Disulfide-phenylazide: A reductively cleavable photoreactive linker for facile modification of nanoparticle surfaces

Article abstract of DOI:10.1039/c2tb00420h

In this study, a reductively cleavable photoreactive linker, disulfide-phenylazide (SPA), was synthesized and used to functionalize inorganic mesoporous silica nanoparticles (MSNs) by simple amidation reaction. Changed zeta potential, FT-IR and UV-vis or fluorescence spectra confirmed the successful photo-responsive functionalization of dextran (DSPA-MSNs) and immobilization of model avidin protein (ASPA-MSNs). With treatment by reducing agents, both DSPA-MSNs (with 10 mM DTT) and ASPA-MSNs (with 10 mM GSH) exhibited the reductive-responsive controllable release of coumarin 460 and bioactive protein. These results implied that the designed photo-affinity functional agent SPA with reductive-responsive cleavage provided a promising method to functionalize inorganic nanoparticles to improve their biocompatibility and biomedical applications. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2tb00420h

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Materials Chemistry B
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PAPER
Disulfide-phenylazide: a reductively cleavable
photoreactive linker for facile modification of
nanoparticle surfaces†
Cite this: J. Mater. Chem. B, 2013, 1,
1125
*
*
Qi Huang, Chunyan Bao, Yao Lin, Jian Chen, Zhenzhen Liu and Linyong Zhu
In this study, a reductively cleavable photoreactive linker, disulfide-phenylazide (SPA), was synthesized and
used to functionalize inorganic mesoporous silica nanoparticles (MSNs) by simple amidation reaction.
Changed zeta potential, FT-IR and UV-vis or fluorescence spectra confirmed the successful photo-
responsive functionalization of dextran (DSPA-MSNs) and immobilization of model avidin protein (ASPA-
MSNs). With treatment by reducing agents, both DSPA-MSNs (with 10 mM DTT) and ASPA-MSNs (with
10 mM GSH) exhibited the reductive-responsive controllable release of coumarin 460 and bioactive
protein. These results implied that the designed photo-affinity functional agent SPA with reductive-
responsive cleavage provided a promising method to functionalize inorganic nanoparticles to improve
their biocompatibility and biomedical applications.
Received 16th November 2012
Accepted 17th December 2012
DOI: 10.1039/c2tb00420h
www.rsc.org/MaterialsB
biomedical applications, the conjugation chemistry should be
simple, efficient, site-selective, and occur under mild biocom-
Introduction
In recent years, inorganic nanoparticles (NPs) such as silicon
dioxide (mesoporous silica, MSNs), semiconductor nanocrystals
(quantum dots, QDs), metal oxides (Fe3O4), and metals (Au, Ag)
have attracted growing interest in biomedical applications due
to their unique intrinsic physical properties and functions.1–3
For instance, besides acting as potential carriers for excellent
drug delivery systems, additional advantages can be provided by
MSNs because of their large surface area and pore volume, and
adjustable pore diameter;4–7 uorescence imaging can greatly
benet from the use of QDs owing to their remarkable and
tunable optical properties;8,9 magnetic resonance imaging
(MRI) of angiogenesis is greatly improved by using magnetic
NPs;10,11 and active participation in therapeutics can be
provided by using Au or Ag NPs.12–15
However, most of the above-mentioned inorganic NPs must
be modied and surface functionalized to avoid the agglomer-
ation of NPs and improve their biocompatibility.16 To date,
various methodologies have been developed.17 Compared to
conventional physical direct encapsulation and ligand changes,
chemical conjugation on the surface of NPs with different
hydrophilic polymers, macromolecules, and biomolecules is
more desirable because it provides a stable linkage and low
toxicity functionalization under in vivo conditions.18–21 For
patible conditions. For example, the Michael addition reaction
between maleimide and thiol, the coupling reaction between
NHS (N-hydroxysuccinimide) activated acid and primary amine,
the click reaction between alkyne and azide are the most
commonly used chemical conjugation methods.22,23 However,
these existing conjugation methods are always limited. Espe-
cially for functionalization with biomacromolecules, like
dextran without suitable active sites and proteins with excessive
active sites for conjugation, complicated multiple steps with low
efficiency are oen necessary. Moreover, the presence of the
toxic catalyst in click chemistry is always unfriendly and greatly
affects the bioactivity of biomacromolecules.
Phenylazide, known as the most popular photoaffinity labeling
agent, is easily photolyzed upon UV irradiation to generate a highly
reactive intermediate, phenylnitrene, which can react with neigh-
boring organic matter to form a covalent bond through C–H
insertion or abstraction and/or C]C addition reaction.24 The het-
erobifunctional nature of phenylazide makes it an excellent
candidate as a coupling agent for material synthesis and photo-
chemical immobilization of various components on the surface of
various substrates.25–29 Recently, in order to extend this covalent
immobilization method to a wide range of materials, several
groups have succeeded in introducing phenylazido groups on
biomacromolecules, such as chitosan, to achieve the controlled
release of drug or bioactive molecules.30,31 It is suggested that
phenylazide would be an excellent candidate as an effective and
photo-responsive linker to functionalize the surface of inorganic
NPs to improve their biomedical applications. However, to the best
of our knowledge, the research in this area is still limited so far.29
Shanghai Key Laboratory of Functional Materials Chemistry, Institute of Fine
Chemicals, East China University of Science and Technology, 130# Meilong Road,
Shanghai, 200237, China. E-mail: baochunyan@ecust.edu.cn; linyongzhu@ecust.
edu.cn; Fax: +86-21-64253742
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c2tb00420h
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In this work, taking MSNs as model inorganic NPs, we purication. Dichloromethane (DCM) was distilled from CaH2
prepared a novel reductively cleavable disulde-phenylazide before use, and NEt3 (TEA) was redistilled from CaH2 and dried
(SPA) linker to functionalize the surface of inorganic NPs. The over KOH pellets.
introduction of photoactive phenylazide enabled the modied
SPA-MSNs to be facilely functionalized by biocompatible
dextran or protein upon light irradiation. As Scheme 1 illus-
trates, upon light irradiation, the generated phenylnitrene
spectra were recorded on a Bruker Avance 500 (400 MHz)
helped to chemisorb biocompatible dextran polymers or
proteins, which are usually difficult to modify on the surface of
inorganic NPs directly. The introduction of a disulde bridge
linkage was designed to remove the dextran or protein wrapping
tion spectra were recorded on a Shimadzu UV-2550 UV-Vis
under reductive stimulus, which endowed the MSNs with
Characterization
Proton and carbon magnetic resonance spectra (1H, 13C NMR)
spectrometer. Mass spectra were recorded on a Micromass
GCTTM and a Micromass LCTTM. FT-IR spectra were obtained
on a Nicolet 380 FT-IR spectrometer with KBr pellets. Absorp-
spectrometer. The steady-state uorescence experiments were
performed on a Varian Cary Eclipse uorescence spectrometer.
potential as a carrier for controlled intracellular release since
glutathione (GSH) can effectively reduce disulde bonds and
exists in the cytoplasm in high concentration (ca. 10 mM).
Irradiations were carried out by using a high-pressure mercury
lamp with an intensity of 6 mW cmꢀ2 at a distance of 10 cm.
Powder XRD diffraction data were collected on a Rigaku D/max-
2550VB/PC diffractometer using Cu Ka radiation operated at
100 mA and 40 kV. Nitrogen adsorption and desorption
Experimental
Materials
All reagents were purchased from commercially available isotherms, surface area (SA), and median pore diameter (MPD)
sources such as Aldrich or TCI and used without further were measured using a Micromeritics ASAP2020M sorption
Scheme 1 Synthesis pathway of the dual stimuli-responsive disulfide-phenylazido (SPA) compound and schematic representation of surface functionalization and
release application of SPA-MSNs. Reagents and conditions: (a) EDC$HCl/NHS/DMAP/CH2Cl2, rt, 2 h, then N-(tert-butoxycarbonyl)cystamine, rt, overnight, 43% yield; (b)
TFA/CH2Cl2, rt, 1 h, 93% yield; (c) succinic anhydride/TEA/1,4-dioxane, reflux, 30 min, 64% yield; (d) EDC$HCl/NHS/DMAP/CH2Cl2, rt, 2 h, then MSNs, 24 h, 98% yield.
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analyzer. Specic surface areas and pore size distributions were
calculated using the Brunauer–Emmett–Teller (BET) and
Barrett–Joyner–Halenda (BJH) methods, respectively. The total
pore volumes were calculated from the nitrogen adsorption–
desorption data. Transmission Electron Micrographs (TEM)
were measured on a JEOL JEM-1400 at 100 kV. Thermogravi-
metric analysis (TGA) was carried out using a TGA/SDTA851e.
Zeta potential was measured using a Malvern Zeta sizer (Zeta
nano ZS).
Synthesis of SPA-graed mesoporous silica NPs (SPA-MSNs)
The aminopropyl modied mesoporous silica NPs (MSNs) were
synthesized as described in the literature34 and 80 mg of this
material was dispersed in 2 mL DCM. Then, SPA (28 mg, 0.07
mmol) was reacted with N-hydroxysuccinimide (NHS) (9 mg,
0.08 mmol), 4-dimethylaminopyridine (DMAP) (9 mg, 0.07
mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) (15 mg, 0.08 mmol) in 1.0 mL DCM, which
was kept stirring at room temperature for 2 hours before adding
to the NPs suspension. Finally, the mixture with the NPs was
stirred at room temperature for another 24 h, followed by
centrifuging (6000 r minꢀ1, 10 min) and washing with DCM, EA
and methanol, respectively. The obtained NPs were nally dried
under high vacuum. All the processes were performed in the
dark.
Synthesis of disulde-phenylazido (SPA) functional molecule
The synthesis route for SPA is shown in Scheme 1. 4-Aazido-
benzoic acid32 and N-(tert-butoxycarbonyl)cystamine33 are
synthesized according to the literature.
N-tert-Butoxycarbonyl-N0-para-azidobenzoic-cystamine
(compound 2). A solution of 4-azidobenzoic acid (2.3 g,
14 mmol), DMAP (1.7 g, 14 mmol), NHS (1.6 g, 14 mmol),
EDC$HCl (3.0 g, 16 mmol) in dry DCM was stirred at r.t. for 2 h,
then N-(tert-butoxycarbonyl)cystamine (3.6 g, 14 mmol) was
added to the above mixture. The reaction was continuously
stirred overnight, and then washed with water, dried over
Na2SO4 and evaporated to yield the crude product. Purication
with column chromatography (ethyl acetate (EA) : petroleum
ether (PE) ¼ 1 : 4) gave the product (2.4 g, 43% yield) as a yellow
Surface functionalization of SPA-MSNs with dextran (DSPA-
MSNs)
Typically, SPA-MSNs (10 mg) were dispersed in deionized water
and were sonicated to form a suspension. Dextran solution
(0.5% w/v, 70 K) in deionized water was added and vigorously
stirred for another 30 min. The mixture was irradiated for 20
min (a high-pressure mercury lamp with an intensity of 6 mW
cmꢀ2) at room temperature and puried by centrifugation
(6000 r minꢀ1, 10 min). Aer washing with deionized water and
methanol three times each, the particles were nally dried
under high vacuum.
1
solid. H NMR (CDCl3, 400 MHz): d ¼ 7.87 (d, J ¼ 8.0 Hz, 2H),
7.08 (d, J ¼ 8.0 Hz, 2H), 3.78 (q, J ¼ 6.0 Hz, 2H), 3.47 (q, J ¼ 6.0
Hz, 2H), 2.98 (t, J ¼ 5.6 Hz, 2H), 2.81 (t, J ¼ 6.8 Hz, 2H), 1.43 (s,
9H); 13C NMR (CDCl3, 100 MHz): 166.7, 143.3, 130.8, 129.0,
119.0, 79.8, 39.5, 39.0, 38.7, 37.9, 28.4; MS (EI): m/z: calcd for
C
16H23N5O3S2 [M + Na]+: 420.1; found 420.1.
N-para-Azidobenzoic-cystamine (compound 3). To a solution
of 2 (2.0 g, 5 mmol) in dry DCM (5 mL), TFA (5 mL) was added.
The loading and release of model dye coumarin 460
The reaction mixture was stirred at room temperature for 1 h, Here, the dye coumarin 460 (commercially purchased from
and then evaporated. Purication with column chromatography Aldrich) was selected as the hydrophobic model for the release
(CH3OH : EA ¼ 1 : 4) gave the product (1.4 g, 93% yield) as a application. Before surface functionalization with dextran, SPA-
yellow viscous solid. 1H NMR (d6-DMSO, 400 MHz): d ¼ 8.73 (t, MSNs (10 mg) were suspended in 10 mL DCM in the dark. Then
J ¼ 6.0 Hz, 1H), 7.97 (s, 2H), 7.91 (d, J ¼ 8.0 Hz, 2H), 7.22 (d, J ¼ 1 mg of coumarin 460 was added and stirred overnight. Then,
8.0 Hz, 2H), 3.57 (q, J ¼ 6.0 Hz, 2H), 3.12 (t, J ¼ 6.8 Hz, 2H), 2.93 dye-loaded SPA-MSNs were collected by centrifugation and
(q, J ¼ 6.8 Hz, 4H); 13C NMR (d6-DMSO, 100 MHz): 165.3, 142.4, dried under vacuum for 12 hours, which was followed by light
130.7, 129.0, 118.9, 38.6, 37.8, 36.7, 34.1; MS (EI): m/z: calcd for irradiation in the presence of dextran for surface functionali-
C
11H15N5OS2 [M + H]+: 298.1; found 298.1.
4-((2-((2-(4-Azidobenzamido)ethyl)disulfanyl)ethyl)amino)-4- determined as 2 wt% by UV-vis absorption calculation. Since the
zation as described above. The loading content of the dye was
oxobutanoic acid (SPA). A solution of 3 (1.3 g, 4 mmol), succinic model molecule coumarin 460 is hydrophobic and therefore
anhydride (0.44 g, 4 mmol) and TEA (1.2 mL, 8 mmol) in insoluble in pure water, a co-solvent of DMF and water (1 : 2)
anhydrous 1,4-dioxane (10 mL) was stirred at 80 ꢁC for 30 min. was used to measure the reducing-responsive release of
Aer cooling to room temperature, a yellow solid was precipi- coumarin 460 from dextran capped SPA-MSNs (DSPA-MSNs).
tated. The solid was ltered off and washed with EA to yield the The detailed procedure is as follows: 5 mg of the above prepared
product (1.1 g, 64% yield) as a yellowish solid. 1H NMR (d6- NPs was suspended and sonicated in 0.5 mL of DMF/water (1/2)
DMSO, 400 MHz): d ¼ 8.68 (t, J ¼ 6.0 Hz, 1H), 8.07 (t, J ¼ 4.0 Hz, co-solvent. The mixture was placed in cellulose ester membrane
1H), 7.91 (d, J ¼ 8.0 Hz, 2H), 7.22 (d, J ¼ 8.0 Hz, 2H), 3.55 (q, J ¼ tubes (molecular weight cut off 3000) which were immersed in
6.0 Hz, 2H), 3.34 (q, J ¼ 6.4 Hz, 2H), 2.90 (t, J ¼ 7.2 Hz, 2H), 2.78 5 mL of the corresponding co-solvent with or without dithio-
(t, J ¼ 7.2 Hz, 2H), 2.41 (t, J ¼ 6.8 Hz, 2H), 2.31 (t, J ¼ 6.4 Hz, 2H); threitol (DTT) and shaken at 37 ꢁC on a shaking table at
13C NMR (d6-DMSO, 100 MHz): 173.8, 171.1, 165.3, 142.3, 200 rpm. At different time intervals, 300 mL of the medium was
130.7, 129.0, 118.9, 38.7, 37.9, 37.2, 37.0, 30.0, 29.1; HRMS (EI): analyzed using uorescence spectroscopy at an excitation
m/z: calcd for C15H19N5O4S2 [M + Na]+: 420.0776; found wavelength of 450 nm by an ultra-micro cell and was then
420.0779.
returned to the medium.
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same conditions. In contrast to the amine modied MSNs with
poor dispersion in physiological solutions, DSPA-MSNs formed
a relatively stable suspension in water, where occulation was
not obvious until aer 2 days.
The immobilization, labeling and release of model protein
avidin
Here, avidin was selected as model for the protein immobili-
zation and release application. Before immobilization,
SPA-MSNs (5 mg) was suspended in 1 mL avidin solution (2 mg/
10 mL, PBS, 0.1 M, pH ¼ 7.4) in dark condition for 30 min. The
mixture was irradiated for 20 min at room temperature and
puried by centrifugation (6000 r minꢀ1, 10 min). Aer washed
with PBS buffer (0.1 M, pH ¼ 7.4) for three times, the particles
were dispersed in 0.5 mL of PBS–SDS buffer (0.1 M, pH ¼ 7.4,
2.5% w/v, sodium dodecyl sulfate polyacrylamide (SDS)). Then
0.5 mL PBS–SDS buffer (0.1 M, pH ¼ 7.4, 2.5% w/v) containing
0.1 mg 655-biotin (commercially purchased from Aldrich) (stock
solution: 1 mg in 1 mL DMSO) was added to the avidin immo-
bilized MSNs (ASPA-MSNs) and allowed the labeling reaction to
Then, to estimate the amount of SPA molecules attached on
the MSNs, FT-IR and UV-Vis spectroscopies were employed.
Fig. 1a shows the FT-IR spectra of SPA, MSNs, and SPA-MSNs.
Comparing with the spectrum of MSNs, the spectrum of SPA-
MSNs not only preserved the characteristics of the MSNs silica
structure but also displayed the characteristic stretching vibra-
tion peak of azido groups at 2310 cmꢀ1, which suggested the
successful conjugation between MSNs and SPA. Meanwhile, the
UV-Vis absorption spectra of SPA, MSNs, and SPA-MSNs are
shown in Fig. 1b. The appearance of maximum absorption at
270 nm of SPA-MSNs, originating from the p–p* transition of
the azido groups, also conrmed the attachment of SPA on
MSNs.27 In addition, by using the Beer–Lambert law, the
amount of SPA attached on the MSNs was determined as
0.34 mmol gꢀ1 calculated from a standard curve. This
ꢁ
proceed for 2 h at 37 C with frequent vortexing. Aer washed
with PBS–SDS buffer (0.1 M, pH ¼ 7.4, 2.5% w/v) for 9 times to
remove the excess biotin, the labeled NPs were collected by
centrifugation (6000 r minꢀ1, 10 min) and were dispersed in
1 mL of PBS–SDS buffer (0.1 M, pH ¼ 7.4, 2.5% w/v). To measure
the reductive-responsive release of protein from ASPA-MSNs,
0.5 mL of the 655-biotin labeled NPs suspension was added into
the corresponding PBS–SDS buffer (0.1 M, pH ¼ 7.4, 2.5% w/v)
in a 1.5 mL centrifuge tube with or without glutathione (GSH,
ꢁ
10 mM). Aer addition, the mixture was shaken at 37 C in a
shaking tables at 200 rpm. At different time intervals, the
supernatant of the mixture was analyzed using uorescence
spectroscopy at the emission of 680 nm in an ultra-micro cell
and was then returned back to the medium.
Results and discussion
Preparation and characterization of SPA-MSNs and the
functionalized DSPA-MSNs
Aminopropyl modied MCM-41 (MSNs) was synthesized using a
previous described sol–gel method.34 For SPA post-conjugation,
MSNs were rstly suspended in DCM solution by sonication,
while an excess amount of EDC/NHS activated SPA in DCM
solution was added to couple with the amine of MSNs by typical
amidation reaction. Aer centrifugal separation and washing
purication, the generated SPA-MSNs were further dispersed in
water by sonication and surface functionalized with dextran
upon light irradiation. The unbound dextran was removed by
repeating the centrifugation in water three times. The success-
ful modication was rstly veried by the changed zeta poten-
tial as listed in Table 1. The zeta potential of MSNs was 14.5 mV
in deionized water, while those of SPA-MSNs and DSPA-MSNs
were changed to 0.66 and ꢀ2.03 mV, respectively, under the
Table 1 Physicochemical characteristic of amine substituted MSNs, SPA-MSNs
and dextran coated DSPA-MSNs
Sample
SBET/m2 gꢀ1 DP/nm VP/cm3 gꢀ1 Zeta potential/mV
MSNs
SPA-MSNs
DSPA-MSNs
480
302
28
6
5
—
0.33
0.2
0.06
14.55
0.66
ꢀ2.31
Fig. 1 (a) FT-IR spectra of SPA, MNSs, and SPA-MSNs, respectively; (b) UV-Vis
absorption spectra of SPA in methanol solution (6 ꢂ 10ꢀ5 mol Lꢀ1), MSNs and
SPA-MSNs in water (0.11 mg mLꢀ1).
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conrmation of the successful attachment of SPA molecules on
MSNs made the further surface functionalization possible.
Aer the successful attachment of SPA on MSNs, the phe-
nylazide group of SPA-MSNs made it photo-reactive with
surrounding C–H bonds through main insertion (singlet) reac-
tions which provided further photo-responsive functionaliza-
tion with high efficiency.35 Here, biocompatible dextran
polymer was selected to functionalize the MSNs, which was
performed easily upon light irradiation aer dispersion in
water. The photoreaction between azido groups and dextran
was monitored by the change in the UV-vis spectrum. As shown
in Fig. 2, with the increasing of UV irradiation time, the
absorption of azido groups at 270 nm gradually decreased and
almost disappeared aer irradiation for 600 s. Thus, to ensure
the photo-chemical reaction between azido groups with dextran
progressed completely, the UV irradiation was performed for 20
min. TEM images of the NPs before (SPA-MSNs) and aer irra-
diation (DSPA-MSNs) were also recorded as shown in Fig. 3.
SPA-MSNs displayed uniform spherical nanoparticles with a
mean diameter of approximately 100 nm, and a highly ordered
mesoporous network with a hexagonal array, which is charac-
teristic of MCM-41 type MSNs. Aer dextran functionalization
upon light irradiation, shells around the particles are clearly
seen in Fig. 3b and the size also increased a little from 100 ꢃ
20 nm to 110 ꢃ 25 nm, which suggested the successful coating
of dextran on the surface of MSNs.
Moreover, small angle XRD measurements were prerformed
to investigate the ordered mesoporous structure of the NPs. As
shown in Fig. 4, three well-resolved diffraction peaks, assigned
as (100), (110), and (200) planes, were clearly observed in the
XRD pattern of SPA-MSNs, which was consistent with the
characteristic diffraction pattern of MCM-41 type MSNs. Aer
dextran functionalization, only one diffraction peak assigned to
the (100) plane survived and the peak intensities weakened a
lot, which further indicated that dextran was coated on the
surface as well as in mesopores. The N2 sorption isotherms of
SPA-MSNs and post-functionalized DSPA-MSNs are also
Fig. 3 TEM images of MSNs before and after functionalization with dextran: (a)
SPA-MSNs, and (b) DSPA-MSNs.
displayed in Fig. 5a. SPA-MSNs showed a typical type IV
isotherm with capillary condensation at 0.2–0.35 P/P0 indicating
uniform mesopores. Aer dextran functionalization upon irra-
diation, DSPA-MSNs showed loss of the hysteresis loop and a
signicantly lower nitrogen adsorption amount which affirmed
that the pores of NPs were blocked by dextran coating. Table 1
shows the detailed changes in surface area (m2 gꢀ1), pore
diameter (nm) and pore volume (cm3 gꢀ1) for the different
functional NPs. Aer dextran functionalization upon light
irradiation, the corresponding surface area and pore volume
were reduced to 28 and 0.06, respectively. Finally, the amount of
coated dextran was determined as 4.5 wt% by the calculation of
thermogravimetric analysis (TGA) as shown in Fig. 5b.
Besides of the light-responsive functionalization behavior of
the prepared SPA-MSNs, the introduction of the disulde bridge
linkage in SPA provided the NPs with reductive-responsive
cleavage behavior, which provided the potential for application
in controlled intracellular release. To estimate the responsive-
ness, a hydrophobic uorescence probe, coumarin 460, was
used as a loading model, which could be loaded in the meso-
porous silica materials by mixing them in DCM solution. Aer
dextran functionalization upon light irradiation, the dye-loaded
NPs were treated with DTT (10 mM), and at different time
Fig. 2 Change of UV-vis absorption spectra of SPA-MSNs (0.18 mg mLꢀ1) reac-
ted with dextran (0.5% w/v, 70 000) for different UV irradiation times. The insert
shows the decrease of the azide absorption at 270 nm with increased irradiation
time.
Fig. 4 XRD patterns of (a) SPA-MSNs and (b) dextran coated DSPA-MSNs.
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Fig. 5 (a) N2 adsorption–desorption isotherms and (b) TG curves of MSNS, SPA-
MSNs, and dextran coated DSPA-MSNs.
Fig. 6 (a) The fluorescence intensity of the dialysis medium from coumarin 460
loaded DSPA-MSNs (0.5% w/v, Mw dextran ¼ 70 000) without and with treatment
of DTT (10 mM) in water; (b) release of coumarin 460 from various DSPA-MSNs
with different molecular weights of dextran in the presence of 10 mM DTT.
intervals, the uorescent intensity of coumarin 460 in the
dialysis medium was tested to monitor the release. As shown in
Fig. 6a, almost three times stronger uorescence intensity was
detected compared with that of physical leaking from NPs protein for immobilization on the surface of SPA-MSNs (ASPA-
without DTT treatment. This can be attributed to the breaking MSNs). To estimate the activity of the immobilized protein upon
of the disulde bridge linkage that made the coated dextran light irradiation, 655-biotin was used for the specic avidin–
polymer leave the NPs, which enhanced the release of the biotin interactions. The obvious uorescence emission of
hydrophobic dye and veried the reduction-responsive 655-biotin of ASPA-MSNs, as shown in Fig. 7a, not only veried
controllable release of the dextran coated DSPA-MSNs. In the protein-immobilization ability of SPA-MSNs, but also
addition, various dextrans with different molecular weights proved that the photo-responsive progress would not destroy
were used to functionalize SPA-MSNs to further evaluate the the bioactivity of the immobilized protein. The reductive-
reductive-responsive release pattern. As shown in Fig. 6b, DSPA- responsive release of the immobilized protein was determined
MSNs with longer chain lengths of dextran showed larger and monitored by analyzing the uorescent intensity of 655-
amounts of release compared to those with shorter chain biotin–avidin in the supernatant by using biological glutathione
lengths of dextran. This is a reasonable result that longer chain (GSH, 10 mM) as a disulde-bond reducing agent, since DTT
lengths of dextran favored the formation of a better shell for would destroy the tertiary structure of the protein and denature
storing more coumarin dye molecules in the mesopores, which the protein, while GSH, which exists in cells in high concen-
improved the upload rate and subsequent release. All the above trations, would selectively cleave the disulde bond in
results indicated that our designed SPA-MSNs could be effec- compounds and maintain the protein bioactivity. As shown in
tively surface functionalized upon light irradiation and cleavage Fig. 7b, more than 8 times stronger uorescence intensity was
upon reductive stimulus that endowed the NPs system with detected compared to that of the NPs without GSH treatment,
effective light-controlled embedding and reductive-responsive and the release rate was determined as 82%. All these results
release behavior.
veried the successful photo-immobilization and controllable
In addition, to extend the biological applications of our dual delivery of protein, which endowed our designed SPA-MSPA
stimuli-responsive inorganic NPs, avidin was used as a model inorganic NPs with promising application in biomedical areas.
1130 | J. Mater. Chem. B, 2013, 1, 1125–1132
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of Shanghai Municipal Education Commission, and Funda-
mental Research Funds for the Central University.
Notes and references
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Acknowledgements
This work was supported by NSFC (21173078, 51273064),
Shanghai Sci. Tech. Comm. (11JC1403300), Innovation Program
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