DOX-UCNPs@mSiO2-TiO2 nanocomposites for near-infrared photocontrolled chemo/photodynamic therapy

Article abstract of DOI:10.1039/c7nj01291h

Currently, incorporating multiple therapeutic functions into one nanostructure has attracted more and more attention for the development of efficient anticancer agents. In this study, a uniform core-shell UCNPs@mSiO₂ nanocomposite was prepared as the carrier to develop the NIR light-controlled chemotherapy associated with photodynamic therapy (PDT). In view of the novel UV emission, the semiconductor photosensitizer TiO₂ was exploited due to its high efficiency and chemical stability. The host modification method was used to integrate TiO₂ doping and mesoporous structure, which can store anticancer drug molecules (doxorubicin, DOX). To improve the utilization of emission, a photolabile o-nitrobenzyl derivative was incorporated to form a sensitive linker (NB linker) as a "gate" to make sure the few leak. Upon NIR irradiation, the UV emission can not only excite TiO₂ to produce reactive oxygen species (ROS), but also induce the breaking of NB linker as well as drug release. The NIR-triggered performances were further demonstrated by the cell experiment using HeLa cells as the model cancer cell. The synergistic effect of chemotherapy and PDT induces enhanced cytotoxicity, which is more powerful than their simple effects added together. Therefore, the novel NIR light-controlled double-therapeutic nanocomposite should be a potential candidate for anticancer agents.

Full text of DOI:10.1039/c7nj01291h

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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DOX-UCNPs@mSiO2-TiO2 nanocomposites for near-infrared
photocontrolled chemo/photodynamic therapy
Yuhua Chen, Ruihan Tong, Na An, Huiming Lin* and Fengyu Qu*
College of Chemistry and Chemical Engineering, Harbin Normal University, P. R.
China, Harbin 150025
Corresponding author: Tel/Fax: +86 0451 88060653.
Eꢀmail address: qufengyu@hrbnu.edu.cn and linhuiming@hrbnu.edu.cn

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ABSTRACT
Currently, the incorporating multiple therapeutic functions into one
nanostructure has attracted more and more attention for the development of efficient
anticancer agents. In this work, uniform coreꢀshell UCNPs@mSiO2 nanocomposite
was prepared as the carrier to construct the NIR controlled chemotherapy associated
with photodynamic therapy (PDT). In view of the novel UV emission, the
semiconductor photosensitiver TiO2 was exploited due to the high efficiency and
chemical stability. The hostꢀmodification method was used to integrate TiO2 doping
and mesoporous structure, which can store anticancer drug molecules (doxorubicin,
DOX). To improve the utilization of emission, a photolabile oꢀnitrobenzyl derivative
was incorporated to form sensitive linker (NB linker) as “gate” to make sure the few
prematurity. Under NIR irradiation, the UV emission can not only excite TiO2 to
produce reactive oxygen species (ROS), but also induce the break of NB linker as
well as drug release. The NIR triggered performances were further demonstrated by
the cell experiment using HeLa cell as the model cancer cell. The synergistic effect
of chemotherapy and PDT induce the enhanced cytotoxicity which is more powerful
than their simple effects added together. Therefore, the novel NIR lightꢀcontrolled
doubleꢀtherapeutic nanocomposite should be a potential candidate in anticancer
agent.

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1 Introduction
With the rapid development of nanotechnology, the used of nanoparticles on
biomedicine brings a revolutionary development on anticancer therapy [1ꢀ9].
Currently, liposomes [2], polymer nanomaterials [10], dendritic nanomaterials [11],
gold nanoparticles [12], magnetic nanoparticles [13], mesoporous silica nanoparticles
(MSNs) [4] etc. have been adopted as nanovehicles to avoid rapid drug metabolism
and effectively to transport drug agents to the specific location. With the excellent
biodegradability and physicochemical stability, tunable pore size, large pore volume
and surface area, and facile modification, MSNs have already been thought to be one
of the most potential candidates as nanocarriers. Besides, with nanoparticle size, the
enhanced permeability and retention effect of MSNs always induces the preferential
accumulation at tumor sites [14]. Currently, some stimulus patterns, including: light
[15], temperature [16], magnetic field [17], electric field [18], ultrasonic [19], pH [20],
ionic strength [21], oxidizingꢀreducing [22], antigenꢀantibody [23], and protease [24]
have been used to manipulate functional MSNs to exhibit the controlled drug release
and to improve drug efficiency.
Among various kinds of stimuli, light is thought as the more promising means
due to the less invasiveness and the controllable active intensity and time.
Considering the minimum photodamage to living organisms, weak background
fluorescence, and high lightꢀpenetration depth in tissues, near infrared (NIR) light,
regarding as transparent window, is widely used in bioꢀpharmaceuticals [25ꢀ27]. It is
known that lanthanide upconversion nanoparticles (UCNPs) could absorb NIR light to

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induce the UVꢀVis emission, which possesses the higher energy in the hope to
manipulate the photoꢀresponsive drug release. In addition, the UVꢀVis emission also
can excite photosensitizer to induce reactive oxygen species (ROS), which would
cause the irreversible damage toward cancer tissue to achieve photodynamic therapy
(PDT). Currently, some semiconcuctors (TiO2 and ZnO) have been reported as
highꢀefficient photosensitizers as they can sensitize the oxygen to produce ROS.
Furthermore, semiconcuctor photosensitizers can overcome some disadvantages of
other photosensitizers, such as poor chemical stability/waterꢀsolubility of organic
photosensitizers (phthalocyanin derivatives) and the difficult modification/assembly
of carbon photosensitizers (CNT, graphene, C60). Considering the match between the
band gap of TiO2/ZnO (3.0ꢀ3.3 eV) and UV emission (330ꢀ370 nm) of NaYF4:Tm,Yb
UCNPs, Zhang et al. used UCNPs as the core and ZnO (TiO2) semiconductor as the
shell for potential application in photodynamic therapy, showing the NIR triggered the
generation of cytotoxic ROS to induce cancer cell death [28ꢀ29]. Based on previous
reports, it is difficult of TiO2 to possess the mesoporous structure due to the quick
hydrolysis of Ti precursors. Lin et al. reported the growth of TiO2 nanoparticles on
UCNPs and Yang et al. used TiO2 nanoparticles as core to coated with UC component
for the NIR acting PDT [30ꢀ31]. However, the lack of porous structure limits their
loading of other therapeutic agents. And the synergistic action of PDT and
chemotherapy is expected the enhanced therapeutic effect.
Here, we reported a facile method to integrate NIR light triggered chemotherapy
and PDT into one nanoplatform. From Scheme 1, UCNPs (NaYF4:Tm,Yb@NaYF4)

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Scheme 1. Illustration of the preparation and controlled release process of
DOXꢀUCNPs@mSiO2ꢀTiO2@NB linker.
were introduced as core and coated with mSiO2 to obtain UCNPs@mSiO2 core–shell
nanoparticles. By a simple host modification method, TiO2 nanoparticles were
injected into the pore of mSiO2 (UCNPs@mSiO2ꢀTiO2). It is possible way to insure
the porous structure and the doping of TiO2 nanoparticles, which was easily adjusted
by varying the amount of tetrabutyl titanate (TBOT). And then, a sensitive
oꢀnitrobenzyl derivative linker (NB linker) was synthesized, and it was exploited as
“gate” to encapsulate the anticancer drug (doxorubicin, DOX) inside the pore. Under
NIR irradiation (980 nm), the UV emission can stimulate TiO2 nanoparticles to
produce ROS inducing PDT. Meanwhile, the UV emission also can make the bond
breaking of NB linker as well as drug release. In addition, folic acid (FA) was grafted
outside to further assure the targeting due to the excess expression of FA receptor on
tumor area. The synergistic reaction of NIRꢀtriggered chemotherapy and PDT would
be expected to reveal the enhanced cytotoxicity to cancer cells.
2. Experimental

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2.1 Materials
Unless specified, all of the chemicals utilized were analytical grade and utilized
without further purification. Yttrium(III)chloridehexahydrate (YCl3·6H2O),
ytterbium(III) chloride hexahydrate (YbCl3·6H2O), thulium(III)chloride hexahydrate
(TmCl3·6H2O), ammonium fluoride(NH4F), sodiun hydroxide (NaOH), methanol,
oleic acid, 1ꢀoctadecene, tetraethylorthosilicate (TEOS), (3ꢀaminopropyl)
triethoxysilane
(APTES),
Nꢀ(3ꢀdimethylaminopropyl)ꢀN0ꢀethylcarbodiimide
hydrochloride (EDC), Nꢀhydroxysuccinimide (NHS), folic acid (FA),
1.3ꢀDiphenylisobenzofuran (DPBF), borane tetrahydrofuran complex solution,
doxorubicin hydrochloride(dox), 1,3ꢀdiphenylisobenzofuran, 3ꢀ(triethoxysilyl)propyl
isocyanate,
hexadecy
ltrimethyl
ammonium
bromide(CTAB)
and
2,6ꢀdimethylnitrobenzene were all purchased from Aladdin. ethanol absolute,
cychohexane, tetrahydrofuran,(THF) was dried with molecular sieve and roasted
prior to use, ethyl acetate, tetrabutyl titanate(TBOT), dimethyl sulfoxide(DMSO),
potassium permanganate (KMnO4), magnesium sulfate anhydrous were all bought
from Sinopharm Chemical Reagent Co., Ltd.
2.2 Synthesis of UCNPs@mSiO2 core shell nanoparticles
0.5 ml of asꢀprepared UCNPs [32ꢀ35] dispersed solution was washed with
cyclohexane twice to remove the excess oleic acid. And the precipitate was dispersed
into 5 mL of CTAB (0.2 M) solution with sonication and vigorous stirring. Later, 5
mL of water was added and the system was stirred for another 4 h at 32 oC. After that,
the PH value was adjusted to 9ꢀ10 using NaOH solution (0.1 M). Subsequently, 200

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uL of TEOS ethanol solution (20%) was added every 30 min under stirring. After
another 24 h, UCNPs@mSiO2 NPs were collected by centrifugation and rinsed with
ethanol repeatedly. At last, the collected products were refluxing in methanol (1 wt%
NaCl) for 3 h at room temperature to remove the template CTAB.
2.3 Synthesis of UCNPs@mSiO2-TiO2 nanoparticles
Firstly, UCNPs@mSiO2 [36ꢀ38] was modified with amino groups by using
(3ꢀaminopropyl)ꢀtrimethoxysilane (APTES). Secondly, 0.05 mmol of amino grafted
UCNPs@mSiO2 was dispersed in 20 mL of ethanol and 0.1 mL of water. Later, a
certain amount of 20 wt% TBOT in ethanol was added dropwise under vigorous
stirring for 30 min. After refluxing for 2 h, these nanoparticles were separated by
centrifugation and washed with ethanol three times. To achieve a crystallized TiO2
shell, the TiO2ꢀUCNs were treated with anhydrous ethanol in sealed autoclave at 180
oC for 24 h. With 75, 100, and 150 uL of TBOT solution, the sample were named
UCNPs@mSiO2ꢀTiO2ꢀ1, UCNPs@mSiO2ꢀTiO2ꢀ2, and UCNPs@mSiO2ꢀTiO2ꢀ3.
2.4 Synthesis of NB linker
As synthesized 2ꢀnitroꢀ1, 3ꢀbenzenedimethanol (1.7977 g, Fig. S1 and 2) was
dissolved in 36 mL of anhydrous THF in the presence of Ar, and then,
3ꢀ(Triethoxysily) propyl isocyanate (3.6251g) in 20 ml of anhydrous THF was added
dropwise with constant pressure drop funnel. Finally, the reaction system was
refluxed for another 12 h in oil bath. At the procedure, the reaction flask was quickly
covered with aluminum foil to avoid the sunlight.
2.5 FA peptide attachment.

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FA
peptide
was
covalently
conjugated
onto
amineꢀfunctionalized
DOXꢀUCNPs@mSiO2ꢀTiO2@NB through the ꢀCOOH group by using crossꢀlinking
reagents EDC and NHS. In this work, 1 mmol of TAT was dissolved in 2 mL of
DMSO followed by the addition of 1 mmol of EDC and 1 mmol of NHS. The mixture
was then stirred at room temperature for 15 min to activate the carboxylic group of FA
peptide. Subsequently, 100 uL of the above solution was added to 5 mL of methanol
solution of aminoꢀfunctionalized DOXꢀUCNPs@mSiO2ꢀTiO2@NB (60 mg), and the
mixture was stirred for 24 h at room temperature. Excess EDC, NHS and FA were
removed by repeatedly washing the nanoparticles with ethanol several times.
2.5 Detection of singlet oxygen.
DPBF was employed as a chemical probe to determine singlet oxygen by measuring
the absorption via UV−Vis spectroscopy. Typically, 2 mL of ethanol solution
containing DPBF (10 mmol Lꢀ1) was added to 2 mL of UCNPs@mSiO2ꢀTiO2 solution
(0.5 mg mLꢀ1) and then transferred into a 5 mL cuvette. The solution was irradiated by
a 980 nm NIR laser (1.5 W cmꢀ2) for different time, and then the solution was
centrifuged at 6000 rpm, and the supernatant was collected by UV−Vis adsorption.
2.6 Drug loading and release
UCNPs@mSiO2ꢀTiO2 (60 mg) and DOX (10 mg) were added to the ethanol solution
(10 mL) and stirred at room temperature for 12 h. Next, 300, 500 and 100 uL of NB
was separately added to the number of different mixed solution and stirred at room
temperature
for
24
h.
The
obtained
solid
(named
as
DOX–UCNPs@mSiO2ꢀTiO2@NB1,
DOX–UCNPs@mSiO2ꢀTiO2@NB2,

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DOX–UCNPs@mSiO2ꢀTiO2@NB3, respectively) was centrifuged, and washed three
or four times with ethanol solution. The loading amount of DOX was measured by
UV/Vis spectroscopy at 480 nm. The loading efficiency (LE wt%) of DOX can be
calculated by using the formula (1). All the data are derived from three times parallel
test.
m(original DOX) — m(residual DOX
Loading Efficiency LE% =
) ×100%
(1)
(
)
m(original DOX)
DOX–UCNPs@mSiO2ꢀTiO2@NB (20 mg) was dispersed in 50 mL of pH 6.5 PBS
and then 980 nm NIR irradiation (1.5W cmꢀ2) was introduced. At the interval time, the
solution was taken out to determine the release amount by UVꢀVis Spectra.
2.7 Cell culture
HeLa cells (cervical cancer cell line) were grown in a monolayer in Dulbecco’s
Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% (v/v) fetal
bovine serum (FBS, Tianhang Bioreagent Co., Zhejiang) and penicillin/streptomycin
(100 U mLꢀ1 and 100 mg mLꢀ1, respectively, Gibco) in a humidified 5% CO2
atmosphere at 37 oC.
2.7.1 Cell cytotoxicities assay. For the MTT assay, HeLa cells were seeded into
96ꢀwell plates at a density of 1 × 104 per well in 100 uL of media and grown overnight.
Then, different NPs were added to the medium, the cells were incubated for 24 h. And
then, the culture medium with nanoparticles was removed and the cells were
resupplied with fresh culture medium. The cells were irradiated with NIR irradiation
(1.5 W cmꢀ2，20 min). The cells were further incubated for 24 h. Afterwards, cells
were incubated in media containing 0.5 mg mLꢀ1 of MTT for 4 h. The medium was

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then replaced with 150 mL of dimethyl sulfoxide (DMSO) per well and the
absorbance was monitored using a microplate reader (BioꢀTekELx800) at the
wavelength of 490 nm. The cytotoxicity was expressed as the percentage of cell
viability compared to untreated cells.
2.7.2 Fluorescence imaging. To analyze the ability of cellular internalization, HeLa
cells were cultured in a 6ꢀwell with the incubation medium (DMEM) for 24 h, and
then treated with FITC modified TfꢀDOXꢀUCNPs@mSiO2ꢀTiO2@NB2 and
DOXꢀUCNPs@mSiO2ꢀTiO2@NB2 at the same final concentration of 500 ꢁg mLꢀ1.
After incubation for 4 h, the medium was removed and the cell was washed twice
with PBS (pH 7.4), Then, the cells were subjected to 980 nm NIR irradiation (1.5 W
cmꢀ2 0ꢀ20 min). The fluorescence imaging experiments were carried out on the Leica
DFC450 C Microsystems Ltd. using a 20× objective.
2.7.3 Flow cytometry. For flow cytometry studies, HeLa cells (1 × 106) were seeded
in 6ꢀwell culture plates and grown overnight. The cells were then treated with
FITCꢀmodified DOXꢀUCNPs@mSiO2ꢀTiO2@NB2 for 4 h. A single cell suspension
was prepared consecutively by rypsinization, washing with PBS, and filtration
through nylon mesh (300 mesh). Thereafter, the cells were analyzed using a flow
cytometer (BD C6) for FITC.
2.8 Characterization
Powder Xꢀray patterns (XRD) were recorded on a SIEMENSD 5005 Xꢀray
diractometer with Cu Ka radiation (40 kV, 30 mA). The nitrogen
adsorption/desorption, surface areas, and median pore diameters were measured using

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a Micromeritics ASAP 2010M sorptometer. Fourier transform infrared (FTIR) spectra
were recorded on a PerkinꢀElmer 580B Infrared Spectrophotometer using the KBr
pellet technique. A UVꢀVis spectrum was used to describe the amount of drug release
(SHIMADZU UV2550 spectrophotometer). Transmission electron microscopy (TEM)
images were recorded on TECNAI F20. Zeta potential was carried out with a
ZetaPALS Zeta Potential Analyzer.
E
Yb
Si
Tm
Ti
Y
F
O
Fig. 1 TEM images of (A) NaYF4:Tm,Yb, (B) NaYF4:Tm,Yb@NaYF4, (C)HRTEM
image, (D) UCNPs@mSiO2ꢀTiO2, (E) STEM and corresponding elemental (Y, Yb,
Tm, F, O, Si, Ti) mappings of UCNPs@mSiO2ꢀTiO2
3 Results and discussion

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3.1 Fabrication and characterization of the materials
The morphologies, particle sizes, and porous structures were investigated through
TEM analysis. As displayed in Fig. 1, NaYF4:Yb,Tm and NaYF4:Yb,Tm@NaYF4
nanoparticles show the dispersed and uniform spherical morphologies with an average
diameter about 20 and 30 nm in size. The highꢀresolution (HR) TEM image (Fig. 1C)
confirms their high crystallinity and the dꢀspacing value of 0.52 nm is ascribed to the
d100 of the hexagonalꢀphase NaYF4. From Fig. 1D, UCNPs@mSiO2ꢀTiO2 reveals the
obvious coreꢀshell structure, and each core was encapsulated by 20 nm silica shell
with wormꢀlike porous structure. The elemental mappings by STEM measurement
clearly reveal that the Y and Fꢀcontaining UCNP is located within the mesoporous
silica shell and Yb3+ as well as Tm3+ are doped only at the interior core of UCNP (Fig.
1E). Moreover, O and Ti distribute homogeneously in the mesoporous silica
framework according to element mapping of O and Ti.
A
B
Fig. 2
(A) The large angle XRD patterns of (a) UCNPs, (b) UCNPs@mSiO2, and
(c)UCNPs@mSiO2ꢀTiO2. (B) Upconversion fluorescence spectra of all the samples
under 980 nm excitation. The inset: the corresponding digital photos.
The XRD patterns of these samples were recorded to further reveal their structures.
As displayed in Fig. 2Aa, the typical diffraction peaks of hexagonalꢀphase NaYF4

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crystals (JCPDS16ꢀ0334) can be found in UCNPs, UCNP@mSiO2, and
UCNPs@mSiO2ꢀTiO2. Besides, the additional diffraction wide peak at 22° appears for
UCNP@mSiO2 and UCNPs@mSiO2ꢀTiO2 owing to the amorphous SiO2 shell. From
Fig. 2Ac, the emerging peaks at 25.4, 37.7, 48.0, and 55.0° is ascribed to (101), (103),
(200), and (211) diffractions of anatase (JDPDSNO.21ꢀ1272), further testifying the
graft of TiO2 nanoparticles. Their wide diffraction peaks further testify their dispersion
and small size that is important for the absorption of UV emission to induce ROS
generation.
The corresponding upconversion luminescence (UCL) spectra under NIR laser
excitation of these samples were recorded as shown in Fig. 2B. Under excitation at
980 nm laser, all of the fluorescence spectra show emission peaks at about 344, 360,
1
1
1
1
450 and 475 nm, which are assigned to the I6ꢀ3F4, D2ꢀ3H6, D2ꢀ3F4 and G4ꢀ3H6
transition from Tm3+, respectively [39]. In addition, NaYF4:Tm,Yb@NaYF4
nanoparticles exhibit the enhanced UCL intensity compared to NaYF4:Tm,Yb owing
to the NaYF4 shell. And the decreased fluorescence of UCNPs@mSiO2 is caused by
the lightꢀscattering effect on both emission and incident light by the silica layer.
Moreover, after the impregnation of TiO2 nanoparticles, the UV emission is inhibited
obviously on account of the adsorption of UV light for TiO2. And the digital photos
(inset) also display the detectable luminescence, suggesting the potential biomedical
applications such as imaging, detection, and sensing.

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A
B
Fig. 3 (A) Absorption spectra changes of DPBF treated with UCNPs@mSiO2ꢀTiO2
after 980 nm irradiation for different time. (B) Decay curves of DPBF treated with
different samples after 980 nm irradiation for different time.
DPBF was employed as a chemical probe to evaluate ROS generation by
measuring the UV−Vis absorption spectroscopy. As shown in Fig. 3A, the typical
absorbance of DPBF at 420 nm decreased obviously with the NIR illumination,
confirming the efficient generation of ROS because DPBF can react ROS irreversibly
to causes the decrease of its characteristic absorption. From Fig. 3B, without NIR
irradiation or dopedꢀTiO2, there is not noteworthy degradation of DPBF. That is to say
the integration of the two factors (NIR irradiation and TiO2) is significant for the ROS
generation. Furthermore, with the increase of dopedꢀTiO2, the degradation of DPBF is
improved due to the enhanced UV absorption (Fig. S3). With the most amount of
dopedꢀTiO2, UCNPs@mSiO2ꢀTiO2ꢀ3 possesses the highest UV absorption and the
most ROS generation. In this paper, the hostꢀmodification method was used to graft
TiO2 NPs into the mesopore that also decreases the porous parameters (Table S1). To
make sure the considerable ROS generation as well as the porous parameters,

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UCNPs@mSiO2ꢀTiO2ꢀ2 was thought as the appropriate and adopted in the following
investigation.
B
A
Fig. 4 (A) FTꢀIR spectra of (a) UCNPs@mSiO2, (b) UCNPs@mSiO2ꢀTiO2, (c)
DOXꢀUCNPs@mSiO2ꢀTiO2, and (d)DOXꢀUCNPs@mSiO2ꢀTiO2ꢀNB. (B) Zeta
potentials of these samples after different treatment (980 nm 1.5 W cmꢀ2).
FTꢀIR spectrometry was used to confirm the drug loading and organic
modification. As shown in Fig. 4Aa, a broad band in the region of 3435 cmꢀ1 is
attributed to the symmetric stretching of Si–OH. In addition, the characteristic peaks
of silica at around 1094 and 809 cmꢀ1 corresponding to vas(Si–O–Si) and vs(Si–O–Si)
also can be found from all the samples. The new peaks at 910, 954 and 1120 cmꢀ1 are
attributed to the vs(TiꢀOꢀSi), vas(TiꢀOꢀSi) and vs(TiꢀO), testifying the doping of TiO2
(Fig. 4Ab). Fig. 4Ac exhibits the torsional vibration adsorption bands of NH2 at 880
cmꢀ1 and obvious absorption peaks of v(C＝O) at 1710 cmꢀ1, testifying the loading of
DOX. From Fig. 4Ad, the absorption peaks at 3014 and 1450 cmꢀ1 are ascribed to
v(CꢀH) and v(CꢀC) of benzene and the present vas(NO2) band at 1525 cmꢀ1 also further
approves the graft of NB linker. In addition，the organic modification was further

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verify by surveying the zetaꢀpotentials of these samples. From Fig. 4B,
UCNPs@mSiO2ꢀTiO2 presents the negative zeta potential (ꢀ21.27±1.11 mV) relying
on the surface SiꢀOH and TiꢀOH with negative charge. After the graft of the sensitive
NB linker, their Zeta potentials increase to ꢀ14.56±0.67, ꢀ5.1±0.75 and ꢀ0.72±0.58 mV
of DOX–UCNPs@mSiO2ꢀTiO2@NB1, DOX–UCNPs@mSiO2ꢀTiO2@NB2, and
DOX–UCNPs@mSiO2ꢀTiO2@NB3.
With
the
most
amount
of
NB,
DOX–UCNPs@mSiO2ꢀTiO2@NB3 possesses the highest Zeta potential owing to the
displacement of negative charge SiꢀOH/TiꢀOH to neutral NB linker.
Table1The porous parameters of the samples
Surface area Pore volume Pore size
Loading
Sample
(m2gꢀ1)
(cm3gꢀ1)
(nm)
efficiency (%)
UCNPs@mSiO2ꢀTiO2
573
169
102
0.66
0.60
0.50
2.35
2.30
2.21
DOXꢀUCNPs@mSiO2ꢀTiO2@NB1
DOXꢀUCNPs@mSiO2ꢀTiO2@NB2
7.24±0.4
11.5±0.3
DOXꢀUCNPs@mSiO2ꢀTiO2@NB3
92
0.27
2.06
13.23±0.5
Moreover,
the
porous
structures
of
UCNPs@mSiO2ꢀTiO2,
and
DOXꢀUCNPs@mSiO2ꢀTiO2@NB1,
DOXꢀUCNPs@mSiO2ꢀTiO2@NB2,
DOXꢀUCNPs@mSiO2ꢀTiO2@NB3 were further studied by small angle XRD (Fig. S4)
and N2 adsorptionꢀdesorption (Fig. S5). All the samples reveal one diffraction peak at
about 2θ=2.26, suggesting their mesoporous structures derived from the mSiO2 shell
that also are verified by the typical IV adsorption isotherm and H4 hysteresis loop in
middle relative pressure region as shown in Fig. S5A. Here, NB silane coupling agent
was exploited as the sensitive linker to seal the drug molecules inside the mesopore,

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so that with the increase of the NB, the DOX loading efficiency of
DOXꢀUCPs@mSiO2ꢀTiO2@NB
increases
as
well.
From
Table
1,
DOXꢀUCNPs@mSiO2ꢀTiO2@NB1,
DOXꢀUCPs@mSiO2ꢀTiO2@NB2,
and
DOXꢀUCNPs@mSiO2ꢀTiO2@NB3 displays 7.24±0.4, 11.5±0.3, 13.23±0.5% drug
loading, respectively. The DOX loading and organic modification also makes the
surface area and pore volume decrease from 573 m2 gꢀ1and 0.66 cm3 gꢀ1 of
UCNPs@mSiO2ꢀTiO2
to
169
m2
gꢀ1 and
0.60
cm3
gꢀ1
of
DOXꢀUCNPs@mSiO2ꢀTiO2@NB1, 102 m2 gꢀ1 and 0.50 cm3 gꢀ1 of
DOXꢀUCNPs@mSiO2ꢀTiO2@NB2, 92 m2 gꢀ1 and 0.27 cm3 gꢀ1 of
DOXꢀUCNPs@mSiO2ꢀTiO2 @NB3, respectively (Table 1).
3.2 Drug release profiles
The in vitro release profiles of DOX from DOXꢀUCNPs@mSiO2ꢀTiO2 @NBs in PBS
buffer (pH 6.5) in response to 980 nm NIR irradiation are shown in Fig. 5A. As can be
seen in Fig. 5A, without NIR irradiation, few DOX molecules leak from
DOXꢀUCNPs@mSiO2ꢀTiO2@NBs
nanocomposites.
After
590
min,
DOXꢀUCNPs@mSiO2ꢀTiO2@NB1 reveals 24.23±1.01 % drug release and that of
DOXꢀUCNPs@mSiO2ꢀTiO2@NB2 and DOXꢀUCNPs@mSiO2ꢀTiO2@NB3 is just
below 10.03±0.76 %. However, DOX can be released freely with the aid of NIR
irradiation. From Fig. 5A, with NIR irradiation, drug release is enhanced markedly.
When the release system are treated by NIR irradiation for 120 min, the release
increase
to
13.11±0.89,
25.06±1.03,
and
33.24±1.35%
for
DOXꢀUCNPs@mSiO2ꢀTiO2@NB1,
DOXꢀUCNPs@mSiO2ꢀTiO2@NB2
and
DOXꢀUCNPs@mSiO2ꢀTiO2@NB3, respectively. After 360 min，the release reaches

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A
B
C
Fig. 5 (A) Release profiles of DOX from DOXꢀUCNPs@mSiO2ꢀTiO2@NB1 (a, d),
DOXꢀUCNPs@mSiO2ꢀTiO2@NB2 (b, e), and DOXꢀUCNPs@mSiO2ꢀTiO2@NB3 (c,
f) with (aꢀc) and without (dꢀf) 980nm irradiation. (B) The DOX release treated with
periodic 980 nm NIR ON/OFF illumination with a periodicity of 15 min (1.5 W cm−2)
(C) Release profiles of DOX from DOXꢀUCNPs@mSiO2ꢀTiO2ꢀNB1 and
DOXꢀUCNPs@mSiO2ꢀTiO2 treated with and without NIR irradiation.
the maximal amount about 88.01±1.50, 77.06±2.03, and 43.76±1.67% for them. In
view of the drug release behaviors displayed in Fig. 5A, the NIR triggered drug
release can be ascribed to the activity of the sensitive NB linker. It is known that, NB
is a typical photolabile group, and the photolysis of NB derivatives in UV emission
was used to manipulate the drug release [40ꢀ41]. Here, NB group was constructed to
form a silica coupling agent which was used as the “gate” to encapsulate the DOX

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inside the mesopore. Without NIR irradiation, there is no UV emission from UCNPs
so that NB “gate” keep inhibiting the leak of drug molecules. When NIR irradiation
was introduced, the UV emission causes the photolysis of NB linker to induce the
“gate” open and drug release. And the “Ladder” pulsatile release profiles as displayed
in Fig. 5B demonstrate that the drug release can be triggered by NIR exposure and the
release amount is highly dependent on the duration of NIR exposure. From Fig. 5C,
without NB “gate” DOXꢀUCNPs@mSiO2ꢀTiO2 does not exhibit the NIR sensitive
drug release. For the above investigation, it is the NB “gate” to make sure the NIR
sensitive drug release. Moreover, photosensitive behavior was further monitor by Zeta
potential change (Fig. 4B). After NIR irradiation, the zeta potential of these
nanocomposites reduce to ꢀ20.13±0.88, ꢀ17.89±0.54, and ꢀ12.57±0.94 mV, because of
the photolysis of NB leaving negative charge ꢀCOOH outside (Fig. S6).
In addition, with the lowest amount of NB DOXꢀUCNPs@mSiO2ꢀTiO2@NB1
exhibits the fastest release, followed by DOXꢀUCNPs@mSiO2ꢀTiO2@NB2 and
DOXꢀUCNPs@mSiO2ꢀTiO2@NB3 as shown in Fig. 5A. This is because the
photolysis of fewest NB “gate” is quickest than others to make the highest release
amount of DOXꢀUCNPs@mSiO2ꢀTiO2@NB1 (88.01±1.50%), while that of
DOXꢀUCNPs@mSiO2ꢀTiO2@NB3 is just 43.76±1.67%. Besides, without NIR,
DOXꢀUCNPs@mSiO2ꢀTiO2@NB1 displays obvious drug leak (24.23±1.01%) owing
to the insufficient NB “gate”. With the increase of NB “gate”, the prematurity
decrease below 10.03±0.76%. To sum up, the amount of “gate” also can be used to
regulate the release performance of the system and the suitable amount of “gate” is

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significant to insure the few prematurity (low side effect) as well as the high drug
release (high drug efficiency) in the focus site.
3.3 Cellular uptake and cytotoxicity
Fig.
6
Fluorescent
images
of
HeLa
cells
incubated
with
DOXꢀUCNPs@mSiO2ꢀTiO2@NB2 without and with FA modification for 4 h
preꢀincubation and then that were exposed to 980 nm irradiation (1.5 W cmꢀ2) for
different time before observations. For each panel, the images from left to right show
cell nuclei, DOXꢀUCNPs@mSiO2ꢀTiO2@NB2 stained with FITC (green), DOX
fluorescence (red) in cells and the overlapping.
To observe the uptake and in vivo drug release of the sample,
DOXꢀUCNPs@mSiO2ꢀTiO2@NB2ꢀFA and DOXꢀUCNPs@mSiO2ꢀTiO2@NB2 was

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preꢀincubated with the HeLa cells for 4 h in the dark. After the incubation, the cells
were washed twice with medium and then irradiated with a 980 nm NIR for 0–20
minutes, and then the cells were examined by fluorescence microscopy. As shown in
Fig. 6, DOXꢀUCNPs@mSiO2ꢀTiO2@NB2ꢀFA can be clearly found in the cytoplasm
of HeLa cells as demonstrated by the green fluorescence from FITC lighting up,
indicating that the nanocomposites have been internalized by HeLa cells through
endocytosis or macropinocytosis as incubated with the cells for 4 h, while there is
few green fluorescence can be found from DOXꢀUCNPs@mSiO2ꢀTiO2@NB2. It is
believed that FAꢀmodification can improve the uptake amount by cancer cells, such
as HeLa cells, owing to the excess expression of FA receptor on tumor area. It is
further confirmed by flow cyotmetry analysis as present in Fig. S7. Furthermore,
with the extension of irradiation time, the intensity of red fluorescence in nucleus is
enhanced, derived from more and more released DOX molecules gathered into the
nucleus. That is helpful to improve the drug efficacy because DOX is a cancer drug
acting on the cell nucleus.
To improve the uptake of these composites by HeLa cells, folic acid (FA) was
grafted outside for the all cell viability experiment. The cell viabilities of HeLa cells
incubated with different amounts of samples were determined by the MTT assay. As
shown in Fig. 7A, with the increase of UCNPs@mSiO2 and
DOXꢀUCNPs@mSiO2@NB2 there is no obvious decrease of the cell viability. Even
when the concentration increases to 250 ug mLꢀ1, the cell viabilities also can remain
above 96.01±2.05% of them revealing the negligible cytotoxicity and the potential

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A
B
Fig. 7 (A) MTT cell viability assay of HeLa after treated by UCNPs@mSiO2,
UCNPs@mSiO2@NB2 and DOXꢀUCNPs@mSiO2@NB2 with irradiation. (B) MTT
cell viability assay of HeLa after treated by UCNPs@mSiO2ꢀTiO2,
UCNPs@mSiO2ꢀTiO2
with
irradiation,
DOXꢀUCNPs@mSiO2ꢀTiO2@NB2,
DOXꢀUCNPs@mSiO2ꢀTiO2@NB2 irradiation. Irradiation experiment: 980 nm
irradiation 1.5 W cmꢀ2 for 20 min.
biomedical application. After the 980 nm irradiation for 20 min, the cell viability of
DOXꢀUCNPs@mSiO2@NB2 drops to 46.26±1.32% (250 ug mLꢀ1). The enhanced
cytotoxicity is caused by the NIR triggered DOX release that is consistent with the
study of the drug release in Fig. 5. Fig. 7B also displays the negligible cytotoxicity of
DOXꢀUCNPs@mSiO2ꢀTiO2@NB and UCNPs@mSiO2ꢀTiO2 under dark condition.
With 980 nm irradiation, the decreased cell viability (70.35±2.03 %) of
UCNPs@mSiO2ꢀTiO2 is attributed to the generation of single oxygen that is
consistent with the above demonstration in Fig. 3. Furthermore,
DOXꢀUCNPs@mSiO2ꢀTiO2@NB2 reveals obvious cytotoxicity depending on the
concentration. When the concentration increases to 250 ug mLꢀ1, the cell viability
drops to 11.56±2.01 % that is derived from the NIR triggered drug release
(chemotherapy) and single oxygen generation (PDT). The synergistic effect of

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chemotherapy and PDT induce the enhanced cytotoxicity to HeLa cells and the
combined therapy is more powerful than their simple effects added together.
4. Conclusion
Herein, the coreꢀshell UCNPs@mSiO2 nanoparticles have been constructed as
the nanocarrier. Via a simple hostꢀmodification method, TiO2 nanoparticles with
adjustable amount can be grafted to the surface (inner and outside) of mSiO2. And
then, antitumor drug DOX was capsulated in the pore of mSiO2 by using the
asꢀprepared oꢀnitrobenzylꢀderivativeꢀincorporated silane coupling agent (NB linker)
as “gate”. Under a single NIR (980 nm) irradiation, the UV emission can excite TiO2
nanoparticles to induce reactive oxygen species generation to achieve PDT.
Simultaneously, the UV emission also can make NB linker broken and DOX release.
The release performance can be regulated by varying the amount of NB linker and
irradiation. To enhance the localization to tumor cancer, FA was connected outside
and the in vitro cytotoxicity of DOXꢀUCNPs@mSiO2ꢀTiO2@NBꢀFA
nanocomposites was surveyed in detail. The NIR controlled cytotoxicity to HeLa
cancer cells is ascribed to the NIR triggered DOX release (chemotherapy) and ROS
generation (PDT). In addition, the synergistic effect of chemotherapy associated with
PDT further improves the cytotoxicity, which is superior to any single function.
Acknowledgments
Financial support for this study was provided by the National Natural Science
Foundation of China (21471041, 21571045), and College Youth Innovation Talents
Training Program of Heilongjang (UNPYSCTꢀ2015053).

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