Fabrication of autofluorescent protein coated mesoporous silica nanoparticles for biological application

Article abstract of DOI:10.1039/c1cc16004d

Glucose sensitive and autofluorescent protein coated mesoporous silica nanoparticles are synthesized through a layer-by-layer technique. The resulting nano-composites can be adhered to the surface of a cell and embedded into the cell membrane. These unique features make this nanocomposite a good candidate as cell marker or drug carrier. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1cc16004d

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 12167–12169
www.rsc.org/chemcomm
COMMUNICATION
Fabrication of autofluorescent protein coated mesoporous silica
nanoparticles for biological applicationw
a
b
b
b
a
b
ab
Yang Yang,* Yi Jia, Liang Gao, Jinbo Fei, Luru Dai, Jie Zhao and Junbai Li*
Received 27th September 2011, Accepted 7th October 2011
DOI: 10.1039/c1cc16004d
Glucose sensitive and autofluorescent protein coated mesoporous
silica nanoparticles are synthesized through a layer-by-layer
technique. The resulting nano-composites can be adhered to
the surface of a cell and embedded into the cell membrane.
These unique features make this nanocomposite a good candidate
as cell marker or drug carrier.
Inorganic, porous, ceramic nanoparticles have attracted special
interest in biomedical fields. They are readily engineered with
the desired size, shape, and porosity. In these nanomaterials,
Scheme 1 (1) Schematic depiction Hb (red) and GOD (blue) immobilized
on the surface of MSN with GA as a cross-linker. (2) The reaction
scheme of coupled enzymatic system catalyzes b-D-glucose.
mesoporous silica nanoparticles (MSNs) possess not only
some common properties such as high specific surface area,
large pore volume and physicochemical stability, but also
several unique features such as facile multifunctionalization
interestingly, these autofluorescent and glucose responsive
composite nanoparticles can be aggregated to cell membranes
selectively. Therefore, as an original purpose, this organic/
inorganic composite nanomaterial was designed as a funda-
mental catalytic nanodevice or biomarker which could lead to
potential applications in biology and medicine.
1
–3
and tunable pore structures. More importantly, nano-sized
MSN particles are particularly significant in medical and
biological fields when they are used as cell markers, gene
transfection reagents or MRI contrast agents to realize the
4
–6
maximization of cellular uptake.
demonstrated the biocompatibility of these silica nanoparticles
Several studies also
As shown in Scheme 1, for protein coated MSN particles
7
–9
preparation, amino-group functionalized MSNs (MSN-NH )
1
in vitro and in vivo.
Therefore, the design of MSN-based
2
4,15
were firstly synthesized according to a published method.
nanomaterials has become a very active field in utilizing these
materials for controlled release, gene transport and expression,
Then, the GA and Hb at pH 7.2 phosphate buffer solution
(PBS) were alternately adsorbed by aldehyde groups of GA.
Each adsorption process was performed for 12 h and at 4 1C to
allow the entire adsorption. Subsequently, the same amount of
GOD was also immobilized on the surface of MSNs using the
same method. In the present work, different protein layer
numbers were immobilized on the surface of MSNs, such as
1
0–13
biomarking and other biotechnological applications.
In the present work, we fabricated the covalently-linked
hemoglobin (Hb) and glucose oxidase (GOD) multilayers on
the surface of MSN particles through a layer-by-layer method
as shown in Scheme 1. Compared with the protein physical
adsorption, the covalently protein coated nanoparticles can
keep higher stability as the biosensor in biological applications.
Herein, glutaraldehyde (GA) was used as crosslinking agent
and the formation of Schiff’s bases between Hb and GOD
layers enabled the composite MSN particles’ autofluorescence
properties. The GOD layers still kept their catalytic activity to
glucose when were coupled to the surface of MSNs. More
MSN@(Hb/GOD), MSN@(Hb/GOD)
GOD) . Fig. 1A shows a TEM image of the MSN particles
with an average diameter of 100–150 nm. The obvious MCM-
1 typical mesoporous channel structure of the nanospheres
2
and MSN@(Hb/
3
4
was observed with parallel stripes. Fig. 1B shows the MSNs
after being coated with a single Hb/GOD protein layer
(
MSN@(Hb/GOD)). A uniform thin layer exists on the outer
surface of the MSN particles and the mesoporous pore
structures are still observed obviously. When the particles
are coated with two controlling protein layers (MSN@(Hb/
a
National Center for Nanoscience and Technology, No. 11,
Bei Yi Tiao, Zhong guan cun, Beijing, 100190, China.
E-mail: yangyang@nanoctr.cn, jbli@iccas.ac.cn;
Fax: +86 10 82612629; Tel: +86 10 82545540
Beijing National Laboratory for Molecular Sciences (BNLMS), Key
Laboratory of Colloid and Interface Sciences, Institute of Chemistry,
Chinese Academy of Sciences, Beijing 100190, China
GOD)
because of the existence of the thicker protein layers
Fig. 1C). However, for the MSN particles coated with three
2
), the mesoporous pores structures become unclear
b
(
controlling protein layers (MSN@(Hb/GOD) ), the meso-
3
w Electronic supplementary information (ESI) available: Movie,
experimental details and Fig. S1–S7. See DOI: 10.1039/c1cc16004d
porous pore structures could not be observed and the particles
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 12167–12169 12167

View Article Online
Fig. 3 CLSM images of MSN@protein particles in Amplex Red and
b-D-glucose solution. The corresponding images were obtained by
excitation at 488 nm (A) and 559 nm (B); the overlapped image
(C) and the pseudo-bright field image (D).
Fig. 1 TEM images of protein coated MSNs by different assembly
layer numbers. (A) MSN; (B) MSN@(Hb/GOD); (C) MSN@(Hb/
2 3
GOD) ; (D) MSN@(Hb/GOD) .
began to aggregate due to relatively large protein amounts
Fig. 1D). Similar results can be proved by SEM images of
time. The fluorescence intensity increased more rapidly with
the increase in glucose solution concentration. The results
proved that the coupled proteins retain their enzymatic activity
and glucose sensitivity when immobilized on the surface of
MSN. The same result was obtained when the coupled reaction
was investigated using a spectrophotometer (Fig. S4 and
corresponding discussion in ESIw).
(
different protein layer coated MSN particles (Fig. S1, ESIw).
The results mentioned above proved that the surface and
thickness of protein layers can be controlled by the number
of assembly layers. In the following experiment, we take
3
MSN@(Hb/GOD) as example (marked as MSN@protein)
except where mentioned especially.
For further investigation, the MSN@protein particles were
observed by CLSM measurements, as shown in Fig. 3. When
the sample was observed by 488 nm laser excitation of CLSM,
we obtained a strong green fluorescent image of MSN@pro-
tein particles (Fig. 3A). Here, it needs to be mentioned that the
above system presented autofluorescent properties without
any external fluorochromes. However, the film consisting of
Hb and GOD did not present strong green fluorescence under
the same conditions except for the absence of GA as cross-
linker (Fig. S5 and S6, ESIw). This phenomenon can be
attributed to the n–p* transition of the CQN bonds in the
Schiff’s bases formed during the crosslinking reaction between
the amino groups of proteins and the aldehyde groups of
Our previous works reported the reaction scheme of the Hb/
1
6
GOD coupled enzymatic system for catalyzing b-D-glucose.
Briefly, GOD catalyzes the oxidation and hydrolysis of
b-D-glucose into gluconic acid and H . In the presence of
Hb, H can react with the fluorogenic reagent Amplex Red
2 2
O
2 2
O
to produce fluorescent compound resorufin. As shown in
Scheme 1, two enzymatic reactions were coupled together.
The coupled proteins still retain their electroactivity by this
1
6
technique. The adsorption/emission peak of resorufin at
B570/585 nm was detected from the UV-vis spectra and
fluorescence spectra and compared to the control experiment
when Amplex Red and b-D-glucose were added into an
MSN@protein dispersed solution (see Fig. S2 and S3, ESIw).
For proving the enzymatic activity and glucose sensitivity of
coupled proteins on the surface of MSN, production of
resorufin from the coupled reaction of GOD and Hb was
investigated using a fluorescence spectrophotometer. The time
scans of enzymatic reaction systems (MSN@protein) were
recorded at 585 nm, as shown in Fig. 2. After adding Amplex
Red and MSN@protein into a different concentration glucose
solution, the fluorescence intensity at 585 nm increased with
1
7,18
GA.
The very-close-coupled protein layer structure also
contributes to autofluorescent properties. The autofluorescent
properties make MSN@proteins become good carrier candidates
to be used in biological tracing because external fluorochromes
could influence the functionality of carriers and the biological
system. Furthermore, when the mixture of b-D-glucose and
Amplex Red was dropped onto the slide which carried
MSN@protein particles and observed by 559 nm laser excitation,
we obtained the red fluorescent image of MSN@protein
particles (Fig. 3B). The red dots of Fig. 3B come from the
catalytic reaction product, resorufin. The result also proved
that glucose catalysis occurs on the surfaces of the MSN
particles (Fig. 3C and D). It is anticipated to apply these
nano-carriers in developing a fluorescence sensor to detect
glucose in vitro.
Furthermore, we investigated the interactions of
MSN@protein nanoparticles with cell membranes. The
MSN@protein particles and the freshly trypsinized Hela cells
were incubated in vitro for 12 h at 37 1C. FM 4-64 marker was
used to stain cell membranes and CLSM was used to investigate
the interaction between particles and cells. From Fig. 4, the
protein coated MSN particles were found to be either
Fig. 2 (A) Real-time monitoring of glucose catalysis at different
concentrations within MSN@protein to analyze glucose consumption
with respect to time using fluorescence spectrofluorometer.
(B) Fluorescence intensity increased with the concentration of glucose
at 585 nm after 10 minutes for glucose consumption.
1
2168 Chem. Commun., 2011, 47, 12167–12169
This journal is c The Royal Society of Chemistry 2011

View Article Online
according to the number of assembly layers. The proteins still
kept their enzymatical activity after being immobilized on the
surface of nanoparticles. After successive addition of glucose
and Amplex Red to the sample, resorufin fluorescence occurs
from MSN particles. The process of enzyme catalysis was
proved by a UV spectrophotometer and a CLSM technique.
In addition, the composite nanoparticles, protein coated
MSN, can be absorbed and embedded into cell membranes.
The nanoparticles present autofluorescence properties because
of formation of Schiff’s bases between protein bilayers. They
also present good biocompatibility and low cytotoxicity. All
these features make these composite nanomaterials good
candidates as cell markers, biosensors or good drug carriers
in potential bioapplications.
Fig. 4 CLSM images of Hela cells stained with FM 4-64 and
MSN@protein suspensions after 12 h co-culturing. The corresponding
images of the MSN@protein nanoparticles (green) (A); FM 4-64
labeled cell membranes (red) (B); the overlapped image (C) and the
pseudo-bright field image (D). In the 3D rebuilding image (E) of the
cells, the main image shows the area of xy section, while the bottom
and the right images represent the corresponding xz and yz orthogonal
sections of the cells.
This work was financially supported by the National Basic
Research Program of China (973 Program 2009CB930101)
and the National Nature Science Foundation of China
(
21003027).
adsorbed or embedded into cell membranes. Fig. 4A shows the
image of MSN@protein particles with green fluorescence
which come from autofluorescence of crosslinking proteins.
Fig. 4B is the image of Hela cell membranes with red fluores-
cence from FM 4-64. While the overlapped image (Fig. 4C)
showed that the MSN@protein particles were either adsorbed
or embedded into cell membranes. In Fig. 4E, the 3D rebuilding
image of the cells also revealed that fluorescence completely
originated from the cell membranes. The top left image shows
the xy horizontal surface of cells, while the bottom and the
right images represent the corresponding xz and yz orthogonal
sections of the cells respectively. In the bottom and the right
images, the yellow dots show that the MSN@protein particles
were surrounded by the cell membrane (red). A 3-D movie of a
single cell after being cultured with MSN@protein nano-
particles is also provided as ESIw to support our hypothesis
Notes and references
1
Q. He, J. Zhang, J. Shi, Z. Zhu, L. Zhang, W. Bu, L. Guo and
Y. Chen, Biomaterials, 2010, 31, 1085–1092.
2 I. I. Slowing, B. G. Trewyn and V. S. Y. Lin, J. Am. Chem. Soc.,
007, 129, 8845–8849.
M. Vallet-Regı, F. Balas and D. Arcos, Angew. Chem., Int. Ed.,
007, 46, 7548–7558.
4 Y.-w. Jun, J.-w. Seo and J. Cheon, Acc. Chem. Res., 2008, 41,
79–189.
2
3
´
2
1
5
Y.-S. Lin, C.-P. Tsai, H.-Y. Huang, C.-T. Kuo, Y. Hung,
D.-M. Huang, Y.-C. Chen and C.-Y. Mou, Chem. Mater., 2005,
1
7, 4570–4573.
6 K.-S. Lee and M. A. El-Sayed, J. Phys. Chem. B, 2006, 110,
9220–19225.
1
7
Z. Tao, M. P. Morrow, T. Asefa, K. K. Sharma, C. Duncan,
A. Anan, H. S. Penefsky, J. Goodisman and A.-K. Souid, Nano
Lett., 2008, 8, 1517–1526.
8
L. Jinwoo, L. Youjin, Y. Jong Kyu, N. Hyon Bin, Y. Taekyung,
K. Hwan, L. Sang-Mok, K. Yoon-Mo, K. Ja Hun, P. Hyun Gyu,
C. Ho Nam, H. Misun, P. Je-Geun, K. Jungbae and H. Taeghwan,
Small, 2008, 4, 143–152.
(see the movie and the caption in ESIw). The result is different
from the polymer or lipid coated MSN systems in our previous
reports in which the MSN composites can be internalized into
1
5,19
cancer cells through an endocytic mechanism.
When
9 J. Lu, M. Liong, J. I. Zink and F. Tamanoi, Small, 2007, 3,
341–1346.
1
0 Y. Klichko, M. Liong, E. Choi, S. Angelos, A. E. Nel,
J. F. Stoddart, F. Tamanoi and J. I. Zink, J. Am. Ceram. Soc.,
another cell line (ESF cells) was selected, we found that
MSN@protein also presented to be cell membrane friendly
1
(
Fig. S7, ESIw). As is well known, cell membranes consist of
2
11 Y. Zhao, J. L. Vivero-Escoto, I. I. Slowing, B. G. Trewyn and
009, 92, S2–S10.
lipid bilayers and membrane proteins. In this work, the system
of MSN@protein might be more favorable to the structure of
the cell membrane and internalized by the cell membrane. We
are currently conducting more studies on the mechanism and
load the aim molecule inside MSN@protein to function inside
the cells. The cytotoxicity of MSN@protein was also investi-
V. S. Y. Lin, Expert Opin. Drug Delivery, 2010, 7, 1013–1029.
2 D. R. Radu, C.-Y. Lai, K. Jeftinija, E. W. Rowe, S. Jeftinija and
1
V. S. Y. Lin, J. Am. Chem. Soc., 2004, 126, 13216–13217.
13 Y.-Z. You, K. K. Kalebaila, S. L. Brock and D. Oupicky, Chem.
Mater., 2008, 20, 3354–3359.
1
1
1
1
4 S. Huh, J. W. Wiench, J.-C. Yoo, M. Pruski and V. S. Y. Lin,
Chem. Mater., 2003, 15, 4247–4256.
5 Y. Yang, X. Yan, Y. Cui, Q. He, D. Li, A. Wang, J. Fei and J. Li,
J. Mater. Chem., 2008, 18, 5731–5737.
6 L. Duan, Q. He, X. Yan, Y. Cui, K. Wang and J. Li, Biochem.
Biophys. Res. Commun., 2007, 354, 357–362.
7 Y. Jia, J. Fei, Y. Cui, Y. Yang, L. Gao and J. Li, Chem. Commun.,
2011, 47, 1175–1177.
18 W. Wei, L. Y. Wang, L. Yuan, Q. Wei, X. D. Yang, Z. G. Su and
2
0
gated by the standard MTT assay. After being incubated
with 0.5 mg MSN@protein samples for 12 h and 24 h, the
viability ratio of the Hela cells reached 91.7% and 89.5%
respectively. It demonstrates that MSN@protein nano-
particles have negligible influence upon the growth of cells
and exhibit low cytotoxicity.
G. H. Ma, Adv. Funct. Mater., 2007, 17, 3153–3158.
In this study, we synthesized Hb and GOD multilayers on
the surface of MSN nanoparticles through GA crosslinking
and layer-by-layer method. The protein layer can be controlled
1
9 Y. Yang, W. Song, A. Wang, P. Zhu, J. Fei and J. Li, Phys. Chem.
Chem. Phys., 2010, 12, 4418–4422.
20 T. Mosmann, J. Immunol. Methods, 1983, 65, 55–63.
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 12167–12169 12169