Colloids and Surfaces B: Biointerfaces 117 (2014) 75–81

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Synthesis of mesoporous silica nanoparticle–oxaliplatin conjugates

for improved anticancer drug delivery
Hongyan He a,b , Haihua Xiao c , Huihui Kuang a,b , Zhigang Xie a , Xuesi Chen a ,
Xiabin Jing a , Yubin Huang a,∗
a
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR
China
b
University of Chinese Academy of Sciences, Beijing 100039, PR China
c
Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN 46556, USA

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

Article history: Mesoporous silica nanoparticles (MSN) with 1,2-bidentate carboxyl groups on the surface reacted with
Received 25 October 2013 1,2-diaminecyclohexano platinum(II) dinitrate (DACH-Pt-(NO3 )2 ) which is an active anticancer species
Received in revised form 17 January 2014 of clinic relevant oxaliplatin to form MSN-Pt. The modification of the parent particles was monitored
Accepted 4 February 2014
by 13 C, 29 Si solid-state NMR, X-ray measurements (XRD) and Fourier transform infrared spectroscopy
Available online 19 February 2014
(FT-IR). After loading with platinum drugs, MSN-Pt exhibited two strong Pt4f signals as indicated by
X-ray photoelectron spectroscopy (XPS). The platinum content in the conjugates was calculated to be
Keywords:
9.7% according to ICP-MS measurements. Confocal laser scanning microscopy (CLSM) displayed that
Mesoporous silica nanoparticles (MSN)
Oxaliplatin
MSN-Pt were uptaken fast by HepG-2 cells and concentrated within endosomes and lysosomes. In vitro
Conjugates MTT assay of MSN-Pt demonstrated an improved cytotoxicity against HepG-2 cells than that of free
Thiol-ene click oxaliplatin. This is due to the fact that MSN-Pt expressed higher platinum intracellular uptake and more
Drug delivery DNA binding (Pt-DNA adducts) than free oxaliplatin. Hence this work highlighted that the platinum
loaded MSN nanoparticles could be a promising future intelligent drug delivery system.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction from the use of MCM-41-type siliceous solids are especially

Cisplatin and its analogs are among the most used antitumor
drugs for clinical chemotherapy [1,2]. However, small molecule has
side effects such as kidney toxicity, nausea, hearing impairment,
and irreversible peripheral nerve damage which caused much pain
to patients [3,4]. To reduce the toxicities of platinum drugs, drug
delivery systems are developed with a capacity of passive tumor
targeting which enables platinum drugs being directed to solid
tumors through the enhanced permeability and retention effect
(EPR) [5,6]. The drug carriers could refrain drug from undesired
release before reaching tumor, and release drug in a controlled and
smart manner. Several type of nanocarriers have been investigated
such as polymer micelles [7,8], polymer capsules [9] and inorganic
particles [10–14].
Over the past decade, especially in recent years, mesoporous
silica nanoparticles (MSN) have attracted much attention because
of their excellent properties. Especially, the examples derived
∗ Corresponding author. Tel.: +86 431 85262769; fax: +86 431 85262769.
E-mail address: ybhuang@ciac.jl.cn (Y. Huang).
attractive because of the properties of the mesoporous materials.
MCM-41-type particles have an ordered arrangement of uniform
two-dimensional (2D) hexagonal p6m mesopores [15]. They have
been proved to be nontoxic, pore size adjustable, physicochemi-
cally stable, easy modified and functionalized [16,17]. Especially,
their high surface areas and straight narrow channels facilitate
adsorption of drugs into their structures [18]. There are some
reports on the loading cisplatin onto the MSN to enhance the
cytotoxic effect [19,20]. For instance, Gu et al. [21] prepared MSN
nanocarriers with high-density of carboxyl groups to combine
with platinum atoms in cisplatin. The complexes greatly improved
growth inhibition effect against MCF-7 and HeLa cells. However,
once again cisplatin could cause serious side effects and resistance
from cancer cells. It is still a big challenge to develop a safer and
more effective drug delivery system for cancer therapy.
Oxaliplatin, oxalate (trans-1,2-diaminocyclohexane) plat-
inum(II), has been developed as the third generation of
platinum(II)-based drugs in the past several decades to over-
come side effects of cisplatin and to reduce cisplatin resistance
[22]. The study on structure–activity relationship of oxaliplatin has
indicated that 1,2-diaminocyclohexane (DACH) works as a stable

http://dx.doi.org/10.1016/j.colsurfb.2014.02.014
0927-7765/© 2014 Elsevier B.V. All rights reserved.
76 H. He et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 75–81
ligand of platinum atom while the oxalic acid is a leaving ligand
in the oxaliplatin molecule [23]. Numerous nanocarriers have
been employed to form complexes with active anticancer species
(DACH-Pt) of oxaliplatin [10,12,24–26]. For instance, Xiao et al.
[7] synthesized a biodegradable and amphiphilic copolymer that
contains pendant 1,2-bidentate carboxyl groups as chelator for
platinum atom. The polymer platinum complex can self-assemble
into micelles and act as pro-drug of oxaliplatin. However, to our
best knowledge, there is no report on loading active species of
oxaliplatin using MSN.
Inspired by the above-mentioned work, we for the first time
designed and synthesized an MSN–oxaliplatin conjugates (MSN-
Pt) by reaction of active anticancer species (DACH-Pt) of oxaliplatin
with 1,2-bidentate carboxyl-modified MSN. To make a complex
with platinum atom, carboxylic groups were essential for MSN. Sev-
eral methods were reported to prepare carboxylic-functionalized
mesoporous silica [27]. One attractive protocol was developed
by Yan et al. [28]. They introduced a high density of carboxyl
groups to MSN by a co-condensation method using tetraethyl
orthosilicate (TEOS) and functional silane precursor which bears
upon itself a reactive anhydride group. The precursor was synthe-
sized via an efficient thiol-ene addition reaction which is featured
by high yields, high regioselectivity and lack of sensitivity to
water and oxygen [29]. However, the functional silane precur-
sor needed to be purified by chromatography on silica gel using
dry dichloromethane as eluent before further use, which was an
extra cost of time and money. Besides, the carboxyl groups func-
tional MSN were not used in drug delivery systems. Herein, we
introduced a more modified, simpler and more convenient route
which was applied to prepare MSN modified with 1,2-bidentate
carboxyl groups as platinum nanocarriers (Fig. 1A). First, parent
particles MSN with thiol groups on the walls (MSN-SH) were syn-
thesized by one-pot method. Subsequently, the thiol groups reacted
with maleic anhydride via efficient thiol-ene addition reaction to
introduce a high density of 1,2-bidentate carboxyl groups. All puri-
fied steps could be easily realized by centrifugation. Thereafter, the
pendant 1,2-bidentate carboxyl group on prepared nanocarriers
could act as a chelator for the active anticancer species (DACH-Pt)
of oxaliplatin to form the oxaliplatin loaded nanoparticles MSN-Pt
(Fig. 1B). Moreover, MSN-Pt can be endocytosed by the cancer cells.
After that, they can release clinic relevant active anticancer species
of oxaliplatin which would undergo further binding with cellular
DNA and the formation of 1, 2-(GpG) crosslinks to inhibit cancer
cell replication.
Fig. 1. (A) Modification of 1,2-bidentate carboxyl groups onto the parent nanoparti-
cles of MSN-SH via thiol-ene click reaction; (B) MSN nanoparticles conjugated with
the oxaliplatin active species and the intracellular release of platinum drugs via acid
mediated hydrolysis to inhibit the replication of DNA.
2. Experimental
2.1. Materials
Tetraethyl orthosilicate (TEOS, Aladdin), n-cetyltr-
imethylammonium bromide (CTAB, YiLi Fine Chemicals Co.
Ltd., Beijing), 3-mercaptopropyltrimethoxysilane (MPTMS,
Aladdin), (3-aminopropyl) trimethoxysilane (APTES, Sigma),
maleic anhydride (Aladdin) were used as received. ((1R,2R)-1,2-
Diaminecyclohexano) platinum(II) dinitrate (DACH-Pt-(NO3 )2
for short) was synthesized as reported [30]. Tetrahydrofuran
(THF) and toluene were purified by distillation from sodium
with benzophenone. 2-(4-Amidinophenyl)-6-indolecarbamidine
dihydrochloride (DAPI), fluorescein isothiocyanate (FITC) and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) were purchased from Sigma–Aldrich Co. Lyso-tracker Red,
Enhanced BCA Protein Assay Kit and Genomic DNA Mini Prepara-
tion Kit with Spin Column were purchased from Beyotime Institute
of Biotechnology. Other reagents were commercially available and
used as received.
2.2. Characterizations
Transmission electron microscopy (TEM) studies were per-
formed on a JEM-1011 electron microscope operating at an acceler-
ation voltage of 100 kV. N2 adsorption–desorption isotherms were
recorded on a Micromeritics ASAP 2020M automated sorption ana-
lyzer. The samples were degassed at 150 ◦ 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 BJH method. Fourier-transform infrared (FT-IR) spectra
were recorded with KBr pellets on a Bio-Rad Win-IR instrument
in the range from 4000 to 400. The solid-state 13 C and 29 Si CP
MAS NMR spectra were recorded on a Bruker AVANCE III 400 WB
spectrometer at 100.62 MHz and 79.50 MHz, respectively. X-ray
measurements (XRD) were performed on a Bruker D8 FOCUS Pow-
der X-ray Diffractometer using Cu K␣ radiation. Inductively coupled
plasma mass spectrometry (ICP-MS, Xseries II, Thermoscientific,
USA) was used for quantitative determination of platinum.
2.3. Synthesis of MSN-COOH
The parent particles MSN-SH modified with thiol groups were
prepared through a co-condensation method according to the pub-
lished method [31].
The 1,2-bidentate carboxyl groups were grafted to the particles
via thiol-ene addition reaction under basic catalysis [28]. Firstly,
MSN-SH (1.0 g) and maleic anhydride (0.98 g, 10 mmol) were dis-
solved in 100 mL of THF and stirred for 0.5 h at room temperature
under nitrogen atmosphere. The addition reaction was initiated by
adding triethylamine (0.28 mL, 2 mmol) and allowed to proceed for
1 h. The products were collected by centrifugation and washed with
water, then aged in deionized water at 80 ◦ C for 24 h to transform
the anhydride unit into 1,2-bidentate carboxyl groups. The final
products were washed with water and ethanol for several times,
dried in a vacuum and named as MSN-COOH.
2.4. Preparation of DACH-Pt loaded nanoparticles MSN-Pt
The MSN-Pt were synthesized according to the published litera-
tures in our lab with some modification [24,30]. Briefly, MSN-COOH
(0.2 g) was dispersed in 20 mL of deionized water, followed by the
addition of Na2 CO3 to neutralize the carboxyl groups. Then they
were collected by centrifugation and washed with water to remove
the redundant Na2 CO3 . Thereafter, the particles were mixed with
aqueous solution of DACH-Pt-(NO3 )2 (20 mL, 0.42 mmol of Pt) and
 H. He et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 75–81
the solution was stirred vigorously for 48 h at 25 ◦ C in a dark and
nitrogen atmosphere. The resulted particles were separated by
centrifugation and washed with deionized water to remove the
unreacted platinum complex. The obtained conjugates were named
as MSN-Pt.
2.5. Synthesis of FITC labeled MSN-Pt (MSN Pt/FITC)
The MSN-Pt were labeled with FITC according to a previously
reported method [32]. Briefly, FITC (5 mg) and APTES (10 ␮L) were
dissolved in 5 mL of ethanol and the mixture was stirred under
dark for 2 h to obtain FITC-APTES stock solution. Next, 30 mg of
MSN-Pt was added to the solution. After the reaction proceeded for
another 24 h, the particles was collected by centrifugation, dialyzed
against deionized water for 48 h and then freeze-dried to obtain
MSN-Pt/FITC.
2.6. In vitro drug release
To evaluate the drug release properties, MSN-Pt (4 mg) was dis-
persed in 5 mL of phosphate buffer (pH 7.4) or acetate buffer (pH
5.0) solutions. The suspensions were then transferred into a pre-
swelled dialysis bag (MWCO = 3500) and dialysis against 25 mL of
the corresponding buffer at 37 ◦ C in a shaking culture incubator
with a speed of 100 rpm. At a predetermined time, 3 mL of solution
was withdrawn from the release medium, and the same volume of
fresh buffer was added to the incubation medium. The taken out
dialysis medium was measured by ICP-MS to calculate the released
amount of platinum.
2.7. Cell lines
HepG-2 cancer cells (a Human liver cancer cell line) was kindly
supplied by the Medical Department of Jilin University in China
and first cultured in Dulbecco’s modified Eagle’s medium (DMEM,
GIBCO), supplemented with 10% heat-inactivated fetal bovine
serum (FBS, GIBCO), 100 U/mL penicillin and 100 ␮g/mL strepto-
mycin at 37 ◦ C in a humidified atmosphere containing 5% CO2 .
2.8. Cytotoxicity of the MSN-COOH and MSN-Pt
The in vitro cytotoxicity of the empty particles MSN-COOH and
the conjugates MSN-Pt were evaluated by MTT assay against HepG-
2 cells. Briefly, cells harvested in a logarithmic growth phase were
seeded in 96-well plates at a density of 105 cells/well and incu-
bated in 100 ␮L of DMEM for 24 h. The medium was then replaced
by empty particles MSN-COOH, oxaliplatin and MSN-Pt at a series
of final equivalent platinum concentration. The incubation was
continued for 48 h. Then, 20 ␮L of MTT solution in PBS with the
concentration of 5 mg/mL was added and the plates were incu-
bated for another 4 h at 37 ◦ C, After that, the medium containing
MTT was removed and 150 ␮L of DMSO was added to each well to
dissolve the MTT formazan crystals. Finally, the plates were shaken
for 10 min, and the absorbance of formazan product was measured
at 492 nm by a microplate reader.
2.9. Internalization and distribution in cells
The internalization and distribution in cells of MSN-Pt/FITC was
determined by CLSM toward HepG-2 cells. Cells were seeded in 6-
well culture plates (a sterile coverslip was put in each well) at a
density of 5 × 104 cells/well and allowed to adhere for 24 h. Then
cells were incubated with free MSN-Pt/FITC at a final Pt concen-
tration of 100 ␮g/mL for 0.5, or 4 h at 37 ◦ C. After the preset time
intervals, the culture medium was removed and cells were washed
for three times with ice-cold PBS and fixed with 4% formaldehyde
77
for 30 min at room temperature. After staining with Lyso-tracker
Red for endosomes and lysosomes and DAPI for the nucleus, the
slides were mounted and observed with an Olympus FV1000 con-
focal laser scanning microscope (CLSM) imaging system (Japan).
2.10. Bioactive platinum content in cells
The content of bioactive platinum which was binding with pro-
tein and DNA in HepG-2 cells was examined by direct platinum
measurements using ICP-MS. HepG-2 cells were seeded in 6-well
plates. These cells were then treated with oxaliplatin or MSN-Pt
with the platinum concentration of 10 ␮M and subsequently incu-
bated at 37 ◦ C for determined time. After washing with PBS three
times, cells were lysed by cell lysis buffer. The platinum contents
in each cell lysis sample were determined by ICP-MS. The corre-
sponding protein content in cells was determined by enhanced
bicinchoninic acid (BCA) assay. The total platinum content was
expressed as ng Pt/mg Protein. In order to evaluate the platinum
contents adducted with DNA, DNA was isolated by using DNA
purification kit after cells were lysed, and DNA was digested with
DNase I before analysis. Total Pt–DNA adducts were expressed as
ng Pt/␮g DNA.
3. Results and discussion
3.1. Synthesis and characterization of MSN-COOH
To prepare MSN modified with 1,2-bidentate carboxyl groups as
platinum nanocarriers, we introduced a convenient route as illus-
trated in Fig. 1A. Firstly, parent particles MSN with thiol groups
on the walls (MSN-SH) were synthesized by one-pot method. Sub-
sequently, we introduce a high density of 1,2-bidentate carboxyl
groups via efficient thiol-ene addition reaction according to the
literature [28]. The advantage of our protocol lies in that it is unnec-
essary to synthesize the functional silane precursor during which
the purifying process is a waste of time. In our method, in fact, syn-
thesis of MSN-COOH is efficiency and all purified steps could be
easily realized by centrifugation.
The parent MSN-SH was prepared by a one-pot, sol–gel
method as reported [31]. The morphology and pore structures
of the parent MSN-SH were characterized by TEM and N2
adsorption–desorption. It was clearly observed that MSN-SH were
uniform spherical particles with a mean diameter of approximately
100 nm (Fig. 2A), and the mesopore channels showed well-ordered
2D hexagonal patterns. The BET isotherm exhibited the characteris-
tic type of IV N2 adsorption/desorption patterns (Fig. 2B), indicating
that the particles possessed uniform mesoporous channels and
narrow pore size distribution [33]. The surface area and cumula-
tive pore volume were calculated to be 965 m2 /g and 1.401 cm3 /g,
respectively, and the average pore size was about 2.75 nm accord-
ing to the BJH pore size distribution. These data indicated that the
MSN-SH of a mesoporous quality was successfully prepared.
In order to introduce the 1,2-bidentate carboxyl groups, thiol
groups on the silica particle walls were used to react with maleic
anhydride easily and efficiently via thiol-ene addition reaction.
The reaction was tracked by 13 C and 29 Si solid-state NMR spec-
troscopy (Fig. 3). The 13 C solid-state NMR spectrum of the obtained
MSN-COOH showed additional resonance signals at about 36,
69 and 176 ppm, which were assigned to characteristic carbon
peaks on 1,2-bidentate carboxyl groups. Meanwhile, the 29 Si solid-
state NMR spectrum of MSN-COOH presented the bulk silicon
peaks (Q region) around −105 ppm and two signals at −58 and
−67 ppm corresponding to the functionalized silica resonances (T
region), indicating that organic groups were covalently attached
to the silica framework [34]. Furthermore, during FT-IR tests,
78 H. He et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 75–81

Fig. 2. Characterization of the parent nanoparticles of MSN-SH: TEM image (A); nitrogen adsorption–desorption isotherm and the inset demonstrates the BJH pore size
distribution of MSN-SH (B).

an additional adsorption peak appeared at 1737 cm−1 , owing to

the C O stretching vibration in carboxyl group (Fig. 4A). All the
results demonstrated that the 1,2-bidentate carboxyl groups were
successfully modified on the mesoporous silica particles.
In order to detect the change on the pore structure of MSN, small
angle XRD patterns of particles before and after functionalization
were studied. As shown in Fig. 4B, both parent MSN-SH and MSN-
COOH exhibited a distinct diffraction peak (1 0 0), which was the
characteristic diffraction pattern of the ordered hexagonal sym-
metry of MCM-41 type [35]. It was worth noting that the (1 0 0)
reflection showed a consistent shift to larger angle after modifica-
tion, indicating a greater d(1 0 0) interplanar spacing [36,37] which
was calculated to be decreased from 2.1 nm (MSN-SH) to 1.7 nm
(MSN-COOH). The results demonstrated that the parent MSN main-
tained the ordered hexagonal symmetry after modification, and
that was beneficial for the subsequent conjugation with platinum
drugs.

3.2. Drug loading and release

Oxaliplatin as the platinum(II) based drug has structure–activity

Fig. 3. 13 C CP-MAS solid-state NMR spectrum of MSN-SH (a) and MSN-COOH (b);
29
Si CP-MAS solid-state NMR spectrum of MSN-COOH (c).
relationship relying on that 1,2-diaminocyclohexane works as a
stable ligand of Pt atom while the oxalic acid is a leaving ligand in
the oxaliplatin molecule. Also, the 1,2-diaminocyclohexane plat-
inum (DACH-Pt) structure is proved to be an active species for
anticancer therapy [38]. In our design, we used DACH-Pt-(NO3 )2
to react with the pendant 1,2-bidentate carboxyl groups on MSN-
COOH walls. This reaction not only formed a linkage between Pt

Fig. 4. (A) FT-IR spectra of MSN-SH (a), MSN-COOH (b) and MSN-Pt (c); (B) the XRD pattern of MSN-SH (a) and MSN-COOH (b).
 H. He et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 75–81
Fig. 5. (A) XPS curve of Pt4f in MSN-Pt (a) and MSN-COOH (b); (B) platinum release profiles of MSN-Pt in buffer solutions at pH 7.4 (a) and 5.0 (b).
complex and MSN, but also created the active species in oxalip-
latin molecular structure conjugating with silica particles. Most
importantly, this structure could make sure to release the active
anticancer species (DACH-Pt) from the conjugates in a denser acidic
condition inside cancer cells.
After conjugated with platinum drugs, IR spectrum of MSN
showed that the C O stretching vibration in carboxyl group shift
from 1737 cm−1 to 1643 cm−1 . The shift means that the carboxyl
group was completely converted to carboxylate ions (Fig. 4A) [28].
In addition, the XPS spectra of MSN-Pt as well as the carrier MSN-
COOH were conducted. As shown in Fig. 5A, the spectra of MSN-Pt
exhibited two strong Pt4f signals in comparison with MSN-COOH.
This suggested that the conjugates were successfully prepared. The
platinum content of MSN-Pt was determined to be 7.9 wt% by ICP-
MS, corresponding to a high equivalent loading of oxaliplatin of
16.1%.
The release profile of platinum drugs from the MSN-Pt in acetate
buffer solution (pH 5.0) and PBS (pH 7.4) were evaluated by dialysis
method. The release amount of platinum was determined by ICP-
MS, and the weight ratio of cumulative released platinum to the
total platinum payload in the MSN-Pt conjugates was calculated.
A sustained and pH-sensitive release manner was demonstrated
(Fig. 5B). Nearly 70% of the loaded platinum was released after 24 h
at pH 5.0, while at pH 7.4 the platinum release was only 20%. The
faster release rate under acidic conditions was the result of the pro-
tonation of the carboxyl groups, which lead to the dissociation of
active anticancer species DACH-Pt. This is beneficial to intracellu-
lar release of anticancer drugs due to the acidic environment in
endosomes and lysosomes (pH 5.0–5.5).
3.3. Cytotoxicity assay
The in vitro cytotoxicities of free oxaliplatin and MSN-Pt were
examined by MTT assay. HepG-2 cells were selected and cultured
with oxaliplatin and MSN-Pt in a series of doses (6.25, 12.5, 25,
50, 100 and 200 ␮M of Pt) for 48 h. The blank particle MSN-COOH
was similarly examined to evaluate its safety. The results are pre-
sented in Fig. 6. First of all, the cell viability after 48 h incubation
with the blank particles was above 90% even at the highest concen-
tration of 1.0 mg mL−1 , which demonstrated that the carrier itself
showed very little toxicity (Fig. 6A). As for the cytotoxicity of the
two drug formulations, free oxaliplatin and MSN-Pt all displayed a
dose-dependent cytotoxicity toward the HepG-2 cells (Fig. 6B). It
is worth noting that the IC50 (the half maximal inhibitory concen-
tration of drug) of MSN-Pt and free oxaliplatin were 25.95 ␮M and
39.29 ␮M, respectively, meaning that MSN-Pt exhibited a some-
what higher cytotoxicity. The improved cytotoxicity is supposed to
be caused by a totally different mechanism of internalization. In
contrast to free oxaliplatin which is known to enter cells by passive
79
diffusion, the MSN-Pt conjugates might be taken up through a more
efficient endocytosis route and then hydrolyzed in late endosomes
or lysosomes. This different route might result in a higher con-
centration of active DACH-Pt species in cells, which have a higher
cytotoxicity capacity than oxaliplatin. This could be further proved
by cellular internalization experiments in the next section.
3.4. Internalization and distribution in cells
Previous reports suggested that MSN were taken up into cells
via endocytosis and mainly located in the acidic compartments
such as endosomal and lysosomal vesicles which should facili-
tate the release of the anticancer drug [39,40]. To correlate the
improved cytotoxicity of the MSN-Pt that was mentioned above,
a co-localization study was carried out to determine whether the
conjugates accumulated within the lysosomes and endosomes after
endocytosis. After cells were incubated with MSN-Pt/FITC for 1 h
or 4 h, the endosomes and lysosomes were labeled with red flores-
cent Lyso-tracker Red. As shown in Fig. 7, the CLSM images clearly
revealed that after 1 h of incubation, the green FITC fluorescence
appeared within HepG-2 cells. With the incubation time prolonged
to 4 h, both the green and red fluorescence became stronger and
they were finely co-localized. It implied that the conjugates could
be taken up by cells through endocytosis efficiently and concen-
trated within endosomes and lysosomes of HepG-2 cells. Under
the acidic environment of the lysosomes, the active DACH-Pt could
subsequently be released quickly, which might be the reason for
the improved cytotoxicity of the MSN-Pt conjugates.
3.5. Bioactive platinum content in cells
To confirm the improved cytotoxicity of the MSN-Pt conju-
gates more directly, the amount of platinum which was uptaken
by HepG-2 cells and adducted with DNA, was assessed by ICP-MS.
The platinum content is expressed as “ng of platinum per mg of
proteins” and “pg of platinum per ␮g of DNA”. The results were
shown in Table 1. When cells were incubated with oxaliplatin and
MSN-Pt at an equivalent initial platinum concentration of 0.25 ␮M
for 1 h, the platinum contents in cells were 0.14 and 42.52 ng of
Table 1
The contents of bioactive platinum drugs in HepG-2 cells.
Sample Cell uptake [ng Pt adduct [pg
Pt/mg Protein] Pt/␮g DNA]
1h 4h 24 h
Oxaliplatin 0.14 0.69 2.72
MSN-Pt 42.52 80.11 9.53
80 H. He et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 75–81

Fig. 6. Cell viability of HepG-2 cells incubated with MSN-COOH (A), free oxaliplatin and MSN-Pt conjugates (B) after 48 h incubation. Data were presented as mean ± standard
deviation (n = 3).

Fig. 7. CLSM images of HepG-2 cells incubated with MSN-Pt/FITC for 1 h and 4 h. For each row, images from left to right were: nucleus stained with DAPI (blue), MSN-Pt/FITC
conjugates (green), late endosomes and lysosomes stained with Lyso-tracker Red (red) and overlaid images. Bar = 10 ␮m. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of the article.)

Pt/mg Protein, respectively. When the incubation time prolonged
to 4 h, the platinum contents in cells increased to 0.69 and 80.11 ng
of Pt/mg Protein, respectively. These data directly indicated that the
platinum delivery through MSN-Pt conjugates into cells has much
higher efficiency than that of oxaliplatin for HepG-2 cells.
It is well-known that platinum(II) drugs exert their cytotoxicity
by binding DNA and cause apoptosis of cancer cells. The amount of
platinum to form Pt-DNA adducts is the most direct measurement
of drug bioactivity. We incubated HepG-2 cells with free oxalip-
latin and MSN-Pt conjugates for 24 h, followed by collecting and
purifying the total DNA of the treated cells. For free oxaliplatin
and MSN-Pt conjugates groups, the platinum contents in DNA were
measured to be 2.72 and 9.53 pg Pt/␮g DNA, respectively. The larger
amount of the platinum in the DNA sample owing by MSN-Pt con-
jugates would be a really reason of their improved therapeutically
effective. More important, the enhanced accumulation of platinum
content in cells and DNA samples has the obvious potential in solv-
ing platinum drug resistance.
4. Conclusions
In this paper, we prepared MSN-Pt conjugates based on oxalipla-
tin drugs with mesoporous silica nanoparticles as drug carriers. The
drug carriers MSN-COOH bearing 1,2-bidentate carboxyl groups
were synthesized via a simple and convenient protocol, and subse-
quent conjugation with platinum drugs were successful to give the
direct binding of oxaliplatin molecules with MSN. The conjugates
were taken up fast by HepG-2 cells and had concentrated within
endosomes and lysosomes. They displayed an improved cytotox-
icity against HepG-2 cells than that of free oxaliplatin. The higher
level of platinum content both in cells and DNA might be the reason
of their improved therapeutically effective. These results support
that the platinum loaded MSN conjugates should have great poten-
tial applications in cancer therapy with reduced side effects and
enhanced therapeutical efficacy.
Acknowledgments
The authors would like to thank the financial support from
National Natural Science Foundation of China (Nos. 51321062,
51273194 and 21174143), the Ministry of Science and Technol-
ogy of China (973 Project, No. 2009CB930102; 863 Project, No.
2012AA021900), “100 Talents Program” of the Chinese Academy
of Sciences (No. KGCX2-YW-802), and Jilin Provincial Science and
Technology Department (No. 20100588).
 H. He et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 75–81 81

References [22] J. Extra, M. Espie, F. Calvo, C. Ferme, L. Mignot, M. Marty, Cancer Chemother.
Pharmacol. 25 (1990) 299–303.
[1] R.A. Alderden, M.D. Hall, T.W. Hambley, J. Chem. Educ. 83 (2006) 728–734. [23] A.S. Abu-Surrah, M. Kettunen, Curr. Med. Chem. 13 (2006) 1337–1357.
[2] R.B. Weiss, M.C. Christian, Drugs 46 (1993) 360–377. [24] R. Wang, X. Hu, S. Wu, H. Xiao, H. Cai, Z. Xie, Y. Huang, X. Jing, Mol. Pharmacol.
[3] N.E. Madias, J.T. Harrington, Am. J. Med. 65 (1978) 307–314. 9 (2012) 3200–3208.
[4] A. Ekborn, G. Laurell, A. Andersson, I. Wallin, S. Eksborg, H. Ehrsson, Hear. Res. [25] H. Xiao, W. Li, R. Qi, L. Yan, R. Wang, S. Liu, Y. Zheng, Z. Xie, Y. Huang, X. Jing, J.
140 (2000) 38–44. Control. Release 163 (2012) 304–314.
[5] M. Yokoyama, T. Okano, Y. Sakurai, H. Ekimoto, C. Shibazaki, K. Kataoka, Cancer [26] A. Paraskar, S. Soni, B. Roy, A.L. Papa, S. Sengupta, Nanotechnology 23 (2012)
Res. 51 (1991) 3229–3236. 075103.
[6] Y. Matsumura, H. Maeda, Cancer Res. 46 (1986) 6387–6392. [27] L. Han, O. Terasaki, S. Che, J. Mater. Chem. 21 (2011) 11033–11039.
[7] H. Xiao, D. Zhou, S. Liu, R. Qi, Y. Zheng, Y. Huang, X. Jing, Macromol. Biosci. 12 [28] Z. Yan, S. Tao, J. Yin, G. Li, J. Mater. Chem. 16 (2006) 2347–2353.
(2012) 367–373. [29] C.E. Hoyle, C.N. Bowman, Angew. Chem. Int. Ed. 49 (2010) 1540–1573.
[8] H. Cabral, N. Nishiyama, K. Kataoka, J. Control. Release 121 (2007) 146–155. [30] H. Xiao, D. Zhou, S. Liu, Y. Zheng, Y. Huang, X. Jing, Acta Biomater. 8 (2012)
[9] D. Zhou, H. Xiao, F. Meng, S. Zhou, J. Guo, X. Li, X. Jing, Y. Huang, Bioconjug. 1859–1868.
Chem. 23 (2012) 2335–2343. [31] C.Y. Lai, B.G. Trewyn, D.M. Jeftinija, K. Jeftinija, S. Xu, S. Jeftinija, V.S.Y. Lin, J. Am.
[10] S.D. Brown, P. Nativo, J.-A. Smith, D. Stirling, P.R. Edwards, B. Venugopal, Chem. Soc. 125 (2003) 4451–4459.
D.J. Flint, J.A. Plumb, D. Graham, N.J. Wheate, J. Am. Chem. Soc. 132 (2010) [32] Y. Zhu, Y. Fang, L. Borchardt, S. Kaskel, Microporous Mesoporous Mater. 141
4678–4684. (2011) 199–206.
[11] S. Dhar, W.L. Daniel, D.A. Giljohann, C.A. Mirkin, S.J. Lippard, J. Am. Chem. Soc. [33] Y. Chen, H. Chen, L. Guo, Q. He, F. Chen, J. Zhou, J. Feng, J. Shi, ACS Nano 4 (2009)
131 (2009) 14652–14653. 529–539.
[12] C. Xu, B. Wang, S. Sun, J. Am. Chem. Soc. 131 (2009) 4216–4217. [34] C. Peng, H. Zhang, J. Yu, Q. Meng, L. Fu, H. Li, L. Sun, X. Guo, J. Phys. Chem. B 109
[13] J.D. Rocca, R.C. Huxford, E. Comstock-Duggan, W. Lin, Angew. Chem. Int. Ed. 50 (2005) 15278–15287.
(2011) 10330–10334. [35] F. Carniato, C. Bisio, G. Paul, G. Gatti, L. Bertinetti, S. Coluccia, L. Marchese, J.
[14] R.P. Feazell, N. Nakayama-Ratchford, H. Dai, S.J. Lippard, J. Am. Chem. Soc. 129 Mater. Chem. 20 (2010) 5504–5509.
(2007) 8438–8439. [36] V. Cauda, C. Argyo, A. Schlossbauer, T. Bein, J. Mater. Chem. 20 (2010)
[15] F. Tang, L. Li, D. Chen, Adv. Mater. 24 (2012) 1504–1534. 4305–4311.
[16] Z. Li, J.C. Barnes, A. Bosoy, J.F. Stoddart, J.I. Zink, Chem. Soc. Rev. 41 (2012) [37] R. Casasús, E. Climent, M.D. Marcos, R. Martínez-Máñez, F. Sancenón,
2590–2605. J. Soto, P. Amorós, J. Cano, E. Ruiz, J. Am. Chem. Soc. 130 (2008)
[17] T.W. Kim, I.I. Slowing, P.W. Chung, V.S.-Y. Lin, ACS Nano 5 (2010) 360–366. 1903–1917.
[18] S.P. Hudson, R.F. Padera, R. Langer, D.S. Kohane, Biomaterials 29 (2008) [38] A. Ibrahim, S. Hirschfeld, M.H. Cohen, D.J. Griebel, G.A. Williams, R. Pazdur,
4045–4055. Oncologist 9 (2004) 8–12.
[19] Z. Tao, B. Toms, J. Goodisman, T. Asefa, ACS Nano 4 (2010) 789–794. [39] I.I. Slowing, J.L. Vivero-Escoto, C.W. Wu, V.S.Y. Lin, Adv. Drug Deliv. Rev. 60
[20] C.H. Lin, S.H. Cheng, W.N. Liao, P.R. Wei, P.J. Sung, C.F. Weng, C.H. Lee, Int. J. (2008) 1278–1288.
Pharm. 429 (2012) 138–147. [40] Y. Klichko, M. Liong, E. Choi, S. Angelos, A.E. Nel, J.F. Stoddart, F. Tamanoi, J.I.
[21] J. Gu, S. Su, Y. Li, Q. He, J. Zhong, J. Shi, J. Phys. Chem. Lett. 1 (2010) 3446–3450. Zink, J. Am. Ceram. Soc. 92 (2009) S2–S10.