Biomaterials 34 (2013) 1364e1371

Contents lists available at SciVerse ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Biomineralization inspired surface engineering of nanocarriers for pH-responsive,

targeted drug delivery
Zhaowei Chen a, b, Zhenhua Li a, b, Youhui Lin a, b, Meili Yin a, Jinsong Ren a, *, Xiaogang Qu a, *
a
State Key Laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun,
Jilin 130022, China
b
Graduate School of the Chinese Academy of Sciences, Beijing 1000039, China

a r t i c l e i n f o a b s t r a c t

Article history: Recent insight into the molecular mechanisms of natural biomineralization has enabled biomimetic
Received 8 October 2012 synthesis of functional organic-inorganic hybrid materials under mild reaction conditions. Here, we
Accepted 24 October 2012 describe a novel method to construct organic-inorganic hybrid on mesoporous silica nanoparticles by
Available online 9 November 2012
utilizing electrostatically absorbed hyaluronic acid (HA) as a reaction site for deposition of calcium
phosphate (CaP) minerals. The addition of another layer of HA on the CaP surfaces not only stabilizes the
Keywords:
nanocomposites but also confers target ability toward CD44 overexpressed cancer cells. The nano-
Biomineralization
materials enable controlled release of loaded anticancer drugs in acidic subcellular environments after
Hyaluronic acid
pH-responsive
receptor mediated endocytosis. More importantly, our study demonstrated that the cancer targeting
Targeted drug delivery nanomaterials dramatically enhanced cellular uptake and cytotoxicity toward breast carcinoma cells.
Cancer therapy These results thus open new opportunities for biomineralization guided nanostructure assemblies with
Mesoporous nanomaterials great potential for biomedical applications.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction utilization as coating materials on various implants to promote

Nature provides spectacular examples of biologically-formed
minerals (biomineralization), such as the single domain nano-
magnets of magnetotactic bacteria, the abrasion-resistant enamel
of teeth and other hard tissues in vertebrates [1e3]. Since bio-
minerals (frequently calcium phosphate) are formed in biological
environments under mild reaction conditions (i.e., aqueous solu-
tion, ambient temperature and pressure, near neutral pH), biomi-
metic approaches could provide new means to directly synthesize
new classes of organic-inorganic functional materials with complex
architectures and versatile properties [2,4e6]. For example, one can
take the ordered deposition of calcium phosphate salts on an
organic matrix by means of a heterogeneous nucleation process, in
which the crystal properties of CaP (such as crystal structures in
morphology and thickness) are readily controllable by tuning the
reaction conditions [4,7e10]. In terms of materials design, bio-
inspired CaP hybrid materials have attracted tremendous interest;
this is mainly due to the fact that these hybrids have a wide range of
very useful properties and potential applications. The favorable
aspects of CaP materials have led to their extensive biological
* Corresponding authors. Tel.: þ86 431 85262625.
E-mail addresses: jren@ciac.jl.cn (J. Ren), xqu@ciac.jl.cn (X. Qu).
bone-implant bonding or as building blocks of 2D/3D scaffolds for
application in tissue engineering [7,11,12]. Clinically, owing to their
“osteoconductive” properties, biomimetic CaP materials have
been widely used as bone substitutes in dentistry and orthopedics
[13e15]. Another striking example is that CaP based nanovectors
used for biomedical applications have attracted increasing research
attention in the past years because of their non-cytotoxicity and
pH-dependent biodegradability [2,16,17]. For instance, Epple et al.
proposed multi-shell CaP-DNA nanoparticles and CaP-DNA/siRNA
nanohybrids for effective transfection of cells [18e20]. Adair et al.
developed CaP nanoparticles that encapsulated both fluorophores
and chemotherapeutics for in vitro imaging and anticancer drug
delivery [21,22]. Despite these promises, the exploring of further
functions of CaP based functional biomimetic materials still
remains a big challenge in this field [2,16].
During the last decades, surface-functionalized, end-capped
mesoporous silica nanoparticles (MSPs) have emerged as appealing
carriers for controlled anticancer drug delivery owing to their low
toxicity, high surface area and large accessible pore volumes [23,24].
One important feature of this system is the controlled release of the
cargo from the host material only at the desired location responding
to specific external stimuli, such as enzymatic activity [25,26],
photo-irradiation [27e29], changes in redox state [30,31], pH
[27,32,33] and temperature [25,28,34,35]. Of the various stimuli

0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biomaterials.2012.10.060
 Z. Chen et al. / Biomaterials 34 (2013) 1364e1371
studied, changing the pH represents an effective strategy for cancer
therapy since extracellular pH in tumor tissue is slightly lower than
that in normal tissue, with endosomes and lysosomes exhibiting
even lower pH values. Notwithstanding these advances, many of the
existing pore-blocking species have disadvantages such as poor
applicability in aqueous solutions and the toxicity of the capping
agents used. This motivates us to design nanocomposites based on
natural materials capped MSPs that could respond to pH variations
for controlled drug release. Especially, the design and synthesis of
CaP based hybrid materials gated MSPs with target ability still
remains a significant challenge until now. Recently, Lee’s group
developed hydroxyapatite (HAp, Ca5(PO4)3OH) covered MSPs by
dispersing urease modified MSPs in acidic solution containing HAp
and urea, in situ CaP mineralization could be triggered on the
surface of MSPs [36]. Although promising, the protocol is compro-
mised by the requirement for tedious processes of the preparation
of nanomaterials and the lack of target ability which would lead to
insufficient uptake at tumor sites and decrease therapeutic effect.
Herein, a simple but efficient rout is developed for constructing
CaP hybrid materials end-capped MSPs, in which each particle is
composed of MSP as the core and natural organic-inorganic hybrid
as the shell. As shown in Scheme 1, to make the structure, we
firstly electrostatically absorb a layer of HA to the surface of
amino functionalized MSPs (designed as MSN@HA), then utilize the
carboxyl groups of HA backbone as the nucleation sites for CaP
heterogeneous nucleation. Thereafter, by precisely controlling the
following growing process, CaP mineral nanoshells with pH tunable
properties could be formed (designed as MSN@CaP). The additional
layer of HA to the mineral surfaces via chelation interactions
produces a colloidal stabilized nanocomposite (designed as
MSN@CaP-HA). In addition to providing colloidal stability, the
outmost layer of HA confers the nanosystem cancer cell target
ability through the special interaction with CD44, which is highly
expressed on various cancer cells [37e42]. Moreover, compared
with other templates or colloidal stabilizing agents, extracellular
matrix HA possesses several advantages which make it an attrac-
tive targeting moiety decorating drug/gene delivery or imaging
vehicles: it is biodegradable, non-immunogenic, non-toxic, and can
be chemically modified [39,43e49]. More importantly, as to the HA
or HA conjugates endocytosis pathway, they mainly accumulated in
endosomal and lysosomal compartments after CD44 mediated
uptake [39,49e51]. Thus, we propose to engineer MSPs with CaP-
HA hybrid shell to overcome one of the biggest challenges in
MSPs’ applications mentioned above, which should afford a new
type of pH-responsive targeted drug delivery platform to enhance
the therapeutic efficiency while minimizing side effects.
Scheme 1. Schematic representation of the biomineralization inspired CaP-HA hybrid
coated MSNs.
1365
2. Materials and methods
2.1. Materials
Nanopure water (18.2 MU; Millpore Co., USA) was used in all experiments and to
prepare all buffers. Tetraethylorthosilicate (TEOS), sodium hydroxide, and rhoda-
mine B were purchased from SigmaeAldrich. N-cetyltrimethylammonium bromide
(CTAB), fluorescein isothiocyanate (FITC) and 3-(2-aminoethylamino)-propyl-
trimethoxysilane were obtained from Alfa Aesar. Doxorubicin (Dox) was purchased
from Sangon (ShangHai, China). Sodium hyaluronate (MW z 10 kDa) and calcium
nitrate were purchased from Aladdin. All the chemicals were used as received
without further purification.
2.2. Apparatus and characterization
FTIR, XRD, SEM, TEM, N2 adsorptionedesorption, and UVevisible spectroscopy
zeta potential were employed to characterize the materials obtained. FT-IR analyses
were carried out on a Bruker Vertex 70 FT-IR Spectrometer. X-ray measurements
were performed on a Bruker D8 FOCUS. Powder X-ray Diffractometer using Cu Ka
radiation. SEM images were obtained with a Hitachi S-4800 FE-SEM. N2 adsorptione
desorption isotherms were recorded on a Micromeritics ASAP 2020M automated
sorption analyzer. The samples were degassed at 40  C for 5 h. The specific surface
areas were calculated from the adsorption data in the low pressure range using the
BET model and pore size was determined following the BJH method. TEM images
were acquired with a TECNAI G2 transmission electron microscope (Philips Elec-
tronic Instruments Co., The Netherlands) at 200 kV. UVeVis spectroscopy was
carried out with Cary 300 UV/Vis spectrophotometer.
2.3. Synthesis of MCM-41-type mesoporous silica nanoparticles
N-cetyltrimethylammonium bromide (CTAB, 0.50 g) was first dissolved in
240 mL of pure water. Sodium hydroxide (1.75 mL, 2 M) was added to CTAB solution,
followed by adjusting the solution temperature to 80  C. TEOS (2.5 mL) added by
dropwise while stirring was continued. The mixture was allowed to stir for 2 h to
give rise to white precipitates. The solid product was filtered, washed with deionized
water and ethanol, and dried in air. To remove the surfactant template (CTAB), the
white powder was refluxed for 24 h in a solution of 3.00 mL of HCl (37%) and
60.00 mL of ethanol followed by extensively washing with deionized water and
ethanol. The resulting surfactant-removed MCM-41 was placed under high vacuum
to remove the remaining solvent in the mesopores. As for fluorescein-labeled
mesoporous silica nanoparticles, fluorescein isothiocyanate (FITC, 2 mg) was reac-
ted with 60 mL of APTES in 1 mL ethanol overnight in the dark. CTAB (0.50 g) was first
dissolved in 240 mL of pure water. Sodium hydroxide (1.75 mL, 2 M) was added to
CTAB solution, followed by adjusting the solution temperature to 80  C. TEOS
(2.44 mL) and APTES-modified dye solution added by dropwise while stirring was
continued. The mixture was allowed to stir for 2 h to give rise to white precipitates.
Finally, the surfactant template, CTAB, was removed by refluxing in acidic ethanol
solution to give FITC-labeled MSP.
2.4. Chemical modification of the MSPs surface (surface amine functionalization)
Extracted MCM-41 nanoparticles (500 mg) were added to dry PhMe (50 mL) and
suspended as a result of stirring and sonication. 3-(2-aminoethylamino)-propyl-
trimethoxysilane (250 mL, 0.2 mmol) was added to the mixture using a micropipette.
The solution was placed under N2 and heated under reflux for 24 h. The nano-
particles were removed from solution by filtration using a fritted funnel and washed
with PhMe, THF, and EtOH, sequentially. The material was dried for 24 h using
vacuum. The presence of amino groups was confirmed by IR spectroscopy using the
KBr pellet method.
2.5. Dye loading and mineralization experiments
For rhodamine B loading, the MSNs (60 mg) were soaked in a solution of
rhodamine B (10 mg) in pure water for 24 h. The nanoparticles were then centri-
fuged and washed thoroughly with pure water to remove unloaded and adsorbed
molecules. Mineralization of the HA-NPs was carried out in the presence of calcium
nitrate and ammonium phosphate using a method similar to that developed by
Schmidt et al. for the liposomes. In brief, the stock solutions (0.1 M calcium nitrate
and 0.1 M ammonium phosphate) were prepared in deionized water. The HA coated
MSN nanoparticles were prepared as following, 20 mg loaded MSNs were dispersed
into 10 mL 0.1 M NaCl solution. Then, 20 mg HA, which were hydrated in 0.1 M NaCl
solution overnight to allow swelling and complete solubilization, were added into
the above solution stirred for 8 h. In order to induce mineralization, 0.03 mL calcium
nitrate solution was slowly added to the solution containing the MSN@HA and
stirred for 1 h. To the resulting solution, 0.02 mL of the ammonium phosphate
solution was slowly added and stirred for 1 h. This procedure of sequential addition
of the stock solutions was repeated 8 times to achieve effective mineralization. Then,
the suspension was allowed to age for 12 h without stirring. The obtained nano-
particles were then dispersed into 10 mL HA (2 mg/mL) solution and stirred for 12 h.
1366 Z. Chen et al. / Biomaterials 34 (2013) 1364e1371
The final substrates were lyophilized to obtain HA stabilized mineralization coated
MSNs. All the washing solutions were collected, and the loading of Rhodamine B
were calculated from the difference in concentrations of the initial and left dyes to
be approximately 38 mmol/g SiO2.
2.6. Dye release experiments
Rhodamine B-loaded MSN@CaP-HA (10 mg) material was dispersed in 5 ml
aqueous buffer solutions (pH 7.4 phosphate buffer and pH 4.5 acetate buffer) at
37 C. Aliquots (50 mL) were taken from the suspension at predetermined time
 absorption band at around 1700 cm1 in the sample can be
intervals, and replaced with an equal volume of the fresh medium. The delivery of
rhodamine dye from the pore to the buffer solution was monitored via the absor-
bance band of the dye centered at 553 nm.
2.7. CaP disintegration experiments
MSN@CaP-HA (10 mg) material was dispersed in 5 ml aqueous buffer solutions
(pH 7.4 phosphate and pH 4.5 acetate buffers) at 37  C, and replaced with an equal
volume of the fresh medium. The release rate of calcium ions was monitored by
taking a 50 mL sample and diluting it using Arsenazo III solution (400 mL, 0.2 mM) in
HBS (HEPES-buffered saline where [HEPES] ¼ 20 mM and [NaCl] ¼ 150 mM at pH 7.4).
The absorbance of Arsenazo III/Ca2þ complex in the solution was measured at
656 nm, and the concentration of calcium ions was calculated based on the standard
curve.
2.8. Dox loading and mineralization experiments
For Dox loading, the MSNs (60 mg) were soaked in a solution of Dox (4 mL, 1 g/L)
in nanopure water for 24 h. The nanoparticles were then centrifuged and washed
thoroughly with nanopure water to remove unloaded and adsorbed molecules. The
mineralization process was almost the same as above. The loading of Dox was
calculated from the difference in concentrations of the initial and left drugs by UV/
Vis spectroscopy to be approximately 56.4 mmol/g SiO2.
2.9. Cell culture
MDA-MB-231 and NIH-3T3 cells were cultured in 25 cm2 flasks in Isceve’s
Modified Dulbecco’s medium (IMDM) (Gibco) containing 10% (v/v) fetal bovine
serum (Gibco) at 37  C in an atmosphere of 5% (v/v) CO2 in air. The media were
changed every three days, and the cells were passaged by trypsinization before
confluence.
2.10. Confocal laser scanning microscopy (CLSM)
FITC exhibits intense green fluorescence under UV light. This property allows the
use of fluorescence to study the distribution of MSN@CaP-HA inside the cells. A
suspension of FITC-MSN@CaP-HA in PBS was introduced to culture medium over-
night to mimic the blood circulation process prior to the cellular uptake. MDA-MB-
231 and NIH3T3 cells were then incubated with nanoparticles in medium for 2.5 h,
and then after washing free nanoparticles, 10 mM lysotracker was added for specific
staining of the lysosomes of cancer cells for further 0.5 h incubation. Finally, the cells
were washed three times with PBS and transferred to serum free medium and then
examined by CLSM.
2.11. Cytotoxicity assays
MTT assays were used to probe cellular viability. MDA-MB-231 and NIH3T3 cells
were seeded at a density of 5000 cells/well (90 mL total volume/well) in 96-well
assay plates. After 24 h, drugs at the indicated concentrations were added and
cells were further incubated for 48 h. To determine toxicity, 10 ml of MTT solution
(BBI) was added to each well of the microtiter plate and the plate was incubated in
the CO2 incubator for an additional 4 h. The cells then were lysed by the addition of
100 ml of DMSO. Absorbance values of formazan were determined with Bio-Rad
model-680 microplate reader at 490 nm (corrected for background absorbance at
630 nm). Six replicates were done for each treatment group.
3. Results and discussion
We first synthesized MSPs functionalized with positively
charged groups that can electrostatically interact with HA by
treating MSPs with 3-(2-aminoethylamino)-propyltrimethox-
ysilane to afford MSNs. The amines of MSNs exhibited an NeH
bending vibration at 1580 cm1 (Fig. 1A-b). As depicted in
Figs. S1 and S2 (see Supporting Information) and Fig. 2a the
spherical particle shape and hexagonally packed mesoporous
structure of the MSNs are confirmed by scanning and transmission
electron microscopy (SEM, X-ray diffraction and TEM, respectively).
The nitrogen adsorptionedesorption isotherms of N2 adsorptione
desorption isotherms of MSNs showed a typical Type IV curve
with a specific surface area of 1320 m2 g1, average pore diameter
of 2.9 nm and a narrow pore distribution (Fig. 1D).
The successful mineralization of MSNs was confirmed by
various spectroscopy methods. As shown in Fig. 1A, the emerging
assigned to C]O stretching of the carboxyl groups contained
within the adsorbed HA molecules. The nucleation of CaP at the
interfaces was supported by the clear peaks corresponding OePeO
bending modes appearing around 600 and 560 cm1, PeO asym-
metric stretching mode at around 1000 cm1 [12]. Consistently, HA
templated mineralization process on MSNs was tracked by zeta
potential measurements in deionized water at each step (Fig. 1C).
The zeta potential of the MSNs decreased from þ16.85 mV to
27.92 mV indicating the adsorption of HA on the interfaces which
is important for the following heterogeneous CaP nucleation. After
mineralization, the obtained nanoparticles exhibited a zeta
potential of 5.87 mV suggesting that mineral deposition almost
completely shielded anionic carboxyl charges. The final zeta
potential reached 20.18 mV, which manifested the anchoring of
another layer of HA. The surrounding of the outmost layer of HA
stabilizes the nanocomposite though electrostatic repulsion among
the HA chains, which is advantageous for potential oncology
application. TEM investigations also provided direct evidence of the
CaP mineral distribution on the HA absorbed MSNs. As shown in
Fig. 2b, mineralized MSNs shows rather rough surfaces and blocked
mesopores, representing the heterogeneous nucleation of CaP
clusters on the exterior surface of MSNs. To confirm the presence of
calcium and phosphate in the shell, energy-dispersive X-ray spec-
troscopy (EDX) was used to analyze the rough surfaces (Fig. 2c).
TEM-associated selected-area electron diffraction (SAED) result
(Fig. 2d) demonstrated that no sharp electron diffraction rings were
found, indicating the formation of amorphous CaP which was the
characteristic product of template-induced mineralization at the
early stage [8,9]. Furthermore, the change of sorption type, together
with a decrease of surface area and pore size distribution, was
expected due to the capping effect of the nanoparticles (Fig. 1D).
Taken together, these results indicated the successful synthesis of
CaP-HA coated MSNs.
To investigate the pH-responsive gating behavior of the hybrid
nanomaterials, rhodamine B was loaded before CaP mineralization.
The resulting particles were then dispersed in buffer at different
pH’s to test their controlled release property. The absorbance
maximum of rhodamine B (553 nm) was plotted as a function of
time to generate a release profile. As shown in Fig. 3A, a lower pH
(pH 4.5) led to the fast release of the loaded rhodamine B molecules
and the release reached a plateau within 24 h with the release
amount of about 82%. In contrast, only negligible rhodamine B was
released from dye loaded MSN@CaP-HA at physiological pH (pH
7.4). The significant difference indicated the induced release in
lower pH was due to the degradation of the mineral composite at
the surface. In a control experiment, monolayer HA absorbed MSNs
exhibited sustained release patterns at both pH 4.5 and 7.4, further
supporting the conclusion that the mineral structure has a direct
effect on the accessibility of the pores and confers the superiority
merely adsorption of monolayer of organic molecules on MSNs.
These results imply that cargo leakage from the biomineralized
system could be significantly reduced during sample storage and
potential blood circulation before accumulating at the tumor area
by the EPR effect.
Next, the disintegration of the mineral coating was investigated
by monitoring the concentration of the released calcium ions as
a function of time. As shown in Fig. 3B, at lysosomal pH (pH 4.5),
 Z. Chen et al. / Biomaterials 34 (2013) 1364e1371 1367

Fig. 1. (A), (B) FTIR spectra of (a) MSPs, (b) MSNs, (c, c’) MSN@HA, (d, d’) MSN@CaP, and (e, e’) MSN@CaP-HA. (C) Zeta potential measured at each step of the coating process in
deionized water. (D) Nitrogen adsorptionedesorption isotherms for MSN (triangle) and mineralized MSN (circle). Corresponding pore size distributions derived from desorption
isotherm measurements and BJH methods. The error bars represent the standard deviation of three measurements.

most of the calcium ions were released within 2 h, implying the fast
disintegration of the mineral shell. However, only insignificant
calcium ions were released at pH 7.4. These findings suggested that
upon receptor mediated endocytosis by cells, the CaP mineral
coatings could be readily dissolved in acidic intracellular
compartments, whereas their dissolution elsewhere in the body
would be rather slow.
We next confirmed the targeting efficiency of the nano-
conjugates. MDA-MB-231 cells, which have a high expression of
CD44, were chosen as target cancer cells, whereas NIH3T3 cells
were used as control cells. It is evident from the flow cytometry
(Fig. 4) that a large shift was observed for MDA-MB-231 cells
treated with the FITC-labeled nanoconjugate (FITC-MSN@CaP-HA),
but no significant shift was observed for NIH-3T3 cells or MDA-MB-
231 cells treated with FITC-MSN@CaP, verifying that HA conjugated
nanoparticles have the capacity to target MDA-MB-231 cells
effectively. Corresponding confocal fluorescence microscopy
images were consistent with the flow cytometry results: a strong
green fluorescence was observed for MDA-MB-231 cells, but no
distinct fluorescence was seen for NIH3T3 cells (Fig. 5a, e). More-
over, competition experiments, using 5 mg/mL free HA, were per-
formed as described previously [43]. As shown in flow cytometry
that the binding and uptake of FITC-MSN@CaP-HA into MDA-MB-
231 cells could be blocked by excess HA (Fig. 4d). These results
could be attributed to the special interaction between HA and CD44
overexpressed on MDA-MB-231 cells. Since no binding occurs
between NIH3T3 cells and the HA on the nanoconjugates, neither
shift nor enhancement was observed. Thus, the adsorption of
another layer HA afforded the nanoconjugates target ability, which
indicated that the design renders the system the ability for target
drug delivery.
Previous reports suggest that HA nanoconjugates were taken up
into cells via CD44 mediated endocytosis and mainly located in the
acidic compartments such as endosomal and lysosomal vesicles
[39,49,50]. Therefore a co-localization study was carried out to
determine if the HA coated mineralized nanoparticles also accu-
mulated within the lysosomes and endosomes after endocytosis.
While incubating cells with FITC doped nanoparticles, the endo-
somes and lysosomes were labeled with red florescent lysotracker
(Fig. 5b, f). After 3 h of incubation, a green fluorescence of FITC was
apparent within MDA-MB-231 cells and co-localized with lyso-
tracker red fluorescence (Fig. 5d), implying that the FITC-labeled
nanoparticles had become highly concentrated within endosomes
and lysosomes of MDA-MB-231 cells. As mentioned above, CaP
mineral coatings could be readily dissolved in acidic intracellular
compartments, thus the enhanced internalization and low pH
inside lysosomes and endosomes should facilitate the release of the
anticancer drugs, which could improve the therapeutic efficiency.
To investigate the toxicity of CaP-HA hybrid coated nano-
particles, cell viability was analyzed by the 3-(4, 5-dimethylthiazol-
2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay in the pres-
ence of MSN@CaP, MSN@CaP-HA, doxorubicin (Dox) loaded
MSN@CaP-HA (Dox@MSN@CaP-HA) and free Dox (Fig. 6). Due to
the fact that MSNs, CaP and HA all exhibit low- or non-cytotoxicity,
the drug carrier: MSN@CaP and MSN@CaP-HA showed a negligible
effect on both kinds of cells, even at high concentrations. However,
growth inhibition of cells was observed after incubation with
both free Dox and Dox@MSN@CaP-HA, which presented dose-
1368 Z. Chen et al. / Biomaterials 34 (2013) 1364e1371

Fig. 2. TEM micrographs of MSNs (a) and mineralized MSNs (b) EDX spectra (c) and TEM-associated SAED patterns of the mineral coating (d).

dependent cytotoxicity behavior. Killing efficiency of
Dox@MSN@CaP-HA on MDA-MB-231 cells was significantly
increased when increasing the amount of nanoparticles. A similar
profile, but to a lower extent, was observed on NIH3T3 cells. In
addition, as shown in Fig. S3, the MTT results of another control cell
line (HEK 293T cells) were similar with that of NIH3T3 cells.
Moreover, at each concentration, Dox@MSN@CaP-HA showed
remarkably higher anticancer efficiency than free Dox. The mech-
anism involved in the augmentation of anticancer efficiency was
related on one hand to the presence of HA attached on the particle
surface and on the other hand to a large amount of CD44 receptors
overexpressed on tumor cell surface. Lesley and co-workers [52]
reported that cooperativity is the primary feature of HA binding
by cell surface CD44. This cooperativity is the result of multiple
binding sites on the repeating disaccharide ligand of HA backbone
and multiple closely arrayed receptors on the cell surface. As
demonstrated above, after receptor mediated endocytosis, the
nanoconjugates mainly accumulated in the endosomal and lyso-
somal compartments, where CaP mineral coatings could be readily
dissolved and facilitated drug release. Synthetically, the enhanced
intracellular uptake and the followed drug release resulted in
enhanced killing efficacy as well as improved targeting specificity

Fig. 3. (A) Release profiles of rhodamine B from mineralized MSNs at (a) pH 4.5, (d) pH 7.4 and HA absorbed MSN at (b) pH 7.4 and (c) pH 4.5. (B) Release profiles of calcium ions
from mineralized MSNs in different pH buffers. The error bars represent the standard deviation of three measurements.
 Z. Chen et al. / Biomaterials 34 (2013) 1364e1371 1369

Fig. 4. Flow cytometry analysis to monitor the binding of FITC doped nanoparticles with MDA-MB-231 cells (target cells) and NIH3T3 (control cells). (a), (e) cells only, (b), (f) FITC-
MSN@CaP, (c), (g) FITC-MSN@CaP-HA, (g), (h) FITC-MSN@CaP-HA after preincubation for 2 h with free HA (5 mg/mL).

Fig. 5. Confocal laser scanning microscopy investigation of the target ability and localization of FITC-MSN@CaP-HA to MDA-MB-231 cells (A) and NIH3T3 cells (B) each sample was
imaged for (a), (e) FITC, (b), (f) lysotracker, (c), (g) bright filed, and (d), (h) overlay. The images were obtained under magnification of 80.

Fig. 6. In vitro viability of MDA-MB-231 cells (target cells) and NIH3T3 cells (control cells) in the presence of MSN@CaP, MSN@CaP-HA, Dox@MSN@CaP-HA, and free DOX. The error
bars represent the standard deviation of three measurements.
1370
Fig. 7. (a), (b) Flow cytogram representing apoptosis assay based on Annexin V-FITC and PI staining. In the flow cytogram, the cells in Q3 region denotes live cells, Q4: apoptotic, Q2:
late apoptotic and Q1: necrotic cells. (c), (d) Laser scanning confocal microscope images of cancer cells. (a), (c) control cancer cells, (b), (d) Dox@MSN@CaP-HA treated cancer cells.
The images were obtained under magnification of 40.
than conventional chemotherapy using Dox only. As for NIH3T3
cells and HEK 293T cells, the relatively low killing efficiency of
Dox@MSN@CaP-HA can be attributed to the minimal internaliza-
tion of the nanovector owing to two possible reasons: (i) lack of
CD44 receptors on the cell surface; (ii) negatively charged surfaces
of the nanoparticles. Summarizing the results, we confirmed our
hypothesis that the CaP-HA hybrid capped MSNs could serve as
a pH-responsive controlled release system for enhanced cancer
target drug delivery.
It is known that DOX suppresses the growth of cancer cells
through intercalation with cellular DNA, inducing apoptosis. To
evaluate the cell apoptosis after treatment by DOX@MSN@CaP-HA
at DOX concentration of 6 mg/mL, Annexin V FITC and PI assay plus
flow cytometry were employed. As shown in Fig. 7b, a significantly
higher proportion of cell population treated with DOX@MSN@CaP-
HA is Annexin V FITC positive as compared to the untreated cells
(Fig. 7a). Fig. 7c, d shows representative morphology and DAPI-
fluorescence photographs of control and treated MDA-MB-231
cells. In the control group, MDA-MB-231 cells grow exuberantly,
with integrated nucleus structure, nucleolus, and clearly distin-
guished karyothecas, and the contents in the nucleus are homo-
geneous. But in the drug group, the majority cells have undergone
morphological changes to nearly spherical shape and shown
hypercondensed chromatin at the nuclear periphery in most cells.
These results support the fact that DOX@MSN@CaP-HA induces
MDA-MB-231 cells to undergo apoptosis.
Z. Chen et al. / Biomaterials 34 (2013) 1364e1371
4. Conclusion
In summary, we have successfully designed and synthesized
a natural organic-inorganic hybrid capped mesoporous silica
nanocontainers that is based on biomineralization mechanism and
responds to pH-stimuli. In preparation process, polyanionic HA was
attached directly to the outlet of the mesopores by the electrostatic
interaction and served as a template for CaP heterogeneous
nucleation to entrap guest molecules within the mesopores. The
attachment of another layer of HA on the mineral surface enhanced
the colloidal stability of the nanoparticles as well as conferred
cancer cell target ability. After CD44 mediated cellar uptake, cargo
release was triggered by the disintegration of the CaP mineral
coating in acidic subcellular compartments. Moreover, we have
successfully demonstrated that the HA nanoconjugates showed
a remarkably enhanced efficiency in killing cancer cells while
sparing normal cells. The low cytotoxicity, targeted cellular uptake
properties, and efficient intracellular pH-stimuli drug release
provide a basis for potential in vivo controlled release biomedical
applications.
Acknowledgments
The authors are grateful for the referee’s helpful comments
on the manuscript. Financial support was provided by the
National Basic Research Program of China (2011CB936004 and
 Z. Chen et al. / Biomaterials 34 (2013) 1364e1371
2012CB720602) and the National Natural Science Foundation of
China (Grants 20831003, 21210002, 20833006, 90913007,
21072182).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biomaterials.2012.10.060.
References
[1] Palmer LC, Newcomb CJ, Kaltz SR, Spoerke ED, Stupp SI. Biomimetic systems
for hydroxyapatite mineralization inspired by bone and enamel. Chem Rev
2008;108:4754e83.
[2] Cai Y, Tang R. Calcium phosphate nanoparticles in biomineralization and
biomaterials. J Mater Chem 2008;18:3775e87.
[3] Xia F, Jiang L. Bio-inspired, smart, multiscale interfacial materials. Adv Mater
2008;20:2842e58.
[4] Haase NR, Shian S, Sandhage KH, Kröger N. Biocatalytic nanoscale coatings
through biomimetic layer-by-layer mineralization. Adv Funct Mater 2011;21:
4243e51.
[5] Murphy WL, Mooney DJ. Bioinspired growth of crystalline carbonate apatite
on biodegradable polymer substrata. J Am Chem Soc 2002;124:1910e7.
[6] Liu K, Jiang L. Multifunctional integration: from biological to bio-inspired
materials. ACS Nano 2011;5:6786e90.
[7] Yeo MG, Kim GH. Preparation and characterization of 3D composite scaffolds
based on rapid-prototyped PCL/b-TCP struts and electrospun PCL coated with
collagen and ha for bone regeneration. Chem Mater 2011;24:903e13.
[8] Schmidt HT, Ostafin AE. Liposome directed growth of calcium phosphate
nanoshells. Adv Mater 2002;14:532e5.
[9] Schmidt HT, Gray BL, Wingert PA, Ostafin AE. Assembly of aqueous-cored
calcium phosphate nanoparticles for drug delivery. Chem Mater 2004;16:
4942e7.
[10] Nudelman F, Pieterse K, George A, Bomans PHH, Friedrich H, Brylka LJ, et al.
The role of collagen in bone apatite formation in the presence of hydroxy-
apatite nucleation inhibitors. Nat Mater 2010;9:1004e9.
[11] Carlo Reis EC, Borges APB, Araújo MVF, Mendes VC, Guan L, Davies JE. Peri-
odontal regeneration using a bilayered PLGA/calcium phosphate construct.
Biomaterials 2011;32:9244e53.
[12] Liu H, Xi P, Xie G, Shi Y, Hou F, Huang L, et al. Simultaneous reduction and
surface functionalization of graphene oxide for hydroxyapatite mineraliza-
tion. J Phys Chem C 2012;116:3334e41.
[13] LeGeros RZ. Calcium phosphate-based osteoinductive materials. Chem Rev
2008;108:4742e53.
[14] Park JK, Shim J-H, Kang KS, Yeom J, Jung HS, Kim JY, et al. Solid free-form
fabrication of tissue-engineering scaffolds with a poly(lactic-co-glycolic
acid) grafted hyaluronic acid conjugate encapsulating an intact bone
morphogenetic proteine2/poly(ethylene glycol) complex. Adv Funct Mater
2011;21:2906e12.
[15] Moreau JL, Xu HHK. Mesenchymal stem cell proliferation and differentiation
on an injectable calcium phosphate-chitosan composite scaffold. Biomaterials
2009;30:2675e82.
[16] Epple M, Ganesan K, Heumann R, Klesing J, Kovtun A, Neumann S, et al.
Application of calcium phosphate nanoparticles in biomedicine. J Mater Chem
2010;20:18e23.
[17] Han S-Y, Han HS, Lee SC, Kang YM, Kim I-S, Park JH. Mineralized hyaluronic acid
nanoparticles as a robust drug carrier. J Mater Chem 2011;21:7996e8001.
[18] Sokolova VV, Radtke I, Heumann R, Epple M. Effective transfection of cells
with multi-shell calcium phosphate-DNA nanoparticles. Biomaterials 2006;
27:3147e53.
[19] Klesing J, Chernousova S, Epple M. Freeze-dried cationic calcium phosphate
nanorods as versatile carriers of nucleic acids (DNA, siRNA). J Mater Chem
2012;22:199e204.
[20] Kozlova D, Chernousova S, Knuschke T, Buer J, Westendorf AM, Epple M. Cell
targeting by antibody-functionalized calcium phosphate nanoparticles.
J Mater Chem 2012;22:396e404.
[21] Kester M, Heakal Y, Fox T, Sharma A, Robertson GP, Morgan TT, et al. Calcium
phosphate nanocomposite particles for in vitro imaging and encapsulated
chemotherapeutic drug delivery to cancer cells. Nano Lett 2008;8:4116e21.
[22] Morgan TT, Muddana HS, Altinoglu EI, Rouse SM, Tabakovic A, Tabouillot T,
et al. Encapsulation of organic molecules in calcium phosphate nano-
composite particles for intracellular imaging and drug delivery. Nano Lett
2008;8:4108e15.
[23] Kim T-W, Slowing II, Chung P-W, Lin VS-Y. Ordered mesoporous polymer-
silica hybrid nanoparticles as vehicles for the intracellular controlled release
of macromolecules. ACS Nano 2010;5:360e6.
1371
[24] Rosenholm JM, Meinander A, Peuhu E, Niemi R, Eriksson JE, Sahlgren C, et al.
Targeting of porous hybrid silica nanoparticles to cancer cells. ACS Nano 2008;
3:197e206.
[25] Chen C, Geng J, Pu F, Yang X, Ren J, Qu X. Polyvalent nucleic acid/mesoporous
silica nanoparticle conjugates: dual stimuli-responsive vehicles for intracel-
lular drug delivery. Angew Chem Int Ed 2011;50:882e6.
[26] Park C, Kim H, Kim S, Kim C. Enzyme responsive nanocontainers with cyclo-
dextrin gatekeepers and synergistic effects in release of guests. J Am Chem Soc
2009;131:16614e5.
[27] Aznar E, Marcos MD, Martinez-Manez RN, Sancenon Fl, Soto J, Amoros P, et al.
pH- and photo-switched release of guest molecules from mesoporous silica
supports. J Am Chem Soc 2009;131:6833e43.
[28] Yang X, Liu X, Liu Z, Pu F, Ren J, Qu X. Near-infrared light-triggered, targeted
drug delivery to cancer cells by aptamer gated nanovehicles. Adv Mater 2012;
24:2890e5.
[29] Mal NK, Fujiwara M, Tanaka Y. Photocontrolled reversible release of guest
molecules from coumarin-modified mesoporous silica. Nature 2003;421:
350e3.
[30] Liu R, Zhao X, Wu T, Feng P. Tunable redox-responsive hybrid nanogated
ensembles. J Am Chem Soc 2008;130:14418e9.
[31] Luo Z, Cai K, Hu Y, Zhao L, Liu P, Duan L, et al. Mesoporous silica nanoparticles
end-capped with collagen: redox-responsive nanoreservoirs for targeted drug
delivery. Angew Chem Int Ed 2011;50:640e3.
[32] Chen C, Pu F, Huang Z, Liu Z, Ren J, Qu X. Stimuli-responsive controlled-release
system using quadruplex DNA-capped silica nanocontainers. Nucleic Acids
Res 2011;39:1638e44.
[33] Luo Z, Cai K, Hu Y, Zhang B, Xu D. Cell-specific intracellular anticancer drug
delivery from mesoporous silica nanoparticles with pH sensitivity. Adv
Healthcare Mater 2012;1:321e5.
[34] Fu Q, Rao GVR, Ista LK, Wu Y, Andrzejewski BP, Sklar LA, et al. Control of
molecular transport through stimuli-responsive ordered mesoporous mate-
rials. Adv Mater 2003;15:1262e6.
[35] Schlossbauer A, Warncke S, Gramlich PME, Kecht J, Manetto A, Carell T, et al.
A programmable DNA-based molecular valve for colloidal mesoporous silica.
Angew Chem Int Ed 2010;49:4734e7.
́
[36] Rim HP, Min KH, Lee HJ, Jeong ́SY, ̃ Lee SC. pH-tunable
́ ́
calcium phosphate
covered mesoporous silica nanocontainers for intracellular controlled release
of guest drugs. Angew Chem Int Ed 2011;50:8853e7.
[37] Ponta H, Sherman L, Herrlich PA. CD44: from adhesion molecules to signalling
regulators. Nat Rev Mol Cell Biol 2003;4:33e45.
[38] Toole BP. Hyaluronan: from extracellular glue to pericellular cue. Nat Rev
Cancer 2004;4:528e39.
[39] Qhattal HSS, Liu X. Characterization of CD44-mediated cancer cell uptake and
intracellular distribution of hyaluronan-grafted liposomes. Mol Pharm 2011;
8:1233e46.
[40] Underhill C. CD44: the hyaluronan receptor. J Cell Sci 1992;103(Pt 2):293e8.
[41] Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B. CD44 is the principal
cell surface receptor for hyaluronate. Cell 1990;61:1303e13.
[42] Platt VM, Szoka Jr FC. Anticancer therapeutics: targeting macromolecules and
nanocarriers to hyaluronan or CD44, a hyaluronan receptor. Mol Pharm 2008;
5:474e86.
[43] Luo Y, Ziebell MR, Prestwich GD. A hyaluronic acidtaxol antitumor bio-
conjugate targeted to cancer cells. Biomacromolecules 2000;1:208e18.
[44] Pouyani T, Prestwich GD. Functionalized derivatives of hyaluronic acid
oligosaccharides: drug carriers and novel biomaterials. Bioconjug Chem 1994;
5:339e47.
[45] Liu G, Choi KY, Bhirde A, Swierczewska M, Yin J, Lee SW, et al. Sticky nano-
particles: a platform for siRNA delivery by a bis(zinc(ii) dipicolylamine)-
functionalized, self-assembled nanoconjugate. Angew Chem Int Ed 2012;51:
445e9.
[46] Lee Y, Lee H, Kim YB, Kim J, Hyeon T, Park H, et al. Bioinspired surface
immobilization of hyaluronic acid on monodisperse magnetite nanocrystals
for targeted cancer imaging. Adv Mater 2008;20:4154e7.
[47] Choi KY, Min KH, Na JH, Choi K, Kim K, Park JH, et al. Self-assembled hya-
luronic acid nanoparticles as a potential drug carrier for cancer therapy:
synthesis, characterization, and in vivo biodistribution. J Mater Chem 2009;
19:4102e7.
[48] Lee H, Lee K, Kim IK, Park TG. Synthesis, characterization, and in vivo diag-
nostic applications of hyaluronic acid immobilized gold nanoprobes. Bioma-
terials 2008;29:4709e18.
[49] Choi KY, Yoon HY, Kim J-H, Bae SM, Park R-W, Kang YM, et al. Smart nano-
carrier based on PEGylated hyaluronic acid for cancer therapy. ACS Nano
2011;5:8591e9.
[50] Stern R. Hyaluronan catabolism: a new metabolic pathway. Eur J Cell Biol
2004;83:317e25.
[51] Robert S. Hyaluronidases in cancer biology. Semin Cancer Biol 2008;18:
275e80.
[52] Lesley J, Hascall VC, Tammi M, Hyman R. Hyaluronan binding by cell surface
CD44. J Biol Chem 2000;275:26967e75.