Article

pubs.acs.org/cm

Large Pore Mesoporous Silica Nanoparticles by Templating with a

Nonsurfactant Molecule, Tannic Acid
Zhe Gao and Ilya Zharov*
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States
*
S Supporting Information

ABSTRACT: We describe a one-pot synthesis of novel large-pore mesoporous silica

nanoparticles using a nonsurfactant template, tannic acid, and protein immobilization on
these particles. The tannic-acid-templated mesoporous silica nanoparticles (TA-MSNPs)
possess tunable large pores (613 nm), unique pore morphology, and a uniform diameter
of ca. 200 nm. TA-MSNPs show high protein adsorption capacity of 77.1 mg/g for
lysozyme, 396.5 mg/g for bovine hemoglobin, and 130.0 mg/g for bovine serum albumin
and rapid protein uptake. They can also adsorb a large amount of m-MDH (421 13 mg/
g) and protect this enzyme from the environment, as demonstrated by its high activity
when encapsulated inside the mesopores of TA-MSNPs.

. INTRODUCTION nm. SBA-15 nanoparticles21 contain mesopores of larger sizes,

Mesoporous silica nanoparticles (MSNPs) attract increasing
attention because of their high surface area, hydrophilic and
easily functionalized surface, and biocompatibility. These
properties are of high interest for a host of applications,1,2
including drug delivery,3 imaging,46 catalysis,7,8 and sensing.9
To fulll this promise, especially in the biomedical area,
monodisperse MSNPs with sizes in the 50200 nm range (to
facilitate cell uptake10,11) and with large mesopores (to allow
for the encapsulation of biomacromolecules) are desired. In
addition, accessible internal volume resulting from intercon-
nected pores would be benecial for these applications.12,13
Numerous strategies for the preparation of MSNPs have
been developed14 using templating agents, usually surfactants.15
Ionic surfactants, such as cetyltrimethylammonium bromide
(CTAB), self-assemble into micelles, which create the voids in
the silica matrix during the preparation of mesoporous silica
materials, such as MCM-41. The formation of the mesoporous
structure of MCM-41 is mainly due to the electrostatic
interactions between the ionic surfactant and silicate species.15
For nonionic surfactants, such as triblock copolymers, the
hydrogen bonding between the surfactants and the silicate is
believed to be the main driving force in the formation of
mesoporous silica materials, such as SBA-15.15 Fluorocarbon
surfactants were also used to synthesize mesoporous silica with
improved stability and novel mesostructures.16 In all these
cases, templating surfactant agents lead to ordered structures
with noninterconnected cylindrical pores.15
Several synthetic protocols for MSNPs with particle diameter
and pore size controlled by using various surfactants and
swelling agents have been reported.1219 For example, MCM-
41 nanoparticles can be prepared with diameters in the 30280
nm range;20 however, the maximum pore size is limited to ca. 6
controlled by triblock-copolymer templates, cosolvents, and
swelling agents in the range of 6 to 50 nm. However, the size of
the smallest SBA-15 particles reported so far is around 500
nm.5,22 It remains a challenge to produce uniform MSNPs with
diameters smaller than 200 nm and pore size larger than 10 nm
in a simple synthetic procedure. Herein, we describe a one-pot
procedure for the synthesis of novel MSNPs with 200 nm
diameter and with large interconnected mesopores tunable in
the 613 nm range using an environmentally friendly
nonsurfactant pore-forming agent, tannic acid (TA).
There are a number of reports on the preparation of
mesoporous silica nanoparticles using nonsurfactant templates,
such as tartaric acid, glucose, maltose,23,24 kanemite,25 and
boron oxide.26 The size of nonsurfactant-templated MSNPs can
be varied in a broad range without the loss of monodispersity,
but the pore size is usually less than 4 nm due to the small size
of the templating molecules.27
TA (whose formal structure is shown in Figure 1) is a
glucoside polymer of gallic acid with multiple phenolic hydroxyl
groups that is found in many plants.28 One of the advantages of
using TA as a porogen is that it is cheap and environmentally
friendly, as opposed to the expensive and toxic surfactants used
in the traditional synthesis of mesoporous materials. TA is an
antioxidant whose antimutagenic and anticarcinogenic proper-
ties has been extensively investigated.29,30 In addition to being
an important dietary supplement,31 TA was used as both the
reducing and the stabilizing agent in the preparation of Au and
Ag nanoparticles.3234 It has been also used as an organic
Received: December 5, 2013
Revised: February 16, 2014
Published: February 17, 2014

2014 American Chemical Society 2030 dx.doi.org/10.1021/cm4039945 | Chem. Mater. 2014, 26, 20302037
Chemistry of Materials
Figure 1. Formal structure of tannic acid (TA) and illustration of TA-
MSNP formation.
ligand in the preparation of engineered lms and particles.3537
To the best of our knowledge, TA has never been used as a
porogen in the preparation of mesoporous silica.
In this paper, we report the synthetic conditions that produce
tannic-acid-templated mesoporous silica nanoparticles (TA-
MSNPs) and describe their unique structure. To illustrate their
potential for biomedical and catalysis applications, we describe
their protein adsorption ability and catalytic activity of an
enzyme encapsulated inside TA-MSNPs.
. EXPERIMENTAL SECTION
Materials. Tannic acid (ACS-grade, Alfa Aesar), tetraethox-
ysilane (TEOS, 99.999+%, Alfa Aesar), ammonium hydroxide
(2830% as NH3, EMD Chemicals, Inc.), ethanol (200 proof,
ACS-grade, Pharmaco-Aaper), 3-aminopropyltriethoxysilane
(99%, Sigma-Aldrich), dansyl choride (Sigma-Aldrich), potas-
sium phosphate monobasic (Sigma-Aldrich), potassium phos-
phate dibasic trihydrate (Mallinckrodt), sodium chloride
(Mallinckrodt), lysozyme (Lz) from chicken egg white
(Sigma-Aldrich), albumin (BSA) from bovine serum (lyophi-
lized powder, Sigma-Aldrich), and hemoglobin (BHb) from
bovine blood (lyophilized powder, Sigma-Aldrich) were all used
as received. Acetonitrile (HPLC grade, VWR Scientic) was
freshly distilled from calcium hydride. Millipore water (18 M
cm) used in all experiments was obtained from a Barnstead E-
pure water purication system.
Instrumentation. Scanning electron microscopy (SEM)
images were recorded using FEI NanoNova and transmission
electron microscopy (TEM) images were recorded using FEI
Philips Tecnai T-12 instruments. Surface area and pore volume
were determined using nitrogen sorption measurements, data
collected using a Micrometrics ASAP 2010 (Norcross, GA)
instrument at 77.3 K. All samples ( 0.1 g) were degassed in
the degassing port of the absorption apparatus at 80 C for
approximately 6 h prior to the nitrogen sorption measurements.
The surface area calculations were based on the Brunauer
EmmettTeller (BET) method by using adsorption data at
relative pressure from 0.05 to 0.20. The pore size distribution
calculations were based on the BarrettJoynerHalenda (BJH)
method. Philips PW3040/00 XPert Pro XRD (Spectris,
England) using Cu K radiation at 45 kV and 40 mA was
used to obtain the X-ray Diraction (XRD) patterns of TA-
MSNPs. The XRD spectra were recorded in the 2 range of
1.810 with a step size of 0.02 in a 2 scattering angle and a
scan speed of 0.01 deg/s. UVvis measurements were
performed using an Ocean Optics USB4000 and a Thermo
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Scientic Evolution 260 B10 instruments. The hydrodynamic
diameter and potential measurements were conducted in
water using a NICOMP 380 ZLS Zeta Potential/Particle Sizer
(PSSNICOMP Particle Sizing Systems). Thermogravimetric
analyses (TGA) were performed using a TA Instruments TGA
2950 Thermogravimetric Analyzer. All TGA experiments were
performed under N2 atmosphere. A Vortex-Genie 2 laboratory
mixer from Scientic Industries was used to redisperse the
protein-loaded SNPs in solution.
Preparation of TA-MSNPs. Tannic acid was dissolved in
50 mL ethanol as follows: 68 mg (4.0 105 mol) of TA4-
MSNP, 136 mg (8.0 105 mol) of TA8-MSNP, 272 mg (1.6
104 mol) of TA16-MSNP, and 544 mg (3.2 104 mol) of
TA32-MSNP. Concentrated ammonium hydroxide (25 mL)
was added to the ethanolic solution with vigorous stirring,
followed by the addition of TEOS (0.3 mL). The onset of
turbidity after the addition of TEOS indicated the start of silica
sphere formation. After 2 h of stirring, the particles were
centrifuged out at 3400 rpm for 40 min and resuspended in
water. To avoid particle agglomeration and removal of silanol
groups that often result from calcination, TA-MSNPs were
puried by water extraction. TA and unreacted TEOS were
removed by repeatedly washing with water and ethanol until
the supernatant was colorless (ca. 10 times). To ascertain the
complete removal of TA, UVvis absorbance of the super-
natant was measured. After the nal rinse, the particles were air-
dried overnight. Typical yields of TA-MSNPs were 5070 mg.
Preparation of 240 nm Silica Spheres. Nonporous SNPs
was prepared by hydrolysis and condensation of TEOS
according to the Stober method.38,39 A total of 250.0 mL of
ethanolic solution containing TEOS (26.7 mL, 0.10 mol) and
an equal volume of an ethanolic solution containing NH4OH
(13.4 mL, 0.20 mol) and water (144 mL, 8.0 mol) were mixed
and vigorously stirred at room temperature. After 24 h, the
mixture was centrifuged and the particles washed in a series of
water/ethanol solutions containing increasing amounts of
water. After the nal rinsing, the supernatant was decanted,
and the silica spheres were air-dried.
Protein Adsorption and Desorption on TA-MSNPs.
Phosphate buer (10 mM, pH 6) was prepared by mixing 0.136
g K2HPO43H2O and 1.280 g KH2PO4 in 1 L of Millipore
water. A total of 30 mg of either Stober-SNPs or TA16-MSNPs
were separately dispersed in 6.0 mL of phosphate buer, and
each colloidal solution was divided into three 2 mL aliquots.
Individual stock solutions (0.1 mM) of lysozyme (Lz), bovine
serum albumin (BSA), and bovine hemogobin (BHb) were
prepared in the same buer, and 2.0 mL of each were added to
an aliquot of nonporous SNPs and TA16-MSNPs, followed by
incubation for 24 h at room temperature. The mixtures were
centrifuged (40 min, 3200 rpm), and the UV absorbance was
recorded for each of the supernatants at 280 nm (Lz), 499 nm
(BHb), or 277 nm (BSA).
The protein adsorption rate studies monitored the amount of
protein adsorbed by TA-MSNPs for 24 h in the phosphate
buer. A total of 40 mg of TA16-MSNPs were suspended in 8
mL phosphate buer and mixed with additional 8 mL of 0.1
mM protein solution in phosphate buer. Aliquots, 1.5 mL
each, were periodically removed and centrifuged, and the
supernatant was analyzed by UV spectroscopy.
The protein release from the loaded TA16-MSNPs was
studied in phosphate buer with 0.1 M KCl by dispersing 10
mg of protein-loaded TA16-MSNPs in 8 mL of phosphate
buer at room temperature by a vortex mixer. At each time
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interval, 1.5 mL of the solution was withdrawn, and the
particles were removed by centrifugation. The protein
concentration in the supernatant was analyzed by UVvis
spectroscopy.
Enzyme Adsorption on TA-MSNPs and Its Activity.
Mitochondrial malic dehydrogenase (m-MDH) was stored as
an ammonium sulfate suspension at 4 C. Before all the
experiments, the enzyme was transferred to phosphate buer by
dialysis (Slide-A-Lyzer Dialysis Cassettes with 3.5K MWCO
cuto, Thermo Scientic) for 18 h at 4 C. The resulting
enzyme solution was concentrated on a centrifuge lter by
centrifugation, and the concentration was calculated using the
calibration curve obtained from the standard BSA assay.
A 100 mM phosphate buer solution was prepared by mixing
15.223 g K2HPO43H2O and 4.536 g KH2PO4 in 1 L Millipore
water and was adjusted to pH 7.5 with 12 M KOH solution.
For m-MDH adsorption experiments, the concentration of the
particles was 2.5 mg/mL and the concentration of the enzyme
was 0.05 mM. The particle suspensions and the m-MDH
solutions were mixed, followed by incubation for 24 h at room
temperature. The solutions were centrifuged (10 min, 12 000
rpm), and the UVvis absorbance was recorded for each of the
supernatants at 280 nm.
For catalytic activity studies, 5 mg/mL TA16-MSNPs or
nonporous 240 nm SNPs were incubated with 0.05 mM m-
MDH solution for 2 days at 4 C to allow for complete
adsorption of m-MDH at low temperature. The m-MDH-
loaded particles were separated from the solution via
centrifugation for 40 min at 6000 rpm and 4 C. The particles
were redipersed in phosphate buer by the vortex mixer for 10
min. The solution was then tested for its catalytic activity. Two
fresh stock solutions were prepared before each experiment:
(1) 0.14 mM -NADH phosphate buer solution and (2) 7.6
mM oxaloacetate phosphate buer solution. A total of 1.4 mL
of solution 1 was added to a cuvette, followed by the addition
of 50 L of solution 2 and 50 L of nanoparticle solution. The
cuvette was shaken by hand and placed into the spectrometer.
The absorbance at 340 nm was recorded at 15 or 30 s intervals
for 5 min. A free m-MDH solution with the same concentration
of the enzyme as the ones loaded in the TA-MSNPs was
prepared. The catalytic activity was measured following the
procedure described above. Blank samples with phosphate
buer instead of the sample solution were also prepared and
used as reference.
The activity of m-MDH immobilized on the TA16-MSNPs
was calculated using the following equation:
(A test (min) Ablank (min)) Vf (mL) df
Units
= when large amounts of ammonia are added to TA in ethanol, a
mL 6.22 Ve(mL)
where Vf is the total volume of the assay, df is the dilution
factor, 6.22 is the millimolar extinction coecient of -NADH
at 340 nm, and Ve is the volume of the enzyme used.
. RESULTS AND DISCUSSION
Synthesis and Characterization of TA-MSNPs. We
discovered that when tannic acid is present during the
formation of silica particles from tetraorthosilicate with
ammonium hydroxide as a base, it acts as a porogen. The
resulting particles possess a highly porous structure as
conrmed by transmission electron microscopy (TEM). The
TEM images (Figure 2) of the pores at the TA-MSNPs
periphery revealed lower density compared to the center of the
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Figure 2. TEM images of tannic-acid-templated silica nanoparticles
produced with dierent amounts of tannic acid: (a) TA4-MSNP, (b)
TA8-MSNP, (c) TA16-MSNP, and (d) TA32-MSNP. Scale bars 50
nm (ac) and 200 nm (d).
particles, suggesting a disordered pore arrangement. The
powder X-ray diraction (XRD) spectra of TA-MSNPs
conrmed the lack of the pore order, as they did not display
any specic peaks. In contrast, the well-ordered hexagonal or
cubic arrangements of the pores in periodic mesoporous silica
materials give diraction patterns in powder XRD experi-
ments.15
The pores in TA-MSNPs are signicantly larger than the TA
dimensions (2.2 1.8 1.2 nm). Therefore, we speculate that
under our reaction conditions, TA self-assembles into branched
supermolecular structures. The disordered pore arrangement in
TA-MSNPs likely results from the inability of TA to form well-
dened micelles.37
TA is a polydentate ligand, which is capable of forming
complexes with metal ions.37,40,41 It is also known that the o-
dihydroxyphenyl groups of the TA are capable of hydrogen
bonding and electrostatic and hydrophobic interactions.42
Thus, supramolecular behavior of TA should depend on its
ionization state. Indeed, we observed that the addition of small
amounts of ammonia to TA in ethanol leads to precipitation,
likely due to the formation of ammonium tannate.43 We found
that with TEOS as precursor under these conditions,
nonporous silica particles are formed. On the other hand,
red solution is formed. It has been suggested that under these
conditions TA undergoes hydrolysis, deprotonation, and
oxidation,44 with the red color attributed to the oxidation
products.45 The supramolecular structure of TA in the presence
of large amounts of ammonia should be the result of
electrostatic interactions between the TA and ammonium
cations, hydrogen bonding and stacking. Although the
exact composition of this supramolecular architecture is still
under investigation, we assume that during the synthesis of TA-
MSNPs under the highly basic conditions, phenolic hydroxyl
groups of TA (pKa 10) are partially deprotonated. Thus,
similar to the anionic surfactants, the negatively charged
supramolecular TA complexes interact electrostatically with
ammonium cations. In addition, protonated hydroxyl groups of
TA should undergo hydrogen bond formation46,47 with soluble
dx.doi.org/10.1021/cm4039945 | Chem. Mater. 2014, 26, 20302037
Chemistry of Materials Article

silicate species. We believe that the electrostatic interactions

and hydrogen bonding between supramolecular TA complexes
and silicate species present in solution result in the formation of
the templated silica framework of TA-MSNPs with large
mesopores. This process is illustratedin Figure 1.
Several batches of TA-MSNPs were prepared to optimize the
amount of ammonia necessary for the formation of mesoporous
particles. We found that the optimum ammonium hydroxide
amount in the synthesis of TA-MSNPs is one-third of the total
reaction solution volume.
Next, we varied the concentration of TA in reaction mixtures
as follows: 5.3 104 M (TA4-MSNP), 10.6 104 M (TA8-
MSNP), 2.13 103 M (TA16-MSNP), and 4.26 103 M
(TA32-MSNP). As shown by TEM (Figure 2), TA4-MSNP,
TA8-MSNP, and TA16-MSNP possess increasing porosity,
similar to most templated materials where increased template
concentrations lead to structures that are more porous.
However, high concentrations of TA prevent the formation
of spherical particles, as illustrated by irregularly shaped rough- Figure 3. Nitrogen adsorptiondesorption isotherms (top) and pore
surface solid agglomerates obtained in the case of TA32-MSNP size distribution curves (bottom). TA4-MSNP, red; TA8-MSNP,
(Figure 2). This observation is in agreement with previous yellow; TA16-MSNP, green; TA32-MSNP, blue.
reports for other mesoporous materials.21
To further characterize the porosity of TA-MSNPs, we nitrogen sorption showed a type II isotherm characteristic of
conducted nitrogen sorption measurements. In order to a macroporous material (pore size >50 nm) with only one
perform these measurements, we removed the TA template hysteresis loop at higher pressure due to the agglomeration of
by extracting TA-MSNPs with water until no TA was the particles. The BET surface area of Ta32-MSNPs is small
detectable in the supernatant by UVvis spectroscopy. This (9.4 m2/g) and comparable to nonporous silica nanoparticles.50
led to an almost complete removal of TA, as indicated by the Although the TA concentration aected the porosity of the
porosity measurements and by thermogravimetric analysis resulting particles, it had little inuence on their size. The
(TGA) discussed below. The BrunauerEmmettTeller scanning electron microscopy (SEM) images (Figure 4)
(BET) surface area, pore volume, and pore parameters of
TA-MSNPs are given in Table 1. They conrm the presence of

Table 1. Structural Properties of TA-MSNPs from TEM and

the Nitrogen Sorption Measurements
BET surface area, pore volume, pore size,
particles size, nm m2/g cm3/g nma
TA4- 209 16 419.9 0.46 5.9
MSNP
TA8- 207 16 445.4 0.82 9.7
MSNP
TA16- 201 11 560.6 1.53 12.9
MSNP
TA32- 9.4 0.12
MSNP
a
BJH adsorption average pore width.

uniform pores, large surface area and pore volume for TA-
MSNPs. In general, an increase in TA concentration resulted in
larger surface area and pore volume. The nitrogen sorption
showed type IV isotherms for TA4-, TA8-, and TA16-MSNPs
with two distinct adsorption steps, characteristic of a
mesoporous structure (Figure 3).48 The small hysteresis
loops around 0.3 P/P0, 0.5 P/P0, and 0.7 P/P0 correspond to
the pore size distribution centered at 6, 10, and 13 nm,
respectively (Figure 3). The hysteresis loop at higher relative
pressure (ca. 0.9 P/P0) can be attributed to interparticle
porosity. The BET surface areas of TA4-, TA8-, and TA16-
MSNP are smaller than those of most MCM-41 particles
(>1000 m2/g)15 but are comparable to that of SBA-15 particles
with similar pore size (419 m2/g).49 The pore volume in TA-
MSNPs is considerably larger compared to other reported
mesoporous nanoparticles. In the case of TA32-MSNPs,
2033
Figure 4. SEM images of tannic-acid-templated silica nanoparticles
produced with dierent amounts of tannic acid: (a) TA4-TEOS, (b)
TA8-TEOS, (c) TA16-TEOS, and (d) TA32-TEOS. Scale bars 500
nm.
demonstrated that TA4-MSNP, TA8-MSNP, and TA16-
MSNP particles are mostly spherical and reasonably mono-
disperse with ca. 200 nm diameter (Table 1) and negligible
aggregation. TA4-MSNPs are not as uniform but possess a
comparable average size with TA8-MSNP and TA16-MSNP. In
addition to electron microscopy, the hydrodynamic diameter of
TA16-MSNPs was measured by dynamic light scattering (DLS)
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Chemistry of Materials
to be 249.2 60.5 nm, in good agreement with SEM data. In
contrast, TA32-MSNPs exhibited agglomeration and their
average size could not be accurately determined.
We also studied the eect of the reaction time on the
nanoparticle morphology and composition by isolating the
products after 2, 6, and 24 h of condensation (TA16-MSNP-2h,
TA16-MSNP-6h, TA16-MSNP-24h, respectively). All of the
reaction times produced monodisperse spherical nanoparticles.
However, although TA16-MSNP-2h and TA16-MSNP-6h
displayed a porous structure, a nonporous structure with
dense core was observed for TA16-MSNP-24h particles
(Supporting Information Figure S1).
The composition of TA-MSNPs (puried by water
extraction) as a function of reaction time was examined using
TGA under N2 atmosphere (Supporting Information Figure
S2). The ca. 1% weight loss observed in the temperature range
35230 C can be attributed to the evaporation of water and
degradation of silanol groups, and the weight loss above 230 C
is likely due to carbonization of residual TA and other possible
volatilizable impurities, which were not completely removed by
water extraction. The weight loss observed for TA16-MSNP-2h
and TA16-MSNP-6h was similar, ca. 3.4%. TGA of a sample
calcinated at 650 C for 1 h, conditions that are expected to
completely remove TA, showed a 2.3% weight loss above 230
C (plot not shown). Comparing the water extracted and
calcinated samples, we concluded that the weight loss
corresponding the residual TA in TA16-MSNP-2h and TA16-
MSNP-6h is ca. 1.1%. This amount of TA corresponds to the
surface coverage of ca. 6.3 103 TA molecules/nm2, which
was not detectable by solid state 13C NMR. In contrast, TA16-
MSNP-24h showed a larger weight loss (ca. 5%) at higher
temperature. This suggests that TA16-MSNP-24h particles
contain more TA. It is possible that at long reaction times,
TEOS condenses at the pore entrances, blocking TA removal
from the pores by water extraction.
Adsorption of Proteins. Mesoporous silica materials have
been used to adsorb various biomolecules with the purpose of
encapsulation or delivery.5153 Applications of conventional
MSNPs such as MCM-41 and SBA-15 in delivery of complex
molecules are limited because of the small pore sizes and
relatively large particles sizes. Because TA-MSNPs contain
remarkably large interconnected mesopores in uniform sub-
200-nm particles, we envision these particles to be an
outstanding candidate for biomacromolecule immobilization.
To support this notion, we investigated the equilibrium
adsorption capacities and adsorption/desorption kinetics of
proteins for TA16-MSNPs in phosphate buer under low and
high ionic strength conditions. Lysozyme (Lz, diameter 3.2 nm,
pI 11.4, MW = 14 KDa),54,66 bovine hemoglobin (BHb,
dimensions 6.4 5.5 5 nm, pI 7.4, MW = 64 KDa),55 and
bovine serum albumin (BSA, dimensions 4 4 14 nm, pI 4.8,
MW = 67 KDa)56,66 were selected as model proteins for their
relatively small size compared to that of the mesopores and for
diverse charge (Table 2). Nonporous silica nanoparticles with
245 14 nm diameter were synthesized by Stober method38
and used for comparison.
We found that positively charged proteins Lz and BHb were
absorbed by both TA16-MSNPs and Stober-SNP, but more
signicantly in the porous TA16-MSNPs (Table 3). The
increased protein adsorption by TA16-MSNPs suggests that the
proteins are adsorbed not only on the external surface of the
particles but also inside the mesopores. However, the 23-fold
increase in adsorption for Lz and 1.5-fold increase in adsorption
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Table 2. Potential of Silica Particles and Model Proteins in
Phosphate Buer and Phosphate Buer/0.1 M KCl Solution
potential, mV
particles 10 mM PBS 10 mM PBS, 0.1 M KCl
TA16-MSNP 25.5 3.6 1.4 0.3
Stober-SNP 49.2 0.2 4.0 0.3
Lz +10.0 0.3 +1.5 1.2
BHb +12.7 0.3 0.7 0.3
BSA 13.6 0.1 2.0 0.2
for BHb are signicantly smaller than expected based on the
60-fold increase in BET surface area compared to Stober-SNPs.
This suggests that not all of the mesopore surfaces are
accessible to the proteins and that a smaller protein (Lz) can
diuse into the TA16-MSNP mesopores easier compared to a
larger protein (BHb).57,58 Interestingly, the negatively charged
BSA was not adsorbed by nonporous Stober-SNPs, but TA16-
MSNPs trapped a noticeable amount of this protein (Table 3).
As discussed above, the amount of TA on the TA-MSNP
surface is negligible. Thus, despite the ability of TA to strongly
interact with proteins,5961 we believe that adsorption of
proteins on TA-MSNPs62,63 is mainly due to the electrostatic
interactions64 and hydrogen bonding65 between the carboxylic
and amino groups of the proteins66 and surface silanols of
TA16-MSNPs, which should be abundant due to the
purication of TA-MSNPs by water extraction (as opposed to
calcination). The nonporous silica nanoparticles also adsorbed
a signicant amount of proteins due to the formation of a
protein corona.67
To distinguish between the eects of electrostatics and
hydrogen bonding in the adsorption of proteins on TA16-
MSNPs, we added 0.1 M KCl to the phosphate buer solutions
to screen the electrostatic interactions. The potentials of silica
particles, which are highly negatively charged at low ionic
strength, and of the proteins were close to zero at high ionic
strength (Table 2). As expected, this resulted in decreased
adsorption for all proteins (Table 3). However, the fact that a
signicant amount of proteins was still adsorbed by the TA16-
MSNPs in the presence of KCl conrmed that hydrogen
bonding plays an important role in the protein adsorption by
TA16-MSNPs.
Finally, we studied the kinetics of protein adsorption/
desorption for TA16-MSNPs. The smallest protein, lysozyme,
reached its adsorption saturation in 3 h, whereas the larger
proteins, BHb and BSA, reached their saturation points in 6 and
8 h, respectively (Figure 5). Because of the large mesopore
openings, the protein adsorption rate of TA16-MSNPs is faster
than that for MCM-41 and SBA-15 particles.65,68 Because the
adsorption capacities of TA16-MSNPs were aected by the
ionic strength of the solution, we hypothesized that proteins
could be released under high ionic strength conditions. Thus,
TA16-MSNPs loaded with proteins in 10 mM phosphate buer
were dispersed in phosphate buer containing 0.1 M KCl, and
protein release was monitored using UVvis spectroscopy
(Supporting Information Figure S3). We found that larger
proteins were not released from TA16-MSNPs, and thus, their
adsorption is nearly irreversible, whereas the small protein,
lysozyme, was slowly released by TA16-MSNPs under these
conditions. These observations may be important for future
applications of TA-MSNPs in drug delivery and enzyme
immobilization. 69 Similarly, Sto ber-SNPs did not show
signicant protein release under these conditions.
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Chemistry of Materials
Table 3. Summary of protein adsorption by TA16-MSNPs and Stober-SNPs in phosphate buer and phosphate buer/KCl
solution
protein adsorption, mg/g
TA-MSNPs
protein phosphate buer 0.1 M KCl
Lz 77 12 31 4
BHb 397 36 305 34
BSA 130 41 32 41
Figure 5. Protein adsorption kinetics for TA-MSNPs. Lz, blue; BHb,
red; BSA, green.
Enzyme Encapsulation. Nanoparticle-immobilized en-
zymes and antibodies are useful in catalysis because of their
improved stability and activity50,70 due to connement
eects.7173 We studied the immobilization of mitochondrial
malic dehydrogenase (m-MDH), which catalyzes conversion of
oxaloacetate to L-malate. m-MDH has the molecular weight of
79 kDa, with 6.4 6.4 4.5 nm dimensions and pI of 8.0
8.5,74 and therefore, it is similar to BHb. We found that TA16-
MSNPs adsorbed 421 13 mg/g of m-MDH, double the
amount of the enzyme that was adsorbed on nonporous Stober-
SNPs (214 19 mg/g), indicating that the enzyme was
immobilized not only on the external surface of TA16-MSNPs
but also inside their mesopores. Moreover, the fact that the
adsorbed amount of m-MDH was comparable with BHb
suggests that proteins possessing comparable size, molecular
weights, and surface charges show similar adsorption capacity in
TA16-MSNPs, regardless of their amino acid sequence. The
loading of m-MDH in TA16-MSNPs is comparable to that
reported for a series of enzymes and MSNPs with 1040 nm
pores.68
We monitored the m-MDH-catalyzed conversion of
oxaloacetate to L-malate in the presence of -NADH at pH
7.5 and room temperature. By measuring the UV absorbance of
-NADH at 340 nm, we followed the conversion of oxaloacetic
acid into maleic acid and calculated the activity of the enzyme
immobilized in the TA16-MSNPs and on nonporous SNPs. A
solution of free m-MDH with the same concentration as the
enzyme adsorbed in TA16-MSNPs was used as a reference.
Figure 6 shows the change in -NADH concentration as a
function of time during the catalytic reaction, which was used to
calculate the conversion rates of -NADH and the catalytic
activity of m-MDH. The average calculated activity of m-MDH
immobilized in TA16-MSNPs was 56.8 2.9, ve times higher
than that of m-MDH adsorbed on nonporous SNPs (11.6
0.4). The amount of the enzyme that was adsorbed by TA16-
MSNPs was only double the amount adsorbed on the surface of
nonporous SNPs (Table 3), which suggests that m-MDH
molecules adsorbed inside the mesopores possess higher
catalytic activity due to connement eects.50
2035
Article
Stober-SNPs
phosphate buer 0.1 M KCl
3.3 3.9 0
273 31 109 22
0.5 0.9 8.2 7.4
Figure 6. Absorbance of -NADH as a function of time during the
conversion of oxaloacetate to L-malate catalyzed by m-MDH
immobilized in TA-MSNPs (blue), on nonporous SNPs (red), and
by m-MDH in solution (green).
The free enzyme has a 4-fold higher catalytic activity (202.4)
compared to the enzyme trapped inside the mesopores because
it is more accessible to the oxaloacetate molecules. However, its
activity decreased 40-fold, and the consumption of -NDAH
decreased from 100% to 5% in 40 min at room temperature. In
contrast, the enzyme encapsulated in the TA16-MSNPs did not
show any reduction in reaction rate, and the conversion
percentage of -NDAH remained at 76% for an extended
period of time, which means that the enzymes are protected by
the silica matrix. These observations are consistent with earlier
reports regarding the encapsulation of enzymes and antibodies
in MSNPs.50,75,76
. CONCLUSIONS
We described the preparation of a new type of templated
mesoporous silica nanoparticles (TA-MSNPs) with unique
pore morphology, large pore volume, and narrow pore size
distribution. The particles with nearly spherical shapes and
relatively monodisperse diameter of ca. 200 nm were
synthesized using tannic acid (TA) as a nonsurfactant, neutral
template in the mixture of ethanol and ammonia. The TA-
MSNPs pore size can be tuned from 6 to 13 nm by varying the
TA concentration. Compared to the conventional MSNPs, the
preparation of TA-MSNPs has several advantages. TA is
nontoxic, unlike the commonly used surfactant templates,
which is particularly important for biomedical applications of
MSNPs. The simple one-pot synthesis of TA-MSNPs using a
cheap and abundant template that can be easily removed by
water extraction is suitable for large-scale production.
TA-MSNPs are a rare example of mesoporous silica
nanoparticles with uniform size, shape, small dimensions and
large mesopores, making them excellent candidates for
biomacromolecule encapsulation. We explored the potential
of TA-MSNPs in protein immobilization. TA-MSNPs showed
high protein adsorption capacity of 77.1 mg/g for Lz, 396.5
mg/g for BHb, and 130.0 mg/g for BSA and rapid protein
dx.doi.org/10.1021/cm4039945 | Chem. Mater. 2014, 26, 20302037
Chemistry of Materials
uptake. The dierential protein adsorption capacity and high
adsorption rates may lead to TA-MSNP applications in protein
separations.
We also studied the catalytic activity of m-MDH
encapsulated (421 13 mg/g) inside TA-MSNPs. We found
that although the activity of the encapsulated m-MDH is four
times lower compared to that in solution, it is higher than that
of m-MDH adsorbed on nonporous SNPs (normalized for
surface area) and does not diminish with time, suggesting
potential applications of TA-MSNPs in biocatalysis.
We are presently studying the mechanism of the pore
formation using TA, selective surface modication of TA-
MSNPs with TA serving as a surface-protecting agent, and
preparation of biodegradable TA-MSNPs for delivery of
therapeutic biomacromolecules.
ASSOCIATED CONTENT
*
S Supporting Information
TEM images of TA-templated silica nanoparticles, TGA plots
for tannic-acid-templated silica nanoparticles, and protein
desorption kinetics for TA-MSNPs and Stober-SNPs. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*I. Zharov. E-mail: i.zharov@utah.edu.
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation
(MWN grant DMR-1008251) and the University of Utah
Research Foundation through a Seed Grant. We thank Prof.
Shelley Minteer (University of Utah) for helpful discussions
and for providing m-MDH and Ms. Fei Wu (University of
Utah) for her help with m-MDH activity measurements.
REFERENCES
(1) Wu, S.; Hung, Y.; Mou, C. Chem. Commun. 2011, 47, 9972
9985.
(2) Wang, Y.; Price, A. D.; Caruso, F. J. Mater. Chem. 2009, 19,
64516464.
(3) Lai, C.-Y.; Trewyn, B. G; Jeftinija, D. M.; Jeftinija, K.; Xu, S.;
Jeftinija, S.; Lin, V. S.Y. J. Am. Chem. Soc. 2003, 125, 44514459.
(4) Vivero-Escoto, J. L.; Slowing, I. I.; Trewyn, B. G.; Lin, V. S.-Y.
Small 2010, 6, 19521967.
(5) Kim, T.; Slowing, I. I.; Chung, P.; Lin, V. S.-Y. ACS Nano 2011, 5,
360366.
(6) Trewyn, B. G.; Giri, S.; Slowing, I. I.; Giri, S.; Chen, H.-T.; Lin, V.
S.-Y. Acc. Chem. Res. 2007, 40, 846853.
(7) Hartmann, M. Chem. Mater. 2005, 17, 45774593.
(8) Mihalcik, D. J.; Lin, W. Angew. Chem., Int. Ed. 2008, 47, 6229
6232.
(9) Villalonga, R.; Dez, P.; Sanchez, A.; Aznar, E.; Martnez-Manez,
R.; Pingarron, J. M. Chem.Eur. J. 2013, 19, 78897894.
(10) Lu, F.; Wu, S. H.; Hung, Y.; Mou, C. Y. Small 2009, 5, 1408
1413.
(11) Tallury, P.; Payton, K.; Santra, S. Nanomedicine 2008, 3, 579
592.
(12) Lu, Y.; Fan, H.; Stump, A.; Ward, T. L.; Rieker, T.; Brinker, C. J.
Nature 1999, 398, 223226.
(13) Kim, M.-H.; Na, H.-K.; Kim, Y.-K.; Ryoo, S.-R.; Cho, H. S.; Lee,
K. E.; Jeon, H.; Ryoo, R.; Min, D.-H. ACS Nano 2011, 5, 35683576.
(14) Tang, F.; Li, L.; Chen, D. Adv. Mater. 2012, 24, 15041534.
2036
Article
(15) Wan, Y.; Zhao, D. Chem. Rev. 2007, 107, 28212860.
(16) Han, Y.; Ying, J. Y. Angew. Chem., Int. Ed. 2005, 44, 288292.
(17) Gao, F.; Botella, P.; Corma, A.; Blesa, J.; Dong, L. J. Phys. Chem.
B 2009, 113, 17961804.
(18) Wang, J. G.; Zhou, H. J.; Sun, P. C.; Ding, D. T.; Chen, T. H.
Chem. Mater. 2010, 22, 38293831.
(19) Kim, M.; Na, H.; Kim, Y.; Ryoo, S.; Cho, H. S.; Lee, K. E.; Jeon,
H.; Ryoo, R.; Min, D. ACS Nano 2011, 5, 35683576.
(20) Lu, F.; Wu, S.; Hung, Y.; Mou, C. Small 2009, 5, 14081413.
(21) Zhao, D.; Feng, J.; Hou, Q.; Melosh, N.; Fredrickson, G. H.;
Chmelka, B. F.; Stucky, G. D. Science 1998, 279, 548552.
(22) Rejman, J.; Oberle, V.; Zuhorn, I. S.; Hoekstra, D. Biochem. J.
2004, 377, 159169.
(23) Wei, Y.; Jin, D.; Ding, T.; Shih, W.-H.; Liu, X.; Cheng, S. Z. D.;
Fu, Q. Adv. Mater. 1998, 10, 313316.
(24) Wei, Y.; Xu, J.; Dong, H.; Dong, J. H.; Qiu, K.; Jansen-Varnum,
S. A. Chem. Mater. 1999, 11, 20232029.
(25) Yanagisawa, T.; Shimizu, T.; Kuroda, K.; Kato, C. Bull. Chem.
Soc. Jpn. 1990, 63, 988992.
(26) Bau, L.; Bartova, B.; Arduini, M.; Mancin, F. Chem. Commun.
2009, 75847586.
(27) Mukherjee, I.; Mylonakis, A.; Guo, Y.; Samuel, S. P.; Li, S.; Wei,
R. Y.; Kojtari, A.; Wei, Y. Microporous Mesoporous Mater. 2009, 122,
168174.
(28) Mori, T.; Rezai-Zadeh, K.; Koyama, N.; Arendash, G. W.;
Yamaguchi, H.; Kakuda, N.; Horikoshi-Sakuraba, Y.; Tan, J.; Town, T.
J. Biol. Chem. 2012, 287, 69126927.
(29) Hagerman, A. E.; Riedl, K. M.; Jones, G. A.; Sovik, K. N.;
Ritchard, N. T.; Hartzfeld, P. W.; Riechel, T. L. J. Agric. Food Chem.
1998, 46, 18871892.
(30) Okuda, T.; Yoshida, T.; Hatano, T. In Phenolic Compounds in
Food and Their Eects on Health II; Huang, M.-T., Ho, C.-T., Lee, C.
Y., Eds.; ACS Symposium Series 507; American Chemical Society:
Washington, DC, 1992; pp 8797.
(31) Pierpoint, W. S. In Flavonoids in Biology and Medicine III. Current
Issues in Flavonoids Research; Das, N. P., Ed.; National University of
Singapore: Singapore, 1990; pp 497514.
(32) Aromal, S. A.; Philip, D. Phys. E (Amsterdam, Neth.) 2012, 44,
16921696.
(33) Sivaraman, S. K.; Elango, I.; Kumar, S.; Santhanam, V. Res.
Commun. 2009, 97, 10551059.
(34) Rainville, L.; Dorais, M.-C.; Boudreau, D. RSC Adv. 2013, 3,
1395313960.
(35) Kozlovskaya, V.; Kharlampieva, E.; Drachuk, I.; Cheng, D.;
Tsukruk, V. V. Soft Matter 2010, 6, 35963608.
(36) Gaelea, A.; Melnig, V.; Popa, M. I. J Mater. Sci. Mater. Med.
2010, 21, 12111223.
(37) Ejima, H.; Richardson, J. J.; Liang, K.; Best, J. P.; van Koeverden,
M. P.; Such, G. K.; Cui, J.; Caruso, F. Science 2013, 341, 154157.
(38) Stober, W.; Fink, A.; Bohn, E. J. Colloid Interface Sci. 1968, 26,
6269.
(39) Wang, W.; Gu, B.; Liang, L.; Hamilton, W. J. Phys. Chem. B
2003, 107, 34003404.
(40) McDonald, M.; Mila, I.; Scalbert, A. J. Agric. Food Chem. 1996,
44, 599606.
(41) Oh, H. I.; Hoff, J. E; Armstrong, G. S.; Haff, L. A. J. Agric. Food
Chem. 1980, 28, 394398.
(42) Van Sumere, C. V.; Albrecht, J.; Dedonder, A.; DePooter, H.,
Pe, I. In The Chemistry and Biochemistry of Plant Proteins; Harborne, J.
B., Van Summere, C. F., Eds.; Academic Press: New York, 1975; pp
212264.
(43) Bellotti, N.; del Amo, B.; Romagnoli, R. Ind. Eng. Chem. Res.
2012, 51, 1662616632.
(44) dIschia, M.; Napolitano, A.; Pezzella, A.; Meredith, P.; Sarna, T.
Angew. Chem., Int. Ed. 2009, 48, 39143921.
(45) Tuominen, A.; Sundman, T. Phytochem. Anal. 2013, 24, 424
435.
(46) Erel-Unal, I.; Sukhishvili, S. A. Macromolecules 2008, 41, 3962
3970.
dx.doi.org/10.1021/cm4039945 | Chem. Mater. 2014, 26, 20302037
Chemistry of Materials Article

(47) Shchepelina, O.; Kozlovskaya, V.; Kharlampieva, E.; Mao, W.;

Alexeev, A.; Tsukruk, V. V. Macromol. Rapid Commun. 2010, 31,
20412046.
(48) Storck, S.; Bretinger, H.; Maier, W. F. Appl. Catal., A 1998, 174,
137146.
(49) Kim, T.-W.; Slowing, I. I.; Chung, P.-W.; Lin, V. S.-Y. ACS Nano
2011, 5, 360366.
(50) Rahman, A. I.; Vejayakumaran, P.; Sipaut, C. S.; Ismail, J.; Chee,
C. K. Mater. Chem. Phys. 2009, 114, 328332.
(51) Popat, A.; Hartonon, S. B.; Stahr, F.; Liu, J.; Qian, S. Z.; Lu, G.
Q. Nanoscale 2011, 3, 28012818.
(52) Sun, Z.; Deng, Y.; Wei, J.; Gu, D.; Tu, B.; Zhao, D. Chem. Mater.
2011, 23, 21762184.
(53) Hudson, S.; Cooney, J.; Magner, E. Angew. Chem., Int. Ed. 2008,
47, 85828594.
(54) Yu, S.; Lee, S. B.; Kang, M.; Martin, C. R. Nano Lett. 2001, 1,
495498.
(55) Leoni, L.; Attiah, D.; Desai, T. A. Sensors 2002, 2 (3), 111120.
(56) Smith, C.; Kirk, R.; West, T.; Bratzel, M.; Cohen, M.; Martin, F.;
Boiarski, A.; Rampersaud, A. A. Diabetes Technol. Ther. 2005, 7, 151
162.
(57) Fan, J.; Lei, J.; Wang, L. M.; Yu, C. Z.; Tu, B.; Zhao, D. Y. Chem.
Commun. 2003, 21402141.
(58) Deere, J.; Magner, E.; Wall, J. G.; Hodnett, B. K. Chem.
Commun. 2001, 465466.
(59) Van Buren, J. P.; Robinson, W. B. J. Agric. Food Chem. 1969, 17,
772777.
(60) Gustavson, K. H. Chemistry of the Tanning Process; Academic
Press: New York, 1956.
(61) Haslam, E. J. Chem. Ecol. 1988, 14, 17891805.
(62) Kisler, J. M.; Daehler, A.; Stevens, G. W.; OConnor, A. J.
Microporous Mesoporous Mater. 2001, 4445, 769774.
(63) Vinu, A.; Murugesan, V.; Tangermann, O.; Hartmann, M. Chem.
Mater. 2004, 16, 30563065.
(64) Lei, C. H.; Shin, Y. S.; Liu, J.; Ackerman, E. J. J. Am. Chem. Soc.
2002, 124, 1124211243.
(65) Han, Y. J.; Stucky, G. D.; Butler, A. J. Am. Chem. Soc. 1999, 121,
98979898.
(66) Katiyar, A.; Ji, L.; Smirniotis, P. G.; Pinto, N. G. Microporous
Mesoporous Mater. 2005, 80, 311320.
(67) Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.;
Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.;
Landfester, K.; Schild, H.; Maskos, M.; Knauer, A. K.; Stauber, R. H.
Nature Nanotechnol. 2013, 8, 772781.
(68) Katiyar, A.; Yadav, S.; Smirniotis, P. G.; Pinto, N. G. J.
Chromatogr. A 2006, 1122, 1320.
(69) Wang, Y.; Caruso, F. Chem. Mater. 2005, 17, 953961.
(70) Bornscheuer, U. T. Angew. Chem., Int. Ed. 2003, 42, 33363337.
(71) Wang, Y.; Caruso, F. Chem. Commun. 2004, 15281529.
(72) Jin, W.; Brennan, J. D. Anal. Chim. Acta 2002, 461, 136.
(73) Wang, Y.; He, J.; Chen, J.; Ren, L.; Jiang, B.; Zhao, J. ACS Appl.
Mater. Interfaces 2012, 4, 27352742.
(74) Righetti, P. G.; Tudor, G. J. Chromatogr. 1981, 220, 115194.
(75) Seelan, S.; Sinha, A. K.; Kato, K.; Yokogawa, Y. Adv. Mater.
2006, 18, 30013004.
(76) Orita, T.; Kato, K.; Tomita, M. J. Ceram. Soc. Jpn. 2011, 119,
238245.

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