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By Yang Zhao, Li-Ning Lin, Yang Lu, Shao-Feng Chen, Liang Dong, and Shu-Hong Yu*
Considering the therapeutic efficiency, the protection of biomedicines from degradation or interaction with the biological
environment before they reach the target sites is critical for
delivery vehicles.[1] Due to the large fraction of voids in their
inner space, hollow materials that can seal drugs, proteins,
enzymes and genes inside for protection purpose have attracted
increasing attention for encapsulation and delivery in biomedical applications.[2] Various materials – such as polymers, carbonates, metals and metal oxides – can be utilized as the shells
of hollow materials.[3] To meet the requirements of therapeutic
vehicles for biocompatibility, biodegradability, high loading efficiency and controlled release, hollow mesoporous silica nanospheres have been used in biomedical application due to their
high surface-to-volume ratio, suitable pore volume and also
the ease of modification of the outer surfaces.[4] The synthesis
methods mainly utilize mono-dispersed hard nanospheres as
templates. Various sizes of products with narrowly distributed
diameters are available by choosing suitable templates and synthesis can be easily scaled-up.[5]
Most of the recent strategies for encapsulation have focused
on loading drugs and genes after the formation of the hollow
mesoporous silica nanospheres. This is because of incompatible techniques involved in the nanosphere synthesis, such as
strong chemical corrosion, calcination at high temperature,
or the use of organic solvent or reactive metals,[6] will induce
the inactivation of therapeutic molecules. Alternatively, a novel
approach of “preloading” for encapsulation has been introduced.[7] This approach is designed to encapsulate biomedicines
during the synthesis of vectors. Especially for hollow materials,
the approach can, in a facile manner, utilize interior voids to
load cargoes and protect them inside. Recent preloading efforts
[∗] Ms. Y. Zhao,[†] Mr. Y. Lu, Dr. S.-F. Chen, Mr. L. Dong,
Prof. Dr. S.-H. Yu
Division of Nanomaterials and Chemistry
Hefei National Laboratory for Physical Sciences at Microscale
Department of Chemistry
National Synchrotron Radiation Laboratory
University of Science and Technology of China
Hefei, Anhui, 230026 (P. R. China)
E-mail: shyu@ustc.edu.cn
Fax: +86 551 3603040
Ms. L.-N. Lin[†]
Department of Materials of Science and Engineering
University of Science and Technology of China
Hefei, Anhui, 230026 (P. R. China)
[†] These authors contributed equally to this work.
DOI: 10.1002/adma.201002395
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Templating Synthesis of Preloaded Doxorubicin in Hollow
Mesoporous Silica Nanospheres for Biomedical
Applications
have mainly been directed towards polyelectrolyte capsules
prepared with inorganic templates using a layer-by-layer (LBL)
approach with mild conditions.[8] For example, Caruso et al.
reported a series of unique polymer capsules based on mesoporous silica particles for encapsulating versatile drugs and
genes, which can solve the problems about water-solubility and
degradation.[9] However, the capsules are usually limited by
their large diameters, which range from micrometers to submicrometers. Hollow materials of nanometer scale with controlled shapes are demanded for effective extravasation against
biological barriers.[10]
Therefore, to incorporate the benefits of preloading cargoes
and suitable diameters, we describe a mild approach to synthesize hollow mesoporous silica nanospheres of around 100 nm
based on porous CaCO3 nanospheres templates. An anticancer
drug, doxorubicin (DOX), is chosen in preloading for tumor
therapy intracellularly.
The synthetic procedures of hollow mesoporous silica@DOX
nanosphere (HMS@DOX) are shown in Scheme 1. Porous
amorphous CaCO3 nanospheres were prepared by a gas diffusion approach.[11] Figure 1A shows that they are uniform monodisperse, available in large scale and stable in absolute ethanol.
The interesting porous structure in Figure 1B enables the
versatile absorption of cargoes for preloading. The Brunauer–
Emmet–Teller (BET) surface area and total pore volume of
CaCO3 nanospheres are 317 and 0.424 m2 g−1, respectively.
First, DOX were encapsulated inside the porous CaCO3
nanospheres for preloading. Then, tetraethoxysilane (TEOS)
was continuously absorbed into the CaCO3. After centrifugation, the TEOS in the CaCO3 particles condensed to form silica
layers. Comparing with porous CaCO3 nanospheres, the TEM
image of the sample in this step (Figure 1C) exhibits substantial
structure and the surface charge dramatically decreases from
+54.01 mV (CaCO3) to +37.22 mV. The results suggest that the
CaCO3 nanospheres have been modified by and external silica
layer. The energy dispersive X-ray spectroscopy (EDS) results in
Figure 1D further confirms the existence of Si, and the massive
molar ratio of Ca to Si suggests the formation of thin external
silica layers.
In the next step, mesoporous silica shells were obtained by
utilizing TEOS as precursor and cetyltrimethylammonium bromide (CTAB) as a template.[12] After centrifugation, a porous
product (Figure 1E) can be obtained. The Ca/Si (Figure 1F)
molar ratio in this product is 7 to 1, based on inductive coupled plasma emission spectrometer (ICP) analysis. The exterior
surface molar ratio of Ca/Si is 3 to 13, based on X-ray photoelectron spectroscopy (XPS) analysis (see the Supporting Information, Table S1). The concentration of Si outside was attributed
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of HMS@DOX (see the SI, Figure S6). The
EDS spectrum in Figure 1H shows no Ca
signal. The low concerntration of Ca in ICP
analysis of the nanosphere composition (Ca/
Si 1:48) and XPS analysis of surface composition (Ca/Si 3:400, see the SI, Table S1) suggest the complete erosion of CaCO3. The differences in the binding energy of C1s, O1s, and
Si2p before and after erosion demonstrate the
consistent oxidation states of the elements in
both of the samples. The results show the stability of HMS@DOX during the erosion (see
Scheme 1. Schematic illustration for the preparation of HMS@DOX nanosphere.
the SI, Table S1). To observe the reservation of
preloaded DOX in HMS@DOX, fluorescent
images were obtained. The obvious red lights
in suspension, precipitated HMS@DOX and the red dots in fluto mesoporous silica shells. The results also demonstrated that
orescent images (see the SI, Figure S7) confirm qualitatively that
most of the Si was immobilized externally rather than internally
the DOX was included into hollow mesoporous silica spheres A
with the DOX.
quantitative loading efficiency of 9.92% for the reserved to origFinally, mild erosion of the CaCO3 core by ethylic acid solution
inal DOX was also measured.
(pH 4) enabled the DOX to be reserved, leading to the formation
Two control experiments were used to illuminate the funcof HMS@DOX. A large-scale image of HMS@DOX (Figure 1G
tion of DOX and TEOS in the synthesis of HMS@DOX.
and Figure S2 in the SI) exhibits typical mesoporous hollow
HMS@D-DOX nanospheres were obtained using a doubled
structures. The shell thickness and internal diameters of HMS@
initial amount of DOX in HMS@DOX in the first step of synDOX are 24 and 92 nm, respectively. Thermogravimetric analysis
thesis. Mesoporous silica nanopheres (MSN) were obtained
(TGA, Figure S3 in the SI) of the weight loss from 150 to 250 °C
with no DOX used during the synthesis. Although resulting
shows that the template CTAB was only 1.4% in HMS@DOX.[13]
in similar products (see the SI, Figure S8a and b) in each step
The type-IV isotherm, shown in Figure S4 (see the SI),[14] proves
as in HMS@DOX, the interior of MSN is obviously more subthe mesoporous property of silica shells. The Brunauer–Emmet–
stantial than HMS@DOX (see the SI, Figure S8c and d). The
Teller (BET) surface area and total pore volume of HMS@DOX
results suggest the hierarchical distribution of DOX and TEOS
are 537 and 0.485 m2 g−1, respectively. The sharp pore size disin porous CaCO3. In the case of HMS@DOX, the initially
tributions indicate that the HMS@DOX has a narrow Brunabsorbed DOX molecules occupy the central interior of the
auer–Joyner–Halenda (BJH) pore size distribution (see the SI,
CaCO3 particles. And the subsequently absorbed TEOS takes
Figure S5). XRD analysis demonstrates the amorphous property
Figure 1. A,B) TEM images of CaCO3 nanospheres. C) TEM image of the sample of HMS@DOX with silica layers after absorbing DOX and TEOS.
D) EDS measured on the marked area shown in (C). E) TEM image of the sample of HMS@DOX after the synthesis of mesoporous silica layer and
before the erosion of CaCO3. F) EDS measured on the marked area shown in ( E). G) TEM images of HMS@DOX nanospheres. The scale bar in inset
image is 140 nm. H) EDS measured on the spheres shown in inset image in (G).
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0.05
0.10
DOX
HMS@DOX
0.15
0.20
Cell Viability (%)
Cell Viability (%)
50
0.5
1.0
1.5
2.0
DOX Concentration for DOX (µg/ml)
release at pH 7.4
HMS@DOX
100
0
Experimental Section
(B)
MSN
100
100
80
80
60
60
40
40
20
20
0
0
5
10
Time (h)
15
20
25
Relative % Release
DOX Concentration in HMS@DOX (µg/ml)
(A)
after being treated with HMS@DOX. The results clearly reveal
the activity of DOX with the mechanisms of killing tumor cells
by DNA damage and topoisomerase II inhibition.[16] Thus,
active preloading of DOX in HMS@DOX can be proved. The
therapeutic effect of HMS@DOX and the release of DOX at pH
7.4 at predetermined time points exhibit a similar trend before
8 h (Figure 2B).
The high therapeutic effect by HMS@DOX was investigated by the uptake behavior in C6 cells. As shown in Figure 3,
increasing amoutns of DOX delivered by HMS@DOX gradually passed through the cytomembrane, assembled in cytoplasm, passed through the nucleus membrane and eventually
assembled in nucleus to kill cells at 0.5, 2, 4 and 8 h, respectively. Also, the nucleus gradually collapsed over time. The
effective uptake of DOX indicates the effective delivery of DOX
by HMS@DOX. Thus, the effective therapy may result from
the enhanced intracellular delivery, the pH-sensitive release and
the protection of DOX extracellular by HMS@DOX.
In summary, we have presented a mild approach for
preloading DOX in hollow mesoporous silica (HMS) spheres
by using porous CaCO3 spheres as templates. With biocompatible mesoporous silica shell, the DOX in HMS@DOX hollow
spheres exhibit greatly enhanced therapeutic effects over the
drug itself towards tumor cell. This might be caused by the protection of DOX extracellular by HMS@DOX, the pH-sensitive
release and the enhanced intracellular delivery. The approach
is suitable for preloading drugs that would be resistant to the
ammonia solution used for silica formation and the weak acidic
solution used for CaCO3 dissolution. Furthermore, the HMS@
DOX has great potential application in cancer therapy, which
worth more investigations in the future.
0
Figure 2. A) Cytotoxicity of DOX, HMS@DOX and MSN on C6 cells for 24 h. The concentration of MSN is consistent with HMS@DOX, which only shows the concentration of DOX in
HMS@DOX and DOX itself. B) Cytotoxicity of HMS@DOX on C6 cells and the release of DOX
from HMS@DOX in pH 7.4 at 0.5, 2, 4, 8, 12 and 24 h. C) The nucleus of blank C6. D) The
nucleus of C6 incubated with HMS@DOX for 12 h.
Adv. Mater. 2010, 22, 5255–5259
COMMUNICATION
up the peripheral interior of CaCO3 particles. First, TEOS in
the periphery condenses to form an external silica layer outside.
Then, mesoporous silica shells are obtained. After the erosion
of CaCO3, the area that once was DOX molecules becomes the
hollow center of HMS@DOX. The DOX that are restrained by
mesoporous silica shells become the preloading cargoes in the
final product. In the case of MSN with no DOX, the interior
CaCO3 are full only of absorbed TEOS which then condenses to
form silica networks. After the erosion, the reserved silica nets
inside become the core of MSN. Doubling the amount of DOX,
the increased area of DOX result in a decreased area covered
of TEOS. With the decreased nucleation sites of the first silica
layers, imperfect and thin mesoporous silica layers will be generated. Then, collapsed shells are obtained after the corrosion.
For biomedical applications, such as drug delivery, the DOX
release, uptake behavior, cytotoxicity and therapeutic effects were
examined. The releases of DOX from HMS@DOX increase
when the environment pH values decrease (see the SI, Figure
S9). The pH-sensitive DOX release might be beneficial at the
reduced pH in intracellular lysosomes, endosomes and certain
cancerous tissues for targeted release and controlled therapy at
the pathological sites.[15] Different therapeutic effects between
DOX and HMS@DOX on C6 cells were observed and the MSN
was introduced as blank sample for HMS@DOX. Figure 2A
exhibits the biocompatibility of MSN to C6 cells at the predetermined concentrations. Although the concentrations of DOX are
all ten times to the DOX in HMS@DOX, the cell viabilities are
similar in both of the samples. The results suggest the greatly
increased therapeutic effect of HMS@DOX over DOX itself.
Furthermore, compared with the normal morphology of C6
nucleus (Figure 2C), the nuclei become fragmented (Figure 2D)
Chemicals:
Calcium
chloride
dehydrate
(CaCl22H2O) of ultrapure grade for molecular biology
was purchased from Sigma-Aldrich Co. (Fluka,
USA). Doxorubicin hydrochloride was obtained from
Bio Basic Inc. (BBI, Canada). All other reagents were
of analytical grade and from Sinopharm Chemical
Reagent Co. Ltd. (SCRC, China).
Synthesis of Porous CaCO3 Particle: 200 mg
CaCl22H2O was dissolved in 100 mL absolute
ethanol in a glass bottle and covered by parafilm
with several pores. Then, the bottle was left in
a desiccator with two glass bottles of ammonia
bicarbonate (NH4HCO3). After gas diffusion reaction
for 3 d, the white nanospheres were centrifugated
and redispersed in absolute ethanol.
Synthesis of Mesoporous Silica Nanospheres
(MSN): 0.5 mL of 14 mg mL−1 CaCO3 nanospheres
and 0.5 mL tetraethoxysilane (TEOS) were dispersed
in 20 mL absolute ethanol for 2 h under stirring.
After the mixture was centrifugated twice to remove
the redundant TEOS and redispersed in 20 mL
absolute ethanol, 0.5 mL of ammonium hydroxide
(25 ∼ 28%, v/v) was added in for condensation
in 12 h. Then the products were redispersed in
absolute ethanol (0.3 mL) containing 7.25 mg
cetyltrimethylammonium bromide (CTAB) solution
for 2 h under stirring. 0.4 mL of 0.1 M NaOH
solution was subsequently added. After 0.5 h,
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Figure 3. A–C), D–F), G–I), J–L) are four series of confocal laser scanning microscopy (CLSM) images of HSM@DOX nanospheres with C6 cells at
the predetermined time points of 0.5, 2, 4 and 8 h, respectively. Each series can be classified to HSM@DOX (red dots), cell nucleus (being dyed in
blue by Hoechst 33258) and the merged images of both above, respectively. The scale bar represents 20 μm.
40 μL TEOS was added. After reaction for 12 h, the white suspended
nanospheres were centrifugated several times to remove the CTAB
molecules and the mesoporous silica nanospheres were obtained
after the erosion of the CaCO3 core in ethylic acid solution (pH 4).
After centrifugation twice, the mesoporous silica nanospheres were
redispersed in deionized water.
Synthesis of Hollow Mesoporous Silica@DOX Nanospheres (HMS@
DOX): First, 0.25 mL of 0.25 mg mL−1 doxorubicin (DOX) and
0.5 mL of 14 mg mL−1 CaCO3 nanospheres were dispersed in 20 mL
absolute ethanol for 2 h under stirring. Then, procedures were follwed
as for the synthesis of mesoporous silica nanospheres. 0.5 mL TEOS
was added under continuous stirring for 2 h. After the mixture was
centrifuged twice to remove the redundant TEOS and redispersed
in 20 mL absolute ethanol, 0.5 mL of ammonium hydroxide (25 ∼
28%, v/v) was added for condensation in 12 h. Then, the products
were redispersed in absolute ethanol (0.3 mL) containing 7.25 mg
cetyltrimethylammonium bromide (CTAB) solution for 2 h under
stirring. 0.4 mL of 0.1 M NaOH solution was subsequently added.
After 0.5 h, 40 μL TEOS was added. After reaction for 12 h, the white
suspended nanospheres were centrifugated several times to remove
the CTAB molecules and the mesoporous silica nanospheres were
obtained after the erosion of the CaCO3 core in ethylic acid solution
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(pH 4). After centrifugation twice, the mesoporous silica nanospheres
were redispersed in deionized water.
Characterization: Surface charges (zeta potentials) were measured on
a Nano-ZS Zetasizer (Malvern Instruments, UK) equipped with a 4.0 mW
internal laser. The morphologies of transmission electron microscope
(TEM) and high-resolution transmission electron microscope (HRTEM)
were observed by transmission electron microscope (TEM, Jeol-2010).
Powder X-ray diffraction (XRD) analysis was performed by Rigaku TTR-III
with Cu Kα radiation. The elements compositions were analyzed by
inductive coupled plasma emission spectrometer (ICP, SCIEX ELAN
DRC-e). X-ray photoelectron spectroscopy (XPS) analysis was performed
on an electron spectrometer (ESCALAB 250, Thermo-VG Scientific).
Fluorescence microscopy images were observed by fluorescence
microscope (IX71, Olympus) and UV light, excited at 470–490 nm
and 365 nm, respectively. Confocal laser scanning microscopy (CLSM)
images were observed by confocal laser scanning microscope (Leica
TCS SP2).
The loading efficiency of DOX was measured by the DOX in upper
solution by centrifugation. The fluorescence of DOX was investigated by
a fluorescence spectrophotometer (F-7000, Hitachi), excited at 468 nm.
The surface area was determined from the N2 adsorption–desorption
isotherms, using an accelerated surface area and porosimetry instrument
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Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
SHY acknowledges funding support from the National Basic Research
Program of China (2010CB934700), International Science & Technology
Cooperation Program of China (2010DFA41170), and the National
Natural Science Foundation of China (No. 50732006).
Received: July 4, 2010
Revised: February 8, 2010
Published online: October 22, 2010
Adv. Mater. 2010, 22, 5255–5259
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(ASAP 2000). The surface area of the activated carbonsampleswas
calculated, using BET equation(m2 g−1).
TGA experiment was performed on a TGA Q5000 under nitrogen
flow in the temperature range from 20 to 1 000 °C at a heating rate of
10 °C min−1.
Release of DOX From HMS@DOX: Series of 7.6 mg HMS@DOX was
suspended into 5 mL phosphate buffer saline (PBS) solution of different
pH (6.0, 7.4, 8.0). The samples were stirred at 37 °C and 200 rpm.
At the given time, predetermined samples were centrifuged and the
supernatant analyzed by a fluorescence spectrophotometer (F-7000,
Hitachi), excited at 468 nm.
MTT Assay in vitro: C6 cells were seeded into 96-well plates and
incubated for 24 h to 70% confluence. After removing the culture
medium, 100 μL of DMEM medium containing HMS@DOX, MSN and
DOX with predetermined concentrations were added to each well. Cells
treated with medium only served as a negative control groups. After 24 h
co-incubation or predetermined times, the medium was replaced with
20 μL of MTT solution (4 mg mL−1) and further cultured for 4 h. After
that, MTT solution was removed and 100 μL of DMSO was added and
the determinate was taken at 490 nm in a spectrophotometric microplate
reader (Bio-tek ELX800, USA).
Internalization of HMS@DOX in vitro: C6 cells were seeded onto
6-well plates at a density of 5 × 104 cells per well and cultivated in 1.0 mL
of DMEM medium respectively. After incubation for 24 h, the culture
medium were removed and DMEM medium of 62.8 μg mL−1 HMS@
DOX containing 0.2 μg mL−1 DOX were then added into each well. For
CLSM observation, after 0.5, 2, 4 and 8 h co-incubation, the cells was
washed by PBS and dyed with 10 μg mL−1 of Hoechst 33258 for 5 min,
following by being washed PBS. At last, the samples were excited with
330–380 nm for nucleus and 470–490 nm for HSM@DOX, respectively.
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