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2014 American Chemical Society 2030 dx.doi.org/10.1021/cm4039945 | Chem. Mater. 2014, 26, 20302037
Chemistry of Materials Article
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 Figure 4. SEM images of tannic-acid-templated silica nanoparticles
with two distinct adsorption steps, characteristic of a produced with dierent amounts of tannic acid: (a) TA4-TEOS, (b)
mesoporous structure (Figure 3).48 The small hysteresis TA8-TEOS, (c) TA16-TEOS, and (d) TA32-TEOS. Scale bars 500
loops around 0.3 P/P0, 0.5 P/P0, and 0.7 P/P0 correspond to nm.
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 demonstrated that TA4-MSNP, TA8-MSNP, and TA16-
porosity. The BET surface areas of TA4-, TA8-, and TA16- MSNP particles are mostly spherical and reasonably mono-
MSNP are smaller than those of most MCM-41 particles disperse with ca. 200 nm diameter (Table 1) and negligible
(>1000 m2/g)15 but are comparable to that of SBA-15 particles aggregation. TA4-MSNPs are not as uniform but possess a
with similar pore size (419 m2/g).49 The pore volume in TA- comparable average size with TA8-MSNP and TA16-MSNP. In
MSNPs is considerably larger compared to other reported addition to electron microscopy, the hydrodynamic diameter of
mesoporous nanoparticles. In the case of TA32-MSNPs, TA16-MSNPs was measured by dynamic light scattering (DLS)
2033 dx.doi.org/10.1021/cm4039945 | Chem. Mater. 2014, 26, 20302037
Chemistry of Materials Article
to be 249.2 60.5 nm, in good agreement with SEM data. In Table 2. Potential of Silica Particles and Model Proteins in
contrast, TA32-MSNPs exhibited agglomeration and their Phosphate Buer and Phosphate Buer/0.1 M KCl Solution
average size could not be accurately determined.
potential, mV
We also studied the eect of the reaction time on the
nanoparticle morphology and composition by isolating the particles 10 mM PBS 10 mM PBS, 0.1 M KCl
products after 2, 6, and 24 h of condensation (TA16-MSNP-2h, TA16-MSNP 25.5 3.6 1.4 0.3
TA16-MSNP-6h, TA16-MSNP-24h, respectively). All of the Stober-SNP 49.2 0.2 4.0 0.3
reaction times produced monodisperse spherical nanoparticles. Lz +10.0 0.3 +1.5 1.2
However, although TA16-MSNP-2h and TA16-MSNP-6h BHb +12.7 0.3 0.7 0.3
displayed a porous structure, a nonporous structure with BSA 13.6 0.1 2.0 0.2
dense core was observed for TA16-MSNP-24h particles
(Supporting Information Figure S1). for BHb are signicantly smaller than expected based on the
The composition of TA-MSNPs (puried by water 60-fold increase in BET surface area compared to Stober-SNPs.
extraction) as a function of reaction time was examined using This suggests that not all of the mesopore surfaces are
TGA under N2 atmosphere (Supporting Information Figure accessible to the proteins and that a smaller protein (Lz) can
S2). The ca. 1% weight loss observed in the temperature range diuse into the TA16-MSNP mesopores easier compared to a
35230 C can be attributed to the evaporation of water and larger protein (BHb).57,58 Interestingly, the negatively charged
degradation of silanol groups, and the weight loss above 230 C BSA was not adsorbed by nonporous Stober-SNPs, but TA16-
is likely due to carbonization of residual TA and other possible MSNPs trapped a noticeable amount of this protein (Table 3).
volatilizable impurities, which were not completely removed by As discussed above, the amount of TA on the TA-MSNP
water extraction. The weight loss observed for TA16-MSNP-2h surface is negligible. Thus, despite the ability of TA to strongly
and TA16-MSNP-6h was similar, ca. 3.4%. TGA of a sample interact with proteins,5961 we believe that adsorption of
calcinated at 650 C for 1 h, conditions that are expected to proteins on TA-MSNPs62,63 is mainly due to the electrostatic
completely remove TA, showed a 2.3% weight loss above 230 interactions64 and hydrogen bonding65 between the carboxylic
C (plot not shown). Comparing the water extracted and and amino groups of the proteins66 and surface silanols of
calcinated samples, we concluded that the weight loss TA16-MSNPs, which should be abundant due to the
corresponding the residual TA in TA16-MSNP-2h and TA16- purication of TA-MSNPs by water extraction (as opposed to
MSNP-6h is ca. 1.1%. This amount of TA corresponds to the calcination). The nonporous silica nanoparticles also adsorbed
surface coverage of ca. 6.3 103 TA molecules/nm2, which a signicant amount of proteins due to the formation of a
was not detectable by solid state 13C NMR. In contrast, TA16- protein corona.67
MSNP-24h showed a larger weight loss (ca. 5%) at higher To distinguish between the eects of electrostatics and
temperature. This suggests that TA16-MSNP-24h particles hydrogen bonding in the adsorption of proteins on TA16-
contain more TA. It is possible that at long reaction times, MSNPs, we added 0.1 M KCl to the phosphate buer solutions
TEOS condenses at the pore entrances, blocking TA removal to screen the electrostatic interactions. The potentials of silica
from the pores by water extraction. particles, which are highly negatively charged at low ionic
Adsorption of Proteins. Mesoporous silica materials have strength, and of the proteins were close to zero at high ionic
been used to adsorb various biomolecules with the purpose of strength (Table 2). As expected, this resulted in decreased
encapsulation or delivery.5153 Applications of conventional adsorption for all proteins (Table 3). However, the fact that a
MSNPs such as MCM-41 and SBA-15 in delivery of complex signicant amount of proteins was still adsorbed by the TA16-
molecules are limited because of the small pore sizes and MSNPs in the presence of KCl conrmed that hydrogen
relatively large particles sizes. Because TA-MSNPs contain bonding plays an important role in the protein adsorption by
remarkably large interconnected mesopores in uniform sub- TA16-MSNPs.
200-nm particles, we envision these particles to be an Finally, we studied the kinetics of protein adsorption/
outstanding candidate for biomacromolecule immobilization. desorption for TA16-MSNPs. The smallest protein, lysozyme,
To support this notion, we investigated the equilibrium reached its adsorption saturation in 3 h, whereas the larger
adsorption capacities and adsorption/desorption kinetics of proteins, BHb and BSA, reached their saturation points in 6 and
proteins for TA16-MSNPs in phosphate buer under low and 8 h, respectively (Figure 5). Because of the large mesopore
high ionic strength conditions. Lysozyme (Lz, diameter 3.2 nm, openings, the protein adsorption rate of TA16-MSNPs is faster
pI 11.4, MW = 14 KDa),54,66 bovine hemoglobin (BHb, than that for MCM-41 and SBA-15 particles.65,68 Because the
dimensions 6.4 5.5 5 nm, pI 7.4, MW = 64 KDa),55 and adsorption capacities of TA16-MSNPs were aected by the
bovine serum albumin (BSA, dimensions 4 4 14 nm, pI 4.8, ionic strength of the solution, we hypothesized that proteins
MW = 67 KDa)56,66 were selected as model proteins for their could be released under high ionic strength conditions. Thus,
relatively small size compared to that of the mesopores and for TA16-MSNPs loaded with proteins in 10 mM phosphate buer
diverse charge (Table 2). Nonporous silica nanoparticles with were dispersed in phosphate buer containing 0.1 M KCl, and
245 14 nm diameter were synthesized by Stober method38 protein release was monitored using UVvis spectroscopy
and used for comparison. (Supporting Information Figure S3). We found that larger
We found that positively charged proteins Lz and BHb were proteins were not released from TA16-MSNPs, and thus, their
absorbed by both TA16-MSNPs and Stober-SNP, but more adsorption is nearly irreversible, whereas the small protein,
signicantly in the porous TA16-MSNPs (Table 3). The lysozyme, was slowly released by TA16-MSNPs under these
increased protein adsorption by TA16-MSNPs suggests that the conditions. These observations may be important for future
proteins are adsorbed not only on the external surface of the applications of TA-MSNPs in drug delivery and enzyme
particles but also inside the mesopores. However, the 23-fold immobilization. 69 Similarly, Sto ber-SNPs did not show
increase in adsorption for Lz and 1.5-fold increase in adsorption signicant protein release under these conditions.
2034 dx.doi.org/10.1021/cm4039945 | Chem. Mater. 2014, 26, 20302037
Chemistry of Materials Article
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 Stober-SNPs
protein phosphate buer 0.1 M KCl phosphate buer 0.1 M KCl
Lz 77 12 31 4 3.3 3.9 0
BHb 397 36 305 34 273 31 109 22
BSA 130 41 32 41 0.5 0.9 8.2 7.4
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ACKNOWLEDGMENTS
This work was supported by the National Science Foundation
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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
(MWN grant DMR-1008251) and the University of Utah Singapore: Singapore, 1990; pp 497514.
Research Foundation through a Seed Grant. We thank Prof. (32) Aromal, S. A.; Philip, D. Phys. E (Amsterdam, Neth.) 2012, 44,
Shelley Minteer (University of Utah) for helpful discussions 16921696.
and for providing m-MDH and Ms. Fei Wu (University of (33) Sivaraman, S. K.; Elango, I.; Kumar, S.; Santhanam, V. Res.
Utah) for her help with m-MDH activity measurements.