TOXICOLOGICAL SCIENCES 122(1), 86–99 (2011)
doi:10.1093/toxsci/kfr078
Advance Access publication April 5, 2011
In Vitro Study and Biocompatibility of Calcined Mesoporous Silica
Microparticles in Mouse Lung
Suhail Al-Salam,* Ghazala Balhaj,† Suleiman Al-Hammadi,† Manjusha Sudhadevi,* Saeed Tariq,‡ Ankush V. Biradar,§
Tewodros Asefa,§,{,1 and Abdul-Kader Souid†,1
*Department of Pathology, †Department of Pediatrics, and ‡Department of Anatomy, Faculty of Medicine and Health Sciences, United Arab Emirates
University, Al Ain, Abu Dhabi 17666, UAE §Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, New
Jersey 08854, {Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854
1
To whom correspondence should be addressed at Department of Pediatrics, Faculty of Medicine and Health Sciences, United Arab Emirates, Al Ain, Abu Dhabi
17666, UAE. Fax +971-3-973422. E-mail: [email protected]. Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610
Taylor Road, Piscataway, NJ 08854. Fax: (732) 445-5312. E-mail: [email protected].
Received December 29, 2010; accepted March 29, 2011
We report on the pneumatocyte structure and function of mouse
lung specimens exposed in vitro to two calcined mesoporous silica
particles, MCM41-cal (spheres, ~300 to 1000 nm in diameter) and
SBA15-cal (irregular rods averaging ~500 nm in diameter and ~1000
nm in length). These mesoporous silica particles are in consideration
for potential medical application as delivery vehicles for genes, drugs,
and bio-imagers. In the study, lung specimens (about 10 mg each)
were excised from male Balb/c mice, immediately immersed in
Krebs-Henseleit buffer, ice-cold, and continuously gassed with
O2:CO2 (95:5). The samples were incubated at 37C in the same
buffer with and without 200 lg/mL MCM41-cal or SBA15-cal for 5–
14 h. The tissues were then rinsed thoroughly and processed for light
and electron microscopy. Normal alveolar morphology was evident in
all the studied specimens. There was no significant difference in the
number of apoptotic cells between the treated and untreated samples.
Despite their relatively large sizes, the particles were abundantly
present in pneumocytes, macrophages, endothelial cells, fibroblasts,
and interstitium. They were seen in different areas of the cytoplasm,
suggesting intracellular movements. Their presence did not appear to
disturb cellular configuration or micro-organelles. Due to their
rigidity and surface charges, some were firmly attached to (indenting)
the nuclear membrane. The rate of respiration (cellular mitochondrial O2 consumption, in lM O2/min/mg) in specimens exposed to 200
lg/mL particles for up to 12 h was the same as untreated specimens.
These findings confirm ‘‘reasonable’’ bioavailability and biocompatibility of calcined mesoporous silicas with mouse lung within at least
5–14 h of exposure time.
Key Words: lung; silica; mesoporous silica; in vitro;
nanomaterials; nanotoxicology.
Silica micro- and nanoparticles are emerging as potential
delivery vehicles for various drugs, genes, small interfering
ribonucleic acids (siRNAs), and nitric oxide for medical
applications (Chen et al., 2009; Das et al., 2010; Hom et al.,
2010; Qhobosheane et al., 2001; Rosenholm et al., 2009;
Stevens et al., 2010; Tao et al., 2010a; Vallet-Regı́ et al., 2007;
Zhou et al., 2010). In light of their impending implementation
in human therapeutics, thorough investigation of their biocompatibility is a burgeoning area of research with much
unmet need (Hudson et al., 2008; Kipen and Laskin, 2005; Lin
et al., 2006; Nel et al., 2006; Service, 2005).
Among many forms of silicas, MCM-41 and SBA-15
mesoporous silicas (Kresge et al., 1992; Zhao et al., 1998)
are by far the two most commonly studied and widely
considered silica-based materials for drug, siRNA, and gene
delivery applications (Hom et al., 2010; Rosenholm et al.,
2009; Slowing et al., 2007; Vallet-Regı́ et al., 2007; Xia et al.,
2009). Their synthesis involves the supramolecular selfassembly of alkoxysilanes around highly hierarchically ordered
aggregates of surfactant or block copolymer cylinderical
micellar templates. After the hydrolysis and condensation of
the alkoxysilanes, the templates are removed either by
calcination at higher temperature or solvent washing (extraction) at room temperature. This produces mesoporous silica
particles that are filled with nanometer size pores and contain
high surface areas. Calcination, which is typically performed
for 2–4 h at > 350C, produces mesoporous particles that are
free of residual organic materials such as surfactant templates
and alkyl groups from the alkoxysilanes. On the other hand,
surfactant removal by solvent extraction leaves residual
surfactants and organic groups in the materials. In our recent
study, we showed that the calcination process, which removes
residual organic groups, has also a profound effect on the
materials’ biocompatibility with murine tissues (Al Shamsi
et al., 2010). Specifically, the calcined materials do not inhibit
cellular bioenergetics of murine tissues, whereas their solventextracted counterparts inhibit pneumatocyte respiration. Besides removing residual organic groups, the calcination process
reduces surface silanol groups on the mesoporous silica
particles, especially when compared with solvent extraction.
These differences in surface silanols and residual surfactants
The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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CALCINED MESOPOROUS SILICA MICROPARTICLES IN MOUSE LUNG
between the calcined and solvent-extracted mesoporous
materials were suggested to be among the reasons for different
cellular bioenergetics that the two materials exhibited on
murine tissues (Al Shamsi et al., 2010). The effect of surface
silanols on biological activity of silica is actually not new.
Silanols have long been cited as culprits in the cytotoxicity of
some other forms of silica materials (Konecny et al., 2001;
Murashov et al., 2006).
In addition to cellular bioenergetics, differences in uptake,
distribution, and other adverse effects (biocompatibility) are
reported for different forms of silica or mesoporous silicas
prepared by various methods (Blumen et al., 2007; Hom et al.,
2010; Hudson et al., 2008; Lin et al., 2006). These differences
may stem from differences in composition, size, structure,
surface areas, surface charges, residual reagents and templates,
and route of administration of the mesoporous materials as well
as the type of cells or tissues studied. For instance, a recent
in vivo study suggested efficient uptake and biocompatibility of
mesoporous silica spheres in murine pneumatocytes and
mesothelioma cells (Blumen et al., 2007). By contrast, Hudson
et al. (2008) described toxicities of mesoporous MCM-41
and SBA-15 in mesothelial cells, e.g., ip and iv injections of
either particle produced pulmonary thromboembolic events
(Hudson et al., 2008). In another study, 15 and 46-nm silicas
(at > 10 lg/mL for 42–72 h) reduced the viability of cultured
bronchoalveolar carcinoma-derived cells (Lin et al., 2006).
Other noted differences in toxicities of silicas were due to
differences in the production of reactive oxygen species
(measured as depletion of cellular glutathione), lipid peroxidation, and membrane damage (Konecny et al., 2001; Lin
et al., 2006; Murashov et al., 2006). The degree of surfaceadsorptive properties of silica particles can also contribute to
their toxicity. For instance, surface-coated (e.g., pegylated or
polyethyleneimine-modified) crystalline silicas (Min-U-Sil 5)
were less harmful compared with their uncoated counterparts
(Beyerle et al., 2010; Galagudza et al., 2010; Radu et al., 2004;
Rosenholm et al., 2009; Xia et al., 2009).
The aforementioned reports as well as others reveal some
contradictory results on the biocompatibility or cytotoxicity of
mesoporous silicas. Furthermore, despite many previous
studies, our knowledge of the biological effects of mesoporous
silicas in various tissues does not match the rapid pace of
research on the development of mesoporous materials with
numerous forms and structures. In particular, the impacts of
composition, method of synthesis, residual materials, surface
charges, sizes, and shapes of mesoporous silica particles on the
particles’ biological activities in different types of cells and
tissues remain to be unexplored. We previously reported on the
effects of solvent-extracted mesoporous silica particles (MCM41 and SBA-15) in myeloid and lymphoid cell lines (Tao et al.,
2008) and, more recently, calcined and solvent-extracted
mesoporous silicas in various murine tissues (Al Shamsi
et al., 2010). More thorough investigation of the biological
effects of mesoporous silica materials also requires studying
87
cellular morphology (by light microscopy), ultrastructure, and
micro-organelles (by transmission electron microscopy [TEM])
and function (e.g., mitochondrial oxygen consumption).
Here we assessed and reported on pneumatocyte structure
and function of mouse lung specimens exposed in vitro to
mesoporous particles, specifically calcined mesoporous silicas,
labeled as MCM41-cal (spheres, 300–1000 nm in diameter) and
SBA15-cal (irregular rods, averaging 500 nm in diameter and
1000 nm in length). The aim of the work was also to explore
the bioavailability and biocompatibility of calcined mesoporous
silicas with the lung. Our in vitro animal model further aimed to
address the potential importance and conflicting reports on
biocompatibility of mesoporous silica materials. The results
showed that the interactions of MCM41-cal and SBA15-cal
with cellular components mouse lung tissues were benign,
within at least 5–14 h of exposure time. Because the lung
tissues was not a cultured pneumatocyte, longer in vitro
exposure experiment (e.g., for days) that can be normally done
with cultured pneumatocytes could not be done. Normal
alveolar morphology and no significant difference in the
number of apoptotic cells between MCM41-cal– and SBA15cal–treated and untreated samples were observed. Despite their
relatively large sizes, the particles were observed to be
abundantly present in pneumocytes, macrophages, endothelial
cells, fibroblasts and interstitium, and different areas of the
cytoplasm, suggesting intracellular movements. Furthermore,
their presence did not appear to disturb cellular configuration or
micro-organelles and the rate of respiration (cellular mitochondrial O2 consumption, in lM O2/min/mg) of the specimens
exposed to 200 lg/mL particles for up to 12 h. These findings
further confirmed reasonable bioavailability and biocompatibility of calcined mesoporous silicas, MCM41-cal or SBA15cal, in vitro in lung specimens within at least 5–14 h of
exposure time.
MATERIALS AND METHODS
Chemicals and reagents. Pd (II) complex of meso-tetra-(4-sulfonatophenyl)tetrabenzoporphyrin sodium salt (Pd phosphor) was purchased from Porphyrin
Products (Logan, UT). The Pd phosphor solution (2.5 mg/mL ¼ 2.0mM) was
prepared in distilled water and stored at 20C in small aliquots. Poly(ethylene
oxide)-block-poly(butylene oxide)-block-poly(ethylene oxide) (Pluronics 123)
[(PEO)20(PPO)70(PEO)20] was purchased from BASF. Cetyltrimethylammonium
bromide (CTAB), tetraethyl orthosilicate (TEOS), and the remaining reagents were
purchased from Sigma-Aldrich (St Louis, MO).
Synthesis of MCM-41. As-synthesized MCM-41 was prepared as described previously (Tao et al., 2008). Briefly, 11.0 mmol of CTAB was
dissolved in 960 mL of Millipore water and 14 mL, 2.0M NaOH solution. After
stirring the solution at 80C for 30 min, 101.2 mmol of TEOS was added. The
resulting solution was stirred for 2 h at 80C prior to extraction of the solid
product by filtration, followed by washing with Millipore water (4 3 80 mL) and
ethanol (4 3 80 mL). Consequently, the product (as-synthesized mesostructured MCM-41) was dried in oven at 80C. The CTAB surfactant template from
the as-synthesized MCM-41 was removed by calcination. Two grams of the assynthesized MCM-41 sample were kept in a tube furnace at 550C for 6 h in an
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AL-SALAM ET AL.
airflow (temperature raised to 550C at a ramp of 10C/min). This produced
calcined MCM-41 (labeled as MCM41-cal). The sample was stored at 25C in
a Dry-Seal vacuum desiccator until use.
Synthesis of SBA-15. As-synthesized SBA-15 was prepared as reported
previously with minor modifications (Tao et al., 2008). A solution containing
12 g of Pluronics-123 [(PEO)20(PPO)70(PEO)20], 313 g Millipore water, and 72 g
HCl (36 wt%) was prepared and stirred vigorously at 40–45C until the Pluronics
template dissolved. TEOS (25.6 g) was added, and stirring of the solution was
continued at 45C for 24 h. The solution was kept in an oven at 80C without
stirring for 24 h; solid particles were then separated by filtration on filter paper.
The particles were washed with Millipore water and ethanol (3 3 80 mL) and
dried at ambient conditions to produce as-synthesized SBA-15 particles. The
Pluronics template from the as-synthesized SBA-15 particles was removed by
calcination as above, giving calcined SBA-15 (labeled as SBA15-cal). The
particles were stored at 25C in a Dry-Seal vacuum desiccator until use.
Tissue processing. All procedures were in compliance with the animal
care guidelines (United Arab Emirates University, Faculty of Medicine and
Health Sciences, UAE). Lung specimens (about 10 mg each) were excised from
anesthetized (10 mL/1.0 g weight of 25% urethane ip) adult male BALB/c
mouse using special scissors (Moria Vannas Wolg Spring, cat. # ST15024-10).
The specimens were immediately chilled in ice-cold Krebs-Henseleit solution
(115mM NaCl, 25mM NaHCO3, 1.23mM NaH2PO4, 1.2mM Na2SO4, 5.9mM
KCl, 1.25mM CaCl2, 1.18mM MgCl2, and 6mM glucose, pH 7.4) gassed with
O2:CO2 (95:5). The samples were then rinsed thoroughly with the same buffer
and transferred to a 250-mL beaker containing 100 mL Krebs-Henseleit buffer
with and without added mesoporous silica particles.
The calcined mesoporous silica particles (200 lg/mL) were added to 100 ml
Krebs-Henseleit buffer and sonicated for 20 s (Branson 2210 Ultrasonics, Branson,
MO) to disperse possible aggregates of the particles. Specimens were incubated in
a water bath at 37C without agitation, with continuous gassing with O2:CO2
(95:5). Samples were then collected 5 h after the incubation, rinsed ‘‘thoroughly’’
with the same buffer, and processed for light and electron microscopy.
For light microscopy analysis, tissues were fixed in 10% neutral formalin,
dehydrated in increasing concentrations of ethanol, cleared with xylene, and
embedded in paraffin. Three-lm sections were prepared from paraffin blocks and
stained with hematoxylin and eosin. Staining for apoptosis was performed using
avidin-biotin immunoperoxidase method that detects activated caspase 3 (Cell
Signaling Technology, Boston, MA). The procedure was performed on 5-lm
paraffin sections using rabbit anti-cleaved caspase 3 antibodies; positive control was
human breast cancer. Positive and negative control sections for apoptosis were used.
For electron microscopy, samples were immersed in McDowell and Trump
fixative for 3 h at 25C. Tissues were rinsed with phosphate buffer saline and fixed
with 1% osmium tetroxide for 1 h. Samples were washed with dH2O, dehydrated
in graded ethanol and propylene oxide, infiltrated, embedded in Agar-100 epoxy
resin, and polymerized at 65C for 24 h. Blocks were trimmed and semithin and
ultrathin sections were cut with Reichert Ultracuts, ultramicrotome. The semithin
sections (1 lm) were stained with 1% aqueous toluidine blue on glass slides. The
ultrathin sections (95 nm) on 200 mesh Cu grids were contrasted with uranyl
acetate followed by lead citrate double stain. The grids were examined and
photographed under a Philips CM10 transmission electron microscope.
Oxygen measurement. The phosphorescence oxygen analyzer was used to
measure cellular respiration as described previously (Al Samri et al., 2011; Al
Shamsi et al., 2010, Lo et al., 1996; Shaban et al., 2010; Souid et al., 2003). Pd
phosphor had an absorption maximum at 625 nm and phosphorescence
emission maximum at 800 nm. Specimens were placed in 1-mL vials that were
sealed from air with crimp top aluminum seals. The mixing of the solution was
achieved with the aid of a parylene-coated stirring bar (1.67 3 2.01 3 4.80
mm; V&P Scientific, Inc., San Diego, CA).
Samples were exposed to light flashes (10/s) from a pulsed light-emitting
diode array with peak output at 625 nm (OTL630A-5-10-66-E, Opto
Technology, Inc, Wheeling, IL). The emitted phosphorescent light was
detected by a Hamamatsu photomultiplier tube (#928) after passing the light
through a wide-band interference filter centered at 800 nm. The amplified
phosphorescence decay was digitized at 1.0 MHz by a 20-MHz A/D converter
(Computer Boards, Inc., Mansfield, MA).
The analytic program used Microsoft Visual Basic 6, Microsoft Access
Database 2007, and Universal Library components (Universal Library for
Measurements Computing Devices, http://www.mccdaq.com/daq-software/universal-library.aspx). The program allowed a direct reading from the PCI-DAS
4020/12 I/O Board (PCI-DAS 4020/12 I/O Board, http://www.mccdaq.com/pcidata-acquisition/PCI-DAS4020-12.aspx). Pulse detection was achieved by
probing 10 phosphorescence intensities exceeding 1.0 volt. Peak detection was
possible by choosing data closest to the pulse decay curve from the 10 highest data
points of a pulse (Shaban et al., 2010).
The phosphorescence decay was exponential. The values of 1/s were linear
with dissolved [O2]: 1/s ¼ 1/so þ kq [O2], where 1/s is the phosphorescence decay
rate in the presence of O2, 1/so is the phosphorescence decay rate in the absence of
O2, and kq is the second-order O2 quenching rate constant/s/lM (Lo et al., 1996).
Inflammation test. For inflammation test, lung specimens were incubated
with and without 200 lg/mL MCM41-cal or SBA15-cal for 5 h. Tissues were
then stained for NF kappa B (p65) as a signaling marker for inflammation using
rabbit polyclonal antibody (Thermo Scientific). The standard streptavidin-biotin
immunoperoxidase method was used (Sintara et al., 2010). Diaminobenzidine
was used as a chromogen. One section of nontreated tissue, one section of
MCM41-cal–treated tissue, and one section of SBA15-cal–treated tissue were
used as negative controls by replacing the primary antibody with phosphate
buffer solution and followed the same procedure used earlier. Sections of
human placenta were used as positive controls as recommended by the
manufacturer.
Instrumentation. The nitrogen gas adsorption-desorption measurements
of the mesoporous materials were carried out on Micromeritics Tristar 3000
volumetric adsorption analyzer after degassing the samples at 160C for 12 h.
TEM images of the mesoporous materials were taken with TOPCON-002B
electron microscope. The sectioned ultramicrotomed nanoparticle-cell specimens were studied using a Philips CM10 transmission electron microscope.
Elemental analyses of the mesoporous materials were carried out at
Quantitative Technologies, Inc., using a Perkin-Elmer 2400 Elemental
Analyzer.
RESULTS
Synthesis and Characterization of Mesoporous Particles
The synthesis of the mesoporous MCM-41– and SBA-15–
type particles was achieved by using the surfactant supramolecular self-assembly method (Kresge et al., 1992; Zhao et al.,
1998). In the synthesis, self-assembled CTAB surfactant or
Pluronics block copolymer micelles were used as soft
templates to produce the respective MCM-41 and SBA-15
materials containing ordered mesoporous structures (Kresge
et al., 1992; Zhao et al., 1998). Because the micellar templates
from Pluronics block copolymer micelles are bigger than those
from CTAB, the former resulted in about twice bigger pore size
mesoporous materials compared with the latter. The surfactant
templates from the materials were removed by calcination
involving heating the samples at 550C for 6 h in an airflow.
This condition was found to be suitable in completely burning
off the organic surfactant templates without damaging the
materials’ structure (see below). The resulting respective
calcined materials were labeled as MCM41-cal and SBA15-cal.
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CALCINED MESOPOROUS SILICA MICROPARTICLES IN MOUSE LUNG
The calcined mesoporous silicas were then characterized by
various methods including TEM, elemental analysis, nitrogen
gas adsorption, and solid-state nuclear magnetic resonance
spectroscopy. The characterization results for both of the
studied MCM41-cal and SBA15-cal particles are compiled in
Table 1. The transmission electron micrographs (TEM) and
29
Si MAS and 13CP-MAS solid-state NMR spectra of the
samples are also shown in Figures 1A and 1B, respectively.
TEM images showed that the calcined MCM-41 materials
possessed spherical or oval-shaped microparticles of ~300 to
1000 nm in size, whereas the calcined SBA-15 materials
consisted of irregularly shaped and micron sized particles of
~500 nm diameter and ~1000 nm long. The materials’ shape
and size did not change in physiological media when observed
by TEM, even after exposure for 24 h in the buffer solutions as
well as within the cells. The materials were further characterized by nitrogen adsorption measurements, which gave a typeIV isotherm with steep capillary condensation steps for both
materials, confirming the presence of highly ordered mesoporous structures and high surface areas in the materials. The
surface areas were obtained to be 1290 and 955 m2/g for
MCM41-cal and SBA15-cal samples, respectively. Their pore
diameters were 59 and 28 Å for of MCM41-cal and SBA15cal, respectively. The carbon, hydrogen, and nitrogen analysis
was by combusting the samples in pure oxygen to convert the
sample’s elements to CO2, H2O, N2, etc. using a Perkin-Elmer
2400 Elemental Analyzer. The product gases were then
separated under steady-state conditions and measured. The
instrument was calibrated prior to sample analysis with a
National Institute of Standards and Technology traceable
organic standard. The results showed that the calcined
mesoporous silica materials contained undetectable C, H, and
N, indicating the successful removal of organic surfactant
templates. This has further ruled out the possibility of any
biological effect by the calcined materials to be attributed to the
surfactant or block copolymer template. We have indeed
previously shown that residual surfactants in mesoporous
silicas can induce biological effect to cells (Al Shamsi et al.,
2010). The elemental analysis was further corroborated by the
13
C CP-MAS solid-state NMR spectroscopy, which also
showed the absence of peaks corresponding to residual
surfactants or organic groups in the materials (Table 1).
Furthermore, the 29Si MAS NMR spectra showed bigger Q4
peak centered at ~110 ppm, indicating the presence of
significant Si(OSi)4 sites. The spectra showed little Q3 and
Q2 peaks centered at ~101 and ~91 ppm, indicating the
presence of less amount of Si(OSi)3(OH) and Si(OSi)2(OH)2
groups, respectively. The latter also suggests the relatively little
amount of surface silanol groups present in the materials,
which is not unexpected as calcined samples would have
significant condensation of their silanols at higher temperature
via a reaction Si-OH þ SiOH / Si-O-Si þ H2O.
Uptake of Mesoporous Particles
To investigate the uptake of the mesoporous particles by the
cells, samples were investigated by light microscopy. The
samples were prepared by fixing the specimens in neutral
formalin, dehydrating them in increasing concentrations of
TABLE 1
Physical Characteristics of MCM41-cal and SBA15-cal
Analysis/characterization
TEM
Powder x-ray diffraction
Nitrogen gas adsorption
13
C CP-MAS solid-state NMRa
Elemental analysis
29
Si MAS solid-state NMR
Nitrogen gas adsorption
MCM41-cal
SBA15-cal
Regular spherical/oval shaped, ~300 to 1000
nm diameter
Highly ordered mesostructures with sharp
low-angle Bragg reflection
Lattice spacing of ~44 Å
Type-IV isotherms with steep capillary
condensation
Large surface area and uniform size
mesopores
No residual CTAB surfactant (template)
Irregular shapes and sizes, mostly rods of
~500 nm diameter and ~1000 nm long
Highly ordered mesostructures with sharp
low-angle Bragg reflection
Lattice spacing of ~106 Å
Type-IV isotherms with steep capillary
condensations
Large surface areas and uniform size
mesopore
No residual Pluronics, P123 block copolymer
templates
No residual ethoxy groups from TEOS
No residual Pluronics, P123 block copolymer
templates
Significant Q4 silicon sites centered at ~110
ppm; significant Q3 centered at ~101 ppm
and some Q1 centered at ~91 ppm
BET surface area ¼ ~955 m2/g
Average BJH pore diameterb ¼ ~59 Å
No residual ethoxy groups from TEOS
No residual CTAB
Significant Q4 silicon sites centered at ~110
ppm; little Q3 and Q2 centered at ~101 and
~91 ppm, respectively
BET surface area ¼ ~1290 m2/g
Average BJH pore diameterb ¼ ~28 Å
Note. BJH, Barrett, Joyner, and Halenda.
a
See Figure 1B.
b
Obtained from the desorption branch of gas adsorption isotherm with BET (Brunaur-Emmett-Teller) method.
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AL-SALAM ET AL.
FIG. 1. TEM images (A) and
29
Si MAS and
13
C CP-MAS solid-state NMR spectra (B) of MCM41-cal and SBA15-cal.
ethanol, clearing them with xylene, and finally embedding them
in paraffin. Three-lm sections were prepared from the paraffin
blocks and stained with hematoxylin and eosin. The staining for
apoptosis was performed on 5-lm paraffin sections using the
avidin-biotin immunoperoxidase method to detect activated
caspase 3 using rabbit anti-cleaved caspase 3 antibodies.
The lung tissue sample was incubated with particle concentrations of 200 lg/mL and incubation times of 5–14 h. This
dosages and incubation times were chosen because they were
found to provide abundant cellular exposures for investigating
biocompatibility in our previous studies (Al Shamsi et al., 2010).
The light microscopy images showed that alveolar structures
were preserved in the untreated specimens (Fig. 2) and in the
specimens treated with 200 lg/mL MCM41-cal (Fig. 3) or
SBA15-cal (Fig. 4) for 5 h. In light microscopy study, MCM41-
cal appeared black in color with a round contour. They were
seen in different areas of the lung parenchyma, approaching
alveolar macrophages (Figs. 3A, 3B, and 3E), in the cytosol of
alveolar pneumocytes (Figs. 3C and 3D), in the cytosol of an
endothelial cell (Fig. 3C), and in the alveolar wall interstitium
(Fig. 3F). There were no noted anomalies in the structure and
shapes as a result of their cellular accumulation (Fig. 5).
SBA15-cal particles also appeared black in color with
a round contour. They were seen in the cytoplasm of alveolar
macrophages (Figs. 4A, 4B, and 4E), type-1 pneumocyte
(Fig. 4C), type-2 pneumocyte (Fig. 4D), and endothelial cells
(Fig. 4F). Alveolar morphology appeared unaltered by the
presence of particles in the cells and interstitium. Moreover,
there was no significant difference in the number of apoptotic
cells between treated and untreated specimens (Fig. 5).
CALCINED MESOPOROUS SILICA MICROPARTICLES IN MOUSE LUNG
FIG. 2. Light microscopy of lung tissue incubated without particles for 5 h.
Specimens were stained with hematoxylin and eosin. (Panels A and B)
Alveolar histology is preserved in untreated lung samples.
Translocation of Mesoporous Silica Particles
To further investigate the translocation of the mesoporous
particles within the cells, the samples were characterized by
TEM. The samples for TEM were prepared from lung
specimens in a Krebs-Henseleit buffer that contained the
calcined mesoporous silica particles. After sonication, the
particles were rinsed with phosphate buffer saline and fixed
with osmium tetroxide for 1 h. They were then embedded
within Agar-100 epoxy resin and polymerized before being
microtomed into thin sections.
The electron microscopy results for the specimens treated
with 200 lg/mL MCM41-cal or SBA15-cal for 5 h are shown
in Figures 6–8. Overall, the results showed that the alveolar
cells were well preserved in all studied specimens. In addition,
the images indicated that the particles are distributed in
different cellular regions of the cells.
Figure 6 shows various TEM images displaying the engulfed
MCM41-cal particles at different locations within the cells.
Additional TEM images for the MCM41-cal in various parts of
the cells are shown in Figure 8. For instance, in Figure 6A, three
MCM41-cal particles were seen attached to the nuclear
membrane of type-2 pneumocyte; the largest particle (with more
surface charges and surface area) was indenting the nuclear
membrane. In addition, two more particles were seen in the
interstitium adjacent to capillaries and one was observed adjacent
to type-1 pneumocyte (Fig. 6A). In Figure 6B, MCM41-cal was
observed in the cytosol of type-1 pneumocyte, not obviously
91
attached to any organelle. In Figure 6C, MCM41-cal was seen in
a fibroblast, adhering to the nucleus. Furthermore, MCM41-cal
was seen bordering (perhaps bridging) a fibroblast and alveolar
cells (Fig. 6D). In Figure 6E, MCM41-cal was seen in the cytosol
of an endothelial cell in the alveolar wall, separating the alveolar
space from a capillary. In addition, intracytoplasmic MCM41-cal
particle showing clear adherence to mitochondria and cell
membrane from intracellular location was observed in Figure
8D. In Figure 8E, a macrophage with intracytoplasmic MCM41cal particle showing adherence to lysosomal membrane and
rough endoplasmic reticulum was seen. Intracytoplasmic
mesoporous MCM41-cal particle showing strict adherence to
cell membrane is also clearly seen in Figure 8F.
Similarly, the calcined mesoporous SBA-15 particles were
observed in different parts within the cells (Figs. 7 and 8). Figure
7A shows that SBA15-cal was in close proximity to a lysosome
of pulmonary macrophage residing in the alveolar space. In
addition, Figure 7B shows two SBA15-cal particles indenting the
nuclear membrane of type-2 pneumocyte. This apparent affinity
to the nuclear membrane could potentially facilitate exchange of
particle contents with nuclear contents if the particles were filled
with drugs or biomolecules. In Figure 7C, SBA15-cal particle
was seen in the cytosol of an endothelial cell, in contact with two
mitochondria. In Figure 7D, two SBA15-cal particles were
observed in the cytosol of an endothelial cell forming a capillary,
demonstrating permissible access to bloodstream. In addition,
one large and one small SBA15-cal particles were seen in the
cytosol of a pulmonary macrophage adjacent to type-1 pneumocyte near the alveolar space (Fig. 7E). One SBA15-cal was also
observed in the cytosol of a pulmonary macrophage, indenting
a lysosome (Fig. 7E). Furthermore, two intracytoplasmic
mesoporous SBA-15 particles were seen strictly adherent to
the nuclear membrane of type-2 pneumocyte with clear
invagination of the nuclear membrane in the area of adherence.
A mesoporous particle was also seen within a vacuole inside the
cytoplasm of type-1 pneumocyte (Fig. 8B). In addition,
a mesoporous particle was seen adherent to the nuclear
membrane of type-2 pneumocyte (Fig. 8C).
Respiration
The term cellular respiration implies delivery of O2 and
metabolic fuels to the mitochondria, oxidation of reduced
metabolic fuels with passage of electrons to O2, and synthesis
of ATP. Impaired respiration thus entails an interference with
any of these metabolic processes, including glucose uptake and
glycolysis. Measurement of cellular mitochondrial oxygen
consumption using Pd phosphor was first introduced by Wilson
and colleagues (Lo et al., 1996). Since then, this method is
widely used to evaluate cellular bioenergetics as a marker of
function (Al Samri et al., 2010; Al Shamsi et al., 2010; Shaban
et al., 2010; Souid et al., 2003; Tao et al., 2008). Detection is
based on the principle that O2 quenches the phosphorescence
of Pd phosphor; thus, the phosphorescence intensity is
inversely related to [O2]. Here we have used this measurement
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AL-SALAM ET AL.
FIG. 3. Light microscopy of lung tissue incubated with 200 lg/mL MCM41-cal for 5 h. Specimens were stained with hematoxylin and eosin. (Panels A and B)
MCM41-cal (thick arrows) attached to the cell membrane of a pulmonary macrophage (arrow head), 310,000. (Panel C) Three MCM41-cal (thick arrows) in the
cytoplasm of an endothelial cell (arrow head) and one (thick arrow) in the cytoplasm of type-2 pneumocyte (thin arrow), 310,000. (Panel D) One MCM41-cal (thick
arrow) in the cytoplasm of type-2 pneumocyte (arrow head) and one MCM41-cal (thick arrow) in the interstitium, 310,000. (Panel E) MCM41-cal (thick arrow) in
the cytoplasm of a pulmonary macrophage (thin arrow), 310,000. (Panel F) MCM41-cal (thick arrows) in the interstitium adjacent to alveolar cells (thin arrow).
to cellular respiration of the lung cells in the presence and
absence of the calcined mesoporous particles. The cellular
respiration data were combined with the above electron
microscopy results and pneumocyte structure to determine
the biocompatibility of the mesoporous silica particles.
In a typical experiment, specimens were incubated in KrebsHenseleit buffer with and without 200 lg/mL MCM41-cal or
SBA15-cal for up to 12 h and then processed for oxygen
measurements. The impact of MCM41-cal and SBA15-cal
treatment on pneumatocyte mitochondrial oxygen consumption
(cellular respiration) was investigated as a measure of metabolic
function. At indicated time points, specimens were removed
from the incubation mixture, weighed, and placed in KrebsHenseleit buffer and their rate of respiration in lM O2/min was
measured. The rate of respiration was found to be about the
same in the calcined mesoporous silica particle–treated and
untreated specimens as seen in Table 2. This indicated that both
the MCM41-cal and SBA15-cal particles did not cause any
inhibition to the cellular respiration or mitochondrial oxygen
consumption.
Inflammation
NF kappa B is expressed in pulmonary macrophages in
untreated and MCM41-cal– and SBA15-cal–treated lung tissue
(Fig. 9). There were no significant differences in the number of
cells expressing NF kappa B between untreated and treated
lung tissues. Moreover, there were no differences in the
intensity of the cytoplasmic expression in the stained cells
between the different conditions.
CALCINED MESOPOROUS SILICA MICROPARTICLES IN MOUSE LUNG
93
FIG. 4. Light microscopy of lung tissue incubated with 200 lg/mL SBA15-cal for 5 h. Specimens were stained with hematoxylin and eosin. (Panel A)
SBA15-cal (thick arrow) in the cytoplasm of a pulmonary macrophage. (Panel B) SBA15-cal (thick arrow) in the cytoplasm of a pulmonary macrophage (arrow
head), 310,000. (Panel C) SBA15-cal (thick arrow) in the cytoplasm of type-1 pneumocyte (arrow head), 310,000. (Panel D) SBA15-cal (thick arrow) in the
cytoplasm of type-2 pneumocyte (arrow head), 310,000. (Panel E) SBA15-cal (thick arrow) in the cytoplasm of a pulmonary macrophage (arrow head), 310,000.
(Panel F) SBA15-cal (thick arrow) in the cytoplasm of an endothelial cell (arrow head), 310,000.
The expression of NF kappa B by pulmonary macrophages
in both untreated lung tissues and in MCM41-cal– and SBA15cal–treated lung tissues was at the physiological level and was
observed to be unrelated to the in vitro procedure or treatment
(as evident by the absence of significant differences in the NF
kappa B expression between the studied tissues).
DISCUSSION
Mesoporous silica ‘‘nano’’ and ‘‘micro’’ materials have
prospective medical uses as delivery vehicles for hydrophobic
drugs (Rosenholm et al., 2009), platinum-based anticancer
agents (Tao et al., 2009, 2010b), siRNAs (Chen et al., 2009;
Hom et al., 2010; Taratula et al., 2009; Xia et al., 2009), and
numerous other bioactive agents (Lin et al., 2005). To
contribute to these prospective medical applications of
mesoporous materials, we here investigated the biocompatibility of two forms of calcined mesoporous silica particles,
MCM41-cal and SBA15-cal, in mouse lung. These micro
materials were prepared using different amphiphilic templates,
CTAB and Pluronics-123. The potential biological applications
of both materials were due to their large surface areas, nanoand microscale sizes, nanometer pores, and suitable properties
for loading and delivering drug and bioactive molecules.
In the first part of the experiment, we have shown by
measurement of mitochondrial oxygen consumption that
these calcined mesoporous silica materials are biocompatible
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AL-SALAM ET AL.
FIG. 5. Light microscopy of lung tissues incubated with and without 200 lg/mL
particles for 5 h. Specimens were stained with rabbit anti-cleaved caspase 3 antibodies.
(Panel A) Untreated specimen; (panel B) specimen treated with MCM41-cal; (panel C)
specimen treated with SBA15-cal; and (panel D) positive control (human breast
cancer). Apoptotic cells have brown cytoplasmic staining (arrows).
with cells within at least 5–14 h of exposure time. The
results of O2 consumption by murine lung specimens
exposed in vitro to MCM41-cal and SBA15-cal for several
hours are compiled in Table 2. The results confirm intact
pneumatocyte respiration (bioenergetics) in lung specimens
treated with the mesoporous silica, MCM41-cal and SBA15cal, particles for 11–12 h.
Compared with solvent-extracted particles, the calcined
MCM-41 and SBA-15 used here have low-density surface
silanols (as evident by 29Si solid-state NMR spectroscopy).
Thus, they are expected to generate less silanol-induced radicals
that can possibly lead to cellular death (Konecny et al., 2001;
Murashov et al., 2006). Moreover, the absence of residual
organics in the calcined materials (Table 1), due to the
calcination procedure, might have also contributed to their
observed biocompatibility in pneumatocytes. Furthermore,
insignificant level of apoptosis in the tissues treated with both
types of materials was observed (Fig. 5), further supporting their
biocompatibility. This conclusion was supported by the finding
that solvent-extracted MCM-41 and SBA-15 particles, whose
surfactant templates were removed by solvent washing, were not
biocompatible with murine lung tissues (Al Shamsi et al., 2010).
In the second analysis, the MCM41-cal and SBA15-cal
particles were observed to be internalized in the lung tissue and
pneumatocytes at 5 h (Figs. 3–8). The particles’ engulfment
took place without the particles being surface functionalized by
organic or polymeric groups as reported by others (Beyerle
et al., 2010; Blumen et al., 2007; Galagudza et al., 2010; Radu
et al., 2004; Rosenholm et al., 2009; Xia et al., 2009). The
uptakes of our calcined mesoporous silica materials were
possibly due to the high binding affinities of phospholipid head
groups on the plasma and nuclear membranes to surface
silanols (Si-OH) or silanolates (Si-O) of silica materials
(Allison et al., 1966; Chunbo et al., 1995; Murashov and
Leszczynski, 1999; Murashov et al., 2006). The TEM images
(Figs. 6–8) further showed that MCM41-cal and SBA15-cal
particles were distributed in different cellular parts and were
abundantly present in pneumocytes, macrophages, endothelial
cells, fibroblasts, and interstitium. They were also seen in
different areas of the cytoplasm, suggesting intracellular movements, and they did not appear to disturb cellular configuration
or micro-organelles. Some of the particles were also observed to
approach and firmly attach to (indent) the nuclear membrane.
Similar observation (particles within the cytoplasm without
getting inside the nucleus) was reported in other cells such as
3T3-L1 fibroblasts for chromophore-functionalized mesoporous
materials (Lin et al., 2005). We did not observe particle
aggregations in our studied cells (even for the rod-shaped
calcined SBA15-cal), although particle aggregation in the
cytosol for rod-shaped CTAB-templated mesoporous silicas of
~450 nm sizes and aspect ratio of ~107 nm was reported by
others (Tsai et al., 2008). Furthermore, despite their shape
differences, our study here showed similar degree of engulfment
for both MCM41-cal and SBA15-cal particles in the lung.
Our biochemical data for the effects of the particles on lung
tissue are primarily based on our novel measurement of cellular
bioenergetics. Cellular mitochondrial oxygen consumption
CALCINED MESOPOROUS SILICA MICROPARTICLES IN MOUSE LUNG
95
FIG. 6. Transmission electron micrographs of lung tissue incubated with
200 lg/mL MCM41-cal for 5 h. (Panel A) Three MCM41-cal (thick arrows) in
the cytoplasm of type-2 pneumocyte (thin arrow) and one MCM41-cal (thick
arrow) in the interstitium adjacent to an alveolar cell (arrow head). (Panel B)
One MCM41-cal (thick arrow) in the cytoplasm of type-1 pneumocyte (arrow
head). (Panel C) MCM41-cal (thick arrow) in the cytoplasm of a fibroblast
(arrow head). (Panel D) MCM41-cal (thick arrow) bordering a fibroblast (arrow
head). (Panel E) MCM41-cal (thick arrow) in the cytoplasm of an endothelial
cell (arrow head). (Panel F) MCM41-cal (thick arrow) attached to the nucleus
of a fibroblast (arrow head).
FIG. 7. Transmission electron micrographs of lung tissue incubated with
200 lg/mL SBA15-cal for 5 h. (Panel A) SBA15-cal (thick arrow) in the
cytoplasm of a pulmonary macrophage (thin arrow). (Panel B) SBA15-cal
(thick arrows) attached to the nucleus of type-2 pneumocyte (thin arrow).
(Panels C and D) SBA15-cal (thick arrows) in the cytoplasm of endothelial
cells (thin arrows). (Panel E) SBA15-cal (thick arrows) in the cytoplasm of
a pulmonary macrophage (thin arrow) adjacent to type-1 pneumocyte (arrow
head). (Panel F) SBA15-cal (thick arrow) in the cytoplasm of a pulmonary
macrophage attached to a lysosome (thin arrow).
(respiration) implies delivery of O2 and metabolic fuels to the
mitochondria, oxidation of reduced metabolic fuels with
release of electrons to O2, and synthesis of ATP. Impaired
respiration thus entails an interference with any of these
processes (Tao et al., 2008). On the other hand, normal cellular
respiration implies well-preserved glucose uptake and glycolysis, steady deliveries of O2 and metabolic fuels to the
mitochondria, intact mechanism and rates of oxidations of
reduced metabolic fuels in the mitochondrial respiratory chain,
normal passage of electrons to O2, and coupled synthesis of
ATP. Thus, the biochemical method and data are appropriate
for assessing biocompatibility. The use of such cellular
bioenergetics to assess nano- and microparticle biocompatibilities is discussed in more detail in our prior publications
(Al Shamsi et al., 2010; Tao et al., 2008).
In addition, we used anti-cleaved caspase 3 staining for
apoptosis to demonstrate whether the mesoporous silica
particles when attached to the cell membrane or internalized
into the cells are not causing injuries that initiate caspases. In
our study, we used the most specific primary antibody against
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AL-SALAM ET AL.
FIG. 8. Transmission electron micrographs of lung tissue treated with the silica particles. (Panel A) Two SBA15-cal particles are seen near the nucleus of type-2
pneumocyte, with clear invagination of the nuclear membrane in the area of adherence (thick arrows). (Panel B) One SBA15-cal particle is seen within a vacuole inside
the cytoplasm of type-1 pneumocyte (thick arrow). (Panel C) One SBA15-cal particle is seen adherent to the nuclear membrane (thick arrow) of type-2 pneumocyte.
(Panel D) Intracytoplasmic MCM41-cal showing clear adherence to the mitochondria and cell membrane (thick arrows) from an intracellular location. (Panel E)
Macrophage with intracytoplasmic MCM41-cal showing adherence to lysosomal membrane (thick arrow) and rough endoplasmic reticulum (arrow head). (Panel F)
Intracytoplasmic MCM41-cal showing strict adherence to the cell membrane (thick arrow).
CALCINED MESOPOROUS SILICA MICROPARTICLES IN MOUSE LUNG
TABLE 2
Lung Respiration in the Presence and Absence of Silica Particles
Cellular
mitochondrial
oxygen consumption (lM O2/min/mg)
Untreated
MCM41-cal
SBA15-cal
0.27 ± 0.03 (n ¼ 4)
0.26 ± 0.06 (n ¼ 4)
0.27 ± 0.06 (n ¼ 5)
Duration
of
incubation
p
Value
11 h
12 h
0.842
0.907
Note. Values are mean ± SD. The difference between untreated and calcined
mesoporous silica particle–treated specimens is not significant. For unit
conversion, 1.0 ml O2 ¼ 1.4276 mg or 0.0446125 mmol.
cleaved caspase 3, which is terminal executioner in apoptotic
pathway. This antibody detects activated capase 3 only; hence,
it clearly detects apoptotic cells. The result showed no
significant increase in apoptosis between MCM41-cal– or
SBA15-cal–treated and untreated lung tissues. It is worth
FIG. 9. Lung tissues stained with NF kappa B (p65): (A) untreated lung tissue,
(B) SBA15-cal–treated lung tissue, and (C) MCM41-cal–treated lung tissue. The
brown cytoplasmic staining in pulmonary macrophages is evident (arrows).
97
noting that the normal respiration in particle-treated lung
tissues rules out activation (Tao et al., 2007), i.e., execution of
apoptosis and mitochondrial disturbances are coupled processes (‘‘mitochondrial cell death pathway’’). Thus, the
biochemical data (normal cellular bioenergetics) presented
support the negative immunohistochemical staining (IHS) for
caspase activation. This finding was also consistent with the
light microscopy results and ultrastructural morphology of the
mesoporous silica–treated tissues.
Histopathological and electron microscopic studies are the
gold standard in assessing tissue changes because they allow
one to see any changes that might occur in the cells. The results
of the light and electron microscopy studied and absence of
caspase staining are consistent with the biochemical data. They
clearly show abundant internalization of particles inside the cell
with well-preserved function and ultrastructure. We thus feel
that absence of ultrastructural and light microscopic changes
together with the absence of significant oxygen consumption
changes and normal cell respiration in the calcined mesoporous
silica–treated lung tissues are further strong indications of intact
cellular bioenergetics and the materials’ biocompatibility,
within at least 5–14 h of exposure time.
The choice of the described in vitro mode of exposure, utilizing
excised lung tissue in this study, was based on our recently
published work (Al Samri et al., 2011; Al Shamsi et al., 2010;
Shaban et al., 2010). Briefly, we developed a novel in vitro system
that sustains the structure and function of various murine tissues
over several hours. This method was validated by assessments of
cellular bioenergetics, histology, ultrastructure, and apoptosis as
function of time for up to 14 h. This approach is utilized here to
investigate the impact of an in vitro exposure of murine lung tissue
to calcined mesoporous silica microparticles. In our previous work
(Al Samri et al., 2011), murine lung was found to be durable
in vitro for at least 14 h. Therefore, it was used here as a model
tissue for the study of the effect of mesoporous particles for 14 h.
These obtained results will be further used for comparative studies
with other tissues in the future.
The choice of excised lung tissue in the study here was also
because the lung is the more likely organ to be exposed to
nanomaterials such as mesoporous silicas. On the other hand,
many nanomaterial delivery systems for drugs for lung cancer
are in development; thus, if proven to be biocompatible, the
studied mesoporous materials may become potential candidate
drug delivery vehicles for such drugs, given their suitable
nanometer structures and high surface areas.
A common route of exposure of lung tissues to such nanoand microparticles is via inhalation. Thus, exposing animal
models to the particles and conducting in vivo studies will
probably give more information about the direct effects of
particles on animals. However, in vitro exposure of the lung
tissue, as shown clearly in the manuscript, provides an
alternative approach to study the effects of these particles.
Our results show that the particles are abundantly internalized
without alternations in cellular structure (measured by light and
98
AL-SALAM ET AL.
electron microscopy) and function (measured by cellular
mitochondrial oxygen consumption). The information gathered
with this reported method is accurate and needs sacrificing very
few animals. If well planned, studies of this type could be
completed with three or less animals. Furthermore, multiple
organs can be studied as function of time using the same
animal. In addition, the reported method has an advantage of
evading the extensive tissue manipulation and collagenase
digestion required for single-cell preparation. Moreover, it
allows a comprehensive analysis (e.g., cellular respiration,
histology, and electron microscopy) in vitro over extended
periods of time. It thus permits the investigation of the effects
of various agents on tissue bioenergetics and cellular
ultrastructure. However, in vitro and in vivo results may not
always be necessarily the same. As previously suggested by
Lin et al. (2006), in vitro systems need to be correlated with
in vivo testing. Nevertheless, the results described in the
manuscript are consistent to other studies showing particle
biocompatibilities at longer exposure time using cell culture or
in vivo systems (Blumen et al., 2007).
The results of IHS for inflammatory markers (using the same
paraffin blocks of lung tissues) with NF kappa B as a signaling
marker of inflammation using a rabbit polyclonal antibody also
showed no significant difference in the expression of NF kappa
B between the MCM41-cal– or SBA15-cal–treated tissues and
untreated tissues. These results clearly support lack of particleinduced inflammation. Moreover, the histology findings do not
reveal inflammatory cell infiltration or morphological changes
suggesting acute inflammation.
In summary, this study shows that the calcined MCM41-cal
and SBA15-cal are bioavailable and biocompatible in mouse lung
within at least 5–14 h of exposure time. This result is supported
by other studies showing particle biocompatibilities at longer
exposure time using cell culture or in vivo systems (Blumen et al.,
2007). The results also show well-preserved pneumatocyte
structure (by light and electron microscopy) and function (by
cellular respiration) after treatment with both types of materials,
confirming their biocompatibility. The particles did not also cause
apoptosis on the cells. The benign properties of the calcined
MCM41-cal and SBA15-cal are likely due to the refined methods
of synthesis and calcination, resulting in fewer surface hydroxyl
groups and free residual surfactants (as evident by the solid-state
NMR data). The results further support the potential uses of these
nanomaterials as drug delivery vehicles for medical applications.
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FUNDING
Lin, W., Huang, Y.-W., Zhou, X.-D., and Ma, Y. (2006). In vitro toxicity of
silica nanoparticles in human lung cancer cells. Toxicol. Appl. Pharmacol.
217, 252–259.
U.S. National Science Foundation (CHE-1004218) to T.A.;
United Arab Emirates University.
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Chen, Y.-C., and Mou, C.-Y. (2005). Well-ordered mesoporous silica
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