio was
varied (1:1, 1:0.5, 1:0.25 and 1:0.125).
The reaction mixture was stirred at room
temperature for 24 h and then transferred to a round bottom flask fitted with a condenser
and refluxed at 80°C for 24 h in an oil bath. The precipitate obtained was cooled,
filtered, washed several times with deionized water, dried and calcined in a muffle
furnace for 6 h at 550°C. Figure 5.2 depicts the various stages involved in the synthesis
process.
136
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica Pluronic P-123 + Solvent
Acidified with HCl to
obtain pH < 1
Stirred well for three hours
Addition of FeCl3 &
TEOS
o
Refluxed at 80 C for 24 hours
Filtered, washed & dried for 8 hours at
o
100 C in vacuum
Physico-chemical characterization
Figure 5.2: Various stages in the pre–synthesis or co–precipitation method for synthesis
of iron oxide incorporated mesoporous SBA–15 silica
5.2.4. Characterization of mesoporous samples
Fourier transform infrared spectroscopy (FT–IR) (Spectrum 100, Perkin Elmer, USA)
was used to confirm the complete removable of surfactant in the synthesized silica
samples. FT–IR analysis was performed between 4000 and 450 cm–1 at a resolution of
4 cm–1 averaging 30 scans for all samples. The moisture–free samples were pelletized
with KBr (IR grade, Merck, Germany) and subjected to analysis.
X–ray diffraction (D8 Focus, Bruker, Germany) was used to characterize the mesoporous
silica. The samples were finely ground using an agate mortar and pestle and the fine
powder was mounted on a polymer slide. The analysis was performed from an angle of
0.5° to 10° (2θ) in steps of 0.001° and the wide angle was done from 10° to 60° in steps
137
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica of 0.001° at the scan rate of 1 second per step. CuKα radiations were used to irradiate the
samples for analysis.
The morphology of the synthesized mesoporous silica was studied using a cold field
emission scanning electron microscope (FE-SEM) (JSM 6701F, JEOL, Japan). The
samples were placed on a conducting alloy stub using carbon paste, sputter coated with a
thin layer of platinum and were imaged using SEM.
The transmission electron
microscope (FE-TEM) images were obtained using a high–resolution transmission
electron microscope (JEM 2100F, JEOL, Japan).
The samples were prepared by
dispersing in ethanol and deposition on a copper grid before imaging. Energy dispersive
X–ray analysis (Oxford Instruments, UK) was carried out to confirm the presence of iron
in the IO–SW. The characteristic peaks were collected for 220 counts at an applied
voltage of 20 kV and a working distance of 15 mm. X–ray photoelectron spectroscopy
(Multilab 2000, Thermo Scientific, UK) was carried out to confirm the oxidation state of
iron in the iron oxide doped SBA–15.
Surface area analysis of mesoporous silica
samples was carried out by BET method (Quantachrome, USA). The outgas temperature
was 300°C and the outgas time was 3 hours and the samples were degassed for 14 hours
prior to analysis.
The magnetic measurements for the IO–SW samples were carried out between the
temperature range 1.8 K–300 K using a superconducting quantum interference device
vibrating sample magnetometer (SVSM, Quantum Design, USA).
138
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica 5.2.5. Animal model and implantation
The tissue response of the biological system against the iron oxide incorporated
mesoporous SBA–15 silica (IO–SW) was carried out using rat models. For implantation,
a pellet of 10 mm x 10 mm x 2 mm was made from the iron oxide incorporated
mesoporous silica. The pellet was sterilized by autoclaving at a temperature of 120°C for
15 minutes and similar pellets were made from mesoporous SBA–15 silica (SW) and
starch for comparison.
Twenty seven Wistar rats, Rattus Norvegicus, each weighing about 200–250 g were used
for animal experiments. The animals were randomly divided into three groups of nine
rats each. All rats were kept in an individual cage and were housed in a temperature–
controlled facility. All the surgical procedures were approved by the Animal Ethics
Committee of SASTRA University (61/SASTRA/IAEC/RPP 02/07/2009).
Every group (9 rats) was assigned randomly to three different time points (2, 4 & 8
weeks) with three rats for each time point. An intra–peritoneal injection of ketamine (30
mg/kg body weight) and xylazine (13 mg/kg body weight) were administered to
anesthetize the animals. The dorsal area of the animals were shaved and sterilized with
70% ethanol solution (Figure 5.3A).
Using a sterile surgical blade No. 22
(Magna Marketing, India), an incision of about 12 mm was made on the dorsum of
animal (Figure 5.3B). A subcutaneous pouch was created on both sides of the incision
and an implant was inserted into each pocket (Figure 5.3C). Upon implantation of the
material into the pouch, the cut was sutured using a non–absorbable surgical black
braided silk thread (Relyonpac, India) (Figure 5.3 D–F). Animals in Groups I, II and III
139
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica were implanted with starch, SW and IO–SW respectively. The sutures were removed
seven days post surgery.
A
B
C
D
E
F
Figure 5.3: Subcutaneous implantation of SW, IO–SW & starch in rat model
5.2.6. Histology studies
At the end of each time point (2, 4 & 8 weeks), rats were euthanized using an overdose of
pentobarbital (75 mg/kg) followed by carbon dioxide asphyxiation. The implant and the
140
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica tissues surrounding the implant were excised. The tissues surrounding the implant were
fixed in 10% formalin solution for seven days. Before embedding in paraffin wax, the
tissue samples were dehydrated in an Automated Tissue Processor (Yorco YSI103LT,
Yorco Scientific, India) by transferring through a series of graded concentrations of
alcohol. The tissue samples were embedded in paraffin using an embedding machine
(EG1150 H&C, Leica Microsystems, Germany) and were sectioned with a microtome
(Rotary Microtome Leica RL2125RT, Leica Microsytems, Germany) stained with
hematoxylin and eosin. The stained samples were viewed under a light microscope to
observe the inflammatory responses on the region of the implant.
The inflammatory response to the implanted materials was determined based on the
averaged number of cell types (neutrophils, lymphocytes, macrophages, and giant cells)
present in the tissue surrounding implant. The average number of inflammatory cells
was determined by counting in a 40X magnification from at least ten different fields by
an independent pathologist.
Tissue responses were evaluated as minimal, mild or
moderate using an evaluation system suggested in literature and shown in Table 5.1 [36]
Table 5.1: Grading of tissue response to the implant
Grade
Minimal
Mild
Moderate
Description
0 – 10 cells (lymphocytes / plasma cells / neutrophils / eosinophils / giant
cells) per field at 40X magnification
10 – 20 cells (lymphocytes / plasma cells / neutrophils / eosinophils / giant
cells) per field at 40X magnification
More than 20 cells (lymphocytes / plasma cells / neutrophils / eosinophils /
giant cells) per field at 40X magnification
141
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica 5.3.
Results & Discussion
5.3.1. Post–synthesis or Grafting technique
The scanning electron micrographs of iron oxide grafted mesoporous silica (IOG–SW)
are shown in Figure 5.3. These images show that the surface of the mesoporous silica is
decorated with the iron oxide nanoparticles. When the grafting process was carried
under sonication instead of stirring, an decrease in the iron oxide nanostructures on the
surface were observed (Figures 5.4 A & B) when compared to those obtained by
sonication (Figures 5.4 C & D).
A
B
C
D
Figure 5.4: FE–SEM images of iron oxide doped mesoporous SW silica (IOG–SW)
through grafting technique
142
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica Figure 5.5 shows the x–ray diffraction patterns obtained for the various samples. The
iron oxide nanoparticles show intense signals at 2θ values 27° and 36° indicating the
presence of Fe3O4 form of iron oxide [JCPDS 85–1436]. The same peaks are observed in
the mesoporous samples IOG–SW prepared by stirring and sonication thereby
confirming the presence of iron oxide in them.
d
Intensity in counts
c
b
a
20
30
40
50
60
2 theta
Figure 5.5: Powder x–ray diffraction pattern of SW (a), iron oxide (b) and IOG–SW
samples prepared under different conditions (c: stirred and d: sonicated) through the
post–synthesis or grafting technique
The post–synthesis or grafting technique primarily promotes the interaction between the
surfaces of mesoporous silica and the iron oxide nanoparticle. Therefore, in the resulting
mesoporous product, the iron oxide nanoparticles mainly occupy the surface of the silica
143
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica framework and a few get localized in the mesopore channels. As the iron oxide is
retained in the mesoporous silica through physical interactions it can be easily separated
during processing or application of the magnetic field (Figure 5.6). Hence, an alternate
method needs to be designed to overcome these limitations.
The co–precipitation
technique could provide a solution to this issue by enabling the entrapment and retention
of the iron oxide nanoparticles in the mesopores.
Figure 5.6: Schematic representation of the removal of surface bound iron oxide due to
weak physical interactions on application of magnetic field
5.3.2. Pre–synthesis or Co–precipitation technique
Figure 5.7, presents the scanning electron micrographs, showing the surface morphology
of iron oxide incorporated mesoporous silica (IO–SW) samples synthesized with
different silica to iron ratios (1:1, 1:0.5, 1:0.25 & 1:0.125) and pH (<1, 3, 5 & 7). An
interesting observation from the scanning electron micrographs is that the characteristic
rod–like morphology of SBA–15 gets heavily distorted as the pH is increased from acidic
to neutral (Figure 5.7). This may be attributed to the fact that a decrease in acidity would
lead to retardation in the reaction rates thereby promoting formation of structures with
greater curvature leading to deviations from the characteristic morphology of SBA–15.
The isoelectric point of silica lies around pH 2. Hence, pH values below its isoelectric
point promote formation of the cationic silicate species that tend to interact effectively
144
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica with the surfactant. However, even at pH below 1, the typical highly elongated rod–like
morphology of SBA–15 is retarded and shorter rods are observed at pH <1 as the silica to
iron ratio changes from 1:0.125 to 1:1. This distortion in morphology can be attributed to
the presence of the iron chloride salt that tends to interfere with the micelle aggregation.
It is seen that as the percentage of iron decreases in the IO–SW, the deviation from the
typical SBA–15 morphology is reduced. At higher pH, both low acidity and presence of
iron chloride tend to destruct the morphology of the mesoporous silica.
pH Si/Fe ratio
1:1
<1
1:0.5
1:0.25
1:0.125
3
5
7
Figure 5.7: Scanning electron micrographs of IO–SW synthesized under various pH and
silica to iron ratios
145
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica The high–resolution transmission electron microscopic images (HR–TEM) for the
IO–SW synthesized under various conditions are shown in Figure 5.8.
A C B D Figure 5.8: TEM images of IO–SW synthesized with 1:1 silica to iron ratio at pH <1 (A),
3 pH (B), 5 pH (C) & 7 pH (D)
The transmission electron micrographs for the samples synthesized with silicon to iron
ratio of 1:1 at various pH shows a marked change in the pore morphology as one moves
from acidic to neutral pH. While the ordered porous framework is observed in the
sample synthesized at pH lesser than 1, the other samples show the absence of oriented
pores. The presence of iron oxide is observed inside the pores of mesoporous silica for
IO–SW synthesized with 1:1 silica to iron ratio at pH <1 while in the other samples, the
146
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica iron oxide seems to be randomly localized. Thus it is evident that the synthesis strategy
at pH < 1 with silica to iron ratio of 1:1 offers high amounts of iron oxide incorporation
without compromising on the textural properties and oriented mesoporous framework.
The FT–IR spectra of the IO–SW with silica to iron ratio of 1:1 synthesized at various
pH as–synthesized are shown in Figure 5.9.
D
%T
C
B
A
4000
3500
3000
2500
2000
1500
1000
500
-1
Wavenumber (cm )
Figure 5.9: FT–IR of IO–SW synthesized at various pH A) <1, B) 3, C) 5 & D) 7
The absorption bands between 1090 and 800 cm–1 show the presence of –Si–O–Si– and
–Si–O– of –Si–OH groups in all the samples confirming the formation of silica. The band
at 590 cm–1 appears only in the iron oxide incorporated samples, which may be attributed
to the Fe–O bond. This band is absent in pristine mesoporous silica indicating the
absence of iron oxide in the samples. Figure 5.9 shows the FT–IR spectra of IO–SW
147
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica synthesized at pH < 1 with various silica to iron ratios of 1:1, 1:0.5, 1:0.25 and 1:0.125.
The characteristic bands for –Si–O–Si– and –Si–OH appear at 1100 cm–1 and 950 cm–1 in
all the samples. The Fe–O band appears at 550 cm–1 in all ratios indicating the presence
of iron oxide in the mesoporous silica.
The x–ray diffraction patterns for the pristine mesoporous silica (SW) and IO–SW
synthesized at a pH below 1 with different silica to iron ratios 1:1, 1:0.5, 1:0.25 and
1:0.125 are shown in Figure 5.10.
e
Intensity in counts
d
c
b
a
20
30
40
50
60
2 theta
Figure 5.10: X–ray diffraction patterns of SW (a) and IO–SW synthesized at pH below 1
with different silica/iron ratios. b) 1:1, c) 1:0.5, d) 1.0.25 & e) 1:0.125
148
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica The peaks at 27° and 36° observed in all the IO–SW samples confirm the presence of
iron oxide in the samples.
The low intensity peaks are due to the presence of
nanostructured iron oxide in the mesoporous samples. The pristine mesoporous SBA–15
silica does not show any peaks in this region indicating the absence of iron oxide.
Figure 5.11 shows the x–ray photoelectron spectra (XPS) of the IO–SW synthesized at
pH <1 using silica to iron ratio of 1:1. The binding energy of 711 eV shows that the iron
exists in the trivalent state in the sample thus confirming the conclusion arrived at from
the x–ray diffraction patterns that the mesoporous silica contains iron in the form of
Fe2O3.
70000
Counts/s
65000
60000
55000
50000
700
702
704
706
708
710
712
714
716
718
720
Binding energy (eV)
Figure 5.11: X–ray photoelectron spectra of IO–SW synthesized at pH <1 with 1:1 silica
to iron ratio
149
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica Table 5.2 summarizes the surface properties derived from the nitrogen adsorption–
desorption isotherms of the various IO–SW samples synthesized at different pH and
silica to iron ratios.
Table 5.2: Surface properties of various IO–SW samples
Name of the
BET Surface Micropore
Pore
Pore
Material
area
area
Volume diameter
2
2
3
(nm)
pH
Ratio
(m /g)
(m /g)
(cm /g)
604
118
0.7657
7.3
<1
1:1
597
98
0.6782
7.1
<1
1:0.5
595
104
0.5979
5.3
<1
1:0.25
598
126
0.5635
5.1
<1
1:0.125
097
7
0.3163
14.1
3
1:1
173
8
0.9373
27.4
3
1:0.5
198
24
0.4557
9.8
3
1:0.25
301
40
0.3447
5.2
3
1:0.125
133
5
0.1201
4.2
5
1:1
176
20
1.2203
31.6
5
1:0.5
261
8
0.2474
4.1
5
1:0.25
508
65
0.9607
8.4
5
1:0.125
167
17
1.2109
33.5
7
1:1
234
29
1.0991
21.9
7
1:0.5
340
40
1.1097
14.9
7
1:0.25
349
33
0.9128
12.0
7
1:0.125
It is interesting to note that the surface area does not vary much with different
percentages of iron loading for the iron oxide incorporated mesoporous samples
synthesized at pH <1. However, in all the other pH conditions (3, 5 and 7), the surface
area was found to decrease dramatically as the content of iron in the sample increased.
Also, the surface area changes were significantly reduced when compared to
conventional mesoporous SBA–15 silica. This may be attributed to the disruptions in the
mesoporous architecture brought about by a combination of the iron precursor as well as
the low acidity. The surface area range of 598–604 m2/g and pore diameter range of
150
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica 5.1–7.3 nm obtained for the samples synthesized at pH < 1 is closely related to the
reported values for conventional SBA–15. This observation is in agreement with the
conclusions drawn from the transmission electron micrographs where non-interruption of
mesoporous frame work in the samples synthesized below pH 1 was observed.
5.3.2.1.
Magnetic properties of IO–SW
The vibrating sample magnetometer (VSM) data obtained at room temperature (300 K)
and low temperature (1.3 K) of IO–SW synthesized at pH <1 with 1:1 ratio of Si/Fe are
shown in figure 5.12.
8
6
1.8 K
300 K
4
0
-2
M (emu/g)
M (emu/g)
2
-4
-6
H (Oe)
-8
-80000
-60000
-40000
-20000
0
20000
40000
60000
80000
H (Oe)
Figure 5.12: VSM of IO–SW synthesized with 1:1 silica to iron ratio at pH <1
151
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica The magnetization curve exhibits zero magnetic memory and zero coercivity indicating a
superparamagnetic behavior for the IO–SW sample. This may be attributed to the small
nanodimensional iron oxide particles distributed in the mesoporous channels.
The
saturation magnetization for the IO–SW sample is 5.5 emu g–1 at 300 K and 5.2 emu g–1
at 1.8 K indicating its potential therapeutic and diagnostic relevance.
5.4.
Tissue compatibility studies
The histopathology slides of the tissue surrounding the implant from different groups at
various time points are shown in Figure 5.13.
Figure 5.13: Light microscopy of subcutaneous nodules of SW (A, B & C) & IO–SW (D,
E & F) at 2 weeks (A & D), 4 weeks (B & E) and 8 weeks (C & F), (L – Lymphocytes,
F– Fibroblasts, P– Plasma cells, IM – Iron laden macrophages, GC– Giant cells). All
images are 40× the original magnification
152
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica Careful observation of the tissue surrounding the implants shows a thin fibrous
encapsulation around the mesoporous silica (SW) and IO–SW implants. While the
structural integrity of SW was maintained even after 8 weeks, the IO–SW had
disintegrated inside the fibrous capsule. The starch control showed mild inflammatory
response after 2 weeks, which then became minimal after 4 and 8 weeks. During the
entire duration of study the tissue surrounding the starch implant had very few
lymphocytes and fibroblasts. In the case of the mesoporous SBA–15 silica (SW), the
tissue in close contact with the implants was characterized by an abundance of
lymphocytes, plasma cells and very few fibroblasts and neutrophils indicating a moderate
tissue response. At the end of even four weeks, the tissue still exhibited a moderate
response which reduced to a mild response after 8 weeks as characterized by a reduction
in the number of lymphocytes. In the case of IO–SW, the tissue in contact with the
implant was characterized by lymphocytes, a few macrophages and fibroblasts eliciting a
moderate inflammatory response. This reduced to a mild response after 4 weeks and
remained mild at the end of 8 weeks post implantation. At the end of 8 weeks, however,
a few iron–laden macrophages were visible in the tissue in the immediate vicinity of the
implant. Table 5.3 summarizes the tissue responses of the three materials over the period
of study.
Table 5.3: Tissue response to SW and IO–SW implants at different time points
Time points
Material
2 weeks
4 weeks
8 weeks
Mild
Minimal
Minimal
Starch
Moderate Moderate
Mild
SW
Mild
Mild
IO–SW Moderate
153
Synthesis of Iron Oxide (Fe2O3) Incorporated Magnetic Mesoporous SBA–15 Silica Based on the inflammatory responses, a material can be classified as a level 1 material if
it elicits a mild inflammatory response in the period of study. A level 2 material elicits
mild to moderate inflammatory response that reduces with time while a level 3 material
causes moderate to severe inflammatory response. Finally a level 4 material is one that
causes severe inflammatory response that does not diminish with time [62]. The in vivo
tissue compatibility studies that were carried out for the SW and IO–SW show that both
these materials can be categorized as level 2 materials and hence can be used for in vivo
applications.
5.5.
Conclusions
This work has successfully incorporated iron oxide in the mesoporous framework
without much compromising the textural properties of the mesoporous silica using a
co–precipitation technique employing ferric chloride as the iron precursor. The silica to
iron ratio of 1:1 was found to have suitable magnetic property that can be explored for
biomedical applications. The in vivo tissue compatibility of these magnetic materials
reported here for the first time show that these materials are well tolerated by the
biological system and the moderate inflammatory response elicited in the first couple of
weeks diminishes with time thus making it a level 2 biomaterial that can be safely used
for short term applications. However, the appearance of iron laden macrophages after
eight weeks indicates potential hemosiderosis–like conditions that need to be further
investigated to understand the long term safety of these materials.
154
5.6.
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